Jasmonate activates secondary cell wall biosynthesis through MYC2-MYB46 module.

Formation of secondary cell wall (SCW) is tightly regulated spatiotemporally by various developmental and environmental signals. Successful fine-tuning of the trade-off between SCW biosynthesis and stress responses requires a better understanding of how plant growth is regulated under environmental stress conditions. However, the current understanding of the interplay between environmental signaling and SCW formation is limited. The lipid-derived plant hormone jasmonate (JA) and its derivatives are important signaling components involved in various physiological processes including plant growth, development, and abiotic/biotic stress responses. Recent studies suggest that JA is involved in SCW formation but the signaling pathway has not been studied for how JA regulates SCW formation. We tested this hypothesis using the transcription factor MYB46, a master switch for SCW biosynthesis, and JA treatments. Both the transcript and protein levels of MYB46, a master switch for SCW formation, were significantly increased by JA treatment, resulting in the upregulation of SCW biosynthesis. We then show that this JA-induced upregulation of MYB46 is mediated by MYC2, a central regulator of JA signaling, which binds to the promoter of MYB46. We conclude that this MYC2-MYB46 module is a key component of the plant response to JA in SCW formation.

Cotton GhNAC4 promotes drought tolerance by regulating secondary cell wall biosynthesis and ribosomal protein homeostasis.

Drought has a severe impact on the quality and yield of cotton. Deciphering the key genes related to drought tolerance is important for understanding the regulation mechanism of drought stress and breeding drought-tolerant cotton cultivars. Several studies have demonstrated that NAC transcription factors are crucial in the regulation of drought stress, however, the related functional mechanisms are still largely unexplored. Here, we identified that NAC transcription factor GhNAC4 positively regulated drought stress tolerance in cotton. The expression of GhNAC4 was significantly induced by abiotic stress and plant hormones. Silencing of GhNAC4 distinctly impaired the resistance to drought stress and overexpressing GhNAC4 in cotton significantly enhanced the stress tolerance. RNA-seq analysis revealed that overexpression of GhNAC4 enriched the expression of genes associated with the biosynthesis of secondary cell walls and ribosomal proteins. We confirmed that GhNAC4 positively activated the expressions of GhNST1, a master regulator reported previously in secondary cell wall formation, and two ribosomal protein-encoding genes GhRPL12 and GhRPL18p, by directly binding to their promoter regions. Overexpression of GhNAC4 promoted the expression of downstream genes associated with the secondary wall biosynthesis, resulting in enhancing secondary wall deposition in the roots, and silencing of GhRPL12 and GhRPL18p significantly impaired the resistance to drought stress. Taken together, our study reveals a novel pathway mediated by GhNAC4 that promotes secondary cell wall biosynthesis to strengthen secondary wall development and regulates the expression of ribosomal protein-encoding genes to maintain translation stability, which ultimately enhances drought tolerance in cotton.

Cell-type-specific transcriptomics uncovers spatial regulatory networks in bioenergy sorghum stems.

Bioenergy sorghum is a low-input, drought-resilient, deep-rooting annual crop that has high biomass yield potential enabling the sustainable production of biofuels, biopower, and bioproducts. Bioenergy sorghum's 4-5 m stems account for ~80% of the harvested biomass. Stems accumulate high levels of sucrose that could be used to synthesize bioethanol and useful biopolymers if information about cell-type gene expression and regulation in stems was available to enable engineering. To obtain this information, laser capture microdissection was used to isolate and collect transcriptome profiles from five major cell types that are present in stems of the sweet sorghum Wray. Transcriptome analysis identified genes with cell-type-specific and cell-preferred expression patterns that reflect the distinct metabolic, transport, and regulatory functions of each cell type. Analysis of cell-type-specific gene regulatory networks (GRNs) revealed that unique transcription factor families contribute to distinct regulatory landscapes, where regulation is organized through various modes and identifiable network motifs. Cell-specific transcriptome data was combined with known secondary cell wall (SCW) networks to identify the GRNs that differentially activate SCW formation in vascular sclerenchyma and epidermal cells. The spatial transcriptomic dataset provides a valuable source of information about the function of different sorghum cell types and GRNs that will enable the engineering of bioenergy sorghum stems, and an interactive web application developed during this project will allow easy access and exploration of the data (https://mc-lab.shinyapps.io/lcm-dataset/).

