Secondary Cell Wall Cellulose Research Articles

PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar.

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. This network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription - quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector-reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis.

In situ infrared imaging of the local orientation of cellulose fibrils in plant secondary cell walls.

The mechanical and chemical properties of plant cell walls greatly rely on the supramolecular assembly of cellulose fibrils. To study the local orientation of cellulose in secondary plant cell walls, diffraction limited infrared (IR) micro-spectroscopic mapping experiments were conducted at different orientation of transverse leaf section of the grass Sorghum bicolor with respect to the polarization direction of the IR radiation. Two-dimensional maps, based on polarization-sensitive absorption bands of cellulose were obtained for different polarization angles. They reveal a significant degree of anisotropy of the cellulose macromolecules as well as of other biopolymers in sclerenchyma and xylem regions of the cross section. Quantification of the signals assigned to polarization sensitive vibrational modes allowed to determine the preferential orientation of the sub-micron cellulose fibrils in single cell walls. A sample of crystalline nano-cellulose comprising both a single microcrystal as well as unordered layers of nanocrystals was used for validation of the approach. The results demonstrate that diffraction limited IR micro-spectroscopy can be used to study hierarchically structured materials with complex anisotropic behavior.

Engineering Curcumin Biosynthesis in Poplar Affects Lignification and Biomass Yield.

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from shoot-tip necrosis and yield penalties. Compared to that of the wild-type (WT), the wood of transgenic poplars had 21% less cellulose, 28% more matrix polysaccharides, 23% more lignin and a significantly altered lignin composition. More specifically, ProCesA8-B:DCS_CURS2 lignin had a reduced syringyl/guaiacyl unit (S/G) ratio, an increased frequency of p-hydroxyphenyl (H) units, a decreased frequency of p-hydroxybenzoates and a higher fraction of phenylcoumaran units. Without, or with alkaline or hot water pretreatment, the saccharification efficiency of the transgenic lines was equal to that of the WT. These differences in (growth) phenotype illustrate that translational research in crops is essential to assess the value of an engineering strategy for applications. Further fine-tuning of this research strategy (e.g., by using more specific promoters or by translating this strategy to other crops such as maize) might lead to transgenic bio-energy crops with cell walls more amenable to deconstruction without settling in yield.

CAGEs are Golgi-localized GT31 enzymes involved in cellulose biosynthesis in Arabidopsis.

SUMMARYCellulose is the main structural component in the plant cell walls. We show that two glycosyltransferase family 31 (GT31) enzymes of Arabidopsis thaliana, here named cellulose synthesis associated glycosyltransferases 1 and 2 (CAGE1 and 2), influence both primary and secondary cell wall cellulose biosynthesis. cage1cage2 mutants show primary cell wall defects manifesting as impaired growth and cell expansion in seedlings and etiolated hypocotyls, along with secondary cell wall defects, apparent as collapsed xylem vessels and reduced xylem wall thickness in the inflorescence stem. Single and double cage mutants also show increased sensitivity to the cellulose biosynthesis inhibitor isoxaben. The cage1cage2 phenotypes were associated with an approximately 30% reduction in cellulose content, an approximately 50% reduction in secondary cell wall CELLULOSE SYNTHASE (CESA) protein levels in stems and reduced cellulose biosynthesis rate in seedlings. CESA transcript levels were not significantly altered in cage1cage2 mutants, suggesting that the reduction in CESA levels was caused by a post‐transcriptional mechanism. Both CAGE1 and 2 localize to the Golgi apparatus and are predicted to synthesize β‐1,3‐galactans on arabinogalactan proteins. In line with this, the cage1cage2 mutants exhibit reduced levels of β‐Yariv binding to arabinogalactan protein linked β‐1,3‐galactan. This leads us to hypothesize that defects in arabinogalactan biosynthesis underlie the cellulose deficiency of the mutants.

