@article{rodriguez_comparative_2026,
	title = {Comparative regulomics of wood formation across dicot and conifer trees},
	copyright = {2026 The Author(s)},
	issn = {2041-1723},
	url = {https://www.nature.com/articles/s41467-026-75624-2},
	doi = {10.1038/s41467-026-75624-2},
	abstract = {Understanding the regulatory program underlying wood formation is key to improving biomass production and carbon sequestration in trees. However, how wood formation evolved and how these programs have been rewired across lineages remains unclear. Here, we present the first high-spatial-resolution evo-devo resource for wood transcriptomes spanning multiple dicots and conifers, representing the two major tree-containing lineages separated by more than 300 million years of evolution. Using orthology-aware co-expression network analysis, we identified genes with conserved and lineage-specific expression patterns. By integrating chromatin accessibility data and transcription factor motif analysis, we further inferred candidate regulatory networks for xylem differentiation and secondary cell wall formation. We demonstrate how this dataset can be used to answer long standing questions in wood biology related to differences in acetylation of cell wall polymers and master regulators of xylem specification across dicot and conifer tree species. The data offer a resource for the tree biology and evo-devo communities, and are publicly available at PlantGenIE.org.},
	language = {en},
	urldate = {2026-07-24},
	journal = {Nature Communications},
	publisher = {Nature Publishing Group},
	author = {Rodriguez, Eduardo and Birkeland, Siri and Chapple, Ellen Dimmen and Fredriksson, Samuel and Carracedo Lorenzo, Zulema and Ahlgren Kalman, Teitur and Kumar, Vikash and Mccann, Jamie and Hill, Jason and Soundiramourtty, Sivagamy and Voxeur, Aline and Røhr, Åsmund Kjendseth and Tuominen, Hannele and Mellerowicz, Ewa J. and Street, Nathaniel R. and Hvidsten, Torgeir R.},
	month = jul,
	year = {2026},
	keywords = {Comparative genomics, Gene regulatory networks, Plant evolution, Plant genetics},
}

Understanding the regulatory program underlying wood formation is key to improving biomass production and carbon sequestration in trees. However, how wood formation evolved and how these programs have been rewired across lineages remains unclear. Here, we present the first high-spatial-resolution evo-devo resource for wood transcriptomes spanning multiple dicots and conifers, representing the two major tree-containing lineages separated by more than 300 million years of evolution. Using orthology-aware co-expression network analysis, we identified genes with conserved and lineage-specific expression patterns. By integrating chromatin accessibility data and transcription factor motif analysis, we further inferred candidate regulatory networks for xylem differentiation and secondary cell wall formation. We demonstrate how this dataset can be used to answer long standing questions in wood biology related to differences in acetylation of cell wall polymers and master regulators of xylem specification across dicot and conifer tree species. The data offer a resource for the tree biology and evo-devo communities, and are publicly available at PlantGenIE.org.

@article{birkeland_updated_2025,
	title = {An updated perspective: what genes make a tree a tree?},
	issn = {1360-1385},
	shorttitle = {An updated perspective},
	url = {https://www.sciencedirect.com/science/article/pii/S1360138525002821},
	doi = {10.1016/j.tplants.2025.09.006},
	abstract = {We learn early on how to tell trees apart from other plants. However, it has proved hard to distinguish trees from other plants at the genetic level, and it is believed that there are no unique ‘tree genes’. With the rapid increase in available tree genomes, we can perform new comparative and evolutionary analyses of plant life histories and growth forms. Here we provide a fresh perspective on the genetic foundation for woodiness and perenniality in angiosperms by analyzing selection pressures and gene family evolution in the rosids using genomic data. We examine genes distinguishing trees from herbs and discuss future directions for uncovering the genetic factors that define a tree in this new era of tree genomics.},
	urldate = {2025-10-24},
	journal = {Trends in Plant Science},
	author = {Birkeland, Siri and Soldado, Eduardo R. and Ranade, Sonali S. and García-Gil, M. Rosario and Choudhary, Shruti and Kumar, Vikash and Tuominen, Hannele and Mellerowicz, Ewa J. and Street, Nathaniel R. and Hvidsten, Torgeir R.},
	month = oct,
	year = {2025},
	keywords = {comparative genomics, plant growth forms, plant life histories, rosids, tree genomics, woodiness},
}

We learn early on how to tell trees apart from other plants. However, it has proved hard to distinguish trees from other plants at the genetic level, and it is believed that there are no unique ‘tree genes’. With the rapid increase in available tree genomes, we can perform new comparative and evolutionary analyses of plant life histories and growth forms. Here we provide a fresh perspective on the genetic foundation for woodiness and perenniality in angiosperms by analyzing selection pressures and gene family evolution in the rosids using genomic data. We examine genes distinguishing trees from herbs and discuss future directions for uncovering the genetic factors that define a tree in this new era of tree genomics.

@article{sivan_carbohydrate-binding_2025,
	title = {Carbohydrate-binding domain {CBM63} of microbial expansin-like {BsEXLX1} facilitates the adsorption of expansin-related proteins to hemicelluloses in plant secondary cell walls},
	volume = {18},
	issn = {2731-3654},
	url = {https://doi.org/10.1186/s13068-025-02674-x},
	doi = {10.1186/s13068-025-02674-x},
	abstract = {Overcoming lignocellulose recalcitrance to enzymatic or chemical processing is a prerequisite for biorefinery applications. Expansins and loosenins are non-lytic proteins that could assist reducing this recalcitrance by disrupting the intermolecular contacts between plant cell wall components. Here, immunolocalization with fluorescence and transmission electron microscopy (TEM) were used to study the ability of a Bacillus subtilis expansin-like protein (BsEXLX1), a Phanerochaete carnosa loosenin protein (PcaLOOL12) and a fusion protein of PcaLOOL12 with the carbohydrate-binding module 63 (CBM63) of BsEXLX1 (i.e., PcaLOOL12-CBM63) to bind secondary cell walls (SCW) of aspen fibres, including fresh aspen wood, milled wood fibres (MWF) and MWF subjected to subcritical water extraction.},
	number = {1},
	urldate = {2025-07-25},
	journal = {Biotechnology for Biofuels and Bioproducts},
	author = {Sivan, Pramod and Dahiya, Deepika and Jabalera, Ylenia and Koitto, Taru and Perez-Jimenez, Raul and Mellerowicz, Ewa J. and Master, Emma and Vilaplana, Francisco},
	month = jul,
	year = {2025},
	keywords = {Expansin, Hemicelluloses, Loosenin, Secondary cell wall},
	pages = {70},
}

Overcoming lignocellulose recalcitrance to enzymatic or chemical processing is a prerequisite for biorefinery applications. Expansins and loosenins are non-lytic proteins that could assist reducing this recalcitrance by disrupting the intermolecular contacts between plant cell wall components. Here, immunolocalization with fluorescence and transmission electron microscopy (TEM) were used to study the ability of a Bacillus subtilis expansin-like protein (BsEXLX1), a Phanerochaete carnosa loosenin protein (PcaLOOL12) and a fusion protein of PcaLOOL12 with the carbohydrate-binding module 63 (CBM63) of BsEXLX1 (i.e., PcaLOOL12-CBM63) to bind secondary cell walls (SCW) of aspen fibres, including fresh aspen wood, milled wood fibres (MWF) and MWF subjected to subcritical water extraction.

@article{biswal_ectopic_2025,
	title = {Ectopic expression of pectate lyase \textit{{Ptxt}}{PL1}-27 in aspen affects leaf cuticle development},
	volume = {28},
	issn = {2589-0042},
	url = {https://www.sciencedirect.com/science/article/pii/S2589004225022242},
	doi = {10.1016/j.isci.2025.113963},
	abstract = {Cuticle - a hydrophobic barrier of cutin and waxes covering the outer cell wall surface of plants - enables survival in terrestrial habitats. However, it is not understood how the hydrophobic cuticle precursors travel through the homogalacturonan-rich hydrophilic cell wall. To elucidate the role of homogalacturonan in cuticle development, we disrupted its integrity by overexpressing a pectate lyase, PtxtPL1-27, in aspen. PtxtPL1-27 had pleiotropic effects on shoot development, including the reduction of cuticle thickness and changes in cutin and wax composition, but the expression of cutin biosynthetic genes was little affected. Despite a reduction in homogalacturonan content in the leaves, labeling with the homogalacturonan-specific antibody JIM5 in the outer epidermal cell wall layer increased and displayed an altered pattern. Moreover, the ultrastructure of cell walls was changed concomitant with lipid accumulation. We propose that the disruption of homogalacturonan integrity affected the cutinsome-dependent transport and polymerization of cutin monomers in the cell wall.},
	number = {12},
	urldate = {2025-11-28},
	journal = {iScience},
	author = {Biswal, Ajaya K. and Banasiak, Alicja and Fernández-Moreno, Josefina-Patricia and Mitra, Madhusree and Harholt, Jesper and Derba-Maceluch, Marta and Majda, Mateusz and Kushwah, Sunita and Kumar, Vikash and Abreu, Ilka and Sivan, Pramod and Pattathil, Sivakumar and Immerzeel, Peter and Gorzsás, András and Moritz, Thomas and Hahn, Michael G. and Scheller, Henrik Vibe and Aharoni, Asaph and Mellerowicz, Ewa J.},
	month = dec,
	year = {2025},
	keywords = {Plant Biology, Plant anatomy, Plant development, Plant physiology},
	pages = {113963},
}

Cuticle - a hydrophobic barrier of cutin and waxes covering the outer cell wall surface of plants - enables survival in terrestrial habitats. However, it is not understood how the hydrophobic cuticle precursors travel through the homogalacturonan-rich hydrophilic cell wall. To elucidate the role of homogalacturonan in cuticle development, we disrupted its integrity by overexpressing a pectate lyase, PtxtPL1-27, in aspen. PtxtPL1-27 had pleiotropic effects on shoot development, including the reduction of cuticle thickness and changes in cutin and wax composition, but the expression of cutin biosynthetic genes was little affected. Despite a reduction in homogalacturonan content in the leaves, labeling with the homogalacturonan-specific antibody JIM5 in the outer epidermal cell wall layer increased and displayed an altered pattern. Moreover, the ultrastructure of cell walls was changed concomitant with lipid accumulation. We propose that the disruption of homogalacturonan integrity affected the cutinsome-dependent transport and polymerization of cutin monomers in the cell wall.

@article{derba-maceluch_glucuronoyl_2025,
	title = {Glucuronoyl {Esterase} {Expressed} in {Aspen} {Xylem} {Affects} γ-{Ester} {Linkages} {Between} {Lignin} and {Glucuronoxylan} {Reducing} {Recalcitrance} and {Accelerating} {Growth}},
	volume = {23},
	copyright = {© 2025 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley \& Sons Ltd.},
	issn = {1467-7652},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.70301},
	doi = {10.1111/pbi.70301},
	abstract = {Wood is the most abundant renewable natural resource composed of different polysaccharides and lignin, but its utilisation is hampered by intermolecular linkages between these components forming lignin-carbohydrate complexes (LCCs) causing recalcitrance. The links between glucuronoxylan and the γ-C of lignin (γ-ester linkages) are thought to contribute to one-third of LCCs, but direct evidence for their natural occurrence and their role in recalcitrance has been scarce so far. To address these issues, Phanerochaete carnosa glucuronoyl esterase (PcGCE), hydrolysing γ-ester linkages, was expressed in cell walls of developing wood in hybrid aspen (Populus tremula L. × tremuloides Michx.). The enzyme reduced HSQC 2D NMR signals corresponding to the γ-esters and xylan in dioxane-extracted LCCs without altering glucuronoxylan content or structure. This increased acid solubility of lignin and decreased lignin content. Reduced wood recalcitrance was shown by increased sugar yields and glucose production rates (by approx. 20\%) in saccharification without pretreatment and increased xylan extractability by subcritical water (by approx. 70\%). Moreover, trees expressing PcGCE exhibited greater primary and secondary growth. Transcriptomics and metabolomics analyses in developing wood suggested that growth could have been induced by a higher transcription of SMR2 and RPOTmp, which was likely triggered by the secondary cell wall integrity signalling. The results provide evidence for the natural existence of LCC γ-esters and their significant contribution to lignocellulose recalcitrance. Furthermore, they show that reducing γ-ester linkages could increase plant productivity.},
	language = {en},
	number = {12},
	urldate = {2025-12-05},
	journal = {Plant Biotechnology Journal},
	author = {Derba-Maceluch, Marta and García Romañach, Laura and Hedenström, Mattias and Mitra, Madhusree and Donev, Evgeniy N. and Urbancsok, János and Yassin, Zakiya and Gandla, Madhavi L. and Sivan, Pramod and Šimura, Jan and Scheepers, Gerhard and Jönsson, Leif J. and Vilaplana, Francisco and Mellerowicz, Ewa J.},
	year = {2025},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.70301},
	keywords = {ce15, glucuronoxylan, glucuronoyl esterase, hardwood genetic engineering, lignin-carbohydrate complexes, lignocellulose, secondary cell wall},
	pages = {5417--5434},
}

Wood is the most abundant renewable natural resource composed of different polysaccharides and lignin, but its utilisation is hampered by intermolecular linkages between these components forming lignin-carbohydrate complexes (LCCs) causing recalcitrance. The links between glucuronoxylan and the γ-C of lignin (γ-ester linkages) are thought to contribute to one-third of LCCs, but direct evidence for their natural occurrence and their role in recalcitrance has been scarce so far. To address these issues, Phanerochaete carnosa glucuronoyl esterase (PcGCE), hydrolysing γ-ester linkages, was expressed in cell walls of developing wood in hybrid aspen (Populus tremula L. × tremuloides Michx.). The enzyme reduced HSQC 2D NMR signals corresponding to the γ-esters and xylan in dioxane-extracted LCCs without altering glucuronoxylan content or structure. This increased acid solubility of lignin and decreased lignin content. Reduced wood recalcitrance was shown by increased sugar yields and glucose production rates (by approx. 20%) in saccharification without pretreatment and increased xylan extractability by subcritical water (by approx. 70%). Moreover, trees expressing PcGCE exhibited greater primary and secondary growth. Transcriptomics and metabolomics analyses in developing wood suggested that growth could have been induced by a higher transcription of SMR2 and RPOTmp, which was likely triggered by the secondary cell wall integrity signalling. The results provide evidence for the natural existence of LCC γ-esters and their significant contribution to lignocellulose recalcitrance. Furthermore, they show that reducing γ-ester linkages could increase plant productivity.

@article{donev_glucuronoyl_2025,
	title = {Glucuronoyl {Esterase} of {Pathogenic} {Phanerochaete} carnosa {Induces} {Immune} {Responses} in {Aspen} {Independently} of {Its} {Enzymatic} {Activity}},
	volume = {n/a},
	copyright = {© 2025 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley \& Sons Ltd.},
	issn = {1467-7652},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.70357},
	doi = {10.1111/pbi.70357},
	abstract = {Microbial enzymes expressed in plants add new functionalities but occasionally trigger undesirable immune responses. Phanerochaete carnosa glucuronoyl esterase (PcGCE) hydrolyses the bond between lignin and 4-O-methyl-α-D-glucuronic acid substituent of glucuronoxylan. PcGCE constitutively expressed in Arabidopsis or hybrid aspen (Populus tremula × tremuloides) improved saccharification but also induced premature leaf senescence. To understand what triggered this senescence, we characterised PcGCE-expressing hybrid aspen by microscopy and omics approaches, supplemented by grafting and recombinant protein application experiments. PcGCE induced massive immune responses followed by senescence in the leaves. Expressing an inactive (PcGCES217A) enzyme has led to similar phenotypes, excluding a possibility that damage-associated molecular patterns (DAMPs) released by glucuronoyl esterase triggered immune responses. Grafting experiments showed that PcGCE transcripts are not mobile but they induce systemic responses. Recombinant PcGCE protein applied to leaves did not induce such responses; thus, PcGCE is probably not perceived as a pathogen-associated molecular pattern (PAMP). We suggest that the observed high expression of PcGCE from the 35S promoter triggers the unfolded protein response. Indeed, restricting PcGCE expression to short-lived xylem cells by using the wood-specific promoter avoided all detrimental effects. Thus, wood-specific expression is a viable strategy for PcGCE deployment in planta, which might be applicable for other stress-inducing proteins.},
	language = {en},
	number = {n/a},
	urldate = {2025-09-19},
	journal = {Plant Biotechnology Journal},
	author = {Donev, Evgeniy N. and Derba-Maceluch, Marta and Liu, Xiao-Kun and Bwanika, Henri Colyn and Dobrowolska, Izabela and Thapa, Mohit and Leśniewska, Joanna and Šimura, Jan and Yi-Lin Tsai, Alex and Krajewski, Konrad S. and Boström, Dan and Kleczkowski, Leszek A. and Eriksson, Maria E. and Ljung, Karin and Master, Emma R. and Mellerowicz, Ewa J.},
	year = {2025},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.70357},
	keywords = {PTI, Populus, biotic stress, glucuronoyl esterase, lignocellulose improvement, transgenic crops, unfolded protein response},
}

Microbial enzymes expressed in plants add new functionalities but occasionally trigger undesirable immune responses. Phanerochaete carnosa glucuronoyl esterase (PcGCE) hydrolyses the bond between lignin and 4-O-methyl-α-D-glucuronic acid substituent of glucuronoxylan. PcGCE constitutively expressed in Arabidopsis or hybrid aspen (Populus tremula × tremuloides) improved saccharification but also induced premature leaf senescence. To understand what triggered this senescence, we characterised PcGCE-expressing hybrid aspen by microscopy and omics approaches, supplemented by grafting and recombinant protein application experiments. PcGCE induced massive immune responses followed by senescence in the leaves. Expressing an inactive (PcGCES217A) enzyme has led to similar phenotypes, excluding a possibility that damage-associated molecular patterns (DAMPs) released by glucuronoyl esterase triggered immune responses. Grafting experiments showed that PcGCE transcripts are not mobile but they induce systemic responses. Recombinant PcGCE protein applied to leaves did not induce such responses; thus, PcGCE is probably not perceived as a pathogen-associated molecular pattern (PAMP). We suggest that the observed high expression of PcGCE from the 35S promoter triggers the unfolded protein response. Indeed, restricting PcGCE expression to short-lived xylem cells by using the wood-specific promoter avoided all detrimental effects. Thus, wood-specific expression is a viable strategy for PcGCE deployment in planta, which might be applicable for other stress-inducing proteins.

@article{renstrom_high-resolution_2025,
	title = {High-resolution imaging of the physical and chemical properties of {Populus} wood using {SilviScan}™ and near-infrared spectroscopy},
	volume = {-1},
	issn = {0928-1541, 2294-1932},
	url = {https://brill.com/view/journals/iawa/aop/article-10.1163-22941932-bja10179/article-10.1163-22941932-bja10179.xml},
	doi = {10.1163/22941932-bja10179},
	abstract = {Summary Spatial information on wood structure and chemistry is crucial for understanding wood functionality. We present a high-throughput and high-resolution near-infrared (NIR) method for combined imaging of the physical and chemical properties of stem sections from Populus trees. Pyrolysis-GC/MS data was used for sensitive and spatially resolved calibration of wood chemistry while SilviScan™ analyses provided reference data for wood physical properties with 25 μm resolution for wood density and 0.2–2.0 mm for microfibril angle (MFA). NIR prediction models were trained and calibrated on material from both field- and greenhouse-grown trees. Thus, the method was developed for NIR imaging of stem samples as small as 4 mm in diameter with an image resolution of 0.03 mm for small-diameter samples and 0.5 mm for samples with multiple annual rings. The NIR model performance, tested against data not used in the training set, reached the coefficient of determination ( R pred 2 ) values for wood density and MFA of 0.60 and 0.72, respectively. The NIR models for wood chemistry showed R pred 2 values of 0.78 and 0.77 for carbohydrates and lignin, respectively. Models for the G-, S- and H-type lignin had R pred 2 values between 0.58 and 0.86. In addition, we developed a prediction model for the determination of tension wood distribution. According to this model, tension wood was frequently observed in young greenhouse samples, which might explain the higher variation found in the chemical and physical properties of wood in greenhouse-grown compared to field-grown trees. The study also demonstrated that NIR-model estimations in image format can capture spatial variations that are not detectable in bulk analyses of wood properties. Examples of the method applied to greenhouse-grown trees highlight the efforts to develop NIR models with good prediction accuracies based on high-resolution data.},
	language = {eng},
	number = {aop},
	urldate = {2025-08-18},
	journal = {IAWA Journal},
	publisher = {Brill},
	author = {Renström, Anna and Scheepers, Gerhard and Yassin, Zakiya and Grahn, Thomas and Sivan, Pramod and Niittylä, Totte and Mellerowicz, Ewa J. and Tuominen, Hannele},
	month = feb,
	year = {2025},
	keywords = {NIR prediction models, NIR-imaging, Py-GC/MS, SilviScan, wood chemistry, wood properties},
	pages = {1--16},
}

Summary Spatial information on wood structure and chemistry is crucial for understanding wood functionality. We present a high-throughput and high-resolution near-infrared (NIR) method for combined imaging of the physical and chemical properties of stem sections from Populus trees. Pyrolysis-GC/MS data was used for sensitive and spatially resolved calibration of wood chemistry while SilviScan™ analyses provided reference data for wood physical properties with 25 μm resolution for wood density and 0.2–2.0 mm for microfibril angle (MFA). NIR prediction models were trained and calibrated on material from both field- and greenhouse-grown trees. Thus, the method was developed for NIR imaging of stem samples as small as 4 mm in diameter with an image resolution of 0.03 mm for small-diameter samples and 0.5 mm for samples with multiple annual rings. The NIR model performance, tested against data not used in the training set, reached the coefficient of determination ( R pred 2 ) values for wood density and MFA of 0.60 and 0.72, respectively. The NIR models for wood chemistry showed R pred 2 values of 0.78 and 0.77 for carbohydrates and lignin, respectively. Models for the G-, S- and H-type lignin had R pred 2 values between 0.58 and 0.86. In addition, we developed a prediction model for the determination of tension wood distribution. According to this model, tension wood was frequently observed in young greenhouse samples, which might explain the higher variation found in the chemical and physical properties of wood in greenhouse-grown compared to field-grown trees. The study also demonstrated that NIR-model estimations in image format can capture spatial variations that are not detectable in bulk analyses of wood properties. Examples of the method applied to greenhouse-grown trees highlight the efforts to develop NIR models with good prediction accuracies based on high-resolution data.

