Secondary Cell Wall Thickening Research Articles

MeGT2.6 increases cellulose synthesis and active gibberellin content to promote cell enlargement in cassava.

Cassava, a pivotal tropical crop, exhibits rapid growth and possesses a substantial biomass. Its stem is rich in cellulose and serves as a crucial carbohydrate storage organ. The height and strength of stems restrict the mechanised operation and propagation of cassava. In this study, the triple helix transcription factor MeGT2.6 was identified through yeast one-hybrid assay using MeCesA1pro as bait, which is critical for cellulose synthesis. Over-expression and loss-of-function lines were generated, and results revealed that MeGT2.6 could promote a significant increase in the plant height, stem diameter, cell size and thickness of SCW of cassava plant. Specifically, MeGT2.6 upregulated the transcription activity of MeGA20ox1 and downregulated the expression level of MeGA2ox1, thereby enhancing the content of active GA3, resulting in a large cell size, high plant height and long stem diameter in cassava. Moreover, MeGT2.6 upregulated the transcription activity of MeCesA1, which promoted the synthesis of cellulose and hemicellulose and produced a thick secondary cell wall. Finally, MeGT2.6 could help supply additional substrates for the synthesis of cellulose and hemicellulose by upregulating the invertase genes (MeNINV1/6). Thus, MeGT2.6 was found to be a multiple regulator; it was involved in GA metabolism and sucrose decomposition and the synthesis of cellulose and hemicellulose.

Cell size and xylem differentiation regulating genes from Salicornia europaea contribute to plant salt tolerance.

Cell wall is involved in plant growth and plays pivotal roles in plant adaptation to environmental stresses. Cell wall remodelling may be crucial to salt adaptation in the euhalophyte Salicornia europaea. However, the mechanism underlying this process is still unclear. Here, full-length transcriptome indicated cell wall-related genes were comprehensively regulated under salinity. The morphology and cell wall components in S. europaea shoot were largely modified under salinity. Through the weighted gene co-expression network analysis, SeXTH2 encoding xyloglucan endotransglucosylase/hydrolases, and two SeLACs encoding laccases were focused. Meanwhile, SeEXPB was focused according to expansin activity and the expression profiling. Function analysis in Arabidopsis validated the functions of these genes in enhancing salt tolerance. SeXTH2 and SeEXPB overexpression led to larger cells and leaves with hemicellulose and pectin content alteration. SeLAC1 and SeLAC2 overexpression led to more xylem vessels, increased secondary cell wall thickness and lignin content. Notably, SeXTH2 transgenic rice exhibited enhanced salt tolerance and higher grain yield. Altogether, these genes may function in the succulence and lignification process in S. europaea. This work throws light on the regulatory mechanism of cell wall remodelling in S. europaea under salinity and provides potential strategies for improving crop salt tolerance and yields.

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

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. 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. 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. 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.

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.

Transcription factor PagMYB31 positively regulates cambium activity and negatively regulates xylem development in poplar.

Wood formation involves consecutive developmental steps, including cell division of vascular cambium, xylem cell expansion, secondary cell wall (SCW) deposition, and programmed cell death. In this study, we identified PagMYB31 as a coordinator regulating these processes in Populus alba × Populus glandulosa and built a PagMYB31-mediated transcriptional regulatory network. PagMYB31 mutation caused fewer layers of cambial cells, larger fusiform initials, ray initials, vessels, fiber and ray cells, and enhanced xylem cell SCW thickening, showing that PagMYB31 positively regulates cambial cell proliferation and negatively regulates xylem cell expansion and SCW biosynthesis. PagMYB31 repressed xylem cell expansion and SCW thickening through directly inhibiting wall-modifying enzyme genes and the transcription factor genes that activate the whole SCW biosynthetic program, respectively. In cambium, PagMYB31 could promote cambial activity through TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/PHLOEM INTERCALATED WITH XYLEM (PXY) signaling by directly regulating CLAVATA3/ESR-RELATED (CLE) genes, and it could also directly activate WUSCHEL HOMEOBOX RELATED4 (PagWOX4), forming a feedforward regulation. We also observed that PagMYB31 could either promote cell proliferation through the MYB31-MYB72-WOX4 module or inhibit cambial activity through the MYB31-MYB72-VASCULAR CAMBIUM-RELATED MADS2 (VCM2)/PIN-FORMED5 (PIN5) modules, suggesting its role in maintaining the homeostasis of vascular cambium. PagMYB31 could be a potential target to manipulate different developmental stages of wood formation.

