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1.  Elevated CO2 and/or ozone modify lignification in the wood of poplars (Populus tremula x alba) 
Journal of Experimental Botany  2012;63(11):4291-4301.
Trees will have to cope with increasing levels of CO2 and ozone in the atmosphere. The purpose of this work was to assess whether the lignification process could be altered in the wood of poplars under elevated CO2 and/or ozone. Young poplars were exposed either to charcoal-filtered air (control), to elevated CO2 (800 μl l−1), to ozone (200 nl l−1) or to a combination of elevated CO2 and ozone in controlled chambers. Lignification was analysed at different levels: biosynthesis pathway activities (enzyme and transcript), lignin content, and capacity to incorporate new assimilates by using 13C labelling. Elevated CO2 and ozone had opposite effects on many parameters (growth, biomass, cambial activity, wood cell wall thickness) except on lignin content which was increased by elevated CO2 and/or ozone. However, this increased lignification was due to different response mechanisms. Under elevated CO2, carbon supply to the stem and effective lignin synthesis were enhanced, leading to increased lignin content, although there was a reduction in the level of some enzyme and transcript involved in the lignin pathway. Ozone treatment induced a reduction in carbon supply and effective lignin synthesis as well as transcripts from all steps of the lignin pathway and some corresponding enzyme activities. However, lignin content was increased under ozone probably due to variations in other major components of the cell wall. Both mechanisms seemed to coexist under combined treatment and resulted in a high increase in lignin content.
doi:10.1093/jxb/ers118
PMCID: PMC3398455  PMID: 22553285
13C labelling; elevated CO2; lignin; ozone; poplar; wood
2.  Transcriptome profiling of radiata pine branches reveals new insights into reaction wood formation with implications in plant gravitropism 
BMC Genomics  2013;14(1):768.
Background
Formation of compression (CW) and opposite wood (OW) in branches and bent trunks is an adaptive feature of conifer trees in response to various displacement forces, such as gravity, wind, snow and artificial bending. Several previous studies have characterized tracheids, wood and gene transcription in artificially or naturally bent conifer trunks. These studies have provided molecular basis of reaction wood formation in response to bending forces and gravity stimulus. However, little is known about reaction wood formation and gene transcription in conifer branches under gravity stress. In this study SilviScan® technology was used to characterize tracheid and wood traits in radiate pine (Pinus radiata D. Don) branches and genes differentially transcribed in CW and OW were investigated using cDNA microarrays.
Results
CW drastically differed from OW in tracheids and wood traits with increased growth, thicker tracheid walls, larger microfibril angle (MFA), higher density and lower stiffness. However, CW and OW tracheids had similar diameters in either radial or tangential direction. Thus, gravity stress largely influenced wood growth, secondary wall deposition, cellulose microfibril orientation and wood properties, but had little impact on primary wall expansion. Microarray gene transcription revealed about 29% of the xylem transcriptomes were significantly altered in CW and OW sampled in both spring and autumn, providing molecular evidence for the drastic variation in tracheid and wood traits. Genes involved in cell division, cellulose biosynthesis, lignin deposition, and microtubules were mostly up-regulated in CW, conferring its greater growth, thicker tracheid walls, higher density, larger MFA and lower stiffness. However, genes with roles in cell expansion and primary wall formation were differentially transcribed in CW and OW, respectively, implicating their similar diameters of tracheid walls and different tracheid lengths. Interestingly, many genes related to hormone and calcium signalling as well as various environmental stresses were exclusively up-regulated in CW, providing important clues for earlier molecular signatures of reaction wood formation under gravity stimulus.
Conclusions
The first comprehensive investigation of tracheid characteristics, wood properties and gene transcription in branches of a conifer species revealed more accurate and new insights into reaction wood formation in response to gravity stress. The identified differentially transcribed genes with diverse functions conferred or implicated drastic CW and OW variation observed in radiata pine branches. These genes are excellent candidates for further researches on the molecular mechanisms of reaction wood formation with a view to plant gravitropism.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-14-768) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-14-768
PMCID: PMC4046691  PMID: 24209714
Compression wood; Tracheid; Conifers; Transcriptome; Microarray; Plant gravitropism; Microfibril angle (MFA); Wood stiffness
3.  Comprehensive Transcriptome Analysis of Developing Xylem Responding to Artificial Bending and Gravitational Stimuli in Betula platyphylla 
PLoS ONE  2014;9(2):e87566.
Betula platyphylla Suk (birch) is a fast-growing woody species that is important in pulp industries and the biofuels. However, as an important pulp species, few studies had been performed on its wood formation. In the present study, we investigated the molecular responses of birch xylem to artificial bending and gravitational stimuli. After trunks of birch trees were subjected to bending for 8 weeks, the cellulose content was significantly greater in tension wood (TW) than in opposite wood (OW) or normal wood (NW), whereas the lignin content in TW was significantly lower than that in OW and NW. In addition, TW grew more rapidly than OW and generated TW-specific fibers with an additional G-layer. Three transcriptome libraries were constructed from TW, OW and NW of B. platyphylla, respectively, after the plants were subjected to artificial bending. Overall, 80,909 nonredundant unigenes with a mean size of 768 nt were assembled. Expression profiles were generated, and 9,684 genes were found to be significantly differentially expressed among the TW, OW and NW libraries. These included genes involved in secondary cell wall structure, wood composition, and cellulose or lignin biosynthesis. Our study showed that during TW formation, genes involved in cellulose synthesis were induced, while the expression of lignin synthesis-related genes decreased, resulting in increased cellulose content and decreased lignin levels in TW. In addition, fasciclin-like arabinogalactan proteins play important role in TW formation. These findings may provide important insights into wood formation at the molecular level.
doi:10.1371/journal.pone.0087566
PMCID: PMC3930542  PMID: 24586282
4.  The poplar PtrWNDs are transcriptional activators of secondary cell wall biosynthesis 
Plant Signaling & Behavior  2010;5(4):469-472.