@article{sivan_modification_2025,
	title = {Modification of xylan in secondary walls alters cell wall biosynthesis and wood formation programs and improves saccharification},
	volume = {23},
	copyright = {© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley \& Sons Ltd.},
	issn = {1467-7652},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.14487},
	doi = {10.1111/pbi.14487},
	abstract = {Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. × tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.},
	language = {en},
	number = {1},
	urldate = {2024-10-25},
	journal = {Plant Biotechnology Journal},
	author = {Sivan, Pramod and Urbancsok, János and Donev, Evgeniy N. and Derba-Maceluch, Marta and Barbut, Félix R. and Yassin, Zakiya and Gandla, Madhavi L. and Mitra, Madhusree and Heinonen, Saara E. and Šimura, Jan and Cermanová, Kateřina and Karady, Michal and Scheepers, Gerhard and Jönsson, Leif J. and Master, Emma R. and Vilaplana, Francisco and Mellerowicz, Ewa J.},
	month = jan,
	year = {2025},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.14487},
	keywords = {Glucuronoxylan, fungal xylanases, lignocellulose, secondary cell wall, transgenic aspen, wood development},
	pages = {174--197},
}

Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. × tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.

@article{barbut_integrity_2024,
	title = {Integrity of xylan backbone affects plant responses to drought},
	volume = {15},
	issn = {1664-462X},
	url = {https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1422701/full},
	doi = {10.3389/fpls.2024.1422701},
	abstract = {{\textless}p{\textgreater}Drought is a major factor affecting crops, thus efforts are needed to increase plant resilience to this abiotic stress. The overlapping signaling pathways between drought and cell wall integrity maintenance responses create a possibility of increasing drought resistance by modifying cell walls. Here, using herbaceous and woody plant model species, {\textless}italic{\textgreater}Arabidopsis{\textless}/italic{\textgreater} and hybrid aspen, respectively, we investigated how the integrity of xylan in secondary walls affects the responses of plants to drought stress. Plants, in which secondary wall xylan integrity was reduced by expressing fungal GH10 and GH11 xylanases or by affecting genes involved in xylan backbone biosynthesis, were subjected to controlled drought while their physiological responses were continuously monitored by RGB, fluorescence, and/or hyperspectral cameras. For {\textless}italic{\textgreater}Arabidopsis{\textless}/italic{\textgreater}, this was supplemented with survival test after complete water withdrawal and analyses of stomatal function and stem conductivity. All {\textless}italic{\textgreater}Arabidopsis{\textless}/italic{\textgreater} xylan-impaired lines showed better survival upon complete watering withdrawal, increased stomatal density and delayed growth inhibition by moderate drought, indicating increased resilience to moderate drought associated with modified xylan integrity. Subtle differences were recorded between xylan biosynthesis mutants ({\textless}italic{\textgreater}irx9{\textless}/italic{\textgreater}, {\textless}italic{\textgreater}irx10{\textless}/italic{\textgreater} and {\textless}italic{\textgreater}irx14{\textless}/italic{\textgreater}) and xylanase-expressing lines. {\textless}italic{\textgreater}irx14{\textless}/italic{\textgreater} was the most drought resistant genotype, and the only genotype with increased lignin content and unaltered xylem conductivity despite its {\textless}italic{\textgreater}irx{\textless}/italic{\textgreater} phenotype. Rosette growth was more affected by drought in GH11- than in GH10-expressing plants. In aspen, mild downregulation of {\textless}italic{\textgreater}GT43B{\textless}/italic{\textgreater} and {\textless}italic{\textgreater}C{\textless}/italic{\textgreater} genes did not affect drought responses and the transgenic plants grew better than the wild-type in drought and well-watered conditions. Both GH10 and GH11 xylanases strongly inhibited stem elongation and root growth in well-watered conditions but growth was less inhibited by drought in GH11-expressing plants than in wild-type. Overall, plants with xylan integrity impairment in secondary walls were less affected than wild-type by moderately reduced water availability but their responses also varied among genotypes and species. Thus, modifying the secondary cell wall integrity can be considered as a potential strategy for developing crops better suited to withstand water scarcity, but more research is needed to address the underlying molecular causes of this variability.{\textless}/p{\textgreater}},
	language = {English},
	urldate = {2024-07-17},
	journal = {Frontiers in Plant Science},
	publisher = {Frontiers},
	author = {Barbut, Félix R. and Cavel, Emilie and Donev, Evgeniy N. and Gaboreanu, Ioana and Urbancsok, János and Pandey, Garima and Demailly, Hervé and Jiao, Dianyi and Yassin, Zakiya and Derba-Maceluch, Marta and Master, Emma R. and Scheepers, Gerhard and Gutierrez, Laurent and Mellerowicz, Ewa J.},
	month = jun,
	year = {2024},
	keywords = {Arabidopsis, Drought stress, Glucuronoxylan, Populus, cell wall integrity, high-throughput phenotyping, hyperspectral imaging, secondary cell wall},
}

\textlessp\textgreaterDrought is a major factor affecting crops, thus efforts are needed to increase plant resilience to this abiotic stress. The overlapping signaling pathways between drought and cell wall integrity maintenance responses create a possibility of increasing drought resistance by modifying cell walls. Here, using herbaceous and woody plant model species, \textlessitalic\textgreaterArabidopsis\textless/italic\textgreater and hybrid aspen, respectively, we investigated how the integrity of xylan in secondary walls affects the responses of plants to drought stress. Plants, in which secondary wall xylan integrity was reduced by expressing fungal GH10 and GH11 xylanases or by affecting genes involved in xylan backbone biosynthesis, were subjected to controlled drought while their physiological responses were continuously monitored by RGB, fluorescence, and/or hyperspectral cameras. For \textlessitalic\textgreaterArabidopsis\textless/italic\textgreater, this was supplemented with survival test after complete water withdrawal and analyses of stomatal function and stem conductivity. All \textlessitalic\textgreaterArabidopsis\textless/italic\textgreater xylan-impaired lines showed better survival upon complete watering withdrawal, increased stomatal density and delayed growth inhibition by moderate drought, indicating increased resilience to moderate drought associated with modified xylan integrity. Subtle differences were recorded between xylan biosynthesis mutants (\textlessitalic\textgreaterirx9\textless/italic\textgreater, \textlessitalic\textgreaterirx10\textless/italic\textgreater and \textlessitalic\textgreaterirx14\textless/italic\textgreater) and xylanase-expressing lines. \textlessitalic\textgreaterirx14\textless/italic\textgreater was the most drought resistant genotype, and the only genotype with increased lignin content and unaltered xylem conductivity despite its \textlessitalic\textgreaterirx\textless/italic\textgreater phenotype. Rosette growth was more affected by drought in GH11- than in GH10-expressing plants. In aspen, mild downregulation of \textlessitalic\textgreaterGT43B\textless/italic\textgreater and \textlessitalic\textgreaterC\textless/italic\textgreater genes did not affect drought responses and the transgenic plants grew better than the wild-type in drought and well-watered conditions. Both GH10 and GH11 xylanases strongly inhibited stem elongation and root growth in well-watered conditions but growth was less inhibited by drought in GH11-expressing plants than in wild-type. Overall, plants with xylan integrity impairment in secondary walls were less affected than wild-type by moderately reduced water availability but their responses also varied among genotypes and species. Thus, modifying the secondary cell wall integrity can be considered as a potential strategy for developing crops better suited to withstand water scarcity, but more research is needed to address the underlying molecular causes of this variability.\textless/p\textgreater

@article{chaudhari_modifying_2024,
	title = {Modifying lignin composition and xylan {O}-acetylation induces changes in cell wall composition, extractability, and digestibility},
	volume = {17},
	issn = {2731-3654},
	url = {https://doi.org/10.1186/s13068-024-02513-5},
	doi = {10.1186/s13068-024-02513-5},
	abstract = {Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.},
	number = {1},
	urldate = {2024-06-10},
	journal = {Biotechnology for Biofuels and Bioproducts},
	author = {Chaudhari, Aniket Anant and Sharma, Anant Mohan and Rastogi, Lavi and Dewangan, Bhagwat Prasad and Sharma, Raunak and Singh, Deepika and Sah, Rajan Kumar and Das, Shouvik and Bhattacharjee, Saikat and Mellerowicz, Ewa J. and Pawar, Prashant Anupama-Mohan},
	month = may,
	year = {2024},
	keywords = {Acetyl Xylan Esterase (AXE), G Lignin, Saccharification, Xylose release},
	pages = {73},
}

Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.

@article{sivan_unraveling_2024,
	title = {Unraveling the unique structural motifs of glucuronoxylan from hybrid aspen wood},
	volume = {343},
	issn = {01448617},
	url = {https://linkinghub.elsevier.com/retrieve/pii/S014486172400660X},
	doi = {10.1016/j.carbpol.2024.122434},
	language = {en},
	urldate = {2024-08-30},
	journal = {Carbohydrate Polymers},
	author = {Sivan, Pramod and Heinonen, Emilia and Escudero, Louis and Gandla, Madhavi Latha and Jiménez-Quero, Amparo and Jönsson, Leif J. and Mellerowicz, Ewa J. and Vilaplana, Francisco},
	month = nov,
	year = {2024},
	pages = {122434},
}

@article{bernacki_biotechnological_2023,
	title = {Biotechnological {Potential} of the {Stress} {Response} and {Plant} {Cell} {Death} {Regulators} {Proteins} in the {Biofuel} {Industry}},
	volume = {12},
	copyright = {http://creativecommons.org/licenses/by/3.0/},
	issn = {2073-4409},
	url = {https://www.mdpi.com/2073-4409/12/16/2018},
	doi = {10.3390/cells12162018},
	abstract = {Production of biofuel from lignocellulosic biomass is relatively low due to the limited knowledge about natural cell wall loosening and cellulolytic processes in plants. Industrial separation of cellulose fiber mass from lignin, its saccharification and alcoholic fermentation is still cost-ineffective and environmentally unfriendly. Assuming that the green transformation is inevitable and that new sources of raw materials for biofuels are needed, we decided to study cell death—a natural process occurring in plants in the context of reducing the recalcitrance of lignocellulose for the production of second-generation bioethanol. “Members of the enzyme families responsible for lysigenous aerenchyma formation were identified during the root hypoxia stress in Arabidopsis thaliana cell death mutants. The cell death regulatory genes, LESION SIMULATING DISEASE 1 (LSD1), PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) conditionally regulate the cell wall when suppressed in transgenic aspen. During four years of growth in the field, the following effects were observed: lignin content was reduced, the cellulose fiber polymerization degree increased and the growth itself was unaffected. The wood of transgenic trees was more efficient as a substrate for saccharification, alcoholic fermentation and bioethanol production. The presented results may trigger the development of novel biotechnologies in the biofuel industry.},
	language = {en},
	number = {16},
	urldate = {2023-08-31},
	journal = {Cells},
	publisher = {Multidisciplinary Digital Publishing Institute},
	author = {Bernacki, Maciej Jerzy and Mielecki, Jakub and Antczak, Andrzej and Drożdżek, Michał and Witoń, Damian and Dąbrowska-Bronk, Joanna and Gawroński, Piotr and Burdiak, Paweł and Marchwicka, Monika and Rusaczonek, Anna and Dąbkowska-Susfał, Katarzyna and Strobel, Wacław Roman and Mellerowicz, Ewa J. and Zawadzki, Janusz and Szechyńska-Hebda, Magdalena and Karpiński, Stanisław},
	month = aug,
	year = {2023},
	note = {Number: 16},
	keywords = {bioethanol, biofuels, cell death, cell wall, lignin, poplar, scarification},
	pages = {2018},
}

Production of biofuel from lignocellulosic biomass is relatively low due to the limited knowledge about natural cell wall loosening and cellulolytic processes in plants. Industrial separation of cellulose fiber mass from lignin, its saccharification and alcoholic fermentation is still cost-ineffective and environmentally unfriendly. Assuming that the green transformation is inevitable and that new sources of raw materials for biofuels are needed, we decided to study cell death—a natural process occurring in plants in the context of reducing the recalcitrance of lignocellulose for the production of second-generation bioethanol. “Members of the enzyme families responsible for lysigenous aerenchyma formation were identified during the root hypoxia stress in Arabidopsis thaliana cell death mutants. The cell death regulatory genes, LESION SIMULATING DISEASE 1 (LSD1), PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) conditionally regulate the cell wall when suppressed in transgenic aspen. During four years of growth in the field, the following effects were observed: lignin content was reduced, the cellulose fiber polymerization degree increased and the growth itself was unaffected. The wood of transgenic trees was more efficient as a substrate for saccharification, alcoholic fermentation and bioethanol production. The presented results may trigger the development of novel biotechnologies in the biofuel industry.

@article{donev_field_2023,
	title = {Field testing of transgenic aspen from large greenhouse screening identifies unexpected winners},
	volume = {21},
	issn = {1467-7652},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.14012},
	doi = {10.1111/pbi.14012},
	abstract = {Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.},
	language = {en},
	number = {5},
	urldate = {2023-04-21},
	journal = {Plant Biotechnology Journal},
	author = {Donev, Evgeniy N. and Derba-Maceluch, Marta and Yassin, Zakiya and Gandla, Madhavi Latha and Pramod, Sivan and Heinonen, Emilia and Kumar, Vikash and Scheepers, Gerhard and Vilaplana, Francisco and Johansson, Ulf and Hertzberg, Magnus and Sundberg, Björn and Winestrand, Sandra and Hörnberg, Andreas and Alriksson, Björn and Jönsson, Leif J. and Mellerowicz, Ewa J.},
	year = {2023},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.14012},
	keywords = {BET analysis, Populus, SilviScan, enzymatic saccharification, field trial, secondary cell wall, subcritical water extraction, transgenic Populus, transgenic trees, wood quality},
	pages = {1005--1021},
}

Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.

@article{urbancsok_flexure_2023,
	title = {Flexure wood formation via growth reprogramming in hybrid aspen involves jasmonates and polyamines and transcriptional changes resembling tension wood development},
	copyright = {New Phytologist© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation},
	issn = {1469-8137},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/nph.19307},
	doi = {10.1111/nph.19307},
	abstract = {Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques. Shaking increased primary and secondary growth and altered wood differentiation by stimulating gelatinous-fiber formation, reducing secondary wall thickness, changing matrix polysaccharides and increasing cellulose, G- and H-lignin contents, cell wall porosity and saccharification yields. Wood-forming tissues exhibited elevated jasmonate, polyamine, ethylene and brassinosteroids and reduced abscisic acid and gibberellin signaling. Transcriptional responses resembled those during tension wood formation but not opposite wood formation and revealed several thigmomorphogenesis-related genes as well as novel gene networks including FLA and XTH genes encoding plasma membrane-bound proteins. Low-intensity stem flexing stimulates growth and induces wood having improved biorefinery properties through molecular and hormonal pathways similar to thigmomorphogenesis in herbaceous plants and largely overlapping with the tension wood program of hardwoods.},
	language = {en},
	urldate = {2023-10-20},
	journal = {New Phytologist},
	author = {Urbancsok, János and Donev, Evgeniy N. and Sivan, Pramod and van Zalen, Elena and Barbut, Félix R. and Derba-Maceluch, Marta and Šimura, Jan and Yassin, Zakiya and Gandla, Madhavi L. and Karady, Michal and Ljung, Karin and Winestrand, Sandra and Jönsson, Leif J. and Scheepers, Gerhard and Delhomme, Nicolas and Street, Nathaniel R. and Mellerowicz, Ewa J.},
	month = oct,
	year = {2023},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/nph.19307},
	keywords = {Populus tremula × tremuloides, flexure wood, jasmonic acid signaling, mechanostimulation, polyamines, saccharification, thigmomorphogenesis, wood development},
}

Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques. Shaking increased primary and secondary growth and altered wood differentiation by stimulating gelatinous-fiber formation, reducing secondary wall thickness, changing matrix polysaccharides and increasing cellulose, G- and H-lignin contents, cell wall porosity and saccharification yields. Wood-forming tissues exhibited elevated jasmonate, polyamine, ethylene and brassinosteroids and reduced abscisic acid and gibberellin signaling. Transcriptional responses resembled those during tension wood formation but not opposite wood formation and revealed several thigmomorphogenesis-related genes as well as novel gene networks including FLA and XTH genes encoding plasma membrane-bound proteins. Low-intensity stem flexing stimulates growth and induces wood having improved biorefinery properties through molecular and hormonal pathways similar to thigmomorphogenesis in herbaceous plants and largely overlapping with the tension wood program of hardwoods.

@article{derba-maceluch_impact_2023,
	title = {Impact of xylan on field productivity and wood saccharification properties in aspen},
	volume = {14},
	issn = {1664-462X},
	url = {https://www.frontiersin.org/articles/10.3389/fpls.2023.1218302},
	doi = {10.3389/fpls.2023.1218302},
	abstract = {Xylan that comprises roughly 25\% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10\% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11\% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10\% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.},
	urldate = {2023-07-18},
	journal = {Frontiers in Plant Science},
	author = {Derba-Maceluch, Marta and Sivan, Pramod and Donev, Evgeniy N. and Gandla, Madhavi Latha and Yassin, Zakiya and Vaasan, Rakhesh and Heinonen, Emilia and Andersson, Sanna and Amini, Fariba and Scheepers, Gerhard and Johansson, Ulf and Vilaplana, Francisco J. and Albrectsen, Benedicte R. and Hertzberg, Magnus and Jönsson, Leif J. and Mellerowicz, Ewa J.},
	year = {2023},
}

Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.

@article{sivan_sequential_2023,
	title = {Sequential extraction of hemicelluloses by subcritical water improves saccharification of hybrid aspen wood grown in greenhouse and field conditions},
	volume = {25},
	issn = {1463-9270},
	url = {https://pubs.rsc.org/en/content/articlelanding/2023/gc/d3gc01020a},
	doi = {10.1039/D3GC01020A},
	abstract = {Fast growing hardwoods are one of the major renewable resources available to produce bio-based materials, platform chemicals and biofuels. However, the industrial processing of lignocellulosic biomass is hindered by the complex molecular structure of the cell wall components and their supramolecular organization. This highlights the necessity of improving green processing strategies to enhance biomass conversion to valuable products from industrial wood production species. In the present study, we implemented a hydrothermal step by sequential subcritical water (SW) in aspen wood prior to saccharification and validated the process for trees grown in greenhouse and field conditions. Subcritical water enables extraction of non-cellulosic cell wall polysaccharides in native polymeric form. A major part of the pectic fraction was easily extracted within the first 10 min, while acetylated xylan was enriched in the subsequent extracts after 20- and 30-min rounds. Prolonged extraction (above 60 min) resulted in partial deacetylation and a reduction of the molar mass of xylan. The analysis of the residues enriched with cellulose and lignin showed several micromorphological changes caused by subcritical water treatment, such as an increased porosity, a loosening of the fibre matrix and a decrease in the macrofibrillar dimensions. These morphological and molecular changes in the organization of cell wall polymers after SW treatment significantly enhanced saccharification yields compared to those of non-treated aspen wood chips from both field and greenhouse conditions. Our study demonstrates that SW can be implemented as pretreatment prior to saccharification reducing the requirements for chemical acid pretreatments. This process enables the extraction of native non-cellulosic cell wall polymers for potential material applications and promotes the subsequent biochemical conversion of the residual biomass into fermentable sugars and platform chemicals in future biorefineries.},
	language = {en},
	number = {14},
	urldate = {2023-07-18},
	journal = {Green Chemistry},
	publisher = {The Royal Society of Chemistry},
	author = {Sivan, Pramod and Heinonen, Emilia and Gandla, Madhavi Latha and Jiménez-Quero, Amparo and Özeren, Hüsamettin Deniz and Jönsson, Leif J. and Mellerowicz, Ewa J. and Vilaplana, Francisco},
	month = jul,
	year = {2023},
	pages = {5634--5646},
}

Fast growing hardwoods are one of the major renewable resources available to produce bio-based materials, platform chemicals and biofuels. However, the industrial processing of lignocellulosic biomass is hindered by the complex molecular structure of the cell wall components and their supramolecular organization. This highlights the necessity of improving green processing strategies to enhance biomass conversion to valuable products from industrial wood production species. In the present study, we implemented a hydrothermal step by sequential subcritical water (SW) in aspen wood prior to saccharification and validated the process for trees grown in greenhouse and field conditions. Subcritical water enables extraction of non-cellulosic cell wall polysaccharides in native polymeric form. A major part of the pectic fraction was easily extracted within the first 10 min, while acetylated xylan was enriched in the subsequent extracts after 20- and 30-min rounds. Prolonged extraction (above 60 min) resulted in partial deacetylation and a reduction of the molar mass of xylan. The analysis of the residues enriched with cellulose and lignin showed several micromorphological changes caused by subcritical water treatment, such as an increased porosity, a loosening of the fibre matrix and a decrease in the macrofibrillar dimensions. These morphological and molecular changes in the organization of cell wall polymers after SW treatment significantly enhanced saccharification yields compared to those of non-treated aspen wood chips from both field and greenhouse conditions. Our study demonstrates that SW can be implemented as pretreatment prior to saccharification reducing the requirements for chemical acid pretreatments. This process enables the extraction of native non-cellulosic cell wall polymers for potential material applications and promotes the subsequent biochemical conversion of the residual biomass into fermentable sugars and platform chemicals in future biorefineries.

