Secondary Cell Wall Composition Research Articles

Deciphering the intricate hierarchical gene regulatory network: unraveling multi-level regulation and modifications driving secondary cell wall formation.

Wood quality is predominantly determined by the amount and the composition of secondary cell walls (SCWs). Consequently, unraveling the molecular regulatory mechanisms governing SCW formation is of paramount importance for genetic engineering aimed at enhancing wood properties. Although SCW formation is known to be governed by a hierarchical gene regulatory network (HGRN), our understanding of how a HGRN operates and regulates the formation of heterogeneous SCWs for plant development and adaption to ever-changing environment remains limited. In this review, we examined the HGRNs governing SCW formation and highlighted the significant key differences between herbaceous Arabidopsis and woody plant poplar. We clarified many confusions in existing literatures regarding the HGRNs and their orthologous gene names and functions. Additionally, we revealed many network motifs including feed-forward loops, feed-back loops, and negative and positive autoregulation in the HGRNs. We also conducted a thorough review of post-transcriptional and post-translational aspects, protein-protein interactions, and epigenetic modifications of the HGRNs. Furthermore, we summarized how the HGRNs respond to environmental factors and cues, influencing SCW biosynthesis through regulatory cascades, including many regulatory chains, wiring regulations, and network motifs. Finally, we highlighted the future research directions for gaining a further understanding of molecular regulatory mechanisms underlying SCW formation.

Natural polymorphisms in ZmIRX15A affect water-use efficiency by modulating stomatal density in maize.

Stomatal density (SD) is closely related to crop drought resistance. Understanding the genetic basis for natural variation in SD may facilitate efforts to improve water-use efficiency. Here, we report a genome-wide association study for SD in maize seedlings, which identified 18 genetic variants that could be resolved to seven candidate genes. A 3-bp insertion variant (InDel1089) in the last exon of Zea mays (Zm) IRX15A (Irregular xylem 15A) had the most significant association with SD and modulated the translation of ZmIRX15A mRNA by affecting its secondary structure. Dysfunction of ZmIRX15A increased SD, leading to an increase in the transpiration rate and CO2 assimilation efficiency. ZmIRX15A encodes a xylan deposition enzyme and its disruption significantly decreased xylan abundance in secondary cell wall composition. Transcriptome analysis revealed a substantial alteration of the expression of genes involved in stomatal complex morphogenesis and drought response in the loss-of-function of ZmIRX15A mutant. Overall, our study provides important genetic insights into the natural variation of leaf SD in maize, and the identified loci or genes can serve as direct targets for enhancing drought resistance in molecular-assisted maize breeding.

Nutrient conditions mediate mycorrhizal effects on biomass production and cell wall chemistry in poplar.

Large scale biofuel production from lignocellulosic feedstock is limited by the financial and environmental costs associated with growing and processing lignocellulosic material and the resilience of these plants to environmental stress. Symbiotic associations with arbuscular (AM) and ectomycorrhizal (EM) fungi represent a potential strategy for expanding feedstock production while reducing nutrient inputs. Comparing AM and EM effects on wood production and chemical composition is a necessary step in developing biofuel feedstocks. Here, we assessed the productivity, biomass allocation, and secondary cell wall composition of greenhouse grown Populus tremuloides inoculated with either AM or EM fungi. Given the long-term goal of reducing nutrient inputs for biofuel production, we further tested the effects of nutrient availability and nitrogen:phosphorus stoichiometry on mycorrhizal responses. Associations with both AM and EM fungi increased plant biomass by 14% to 74% depending on the nutrient conditions but had minimal effects on secondary cell wall composition. Mycorrhizal plants, especially those inoculated with EM fungi, also allocated a greater portion of their biomass to roots, which could be beneficial in the field where plants are likely to experience both water and nutrient stress. Leaf nutrient content was weakly, but positively correlated with wood production in mycorrhizal plants. Surprisingly, phosphorus played a larger role in EM plants compared to AM plants. Relative nitrogen and phosphorus availability was correlated with shifts in secondary cell wall composition. For AM associations the benefit of increased wood biomass may be partially offset by increased lignin content, a trait that affects downstream processing of lignocellulosic tissue for biofuels. By comparing AM and EM effects on the productivity and chemical composition of lignocellulosic tissue, this work links broad functional diversity in mycorrhizal associations to key biofuels traits and highlights the importance of considering both biotic and abiotic factors when developing strategies for sustainable biofuel production.