Secondary cell walls, consisting of cellulose, hemicelluloses and lignin, make up the bulk of wood biomass. It is therefore expected that dissection of the molecular mechanisms underlying secondary wall biosynthesis and its regulation will be instrumental to unravel the process of wood formation in tree species. Wood formation requires the coordinated activation of genes in the secondary wall biosynthetic program that is essential for the biosynthesis and assembly of wood components. It has recently been discovered that a group of poplar (Populus trichocarpa) wood-associated NAC domain transcription factors, PtrWNDs, which are functional orthologs of the Arabidopsis SND1, are capable of turning on the entire secondary wall biosynthetic program when expressed in Arabidopsis. In addition, two of the PtrWNDs were found to be able to activate the promoters of poplar wood biosynthetic genes and a number of other poplar wood-associated transcription factors. Further testing reveals that the promoters of these poplar wood-associated transcription factors are also activated by other PtrWNDs. It is therefore proposed that PtrWNDs are master transcriptional switches regulating a cascade of downstream transcription factors and thereby mediate the coordinated activation of wood biosynthetic genes during wood formation.
PMCID: PMC2958599  PMID: 20383071
NAC domain transcription factor; Populus trichocarpa; secondary wall biosynthesis; transcriptional regulation; wood formation
5.  Ectomycorrhizal Colonization and Diversity in Relation to Tree Biomass and Nutrition in a Plantation of Transgenic Poplars with Modified Lignin Biosynthesis 
PLoS ONE  2013;8(3):e59207.
Wood from biomass plantations with fast growing tree species such as poplars can be used as an alternative feedstock for production of biofuels. To facilitate utilization of lignocellulose for saccharification, transgenic poplars with modified or reduced lignin contents may be useful. However, the potential impact of poplars modified in the lignification pathway on ectomycorrhizal (EM) fungi, which play important roles for plant nutrition, is not known. The goal of this study was to investigate EM colonization and community composition in relation to biomass and nutrient status in wildtype (WT, Populus tremula × Populus alba) and transgenic poplar lines with suppressed activities of cinnamyl alcohol dehydrogenase, caffeate/5-hydroxyferulate O-methyltransferase, and cinnamoyl-CoA reductase in a biomass plantation. In different one-year-old poplar lines EM colonization varied from 58% to 86%, but the EM community composition of WT and transgenic poplars were indistinguishable. After two years, the colonization rate of all lines was increased to about 100%, but separation of EM communities between distinct transgenic poplar genotypes was observed. The differentiation of the EM assemblages was similar to that found between different genotypes of commercial clones of Populus × euramericana. The transgenic poplars exhibited significant growth and nutrient element differences in wood, with generally higher nutrient accumulation in stems of genotypes with lower than in those with higher biomass. A general linear mixed model simulated biomass of one-year-old poplar stems with high accuracy (adjusted R2 = 97%) by two factors: EM colonization and inverse wood N concentration. These results imply a link between N allocation and EM colonization, which may be crucial for wood production in the establishment phase of poplar biomass plantations. Our data further support that multiple poplar genotypes regardless whether generated by transgenic approaches or conventional breeding increase the variation in EM community composition in biomass plantations.
doi:10.1371/journal.pone.0059207
PMCID: PMC3596300  PMID: 23516610
6.  WGDB: Wood Gene Database with search interface 
Bioinformation  2014;10(1):39-42.
Wood quality can be defined in terms of particular end use with the involvement of several traits. Over the last fifteen years researchers have assessed the wood quality traits in forest trees. The wood quality was categorized as: cell wall biochemical traits, fibre properties include the microfibril angle, density and stiffness in loblolly pine [1]. The user friendly and an open-access database has been developed named Wood Gene Database (WGDB) for describing the wood genes along the information of protein and published research articles. It contains 720 wood genes from species namely Pinus, Deodar, fast growing trees namely Poplar, Eucalyptus. WGDB designed to encompass the majority of publicly accessible genes codes for cellulose, hemicellulose and lignin in tree species which are responsive to wood formation and quality. It is an interactive platform for collecting, managing and searching the specific wood genes; it also enables the data mining relate to the genomic information specifically in Arabidopsis thaliana, Populus trichocarpa, Eucalyptus grandis, Pinus taeda, Pinus radiata, Cedrus deodara, Cedrus atlantica. For user convenience, this database is cross linked with public databases namely NCBI, EMBL & Dendrome with the search engine Google for making it more informative and provides bioinformatics tools named BLAST,COBALT.
Availability
The database is freely available on www.wgdb.in
doi:10.6026/97320630010039
PMCID: PMC3916818  PMID: 24516325
Wood; Cellulose; Pinus; Cedrus; Poplar; Eucalyptus
7.  FTIR-ATR-based prediction and modelling of lignin and energy contents reveals independent intra-specific variation of these traits in bioenergy poplars 
Plant Methods  2011;7:9.