The DUF288 domain containing proteins GhSTLs participate in cotton fiber cellulose synthesis and impact on fiber elongation

Cotton is one of the most important economic crops in the world, with over 90 % cellulose in the mature fiber. However, the cellulose synthesis mechanism in cotton fibers is poorly understood. Here, we identified four DUF288 domain containing proteins, which we designated GhSTL1-4. These four GhSTL genes are highly expressed in 6 days post anthesis (dpa) and 20 dpa cotton fibers. They are localized to the Golgi apparatus, and can rescue the growth defects in primary cell wall (PCW) and secondary cell wall (SCW) of cellulose synthesis of the Arabidopsis stl1stl2 double mutant at varying degrees. Silencing of GhSTLs resulted in reduced cellulose content and shorter fibers. In addition, split-ubiquitin membrane yeast two-hybrid analysis showed that GhSTL1 and GhSTL4 can interact with PCW-related GhCesA6-1/6-3 and SCW-associated GhCesA7-1/7-2. GhSTL3 can interact with SCW-related GhCesA4-3. These interactions are further confirmed by firefly luciferase complementation imaging assay. Together, we demonstrate that GhSTLs can selectively interact with both the PCW and SCW-associated GhCesAs and impact on cellulose synthesis and fiber development. Our findings provide insights into the mechanism underlying cellulose biosynthesis in cotton fibers, and offer potential candidate genes to coordinate PCW and SCW cellulose synthesis of cotton fibers for developing elite cotton varieties with enhanced fiber quality.

Functional understanding of secondary cell wall cellulose synthases in Populus trichocarpa via the Cas9/gRNA-induced gene knockouts.

Summary Plant cellulose is synthesized by a large plasma membrane‐localized cellulose synthase (CesA) complex. However, an overall functional determination of secondary cell wall (SCW) CesAs is still lacking in trees, especially one based on gene knockouts.Here, the Cas9/gRNA‐induced knockouts of PtrCesA4, 7A, 7B, 8A and 8B genes were produced in Populus trichocarpa. Based on anatomical, immunohistochemical and wood composition evidence, we gained a comprehensive understanding of five SCW PtrCesAs at the genetic level.Complete loss of PtrCesA4, 7A/B or 8A/B led to similar morphological abnormalities, indicating similar and nonredundant genetic functions. The absence of the gelatinous (G) layer, one‐layer‐walled fibres and a 90% decrease in cellulose in these mutant woods revealed that the three classes of SCW PtrCesAs are essential for multilayered SCW structure and wood G‐fibre. In addition, the mutant primary and secondary phloem fibres lost the n(G + L)‐ and G‐layers and retained the thicker S‐layers (L, lignified; S, secondary). Together with polysaccharide immunolocalization data, these findings suggest differences in the role of SCW PtrCesAs‐synthesized cellulose in wood and phloem fibre wall structures.Overall, this functional understanding of the SCW PtrCesAs provides further insights into the impact of lacking cellulose biosynthesis on growth, SCW, wood G‐fibre and phloem fibre wall structures in the tree.

Advances in the role of auxin for transcriptional regulation of lignin biosynthesis.

Lignin is a natural polymer interlaced with cellulose and hemicellulose in secondary cell walls (SCWs). Auxin acts via its signalling transduction to regulate most of plant physiological processes. Lignification responds to auxin signals likewise and affects the development of anther and secondary xylem in plants. In this review, the research advances of AUXIN RESPONSE FACTOR (ARF)-dependent signalling pathways regulating lignin formation are discussed in detail. In an effort to facilitate the understanding of several key regulators in this process, we present a regulatory framework that comprises protein-protein interactions at the top and protein-gene regulation divided into five tiers. This characterises the regulatory roles of auxin in lignin biosynthesis and links auxin signalling transduction to transcriptional cascade of lignin biosynthesis. Our works further point to several of significant problems that need to be resolved in the future to gain a better understanding of the underlying mechanisms through which auxin regulates lignin biosynthesis.

Functions and regulatory framework of ZmNST3 in maize under lodging and drought stress.

The growth and development of maize are negatively affected by various abiotic stresses including drought, high salinity, extreme temperature, and strong wind. Therefore, it is important to understand the molecular mechanisms underlying abiotic stress resistance in maize. In the present work, we identified that a novel NAC transcriptional factor, ZmNST3, enhances maize lodging resistance and drought stress tolerance. ChIP-Seq and expression of target genes analysis showed that ZmNST3 could directly regulate the expression of genes related to cell wall biosynthesis which could subsequently enhance lodging resistance. Furthermore, we also demonstrated that ZmNST3 affected the expression of genes related to the synthesis of antioxidant enzyme secondary metabolites that could enhance drought resistance. More importantly, we are the first to report that ZmNST3 directly binds to the promoters of CESA5 and Dynamin-Related Proteins2A (DRP2A) and activates the expression of genes related to secondary cell wall cellulose biosynthesis. Additionally, we revealed that ZmNST3 directly binds to the promoters of GST/GlnRS and activates genes which could enhance the production of antioxidant enzymes in vivo. Overall, our work contributes to a comprehensive understanding of the regulatory network of ZmNST3 in regulating maize lodging and drought stress resistance.