AUXIN RESPONSE FACTORS 6 and 17 control the flag leaf angle in rice by regulating secondary cell wall biosynthesis of lamina joints.

Flag leaf angle impacts the photosynthetic capacity of densely grown plants and is thus an important agronomic breeding trait for crop architecture and yield. The hormone auxin plays a key role in regulating this trait, yet the underlying molecular and cellular mechanisms remain unclear. Here, we report that two rice (Oryza sativa) auxin response factors (ARFs), OsARF6 and OsARF17, which are highly expressed in lamina joint tissues, control flag leaf angle in response to auxin. Loss-of-function double osarf6 osarf17 mutants displayed reduced secondary cell wall levels of lamina joint sclerenchymatous cells (Scs), resulting in an exaggerated flag leaf angle and decreased grain yield under dense planting conditions. Mechanical measurements indicated that the mutant lamina joint tissues were too weak to support the weight of the flag leaf blade, resembling the phenotype of the rice increased leaf angle1 (ila1) mutant. We demonstrate that OsARF6 and OsARF17 directly bind to the ILA1 promoter independently and synergistically to activate its expression. In addition, auxin-induced ILA1 expression was dependent on OsARF6 and OsARF17. Collectively, our study reveals a mechanism that integrates auxin signaling with the secondary cell wall composition to determine flag leaf angle, providing breeding targets in rice, and potentially other cereals, for this key trait.

Nano-indentation reveals a potential role for gradients of cell wall stiffness in directional movement of the resurrection plant Selaginella lepidophylla

As a physical response to water loss during drought, inner Selaginella lepidophylla stems curl into a spiral shape to prevent photoirradiation damage to their photosynthetic surfaces. Curling is reversible and involves hierarchical deformation, making S. lepidophylla an attractive model with which to study water-responsive actuation. Investigation at the organ and tissue level has led to the understanding that the direction and extent of stem curling can be partially attributed to stiffness gradients between adaxial and abaxial stem sides at the nanoscale. Here, we examine cell wall elasticity to understand how it contributes to the overall stem curling. We compare the measured elastic moduli along the stem length and between adaxial and abaxial stem sides using atomic force microscopy nano-indentation testing. We show that changes in cortex secondary cell wall development lead to cell wall stiffness gradients from stem tip to base, and also between adaxial and abaxial stem sides. Changes in cortical cell wall morphology and secondary cell wall composition are suggested to contribute to the observed stiffness gradients.

Three-dimensional functional gradients direct stem curling in the resurrection plant Selaginella lepidophylla.

Upon hydration and dehydration, the vegetative tissue of Selaginella lepidophylla can reversibly swell and shrink to generate complex morphological transformations. Here, we investigate how structural and compositional properties at tissue and cell wall levels in S. lepidophylla lead to different stem curling profiles between inner and outer stems. Our results show that directional bending in both stem types is associated with cross-sectional gradients of tissue density, cell orientation and secondary cell wall composition between adaxial and abaxial stem sides. In inner stems, longitudinal gradients of cell wall thickness and composition affect tip-to-base tissue swelling and shrinking, allowing for more complex curling as compared to outer stems. Together, these features yield three-dimensional functional gradients that allow the plant to reproducibly deform in predetermined patterns that vary depending on the stem type. This study is the first to demonstrate functional gradients at different hierarchical levels combining to operate in a three-dimensional context.