@article{derba-maceluch_xylan_2023,
	title = {Xylan glucuronic acid side chains fix suberin-like aliphatic compounds to wood cell walls},
	volume = {238},
	issn = {1469-8137},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/nph.18712},
	doi = {10.1111/nph.18712},
	abstract = {Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin–carbohydrate complex assembly.},
	language = {en},
	number = {1},
	urldate = {2023-03-10},
	journal = {New Phytologist},
	author = {Derba-Maceluch, Marta and Mitra, Madhusree and Hedenström, Mattias and Liu, Xiaokun and Gandla, Madhavi L. and Barbut, Félix R. and Abreu, Ilka N. and Donev, Evgeniy N. and Urbancsok, János and Moritz, Thomas and Jönsson, Leif J. and Tsang, Adrian and Powlowski, Justin and Master, Emma R. and Mellerowicz, Ewa J.},
	month = jan,
	year = {2023},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/nph.18712},
	keywords = {Populus, lignin-carbohydrate complexes (LCCs), lignin–carbohydrate complexes, saccharification, suberin, wood cell wall, xylan},
	pages = {297--312},
}

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin–carbohydrate complex assembly.

@article{majda_elongation_2021,
	title = {Elongation of wood fibers combines features of diffuse and tip growth},
	issn = {0028-646X, 1469-8137},
	url = {https://onlinelibrary.wiley.com/doi/10.1111/nph.17468},
	doi = {10/gj3qbx},
	language = {en},
	urldate = {2021-06-03},
	journal = {New Phytologist},
	author = {Majda, Mateusz and Kozlova, Liudmila and Banasiak, Alicja and Derba‐Maceluch, Marta and Iashchishyn, Igor A. and Morozova‐Roche, Ludmilla A. and Smith, Richard S. and Gorshkova, Tatyana and Mellerowicz, Ewa J.},
	month = may,
	year = {2021},
	pages = {nph.17468},
}

@article{pramod_saccharification_2021,
	title = {Saccharification {Potential} of {Transgenic} {Greenhouse}- and {Field}-{Grown} {Aspen} {Engineered} for {Reduced} {Xylan} {Acetylation}},
	volume = {12},
	issn = {1664-462X},
	url = {https://www.frontiersin.org/article/10.3389/fpls.2021.704960},
	doi = {10.3389/fpls.2021.704960},
	abstract = {High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results.},
	urldate = {2021-09-30},
	journal = {Frontiers in Plant Science},
	author = {Pramod, Sivan and Gandla, Madhavi Latha and Derba-Maceluch, Marta and Jönsson, Leif J. and Mellerowicz, Ewa J. and Winestrand, Sandra},
	year = {2021},
	pages = {1722},
}

High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results.

@article{dominguez_sucrose_2021,
	title = {Sucrose synthase determines carbon allocation in developing wood and alters carbon flow at the whole tree level in aspen},
	volume = {229},
	issn = {0028-646X, 1469-8137},
	url = {https://onlinelibrary.wiley.com/doi/10.1111/nph.16721},
	doi = {10.1111/nph.16721},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {New Phytologist},
	author = {Dominguez, Pia Guadalupe and Donev, Evgeniy and Derba‐Maceluch, Marta and Bünder, Anne and Hedenström, Mattias and Tomášková, Ivana and Mellerowicz, Ewa J. and Niittylä, Totte},
	month = jan,
	year = {2021},
	pages = {186--198},
}

@article{kushwah_arabidopsis_2020,
	title = {Arabidopsis \textit{{XTH4}} and \textit{{XTH9}} {Contribute} to {Wood} {Cell} {Expansion} and {Secondary} {Wall} {Formation}},
	volume = {182},
	issn = {0032-0889, 1532-2548},
	url = {https://academic.oup.com/plphys/article/182/4/1946-1965/6116228},
	doi = {10.1104/pp.19.01529},
	language = {en},
	number = {4},
	urldate = {2021-06-07},
	journal = {Plant Physiology},
	author = {Kushwah, Sunita and Banasiak, Alicja and Nishikubo, Nobuyuki and Derba-Maceluch, Marta and Majda, Mateusz and Endo, Satoshi and Kumar, Vikash and Gomez, Leonardo and Gorzsas, Andras and McQueen-Mason, Simon and Braam, Janet and Sundberg, Björn and Mellerowicz, Ewa J.},
	month = apr,
	year = {2020},
	pages = {1946--1965},
}

@article{derba-maceluch_cell_2020,
	title = {Cell {Wall} {Acetylation} in {Hybrid} {Aspen} {Affects} {Field} {Performance}, {Foliar} {Phenolic} {Composition} and {Resistance} to {Biological} {Stress} {Factors} in a {Construct}-{Dependent} {Fashion}},
	volume = {11},
	issn = {1664-462X},
	url = {https://www.frontiersin.org/article/10.3389/fpls.2020.00651/full},
	doi = {10.3389/fpls.2020.00651},
	urldate = {2021-06-07},
	journal = {Frontiers in Plant Science},
	author = {Derba-Maceluch, Marta and Amini, Fariba and Donev, Evgeniy N. and Pawar, Prashant Mohan-Anupama and Michaud, Lisa and Johansson, Ulf and Albrectsen, Benedicte R. and Mellerowicz, Ewa J.},
	month = may,
	year = {2020},
	pages = {651},
}

@incollection{popper_expression_2020,
	address = {New York, NY},
	title = {Expression of {Cell} {Wall}–{Modifying} {Enzymes} in {Aspen} for {Improved} {Lignocellulose} {Processing}},
	volume = {2149},
	isbn = {978-1-0716-0619-3 978-1-0716-0621-6},
	url = {http://link.springer.com/10.1007/978-1-0716-0621-6_9},
	doi = {10.1007/978-1-0716-0621-6_9},
	language = {en},
	urldate = {2021-06-07},
	booktitle = {The {Plant} {Cell} {Wall}},
	publisher = {Springer New York},
	author = {Derba-Maceluch, Marta and Mellerowicz, Ewa J.},
	editor = {Popper, Zoë A.},
	year = {2020},
	note = {Series Title: Methods in Molecular Biology},
	pages = {145--164},
}

@article{baison_genetic_2020,
	title = {Genetic control of tracheid properties in {Norway} spruce wood},
	volume = {10},
	issn = {2045-2322},
	url = {http://www.nature.com/articles/s41598-020-72586-3},
	doi = {10.1038/s41598-020-72586-3},
	abstract = {Abstract
            
              Through the use of genome-wide association studies (GWAS) mapping it is possible to establish the genetic basis of phenotypic trait variation. Our GWAS study presents the first such effort in Norway spruce (
              Picea abies
              (L). Karst.) for the traits related to wood tracheid characteristics. The study employed an exome capture genotyping approach that generated 178 101 Single Nucleotide Polymorphisms (SNPs) from 40 018 probes within a population of 517 Norway spruce mother trees. We applied a least absolute shrinkage and selection operator (LASSO) based association mapping method using a functional multi-locus mapping approach, with a stability selection probability method as the hypothesis testing approach to determine significant Quantitative Trait Loci (QTLs). The analysis has provided 30 significant associations, the majority of which show specific expression in wood-forming tissues or high ubiquitous expression, potentially controlling tracheids dimensions, their cell wall thickness and microfibril angle. Among the most promising candidates based on our results and prior information for other species are:
              Picea abies BIG GRAIN 2
              (
              PabBG2)
              with a predicted function in auxin transport and sensitivity, and
              MA\_373300g0010
              encoding a protein similar to wall-associated receptor kinases, which were both associated with cell wall thickness. The results demonstrate feasibility of GWAS to identify novel candidate genes controlling industrially-relevant tracheid traits in Norway spruce.},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {Scientific Reports},
	author = {Baison, J. and Zhou, Linghua and Forsberg, Nils and Mörling, Tommy and Grahn, Thomas and Olsson, Lars and Karlsson, Bo and Wu, Harry X. and Mellerowicz, Ewa J. and Lundqvist, Sven-Olof and García-Gil, María Rosario},
	month = dec,
	year = {2020},
	pages = {18089},
}

Abstract Through the use of genome-wide association studies (GWAS) mapping it is possible to establish the genetic basis of phenotypic trait variation. Our GWAS study presents the first such effort in Norway spruce ( Picea abies (L). Karst.) for the traits related to wood tracheid characteristics. The study employed an exome capture genotyping approach that generated 178 101 Single Nucleotide Polymorphisms (SNPs) from 40 018 probes within a population of 517 Norway spruce mother trees. We applied a least absolute shrinkage and selection operator (LASSO) based association mapping method using a functional multi-locus mapping approach, with a stability selection probability method as the hypothesis testing approach to determine significant Quantitative Trait Loci (QTLs). The analysis has provided 30 significant associations, the majority of which show specific expression in wood-forming tissues or high ubiquitous expression, potentially controlling tracheids dimensions, their cell wall thickness and microfibril angle. Among the most promising candidates based on our results and prior information for other species are: Picea abies BIG GRAIN 2 ( PabBG2) with a predicted function in auxin transport and sensitivity, and MA_373300g0010 encoding a protein similar to wall-associated receptor kinases, which were both associated with cell wall thickness. The results demonstrate feasibility of GWAS to identify novel candidate genes controlling industrially-relevant tracheid traits in Norway spruce.

@article{kumar_genome-wide_2020,
	title = {Genome-{Wide} {Identification} of {Populus} {Malectin}/{Malectin}-{Like} {Domain}-{Containing} {Proteins} and {Expression} {Analyses} {Reveal} {Novel} {Candidates} for {Signaling} and {Regulation} of {Wood} {Development}},
	volume = {11},
	issn = {1664-462X},
	url = {https://www.frontiersin.org/articles/10.3389/fpls.2020.588846/full},
	doi = {10/gjcxjw},
	abstract = {Malectin domain (MD) is a ligand-binding protein motif of pro- and eukaryotes. It is particularly abundant in Viridiplantae, where it occurs as either a single (MD, PF11721) or tandemly duplicated domain (PF12819) called malectin-like domain (MLD). In herbaceous plants, MD- or MLD-containing proteins (MD proteins) are known to regulate development, reproduction, and resistance to various stresses. However, their functions in woody plants have not yet been studied. To unravel their potential role in wood development, we carried out genome-wide identification of MD proteins in the model tree species black cottonwood (
              Populus trichocarpa
              ), and analyzed their expression and co-expression networks.
              P. trichocarpa
              had 146
              MD
              genes assigned to 14 different clades, two of which were specific to the genus
              Populus
              . 87\% of these genes were located on chromosomes, the rest being associated with scaffolds. Based on their protein domain organization, and in agreement with the exon-intron structures, the
              MD
              genes identified here could be classified into five superclades having the following domains: leucine-rich repeat (LRR)-MD-protein kinase (PK), MLD-LRR-PK, MLD-PK (
              Cr
              RLK1L), MLD-LRR, and MD-Kinesin. Whereas the majority of
              MD
              genes were highly expressed in leaves, particularly under stress conditions, eighteen showed a peak of expression during secondary wall formation in the xylem and their co-expression networks suggested signaling functions in cell wall integrity, pathogen-associated molecular patterns, calcium, ROS, and hormone pathways. Thus,
              P. trichocarpa MD
              genes having different domain organizations comprise many genes with putative foliar defense functions, some of which could be specific to
              Populus
              and related species, as well as genes with potential involvement in signaling pathways in other tissues including developing wood.},
	urldate = {2021-06-03},
	journal = {Frontiers in Plant Science},
	author = {Kumar, Vikash and Donev, Evgeniy N. and Barbut, Félix R. and Kushwah, Sunita and Mannapperuma, Chanaka and Urbancsok, János and Mellerowicz, Ewa J.},
	month = dec,
	year = {2020},
	pages = {588846},
}

Malectin domain (MD) is a ligand-binding protein motif of pro- and eukaryotes. It is particularly abundant in Viridiplantae, where it occurs as either a single (MD, PF11721) or tandemly duplicated domain (PF12819) called malectin-like domain (MLD). In herbaceous plants, MD- or MLD-containing proteins (MD proteins) are known to regulate development, reproduction, and resistance to various stresses. However, their functions in woody plants have not yet been studied. To unravel their potential role in wood development, we carried out genome-wide identification of MD proteins in the model tree species black cottonwood ( Populus trichocarpa ), and analyzed their expression and co-expression networks. P. trichocarpa had 146 MD genes assigned to 14 different clades, two of which were specific to the genus Populus . 87% of these genes were located on chromosomes, the rest being associated with scaffolds. Based on their protein domain organization, and in agreement with the exon-intron structures, the MD genes identified here could be classified into five superclades having the following domains: leucine-rich repeat (LRR)-MD-protein kinase (PK), MLD-LRR-PK, MLD-PK ( Cr RLK1L), MLD-LRR, and MD-Kinesin. Whereas the majority of MD genes were highly expressed in leaves, particularly under stress conditions, eighteen showed a peak of expression during secondary wall formation in the xylem and their co-expression networks suggested signaling functions in cell wall integrity, pathogen-associated molecular patterns, calcium, ROS, and hormone pathways. Thus, P. trichocarpa MD genes having different domain organizations comprise many genes with putative foliar defense functions, some of which could be specific to Populus and related species, as well as genes with potential involvement in signaling pathways in other tissues including developing wood.

@article{wang_hybrid_2020,
	title = {Hybrid {Aspen} {Expressing} a {Carbohydrate} {Esterase} {Family} 5 {Acetyl} {Xylan} {Esterase} {Under} {Control} of a {Wood}-{Specific} {Promoter} {Shows} {Improved} {Saccharification}},
	volume = {11},
	issn = {1664-462X},
	url = {https://www.frontiersin.org/article/10.3389/fpls.2020.00380/full},
	doi = {10.3389/fpls.2020.00380},
	urldate = {2021-06-07},
	journal = {Frontiers in Plant Science},
	author = {Wang, Zhao and Pawar, Prashant Mohan-Anupama and Derba-Maceluch, Marta and Hedenström, Mattias and Chong, Sun-Li and Tenkanen, Maija and Jönsson, Leif J. and Mellerowicz, Ewa J.},
	month = apr,
	year = {2020},
	pages = {380},
}

@article{kumar_poplar_2019,
	title = {Poplar carbohydrate‐active enzymes: whole‐genome annotation and functional analyses based on {RNA} expression data},
	volume = {99},
	issn = {0960-7412, 1365-313X},
	shorttitle = {Poplar carbohydrate‐active enzymes},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/tpj.14417},
	doi = {10.1111/tpj.14417},
	language = {en},
	number = {4},
	urldate = {2021-06-07},
	journal = {The Plant Journal},
	author = {Kumar, Vikash and Hainaut, Matthieu and Delhomme, Nicolas and Mannapperuma, Chanaka and Immerzeel, Peter and Street, Nathaniel R. and Henrissat, Bernard and Mellerowicz, Ewa J.},
	month = aug,
	year = {2019},
	pages = {589--609},
}

@article{ratke_downregulating_2018,
	title = {Downregulating aspen xylan biosynthetic {GT43} genes in developing wood stimulates growth via reprograming of the transcriptome},
	volume = {219},
	issn = {0028646X},
	url = {http://doi.wiley.com/10.1111/nph.15160},
	doi = {10/gdmzzr},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {New Phytologist},
	author = {Ratke, Christine and Terebieniec, Barbara K. and Winestrand, Sandra and Derba-Maceluch, Marta and Grahn, Thomas and Schiffthaler, Bastian and Ulvcrona, Thomas and Özparpucu, Merve and Rüggeberg, Markus and Lundqvist, Sven-Olof and Street, Nathaniel R. and Jönsson, Leif J. and Mellerowicz, Ewa J.},
	month = jul,
	year = {2018},
	pages = {230--245},
}

@article{donev_engineering_2018,
	title = {Engineering {Non}-cellulosic {Polysaccharides} of {Wood} for the {Biorefinery}},
	volume = {9},
	issn = {1664-462X},
	url = {https://www.frontiersin.org/article/10.3389/fpls.2018.01537/full},
	doi = {10/gjcq6g},
	urldate = {2021-06-07},
	journal = {Frontiers in Plant Science},
	author = {Donev, Evgeniy and Gandla, Madhavi Latha and Jönsson, Leif J. and Mellerowicz, Ewa J.},
	month = oct,
	year = {2018},
	pages = {1537},
}

@article{felten_ethylene_2018,
	title = {Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen},
	volume = {218},
	issn = {0028646X},
	url = {http://doi.wiley.com/10.1111/nph.15078},
	doi = {10.1111/nph.15078},
	language = {en},
	number = {3},
	urldate = {2021-06-07},
	journal = {New Phytologist},
	author = {Felten, Judith and Vahala, Jorma and Love, Jonathan and Gorzsás, András and Rüggeberg, Markus and Delhomme, Nicolas and Leśniewska, Joanna and Kangasjärvi, Jaakko and Hvidsten, Torgeir R. and Mellerowicz, Ewa J. and Sundberg, Björn},
	month = may,
	year = {2018},
	pages = {999--1014},
}

@article{hayatgheibi_genetic_2018,
	title = {Genetic control of transition from juvenile to mature wood with respect to microfibril angle in {Norway} spruce ( \textit{{Picea} abies} ) and lodgepole pine ( \textit{{Pinus} contorta} )},
	volume = {48},
	issn = {0045-5067, 1208-6037},
	url = {http://www.nrcresearchpress.com/doi/10.1139/cjfr-2018-0140},
	doi = {10.1139/cjfr-2018-0140},
	abstract = {Genetic control of microfibril angle (MFA) transition from juvenile wood to mature wood was evaluated in Norway spruce (Picea abies (L.) Karst) and lodgepole pine (Pinus contorta Douglas ex Loudon). Increment cores were collected at breast height (1.3 m) from 5664 trees in two 21-year-old Norway spruce progeny trials in southern Sweden and from 823 trees in two lodgepole pine progeny trials, aged 34–35 years, in northern Sweden. Radial variations in MFA from pith to bark were measured for each core using SilviScan. To estimate MFA transition from juvenile wood to mature wood, a threshold level of MFA 20° was considered, and six different regression functions were fitted to the MFA profile of each tree after exclusion of outliers, following three steps. The narrow-sense heritability estimates (h
              2
              ) obtained for MFA transition were highest based on the slope function, ranging from 0.21 to 0.23 for Norway spruce and from 0.34 to 0.53 for lodgepole pine, while h
              2
              were mostly non-significant based on the logistic function, under all exclusion methods. Results of this study indicate that it is possible to select for an earlier MFA transition from juvenile wood to mature wood in Norway spruce and lodgepole pine selective breeding programs, as the genetic gains (ΔG) obtained in direct selection of this trait were very high in both species.},
	language = {en},
	number = {11},
	urldate = {2021-06-07},
	journal = {Canadian Journal of Forest Research},
	author = {Hayatgheibi, Haleh and Forsberg, Nils Erik Gustaf and Lundqvist, Sven-Olof and Mörling, Tommy and Mellerowicz, Ewa J. and Karlsson, Bo and Wu, Harry X. and García-Gil, M. Rosario},
	month = nov,
	year = {2018},
	pages = {1358--1365},
}

Genetic control of microfibril angle (MFA) transition from juvenile wood to mature wood was evaluated in Norway spruce (Picea abies (L.) Karst) and lodgepole pine (Pinus contorta Douglas ex Loudon). Increment cores were collected at breast height (1.3 m) from 5664 trees in two 21-year-old Norway spruce progeny trials in southern Sweden and from 823 trees in two lodgepole pine progeny trials, aged 34–35 years, in northern Sweden. Radial variations in MFA from pith to bark were measured for each core using SilviScan. To estimate MFA transition from juvenile wood to mature wood, a threshold level of MFA 20° was considered, and six different regression functions were fitted to the MFA profile of each tree after exclusion of outliers, following three steps. The narrow-sense heritability estimates (h 2 ) obtained for MFA transition were highest based on the slope function, ranging from 0.21 to 0.23 for Norway spruce and from 0.34 to 0.53 for lodgepole pine, while h 2 were mostly non-significant based on the logistic function, under all exclusion methods. Results of this study indicate that it is possible to select for an earlier MFA transition from juvenile wood to mature wood in Norway spruce and lodgepole pine selective breeding programs, as the genetic gains (ΔG) obtained in direct selection of this trait were very high in both species.

@article{allario_ptxtpme1_2018,
	title = {{PtxtPME1} and homogalacturonans influence xylem hydraulic properties in poplar},
	volume = {163},
	copyright = {© 2018 Scandinavian Plant Physiology Society},
	issn = {1399-3054},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/ppl.12702},
	doi = {10/gcxrch},
	abstract = {While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under- or over-express a xylem-abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under- and over-expressing aspen lines developed xylem with lower-specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.},
	language = {en},
	number = {4},
	urldate = {2021-06-21},
	journal = {Physiologia Plantarum},
	author = {Allario, Thierry and Tixier, Aude and Awad, Hosam and Lemaire, Cedric and Brunel, Nicole and Badel, Eric and Barigah, Têtè S. and Julien, Jean-Louis and Peyret, Pierre and Mellerowicz, Ewa J. and Cochard, Herve and Herbette, Stephane},
	year = {2018},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/ppl.12702},
	pages = {502--515},
}

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under- or over-express a xylem-abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under- and over-expressing aspen lines developed xylem with lower-specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.