Chitinase-like1 Plays a Role in Stalk Tensile Strength in Maize.

Stalk lodging in maize (Zea mays) causes significant yield losses due to breaking of stalk tissue below the ear node before harvest. Here, we identified the maize brittle stalk4 (bk4) mutant in a Mutator F2 population. This mutant was characterized by highly brittle aerial parts that broke easily from mechanical disturbance or in high-wind conditions. The bk4 plants displayed a reduction in average stalk diameter and mechanical strength, dwarf stature, senescence at leaf tips, and semisterility of pollen. Histological studies demonstrated a reduction in lignin staining of cells in the bk4 mutant leaves and stalk, and deformation of vascular bundles in the stalk resulting in the loss of xylem and phloem tissues. Biochemical characterization showed a significant reduction in p-coumaric acid, Glc, Man, and cellulose contents. The candidate gene responsible for bk4 phenotype is Chitinase-like1 protein (Ctl1), which is expressed at its highest levels in elongated internodes. Expression levels of secondary cell wall cellulose synthase genes (CesA) in the bk4 single mutant, and phenotypic observations in double mutants combining bk4 with bk2 or null alleles for two CesA genes, confirmed interaction of ZmCtl1 with CesA genes. Overexpression of ZmCtl1 enhanced mechanical stalk strength without affecting plant stature, senescence, or fertility. Biochemical characterization of ZmCtl1 overexpressing lines supported a role for ZmCtl1 in tensile strength enhancement. Conserved identity of CTL1 peptides across plant species and analysis of Arabidopsis (Arabidopsis thaliana) ctl1-1 ctl2-1 double mutants indicated that Ctl1 might have a conserved role in plants.

Convergent evolution of hetero-oligomeric cellulose synthesis complexes in mosses and seed plants.

In seed plants, cellulose is synthesized by rosette-shaped cellulose synthesis complexes (CSCs) that are obligate hetero-oligomeric, comprising three non-interchangeable cellulose synthase (CESA) isoforms. The moss Physcomitrella patens has rosette CSCs and seven CESAs, but its common ancestor with seed plants had rosette CSCs and a single CESA gene. Therefore, if P.patens CSCs are hetero-oligomeric, then CSCs of this type evolved convergently in mosses and seed plants. Previous gene knockout and promoter swap experiments showed that PpCESAs from class A (PpCESA3 and PpCESA8) and class B (PpCESA6 and PpCESA7) have non-redundant functions in secondary cell wall cellulose deposition in leaf midribs, whereas the two members of each class are redundant. Based on these observations, we proposed the hypothesis that the secondary class A and class B PpCESAs associate to form hetero-oligomeric CSCs. Here we show that transcription of secondary class A PpCESAs is reduced when secondary class B PpCESAs are knocked out and vice versa, as expected for genes encoding isoforms that occupy distinct positions within the same CSC. The class A and class B isoforms co-accumulate in developing gametophores and co-immunoprecipitate, suggesting that they interact to form a complex in planta. Finally, secondary PpCESAs interact with each other, whereas three of four fail to self-interact when expressed in two different heterologous systems. These results are consistent with the hypothesis that obligate hetero-oligomeric CSCs evolved independently in mosses and seed plants and we propose the constructive neutral evolution hypothesis as a plausible explanation for convergent evolution of hetero-oligomeric CSCs.

PagMYB216 is involved in the regulation of cellulose synthesis in Populus

Cellulose accounts for most of the structural material in secondary cell walls. Although the synthesis of cellulose has been extensively investigated, the regulation of cellulose synthesis in woody plants remains elusive. In this study, we showed PagMYB216 to be involved in the regulation of cellulose synthesis in Populus. Subcellular localization and transcriptional activation analyses suggest that PagMYB216 is a nuclear-localized transcriptional activator. Transient transcriptional assays revealed that PagMYB216 could activate the expression of secondary cell wall cellulose synthesis genes (CESAs) through binding to the GAMYB motif. Overexpression of PagMYB216 in Populus resulted in enhanced expression of secondary wall CESAs and increased cellulose content in stems. In contrast, the dominant repression of PagMYB216 transgenic poplar plants led to the downregulation of secondary wall CESAs and decreased cellulose content. In addition, cellulose deposition and secondary wall thickness were increased in stems from PagMYB216 overexpression poplar plants and decreased in stems from PagMYB216 dominant repression poplar plants. Taken together, our results suggest that PagMYB216 is involved in the modulation of cellulose synthesis in Populus by controlling the expression of secondary wall CESAs.