COBRA-LIKE4 Modulates Cellulose Synthase Velocity and Facilitates Cellulose Deposition in the Secondary Cell Wall.

Cellulose is a critical component of secondary cell walls and woody tissues of plants. Cellulose synthase (CESA) complexes (CSCs) produce cellulose as they move within the plasma membrane, extruding glucan chains into the cell wall that coalesce and crystallize into cellulose fibrils. Here we examine COBRA-LIKE4 (COBL4), a GPI-anchored protein on the outer leaflet of the plasma membrane that is required for normal cellulose deposition in secondary cell walls. Characterization of the Arabidopsis (Arabidopsis thaliana) cobl4 mutant alleles called irregular xylem6, irx6-2 and irx6-3, showed reduced ⍺-cellulose content and lower crystallinity, supporting a role for COBL4 in maintaining cellulose quantity and quality. In live-cell imaging, mNeon Green-tagged CESA7 moved in the plasma membrane at higher speeds in the irx6-2 background compared to wild type. To test conservation of COBL4 function between herbaceous and woody plants, poplar (Populus trichocarpa) COBL4 homologs PtCOBL4a and PtCOBL4b were transformed into, and rescued, the Arabidopsis irx6 mutants. Using the Arabidopsis secondary cell wall-inducible VND7-GR system to study poplar COBL4 dynamics, YFP-tagged PtCOBL4a localized to the plasma membrane in regions of high cellulose deposition in secondary cell wall bands. As predicted for a lipid-linked protein, COBL4 was more mobile in the plane of the plasma membrane than CESA7 or a control plasma membrane marker. Following programmed cell death, COBL4 anchored to the secondary cell wall bands. These data support a role for COBL4 as a modulator of cellulose organization in the secondary cell wall, influencing cellulose production and CSC velocity at the plasma membrane.

Elongated galactan side chains mediate cellulose-pectin interactions in engineered Arabidopsis secondary cell walls.

The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi-dimensional solid-state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin-cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell-specific expression strategies to avoid negative effects on plant growth.

VND Genes Redundantly Regulate Cell Wall Thickening during Parasitic Nematode Infection.

Plant parasitic root-knot nematodes are major agricultural pests worldwide, as they infect plant roots and cause substantial damages to crop plants. Root-knot nematodes induce specialized feeding cells known as giant cells (GCs) in the root vasculature, which serve as nutrient reservoirs for the infecting nematodes. Here, we show that the cell walls of GCs thicken to form pitted patterns that superficially resemble metaxylem cells. Interestingly, VASCULAR-RELATED NAC-DOMAIN1 (VND1) was found to be upregulated, while the xylem-type programmed cell death marker XYLEM CYSTEINE PEPTIDASE 1 was downregulated upon nematode infection. The vnd2 and vnd3 mutants showed reduced secondary cell wall pore size, while the vnd1 vnd2 vnd3 triple mutant produced significantly fewer nematode egg masses when compared with the wild type. These results suggest that the GC development pathway likely shares common signaling modules with the metaxylem differentiation pathway and VND1, VND2, and VND3 redundantly regulate plant-nematode interaction through secondary cell wall formation.

An essential role for mannan degradation in both cell growth and secondary cell wall formation.

Coordination of secondary cell wall deposition and cell expansion during plant growth is required for cell development, particularly in vascular tissues. Yet the fundamental coordination process has received little attention. We observed that the Arabidopsis endo-1,4-mannanase gene, AtMAN6, is involved in the formation of cell walls in vascular tissues. In the inflorescence stem, the man6 mutant had smaller vessel cells with thicker secondary cell walls and shorter fiber cells. Elongation growth was reduced in the root, and secondary cell wall deposition in vessel cells occurred early. Overexpression of AtMAN6 resulted in the inverse phenotypes of the man6 mutant. AtMAN6 was discovered on the plasma membrane and was specifically expressed in vessel cells during its early development. The AtMAN6 protein degraded galactoglucomannan to produce oligosaccharides, which caused secondary cell wall deposition in vessel and fiber cells to be suppressed. Transcriptome analysis revealed that the expression of genes involved in the regulation of secondary cell wall synthesis was changed in both man6 mutant and AtMAN6 overexpression plants. AtMAN6's C-terminal cysteine repeat motif (CCRM) was found to facilitate homodimerization and is required for its activity. According to the findings, the oligosaccharides produced by AtMAN6 hydrolysis may act as a signal to mediate this coordination between cell growth and secondary cell wall deposition.