Background
There is an increasing demand for renewable resources to replace fossil fuels. However, different applications such as the production of secondary biofuels or combustion for energy production require different wood properties. Therefore, high-throughput methods are needed for rapid screening of wood in large scale samples, e.g., to evaluate the outcome of tree breeding or genetic engineering. In this study, we investigated the intra-specific variability of lignin and energy contents in extractive-free wood of hybrid poplar progenies (Populus trichocarpa × deltoides) and tested if the range was sufficient for the development of quantitative prediction models based on Fourier transform infrared spectroscopy (FTIR). Since lignin is a major energy-bearing compound, we expected that the energy content of wood would be positively correlated with the lignin content.
Results
Lignin contents of extractive-free poplar wood samples determined by the acetyl bromide method ranged from 23.4% to 32.1%, and the calorific values measured with a combustion calorimeter varied from 17260 to 19767 J g-1. For the development of calibration models partial least square regression and cross validation was applied to correlate FTIR spectra determined with an attenuated total reflectance (ATR) unit to measured values of lignin or energy contents. The best models with high coefficients of determination (R2 (calibration) = 0.91 and 0.90; R2 (cross-validation) = 0.81 and 0.79) and low root mean square errors of cross validation (RMSECV = 0.77% and 62 J g-1) for lignin and energy determination, respectively, were obtained after data pre-processing and automatic wavenumber restriction. The calibration models were validated by analyses of independent sets of wood samples yielding R2 = 0.88 and 0.86 for lignin and energy contents, respectively.
Conclusions
These results show that FTIR-ATR spectroscopy is suitable as a high-throughput method for lignin and energy estimations in large data sets. Our study revealed that the intra-specific variations in lignin and energy contents were unrelated to each other and that the lignin content, therefore, was no predictor of the energy content. Employing principle component analyses we showed that factor loadings for the energy content were mainly associated with carbohydrate ring vibrations, whereas those for lignin were mainly related to aromatic compounds. Therefore, our analysis suggests that it may be possible to optimize the energy content of trees without concomitant increase in lignin.
doi:10.1186/1746-4811-7-9
PMCID: PMC3094334  PMID: 21477346
Bioenergy; heat value; intraspecific variation; lignin; high throughput method; FTIR spectroscopy
8.  Transcriptional regulation of lignin biosynthesis 
Plant Signaling & Behavior  2009;4(11):1028-1034.
Lignin is the second most abundant plant biopolymer mainly present in the secondary walls of tracheary elements and fibers in wood. Understanding how lignin is biosynthesized has long been an interest to plant biologists and will have a significant impact on tree biotechnology. Lignin is polymerized from monolignols that are synthesized through the lignin biosynthetic pathway. To make lignin, all the genes in the lignin biosynthetic pathway need to be coordinately turned on. It has been shown that a common cis-element, namely the AC element, is present in the majority of the lignin biosynthetic genes and required for their expression in lignifying cells. Important progress has been made in the identification of transcription factors that bind to the AC elements and are potentially involved in the coordinated regulation of lignin biosynthesis. The Arabidopsis MYB58 and MYB63 as well as their poplar ortholog PtrMYB28 are transcriptional activators of the lignin biosynthetic pathway, whereas the eucalyptus egMYB2 and pine PtMYB4 transcription factors are likely Arabidopsis MYB46 orthologs involved in the regulation of the entire secondary wall biosynthetic program. It was found that the transcriptional regulation of lignin biosynthesis is under the control of the same transcriptional network regulating the biosynthesis of other secondary wall components, including cellulose and xylan. The identification of transcription factors directly activating lignin biosynthetic genes provides unprecedented tools to potentially manipulate the amount of lignin in wood and other plant products based on our needs.
PMCID: PMC2819510  PMID: 19838072
lignin; lignin biosynthesis; MYB; secondary wall biosynthesis; transcription factor; transcriptional regulation
9.  Anatomy and lignin distribution in reaction phloem fibres of several Japanese hardwoods 
Annals of Botany  2012;110(4):897-904.
Background and Aims
Although tension wood formation and the structure of gelatinous fibres (G-fibres) have been widely investigated, studies of the influence of the reaction phenomenon on phloem fibres have been few and incomplete in comparison with those of xylem wood fibres. This study was undertaken to clarify the influence of stem inclination on phloem fibres using several Japanese hardwood species that produce different G-fibre types in tension wood.
Methods
Eight hardwood species were inclined at 30–45° at the beginning of April. Specimens were collected in July and December. The cell-wall structure and lignin distribution of phloem fibres on both the tension and opposite sides were compared by light microscopy, ultraviolet microscopy, confocal laser scanning microscopy after staining with acriflavine, and transmission electron microscopy after staining with potassium permanganate.
Key Results
Three types of changes were found in tension-side phloem fibres: (1) increases in the proportion of the syringyl unit in lignin in the S1 and S2 layers and compound middle lamella (Cercidiphyllum japonicum), (2) formation of unlignified gelatinous layers (Melia azedarach and Acer rufinerve) and (3) increases in the number of layers (n) in the multi-layered structure of S1 + S2 + n (G + L) (Mallotus japonicus). Other species showed no obvious change in cell-wall structure or lignin distribution.