@article{pawar_qtl_2018,
	title = {{QTL} {Mapping} of {Wood} {FT}-{IR} {Chemotypes} {Shows} {Promise} for {Improving} {Biofuel} {Potential} in {Short} {Rotation} {Coppice} {Willow} ({Salix} spp.)},
	volume = {11},
	issn = {1939-1242},
	url = {https://doi.org/10.1007/s12155-018-9901-8},
	doi = {10.1007/s12155-018-9901-8},
	abstract = {An increasing interest to convert lignocellulosic biomass into biofuels has highlighted the potential of using willows for this purpose, due to its fast growth in short rotation coppice systems. Here, we use a mapping population of 463 individuals of a cross between Salix viminalis and S. viminalis × S. schwerinii to investigate the genetic background of different wood chemical traits, information of importance for breeding towards different uses of wood. Furthermore, using a subset of the mapping population, the correlation between biogas production and chemical traits was investigated. The phenotyping of wood was carried by Furrier-transformed-Infrared spectrometry (FT-IR) and water content analysis. Quantitative trait loci (QTLs) analysis was used to identify regions in the genome of importance for the phenotypic variation of these chemical traits. We found 27 QTLs for various traits. On linkage group (LG) VI-1, QTLs for signals assigned to G-lignin, lignin, and the S/G ratio were collocated and on LG XIV we found a cluster of QTLs representing signals assigned to lignin, cellulose, hemicellulose, and water. The QTLs explained from 3.4 to 6.9\% of the phenotypic variation indicating a quantitative genetic background where many genes influence the traits. For the biogas production, a positive and negative correlation was seen with the signals assigned to acetyl and lignin, respectively. This study represents a first step in the understanding of the genetic background of wood chemical traits for willows, information needed for complementary studies, mapping of important genes, and for breeding of varieties for biofuel production purposes.},
	language = {en},
	number = {2},
	urldate = {2021-06-07},
	journal = {BioEnergy Research},
	author = {Pawar, Prashant Mohan-Anupama and Schnürer, Anna and Mellerowicz, Ewa J. and Rönnberg-Wästljung, Ann Christin},
	month = jun,
	year = {2018},
	pages = {351--363},
}

An increasing interest to convert lignocellulosic biomass into biofuels has highlighted the potential of using willows for this purpose, due to its fast growth in short rotation coppice systems. Here, we use a mapping population of 463 individuals of a cross between Salix viminalis and S. viminalis × S. schwerinii to investigate the genetic background of different wood chemical traits, information of importance for breeding towards different uses of wood. Furthermore, using a subset of the mapping population, the correlation between biogas production and chemical traits was investigated. The phenotyping of wood was carried by Furrier-transformed-Infrared spectrometry (FT-IR) and water content analysis. Quantitative trait loci (QTLs) analysis was used to identify regions in the genome of importance for the phenotypic variation of these chemical traits. We found 27 QTLs for various traits. On linkage group (LG) VI-1, QTLs for signals assigned to G-lignin, lignin, and the S/G ratio were collocated and on LG XIV we found a cluster of QTLs representing signals assigned to lignin, cellulose, hemicellulose, and water. The QTLs explained from 3.4 to 6.9% of the phenotypic variation indicating a quantitative genetic background where many genes influence the traits. For the biogas production, a positive and negative correlation was seen with the signals assigned to acetyl and lignin, respectively. This study represents a first step in the understanding of the genetic background of wood chemical traits for willows, information needed for complementary studies, mapping of important genes, and for breeding of varieties for biofuel production purposes.

@article{armezzani_transcriptional_2018,
	title = {Transcriptional induction of cell wall remodelling genes is coupled to microtubule-driven growth isotropy at the shoot apex in {Arabidopsis}},
	issn = {1477-9129, 0950-1991},
	url = {https://journals.biologists.com/dev/article/doi/10.1242/dev.162255/264733/Transcriptional-induction-of-cell-wall-remodelling},
	doi = {10/gdhnxm},
	abstract = {The shoot apical meristem of higher plants continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Cell growth, driven by turgor pressure, largely depends on the cell walls, which allow cell expansion through synthesis and structural changes. A previous study revealed a major contribution of wall isotropy in organ emergence, through the disorganization of cortical microtubules. We show here that this disorganization is coupled with the transcriptional control of genes involved in wall remodelling. Some of these genes are induced when microtubules are disorganized and cells shift to isotropic growth. Mechanical modelling shows that this coupling has the potential to compensate for reduced cell expansion rates induced by the shift to isotropic growth. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module activated during organ outgrowth, linking microtubule arrangements to cell wall remodelling.},
	language = {en},
	urldate = {2021-06-07},
	journal = {Development},
	author = {Armezzani, Alessia and Abad, Ursula and Ali, Olivier and Robin, Amélie Andres and Vachez, Laetitia and Larrieu, Antoine and Mellerowicz, Ewa J. and Taconnat, Ludivine and Battu, Virginie and Stanislas, Thomas and Liu, Mengying and Vernoux, Teva and Traas, Jan and Sassi, Massimiliano},
	month = jan,
	year = {2018},
	pages = {dev.162255},
}

The shoot apical meristem of higher plants continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Cell growth, driven by turgor pressure, largely depends on the cell walls, which allow cell expansion through synthesis and structural changes. A previous study revealed a major contribution of wall isotropy in organ emergence, through the disorganization of cortical microtubules. We show here that this disorganization is coupled with the transcriptional control of genes involved in wall remodelling. Some of these genes are induced when microtubules are disorganized and cells shift to isotropic growth. Mechanical modelling shows that this coupling has the potential to compensate for reduced cell expansion rates induced by the shift to isotropic growth. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module activated during organ outgrowth, linking microtubule arrangements to cell wall remodelling.

@article{escamez_collection_2017,
	title = {A collection of genetically engineered {Populus} trees reveals wood biomass traits that predict glucose yield from enzymatic hydrolysis},
	volume = {7},
	issn = {2045-2322},
	url = {http://www.nature.com/articles/s41598-017-16013-0},
	doi = {10/gcmn8b},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {Scientific Reports},
	author = {Escamez, Sacha and Latha Gandla, Madhavi and Derba-Maceluch, Marta and Lundqvist, Sven-Olof and Mellerowicz, Ewa J. and Jönsson, Leif J. and Tuominen, Hannele},
	month = dec,
	year = {2017},
	pages = {15798},
}

@article{sundell_aspwood_2017,
	title = {{AspWood}: {High}-{Spatial}-{Resolution} {Transcriptome} {Profiles} {Reveal} {Uncharacterized} {Modularity} of {Wood} {Formation} in {Populus} tremula},
	volume = {29},
	issn = {1040-4651, 1532-298X},
	shorttitle = {{AspWood}},
	url = {https://academic.oup.com/plcell/article/29/7/1585-1604/6099151},
	doi = {10/gbshnb},
	language = {en},
	number = {7},
	urldate = {2021-06-07},
	journal = {The Plant Cell},
	author = {Sundell, David and Street, Nathaniel R. and Kumar, Manoj and Mellerowicz, Ewa J. and Kucukoglu, Melis and Johnsson, Christoffer and Kumar, Vikash and Mannapperuma, Chanaka and Delhomme, Nicolas and Nilsson, Ove and Tuominen, Hannele and Pesquet, Edouard and Fischer, Urs and Niittylä, Totte and Sundberg, Björn and Hvidsten, Torgeir R.},
	month = jul,
	year = {2017},
	pages = {1585--1604},
}

@article{lesniewska_defense_2017,
	title = {Defense {Responses} in {Aspen} with {Altered} {Pectin} {Methylesterase} {Activity} {Reveal} the {Hormonal} {Inducers} of {Tyloses}},
	volume = {173},
	issn = {0032-0889, 1532-2548},
	url = {https://academic.oup.com/plphys/article/173/2/1409-1419/6116100},
	doi = {10.1104/pp.16.01443},
	language = {en},
	number = {2},
	urldate = {2021-06-07},
	journal = {Plant Physiology},
	author = {Leśniewska, Joanna and Öhman, David and Krzesłowska, Magdalena and Kushwah, Sunita and Barciszewska-Pacak, Maria and Kleczkowski, Leszek A. and Sundberg, Björn and Moritz, Thomas and Mellerowicz, Ewa J.},
	month = feb,
	year = {2017},
	pages = {1409--1419},
}

@article{pawar_downregulation_2017,
	title = {Downregulation of {RWA} genes in hybrid aspen affects xylan acetylation and wood saccharification},
	volume = {214},
	copyright = {© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust},
	issn = {1469-8137},
	url = {https://nph.onlinelibrary.wiley.com/doi/abs/10.1111/nph.14489},
	doi = {https://doi.org/10.1111/nph.14489},
	abstract = {High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25\% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.},
	language = {en},
	number = {4},
	urldate = {2021-06-07},
	journal = {New Phytologist},
	author = {Pawar, Prashant Mohan-Anupama and Ratke, Christine and Balasubramanian, Vimal K. and Chong, Sun-Li and Gandla, Madhavi Latha and Adriasola, Mathilda and Sparrman, Tobias and Hedenström, Mattias and Szwaj, Klaudia and Derba-Maceluch, Marta and Gaertner, Cyril and Mouille, Gregory and Ezcurra, Ines and Tenkanen, Maija and Jönsson, Leif J. and Mellerowicz, Ewa J.},
	year = {2017},
	note = {\_eprint: https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/nph.14489},
	keywords = {Cas1p, Populus, REDUCED CELL WALL ACETYLATION, saccharification, wood acetylation, xylan, xylan acetylation, xylem},
	pages = {1491--1505},
}

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.

@article{pawar_downregulation_2017,
	title = {Downregulation of {\textless}span style="font-variant:small-caps;"{\textgreater}{RWA}{\textless}/span{\textgreater} genes in hybrid aspen affects xylan acetylation and wood saccharification},
	volume = {214},
	issn = {0028-646X, 1469-8137},
	shorttitle = {Downregulation of {\textless}span style="font-variant},
	url = {https://onlinelibrary.wiley.com/doi/10.1111/nph.14489},
	doi = {10.1111/nph.14489},
	language = {en},
	number = {4},
	urldate = {2021-06-07},
	journal = {New Phytologist},
	author = {Pawar, Prashant Mohan‐Anupama and Ratke, Christine and Balasubramanian, Vimal K. and Chong, Sun‐Li and Gandla, Madhavi Latha and Adriasola, Mathilda and Sparrman, Tobias and Hedenström, Mattias and Szwaj, Klaudia and Derba‐Maceluch, Marta and Gaertner, Cyril and Mouille, Gregory and Ezcurra, Ines and Tenkanen, Maija and Jönsson, Leif J. and Mellerowicz, Ewa J.},
	month = jun,
	year = {2017},
	pages = {1491--1505},
}

@article{pawar_muro_2017,
	title = {In muro deacetylation of xylan affects lignin properties and improves saccharification of aspen wood},
	volume = {10},
	issn = {1754-6834},
	url = {http://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-017-0782-4},
	doi = {10.1186/s13068-017-0782-4},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {Biotechnology for Biofuels},
	author = {Pawar, Prashant Mohan-Anupama and Derba-Maceluch, Marta and Chong, Sun-Li and Gandla, Madhavi Latha and Bashar, Shamrat Shafiul and Sparrman, Tobias and Ahvenainen, Patrik and Hedenström, Mattias and Özparpucu, Merve and Rüggeberg, Markus and Serimaa, Ritva and Lawoko, Martin and Tenkanen, Maija and Jönsson, Leif J. and Mellerowicz, Ewa J.},
	month = dec,
	year = {2017},
	pages = {98},
}

@article{majda_mechanochemical_2017,
	title = {Mechanochemical {Polarization} of {Contiguous} {Cell} {Walls} {Shapes} {Plant} {Pavement} {Cells}},
	volume = {43},
	issn = {15345807},
	url = {https://linkinghub.elsevier.com/retrieve/pii/S1534580717308304},
	doi = {10/gcjnxj},
	language = {en},
	number = {3},
	urldate = {2021-06-07},
	journal = {Developmental Cell},
	author = {Majda, Mateusz and Grones, Peter and Sintorn, Ida-Maria and Vain, Thomas and Milani, Pascale and Krupinski, Pawel and Zagórska-Marek, Beata and Viotti, Corrado and Jönsson, Henrik and Mellerowicz, Ewa J. and Hamant, Olivier and Robert, Stéphanie},
	month = nov,
	year = {2017},
	pages = {290--304.e4},
}

@article{fernandez-moreno_efficient_2016,
	title = {An efficient method for medium throughput screening of cuticular wax composition in different plant species},
	volume = {12},
	issn = {1573-3882, 1573-3890},
	url = {http://link.springer.com/10.1007/s11306-016-0982-0},
	doi = {10.1007/s11306-016-0982-0},
	language = {en},
	number = {4},
	urldate = {2021-06-07},
	journal = {Metabolomics},
	author = {Fernandez-Moreno, Josefina-Patricia and Malitsky, Sergey and Lashbrooke, Justin and Biswal, Ajaya Kumar and Racovita, Radu C. and Mellerowicz, Ewa J. and Jetter, Reinhard and Orzaez, Diego and Aharoni, Asaph and Granell, Antonio},
	month = apr,
	year = {2016},
	pages = {73},
}

@article{pawar_expression_2016,
	title = {Expression of fungal acetyl xylan esterase in \textit{{Arabidopsis} thaliana} improves saccharification of stem lignocellulose},
	volume = {14},
	issn = {14677644},
	url = {http://doi.wiley.com/10.1111/pbi.12393},
	doi = {10.1111/pbi.12393},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {Plant Biotechnology Journal},
	author = {Pawar, Prashant Mohan-Anupama and Derba-Maceluch, Marta and Chong, Sun-Li and Gómez, Leonardo D. and Miedes, Eva and Banasiak, Alicja and Ratke, Christine and Gaertner, Cyril and Mouille, Grégory and McQueen-Mason, Simon J. and Molina, Antonio and Sellstedt, Anita and Tenkanen, Maija and Mellerowicz, Ewa J.},
	month = jan,
	year = {2016},
	pages = {387--397},
}

@article{chen_genetic_2016,
	title = {Genetic analysis of fiber dimensions and their correlation with stem diameter and solid-wood properties in {Norway} spruce},
	volume = {12},
	issn = {1614-2942, 1614-2950},
	url = {http://link.springer.com/10.1007/s11295-016-1065-0},
	doi = {10.1007/s11295-016-1065-0},
	language = {en},
	number = {6},
	urldate = {2021-06-07},
	journal = {Tree Genetics \& Genomes},
	author = {Chen, Zhi-Qiang and Karlsson, Bo and Mörling, Tommy and Olsson, Lars and Mellerowicz, Ewa J. and Wu, Harry X. and Lundqvist, Sven-Olof and Gil, María Rosario García},
	month = dec,
	year = {2016},
	pages = {123},
}

@article{chen_method_2016,
	title = {Method for accurate fiber length determination from increment cores for large-scale population analyses in {Norway} spruce},
	volume = {70},
	issn = {1437-434X, 0018-3830},
	url = {https://www.degruyter.com/document/doi/10.1515/hf-2015-0138/html},
	doi = {10/f3rwht},
	abstract = {Abstract
            
              Fiber (tracheid) length is an important trait targeted for genetic and silvicultural improvement. Such studies require large-scale non-destructive sampling, and accurate length determination. The standard procedure for non-destructive sampling is to collect increment cores, singularize their cells by maceration, measure them with optical analyzer and apply various corrections to suppress influence of non-fiber particles and cut fibers, as fibers are cut by the corer. The recently developed expectation-maximization method (EM) not only addresses the problem of non-fibers and cut fibers, but also corrects for the sampling bias. Here, the performance of the EM method has been evaluated by comparing it with length-weighing and squared length-weighing, both implemented in fiber analyzers, and with microscopy data for intact fibers, corrected for sampling bias, as the reference. This was done for 12-mm increment cores from 16 Norway spruce (
              Picea abies
              (L.) Karst) trees on fibers from rings 8–11 (counted from pith), representing juvenile wood of interest in breeding programs. The EM-estimates provided mean-fiber-lengths with bias of only +2.7\% and low scatter. Length-weighing and length
              2
              -weighing gave biases of -7.3\% and +9.3\%, respectively, and larger scatter. The suggested EM approach constitutes a more accurate non-destructive method for fiber length (FL) determination, expected to be applicable also to other conifers.},
	number = {9},
	urldate = {2021-06-07},
	journal = {Holzforschung},
	author = {Chen, Zhi-Qiang and Abramowicz, Konrad and Raczkowski, Rafal and Ganea, Stefana and Wu, Harry X. and Lundqvist, Sven-Olof and Mörling, Tommy and de Luna, Sara Sjöstedt and García Gil, María Rosario and Mellerowicz, Ewa J.},
	month = sep,
	year = {2016},
	pages = {829--838},
}

Abstract Fiber (tracheid) length is an important trait targeted for genetic and silvicultural improvement. Such studies require large-scale non-destructive sampling, and accurate length determination. The standard procedure for non-destructive sampling is to collect increment cores, singularize their cells by maceration, measure them with optical analyzer and apply various corrections to suppress influence of non-fiber particles and cut fibers, as fibers are cut by the corer. The recently developed expectation-maximization method (EM) not only addresses the problem of non-fibers and cut fibers, but also corrects for the sampling bias. Here, the performance of the EM method has been evaluated by comparing it with length-weighing and squared length-weighing, both implemented in fiber analyzers, and with microscopy data for intact fibers, corrected for sampling bias, as the reference. This was done for 12-mm increment cores from 16 Norway spruce ( Picea abies (L.) Karst) trees on fibers from rings 8–11 (counted from pith), representing juvenile wood of interest in breeding programs. The EM-estimates provided mean-fiber-lengths with bias of only +2.7% and low scatter. Length-weighing and length 2 -weighing gave biases of -7.3% and +9.3%, respectively, and larger scatter. The suggested EM approach constitutes a more accurate non-destructive method for fiber length (FL) determination, expected to be applicable also to other conifers.

@article{krzeslowska_pectinous_2016,
	title = {Pectinous cell wall thickenings formation – {A} common defense strategy of plants to cope with {Pb}},
	volume = {214},
	issn = {02697491},
	url = {https://linkinghub.elsevier.com/retrieve/pii/S0269749116302858},
	doi = {10.1016/j.envpol.2016.04.019},
	language = {en},
	urldate = {2021-06-07},
	journal = {Environmental Pollution},
	author = {Krzesłowska, Magdalena and Rabęda, Irena and Basińska, Aneta and Lewandowski, Michał and Mellerowicz, Ewa J. and Napieralska, Anna and Samardakiewicz, Sławomir and Woźny, Adam},
	month = jul,
	year = {2016},
	pages = {354--361},
}

@article{chong_active_2015,
	title = {Active fungal {GH115} alpha-glucuronidase produced in {Arabidopsis} thaliana affects only the {UX1}-reactive glucuronate decorations on native glucuronoxylans},
	volume = {15},
	issn = {1472-6750 (Electronic) 1472-6750 (Linking)},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26084671},
	doi = {10.1186/s12896-015-0154-8},
	abstract = {BACKGROUND: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of alpha-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 alpha-glucuronidases cleave the internal alpha-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 alpha-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2. RESULTS: The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70\% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60\% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta. CONCLUSIONS: Active GH115 alpha-glucuronidase has been produced for the first time in plants. The cell wall-targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {BMC Biotechnol},
	author = {Chong, S. L. and Derba-Maceluch, M. and Koutaniemi, S. and Gomez, L. D. and McQueen-Mason, S. J. and Tenkanen, M. and Mellerowicz, E. J.},
	month = jun,
	year = {2015},
	note = {Edition: 2015/06/19},
	keywords = {Arabidopsis Proteins/genetics/metabolism, Arabidopsis/enzymology, Cell Wall/enzymology, Gene Expression Regulation, Enzymologic, Glucuronates/metabolism, Glucuronic Acid/metabolism, Glycoside Hydrolases/*biosynthesis/genetics, Glycosyltransferases/genetics/metabolism, Lignin/genetics/*metabolism, Oligosaccharides/metabolism, Polysaccharides/metabolism, Schizophyllum/*enzymology, Xylans/*metabolism},
	pages = {56},
}

BACKGROUND: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of alpha-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 alpha-glucuronidases cleave the internal alpha-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 alpha-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2. RESULTS: The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta. CONCLUSIONS: Active GH115 alpha-glucuronidase has been produced for the first time in plants. The cell wall-targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.

@article{gorshkova_aspen_2015,
	title = {Aspen {Tension} {Wood} {Fibers} {Contain} beta-(1---{\textgreater} 4)-{Galactans} and {Acidic} {Arabinogalactans} {Retained} by {Cellulose} {Microfibrils} in {Gelatinous} {Walls}},
	volume = {169},
	issn = {1532-2548 (Electronic) 0032-0889 (Linking)},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26378099},
	doi = {10.1104/pp.15.00690},
	abstract = {Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula x Populus tremuloides). beta-(1--{\textgreater}4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. beta-(1--{\textgreater}4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, beta-(1--{\textgreater}4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high beta-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.},
	language = {en},
	number = {3},
	urldate = {2021-06-07},
	journal = {Plant Physiol},
	author = {Gorshkova, T. and Mokshina, N. and Chernova, T. and Ibragimova, N. and Salnikov, V. and Mikshina, P. and Tryfona, T. and Banasiak, A. and Immerzeel, P. and Dupree, P. and Mellerowicz, E. J.},
	month = nov,
	year = {2015},
	note = {Edition: 2015/09/18},
	keywords = {*Models, Biological, Biopolymers/chemistry/metabolism, Cell Wall/chemistry/metabolism, Cellulose/chemistry/*metabolism, Galactans/chemistry/*metabolism, Galactose/metabolism, Gelatin/chemistry/metabolism, Glucans/chemistry/metabolism, Microfibrils/chemistry/*metabolism, Pectins/chemistry/metabolism, Polysaccharides/chemistry/*metabolism, Populus/chemistry/cytology/*metabolism, Wood/chemistry/cytology/metabolism, Xylans/chemistry/metabolism, beta-Galactosidase/metabolism},
	pages = {2048--63},
}

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula x Populus tremuloides). beta-(1–\textgreater4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. beta-(1–\textgreater4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, beta-(1–\textgreater4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high beta-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.