Xylan in the Middle: Understanding Xylan Biosynthesis and Its Metabolic Dependencies Toward Improving Wood Fiber for Industrial Processing.

Lignocellulosic biomass, encompassing cellulose, lignin and hemicellulose in plant secondary cell walls (SCWs), is the most abundant source of renewable materials on earth. Currently, fast-growing woody dicots such as Eucalyptus and Populus trees are major lignocellulosic (wood fiber) feedstocks for bioproducts such as pulp, paper, cellulose, textiles, bioplastics and other biomaterials. Processing wood for these products entails separating the biomass into its three main components as efficiently as possible without compromising yield. Glucuronoxylan (xylan), the main hemicellulose present in the SCWs of hardwood trees carries chemical modifications that are associated with SCW composition and ultrastructure, and affect the recalcitrance of woody biomass to industrial processing. In this review we highlight the importance of xylan properties for industrial wood fiber processing and how gaining a greater understanding of xylan biosynthesis, specifically xylan modification, could yield novel biotechnology approaches to reduce recalcitrance or introduce novel processing traits. Altering xylan modification patterns has recently become a focus of plant SCW studies due to early findings that altered modification patterns can yield beneficial biomass processing traits. Additionally, it has been noted that plants with altered xylan composition display metabolic differences linked to changes in precursor usage. We explore the possibility of using systems biology and systems genetics approaches to gain insight into the coordination of SCW formation with other interdependent biological processes. Acetyl-CoA, s-adenosylmethionine and nucleotide sugars are precursors needed for xylan modification, however, the pathways which produce metabolic pools during different stages of fiber cell wall formation still have to be identified and their co-regulation during SCW formation elucidated. The crucial dependence on precursor metabolism provides an opportunity to alter xylan modification patterns through metabolic engineering of one or more of these interdependent pathways. The complexity of xylan biosynthesis and modification is currently a stumbling point, but it may provide new avenues for woody biomass engineering that are not possible for other biopolymers.

Ectopic expression of SOD and APX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance

Hydrogen peroxide (H2O2) is known to accumulate in plants during abiotic stress conditions and also acts as a signalling molecule. In this study, Arabidopsis thaliana transgenics overexpressing cytosolic CuZn-superoxide dismutase (PaSOD) from poly-extremophile high-altitude Himalayan plant Potentilla atrosanguinea, cytosolic ascorbate peroxidase (RaAPX) from Rheum australe and dual transgenics overexpressing both the genes were developed and analyzed under salt stress. In comparison to wild-type (WT) or single transgenics, the performance of dual transgenics under salt stress was better with higher biomass accumulation and cellulose content. We identified genes involved in cell wall biosynthesis, including nine cellulose synthases (CesA), seven cellulose synthase-like proteins together with other wall-related genes. RNA-seq analysis and qPCR revealed differential regulation of genes (CesA 4, 7 and 8) and transcription factors (MYB46 and 83) involved in secondary cell wall cellulose biosynthesis, amongst which most of the cellulose biosynthesis gene showed upregulation in single (PaSOD line) and dual transgenics at 100mM salt stress. A positive correlation between cellulose content and H2O2 accumulation was observed in these transgenic lines. Further, cellulose content was 1.6-2 folds significantly higher in PaSOD and dual transgenic lines, 1.4 fold higher in RaAPX lines as compared to WT plants under stress conditions. Additionally, transgenics overexpressing PaSOD and RaAPX also displayed higher amounts of phenolics as compared to WT. The novelty of present study is that H2O2 apart from its role in signalling, italso provides mechanical strength to plants and aid in plant biomass production during salt stress by transcriptional activation of cellulose biosynthesis pathway. This modulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create plants with enhanced cellulose content for biofuel use.

Secondary Wall Regulating NACs Differentially Bind at the Promoter at a CELLULOSE SYNTHASE A4 Cis-eQTL.