Conclusions
Phloem fibres of the tree species examined in our study showed three types of changes in lignin distribution and cell-wall structure. The reaction phenomenon may vary with tree species and may not be closely related to G-fibre type in tension wood.
doi:10.1093/aob/mcs144
PMCID: PMC3423802  PMID: 22778147
Phloem fibre; lignification; reaction phloem; Cercidiphyllum japonicum; Melia azedarach; Acer rufinerve; Mallotus japonicus; Celtis sinensis; Aphananthe aspera; Corylus sieboldiana; Magnolia salicifolia
10.  N-fertilization has different effects on the growth, carbon and nitrogen physiology, and wood properties of slow- and fast-growing Populus species 
Journal of Experimental Botany  2012;63(17):6173-6185.
To investigate how N-fertilization affects the growth, carbon and nitrogen (N) physiology, and wood properties of poplars with contrasting growth characteristics, slow-growing (Populus popularis, Pp) and fast-growing (P. alba×P. glandulosa, Pg) poplar saplings were exposed to different N levels. Above-ground biomass, leaf area, photosynthetic rates (A), instantaneous photosynthetic nitrogen use efficiency (PNUE i), chlorophyll and foliar sugar concentrations were higher in Pg than in Pp. Foliar nitrate reductase (NR) activities and root glutamate synthase (GOGAT) activities were higher in Pg than in Pp as were the N amount and NUE of new shoots. Lignin contents and calorific values of Pg wood were less than that of Pp wood. N-fertilization reduced root biomass of Pg more than of Pp, but increased leaf biomass, leaf area, A, and PNUEi of Pg more than of Pp. Among 13 genes involved in the transport of ammonium or nitrate or in N assimilation, transcripts showed more pronounced changes to N-fertilization in Pg than in Pp. Increases in NR activities and N contents due to N-fertilization were larger in Pg than in Pp. In both species, N-fertilization resulted in lower calorific values as well as shorter and wider vessel elements/fibres. These results suggest that growth, carbon and N physiology, and wood properties are more sensitive to increasing N availability in fast-growing poplars than in slow-growing ones, which is probably due to prioritized resource allocation to the leaves and accelerated N physiological processes in fast-growing poplars under higher N levels.
doi:10.1093/jxb/ers271
PMCID: PMC3481209  PMID: 23028021
Amino acids; ammonium transporter; bioenergy; carbohydrates; gene expression; nitrate transporter
11.  Transcriptome profiling of Pinus radiata juvenile wood with contrasting stiffness identifies putative candidate genes involved in microfibril orientation and cell wall mechanics 
BMC Genomics  2011;12:480.
Background
The mechanical properties of wood are largely determined by the orientation of cellulose microfibrils in secondary cell walls. Several genes and their allelic variants have previously been found to affect microfibril angle (MFA) and wood stiffness; however, the molecular mechanisms controlling microfibril orientation and mechanical strength are largely uncharacterised. In the present study, cDNA microarrays were used to compare gene expression in developing xylem with contrasting stiffness and MFA in juvenile Pinus radiata trees in order to gain further insights into the molecular mechanisms underlying microfibril orientation and cell wall mechanics.
Results
Juvenile radiata pine trees with higher stiffness (HS) had lower MFA in the earlywood and latewood of each ring compared to low stiffness (LS) trees. Approximately 3.4 to 14.5% out of 3, 320 xylem unigenes on cDNA microarrays were differentially regulated in juvenile wood with contrasting stiffness and MFA. Greater variation in MFA and stiffness was observed in earlywood compared to latewood, suggesting earlywood contributes most to differences in stiffness; however, 3-4 times more genes were differentially regulated in latewood than in earlywood. A total of 108 xylem unigenes were differentially regulated in juvenile wood with HS and LS in at least two seasons, including 43 unigenes with unknown functions. Many genes involved in cytoskeleton development and secondary wall formation (cellulose and lignin biosynthesis) were preferentially transcribed in wood with HS and low MFA. In contrast, several genes involved in cell division and primary wall synthesis were more abundantly transcribed in LS wood with high MFA.
Conclusions
Microarray expression profiles in Pinus radiata juvenile wood with contrasting stiffness has shed more light on the transcriptional control of microfibril orientation and the mechanical properties of wood. The identified candidate genes provide an invaluable resource for further gene function and association genetics studies aimed at deepening our understanding of cell wall biomechanics with a view to improving the mechanical properties of wood.
doi:10.1186/1471-2164-12-480
PMCID: PMC3224210  PMID: 21962175
12.  Investigation of tension wood formation and 2,6-dichlorbenzonitrile application in short rotation coppice willow composition and enzymatic saccharification 
Background
Short rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment.
Results
Glucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin.
Conclusions
Final glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.
doi:10.1186/1754-6834-4-13
PMCID: PMC3115855  PMID: 21609446
13.  Enhancement of total sugar and lignin yields through dissolution of poplar wood by hot water and dilute acid flowthrough pretreatment 
Background
Pretreatment is a vital but expensive step in biomass biofuel production. Overall, most of this past effort has been directed at maximizing sugar yields from hemicellulose and cellulose through trials with different chemicals, operating conditions, and equipment configurations. Flowthrough pretreatment provides a promising platform to dissolution of lignocellulosic biomass to generate high yields of fermentable sugars and lignin for biofuels productions.