NST3 induces ectopic transdifferentiation, forming secondary walls with diverse patterns and composition in Arabidopsis thaliana.

BACKGROUND AND AIMS: The master transcription factor NAC SECONDARY WALL THICKENING PROMOTING FACTOR3 (NST3), also known as SND1, plays a pivotal role in regulating secondary cell wall (SCW) development in interfascicular and xylary fibers in Arabidopsis thaliana. Despite progress in understanding SCW assembly in xylem vessel-like cells, the mechanisms behind its assembly across different cell types remain unclear. Overexpressing NST3 or its homolog NST1 leads to reduced fertility, posing challenges for studying their impact on secondary wall formation. This study aimed at developing a tightly regulated dexamethasone (DEX)-inducible expression system for NST3 and NST1 to elucidate the structure and assembly of diverse SCWs. METHODS: Using the DEX-inducible system, we characterized ectopically formed SCWs for their diverse patterns, mesoscale organization, cellulose microfibril orientation, and molecular composition using spinning disk confocal microscopy, field emission scanning electron microscopy (FESEM), vibrational sum-frequency generation (SFG) spectroscopy and, histochemical staining and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. KEY RESULTS: Upon DEX treatment, NST3 and NST1 transgenic hypocotyls underwent time-dependent transdifferentiation, progressing from protoxylem-like to metaxylem-like cells. NST3-induced plants exhibited normal growth but had rough secondary wall surfaces with delaminating S2 and S3 layers. Mesoscale examination of induced SCWs in epidermal cells revealed that macrofibril thickness and orientation were comparable to xylem vessels, while wall thickness resembled that of interfascicular fibers. Additionally, induced epidermal cells formed SCWs with altered cellulose and lignin contents. CONCLUSIONS: These findings suggest NST3 and/or NST1 induce SCWs with shared characteristics of both xylem and fiber-like cells forming loosely arranged cell wall layers and cellulose organized at multiple angles relative to the cell growth axis and with varied cellulose and lignin abundance. This inducible system opens avenues to explore ectopic SCWs for bioenergy and bioproducts, offering valuable insights into SCW patterning across diverse cell types and developmental stages.

Analyses of high spatial resolution datasets identify genes associated with multi-layered secondary cell wall thickening in Pinus bungeana.

BACKGROUND AND AIMS: Secondary cell wall (SCW) thickening is a major cellular developmental stage determining wood structure and properties. Although the molecular regulation of cell wall deposition during tracheary element differentiation has been well established in primary growth systems, less is known about the gene regulatory processes involved in the multi-layered SCW thickening of mature trees. METHODS: Using third-generation [long-read single-molecule real-time (SMRT)] and second-generation [short-read sequencing by synthesis (SBS)] sequencing methods, we established a Pinus bungeana transcriptome resource with comprehensive functional and structural annotation for the first time. Using these approaches, we generated high spatial resolution datasets for the vascular cambium, xylem expansion regions, early SCW thickening, late SCW thickening and mature xylem tissues of 71-year-old Pinus bungeana trees. KEY RESULTS: A total of 79 390 non-redundant transcripts, 31 808 long non-coding RNAs and 5147 transcription factors were annotated and quantified in different xylem tissues at all growth and differentiation stages. Furthermore, using this high spatial resolution dataset, we established a comprehensive transcriptomic profile and found that members of the NAC, WRKY, SUS, CESA and LAC gene families are major players in early SCW formation in tracheids, whereas members of the MYB and LBD transcription factor families are highly expressed during late SCW thickening. CONCLUSIONS: Our results provide new molecular insights into the regulation of multi-layered SCW thickening in conifers. The high spatial resolution datasets provided can serve as important gene resources for improving softwoods.