@article{rabeda_colocalization_2015,
	title = {Colocalization of low-methylesterified pectins and {Pb} deposits in the apoplast of aspen roots exposed to lead},
	volume = {205},
	issn = {1873-6424 (Electronic) 0269-7491 (Linking)},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26123720},
	doi = {10.1016/j.envpol.2015.05.048},
	abstract = {Low-methylesterified homogalacturonans have been suggested to play a role in the binding and immobilization of Pb in CW. Using root apices of hybrid aspen, a plant with a high phytoremediation potential, as a model, we demonstrated that the in situ distribution pattern of low-methylesterified homogalacturonan, pectin epitope (JIM5-P), reflects the pattern of Pb occurrence. The region which indicated high JIM5-P level corresponded with "Pb accumulation zone". Moreover, JIM5-P was especially abundant in cell junctions, CWs lining the intercellular spaces and the corners of intercellular spaces indicating the highest accumulation of Pb. Furthermore, JIM5-P and Pb commonly co-localized. The observations indicate that low-methylesterified homogalacturonan is the CW polymer that determines the capacity of CW for Pb sequestration. Our results suggest a promising directions for CW modification for enhancing the efficiency of plant roots in Pb accumulation, an important aspect in the phytoremediation of soils contaminated with trace metals.},
	language = {en},
	urldate = {2021-06-07},
	journal = {Environ Pollut},
	author = {Rabeda, I. and Bilski, H. and Mellerowicz, E. J. and Napieralska, A. and Suski, S. and Wozny, A. and Krzeslowska, M.},
	month = oct,
	year = {2015},
	note = {Edition: 2015/07/01},
	keywords = {Antibodies, Monoclonal/metabolism, Biodegradation, Environmental, Biomarkers/blood, Cell wall, Esterification, Heavy metal, Lead/*metabolism, Pectins/*metabolism, Phytoremediation, Plant Roots/metabolism, Populus, Populus/*metabolism, Soil Pollutants/*metabolism, Tolerance},
	pages = {315--26},
}

Low-methylesterified homogalacturonans have been suggested to play a role in the binding and immobilization of Pb in CW. Using root apices of hybrid aspen, a plant with a high phytoremediation potential, as a model, we demonstrated that the in situ distribution pattern of low-methylesterified homogalacturonan, pectin epitope (JIM5-P), reflects the pattern of Pb occurrence. The region which indicated high JIM5-P level corresponded with "Pb accumulation zone". Moreover, JIM5-P was especially abundant in cell junctions, CWs lining the intercellular spaces and the corners of intercellular spaces indicating the highest accumulation of Pb. Furthermore, JIM5-P and Pb commonly co-localized. The observations indicate that low-methylesterified homogalacturonan is the CW polymer that determines the capacity of CW for Pb sequestration. Our results suggest a promising directions for CW modification for enhancing the efficiency of plant roots in Pb accumulation, an important aspect in the phytoremediation of soils contaminated with trace metals.

@article{latha_gandla_expression_2015,
	title = {Expression of a fungal glucuronoyl esterase in {Populus}: effects on wood properties and saccharification efficiency},
	volume = {112},
	issn = {1873-3700 (Electronic) 0031-9422 (Linking)},
	shorttitle = {Expression of a fungal glucuronoyl esterase in {Populus}},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/24997793},
	doi = {10.1016/j.phytochem.2014.06.002},
	abstract = {The secondary walls of angiosperms contain large amounts of glucuronoxylan that is thought to be covalently linked to lignin via ester bonds between 4-O-methyl-alpha-D-glucuronic acid (4-O-Me-GlcA) moieties in glucuronoxylan and alcohol groups in lignin. This linkage is proposed to be hydrolysed by glucuronoyl esterases (GCEs) secreted by wood-degrading fungi. We report effects of overexpression of a GCE from the white-rot basidiomycete Phanerochaete carnosa, PcGCE, in hybrid aspen (Populus tremula L. x tremuloides Michx.) on the wood composition and the saccharification efficiency. The recombinant enzyme, which was targeted to the plant cell wall using the signal peptide from hybrid aspen cellulase PttCel9B3, was constitutively expressed resulting in the appearance of GCE activity in protein extracts from developing wood. Diffuse reflectance FT-IR spectroscopy and pyrolysis-GC/MS analyses showed significant alternation in wood chemistry of transgenic plants including an increase in lignin content and S/G ratio, and a decrease in carbohydrate content. Sequential wood extractions confirmed a massive (+43\%) increase of Klason lignin, which was accompanied by a ca. 5\% decrease in cellulose, and ca. 20\% decrease in wood extractives. Analysis of the monosaccharide composition using methanolysis showed a reduction of 4-O-Me-GlcA content without a change in Xyl contents in transgenic lines, suggesting that the covalent links between 4-O-Me-GlcA moieties and lignin protect these moieties from degradation. Enzymatic saccharification without pretreatment resulted in significant decreases of the yields of Gal, Glc, Xyl and Man in transgenic lines, consistent with their increased recalcitrance caused by the increased lignin content. In contrast, the enzymatic saccharification after acid pretreatment resulted in Glc yields similar to wild-type despite of their lower cellulose content. These data indicate that whereas PcGCE expression in hybrid aspen increases lignin deposition, the inhibitory effects of lignin are efficiently removed during acid pretreatment, and the extent of wood cellulose conversion during hydrolysis after acid pretreatment is improved in the transgenic lines possible due to reduced cell wall cross-links between cell wall biopolymers by PcGCE.},
	language = {en},
	urldate = {2021-06-07},
	journal = {Phytochemistry},
	author = {Latha Gandla, M. and Derba-Maceluch, M. and Liu, X. and Gerber, L. and Master, E. R. and Mellerowicz, E. J. and Jonsson, L. J.},
	month = apr,
	year = {2015},
	note = {Edition: 2014/07/07},
	keywords = {Ce15, Enzymatic saccharification, Esterases/*genetics/metabolism, Gene Expression, Glucuronic Acid/*metabolism, Glucuronoyl esterase, Hybrid aspen, Hydrolysis, Phanerochaete/*enzymology/*genetics, Polysaccharides/metabolism, Populus, Populus/*genetics, Secondary cell wall, Wood/*chemistry/*metabolism},
	pages = {210--20},
}

The secondary walls of angiosperms contain large amounts of glucuronoxylan that is thought to be covalently linked to lignin via ester bonds between 4-O-methyl-alpha-D-glucuronic acid (4-O-Me-GlcA) moieties in glucuronoxylan and alcohol groups in lignin. This linkage is proposed to be hydrolysed by glucuronoyl esterases (GCEs) secreted by wood-degrading fungi. We report effects of overexpression of a GCE from the white-rot basidiomycete Phanerochaete carnosa, PcGCE, in hybrid aspen (Populus tremula L. x tremuloides Michx.) on the wood composition and the saccharification efficiency. The recombinant enzyme, which was targeted to the plant cell wall using the signal peptide from hybrid aspen cellulase PttCel9B3, was constitutively expressed resulting in the appearance of GCE activity in protein extracts from developing wood. Diffuse reflectance FT-IR spectroscopy and pyrolysis-GC/MS analyses showed significant alternation in wood chemistry of transgenic plants including an increase in lignin content and S/G ratio, and a decrease in carbohydrate content. Sequential wood extractions confirmed a massive (+43%) increase of Klason lignin, which was accompanied by a ca. 5% decrease in cellulose, and ca. 20% decrease in wood extractives. Analysis of the monosaccharide composition using methanolysis showed a reduction of 4-O-Me-GlcA content without a change in Xyl contents in transgenic lines, suggesting that the covalent links between 4-O-Me-GlcA moieties and lignin protect these moieties from degradation. Enzymatic saccharification without pretreatment resulted in significant decreases of the yields of Gal, Glc, Xyl and Man in transgenic lines, consistent with their increased recalcitrance caused by the increased lignin content. In contrast, the enzymatic saccharification after acid pretreatment resulted in Glc yields similar to wild-type despite of their lower cellulose content. These data indicate that whereas PcGCE expression in hybrid aspen increases lignin deposition, the inhibitory effects of lignin are efficiently removed during acid pretreatment, and the extent of wood cellulose conversion during hydrolysis after acid pretreatment is improved in the transgenic lines possible due to reduced cell wall cross-links between cell wall biopolymers by PcGCE.

@article{chong_glucuronic_2015,
	title = {Glucuronic acid in {Arabidopsis} thaliana xylans carries a novel pentose substituent},
	volume = {79},
	issn = {1879-0003 (Electronic) 0141-8130 (Linking)},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26047894},
	doi = {10/f3pws3},
	abstract = {Glucuronic acids in Arabidopsis thaliana xylans exist in 4-O-methylated (MeGlcA) and non-methylated (GlcA) forms at a ratio of about 3:2. The matrix-assisted laser desorption/ionization mass spectrometry analysis of the endoxylanase liberated acidic oligosaccharides from the Arabidopsis inflorescence stem showed that two peaks with GlcA (GlcA-Xyl4Ac1 and GlcA-Xyl5Ac2) had abnormally high intensities, as well as different tandem mass spectra, than their 4-O-methylated counterparts. These peaks were interestingly enriched in the xylan biosynthesis mutant irx7 and irx9-1. Multi-stages fragmentation analysis using negative ion electrospray-ion trap mass spectrometry indicated that this GlcA was further carrying a pentose residue in the glucuronoxylan-derived oligosaccharide from irx9-1. The structure was also identified in Arabidopsis wild type. The results prove evidence of a new pentose substitution on the GlcA residue of Arabidopsis GX, which is likely present in the primary walls.},
	language = {en},
	urldate = {2021-06-07},
	journal = {Int J Biol Macromol},
	author = {Chong, S. L. and Koutaniemi, S. and Juvonen, M. and Derba-Maceluch, M. and Mellerowicz, E. J. and Tenkanen, M.},
	month = aug,
	year = {2015},
	note = {Edition: 2015/06/07},
	keywords = {Arabidopsis Proteins/chemistry/*genetics, Arabidopsis thaliana, Arabidopsis/*chemistry/genetics, Endo-1,4-beta Xylanases/genetics, Gene Expression Regulation, Plant, Glucuronic Acid/*chemistry/genetics, Glucuronoxylan, Oligosaccharides/chemistry, Pentoses/*chemistry/genetics, Pentosyltransferases/chemistry/*genetics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tandem mass spectrometry, Xylans/biosynthesis/chemistry/*genetics},
	pages = {807--12},
}

Glucuronic acids in Arabidopsis thaliana xylans exist in 4-O-methylated (MeGlcA) and non-methylated (GlcA) forms at a ratio of about 3:2. The matrix-assisted laser desorption/ionization mass spectrometry analysis of the endoxylanase liberated acidic oligosaccharides from the Arabidopsis inflorescence stem showed that two peaks with GlcA (GlcA-Xyl4Ac1 and GlcA-Xyl5Ac2) had abnormally high intensities, as well as different tandem mass spectra, than their 4-O-methylated counterparts. These peaks were interestingly enriched in the xylan biosynthesis mutant irx7 and irx9-1. Multi-stages fragmentation analysis using negative ion electrospray-ion trap mass spectrometry indicated that this GlcA was further carrying a pentose residue in the glucuronoxylan-derived oligosaccharide from irx9-1. The structure was also identified in Arabidopsis wild type. The results prove evidence of a new pentose substitution on the GlcA residue of Arabidopsis GX, which is likely present in the primary walls.

@article{ratke_populus_2015,
	title = {Populus {GT43} family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification},
	volume = {13},
	issn = {1467-7652 (Electronic) 1467-7644 (Linking)},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/25100045},
	doi = {10/f3nk7x},
	abstract = {The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9-L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {Plant Biotechnol J},
	author = {Ratke, C. and Pawar, P. M. and Balasubramanian, V. K. and Naumann, M. and Duncranz, M. L. and Derba-Maceluch, M. and Gorzsas, A. and Endo, S. and Ezcurra, I. and Mellerowicz, E. J.},
	month = jan,
	year = {2015},
	note = {Edition: 2014/08/08},
	keywords = {*Genes, Plant, *Multigene Family, *Promoter Regions, Genetic, Cell Wall/*metabolism, Cloning, Molecular, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Genetic Vectors/metabolism, Glucuronidase/metabolism, Plants, Genetically Modified, Populus/*genetics, Transcriptional Activation/genetics, Wood/genetics/*metabolism, Xylans/*biosynthesis, genetic engineering, poplar, secondary wall, transgenic trees, wood development, xylan biosynthesis},
	pages = {26--37},
}

The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9-L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.

@article{derbamaceluch_suppression_2015,
	title = {Suppression of xylan endotransglycosylase \textit{{PtxtXyn10A}} affects cellulose microfibril angle in secondary wall in aspen wood},
	volume = {205},
	issn = {0028-646X, 1469-8137},
	url = {https://onlinelibrary.wiley.com/doi/10.1111/nph.13099},
	doi = {10/f3n8td},
	language = {en},
	number = {2},
	urldate = {2021-06-08},
	journal = {New Phytologist},
	author = {Derba‐Maceluch, Marta and Awano, Tatsuya and Takahashi, Junko and Lucenius, Jessica and Ratke, Christine and Kontro, Inkeri and Busse‐Wicher, Marta and Kosik, Ondrej and Tanaka, Ryo and Winzéll, Anders and Kallas, Åsa and Leśniewska, Joanna and Berthold, Fredrik and Immerzeel, Peter and Teeri, Tuula T. and Ezcurra, Ines and Dupree, Paul and Serimaa, Ritva and Mellerowicz, Ewa J.},
	month = jan,
	year = {2015},
	pages = {666--681},
}

@article{pawar_acetylation_2013,
	title = {Acetylation of woody lignocellulose: significance and regulation},
	volume = {4},
	issn = {1664-462X},
	shorttitle = {Acetylation of woody lignocellulose},
	url = {http://journal.frontiersin.org/article/10.3389/fpls.2013.00118/abstract},
	doi = {10/f236bp},
	urldate = {2021-06-08},
	journal = {Frontiers in Plant Science},
	author = {Pawar, Prashant Mohan-Anupama and Koutaniemi, Sanna and Tenkanen, Maija and Mellerowicz, Ewa J.},
	year = {2013},
}

@article{mahboubi_aspen_2013,
	title = {Aspen {SUCROSE} {TRANSPORTER3} {Allocates} {Carbon} into {Wood} {Fibers}},
	volume = {163},
	issn = {0032-0889, 1532-2548},
	url = {https://academic.oup.com/plphys/article/163/4/1729-1740/6111119},
	doi = {10/f24j68},
	language = {en},
	number = {4},
	urldate = {2021-06-08},
	journal = {PLANT PHYSIOLOGY},
	author = {Mahboubi, A. and Ratke, C. and Gorzsas, A. and Kumar, M. and Mellerowicz, E. J. and Niittylä, T.},
	month = dec,
	year = {2013},
	pages = {1729--1740},
}

@article{manabe_reduced_2013,
	title = {Reduced {Wall} {Acetylation} {Proteins} {Play} {Vital} and {Distinct} {Roles} in {Cell} {Wall} \textit{{O}} -{Acetylation} in {Arabidopsis}},
	volume = {163},
	issn = {1532-2548},
	url = {https://academic.oup.com/plphys/article/163/3/1107/6112718},
	doi = {10/f2zn5j},
	abstract = {Abstract
            The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20\% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63\% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls.},
	language = {en},
	number = {3},
	urldate = {2021-06-08},
	journal = {Plant Physiology},
	author = {Manabe, Yuzuki and Verhertbruggen, Yves and Gille, Sascha and Harholt, Jesper and Chong, Sun-Li and Pawar, Prashant Mohan-Anupama and Mellerowicz, Ewa J. and Tenkanen, Maija and Cheng, Kun and Pauly, Markus and Scheller, Henrik Vibe},
	month = oct,
	year = {2013},
	pages = {1107--1117},
}

Abstract The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls.

@article{miedes_xyloglucan_2013,
	title = {Xyloglucan endotransglucosylase/hydrolase ({XTH}) overexpression affects growth and cell wall mechanics in etiolated {Arabidopsis} hypocotyls},
	volume = {64},
	issn = {1460-2431, 0022-0957},
	url = {https://academic.oup.com/jxb/article-lookup/doi/10.1093/jxb/ert107},
	doi = {10/f244cc},
	language = {en},
	number = {8},
	urldate = {2021-06-08},
	journal = {Journal of Experimental Botany},
	author = {Miedes, Eva and Suslov, Dmitry and Vandenbussche, Filip and Kenobi, Kim and Ivakov, Alexander and Van Der Straeten, Dominique and Lorences, Ester P. and Mellerowicz, Ewa J. and Verbelen, Jean-Pierre and Vissenberg, Kris},
	month = may,
	year = {2013},
	pages = {2481--2497},
}

@article{tsai_constitutive_2012,
	title = {Constitutive expression of a fungal glucuronoyl esterase in {Arabidopsis} reveals altered cell wall composition and structure: {Transgenic} expression of {PcGCE} in {Arabidopsis}},
	volume = {10},
	issn = {14677644},
	shorttitle = {Constitutive expression of a fungal glucuronoyl esterase in {Arabidopsis} reveals altered cell wall composition and structure},
	url = {http://doi.wiley.com/10.1111/j.1467-7652.2012.00735.x},
	doi = {10/f2zwvt},
	language = {en},
	number = {9},
	urldate = {2021-06-08},
	journal = {Plant Biotechnology Journal},
	author = {Tsai, Alex Y.-L. and Canam, Thomas and Gorzsás, András and Mellerowicz, Ewa J. and Campbell, Malcolm M. and Master, Emma R.},
	month = dec,
	year = {2012},
	pages = {1077--1087},
}

@article{gorshkova_plant_2012,
	title = {Plant {Fiber} {Formation}: {State} of the {Art}, {Recent} and {Expected} {Progress}, and {Open} {Questions}},
	volume = {31},
	issn = {0735-2689, 1549-7836},
	shorttitle = {Plant {Fiber} {Formation}},
	url = {http://www.tandfonline.com/doi/abs/10.1080/07352689.2011.616096},
	doi = {10/f2z5m4},
	language = {en},
	number = {3},
	urldate = {2021-06-08},
	journal = {Critical Reviews in Plant Sciences},
	author = {Gorshkova, Tatyana and Brutch, Nina and Chabbert, Brigitte and Deyholos, Michael and Hayashi, Takahisa and Lev-Yadun, Simcha and Mellerowicz, Ewa J. and Morvan, Claudine and Neutelings, Godfrey and Pilate, Gilles},
	month = may,
	year = {2012},
	pages = {201--228},
}

@article{mellerowicz_tensional_2012,
	title = {Tensional stress generation in gelatinous fibres: a review and possible mechanism based on cell-wall structure and composition},
	volume = {63},
	issn = {0022-0957, 1460-2431},
	shorttitle = {Tensional stress generation in gelatinous fibres},
	url = {https://academic.oup.com/jxb/article-lookup/doi/10.1093/jxb/err339},
	doi = {10/cn6bx8},
	language = {en},
	number = {2},
	urldate = {2021-06-08},
	journal = {Journal of Experimental Botany},
	author = {Mellerowicz, E. J. and Gorshkova, T. A.},
	month = jan,
	year = {2012},
	pages = {551--565},
}

@article{raiola_pectin_2011,
	title = {Pectin {Methylesterase} {Is} {Induced} in \textit{{Arabidopsis}} upon {Infection} and {Is} {Necessary} for a {Successful} {Colonization} by {Necrotrophic} {Pathogens}},
	volume = {24},
	issn = {0894-0282, 1943-7706},
	url = {https://apsjournals.apsnet.org/doi/10.1094/MPMI-07-10-0157},
	doi = {10/fwswvk},
	abstract = {The ability of bacterial or fungal necrotrophs to produce enzymes capable of degrading pectin is often related to a successful initiation of the infective process. Pectin is synthesized in a highly methylesterified form and is subsequently de-esterified in muro by pectin methylesterase. De-esterification makes pectin more susceptible to the degradation by pectic enzymes such as endopolygalacturonases (endoPG) and pectate lyases secreted by necrotrophic pathogens during the first stages of infection. We show that, upon infection, Pectobacterium carotovorum and Botrytis cinerea induce in Arabidopsis a rapid expression of AtPME3 that acts as a susceptibility factor and is required for the initial colonization of the host tissue.},
	language = {en},
	number = {4},
	urldate = {2021-06-08},
	journal = {Molecular Plant-Microbe Interactions®},
	author = {Raiola, Alessandro and Lionetti, Vincenzo and Elmaghraby, Ibrahim and Immerzeel, Peter and Mellerowicz, Ewa J. and Salvi, Giovanni and Cervone, Felice and Bellincampi, Daniela},
	month = apr,
	year = {2011},
	pages = {432--440},
}

The ability of bacterial or fungal necrotrophs to produce enzymes capable of degrading pectin is often related to a successful initiation of the infective process. Pectin is synthesized in a highly methylesterified form and is subsequently de-esterified in muro by pectin methylesterase. De-esterification makes pectin more susceptible to the degradation by pectic enzymes such as endopolygalacturonases (endoPG) and pectate lyases secreted by necrotrophic pathogens during the first stages of infection. We show that, upon infection, Pectobacterium carotovorum and Botrytis cinerea induce in Arabidopsis a rapid expression of AtPME3 that acts as a susceptibility factor and is required for the initial colonization of the host tissue.

