C2H2 zinc finger protein PagIDD15A regulates secondary wall thickening and lignin biosynthesis in poplar.

Wood production is largely determined by the activity of cambial cell proliferation, and the secondary cell wall (SCW) thickening of xylem cells determines the wood property. In this study, we identified an INDETERMINATE DOMAIN (IDD) type C2H2 zinc finger transcription factor PagIDD15A as a regulator of wood formation in Populus alba × Populus glandulosa. Downregulation of PagIDD15A expression by RNA interference (RNAi) inhibited xylem development and xylem cell secondary wall thickening. RNA-seq analysis showed that PagPAL1, PagCCR2 and PagCCoAOMT1 were downregulated in the differentiating xylem of the PagIDD15A-RNAi transgenic plants, showing that PagIDD15A may regulate SCW biosynthesis through inhibiting lignin biosynthesis. The downregulation of PagVND6-B2, PagMYB10 and PagMYC4 and upregulation of PagWRKY12 in the differentiating xylem of RNAi transgenic plants suggest that PagIDD15A may also regulate these transcription factor (TF) genes to affect SCW thickening. RT-qPCR analysis in the phloem-cambium of RNAi transgenic demonstrates that PagIDD15A may regulate the expression of the genes associated with cell proliferation, including, PagSHR (SHORTROOT), PagSCR (SCARECROW), PagCYCD3;1 (CYCLIN D3;1) and PagSMR4 (SIAMESE-RELATED4), to affect the cambial activity. This study provides the knowledge of the IDD-type C2H2 zinc finger protein in regulating wood formation.

Tubulin participates in establishing protoxylem vessel reinforcement patterns and hydraulic conductivity in maize.

Water transportation to developing tissues relies on the structure and function of plant xylem cells. Plant microtubules govern the direction of cellulose microfibrils and guide secondary cell wall formation and morphogenesis. However, the relevance of microtubule-determined xylem wall thickening patterns in plant hydraulic conductivity remains unclear. In the present study, we identified a maize (Zea mays) semi-dominant mutant, designated drought-overly-sensitive1 (ZmDos1), the upper leaves of which wilted even when exposed to well-watered conditions during growth; the wilting phenotype was aggravated by increased temperatures and decreased humidity. Protoxylem vessels in the stem and leaves of the mutant showed altered thickening patterns of the secondary cell wall (from annular to spiral), decreased inner diameters, and limited water transport efficiency. The causal mutation for this phenotype was found to be a G-to-A mutation in the maize gene α-tubulin4, resulting in a single amino acid substitution at position 196 (E196K). Ectopic expression of the mutant α-tubulin4 in Arabidopsis (Arabidopsis thaliana) changed the orientation of microtubule arrays, suggesting a determinant role of this gene in microtubule assembly and secondary cell wall thickening. Our findings suggest that the spiral wall thickenings triggered by the α-tubulin mutation are stretched during organ elongation, causing a smaller inner diameter of the protoxylem vessels and affecting water transport in maize. This study underscores the importance of tubulin-mediated protoxylem wall thickening in regulating plant hydraulics, improves our understanding of the relationships between protoxylem structural features and functions, and offers candidate genes for the genetic enhancement of maize.

GhMYB30-GhMUR3 affects fiber elongation and secondary wall thickening in cotton.

Xyloglucan, an important hemicellulose, plays a crucial role in maintaining cell wall structure and cell elongation. However, the effects of xyloglucan on cotton fiber development are not well understood. GhMUR3 encodes a xyloglucan galactosyltransferase that is essential for xyloglucan synthesis and is highly expressed during fiber elongation. In this study, we report that GhMUR3 participates in cotton fiber development under the regulation of GhMYB30. Overexpression GhMUR3 affects the fiber elongation and cell wall thickening. Transcriptome showed that the expression of genes involved in secondary cell wall synthesis was prematurely activated in OE-MUR3 lines. In addition, GhMYB30 was identified as a key regulator of GhMUR3 by Y1H, Dual-Luc, and electrophoretic mobility shift assay (EMSA) assays. GhMYB30 directly bound the GhMUR3 promoter and activated GhMUR3 expression. Furthermore, DAP-seq of GhMYB30 was performed to identify its target genes in the whole genome. The results showed that many target genes were associated with fiber development, including cell wall synthesis-related genes, BR-related genes, reactive oxygen species pathway genes, and VLCFA synthesis genes. It was demonstrated that GhMYB30 may regulate fiber development through multiple pathways. Additionally, GhMYB46 was confirmed to be a target gene of GhMYB30 by EMSA, and GhMYB46 was significantly increased in GhMYB30-silenced lines, indicating that GhMYB30 inhibited GhMYB46 expression. Overall, these results revealed that GhMUR3 under the regulation of GhMYB30 and plays an essential role in cotton fiber elongation and secondary wall thickening. Additionally, GhMYB30 plays an important role in the regulation of fiber development and regulates fiber secondary wall synthesis by inhibiting the expression of GhMYB46.