Ginseng-derived patatin-related phospholipase PgpPLAIIIβ alters plant growth and lignification of xylem in hybrid poplars

Patatin-liked phospholipase A (pPLAs) are major lipid acyl hydrolases that participate in various biological functions in plant growth and development. Previously, a ginseng-derived pPLAIII homolog was reported to reduce lignin content in Arabidopsis. This led us to evaluate its possible usefulness as a biomass source in wood plant. Herein, we report that there are six members in the pPLAIII gene family in poplar. Overexpression of pPLAIIIβ derived from ginseng resulted in a reduced plant height with radially expanded stem growth in hybrid poplars. Compared with the wild type (WT), the chlorophyll content was increased in the overexpression poplar lines, whereas the leaf size was smaller. The secondary cell wall structure in overexpression lines was also altered, exhibiting reduced lignification in the xylem. Two transcription factors, MYB92 and MYB152, which control lignin biosynthesis, were downregulated in the overexpression lines. The middle xylem of the overexpression line showed heavy thickening, making it thicker than the other xylem parts and the WT xylem, which rather could have been contributed by the presence of more cellulose in the selected surface area. Taken together, the results suggest that PgpPLAIIIβ plays a role not only in cell elongation patterns, but also in determining the secondary cell wall composition.

Sweet Sorghum Originated through Selection of Dry, a Plant-Specific NAC Transcription Factor Gene.

Sorghum (Sorghum bicolor) is the fifth most popular crop worldwide and a C4 model plant. Domesticated sorghum comes in many forms, including sweet cultivars with juicy stems and grain sorghum with dry, pithy stems at maturity. The Dry locus, which controls the pithy/juicy stem trait, was discovered over a century ago. Here, we found that Dry gene encodes a plant-specific NAC transcription factor. Dry was either deleted or acquired loss-of-function mutations in sweet sorghum, resulting in cell collapse and altered secondary cell wall composition in the stem. Twenty-three Dry ancestral haplotypes, all with dry, pithy stems, were found among wild sorghum and wild sorghum relatives. Two of the haplotypes were detected in domesticated landraces, with four additional dry haplotypes with juicy stems detected in improved lines. These results imply that selection for Dry gene mutations was a major step leading to the origin of sweet sorghum. The Dry gene is conserved in major cereals; fine-tuning its regulatory network could provide a molecular tool to control crop stem texture.

A multi-omics approach reveals function of Secretory Carrier-Associated Membrane Proteins in wood formation of\u200b \u200b\u200bPopulus\u200b\u200b \u200btrees

BackgroundSecretory Carrier-Associated Membrane Proteins (SCAMPs) are highly conserved 32–38 kDa proteins that are involved in membrane trafficking. A systems approach was taken to elucidate function of SCAMPs in wood formation of Populus trees. Phenotypic and multi-omics analyses were performed in woody tissues of transgenic Populus trees carrying an RNAi construct for Populus tremula x tremuloides SCAMP3 (PttSCAMP3; Potri.019G104000).ResultsThe woody tissues of the transgenic trees displayed increased amounts of both polysaccharides and lignin oligomers, indicating increased deposition of both the carbohydrate and lignin components of the secondary cell walls. This coincided with a tendency towards increased wood density as well as significantly increased thickness of the suberized cork in the transgenic lines. Multivariate OnPLS (orthogonal projections to latent structures) modeling of five different omics datasets (the transcriptome, proteome, GC-MS metabolome, LC-MS metabolome and pyrolysis-GC/MS metabolome) collected from the secondary xylem tissues of the stem revealed systemic variation in the different variables in the transgenic lines, including changes that correlated with the changes in the secondary cell wall composition. The OnPLS model also identified a rather large number of proteins that were more abundant in the transgenic lines than in the wild type. Several of these were related to secretion and/or endocytosis as well as both primary and secondary cell wall biosynthesis.ConclusionsPopulus SCAMP proteins were shown to influence accumulation of secondary cell wall components, including polysaccharides and phenolic compounds, in the woody tissues of Populus tree stems. Our multi-omics analyses combined with the OnPLS modelling suggest that this function is mediated by changes in membrane trafficking to fine-tune the abundance of cell wall precursors and/or proteins involved in cell wall biosynthesis and transport. The data provides a multi-level source of information for future studies on the function of the SCAMP proteins in plant stem tissues.