Arabidopsis thaliana CELLULOSE SYNTHASE A4/7/8 (CESA4/7/8) are three non-redundant subunits of the secondary cell wall cellulose synthase complex. Transcript abundance of these genes can vary among genotypes and expression quantitative trait loci (eQTL) were identified in a recombinant population of the accessions Bay-0 and Shahdara. Genetic mapping and analysis of the transcript levels of CESAs between two distinct near isogenic lines (NILs) confirmed a change in CESA4 expression that segregates within that interval. We sequenced the promoters and identified 16 polymorphisms differentiating CESA4Sha and CESA4Bay. In order to determine which of these SNPs could be responsible for this eQTL, we screened for transcription factor protein affinity with promoter fragments of CESA4Bay, CESA4Sha, and the reference genome CESA4Col. The wall thickening activator proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (NST2) and NST3 exhibited a decrease in binding with the CESA4Sha promoter with a tracheary element-regulating cis-element (TERE) polymorphism. While NILs harboring the TERE polymorphisms exhibited significantly different CESA4 expression, cellulose crystallinity and cell wall thickness were indistinguishable. These results suggest that the TERE polymorphism resulted in differential transcription factor binding and CESA4 expression; yet A. thaliana is able to tolerate this transcriptional variability without compromising the structural elements of the plant, providing insight into the elasticity of gene regulation as it pertains to cell wall biosynthesis and regulation. We also explored available DNA affinity purification sequencing data to resolve a core binding site, C(G/T)TNNNNNNNA(A/C)G, for secondary wall NACs referred to as the VNS element.

Genome-Wide Identification of R2R3-MYB Transcription Factors Regulating Secondary Cell Wall Thickening in Cotton Fiber Development.

MYB proteins represent one of the largest transcription factor (TF) families in plants, some of which act as key transcriptional regulators of secondary cell wall (SCW) biosynthesis. Cotton (Gossypium hirsutum) fiber is thought to be an ideal single-cell model to study cell elongation and SCW biosynthesis. However, little knowledge regarding the TFs controlling fiber SCW biosynthesis, particularly for R2R3-MYBs is known. By far, no comprehensive genome-wide analysis of the secondary wall-associated R2R3-MYBs has been reported in cultivated tetraploid upland cotton. In this study, we identified 419 R2R3-MYB genes by systematically examining the cotton genome. A combination of phylogenetic, RNA-seq and co-expression analyses indicated that 36 R2R3-MYBs were either preferentially or highly expressed in 20 day post anthesis (dpa) fibers and are putative SCW regulators. Among these MYB genes, 22 MYBs are homologs of known SCW MYB proteins and the other 14 MYBs are novel proteins without prior reported SCW biosynthesis-related functions. Finally, we highlighted on the roles of two MYBs named GhMYB46_D13 and GhMYB46_D9, both of which displayed the highest expression in 20 dpa fibers. Expression of GhMYB46_D13 or GhMYB46_D9 individually in Arabidopsis resulted in ectopic SCW deposition in transgenic plants. Furthermore, both GhMYB46_D13 and GhMYB46_D9 were able to activate the cotton fiber SCW cellulose synthase gene promoters. Thus, we have identified 36 R2R3-MYBs as potential SCW regulators in cotton fibers that represent strong candidates for further functional studies during fiber development and SCW thickening.

Functional Analysis of Cellulose Synthase CesA4 and CesA6 Genes in Switchgrass (Panicum virgatum) by Overexpression and RNAi-Mediated Gene Silencing.

Switchgrass (Panicum virgatum L.) is a leading lignocellulosic bioenergy feedstock. Cellulose is a major component of the plant cell walls and the primary substrate for saccharification. Accessibility of cellulose to enzymatic breakdown into fermentable sugars is limited by the presence of lignin in the plant cell wall. In this study, putatively novel switchgrass secondary cell wall cellulose synthase PvCesA4 and primary cell wall PvCesA6 genes were identified and their functional role in cellulose synthesis and cell wall composition was examined by overexpression and knockdown of the individual genes in switchgrass. The endogenous expression of PvCesA4 and PvCesA6 genes varied among including roots, leaves, stem, and reproductive tissues. Increasing or decreasing PvCesA4 and PvCesA6 expression to extreme levels in the transgenic lines resulted in decreased biomass production. PvCesA6-overexpressing lines had reduced lignin content and syringyl/guaiacyl lignin monomer ratio accompanied by increased sugar release efficiency, suggesting an impact of PvCesA6 expression levels on lignin biosynthesis. Cellulose content and cellulose crystallinity were decreased, while xylan content was increased in PvCesA4 and PvCesA6 overexpression or knockdown lines. The increase in xylan content suggests that the amount of non-cellulosic cell wall polysaccharide was modified in these plants. Taken together, the results show that the manipulation of the cellulose synthase genes alters the cell wall composition and availability of cellulose as a bioprocessing substrate.