GhHB14_D10 and GhREV_D5, two HD-ZIP III transcription factors, play a regulatory role in cotton fiber secondary cell wall biosynthesis.

KEY MESSAGE: GhHB14_D10 and GhREV_D5 regulated secondary cell wall formation and played an important role in fiber development. Cotton serves as an important source of natural fiber, and the biosynthesis of the secondary cell wall plays a pivotal role in determining cotton fiber quality. Nevertheless, the intricacies of this mechanism in cotton fiber remain insufficiently elucidated. This study investigates the functional roles of GhHB14_D10 and GhREV_D5, two HD-ZIP III transcription factors, in secondary cell wall biosynthesis in cotton fibers. Both GhHB14_D10 and GhREV_D5 were found to be localized in the nucleus with transcriptional activation activity. Ectopic overexpression of GhHB14_D10 and GhREV_D5 in Arabidopsis resulted in changed xylem differentiation, secondary cell wall deposition, and expression of genes related to the secondary cell wall. Silencing of GhHB14_D10 and GhREV_D5 in cotton led to enhanced fiber length, reduced cell wall thickness, cellulose contents and expression of secondary cell wall-related genes. Moreover, GhHB14_D10's direct interaction with GhREV_D5, and transcriptional regulation of cellulose biosynthesis genes GhCesA4-4 and GhCesA7-2 revealed their collaborative roles in secondary cell wall during cotton fiber development. Overall, these results shed light on the roles of GhHB14_D10 and GhREV_D5 in secondary cell wall biosynthesis, offering a strategy for the genetic improvement of cotton fiber quality.

Abscisic acid regulates secondary cell-wall formation and lignin deposition in Arabidopsis thaliana through phosphorylation of NST1.

Plant secondary cell-wall (SCW) deposition and lignification are affected by both seasonal factors and abiotic stress, and these responses may involve the hormone abscisic acid (ABA). However, the mechanisms involved are not clear. Here we show that mutations that limit ABA synthesis or signaling reduce the extent of SCW thickness and lignification in Arabidopsis thaliana through the core ABA-signaling pathway involving SnRK2 kinases. SnRK2.2. 3 and 6 physically interact with the SCW regulator NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 (NST1), a NAC family transcription factor that orchestrates the transcriptional activation of a suite of downstream SCW biosynthesis genes, some of which are involved in the biosynthesis of cellulose and lignin. This interaction leads to phosphorylation of NST1 at Ser316, a residue that is highly conserved among NST1 proteins from dicots, but not monocots, and is required for transcriptional activation of downstream SCW-related gene promoters. Loss of function of NST1 in the snd1 mutant background results in lack of SCWs in the interfascicular fiber region of the stem, and the Ser316Ala mutant of NST1 fails to complement this phenotype and ABA-induced lignin pathway gene expression. The discovery of NST1 as a key substrate for phosphorylation by SnRK2 suggests that the ABA-mediated core-signaling cascade provided land plants with a hormone-modulated, competitive desiccation-tolerance strategy allowing them to differentiate water-conducting and supporting tissues built of cells with thicker cell walls.

Deciphering the Molecular Mechanism of the Intermediate Secondary Growth and Internode Elongation of the Castor Bean (Ricinus communis L.) by the Combined Analysis of the Transcriptome and Metabolome.

The length of internodes plays a crucial role in determining the height of the castor plant (Ricinus communis L.). However, the specific mechanisms underlying internode elongation, particularly in the main stem of the castor plant, remain uncertain. To further investigate this, we conducted a study focusing on the internode tissue of the dwarf castor variety 071113, comparing it with the control high-stalk Zhuansihao. Our study included a cytological observation, physiological measurement, transcriptome sequencing, and metabolic determination. Our integrated findings reveal that the dwarf variety 071113 undergoes an earlier lignification development in the main stem and has a more active lignin synthesis pathway during internode intermediate development. In addition, the dwarf variety exhibited lower levels of the plant hormone indole-3-acetic acid (IAA), which had an impact on the development process. Furthermore, we identified specific enzymes and regulators that were enriched in the pathways of the cell cycle, auxin signal transduction, and secondary cell wall synthesis. Using these findings, we developed a model that explained the intermediate secondary growth observed in castor internode elongation and enhanced our comprehension of the dwarfing mechanism of the 071113 variety. This research provides a theoretical groundwork for the future breeding of dwarf castor varieties.