@article{wang_reduced_2011,
	title = {Reduced {Expression} of the {SHORT}-{ROOT} {Gene} {Increases} the {Rates} of {Growth} and {Development} in {Hybrid} {Poplar} and {Arabidopsis}},
	volume = {6},
	issn = {1932-6203},
	url = {https://dx.plos.org/10.1371/journal.pone.0028878},
	doi = {10/cx2jsh},
	language = {en},
	number = {12},
	urldate = {2021-06-08},
	journal = {PLoS ONE},
	author = {Wang, Jiehua and Andersson-Gunnerås, Sara and Gaboreanu, Ioana and Hertzberg, Magnus and Tucker, Matthew R. and Zheng, Bo and Leśniewska, Joanna and Mellerowicz, Ewa J. and Laux, Thomas and Sandberg, Göran and Jones, Brian},
	editor = {Schönbach, Christian},
	month = dec,
	year = {2011},
	pages = {e28878},
}

@article{nishikubo_xyloglucan_2011,
	title = {Xyloglucan \textit{endo} -{Transglycosylase}-{Mediated} {Xyloglucan} {Rearrangements} in {Developing} {Wood} of {Hybrid} {Aspen}},
	volume = {155},
	issn = {1532-2548},
	url = {https://academic.oup.com/plphys/article/155/1/399/6111559},
	doi = {10/fswhm6},
	abstract = {Abstract
            Xyloglucan endo-transglycosylases (XETs) encoded by xyloglucan endo-transglycosylases/hydrolase (XTH) genes modify the xyloglucan-cellulose framework of plant cell walls, thereby regulating their expansion and strength. To evaluate the importance of XET in wood development, we studied xyloglucan dynamics and XTH gene expression in developing wood and modified XET activity in hybrid aspen (Populus tremula × tremuloides) by overexpressing PtxtXET16-34. We show that developmental modifications during xylem differentiation include changes from loosely to tightly bound forms of xyloglucan and increases in the abundance of fucosylated xyloglucan epitope recognized by the CCRC-M1 antibody. We found that at least 16 Populus  XTH genes, all likely encoding XETs, are expressed in developing wood. Five genes were highly and ubiquitously expressed, whereas PtxtXET16-34 was expressed more weakly but specifically in developing wood. Transgenic up-regulation of XET activity induced changes in cell wall xyloglucan, but its effects were dependent on developmental stage. For instance, XET overexpression increased abundance of the CCRC-M1 epitope in cambial cells and xylem cells in early stages of differentiation but not in mature xylem. Correspondingly, an increase in tightly bound xyloglucan content was observed in primary-walled xylem but a decrease was seen in secondary-walled xylem. Thus, in young xylem cells, XET activity limits xyloglucan incorporation into the tightly bound wall network but removes it from cell walls in older cells. XET overexpression promoted vessel element growth but not fiber expansion. We suggest that the amount of nascent xyloglucan relative to XET is an important determinant of whether XET strengthens or loosens the cell wall.},
	language = {en},
	number = {1},
	urldate = {2021-06-08},
	journal = {Plant Physiology},
	author = {Nishikubo, Nobuyuki and Takahashi, Junko and Roos, Alexandra A. and Derba-Maceluch, Marta and Piens, Kathleen and Brumer, Harry and Teeri, Tuula T. and Stålbrand, Henrik and Mellerowicz, Ewa J.},
	month = jan,
	year = {2011},
	pages = {399--413},
}

Abstract Xyloglucan endo-transglycosylases (XETs) encoded by xyloglucan endo-transglycosylases/hydrolase (XTH) genes modify the xyloglucan-cellulose framework of plant cell walls, thereby regulating their expansion and strength. To evaluate the importance of XET in wood development, we studied xyloglucan dynamics and XTH gene expression in developing wood and modified XET activity in hybrid aspen (Populus tremula × tremuloides) by overexpressing PtxtXET16-34. We show that developmental modifications during xylem differentiation include changes from loosely to tightly bound forms of xyloglucan and increases in the abundance of fucosylated xyloglucan epitope recognized by the CCRC-M1 antibody. We found that at least 16 Populus  XTH genes, all likely encoding XETs, are expressed in developing wood. Five genes were highly and ubiquitously expressed, whereas PtxtXET16-34 was expressed more weakly but specifically in developing wood. Transgenic up-regulation of XET activity induced changes in cell wall xyloglucan, but its effects were dependent on developmental stage. For instance, XET overexpression increased abundance of the CCRC-M1 epitope in cambial cells and xylem cells in early stages of differentiation but not in mature xylem. Correspondingly, an increase in tightly bound xyloglucan content was observed in primary-walled xylem but a decrease was seen in secondary-walled xylem. Thus, in young xylem cells, XET activity limits xyloglucan incorporation into the tightly bound wall network but removes it from cell walls in older cells. XET overexpression promoted vessel element growth but not fiber expansion. We suggest that the amount of nascent xyloglucan relative to XET is an important determinant of whether XET strengthens or loosens the cell wall.

@article{nishikubo_xyloglucan_2011,
	title = {Xyloglucan endo-{Transglycosylase}-{Mediated} {Xyloglucan} {Rearrangements} in {Developing} {Wood} of {Hybrid} {Aspen}},
	volume = {155},
	issn = {0032-0889},
	url = {https://doi.org/10.1104/pp.110.166934},
	doi = {10/fswhm6},
	abstract = {Xyloglucan endo-transglycosylases (XETs) encoded by xyloglucan endo-transglycosylases/hydrolase (XTH) genes modify the xyloglucan-cellulose framework of plant cell walls, thereby regulating their expansion and strength. To evaluate the importance of XET in wood development, we studied xyloglucan dynamics and XTH gene expression in developing wood and modified XET activity in hybrid aspen (Populus tremula × tremuloides) by overexpressing PtxtXET16-34. We show that developmental modifications during xylem differentiation include changes from loosely to tightly bound forms of xyloglucan and increases in the abundance of fucosylated xyloglucan epitope recognized by the CCRC-M1 antibody. We found that at least 16 Populus  XTH genes, all likely encoding XETs, are expressed in developing wood. Five genes were highly and ubiquitously expressed, whereas PtxtXET16-34 was expressed more weakly but specifically in developing wood. Transgenic up-regulation of XET activity induced changes in cell wall xyloglucan, but its effects were dependent on developmental stage. For instance, XET overexpression increased abundance of the CCRC-M1 epitope in cambial cells and xylem cells in early stages of differentiation but not in mature xylem. Correspondingly, an increase in tightly bound xyloglucan content was observed in primary-walled xylem but a decrease was seen in secondary-walled xylem. Thus, in young xylem cells, XET activity limits xyloglucan incorporation into the tightly bound wall network but removes it from cell walls in older cells. XET overexpression promoted vessel element growth but not fiber expansion. We suggest that the amount of nascent xyloglucan relative to XET is an important determinant of whether XET strengthens or loosens the cell wall.},
	number = {1},
	urldate = {2021-06-08},
	journal = {Plant Physiology},
	author = {Nishikubo, Nobuyuki and Takahashi, Junko and Roos, Alexandra A. and Derba-Maceluch, Marta and Piens, Kathleen and Brumer, Harry and Teeri, Tuula T. and Stålbrand, Henrik and Mellerowicz, Ewa J.},
	month = jan,
	year = {2011},
	pages = {399--413},
}

Xyloglucan endo-transglycosylases (XETs) encoded by xyloglucan endo-transglycosylases/hydrolase (XTH) genes modify the xyloglucan-cellulose framework of plant cell walls, thereby regulating their expansion and strength. To evaluate the importance of XET in wood development, we studied xyloglucan dynamics and XTH gene expression in developing wood and modified XET activity in hybrid aspen (Populus tremula × tremuloides) by overexpressing PtxtXET16-34. We show that developmental modifications during xylem differentiation include changes from loosely to tightly bound forms of xyloglucan and increases in the abundance of fucosylated xyloglucan epitope recognized by the CCRC-M1 antibody. We found that at least 16 Populus  XTH genes, all likely encoding XETs, are expressed in developing wood. Five genes were highly and ubiquitously expressed, whereas PtxtXET16-34 was expressed more weakly but specifically in developing wood. Transgenic up-regulation of XET activity induced changes in cell wall xyloglucan, but its effects were dependent on developmental stage. For instance, XET overexpression increased abundance of the CCRC-M1 epitope in cambial cells and xylem cells in early stages of differentiation but not in mature xylem. Correspondingly, an increase in tightly bound xyloglucan content was observed in primary-walled xylem but a decrease was seen in secondary-walled xylem. Thus, in young xylem cells, XET activity limits xyloglucan incorporation into the tightly bound wall network but removes it from cell walls in older cells. XET overexpression promoted vessel element growth but not fiber expansion. We suggest that the amount of nascent xyloglucan relative to XET is an important determinant of whether XET strengthens or loosens the cell wall.

@article{ibatullin_real-time_2009,
	title = {A {Real}-{Time} {Fluorogenic} {Assay} for the {Visualization} of {Glycoside} {Hydrolase} {Activity} in {Planta}},
	volume = {151},
	issn = {0032-0889, 1532-2548},
	url = {https://academic.oup.com/plphys/article/151/4/1741-1750/6109797},
	doi = {10/b6s8sm},
	language = {en},
	number = {4},
	urldate = {2021-06-08},
	journal = {Plant Physiology},
	author = {Ibatullin, Farid M. and Banasiak, Alicja and Baumann, Martin J. and Greffe, Lionel and Takahashi, Junko and Mellerowicz, Ewa J. and Brumer, Harry},
	month = dec,
	year = {2009},
	pages = {1741--1750},
}

@article{takahashi_korrigan1_2009,
	title = {{KORRIGAN1} and its {Aspen} {Homolog} {PttCel9A1} {Decrease} {Cellulose} {Crystallinity} in {Arabidopsis} {Stems}},
	volume = {50},
	issn = {1471-9053, 0032-0781},
	url = {https://academic.oup.com/pcp/article-lookup/doi/10.1093/pcp/pcp062},
	doi = {10/djcxr3},
	language = {en},
	number = {6},
	urldate = {2021-06-08},
	journal = {Plant and Cell Physiology},
	author = {Takahashi, Junko and Rudsander, Ulla J. and Hedenström, Mattias and Banasiak, Alicja and Harholt, Jesper and Amelot, Nicolas and Immerzeel, Peter and Ryden, Peter and Endo, Satoshi and Ibatullin, Farid M. and Brumer, Harry and del Campillo, Elena and Master, Emma R. and Vibe Scheller, Henrik and Sundberg, Björn and Teeri, Tuula T. and Mellerowicz, Ewa J.},
	month = jun,
	year = {2009},
	pages = {1099--1115},
}

@article{meng_udp-glucose_2009,
	title = {{UDP}-{Glucose} {Pyrophosphorylase} is not {Rate} {Limiting}, but is {Essential} in {Arabidopsis}},
	volume = {50},
	issn = {0032-0781, 1471-9053},
	url = {https://academic.oup.com/pcp/article-lookup/doi/10.1093/pcp/pcp052},
	doi = {10/cwnt2d},
	language = {en},
	number = {5},
	urldate = {2021-06-08},
	journal = {Plant and Cell Physiology},
	author = {Meng, M. and Geisler, M. and Johansson, H. and Harholt, J. and Scheller, H. V. and Mellerowicz, E. J. and Kleczkowski, L. A.},
	month = may,
	year = {2009},
	pages = {998--1011},
}

@article{goue_microgenomic_2008,
	title = {Microgenomic analysis reveals cell type‐specific gene expression patterns between ray and fusiform initials within the cambial meristem of \textit{{Populus}}},
	volume = {180},
	issn = {0028-646X, 1469-8137},
	url = {https://onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.2008.02556.x},
	doi = {10/cpqv9f},
	language = {en},
	number = {1},
	urldate = {2021-06-10},
	journal = {New Phytologist},
	author = {Goué, Nadia and Lesage‐Descauses, Marie‐Claude and Mellerowicz, Ewa J. and Magel, Elisabeth and Label, Philippe and Sundberg, Björn},
	month = oct,
	year = {2008},
	pages = {45--56},
}

@article{siedlecka_pectin_2008,
	title = {Pectin {Methyl} {Esterase} {Inhibits} {Intrusive} and {Symplastic} {Cell} {Growth} in {Developing} {Wood} {Cells} of \textit{{Populus}}},
	volume = {146},
	issn = {1532-2548},
	url = {https://academic.oup.com/plphys/article/146/2/323/6107207},
	doi = {10/bz7b7b},
	abstract = {Abstract
            Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula × tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up- and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.},
	language = {en},
	number = {2},
	urldate = {2021-06-10},
	journal = {Plant Physiology},
	author = {Siedlecka, Anna and Wiklund, Susanne and Péronne, Marie-Amélie and Micheli, Fabienne and Leśniewska, Joanna and Sethson, Ingmar and Edlund, Ulf and Richard, Luc and Sundberg, Björn and Mellerowicz, Ewa J.},
	month = feb,
	year = {2008},
	pages = {323--324},
}

Abstract Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula × tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up- and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.

@article{wiklund_visualization_2008,
	title = {Visualization of {GC}/{TOF}-{MS}-{Based} {Metabolomics} {Data} for {Identification} of {Biochemically} {Interesting} {Compounds} {Using} {OPLS} {Class} {Models}},
	volume = {80},
	issn = {0003-2700, 1520-6882},
	url = {https://pubs.acs.org/doi/10.1021/ac0713510},
	doi = {10/bjbc4d},
	language = {en},
	number = {1},
	urldate = {2021-06-10},
	journal = {Analytical Chemistry},
	author = {Wiklund, Susanne and Johansson, Erik and Sjöström, Lina and Mellerowicz, Ewa J. and Edlund, Ulf and Shockcor, John P. and Gottfries, Johan and Moritz, Thomas and Trygg, Johan},
	month = jan,
	year = {2008},
	pages = {115--122},
}

@article{mellerowicz_xyloglucan_2008,
	title = {Xyloglucan: {The} {Molecular} {Muscle} of {Trees}},
	volume = {102},
	issn = {1095-8290, 0305-7364},
	shorttitle = {Xyloglucan},
	url = {https://academic.oup.com/aob/article-lookup/doi/10.1093/aob/mcn170},
	doi = {10/d76fhs},
	language = {en},
	number = {5},
	urldate = {2021-06-10},
	journal = {Annals of Botany},
	author = {Mellerowicz, Ewa J. and Immerzeel, Peter and Hayashi, Takahisa},
	month = nov,
	year = {2008},
	pages = {659--665},
}

@article{meng_differential_2007,
	title = {Differential tissue/organ-dependent expression of two sucrose- and cold-responsive genes for {UDP}-glucose pyrophosphorylase in {Populus}},
	volume = {389},
	issn = {03781119},
	url = {https://linkinghub.elsevier.com/retrieve/pii/S0378111906007062},
	doi = {10/fn8z4c},
	language = {en},
	number = {2},
	urldate = {2021-06-10},
	journal = {Gene},
	author = {Meng, Meng and Geisler, Matt and Johansson, Henrik and Mellerowicz, Ewa J. and Karpinski, Stanislaw and Kleczkowski, Leszek A.},
	month = mar,
	year = {2007},
	pages = {186--195},
}

@article{gray-mitsumune_ectopic_2007,
	title = {Ectopic expression of a wood-abundant expansin {PttEXPA1} promotes cell expansion in primary and secondary tissues in aspen},
	volume = {0},
	issn = {1467-7644, 1467-7652},
	url = {http://doi.wiley.com/10.1111/j.1467-7652.2007.00295.x},
	doi = {10/cz7ht2},
	language = {en},
	number = {0},
	urldate = {2021-06-10},
	journal = {Plant Biotechnology Journal},
	author = {Gray-Mitsumune, Madoka and Blomquist, Kristina and McQueen-Mason, Simon and Teeri, Tuula T. and Sundberg, Björn and Mellerowicz, Ewa J.},
	month = oct,
	year = {2007},
	pages = {071003005211001--???},
}

@article{nishikubo_xyloglucan_2007,
	title = {Xyloglucan {Endo}-transglycosylase ({XET}) {Functions} in {Gelatinous} {Layers} of {Tension} {Wood} {Fibers} in {Poplar}—{A} {Glimpse} into the {Mechanism} of the {Balancing} {Act} of {Trees}},
	volume = {48},
	issn = {1471-9053, 0032-0781},
	url = {https://academic.oup.com/pcp/article-lookup/doi/10.1093/pcp/pcm055},
	doi = {10/dm4c3z},
	language = {en},
	number = {6},
	urldate = {2021-06-10},
	journal = {Plant and Cell Physiology},
	author = {Nishikubo, Nobuyuki and Awano, Tatsuya and Banasiak, Alicja and Bourquin, Veronica and Ibatullin, Farid and Funada, Ryo and Brumer, Harry and Teeri, Tuula T. and Hayashi, Takahisa and Sundberg, Björn and Mellerowicz, Ewa J.},
	month = jun,
	year = {2007},
	pages = {843--855},
}

@article{andersson-gunneras_biosynthesis_2006,
	title = {Biosynthesis of cellulose-enriched tension wood in {Populus}: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis},
	volume = {45},
	issn = {1365-313X},
	shorttitle = {Biosynthesis of cellulose-enriched tension wood in {Populus}},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-313X.2005.02584.x},
	doi = {10/fkwhm3},
	abstract = {Stems and branches of angiosperm trees form tension wood (TW) when exposed to a gravitational stimulus. One of the main characteristics of TW, which distinguishes it from normal wood, is the formation of fibers with a thick inner gelatinous cell wall layer mainly composed of crystalline cellulose. Hence TW is enriched in cellulose, and deficient in lignin and hemicelluloses. An expressed sequence tag library made from TW-forming tissues in Populus tremula (L.) × tremuloides (Michx.) and data from transcript profiling using microarray and metabolite analysis were obtained during TW formation in Populus tremula (L.) in two growing seasons. The data were examined with the aim of identifying the genes responsible for the change in carbon (C) flow into various cell wall components, and the mechanisms important for the formation of the gelatinous cell wall layer (G-layer). A specific effort was made to identify carbohydrate-active enzymes with a putative function in cell wall biosynthesis. An increased C flux to cellulose was suggested by a higher abundance of sucrose synthase transcripts. However, genes related to the cellulose biosynthetic machinery were not generally affected, although the expression of secondary wall-specific CesA genes was modified in both directions. Other pathways for which the data suggested increased activity included lipid and glucosamine biosynthesis and the pectin degradation machinery. In addition, transcripts encoding fasciclin-like arabinogalactan proteins were particularly increased and found to lack true Arabidopsis orthologs. Major pathways for which the transcriptome and metabolome analysis suggested decreased activity were the pathway for C flux through guanosine 5′-diphosphate (GDP) sugars to mannans, the pentose phosphate pathway, lignin biosynthesis, and biosynthesis of cell wall matrix carbohydrates. Several differentially expressed auxin- and ethylene-related genes and transcription factors were also identified.},
	language = {en},
	number = {2},
	urldate = {2021-06-11},
	journal = {The Plant Journal},
	author = {Andersson-Gunnerås, Sara and Mellerowicz, Ewa J. and Love, Jonathan and Segerman, Bo and Ohmiya, Yasunori and Coutinho, Pedro M. and Nilsson, Peter and Henrissat, Bernard and Moritz, Thomas and Sundberg, Björn},
	year = {2006},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2005.02584.x},
	keywords = {cell walls, cellulose, development, hemicellulose, lignin, poplar},
	pages = {144--165},
}

Stems and branches of angiosperm trees form tension wood (TW) when exposed to a gravitational stimulus. One of the main characteristics of TW, which distinguishes it from normal wood, is the formation of fibers with a thick inner gelatinous cell wall layer mainly composed of crystalline cellulose. Hence TW is enriched in cellulose, and deficient in lignin and hemicelluloses. An expressed sequence tag library made from TW-forming tissues in Populus tremula (L.) × tremuloides (Michx.) and data from transcript profiling using microarray and metabolite analysis were obtained during TW formation in Populus tremula (L.) in two growing seasons. The data were examined with the aim of identifying the genes responsible for the change in carbon (C) flow into various cell wall components, and the mechanisms important for the formation of the gelatinous cell wall layer (G-layer). A specific effort was made to identify carbohydrate-active enzymes with a putative function in cell wall biosynthesis. An increased C flux to cellulose was suggested by a higher abundance of sucrose synthase transcripts. However, genes related to the cellulose biosynthetic machinery were not generally affected, although the expression of secondary wall-specific CesA genes was modified in both directions. Other pathways for which the data suggested increased activity included lipid and glucosamine biosynthesis and the pectin degradation machinery. In addition, transcripts encoding fasciclin-like arabinogalactan proteins were particularly increased and found to lack true Arabidopsis orthologs. Major pathways for which the transcriptome and metabolome analysis suggested decreased activity were the pathway for C flux through guanosine 5′-diphosphate (GDP) sugars to mannans, the pentose phosphate pathway, lignin biosynthesis, and biosynthesis of cell wall matrix carbohydrates. Several differentially expressed auxin- and ethylene-related genes and transcription factors were also identified.

@article{geisler-lee_poplar_2006,
	title = {Poplar carbohydrate-active enzymes. {Gene} identification and expression analyses},
	volume = {140},
	issn = {0032-0889},
	doi = {10/bj9bkd},
	abstract = {Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr.\&Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar ( Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis ( Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants.},
	language = {English},
	number = {3},
	journal = {Plant Physiology},
	publisher = {Amer Soc Plant Biologists},
	author = {Geisler-Lee, J. and Geisler, M. and Coutinho, P. M. and Segerman, B. and Nishikubo, N. and Takahashi, J. and Aspeborg, H. and Djerbi, S. and Master, E. and Andersson-Gunneras, S. and Sundberg, B. and Karpinski, S. and Teeri, T. T. and Kleczkowski, L. A. and Henrissat, B. and Mellerowicz, E. J.},
	month = mar,
	year = {2006},
	note = {Place: Rockville
WOS:000235868900014},
	keywords = {arabidopsis, callose synthase, cell-walls, cellulose, family, hybrid aspen, multiple sequence alignment, pectin methylesterases, populus, sucrose},
	pages = {946--962},
}

Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr.&Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar ( Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis ( Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants.