PtoMYB031, the R2R3 MYB transcription factor involved in secondary cell wall biosynthesis in poplar.

The biosynthesis of the secondary cell wall (SCW) is orchestrated by an intricate hierarchical transcriptional regulatory network. This network is initiated by first-layer master switches, SCW-NAC transcription factors, which in turn activate the second-layer master switches MYBs. These switches play a crucial role in regulating xylem specification and differentiation during SCW formation. However, the roles of most MYBs in woody plants are yet to be fully understood. In this study, we identified and isolated the R2R3-MYB transcription factor, PtoMYB031, from Populus tomentosa. We explored its expression, mainly in xylem tissues, and its role as a transcriptional repressor in the nucleus. We used overexpression and RNA interference techniques in poplar, along with Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays, to analyze the regulatory effects of PtoMYB031. Overexpression of PtoMYB031 in poplar significantly reduced lignin, cellulose, and hemicellulose content, and inhibited vascular development in stems, resulting in decreased SCW thickness in xylem tissues. Gene expression analysis showed that structural genes involved in SCW biosynthesis were downregulated in PtoMYB031-OE lines. Conversely, RNA interference of PtoMYB031 increased these compounds. Additionally, PtoMYB031 was found to recruit the repressor PtoZAT11, forming a transcriptional inhibition complex. Our findings provide new insights into how PtoMYB031, through its interaction with PtoZAT11, forms a complex that can suppress the expression of key regulatory genes, PtoWND1A and PtoWND2B, in SCW biosynthesis. This study enhances our understanding of the transcriptional regulation involved in SCW formation in poplar, highlighting the significant role of PtoMYB031.

Microtubule-associated phase separation of MIDD1 tunes cell wall spacing in xylem vessels in Arabidopsis thaliana.

Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana. Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.

UBC6, a ubiquitin-conjugating enzyme, participates in secondary cell wall thickening in the inflorescence stem of Arabidopsis

Secondary cell wall (SCW) thickening in plant inflorescence stems is a complicated cellular process that is essential for stem strength and biomass. Although Arabidopsis NAC transcription factor (TF) 1 (NST1) regulates the SCW thickening in anther walls, the single T-DNA-insertion mutant (nst1) does not show disrupted SCW thickening in anther endothecium, interfascicular fibers or xylem. To better understand the regulatory mechanism of this process, we generated an ethyl methanesulfonate (EMS)-mutagenized Arabidopsis population with the nst1 background. scd5 (SCW-defective mutant 5) was isolated in a forward genetic screen from the EMS mutant library, which displayed not only less lignin deposition in the interfascicular fiber and xylem than the wild type but also a pendent inflorescence stem. The EMS-induced mutation associated with the scd5 phenotype was found in the 5th exon of At2G46030 that encodes a ubiquitin-conjugating enzyme (UBC6), we thereby renamed the allele nst1 ubc6. Overexpressing UBC6 in nst1 ubc6 rescued the defective SCW, whereas disrupting UBC6 in nst1 by the CRISPR/Cas9 system caused a phenotype similar to that observed in nst1 ubc6. UBC6 was localized to the nucleus and plasma membrane, and possessed E2 ubiquitin-conjugating activity in vitro. MYB7 and MYB32 are considered as transcription repressors in the phenylpropanoid pathway and are involved in NAC TF-related transcriptional regulation in SCW thickening. UBC6 can interact with MYB7 and MYB32 and positively mediate the degradation of MYB7 and MYB32 by the 26S proteasome. Overall, these results indicated the contribution of UBC6 to SCW thickening in Arabidopsis inflorescence stems.