FvNST1b NAC Protein Induces Secondary Cell Wall Formation in Strawberry.

Secondary cell wall thickening plays a crucial role in plant growth and development. Diploid woodland strawberry (Fragaria vesca) is an excellent model for studying fruit development, but its molecular control of secondary wall thickening is largely unknown. Previous studies have shown that Arabidopsis NAC secondary wall thickening promoting factor1 (AtNST1) and related proteins are master regulators of xylem fiber cell differentiation in multiple plant species. In this study, a NST1-like gene, FvNST1b, was isolated and characterized from strawberry. Sequence alignment and phylogenetic analysis showed that the FvNST1b protein contains a highly conserved NAC domain, and it belongs to the same family as AtNST1. Overexpression of FvNST1b in wild-type Arabidopsis caused extreme dwarfism, induced ectopic thickening of secondary walls in various tissues, and upregulated the expression of genes related to secondary cell wall synthesis. In addition, transient overexpression of FvNST1b in wild-type Fragaria vesca fruit produced cells resembling tracheary elements. These results suggest that FvNST1b positively regulates secondary cell wall formation as orthologous genes from other species.

Same Locus for Non-shattering Seed Pod in Two Independently Domesticated Legumes, Vigna angularis and Vigna unguiculata.

Loss of pod shattering is one of the most important domestication-related traits in legume crops. The non-shattering phenotypes have been achieved either by disturbed formation of abscission layer between the valves, or by loss of helical tension in sclerenchyma of endocarp, that split open the pods to disperse the seeds. During domestication, azuki bean (Vigna angularis) and yard-long bean (Vigna unguiculata cv-gr. Sesquipedalis) have reduced or lost the sclerenchyma and thus the shattering behavior of seed pods. Here we performed fine-mapping with backcrossed populations and narrowed the candidate genomic region down to 4 kbp in azuki bean and 13 kbp in yard-long bean. Among the genes located in these regions, we found MYB26 genes encoded truncated proteins in azuki bean, yard-long bean, and even cowpea. As such, our findings indicate that independent domestication on the two legumes has selected the same locus for the same traits. We also argue that MYB26 could be a target gene for improving shattering phenotype in other legumes, such as soybean.

Modulation of NAC transcription factor NST1 activity by XYLEM NAC DOMAIN1 regulates secondary cell wall formation in Arabidopsis.

In Arabidopsis, secondary cell walls (SCW) are formed in fiber cells and vessel cells in vascular tissue for providing plants with mechanical strength and channels for the long distance transportation of water and nutrients. NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) acts as a key gene for the initiation of SCW formation through a hierarchical transcription network. In this study, we report that NST activity is modulated by the NAC domain transcription factor XYLEM NAC DOMAIN1 (XND1) during plant growth. Using yeast two-hybrid screening and in vivo protein interaction analysis, XND1 was identified as an NST-interacting protein that modulates NST1 activity. XND1 and NST1 were co-localized in the nucleus and the interaction of XND1 with NST1 resulted in inhibition of NST1 transactivation activity. In the process of inflorescence growth, XND1 was expressed with a similar pattern to NST1. Up-regulation of XND1 in fiber cells repressed SCW formation. The study demonstrates that NST1 activity is modulated by XND1 in the regulation of secondary cell walls formation.

Knockdown of Arabidopsis ROOT UVB SENSITIVE4 Disrupts Anther Dehiscence by Suppressing Secondary Thickening in the Endothecium.