@article{gray-mitsumune_expansins_2004,
	title = {Expansins {Abundant} in {Secondary} {Xylem} {Belong} to {Subgroup} {A} of the α-{Expansin} {Gene} {Family}},
	volume = {135},
	issn = {0032-0889},
	url = {https://doi.org/10.1104/pp.104.039321},
	doi = {10/dd876s},
	abstract = {Differentiation of xylem cells in dicotyledonous plants involves expansion of the radial primary cell walls and intrusive tip growth of cambial derivative cells prior to the deposition of a thick secondary wall essential for xylem function. Expansins are cell wall-residing proteins that have an ability to plasticize the cellulose-hemicellulose network of primary walls. We found expansin activity in proteins extracted from the cambial region of mature stems in a model tree species hybrid aspen (Populus tremula × Populus tremuloides Michx). We identified three α-expansin genes (PttEXP1, PttEXP2, and PttEXP8) and one β-expansin gene (PttEXPB1) in a cambial region expressed sequence tag library, among which PttEXP1 was most abundantly represented. Northern-blot analyses in aspen vegetative organs and tissues showed that PttEXP1 was specifically expressed in mature stems exhibiting secondary growth, where it was present in the cambium and in the radial expansion zone. By contrast, PttEXP2 was mostly expressed in developing leaves. In situ reverse transcription-PCR provided evidence for accumulation of mRNA of PttEXP1 along with ribosomal rRNA at the tips of intrusively growing xylem fibers, suggesting that PttEXP1 protein has a role in intrusive tip growth. An examination of tension wood and leaf cDNA libraries identified another expansin, PttEXP5, very similar to PttEXP1, as the major expansin in developing tension wood, while PttEXP3 was the major expansin expressed in developing leaves. Comparative analysis of expansins expressed in woody stems in aspen, Arabidopsis, and pine showed that the most abundantly expressed expansins share sequence similarities, belonging to the subfamily A of α-expansins and having two conserved motifs at the beginning and end of the mature protein, RIPVG and KNFRV, respectively. This conservation suggests that these genes may share a specialized, not yet identified function.},
	number = {3},
	urldate = {2021-06-15},
	journal = {Plant Physiology},
	author = {Gray-Mitsumune, Madoka and Mellerowicz, Ewa J. and Abe, Hisashi and Schrader, Jarmo and Winzéll, Anders and Sterky, Fredrik and Blomqvist, Kristina and McQueen-Mason, Simon and Teeri, Tuula T. and Sundberg, Björn},
	month = jul,
	year = {2004},
	pages = {1552--1564},
}

Differentiation of xylem cells in dicotyledonous plants involves expansion of the radial primary cell walls and intrusive tip growth of cambial derivative cells prior to the deposition of a thick secondary wall essential for xylem function. Expansins are cell wall-residing proteins that have an ability to plasticize the cellulose-hemicellulose network of primary walls. We found expansin activity in proteins extracted from the cambial region of mature stems in a model tree species hybrid aspen (Populus tremula × Populus tremuloides Michx). We identified three α-expansin genes (PttEXP1, PttEXP2, and PttEXP8) and one β-expansin gene (PttEXPB1) in a cambial region expressed sequence tag library, among which PttEXP1 was most abundantly represented. Northern-blot analyses in aspen vegetative organs and tissues showed that PttEXP1 was specifically expressed in mature stems exhibiting secondary growth, where it was present in the cambium and in the radial expansion zone. By contrast, PttEXP2 was mostly expressed in developing leaves. In situ reverse transcription-PCR provided evidence for accumulation of mRNA of PttEXP1 along with ribosomal rRNA at the tips of intrusively growing xylem fibers, suggesting that PttEXP1 protein has a role in intrusive tip growth. An examination of tension wood and leaf cDNA libraries identified another expansin, PttEXP5, very similar to PttEXP1, as the major expansin in developing tension wood, while PttEXP3 was the major expansin expressed in developing leaves. Comparative analysis of expansins expressed in woody stems in aspen, Arabidopsis, and pine showed that the most abundantly expressed expansins share sequence similarities, belonging to the subfamily A of α-expansins and having two conserved motifs at the beginning and end of the mature protein, RIPVG and KNFRV, respectively. This conservation suggests that these genes may share a specialized, not yet identified function.

@article{gray-mitsumune_liquid_2004,
	title = {Liquid {Phase} {Fluorescence} in situ {RT}-{PCR} {Analysis} for {Gene} {Expression} {Analysis} in {Woody} {Stems}},
	volume = {6},
	issn = {1438-8677},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1055/s-2003-44747},
	doi = {10/bf7fs7},
	abstract = {Abstract: We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen (Populus tremula L. ×tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.},
	language = {en},
	number = {1},
	urldate = {2021-06-15},
	journal = {Plant Biology},
	author = {Gray-Mitsumune, M. and Abe, H. and Takahashi, J. and Sundberg, B. and Mellerowicz, E. J.},
	year = {2004},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1055/s-2003-44747},
	keywords = {Confocal laser scanning microscopy, Populus, in situ RT-PCR, vascular cambium, wood, xylem},
	pages = {47--54},
}

Abstract: We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen (Populus tremula L. ×tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.

@article{bourquin_xyloglucan_2002,
	title = {Xyloglucan {Endotransglycosylases} {Have} a {Function} during the {Formation} of {Secondary} {Cell} {Walls} of {Vascular} {Tissues}},
	volume = {14},
	issn = {1040-4651},
	url = {https://doi.org/10.1105/tpc.007773},
	doi = {10/cfv573},
	abstract = {Xyloglucan transglycosylases (XETs) have been implicated in many aspects of cell wall biosynthesis, but their function in vascular tissues, in general, and in the formation of secondary walls, in particular, is less well understood. Using an in situ XET activity assay in poplar stems, we have demonstrated XET activity in xylem and phloem fibers at the stage of secondary wall formation. Immunolocalization of fucosylated xylogucan with CCRC-M1 antibodies showed that levels of this species increased at the border between the primary and secondary wall layers at the time of secondary wall deposition. Furthermore, one of the most abundant XET isoforms in secondary vascular tissues (PttXET16A) was cloned and immunolocalized to fibers at the stage of secondary wall formation. Together, these data strongly suggest that XET has a previously unreported role in restructuring primary walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers. We also observed that xylogucan is incorporated at a high level in the inner layer of nacreous walls of mature sieve tube elements.},
	number = {12},
	urldate = {2021-10-19},
	journal = {The Plant Cell},
	author = {Bourquin, Veronica and Nishikubo, Nobuyuki and Abe, Hisashi and Brumer, Harry and Denman, Stuart and Eklund, Marlin and Christiernin, Maria and Teeri, Tunla T. and Sundberg, Björn and Mellerowicz, Ewa J.},
	month = dec,
	year = {2002},
	pages = {3073--3088},
}

Xyloglucan transglycosylases (XETs) have been implicated in many aspects of cell wall biosynthesis, but their function in vascular tissues, in general, and in the formation of secondary walls, in particular, is less well understood. Using an in situ XET activity assay in poplar stems, we have demonstrated XET activity in xylem and phloem fibers at the stage of secondary wall formation. Immunolocalization of fucosylated xylogucan with CCRC-M1 antibodies showed that levels of this species increased at the border between the primary and secondary wall layers at the time of secondary wall deposition. Furthermore, one of the most abundant XET isoforms in secondary vascular tissues (PttXET16A) was cloned and immunolocalized to fibers at the stage of secondary wall formation. Together, these data strongly suggest that XET has a previously unreported role in restructuring primary walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers. We also observed that xylogucan is incorporated at a high level in the inner layer of nacreous walls of mature sieve tube elements.

@article{mellerowicz_unravelling_2001,
	title = {Unravelling cell wall formation in the woody dicot stem},
	volume = {47},
	issn = {0167-4412},
	abstract = {Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.},
	language = {eng},
	number = {1-2},
	journal = {Plant Molecular Biology},
	author = {Mellerowicz, E. J. and Baucher, M. and Sundberg, B. and Boerjan, W.},
	month = sep,
	year = {2001},
	keywords = {Cell Wall, Cellulose, Enzymes, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Lignin, Plant Stems, Trees, Wood},
	pages = {239--274},
}

Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.

@article{mellerowicz_prfll_1998,
	title = {{PRFLL}– a {Pinus} radiata homologue of {FLORICAULA} and {LEAFY} is expressed in buds containing vegetative shoot  and undifferentiated male cone primordia},
	volume = {206},
	issn = {1432-2048},
	url = {https://doi.org/10.1007/s004250050440},
	doi = {10.1007/s004250050440},
	abstract = {In angiosperms, the formation of the flower meristem is controlled by partially redundant flower meristem identity genes of which FLORICAULA (FLO)/LEAFY (LFY ) plays a central role. It is not known whether formation of reproductive organs of pre-angiosperm species is similarly regulated. Recently, a FLO/LFY-like cDNA, NEEDLY (NLY ), has been cloned in a conifer species Pinus radiata (D. Don). Here we report cloning of a different Pinusradiata FLO/LFY-l ike cDNA, PRFLL. PRFLL had two large regions of high similarity to angiosperm FLO/LFY orthologues: amino acids 61–126 and 247–406 (50\% and 81\% identity, and 75\% and 88\% similarity, respectively, to LFY) and shorter regions of local similarity. Overall identity was 53\% to LFY and 61\% to NLY. Phylogenetic analysis of deduced protein sequences including partial LFY-like sequences from Pseudotsuga menziesii indicated that conifer proteins constituted a separate clade that could be divided into two groups represented by NLY and PRFLL. In contrast to angiosperms, both conifers had two paralogous proteins resembling LFY. Northern hybridisation analysis revealed expression of PRFLL in vegetative buds of juvenile, adolescent and mature trees. The transcript was not detected in vascular cambium, roots or secondary needles. To follow PRFLL expression during the early stages of cone development we analysed a temporal series of buds containing cone primordia, and developing cones, using Northern hybridisation and confocal microscopy in parallel. PRFLL mRNA was detected in buds from dominant and subordinate branches, in which cone and shoot primordia develop, and in developing male cones but not in developing female cones. Expression was particularly high in buds containing axillary primordia prior to their differentiation as male cone primordia. This is consistent with PRFLL being involved in determination of the male cone primordium identity.},
	language = {en},
	number = {4},
	urldate = {2024-10-02},
	journal = {Planta},
	author = {Mellerowicz, Ewa J. and Horgan, Kathryn and Walden, Adrian and Coker, Astrid and Walter, Christian},
	month = oct,
	year = {1998},
	keywords = {Conifer (flowering), Flowering, Key words: Cone development, LEAFY, Meristem identity, Pinus (flowering)},
	pages = {619--629},
}

In angiosperms, the formation of the flower meristem is controlled by partially redundant flower meristem identity genes of which FLORICAULA (FLO)/LEAFY (LFY ) plays a central role. It is not known whether formation of reproductive organs of pre-angiosperm species is similarly regulated. Recently, a FLO/LFY-like cDNA, NEEDLY (NLY ), has been cloned in a conifer species Pinus radiata (D. Don). Here we report cloning of a different Pinusradiata FLO/LFY-l ike cDNA, PRFLL. PRFLL had two large regions of high similarity to angiosperm FLO/LFY orthologues: amino acids 61–126 and 247–406 (50% and 81% identity, and 75% and 88% similarity, respectively, to LFY) and shorter regions of local similarity. Overall identity was 53% to LFY and 61% to NLY. Phylogenetic analysis of deduced protein sequences including partial LFY-like sequences from Pseudotsuga menziesii indicated that conifer proteins constituted a separate clade that could be divided into two groups represented by NLY and PRFLL. In contrast to angiosperms, both conifers had two paralogous proteins resembling LFY. Northern hybridisation analysis revealed expression of PRFLL in vegetative buds of juvenile, adolescent and mature trees. The transcript was not detected in vascular cambium, roots or secondary needles. To follow PRFLL expression during the early stages of cone development we analysed a temporal series of buds containing cone primordia, and developing cones, using Northern hybridisation and confocal microscopy in parallel. PRFLL mRNA was detected in buds from dominant and subordinate branches, in which cone and shoot primordia develop, and in developing male cones but not in developing female cones. Expression was particularly high in buds containing axillary primordia prior to their differentiation as male cone primordia. This is consistent with PRFLL being involved in determination of the male cone primordium identity.

@article{lloyd_changes_1996,
	title = {Changes in nuclear genome size and relative ribosomal {RNA} gene content in cambial region cells of {Abies} balsamea shoots during the development of dormancy},
	volume = {74},
	issn = {0008-4026},
	url = {https://cdnsciencepub.com/doi/abs/10.1139/b96-035},
	doi = {10.1139/b96-035},
	abstract = {The relationship between nuclear genome size, measured cytophotometrically, and relative ribosomal RNA gene (rDNA) content, determined as the ratio of the hybridization signals from a 25S rRNA gene probe and a randomly labelled total genomic DNA probe, was investigated in cambial region cells of balsam fir (Abies balsamea (L.) Mill.) shoots during the onset of dormancy and the transition between the dormancy stages of rest and quiescence. The dormancy status was manipulated by exposing potted trees for 13 weeks, starting August 14 when the cambium was still active, to one of the following environments: (i) the declining temperature and photoperiod of the natural environment (denoted N-N), (ii) simulated natural temperature and 15-h photoperiod (sN-15), (iii) simulated natural temperature and 8-h photoperiod (sN-8), and (iv) warm temperature and 15-h photoperiod (sN-15). On November 12, the trees were transferred to a greenhouse having environmental conditions favorable for growth to assess their ability to reactivate. The entire activity –rest–quiescence transition occurred in the N-N environment and was associated with an increase in nuclear genome size and a decrease in the relative rDNA content. The sN-8 environment also induced the transition but not completely, and the associated increase and decrease in nuclear genome size and relative rDNA content, respectively, were smaller than in the N-N environment. The sN-15 environment delayed the onset of rest, inhibited the changeover to quiescence, increased then decreased the nuclear genome size, and did not affect the relative rDNA content. The W-15 environment, which induced budbreak and the formation of a false ring, also prevented the rest–quiescence transition and transiently increased the nuclear genome size without altering the relative rDNA content. The data suggest that the normal rest–quiescence progression in shoot cambial cells is associated with an increase in nuclear genome size, which is caued by amplification of a fraction that is not rDNA but is recognized by our genomic probe. Keywords: dormancy, genome size, ribosomal RNA genes, vascular cambium.},
	number = {2},
	urldate = {2024-10-02},
	journal = {Canadian Journal of Botany},
	publisher = {NRC Research Press},
	author = {Lloyd, A. D. and Little, C. H. A. and Mellerowicz, E. J. and Riding, R. T.},
	month = feb,
	year = {1996},
	pages = {290--298},
}

The relationship between nuclear genome size, measured cytophotometrically, and relative ribosomal RNA gene (rDNA) content, determined as the ratio of the hybridization signals from a 25S rRNA gene probe and a randomly labelled total genomic DNA probe, was investigated in cambial region cells of balsam fir (Abies balsamea (L.) Mill.) shoots during the onset of dormancy and the transition between the dormancy stages of rest and quiescence. The dormancy status was manipulated by exposing potted trees for 13 weeks, starting August 14 when the cambium was still active, to one of the following environments: (i) the declining temperature and photoperiod of the natural environment (denoted N-N), (ii) simulated natural temperature and 15-h photoperiod (sN-15), (iii) simulated natural temperature and 8-h photoperiod (sN-8), and (iv) warm temperature and 15-h photoperiod (sN-15). On November 12, the trees were transferred to a greenhouse having environmental conditions favorable for growth to assess their ability to reactivate. The entire activity –rest–quiescence transition occurred in the N-N environment and was associated with an increase in nuclear genome size and a decrease in the relative rDNA content. The sN-8 environment also induced the transition but not completely, and the associated increase and decrease in nuclear genome size and relative rDNA content, respectively, were smaller than in the N-N environment. The sN-15 environment delayed the onset of rest, inhibited the changeover to quiescence, increased then decreased the nuclear genome size, and did not affect the relative rDNA content. The W-15 environment, which induced budbreak and the formation of a false ring, also prevented the rest–quiescence transition and transiently increased the nuclear genome size without altering the relative rDNA content. The data suggest that the normal rest–quiescence progression in shoot cambial cells is associated with an increase in nuclear genome size, which is caued by amplification of a fraction that is not rDNA but is recognized by our genomic probe. Keywords: dormancy, genome size, ribosomal RNA genes, vascular cambium.

@article{mellerowicz_nuclear_1995,
	title = {Nuclear and cytoplasmic changes associated with maturation in the vascular cambium of {Larix} laricina},
	volume = {15},
	issn = {0829-318X},
	url = {https://doi.org/10.1093/treephys/15.7-8.443},
	doi = {10.1093/treephys/15.7-8.443},
	abstract = {We studied the effects of apical maturation on the vascular cambium of juvenile and mature scions of Larix laricina (Du Roi) K. Koch that had been grafted on seedling rootstocks. Comparisons between the juvenile and mature cambium in nuclear genome size, nuclear shape, DNA concentration, number and volume of nucleoli per nucleus, and concentration of extranuclear RNAs, proteins and insoluble carbohydrates were conducted on four occasions during the annual cycle of cambial activity and dormancy. All investigated variables exhibited strong annual oscillations, whereas differences between the two maturation stages were less prominent. Many of the differences between the two phases could be explained by delayed spring reactivation and accelerated onset of dormancy in the mature cambium compared with the juvenile cambium. At the time of reactivation and during activity, the mature cambium exhibited lower genome size, lower DNA concentration, fewer nucleoli per nucleus and a higher extranuclear concentration of insoluble carbohydrates than the juvenile cambium. The dormant mature cambium contained more extranuclear RNAs than the dormant juvenile cambium. The observed differences provide circumstantial evidence of changes in chromatin organization or functioning, or both, during maturation.},
	number = {7-8},
	urldate = {2024-10-02},
	journal = {Tree Physiology},
	author = {Mellerowicz, E. J. and Riding, R. T. and Greenwood, M. S.},
	month = jul,
	year = {1995},
	pages = {443--449},
}

We studied the effects of apical maturation on the vascular cambium of juvenile and mature scions of Larix laricina (Du Roi) K. Koch that had been grafted on seedling rootstocks. Comparisons between the juvenile and mature cambium in nuclear genome size, nuclear shape, DNA concentration, number and volume of nucleoli per nucleus, and concentration of extranuclear RNAs, proteins and insoluble carbohydrates were conducted on four occasions during the annual cycle of cambial activity and dormancy. All investigated variables exhibited strong annual oscillations, whereas differences between the two maturation stages were less prominent. Many of the differences between the two phases could be explained by delayed spring reactivation and accelerated onset of dormancy in the mature cambium compared with the juvenile cambium. At the time of reactivation and during activity, the mature cambium exhibited lower genome size, lower DNA concentration, fewer nucleoli per nucleus and a higher extranuclear concentration of insoluble carbohydrates than the juvenile cambium. The dormant mature cambium contained more extranuclear RNAs than the dormant juvenile cambium. The observed differences provide circumstantial evidence of changes in chromatin organization or functioning, or both, during maturation.

@article{zhong_seasonal_1995,
	title = {Seasonal variation in the nuclear genome size of ray cells in the vascular cambium of {Fraxinus} americana},
	volume = {93},
	issn = {1399-3054},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1399-3054.1995.tb02233.x},
	doi = {10.1111/j.1399-3054.1995.tb02233.x},
	abstract = {The nuclear DNA content in ray cells from the 1-year-old vascular cambium of white ash (Fraxinus americana L.) trees was determined at intervals during the annual cycle of cambial activity and dormancy by using Feulgen microspectrophotometry. By 10 September, these cells had entered dormancy in G1 with a normal DNA distribution and a minimal average DNA content of 2.65 pg. The average amount of DNA increased to 3.51 pg by 30 November, remained at this elevated value until at least 30 March, when the cambium was still dormant, then declined to the minimum level on 1 May and 10 June, when the cells were mitotically active. The springtime decline appeared to occur both before and during cell division. Between 1 May and 10 June, the prophase (4C) and telophase (2C) DNA contents decreased significantly. The amount of nuclear DNA measured by microspectrophotometry was verified by using flow cytometry and image analysis. The results support the view that there is an annual oscillation in the nuclear genome size of shoot meristematic cells in tree species native to the northern temperate zone.},
	language = {en},
	number = {2},
	urldate = {2024-10-02},
	journal = {Physiologia Plantarum},
	author = {Zhong, Y. and Mellerowicz, E. J. and Lloyd, A. D. and Leinhos, V. and Riding, R. T. and Little, C. H. A.},
	year = {1995},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1399-3054.1995.tb02233.x},
	keywords = {DNA amplification and loss, Fraxinus americana, flow cytometry, genome size, image analysis, microspectrophotometry, periodicity, protoplast, ray cell, vascular cambium, white ash},
	pages = {305--311},
}

The nuclear DNA content in ray cells from the 1-year-old vascular cambium of white ash (Fraxinus americana L.) trees was determined at intervals during the annual cycle of cambial activity and dormancy by using Feulgen microspectrophotometry. By 10 September, these cells had entered dormancy in G1 with a normal DNA distribution and a minimal average DNA content of 2.65 pg. The average amount of DNA increased to 3.51 pg by 30 November, remained at this elevated value until at least 30 March, when the cambium was still dormant, then declined to the minimum level on 1 May and 10 June, when the cells were mitotically active. The springtime decline appeared to occur both before and during cell division. Between 1 May and 10 June, the prophase (4C) and telophase (2C) DNA contents decreased significantly. The amount of nuclear DNA measured by microspectrophotometry was verified by using flow cytometry and image analysis. The results support the view that there is an annual oscillation in the nuclear genome size of shoot meristematic cells in tree species native to the northern temperate zone.

@article{lloyd_fluctuations_1994,
	title = {Fluctuations in ribosomal {RNA} gene content and nucleolar activity in the cambial region of {Abies} balsamea ({Pinaceae}) shoots during reactivation},
	volume = {81},
	copyright = {© 1994 Botanical Society of America},
	issn = {1537-2197},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/j.1537-2197.1994.tb15622.x},
	doi = {10.1002/j.1537-2197.1994.tb15622.x},
	abstract = {Tissue was collected from the vascular cambial region of 1-year-old balsam fir shoots over an 11-week period during which cambial reactivation occurred. The amount of rDNA (ribosomal RNA genes) relative to total genomic DNA was determined by quantitative slot blots for three trees, one of which showed a 3-week delay in reactivation. In addition, nucleolar activity was estimated by measuring nucleolar volume, number, and staining intensity. Relative rRNA gene content increased transiently prior to the onset of cambial cell periclinal division. Nucleolar volume also increased transiently, but 1–2 weeks prior to the maximal relative rDNA value. The increases in relative rDNA and nucleolar activity were delayed in the tree in which reactivation was late. We interpret these changes as reflecting the amplification and loss of genes encoding rRNA to facilitate cambial cell reactivation.},
	language = {en},
	number = {11},
	urldate = {2024-10-02},
	journal = {American Journal of Botany},
	author = {Lloyd, Andrew D. and Mellerowicz, Ewa J. and Chow, Cynthia H. and Riding, Richard T. and Little, C. H. Anthony},
	year = {1994},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1537-2197.1994.tb15622.x},
	pages = {1384--1389},
}

Tissue was collected from the vascular cambial region of 1-year-old balsam fir shoots over an 11-week period during which cambial reactivation occurred. The amount of rDNA (ribosomal RNA genes) relative to total genomic DNA was determined by quantitative slot blots for three trees, one of which showed a 3-week delay in reactivation. In addition, nucleolar activity was estimated by measuring nucleolar volume, number, and staining intensity. Relative rRNA gene content increased transiently prior to the onset of cambial cell periclinal division. Nucleolar volume also increased transiently, but 1–2 weeks prior to the maximal relative rDNA value. The increases in relative rDNA and nucleolar activity were delayed in the tree in which reactivation was late. We interpret these changes as reflecting the amplification and loss of genes encoding rRNA to facilitate cambial cell reactivation.