Comparative Root Transcriptome Profiling and Gene Regulatory Network Analysis between Eastern and Western Carrot (Daucus carota L.) Cultivars Reveals Candidate Genes for Vascular Tissue Patterning.

Carrot (Daucus carota L.) is a highly consumed vegetable rich in carotenoids, known for their potent antioxidant, anti-inflammatory, and immune-protecting properties. While genetic and molecular studies have largely focused on wild and Western carrot cultivars (cvs), little is known about the evolutionary interactions between closely related Eastern and Western cvs. In this study, we conducted comparative transcriptome profiling of root tissues from Eastern (UHSBC-23-1) and Western (UHSBC-100) carrot cv. to better understand differentially expressed genes (DEGs) associated with storage root development and vascular cambium (VC) tissue patterning. Through reference-guided TopHat mapping, we achieved an average mapping rate of 73.87% and identified a total of 3544 DEGs (p < 0.05). Functional annotation and gene ontology classification revealed 97 functional categories, including 33 biological processes, 19 cellular components, 45 metabolic processes, and 26 KEGG pathways. Notably, Eastern cv. exhibited enrichment in cell wall, plant-pathogen interaction, and signal transduction terms, while Western cv. showed dominance in photosynthesis, metabolic process, and carbon metabolism terms. Moreover, constructed gene regulatory network (GRN) for both cvs. obtained orthologs with 1222 VC-responsive genes of Arabidopsis thaliana. In Western cv, GRN revealed VC-responsive gene clusters primarily associated with photosynthetic processes and carbon metabolism. In contrast, Eastern cv. exhibited a higher number of stress-responsive genes, and transcription factors (e.g., MYB15, WRKY46, AP2/ERF TF connected via signaling pathways with NAC036) were identified as master regulators of xylem vessel differentiation and secondary cell wall thickening. By elucidating the comparative transcriptome profiles of Eastern and Western cvs. for the first time, our study provides valuable insights into the differentially expressed genes involved in root development and VC tissue patterning. The identification of key regulatory genes and their roles in these processes represents a significant advancement in our understanding of the evolutionary relations and molecular mechanisms underlying secondary growth of carrot and regulation by vascular cambium.

Stone cell formation in the pedicel of pears and apples.

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.

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.

The transcription factor ERF108 interacts with AUXIN RESPONSE FACTORs to mediate cotton fiber secondary cell wall biosynthesis.

Phytohormones play indispensable roles in plant growth and development. However, the molecular mechanisms underlying phytohormone-mediated regulation of fiber secondary cell wall (SCW) formation in cotton (Gossypium hirsutum) remain largely underexplored. Here, we provide mechanistic evidence for functional interplay between the AP2/ERF transcription factor GhERF108 and auxin response factors GhARF7-1 and GhARF7-2 in dictating the ethylene-auxin signaling crosstalk that regulates fiber SCW biosynthesis. Specifically, in vitro cotton ovule culture revealed that ethylene and auxin promote fiber SCW deposition. GhERF108 RNAi cotton displayed remarkably reduced cell wall thickness compared with controls. GhERF108 interacted with GhARF7-1 and GhARF7-2 to enhance the activation of the MYB transcription factor gene GhMYBL1 (MYB domain like protein 1) in fibers. GhARF7-1 and GhARF7-2 respond to auxin signals that promote fiber SCW thickening. GhMYBL1 RNAi and, GhARF7-1 and GhARF7-2 VIGS cotton displayed similar defects in fiber SCW formation as GhERF108 RNAi cotton. Moreover, the ethylene and auxin responses were reduced in GhMYBL1 RNAi plants. GhMYBL1 directly binds to the promoters of GhCesA4-1, GhCesA4-2, and GhCesA8-1 and activates their expression to promote cellulose biosynthesis, thereby boosting fiber SCW formation. Collectively, our findings demonstrate that the collaboration between GhERF108 and GhARF7-1 or GhARF7-2 establishes ethylene-auxin signaling crosstalk to activate GhMYBL1, ultimately leading to the activation of fiber SCW biosynthesis.