Results
Dissolution of xylan, lignin, and cellulose from poplar wood were significantly enhanced by water-only and dilute acid (0.05% w/w, H2SO4) flowthrough pretreatment when the temperature was raised from 200°C to 280°C over a range of flow rates 10-62.5 mL/min, resulting in more than 98% solid removal. Up to 40% of original xylan was converted to xylose in the hydrolyzate and the rest xylan was solubilized into xylooligomers with negligible furfural formation. Up to 100% cellulose was removed into hydrolyzate with the highest glucose yield of 60% and low 5-hydroxymethylfurfural (5-HMF) formation. The maximal recovered insoluble lignin and soluble lignin were 98% and 15% of original lignin, respectively. In addition, enzymatic hydrolysis of pretreated whole slurries was characterized under various enzyme loadings with or without Bovine serum albumin (BSA) treatment. More than 90% glucose yield and 95% xylose yield were obtained from enzymatic hydrolysis of dilute acid pretreated whole slurries with 10 mg protein Ctec 2 with 2 mg Htec2/g glucan + xylan.
Conclusions
Nearly complete dissolution of whole biomass was realized through water-only and dilute acid flowthrough pretreatment under tested conditions. Temperature was considered as the most significant factor for cellulose degradation. The cellulose removal significantly increased as temperature reached 240°C for water-only and 220°C for dilute acid. Dilute acid pretreatment resulted in higher yields of recovered xylan and cellulose as monomeric sugars in the hydrolyzate than that for water-only pretreatment. Enzymes readily hydrolyzed the degraded cellulose and xylooligomers in pretreatment hydrolysate. Results suggested that kinetics controlled the flowthrough pretreatment of biomass dissolution, which was also affected by flow rate to certain extent.
doi:10.1186/1754-6834-7-76
PMCID: PMC4040120  PMID: 24936209
Hot water; dilute acid; flowthrough pretreatment; Severity parameter; poplar wood; Enzymatic hydrolysis
14.  Expression analysis of LIM gene family in poplar, toward an updated phylogenetic classification 
BMC Research Notes  2012;5:102.
Background
Plant LIM domain proteins may act as transcriptional activators of lignin biosynthesis and/or as actin binding and bundling proteins. Plant LIM genes have evolved in phylogenetic subgroups differing in their expression profiles: in the whole plant or specifically in pollen. However, several poplar PtLIM genes belong to uncharacterized monophyletic subgroups and the expression patterns of the LIM gene family in a woody plant have not been studied.
Findings
In this work, the expression pattern of the twelve duplicated poplar PtLIM genes has been investigated by semi quantitative RT-PCR in different vegetative and reproductive tissues. As in other plant species, poplar PtLIM genes were widely expressed in the tree or in particular tissues. Especially, PtXLIM1a, PtXLIM1b and PtWLIM1b genes were preferentially expressed in the secondary xylem, suggesting a specific function in wood formation. Moreover, the expression of these genes and of the PtPLIM2a gene was increased in tension wood. Western-blot analysis confirmed the preferential expression of PtXLIM1a protein during xylem differentiation and tension wood formation. Genes classified within the pollen specific PLIM2 and PLIM2-like subgroups were all strongly expressed in pollen but also in cottony hairs. Interestingly, pairs of duplicated PtLIM genes exhibited different expression patterns indicating subfunctionalisations in specific tissues.
Conclusions
The strong expression of several LIM genes in cottony hairs and germinating pollen, as well as in xylem fibers suggests an involvement of plant LIM domain proteins in the control of cell expansion. Comparisons of expression profiles of poplar LIM genes with the published functions of closely related plant LIM genes suggest conserved functions in the areas of lignin biosynthesis, pollen tube growth and mechanical stress response. Based on these results, we propose a novel nomenclature of poplar LIM domain proteins.
doi:10.1186/1756-0500-5-102
PMCID: PMC3392731  PMID: 22339987
15.  Functional Characterization of the Poplar R2R3-MYB Transcription Factor PtoMYB216 Involved in the Regulation of Lignin Biosynthesis during Wood Formation 
PLoS ONE  2013;8(10):e76369.
Because of the importance of wood in many industrial applications, tremendous studies have been performed on wood formation, especially in lignin biosynthesis. MYB transcription factors (TFs), which consist of a large family of plant TFs, have been reported to directly regulate lignin biosynthetic genes in a number of plants. In this study, we describe the cloning and functional characterization of PtoMYB216, a cDNA isolated from Chinese white poplar (Populus tomentosa Carr.). PtoMYB216 encodes a protein belonging to the R2R3-MYB family and displays significant similarity with other MYB factors shown to regulate lignin synthesis in Arabidopsis. Gene expression profiling studies showed that PtoMYB216 mRNA is specifically expressed during secondary wall formation in wood. The 1.8-kb promoter sequence of PtoMYB216 was fused to the GUS coding sequence and introduced into wild-type A. thaliana. GUS expression was shown to be restricted to tissues undergoing secondary cell wall formation. Overexpression of PtoMYB216 specifically activated the expression of the upstream genes in the lignin biosynthetic pathway and resulted in ectopic deposition of lignin in cells that are normally unligninified. These results suggest that PtoMYB216 is specific transcriptional activators of lignin biosynthesis and involved in the regulation of wood formation in poplar.
doi:10.1371/journal.pone.0076369
PMCID: PMC3810269  PMID: 24204619
16.  Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa 
Planta  2009;230(3):589-597.
Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare lignification in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-μm. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm−1, differences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of fibers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood saccharification. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-009-0963-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00425-009-0963-x
PMCID: PMC2715566  PMID: 19526248
Populus trichocarpa (black cottonwood); Cell wall; Lignin; Microspectroscopy; Raman imaging; Transgenic
17.  Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway 
BMC Genomics  2011;12(Suppl 3):S13.
Background
Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem.
Results
In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing. Differential expression of the small RNAs between secondary xylem tissues with contrasting lignin content suggests that a cascade of miRNAs play an interconnected role in regulating the lignin biosynthetic pathway in Acacia species.
Conclusions
Our study critically demonstrated the roles of small RNAs during secondary wall formation. Comparison of the expression pattern of small RNAs between secondary xylem tissues with contrasting lignin content strongly indicated that small RNAs play a key regulatory role during lignin biosynthesis. Our analyses suggest an evolutionary mechanism for miRNA targets on the basis of the length of their 5’ and 3’ UTRs and their cellular roles. The results obtained can be used to better understand the roles of small RNAs during lignin biosynthesis and for the development of gene constructs for silencing of specific genes involved in monolignol biosynthesis with minimal effect on plant fitness and viability. For the first time, small RNAs were proven to play an important regulatory role during lignin biosynthesis in A. mangium.
doi:10.1186/1471-2164-12-S3-S13
PMCID: PMC3333172  PMID: 22369296
18.  Proteomics of Plasma Membranes from Poplar Trees Reveals Tissue Distribution of Transporters, Receptors, and Proteins in Cell Wall Formation* 
By exploiting the abundant tissues available from Populus trees, 3–4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as “housekeeping” proteins, a typical example being P-type H+-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.
doi:10.1074/mcp.M900289-MCP200
PMCID: PMC2830847  PMID: 19955078
19.  Degradation of 14C-Labeled Lignins and 14C-Labeled Aromatic Acids by Fusarium solani 
Abilities of isolate AF-W1 of Fusarium solani to degrade the side chain and the ring structure of synthetic dehydrogenative polymerizates, aromatic acids, or lignin in sound wood were investigated under several conditions of growth substrate or basal medium and pH. Significant transformations of lignins occurred in 50 days in both unextracted and extracted sound wood substrates with 3% malt as the growth substrate and the pH buffered initially at 4.0 with 2,2-dimethylsuccinate. Degradation of lignin in such woods also occurred under unbuffered pH conditions when a basal medium of either 3% malt or powdered cellulose in deionized water was present. Decomposition of the lignin in these woods did not occur in cultures where d-glucose was present as a growth substrate. F. solani significantly transformed, as measured as evolved 14CO2, both synthetic side chain (β, γ)-14C- and U-ring-14C-labeled lignins in 30 days under liquid culture conditions of only distilled deionized water and no pH adjustment. Degradation of dehydrogenative polymerizates by F. solani was reduced drastically when D2 was the liquid medium. AF-W1 also cleaved the α-14C from p-hydroxybenzoic acid and evolved 14CO2 from the substrate, [3-14C]cinnamic acid. Thus, the fungus cleaved side chain carbon from substrate that originally lacked hydroxyl substitution on the aromatic nucleus. Surprisingly, small amounts of 14C cleaved from aromatic acids by F. solani were incorporated into cell mass. Initial buffering of the culture medium to pH 4.0 or 5.0 with 0.1 M 2,2-dimethylsuccinate significantly increased F. solani degradation of all lignins or aromatic acids. Results indicated that AF-W1 used lignin as a sole carbon source.
PMCID: PMC291584  PMID: 16345616
20.  Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock 
Background
Biofuel production from lignocellulosic material is hampered by biomass recalcitrance towards enzymatic hydrolysis due to the compact architecture of the plant cell wall and the presence of lignin. The purpose of this work is to study the ability of an industrially available laccase-mediator system to modify and remove lignin during pretreatment of wood (Eucalyptus globulus) feedstock, thus improving saccharification, and to analyze the chemical modifications produced in the whole material and especially in the recalcitrant lignin moiety.
Results
Up to 50% lignin removal from ground eucalypt wood was attained by pretreatment with recombinant Myceliophthora thermophila laccase and methyl syringate as mediator, followed by alkaline peroxide extraction in a multistage sequence. The lignin removal directly correlated with increases (approximately 40%) in glucose and xylose yields after enzymatic hydrolysis. The pretreatment using laccase alone (without mediator) removed up to 20% of lignin from eucalypt wood. Pyrolysis-gas chromatography/mass spectrometry of the pretreated wood revealed modifications of the lignin polymer, as shown by lignin markers with shortened side chains and increased syringyl-to-guaiacyl ratio. Additional information on the chemical modifications produced was obtained by two-dimensional nuclear magnetic resonance of the whole wood swollen in dimethylsulfoxide-d6. The spectra obtained revealed the removal of guaiacyl and syringyl lignin units, although with a preferential removal of the former, and the lower number of aliphatic side-chains per phenylpropane unit (involved in main β-O-4ʹ and β-βʹ inter-unit linkages), in agreement with the pyrolysis-gas chromatography/mass spectrometry results, without a substantial change in the wood polysaccharide signals. However, the most noticeable modification observed in the spectra was the formation of Cα-oxidized syringyl lignin units during the enzymatic treatment. Further insight into the modifications of lignin structure, affecting other inter-unit linkages and oxidized structures, was attained by nuclear magnetic resonance of the lignins isolated from the eucalypt feedstock after the enzymatic pretreatments.