@article{mellerowicz_nucleolar_1993,
	title = {Nucleolar activity in the fusiform cambial cells of {Abies} balsamea ({Pinaceae}): effect of season and age},
	volume = {80},
	copyright = {© 1993 Botanical Society of America},
	issn = {1537-2197},
	shorttitle = {Nucleolar activity in the fusiform cambial cells of {Abies} balsamea ({Pinaceae})},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/j.1537-2197.1993.tb15349.x},
	doi = {10.1002/j.1537-2197.1993.tb15349.x},
	abstract = {Nucleolar involvement in the regulation of the activity-rest-quiescence cycle of the vascular cambium was assessed by determining the seasonal variation in number, diameter, and volume of nucleoli in fusiform cells of Abies balsamea (L.) Mill. The cells were isolated from 1- and 19-yr-old cambia and stained with either silver nitrate or Feulgen + naphthol yellow-S. The ability of fusiform cells to incorporate [5-3H]-uridine into nuclei and nucleoli was also determined. In the 1-yr-old cambium, the activity of the nucleoli, as evidenced by their diameter, total volume per cell, and intensity of staining with silver nitrate, exhibited two maxima during the year—a large one during cambial reactivation in April-May and a small one during the rest-quiescence transition in October. Incorporation of radiolabeled uridine at 20 C was low at the end of the active period and increased during the rest–quiescence transition, suggesting that the quiescent, but not the resting, cambium can rapidly resume nucleolar activity when the temperature is permissive. The number of nucleoli per cell varied between two and eight, and was higher during the dormant than the active period. The increase in number took place during the autumnal activity–rest–quiescence transition, when cambial cells were arrested in the G1 phase of the cell cycle. Similar seasonal changes in nucleolar morphology were observed in the 19-yr-old cambium. Nucleolar diameter and total nucleolar volume were larger in the 19-yr-old cambium than in the 1-yr-old cambium, whereas nucleolar number was lower. Th results suggest that repression of rRNA genes underlies the development of rest when the cambium will not produce new cells.},
	language = {en},
	number = {10},
	urldate = {2024-10-02},
	journal = {American Journal of Botany},
	author = {Mellerowicz, E. J. and Riding, R. T. and Little, C. H. A.},
	year = {1993},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1537-2197.1993.tb15349.x},
	pages = {1168--1174},
}

Nucleolar involvement in the regulation of the activity-rest-quiescence cycle of the vascular cambium was assessed by determining the seasonal variation in number, diameter, and volume of nucleoli in fusiform cells of Abies balsamea (L.) Mill. The cells were isolated from 1- and 19-yr-old cambia and stained with either silver nitrate or Feulgen + naphthol yellow-S. The ability of fusiform cells to incorporate [5-3H]-uridine into nuclei and nucleoli was also determined. In the 1-yr-old cambium, the activity of the nucleoli, as evidenced by their diameter, total volume per cell, and intensity of staining with silver nitrate, exhibited two maxima during the year—a large one during cambial reactivation in April-May and a small one during the rest-quiescence transition in October. Incorporation of radiolabeled uridine at 20 C was low at the end of the active period and increased during the rest–quiescence transition, suggesting that the quiescent, but not the resting, cambium can rapidly resume nucleolar activity when the temperature is permissive. The number of nucleoli per cell varied between two and eight, and was higher during the dormant than the active period. The increase in number took place during the autumnal activity–rest–quiescence transition, when cambial cells were arrested in the G1 phase of the cell cycle. Similar seasonal changes in nucleolar morphology were observed in the 19-yr-old cambium. Nucleolar diameter and total nucleolar volume were larger in the 19-yr-old cambium than in the 1-yr-old cambium, whereas nucleolar number was lower. Th results suggest that repression of rRNA genes underlies the development of rest when the cambium will not produce new cells.

@article{mellerowicz_does_1992,
	title = {Does {DNA} {Endoreduplication} {Occur} {During} {Differentiation} of {Secondary} {Xylem} and {Phloem} in {Abies} balsamea?},
	volume = {153},
	issn = {1058-5893},
	url = {https://www.journals.uchicago.edu/doi/10.1086/297003},
	doi = {10.1086/297003},
	abstract = {The amount of nuclear DNA was determined microspectrophotometrically in cells of the vascular cambium and differentiating secondary xylem and phloem in Abies balsamea (L.) Mill. Feulgen-stained radial sections were prepared from 1-yr-old shoots of mature trees sampled during (May 21) and after (June 5) the period of high mitotic activity of the cambium. Differentiating xylem and phloem cells had an unreplicated DNA level, indicating that differentiation proceeded from the G$_{\textrm{1}}$ phase of the cell cycle. The DNA content did not change during differentiation, indicating a lack of endoreduplication, whereas the nuclear size increased during cell expansion and decreased during cell maturation until the nuclei broke down. The genome size in fusiform cambial cells and differentiating xylem and phloem elements decreased between the two sampling dates. This decrease agrees with our previous observation that the genome size in fusiform cambial cells decreases during the growing season and increases during dormancy, probably as a result of loss of amplified sequences followed by DNA amplification.},
	number = {1},
	urldate = {2024-10-02},
	journal = {International Journal of Plant Sciences},
	publisher = {The University of Chicago Press},
	author = {Mellerowicz, E. J. and Riding, R. T.},
	month = mar,
	year = {1992},
	pages = {26--30},
}

The amount of nuclear DNA was determined microspectrophotometrically in cells of the vascular cambium and differentiating secondary xylem and phloem in Abies balsamea (L.) Mill. Feulgen-stained radial sections were prepared from 1-yr-old shoots of mature trees sampled during (May 21) and after (June 5) the period of high mitotic activity of the cambium. Differentiating xylem and phloem cells had an unreplicated DNA level, indicating that differentiation proceeded from the G$_{\textrm{1}}$ phase of the cell cycle. The DNA content did not change during differentiation, indicating a lack of endoreduplication, whereas the nuclear size increased during cell expansion and decreased during cell maturation until the nuclei broke down. The genome size in fusiform cambial cells and differentiating xylem and phloem elements decreased between the two sampling dates. This decrease agrees with our previous observation that the genome size in fusiform cambial cells decreases during the growing season and increases during dormancy, probably as a result of loss of amplified sequences followed by DNA amplification.

@article{mellerowicz_periodicity_1992,
	title = {Periodicity of cambial activity in {Abies} balsamea. {I}. {Effects} of temperature and photoperiod on cambial dormancy and frost hardiness},
	volume = {85},
	issn = {1399-3054},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1399-3054.1992.tb05820.x},
	doi = {10.1111/j.1399-3054.1992.tb05820.x},
	abstract = {The relationship between from hardiness and growth potential, and their dependence on temperature and photoperiod, was investigated in the one-year-old cambium of balsam fir [Abies balsamea (L.) Mill.]. Six-year-old trees were exposed for 9 weeks to either the natural environment or one of 4 controlled environments in the fall (18 September-18 November), spring (12 April–14 June) and summer (19 July – 19 September). The 4 controlled environments were (1) WS, warm temperature (24/20°C in day/night) + short day (8 h). (2) WL. warm temperature (24/20°C) + long day (8 h + 1 h night break), (3) CS. cold temperature (9/5°C) + short day (8 h) and (4) CL, cold temperature (9/5°C) + long day (8 h + 1 h night break). At the beginning and end of each exposure, cambial activity was measured by recording the number of xylem, cambium and phloem cells, frost hardiness was estimated from the cambium's ability to survive freezing to –40°C, and cambial growth potential was deduced from the duration of the cell cycle and the production of xylem, cambium and phloem cells in cuttings cultured for 4 weeks with exogenous indole-3-acetic acid (IAA) under environmental conditions favourable for cambial activity. In the natural environment, frost hardening began in September and was completed in November, while dehardening occurred when the cambium reactivated. CL, CS, and to a lesser extent WS, promoted hardening in the summer and fall, but did not prevent dehardening in the spring. The cambial growth potential in the natural environment declined from a maximum in April to a low level in June, reached a minimum in September, then increased to a high level in November. This potential was promoted by CL and CS on all dates by WL in the summer and fall. The ratio of xylem to phloem induced by IAA treatment was greatest in June and least in September in cuttings from trees exposed to the natural environment, and was increased by CL and CS in the fall. The cambium in intact branches of trees protected from chilling during the fall and winter resumed cell cycling after less than 9 weeks of dormancy, but produced mostly or only phloem in the subsequent growing period. It is concluded that the frost hardiness of the cambium, the IAA-induced cycling of cambial cells, and IAA-induced xylem to phloem ratio vary independently with season, temperature and photoperiod, and that the periodicity of these processes is regulated endogenously.},
	language = {en},
	number = {3},
	urldate = {2024-10-02},
	journal = {Physiologia Plantarum},
	author = {Mellerowicz, E. J. and Coleman, W. K. and Riding, R. T. and Little, C. H. A.},
	year = {1992},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1399-3054.1992.tb05820.x},
	keywords = {Abies balsamea, IAA sensitivity, balsam fir, dormancy, frost hardiness, phloem, photoperiod, quiescence, rest, temperature, vascular cambium, xylem},
	pages = {515--525},
}

The relationship between from hardiness and growth potential, and their dependence on temperature and photoperiod, was investigated in the one-year-old cambium of balsam fir [Abies balsamea (L.) Mill.]. Six-year-old trees were exposed for 9 weeks to either the natural environment or one of 4 controlled environments in the fall (18 September-18 November), spring (12 April–14 June) and summer (19 July – 19 September). The 4 controlled environments were (1) WS, warm temperature (24/20°C in day/night) + short day (8 h). (2) WL. warm temperature (24/20°C) + long day (8 h + 1 h night break), (3) CS. cold temperature (9/5°C) + short day (8 h) and (4) CL, cold temperature (9/5°C) + long day (8 h + 1 h night break). At the beginning and end of each exposure, cambial activity was measured by recording the number of xylem, cambium and phloem cells, frost hardiness was estimated from the cambium's ability to survive freezing to –40°C, and cambial growth potential was deduced from the duration of the cell cycle and the production of xylem, cambium and phloem cells in cuttings cultured for 4 weeks with exogenous indole-3-acetic acid (IAA) under environmental conditions favourable for cambial activity. In the natural environment, frost hardening began in September and was completed in November, while dehardening occurred when the cambium reactivated. CL, CS, and to a lesser extent WS, promoted hardening in the summer and fall, but did not prevent dehardening in the spring. The cambial growth potential in the natural environment declined from a maximum in April to a low level in June, reached a minimum in September, then increased to a high level in November. This potential was promoted by CL and CS on all dates by WL in the summer and fall. The ratio of xylem to phloem induced by IAA treatment was greatest in June and least in September in cuttings from trees exposed to the natural environment, and was increased by CL and CS in the fall. The cambium in intact branches of trees protected from chilling during the fall and winter resumed cell cycling after less than 9 weeks of dormancy, but produced mostly or only phloem in the subsequent growing period. It is concluded that the frost hardiness of the cambium, the IAA-induced cycling of cambial cells, and IAA-induced xylem to phloem ratio vary independently with season, temperature and photoperiod, and that the periodicity of these processes is regulated endogenously.

@article{mellerowicz_periodicity_1992,
	title = {Periodicity of cambial activity in {Abies} balsamea. {II}. {Effects} of temperature and photoperiod on the size of the nuclear genome in fusiform cambial cells},
	volume = {85},
	issn = {1399-3054},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1399-3054.1992.tb05821.x},
	doi = {10.1111/j.1399-3054.1992.tb05821.x},
	abstract = {Previously, we showed that the size of the nuclear genome, measured cytophotometrically in Feulgen-stained fusiform cambial cells of Abies balsamea (L.) Mill., oscillates annually between a maximum in spring and a minimum in late summer, the labile, extra DNA being synthesized during the fall. To determine it the oscillation is induced by the concomitant seasonal changes in temperature and photoperiod, genome size was measured in cambial cells obtained from one-year-old branches of 6-year-old potted trees at the beginning and end of 9 weeks of exposure during the fall, spring and summer to either the natural environment or one of 4 controlled environments, viz. (1) WS, warm temperature (24/20°C in day/night) and short photoperiod (8 h). (2) WL, warm temperature (24/20°C) and long photoperiod (8 h + 1 h night break), (3) CS, cold temperature (9/5°C) and short photoperiod (8 h). and (4) CL, cold temperature (9/5°C) and long photoperiod (8 h + 1 h night break). Overall, genome size (2C) varied between 20 and 34 pg. In the fall, when the cambium was initially dormant, the genome size increased in the natural environment, did not change under short days (WS and CS), and decreased under long days (WL and CL). The cambium reactivated in both WS and WL conditions. In the spring, while the cambium reactivated, the size of the genome decreased in the natural, WS and WL conditions, but not in the CS environment. In the CL conditions, the genome size started to decrease at the end of the 9-week exposure period. The decrease apparently occurred between prophase and telophase, which suggests that the extra DNA is extrachromosomal. In the summer, while the cambium ceased activity, the genome size did not change in the WS, WL and natural environments, whereas it decreased in the CS and CL conditions. The results indicate that increasing temperature and lengthening photoperiod in the spring induce the loss of the extra DNA. However, the environmental conditions that promote DNA synthesis in the fall remain unknown. Genome size varied independently of cambial growth potential and frost hardiness measured previously in the same experimental trees, indicating that the regulation of these processes does not directly involve the extra DNA. However, the finding that cambial cells cycled in the CS and CL environments only in the spring, when their genome size was large, suggests that the extra DNA is important for cambial growth at low temperatures.},
	language = {en},
	number = {3},
	urldate = {2024-10-02},
	journal = {Physiologia Plantarum},
	author = {Mellerowicz, E. J. and Riding, R. T. and Little, C. H. A.},
	year = {1992},
	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1399-3054.1992.tb05821.x},
	keywords = {Abies balsamea, DNA amplification, balsam fir, cytophotometry, differential DNA replication, extrachromosomal DNA, genome rearrangements, genome size, periodicity, photoperiod, temperature, vascular cambium},
	pages = {526--530},
}

Previously, we showed that the size of the nuclear genome, measured cytophotometrically in Feulgen-stained fusiform cambial cells of Abies balsamea (L.) Mill., oscillates annually between a maximum in spring and a minimum in late summer, the labile, extra DNA being synthesized during the fall. To determine it the oscillation is induced by the concomitant seasonal changes in temperature and photoperiod, genome size was measured in cambial cells obtained from one-year-old branches of 6-year-old potted trees at the beginning and end of 9 weeks of exposure during the fall, spring and summer to either the natural environment or one of 4 controlled environments, viz. (1) WS, warm temperature (24/20°C in day/night) and short photoperiod (8 h). (2) WL, warm temperature (24/20°C) and long photoperiod (8 h + 1 h night break), (3) CS, cold temperature (9/5°C) and short photoperiod (8 h). and (4) CL, cold temperature (9/5°C) and long photoperiod (8 h + 1 h night break). Overall, genome size (2C) varied between 20 and 34 pg. In the fall, when the cambium was initially dormant, the genome size increased in the natural environment, did not change under short days (WS and CS), and decreased under long days (WL and CL). The cambium reactivated in both WS and WL conditions. In the spring, while the cambium reactivated, the size of the genome decreased in the natural, WS and WL conditions, but not in the CS environment. In the CL conditions, the genome size started to decrease at the end of the 9-week exposure period. The decrease apparently occurred between prophase and telophase, which suggests that the extra DNA is extrachromosomal. In the summer, while the cambium ceased activity, the genome size did not change in the WS, WL and natural environments, whereas it decreased in the CS and CL conditions. The results indicate that increasing temperature and lengthening photoperiod in the spring induce the loss of the extra DNA. However, the environmental conditions that promote DNA synthesis in the fall remain unknown. Genome size varied independently of cambial growth potential and frost hardiness measured previously in the same experimental trees, indicating that the regulation of these processes does not directly involve the extra DNA. However, the finding that cambial cells cycled in the CS and CL environments only in the spring, when their genome size was large, suggests that the extra DNA is important for cambial growth at low temperatures.

@article{mellerowicz_nuclear_1990,
	title = {Nuclear size and shape changes in fusiform cambial cells of {Abies} balsamea during the annual cycle of activity and dormancy},
	volume = {68},
	issn = {0008-4026},
	url = {https://cdnsciencepub.com/doi/abs/10.1139/b90-243},
	doi = {10.1139/b90-243},
	abstract = {Interphase, prophase, and telophase nuclei were measured at different times during the year in fusiform cambial cells located at the base of the main stem of 6-year-old trees and in 1-year-old shoots from the top of 21-year-old trees. In both materials, the size and shape of nuclei exhibited seasonal changes that were independent of the cell cycle. Nuclear length was greater, whereas nuclear width and depth were smaller, when the cambium was dormant than when it was active. Nuclear volume gradually declined during the growing period and increased during the rest stage of dormancy. Nuclei had the highest surface to volume ratio in the quiescence stage of dormancy. During cambial activity, the projected area of prophase nuclei decreased, whereas it did not change for telophase nuclei. Nuclear volumes from the present investigation were combined with nuclear DNA contents previously recorded to estimate seasonal changes in nuclear DNA concentration which appear to be associated with the annual cycle of cambial activity and dormancy. Key words: balsam fir, periodicity, nucleus, xylogenesis, rest.},
	number = {9},
	urldate = {2024-10-02},
	journal = {Canadian Journal of Botany},
	publisher = {NRC Research Press},
	author = {Mellerowicz, E. J. and Riding, R. T. and Little, C. H. A.},
	month = sep,
	year = {1990},
	pages = {1857--1863},
}

Interphase, prophase, and telophase nuclei were measured at different times during the year in fusiform cambial cells located at the base of the main stem of 6-year-old trees and in 1-year-old shoots from the top of 21-year-old trees. In both materials, the size and shape of nuclei exhibited seasonal changes that were independent of the cell cycle. Nuclear length was greater, whereas nuclear width and depth were smaller, when the cambium was dormant than when it was active. Nuclear volume gradually declined during the growing period and increased during the rest stage of dormancy. Nuclei had the highest surface to volume ratio in the quiescence stage of dormancy. During cambial activity, the projected area of prophase nuclei decreased, whereas it did not change for telophase nuclei. Nuclear volumes from the present investigation were combined with nuclear DNA contents previously recorded to estimate seasonal changes in nuclear DNA concentration which appear to be associated with the annual cycle of cambial activity and dormancy. Key words: balsam fir, periodicity, nucleus, xylogenesis, rest.

@article{mellerowicz_genomic_1989,
	title = {Genomic variability in the vascular cambium of {Abies} balsamea},
	volume = {67},
	issn = {0008-4026},
	url = {https://cdnsciencepub.com/doi/abs/10.1139/b89-132},
	doi = {10.1139/b89-132},
	abstract = {DNA content was measured cytophotometrically in Feulgen-stained nuclei of fusiform cambial initials from 1- (twig) and 19-year-old (stem base) cambia of balsam fir (Abies balsamea) trees throughout the season. Telophase (2C) and prophase (4C) DNA contents varied with both cambial age and time of year. Maximum DNA contents were observed at the beginning of cambial activity in April. They were lower in the 1-year-old cambium (42.3 (2C) and 84.3 (4C) pg DNA) than in the 19-year-old cambium (59.4 (2C) and 111.1 (4C) pg DNA). The DNA levels decreased during the cambial growing season, attaining minimal values in September of 28.3 (2C) and 53.5 (4C) pg and 37.9 (2C) and 74.5 (4C) pg in the 1- and 19-year-old cambia, respectively. In the autumn, cells accumulated in the G1 phase of the cell cycle. DNA synthesis then took place increasing the 2C levels from the minimum values to the maximum values. This DNA synthesis in 1-year-old cambia started before the beginning of the transition from the resting to the quiescent stage of dormancy.},
	number = {4},
	urldate = {2024-10-02},
	journal = {Canadian Journal of Botany},
	publisher = {NRC Research Press},
	author = {Mellerowicz, E. J. and Riding, R. T. and Little, C. H. A.},
	month = apr,
	year = {1989},
	pages = {990--996},
}

DNA content was measured cytophotometrically in Feulgen-stained nuclei of fusiform cambial initials from 1- (twig) and 19-year-old (stem base) cambia of balsam fir (Abies balsamea) trees throughout the season. Telophase (2C) and prophase (4C) DNA contents varied with both cambial age and time of year. Maximum DNA contents were observed at the beginning of cambial activity in April. They were lower in the 1-year-old cambium (42.3 (2C) and 84.3 (4C) pg DNA) than in the 19-year-old cambium (59.4 (2C) and 111.1 (4C) pg DNA). The DNA levels decreased during the cambial growing season, attaining minimal values in September of 28.3 (2C) and 53.5 (4C) pg and 37.9 (2C) and 74.5 (4C) pg in the 1- and 19-year-old cambia, respectively. In the autumn, cells accumulated in the G1 phase of the cell cycle. DNA synthesis then took place increasing the 2C levels from the minimum values to the maximum values. This DNA synthesis in 1-year-old cambia started before the beginning of the transition from the resting to the quiescent stage of dormancy.