Jasmonic acid regulates lignin deposition in poplar through JAZ5-MYB/NAC interaction.

Jasmonic acid (JA) is a phytohormone involved in plant defense, growth, and development, etc. However, the regulatory mechanisms underlying JA-mediated lignin deposition and secondary cell wall (SCW) formation remain poorly understood. In this study, we found that JA can inhibit lignin deposition and SCW thickening in poplar trees through exogenous MeJA treatment and observation of the phenotypes of a JA synthesis mutant, opdat1. Hence, we identified a JA signal inhibitor PtoJAZ5, belonging to the TIFY gene family, which is involved in the regulation of secondary vascular development of Populus tomentosa. RT-qPCR and GUS staining revealed that PtoJAZ5 was highly expressed in poplar stems, particularly in developing xylem. Overexpression of PtoJAZ5 inhibited SCW thickening and down-regulated the expression of SCW biosynthesis-related genes. Further biochemical analysis showed that PtoJAZ5 interacted with multiple SCW switches NAC/MYB transcription factors, including MYB3 and WND6A, through yeast two-hybrid and bimolecular fluorescent complementation experiments. Transcriptional activation assays demonstrated that MYB3-PtoJAZ5 and WND6A-PtoJAZ5 complexes regulated the expression of lignin synthetic genes. Our results suggest that PtoJAZ5 plays a negative role in JA-induced lignin deposition and SCW thickening in poplar and provide new insights into the molecular mechanisms underlying JA-mediated regulation of SCW formation.

High temperature inhibits vascular development via the PIF4-miR166-HB15 module in Arabidopsis

The plant vascular system is an elaborate network of conducting and supporting tissues that extends throughout the plant body, and its structure and function must be orchestrated with different environmental conditions. Under high temperature, plants display thin and lodging stems that may lead to decreased yield and quality of crops. However, the molecular mechanism underlying high-temperature-mediated regulation of vascular development is not known. Here, we show that Arabidopsis plants overexpressing the basic-helix-loop-helix (bHLH) transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4), a central regulator of high-temperature signaling, display fewer vascular bundles (VBs) and decreased secondary cell wall (SCW) thickening, mimicking the lodging inflorescence stems of high-temperature-grown wild-type plants. Rising temperature and elevated PIF4 expression reduced the expression of MIR166 and, concomitantly, elevated the expression of the downstream class III homeodomain leucine-zipper (HD-ZIP III) family gene HB15. Consistently, knockdown of miR166 and overexpression of HB15 led to inhibition of vascular development and SCW formation, whereas the hb15 mutant displayed the opposite phenotype in response to high temperature. Moreover, invitro and invivo assays verified that PIF4 binds to the promoters of several MIR166 genes and represses their expression. Our study establishes a direct functional link between PIF4 and the miR166-HB15 module in modulating vascular development and SCW thickening and consequently stem-lodging susceptibility at elevated temperatures.

A cell wall-localized β-1,3-glucanase promotes fiber cell elongation and secondary cell wall deposition.