Conclusions
This work shows the potential of an oxidative enzymatic pretreatment to delignify and improve cellulase saccharification of a hardwood feedstock (eucalypt wood) when applied directly on the ground lignocellulosic material, and reveals the main chemical changes in the pretreated material, and its recalcitrant lignin moiety, behind the above results.
doi:10.1186/1754-6834-7-6
PMCID: PMC3917704  PMID: 24401177
2D NMR; Analytical pyrolysis; Bioethanol; Eucalyptus globulus; Enzymatic delignification; Laccase; Lignin; Lignocellulose; Pretreatment; Saccharification
21.  Imbalanced Lignin Biosynthesis Promotes the Sexual Reproduction of Homothallic Oomycete Pathogens 
PLoS Pathogens  2009;5(1):e1000264.
Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-l-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-l-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment.
Author Summary
Lignin is an essential component of wood and the second most abundant terrestrial biopolymer. Plants synthesize lignin to strengthen cell walls and to resist pathogen attack. Because the technological value of plants is determined by the amount and composition of lignin, genetic engineering approaches frequently aim at altering these parameters. However, data showing how plants with modified lignin content interact with the biotic environment are still scarce. This study is the first of its kind to evaluate how a model plant, which was mutated for a key enzyme in lignin biosynthesis, interacts with pathogens from different kingdoms. We found that the mutants were generally more susceptible to bacteria and fungi. Additionally, the mutation altered the plant physiology in a manner that elevated sexual reproduction of an oomycete pathogen. Oospores resulting from this mode of reproduction are the most persisting propagules of this pathogen. Their elevated formation correlated with the accumulation of a soluble phenylpropanoid derivative in the mutants, which we synthesized and found to be able to stimulate sexual oomycete reproduction in vitro. Our study indicates that interfering with lignin composition may drastically alter the outcome of plant–pathogen interactions.
doi:10.1371/journal.ppat.1000264
PMCID: PMC2613516  PMID: 19148278
22.  Structural Characterization of Lignin during Pinus taeda Wood Treatment with Ceriporiopsis subvermispora 
Pinus taeda wood chips were biotreated with Ceriporiopsis subvermispora under solid-state fermentation for periods varying from 15 to 90 days. Milled wood lignins extracted from sound and biotreated wood samples were characterized by wet-chemical and spectroscopic techniques. Treatment of the lignins by derivatization followed by reductive cleavage (DFRC) made it possible to detect DFRC monomers and dimers that are diagnostic of the occurrence of arylglycerol-β-O-aryl and β-β, β-5, β-1, and 4-O-5 units in the lignin structure. Quantification of these DFRC products indicated that β-O-aryl cleavage was a significant route for lignin biodegradation but that β-β, β-5, β-1, and 4-O-5 linkages were more resistant to the biological attack. The amount of aromatic hydroxyls did not increase with the split of β-O-4 linkages, suggesting that the β-O-4 cleavage products remain as quinone-type structures as detected by UV and visible spectroscopy. Nuclear magnetic resonance techniques also indicated the formation of new substructures containing nonoxygenated, saturated aliphatic carbons (CH2 and CH3) in the side chains of lignins extracted from biotreated wood samples.
doi:10.1128/AEM.70.7.4073-4078.2004
PMCID: PMC444757  PMID: 15240285
23.  Induction of Cambial Reactivation by Localized Heating in a Deciduous Hardwood Hybrid Poplar (Populus sieboldii × P. grandidentata) 
Annals of Botany  2007;100(3):439-447.
Background and Aims
The timing of cambial reactivation plays an important role in the control of both the quantity and the quality of wood. The effect of localized heating on cambial reactivation in the main stem of a deciduous hardwood hybrid poplar (Populus sieboldii × P. grandidentata) was investigated.
Methods
Electric heating tape (20–22 °C) was wrapped at one side of the main stem of cloned hybrid poplar trees at breast height in winter. Small blocks were collected from both heated and non-heated control portions of the stem for sequential observations of cambial activity and for studies of the localization of storage starch around the cambium from dormancy to reactivation by light microscopy.
Key Results
Cell division in phloem began earlier than cambial reactivation in locally heated portions of stems. Moreover, the cambial reactivation induced by localized heating occurred earlier than natural cambial reactivation. In heated stems, well-developed secondary xylem was produced that had almost the same structure as the natural xylem. When cambial reactivation was induced by heating, the buds of trees had not yet burst, indicating that there was no close temporal relationship between bud burst and cambial reactivation. In heated stems, the amount of storage starch decreased near the cambium upon reactivation of the cambium. After cambial reactivation, storage starch disappeared completely. Storage starch appeared again, near the cambium, during xylem differentiation in heated stems.