Ectopic Expression of PtrLBD39 Retarded Primary and Secondary Growth in Populus trichocarpa.

Primary and secondary growth of trees are needed for increments in plant height and stem diameter, respectively, affecting the production of woody biomass for applications in timber, pulp/paper, and related biomaterials. These two types of growth are believed to be both regulated by distinct transcription factor (TF)-mediated regulatory pathways. Notably, we identified PtrLBD39, a highly stem phloem-specific TF in Populus trichocarpa and found that the ectopic expression of PtrLBD39 in P. trichocarpa markedly retarded both primary and secondary growth. In these overexpressing plants, the RNA-seq, ChIP-seq, and weighted gene co-expression network analysis (WGCNA) revealed that PtrLBD39 directly or indirectly regulates TFs governing vascular tissue development, wood formation, hormonal signaling pathways, and enzymes responsible for wood components. This regulation led to growth inhibition, decreased fibrocyte secondary cell wall thickness, and reduced wood production. Therefore, our study indicates that, following ectopic expression in P. trichocarpa, PtrLBD39 functions as a repressor influencing both primary and secondary growth.

Bulked Segregant RNA-Seq Reveals Different Gene Expression Patterns and Mutant Genes Associated with the Zigzag Pattern of Tea Plants (Camellia sinensis).

The unique zigzag-patterned tea plant is a rare germplasm resource. However, the molecular mechanism behind the formation of zigzag stems remains unclear. To address this, a BC1 genetic population of tea plants with zigzag stems was studied using histological observation and bulked segregant RNA-seq. The analysis revealed 1494 differentially expressed genes (DEGs) between the upright and zigzag stem groups. These DEGs may regulate the transduction and biosynthesis of plant hormones, and the effects on the phenylpropane biosynthesis pathways may cause the accumulation of lignin. Tissue sections further supported this finding, showing differences in cell wall thickness between upright and curved stems, potentially due to lignin accumulation. Additionally, 262 single-nucleotide polymorphisms (SNPs) across 38 genes were identified as key SNPs, and 5 genes related to zigzag stems were identified through homologous gene function annotation. Mutations in these genes may impact auxin distribution and content, resulting in the asymmetric development of vascular bundles in curved stems. In summary, we identified the key genes associated with the tortuous phenotype by using BSR-seq on a BC1 population to minimize genetic background noise.

GhMYB52 Like: A Key Factor That Enhances Lint Yield by Negatively Regulating the Lignin Biosynthesis Pathway in Fibers of Upland Cotton (Gossypium hirsutum L.).

In the context of sustainable agriculture and biomaterial development, understanding and enhancing plant secondary cell wall formation are crucial for improving crop fiber quality and biomass conversion efficiency. This is especially critical for economically important crops like upland cotton (Gossypium hirsutum L.), for which fiber quality and its processing properties are essential. Through comprehensive genome-wide screening and analysis of expression patterns, we identified a particularly high expression of an R2R3 MYB transcription factor, GhMYB52 Like, in the development of the secondary cell wall in cotton fiber cells. Utilizing gene-editing technology to generate a loss-of-function mutant to clarify the role of GhMYB52 Like, we revealed that GhMYB52 Like does not directly contribute to cellulose synthesis in cotton fibers but instead represses a subset of lignin biosynthesis genes, establishing it as a lignin biosynthesis inhibitor. Concurrently, a substantial decrease in the lint index, a critical measure of cotton yield, was noted in parallel with an elevation in lignin levels. This study not only deepens our understanding of the molecular mechanisms underlying cotton fiber development but also offers new perspectives for the molecular improvement of other economically important crops and the enhancement of biomass energy utilization.

GhCASPL1 regulates secondary cell wall thickening in cotton fibers by stabilizing the cellulose synthase complex on the plasma membrane.