β-1,3-glucanase functions in plant physiological and developmental processes. However, how β-1,3-glucanase participates in cell wall development remains largely unknown. Here, we answered this question by examining the role of GhGLU18, a β-1,3-glucanase, in cotton (Gossypium hirsutum) fibers, in which the content of β-1,3-glucan changes dynamically from 10% of the cell wall mass at the onset of secondary wall deposition to <1% at maturation. GhGLU18 was specifically expressed in cotton fiber with higher expression in late fiber elongation and secondary cell wall (SCW) synthesis stages. GhGLU18 largely localized to the cell wall and was able to hydrolyze β-1,3-glucan in vitro. Overexpression of GhGLU18 promoted polysaccharide accumulation, cell wall reconstruction, and cellulose synthesis, which led to increased fiber length and strength with thicker cell walls and shorter pitch of the fiber helix. However, GhGLU18-suppressed cotton resulted in opposite phenotypes. Additionally, GhGLU18 was directly activated by GhFSN1 (fiber SCW-related NAC1), a NAC transcription factor reported previously as the master regulator in SCW formation during fiber development. Our results demonstrate that cell wall-localized GhGLU18 promotes fiber elongation and SCW thickening by degrading callose and enhancing polysaccharide metabolism and cell wall synthesis.

Two R2R3-MYB transcription factors from Chinese cedar (Cryptomeria fortunei Hooibrenk) are involved in the regulation of secondary cell wall formation

As the most abundant renewable energy source, wood comprises the secondary cell wall (SCW). SCW biosynthesis involves lignin and cellulose deposition. Increasing studies have illustrated that R2R3-MYB transcription factors (TFs) play pivotal roles in affecting lignin accumulation and SCW formation. Nevertheless, the regulatory roles of R2R3-MYBs are still unresolved in Cryptomeria fortunei Hooibrenk cambium and wood formation. To dissect the potentials of CfMYBs, we successfully cloned and intensively studied the functions of CfMYB4 and CfMYB5 in SCW formation and abiotic stress response. They both contained the conserved MYB domain capable of forming a special structure that could bind to the core motifs of downstream genes. The phylogenetic tree implied that two CfMYBs clustered into different evolutionary branches. They were predominantly expressed in the stem and were localized to the nucleus. Furthermore, CfMYB4 functioned as an activator to enhance lignin and cellulose accumulation, and increase the SCW thickness by elevating the expression levels of SCW-related genes. By contrast, CfMYB5 negatively regulated lignin and cellulose biosynthesis, and decreased SCW formation by reducing the expression of SCW biosynthetic genes. Our data not only highlight the regulatory functions of CfMYBs in lignin deposition but also provide critical insights into the development of strategies for the genetic improvement of Cryptomeria fortunei wood biomass.

Ectopic Expression of FvVND4c Promotes Secondary Cell Wall Thickening and Flavonoid Accumulation in Fragaria vesca.

Secondary cell wall (SCW) thickening has a significant effect on the growth and development of plants, as well as in the resistance to various biotic and abiotic stresses. Lignin accounts for the strength of SCW. It is synthesized through the phenylpropanoid pathway that also leads to flavonoid synthesis. The coupling strategies for lignin and flavonoid syntheses are diverse in plants. How their syntheses are balanced by transcriptional regulation in fleshy fruits is still unclear. The diploid strawberry (Fragaria vesca) is a model for fleshy fruits research due to its small genome and wide scope of genetic transformation. SCW thickening is regulated by a multilevel transcriptional regulatory network wherein vascular-related NAC domains (VNDs) act as key regulators. In this study, we systematically characterized VNDs in Fragaria vesca and explored their functions. The overexpression of FvVND4c in diploid strawberry fruits resulted in SCW thickening and fruit color changes accompanied with the accumulation of lignin and flavonoids. Genes related to these phenotypes were also induced upon FvVND4c overexpression. Among the induced genes, we found FvMYB46 to be a direct downstream regulator of FvVND4c. The overexpression of FvMYB46 resulted in similar phenotypes as FvVND4c, except for the color change. Transcriptomic analyses suggest that both FvVND4c and FvMYB46 act on phenylpropanoid and flavonoid biosynthesis pathways, and induce lignin synthesis for SCW. These results suggest that FvVND4c and FvMYB46 cooperatively regulate SCW thickening and flavonoid accumulation in Fragaria vesca.

Retraction Note: Overexpression of PsnSuSy1, 2 genes enhances secondary cell wall thickening, vegetative growth, and mechanical strength in transgenic tobacco.