Domain swaps of Arabidopsis secondary wall cellulose synthases to elucidate their class specificity.

Cellulose microfibrils are synthesized by membrane‐embedded cellulose synthesis complexes (CSCs), currently modeled as hexamers of cellulose synthase (CESA) trimers. The three paralogous CESAs involved in secondary cell wall (SCW) cellulose biosynthesis in Arabidopsis (CESA4, CESA7, CESA8) are similar, but nonredundant, with all three isoforms required for assembly and function of the CSC. The molecular basis of protein–protein recognition among the isoforms is not well understood. To investigate the locations of the interfaces that are responsible for isoform recognition, we swapped three domains between the Arabidopsis CESAs required for SCW synthesis (CESA4, CESA7, and CESA8): N‐terminus, central domain containing the catalytic core, and C‐terminus. Chimeric genes with all pairwise permutations of the domains were tested for in vivo functionality within knockout mutant backgrounds of cesa4, cesa7, and cesa8. Immunoblotting with isoform‐specific antibodies confirmed the anticipated protein expression in transgenic plants. The percent recovery of stem height and crystalline cellulose content was assayed, as compared to wild type, the mutant background lines, and other controls. Retention of the native central domain was sufficient for CESA8 chimeras to function, with neither its N‐terminal nor C‐terminal domains required. The C‐terminal domain is required for class‐specific function of CESA4 and CESA7, and CESA7 also requires its own N‐terminus. Across all isoforms, the results indicate that the central domain, as well as the N‐ and C‐terminal regions, contributes to class‐specific function variously in Arabidopsis CESA4, CESA7, and CESA8.

Expression Analysis of Cell Wall-Related Genes in Cannabis sativa: The “Ins and Outs” of Hemp Stem Tissue Development

Textile hemp (Cannabis sativa L.) is a multipurpose crop producing biomass with uses in e.g., the textile, biocomposite, and construction sectors. It was previously shown that the hypocotyl of hemp is useful to study the kinetics of secondary tissue development, where primary and secondary growths are temporally uncoupled. We here sought to demonstrate that the stem of adult hemp plants is an additional suitable model to study the heterogeneous lignification of the tissues and the mechanisms underlying secondary cell wall formation in bast fibres. A targeted quantitative PCR analysis carried out on a set of twenty genes involved in cell wall biosynthesis clearly showed differences in expression in the core and cortical tissues along four stem regions spanning from elongation to cell wall thickening. Genes involved in phenylpropanoid biosynthesis and secondary cell wall cellulose synthases were expressed at higher levels in core tissues at the bottom, while specific genes, notably a class III peroxidase and a gene partaking in lignan biosynthesis, were highly expressed in the cortex of elongating internodes. The two systems, the hypocotyl and the adult stem of textile hemp, are equally valid and complementary to address questions related to lignification and secondary cell wall deposition.

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis.

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis (Arabidopsis thaliana) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis.

Comparison and validation of Fourier transform infrared spectroscopic methods for monitoring secondary cell wall cellulose from cotton fibers

The amount of secondary cell wall (SCW) cellulose in the fiber affects the quality and commercial value of cotton. Accurate assessments of SCW cellulose are essential for improving cotton fibers. Fourier transform infrared (FT-IR) spectroscopy enables distinguishing SCW from other cell wall components in a rapid and non-invasive way. Thus it has been used for monitoring SCW development in model plants. Recently, several FT-IR methods have been proposed for monitoring cotton fiber development. However, they are rarely utilized for assessing SCW cellulose from cotton fiber due to limited validation with various cotton species grown in different conditions. Thus, we compared and validated three FT-IR methods including two previously proposed methods analyzing entire spectra or specific bands as well as a new method analyzing FT-IR spectral regions corresponding to cellulose with various cotton fibers grown in planta and in vitro. Comparisons of the FT-IR methods with reference methods showed that the two FT-IR methods analyzing the entire spectra or cellulose regions by principal component analysis monitored SCW qualitatively, whereas the FT-IR method analyzing specific bands (708, 730, and 800 cm−1) by a simple algorithm allowed the monitoring of SCW cellulose levels quantitatively. The quantitative FT-IR method is a potential substitute for lengthy and laborious chemical assays for monitoring SCW cellulose levels from cotton fibers, and it can be used for a better understanding of cotton fiber SCW development and as a part of the quality assessment tools used to guide choices for improving fiber quality.