Cotton (Gossypium hirsutum) fibers are elongated single cells that rapidly accumulate cellulose during secondary cell wall (SCW) thickening, which requires cellulose synthase complex (CSC) activity. Here, we describe the CSC-interacting factor CASPARIAN STRIP MEMBRANE DOMAIN-LIKE1 (GhCASPL1), which contributes to SCW thickening by influencing CSC stability on the plasma membrane. GhCASPL1 is preferentially expressed in fiber cells during SCW biosynthesis and encodes a MARVEL domain protein. The ghcaspl1 ghcaspl2 mutant exhibited reduced plant height and produced mature fibers with fewer natural twists, lower tensile strength, and a thinner SCW compared to the wild type. Similarly, the Arabidopsis (Arabidopsis thaliana) caspl1 caspl2 double mutant showed a lower cellulose content and thinner cell walls in the stem vasculature than the wild type but normal plant morphology. Introducing the cotton gene GhCASPL1 successfully restored the reduced cellulose content of the Arabidopsis caspl1 caspl2 mutant. Detergent treatments, ultracentrifugation assays, and enzymatic assays showed that the CSC in the ghcaspl1 ghcaspl2 double mutant showed reduced membrane binding and decreased enzyme activity compared to the wild type. GhCASPL1 binds strongly to phosphatidic acid (PA), which is present in much higher amounts in thickening fiber cells compared to ovules and leaves. Mutating the PA-binding site in GhCASPL1 resulted in the loss of its colocalization with GhCesA8, and it failed to localize to the plasma membrane. PA may alter membrane structure to facilitate protein-protein interactions, suggesting that GhCASPL1 and PA collaboratively stabilize the CSC. Our findings shed light on CASPL functions and the molecular machinery behind SCW biosynthesis in cotton fibers.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue.

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Functional investigation of five R2R3-MYB transcription factors associated with wood development in Eucalyptus using DAP-seq-ML.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

Functional Diversification and the Plant Secondary Cell Wall.

Much evidence exists suggesting the presence of genetic functional diversification in plants, though literature associated with the role of functional diversification in the evolution of the plant secondary cell wall (SCW) has sparsely been compiled and reviewed in a recent context. This review aims to elucidate, through the examination of gene phylogenies associated with its biosynthesis and maintenance, the role of functional diversification in shaping the critical, dynamic, and characteristic organelle, the secondary cell wall. It will be asserted that gene families resulting from gene duplication and subsequent functional divergence are present and are heavily involved in SCW biosynthesis and maintenance. Furthermore, diversification will be presented as a significant driver behind the evolution of the many functional characteristics of the SCW. The structure and function of the plant cell wall and its constituents will first be explored, followed by a discussion on the phenomenon of gene duplication and the resulting genetic functional divergence that can emerge. Finally, the major constituents of the SCW and their individual relationships with duplication and divergence will be reviewed to the extent of current knowledge on the subject.

Stone cell formation in the pedicel of pears and apples.

MAIN CONCLUSION: For the first time, stone cells in pear and apple pedicel were studied. The lignification of the pedicel outer part was correlated with flesh, and the secondary cell wall biosynthesis genes were activated. Fruit pedicels act as bridges between the fruit and the shoot. They have secondary thickened cell walls that presumably function in mechanical support, water and nutrient transport. Stone cells are cells with a secondary cell wall thickening. In pears, yet not in apples, the stone cells affect the flesh texture. There have been few reports on stone cell formation in pear and apple pedicels; therefore, we studied these cells for the first time. The apple pedicel had few stone cells in the cortex. The formation of stone cells in pear continued until seven weeks after flowering (WAF), and the density was significantly higher than in apple. The stone cell formation degree (SFD) of pear was 3.6-7.1 times higher than that of apple. Total lignin and lignin non-condensed structure (G and S units) content in the pear pedicle outer part was 1.5-2.7 times higher than that of the apple at harvest. The SFD of the pedicel outer part had a positive correlation with the G and S units content of the flesh. The total lignin and G and S units content between flesh and the pedicel outer part were positively correlated. Correlation analysis revealed a positive relationship between fruit and pedicel formation of the stone cells. The WGCNA showed that NST3 was linked to NAC028, MYB46, CESA, POD, LAC, and VSR6. These genes were highly expressed in the outer part of the pear pedicel, while they were suppressed in that issue of the apple at 4 WAF.