Retraction Note: Overexpression of PsnSuSy1, 2 genes enhances secondary cell wall thickening, vegetative growth, and mechanical strength in transgenic tobacco.

The RLCK–VND6 module coordinates secondary cell wall formation and adaptive growth in rice

The orderly deposition of secondary cell wall (SCW) in plants is implicated in various biological programs and is precisely controlled. Although many positive and negative regulators of SCW have been documented, the molecular mechanisms underlying SCW formation coordinated with distinct cellular physiological processes during plant adaptive growth remain largely unclear. Here, we report the identification of Cellulose Synthase co-expressed Kinase1 (CSK1), which encodes a receptor-like cytoplasmic kinase, as a negative regulator of SCW formation and its signaling cascade in rice. Transcriptome deep sequencing of developing internodes and genome-wide co-expression assays revealed that CSK1 is co-expressed with cellulose synthase genes and is responsive to various stress stimuli. The increased SCW thickness and vigorous vessel transport in csk1 indicate that CSK1 functions as a negative regulator of SCW biosynthesis. Through observation of green fluorescent protein-tagged CSK1 in rice protoplasts and stable transgenic plants, we found that CSK1 is localized in the nucleus and cytoplasm adjacent to the plasma membrane. Biochemical and molecular assays demonstrated that CSK1 phosphorylates VASCULAR-RELATED NAC-DOMAIN 6 (VND6), a master SCW-associated transcription factor, in the nucleus, which reduces the transcription of a suite of SCW-related genes, thereby attenuating SCW accumulation. Consistently, genetic analyses show that CSK1 functions upstream of VND6 in regulating SCW formation. Interestingly, our physiological analyses revealed that CSK1 and VND6 are involved in abscisic acid-mediated regulation of cell growth and SCW deposition. Taken together, these results indicate that the CSK1–VND6 module is an important component of the SCW biosynthesis machinery, which coordinates SCW accumulation and adaptive growth in rice. Our study not only identifies a new regulator of SCW biosynthesis but also reveals a fine-tuned mechanism for precise control of SCW deposition, offering tools for rationally tailoring agronomic traits.

Auxin inhibits lignin and cellulose biosynthesis in stone cells of pear fruit via the PbrARF13-PbrNSC-PbrMYB132 transcriptional regulatory cascade.

Stone cells are often present in pear fruit, and they can seriously affect the fruit quality when present in large numbers. The plant growth regulator NAA, a synthetic auxin, is known to play an active role in fruit development regulation. However, the genetic mechanisms of NAA regulation of stone cell formation are still unclear. Here, we demonstrated that exogenous application of 200 μM NAA reduced stone cell content and also significantly decreased the expression level of PbrNSC encoding a transcriptional regulator. PbrNSC was shown to bind to an auxin response factor, PbrARF13. Overexpression of PbrARF13 decreased stone cell content in pear fruit and secondary cell wall (SCW) thickness in transgenic Arabidopsis plants. In contrast, knocking down PbrARF13 expression using virus-induced gene silencing had the opposite effect. PbrARF13 was subsequently shown to inhibit PbrNSC expression by directly binding to its promoter, and further to reduce stone cell content. Furthermore, PbrNSC was identified as a positive regulator of PbrMYB132 through analyses of co-expression network of stone cell formation-related genes. PbrMYB132 activated the expression of gene encoding cellulose synthase (PbrCESA4b/7a/8a) and lignin laccase (PbrLAC5) binding to their promotors. As expected, overexpression or knockdown of PbrMYB132 increased or decreased stone cell content in pear fruit and SCW thickness in Arabidopsis transgenic plants. In conclusion, our study shows that the 'PbrARF13-PbrNSC-PbrMYB132' regulatory cascade mediates the biosynthesis of lignin and cellulose in stone cells of pear fruit in response to auxin signals and also provides new insights into plant SCW formation.