Altering carbon allocation in hybrid poplar (Populus alba×grandidentata) impacts cell wall growth and development.

Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of oligosaccharides (RFOs) that function as transport carbohydrates in the phloem, as storage compounds in sink tissues and as soluble metabolites that combat both abiotic and biotic stress in several plant species. Hybrid poplar (Populus alba × grandidentata) overexpressing the Arabidopsis thaliana GolS3 (AtGolS3) gene showed clear effects on development; the extreme overexpressing lines were stunted and had cell wall traits characteristic of tension wood, whereas lines with only moderate up‐regulation grew normally and had moderately altered secondary cell wall composition and ultrastructure. Stem cross‐sections of the developing xylem revealed a significant increase in the number of vessels, as well as the clear presence of a G‐layer in the fibres. Furthermore, AtGolS3‐OE lines possessed higher cellulose and lower lignin contents, an increase in cellulose crystallinity, and significantly altered hemicellulose‐derived carbohydrates, notably manifested by their mannose and xylose contents. In addition, the transgenic plants displayed elevated xylem starch content. Transcriptome interrogation of the transgenic plants showed a significant up‐regulation of genes involved in the synthesis of myo‐inositol, along with genes involved in sucrose degradation. The results suggest that the overexpression of GolS and its product galactinol may serve as a molecular signal that initiates metabolic changes, culminating in a change in cell wall development and potentially the formation of tension wood.

Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in Sorghum bicolor.

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses.

Secondary cell wall composition and candidate gene expression in developing willow (Salix purpurea) stems

The properties of the secondary cell wall (SCW) in willow largely determine the suitability of willow biomass feedstock for potential bioenergy and biofuel applications. SCW development has been little studied in willow and it is not known how willow compares with model species, particularly the closely related genus Populus. To address this and relate SCW synthesis to candidate genes in willow, a tractable bud culture-derived system was developed in Salix purpurea, and cell wall composition and RNA-Seq transcriptome were followed in stems during early development. A large increase in SCW deposition in the period 0–2 weeks after transfer to soil was characterised by a big increase in xylan content, but no change in the frequency of substitution of xylan with glucuronic acid, and increased abundance of putative transcripts for synthesis of SCW cellulose, xylan and lignin. Histochemical staining and immunolabeling revealed that increased deposition of lignin and xylan was associated with xylem, xylem fibre cells and phloem fibre cells. Transcripts orthologous to those encoding xylan synthase components IRX9 and IRX10 and xylan glucuronyl transferase GUX1 in Arabidopsis were co-expressed, and showed the same spatial pattern of expression revealed by in situ hybridisation at four developmental stages, with abundant expression in proto-xylem, xylem fibre and ray parenchyma cells and some expression in phloem fibre cells. The results show a close similarity with SCW development in Populus species, but also give novel information on the relationship between spatial and temporal variation in xylan-related transcripts and xylan composition.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-014-2034-1) contains supplementary material, which is available to authorized users.

Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana

BackgroundSecond-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield. Saccharification models were built to elucidate which cell wall parameters played a role in cell wall recalcitrance.ResultsAlthough lignin is a key polymer providing the strength necessary for the plant’s ability to grow upward, a reduction in lignin content down to 64% of the wild-type level in Arabidopsis was tolerated without any obvious growth penalty. In contrast to common perception, we found that a reduction in lignin was not compensated for by an increase in cellulose, but rather by an increase in matrix polysaccharides. In most lignin mutants, the saccharification yield was improved by up to 88% cellulose conversion for the cinnamoyl-coenzyme A reductase1 mutants under pretreatment conditions, whereas the wild-type cellulose conversion only reached 18%. The saccharification models and Pearson correlation matrix revealed that the lignin content was the main factor determining the saccharification yield. However, also lignin composition, matrix polysaccharide content and composition, and, especially, the xylose, galactose, and arabinose contents influenced the saccharification yield. Strikingly, cellulose content did not significantly affect saccharification yield.ConclusionsAlthough the lignin content had the main effect on saccharification, also other cell wall factors could be engineered to potentially increase the cell wall processability, such as the galactose content. Our results contribute to a better understanding of the effect of lignin perturbations on plant cell wall composition and its influence on saccharification yield, and provide new potential targets for genetic improvement.