Conclusions
The results suggest that, in deciduous diffuse-porous hardwood poplar growing in a temperate zone, the temperature in the stem is a limiting factor for reactivation of phloem and cambium. An increase in temperature might induce the conversion of storage starch to sucrose for the activation of cambial cell division and secondary xylem. Localized heating in poplar stems provides a useful experimental system for studies of cambial biology.
doi:10.1093/aob/mcm130
PMCID: PMC2533603  PMID: 17621596
Populus sieboldii × Populus grandidentata; localized heating, cambial reactivation; model system; storage starch; xylem differentiation
24.  Integrative Analysis of Transgenic Alfalfa (Medicago sativa L.) Suggests New Metabolic Control Mechanisms for Monolignol Biosynthesis 
PLoS Computational Biology  2011;7(5):e1002047.
The entanglement of lignin polymers with cellulose and hemicellulose in plant cell walls is a major biological barrier to the economically viable production of biofuels from woody biomass. Recent efforts of reducing this recalcitrance with transgenic techniques have been showing promise for ameliorating or even obviating the need for costly pretreatments that are otherwise required to remove lignin from cellulose and hemicelluloses. At the same time, genetic manipulations of lignin biosynthetic enzymes have sometimes yielded unforeseen consequences on lignin composition, thus raising the question of whether the current understanding of the pathway is indeed correct. To address this question systemically, we developed and applied a novel modeling approach that, instead of analyzing the pathway within a single target context, permits a comprehensive, simultaneous investigation of different datasets in wild type and transgenic plants. Specifically, the proposed approach combines static flux-based analysis with a Monte Carlo simulation in which very many randomly chosen sets of parameter values are evaluated against kinetic models of lignin biosynthesis in different stem internodes of wild type and lignin-modified alfalfa plants. In addition to four new postulates that address the reversibility of some key reactions, the modeling effort led to two novel postulates regarding the control of the lignin biosynthetic pathway. The first posits functionally independent pathways toward the synthesis of different lignin monomers, while the second postulate proposes a novel feedforward regulatory mechanism. Subsequent laboratory experiments have identified the signaling molecule salicylic acid as a potential mediator of the postulated control mechanism. Overall, the results demonstrate that mathematical modeling can be a valuable complement to conventional transgenic approaches and that it can provide biological insights that are otherwise difficult to obtain.
Author Summary
Cellulose-based biofuels presently offer the most environmentally attractive and technologically promising alternative to fossil fuels. To be viable, biofuels must be derived from non-food crops, such as grasses, wood, bark, and plant residues. Techniques for releasing the energy stored in these renewable materials must first untangle a very recalcitrant scaffold of interlinking molecules inside the plant cell walls, which is very costly. Much of the recalcitrance is due to the natural polymer lignin, which hardens the cell walls and is composed of three different building blocks, called monolignols. Modern transgenic techniques have yielded plant lines whose cell walls are easier to break down, but some of these modified plants have exhibited unexplained and undesired features. Here, we present new computational methods for analyzing monolignol biosynthesis in unprecedented detail. The analysis simultaneously accounts for lignin biosynthesis in various transgenic lines and different developmental stages and yields six novel, testable postulates regarding the metabolic control of the pathway. The results suggest new, targeted experiments towards a better understanding of monolignol biosynthesis and issues of recalcitrance reduction. More generally, the results highlight the genuine benefits of using computational methods as companions and complements to experimental studies.
doi:10.1371/journal.pcbi.1002047
PMCID: PMC3098223  PMID: 21625579
25.  Habituation to thaxtomin A in hybrid poplar cell suspensions provides enhanced and durable resistance to inhibitors of cellulose synthesis 
BMC Plant Biology  2010;10:272.
Background
Thaxtomin A (TA), a phytotoxin produced by the phytopathogen Streptomyces scabies, is essential for the development of potato common scab disease. TA inhibits cellulose synthesis but its actual mode of action is unknown. Addition of TA to hybrid poplar (Populus trichocarpa x Populus deltoides) cell suspensions can activate a cellular program leading to cell death. In contrast, it is possible to habituate hybrid poplar cell cultures to grow in the presence of TA levels that would normally induce cell death. The purpose of this study is to characterize TA-habituated cells and the mechanisms that may be involved in enhancing resistance to TA.
Results
Habituation to TA was performed by adding increasing levels of TA to cell cultures at the time of subculture over a period of 12 months. TA-habituated cells were then cultured in the absence of TA for more than three years. These cells displayed a reduced size and growth compared to control cells and had fragmented vacuoles filled with electron-dense material. Habituation to TA was associated with changes in the cell wall composition, with a reduction in cellulose and an increase in pectin levels. Remarkably, high level of resistance to TA was maintained in TA-habituated cells even after being cultured in the absence of TA. Moreover, these cells exhibited enhanced resistance to two other inhibitors of cellulose biosynthesis, dichlobenil and isoxaben. Analysis of gene expression in TA-habituated cells using an Affymetrix GeneChip Poplar Genome Array revealed that durable resistance to TA is associated with a major and complex reprogramming of gene expression implicating processes such as cell wall synthesis and modification, lignin and flavonoid synthesis, as well as DNA and chromatin modifications.
Conclusions
We have shown that habituation to TA induced durable resistance to the bacterial toxin in poplar cells. TA-habituation also enhanced resistance to two other structurally different inhibitors of cellulose synthesis that were found to target different proteins. Enhanced resistance was associated with major changes in the expression of numerous genes, including some genes that are involved in DNA and chromatin modifications, suggesting that epigenetic changes might be involved in this process.
doi:10.1186/1471-2229-10-272
PMCID: PMC3016406  PMID: 21143977

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