Transcriptional Regulation of Grass Secondary Cell Wall Biosynthesis: Playing Catch-Up with Arabidopsis thaliana

Secondary cell wall synthesis occurs in specialized cell types following completion of cell enlargement. By virtue of mechanical strength provided by a wall thickened with cellulose, hemicelluloses, and lignin, these cells can function as water-conducting vessels and provide structural support. Several transcription factor families regulate genes encoding wall synthesis enzymes. Certain NAC and MYB proteins directly bind to the SNBE and AC elements upstream of structural genes and other transcription factors. The most detailed model of this regulatory network is established predominantly for a eudicot, Arabidopsis thaliana. In grasses, both the patterning and the composition of secondary cell walls are distinct from that of eudicots. These differences suggest transcriptional regulation is similarly distinct. Putative rice and maize orthologs of several eudicot cell wall regulators genetically complement mutants of A. thaliana or result in wall defects when constitutively overexpressed; nevertheless, aside from a maize, ZmMYB31, and a switchgrass protein, PvMYB4, function has not been tested in a grass. Similar to the seminal work conducted in A. thaliana, gene expression profiling in maize, rice, and other grasses implicates additional genes as regulators. Characterization of these genes will continue to elucidate the relationship between the transcription regulatory networks of eudicots and grasses.

High nitrogen fertilization and stem leaning have overlapping effects on wood formation in poplar but invoke largely distinct molecular pathways

Previous studies indicated that high nitrogen fertilization may impact secondary xylem development and alter fibre anatomy and composition. The resulting wood shares some resemblance with tension wood, which has much thicker cell walls than normal wood due to the deposition of an additional layer known as the G-layer. This report compares the short-term effects of high nitrogen fertilization and tree leaning to induce tension wood, either alone or in combination, upon wood formation in young trees of Populus trichocarpa (Torr. & Gray) × P. deltoides Bartr. ex Marsh. Fibre anatomy, chemical composition and transcript profiles were examined in newly formed secondary xylem. Each of the treatments resulted in thicker cell walls relative to the controls. High nitrogen and tree leaning had overlapping effects on chemical composition based on Fourier transform infrared analysis, specifically indicating that secondary cell wall composition was shifted in favour of cellulose and hemicelluloses relative to lignin content. In contrast, the high-nitrogen trees had shorter fibres, whilst the leaning trees had longer fibres that the controls. Microarray transcript profiling carried out after 28 days of treatment identified 180 transcripts that accumulated differentially in one or more treatments. Only 10% of differentially expressed transcripts were affected in all treatments relative to the controls. Several of the affected transcripts were related to carbohydrate metabolism, secondary cell wall formation, nitrogen metabolism and osmotic stress. RT-qPCR analyses at 1, 7 and 28 days showed that several transcripts followed very different accumulation profiles in terms of rate and level of accumulation, depending on the treatment. Our findings suggest that high nitrogen fertilization and tension wood induction elicit largely distinct and molecular pathways with partial overlap. When combined, the two types of environmental cue yielded additive effects.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its consequences for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Mutations of the secondary cell wall.

It has not been possible to isolate a number of crucial enzymes involved in plant cell wall synthesis. Recent progress in identifying some of these steps has been overcome by the isolation of mutants defective in various aspects of cell wall synthesis and the use of these mutants to identify the corresponding genes. Secondary cell walls offer numerous advantages for genetic analysis of plant cell walls. It is possible to recover very severe mutants since the plants remain viable. In addition, although variation in secondary cell wall composition occurs between different species and between different cell types, the composition of the walls is relatively simple compared to primary cell walls. Despite these advantages, relatively few secondary cell wall mutations have been described to date. The only secondary cell wall mutations characterised to date, in which the basis of the abnormality is known, have defects in either the control of secondary cell wall deposition or secondary cell wall cellulose or lignin biosynthesis. These mutants have, however, provided essential information on secondary cell wall biosynthesis.