ROOT UV-B SENSITIVE4 (RUS4) encodes a protein with no known function that contains a conserved Domain of Unknown Function 647 (DUF647). The DUF647-containing proteins RUS1 and RUS2 have previously been associated with root UV-B-sensing pathway that plays a major role in Arabidopsis early seedling morphogenesis and development. Here, we show that RUS4 knockdown Arabidopsis plants, referred to as amiR-RUS4, were severely reduced in male fertility with indehiscent anthers. Light microscopy of anther sections revealed a significantly reduced secondary wall thickening in the endothecium of amiR-RUS4 anthers. We further show that the transcript abundance of the NAC domain genes NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST2, which have been shown to regulate the secondary cell wall thickenings in the anther endothecium, were dramatically reduced in the amiR-RUS4 floral buds. Expression of the secondary cell wall-associated MYB transcription factor genes MYB103 and MYB85 were also strongly reduced in floral buds of the amiR-RUS4 plants. Overexpression of RUS4 led to increased secondary thickening in the endothecium. However, the rus4-2 mutant exhibited no obvious phenotype. Promoter-GUS analysis revealed that the RUS4 promoter was highly active in the anthers, supporting its role in anther development. Taken together, these results suggest that RUS4, probably functions redundantly with other genes, may play an important role in the secondary thickening formation in the anther endothecium by indirectly affecting the expression of secondary cell wall biosynthetic genes.

TCP24 modulates secondary cell wall thickening and anther endothecium development.

miR319-targeted TCP genes are believed to regulate cell division in leaves and floral organs. However, it remains unknown whether these genes are involved in cell wall development. Here, we report that TCP24 negatively regulates secondary wall thickening in floral organs and roots. The overexpression of the miR319a-resistant version of TCP24 in Arabidopsis disrupted the thickening of secondary cell walls in the anther endothecium, leading to male sterility because of arrested anther dehiscence and pollen release. Several genes linked to secondary cell wall biogenesis and thickening were down-regulated in these transgenic plants. By contrast, the inhibition of TCP24 using the ectopic expression of a TCP24-SRDX repressor fusion protein, or the silencing of TCP genes by miR319a overexpression, increased cell wall lignification and the enhanced secondary cell wall thickening. Our results suggest that TCP24 acts as an important regulator of secondary cell wall thickening and modulates anther endothecium development.

Arabidopsis C3H14 and C3H15 have overlapping roles in the regulation of secondary wall thickening and anther development.

Plant tandem CCCH zinc finger (TZF) proteins play diverse roles in developmental and adaptive processes. Arabidopsis C3H14 has been shown to act as a potential regulator of secondary wall biosynthesis. However, there is lack of direct evidence to support its functions in Arabidopsis. It is demonstrated here that C3H14 and its homologue C3H15 redundantly regulate secondary wall formation and that they additionally function in anther development. Plants with double, but not single, T-DNA mutants for C3H14 or C3H15 have few pollen grains and thinner stem secondary walls than the wild type. Plants homozygous for c3h14 and heterozygous for c3h15 [c3h14 c3h15(±)] have slightly thinner secondary walls than plants heterozygous for c3h14 and homozygous for c3h15 [c3h14(±) c3h15], and c3h14(±) c3h15 have lower fertility. Overexpression of C3H14 or C3H15 led to increased secondary wall thickness in stems and the ectopic deposition of secondary walls in various tissues, but did not affect anther morphology. Transcript profiles from the C3H14/15 overexpression and c3h14 c3h15 plants revealed marked changes in the expression of many genes associated with cell wall metabolism and pollen formation. Subcellular localization and biochemical analyses suggest that C3H14/15 might function at both the transcriptional and post-transcriptional levels.

R2R3-MYB gene pairs in Populus: evolution and contribution to secondary wall formation and flowering time.

In plants, the R2R3-MYB gene family contains many pairs of paralogous genes, which play the diverse roles in developmental processes and environmental responses. The paper reports the characterization of 81 pairs of Populus R2R3-MYB genes. Chromosome placement, phylogenetic, and motif structure analyses showed that these gene pairs resulted from multiple types of gene duplications and had five different gene fates. Tissue expression patterns revealed that most duplicated genes were specifically expressed in the tissues examined. qRT-PCR confirmed that nine pairs were highly expressed in xylem, of which three pairs (PdMYB10/128, PdMYB90/167, and PdMYB92/125) were further functionally characterized. The six PdMYBs were localized to the nucleus and had transcriptional activities in yeast. The heterologous expression of PdMYB10 and 128 in Arabidopsis increased stem fibre cell-wall thickness and delayed flowering. In contrast, overexpression of PdMYB90, 167, 92, and 125 in Arabidopsis decreased stem fibre and vessel cell-wall thickness and promoted flowering. Cellulose, xylose, and lignin contents were changed in overexpression plants. The expression levels of several genes involved in secondary wall formation and flowering were affected by the overexpression of the six PdMYBs in Arabidopsis. This study addresses the diversity of gene duplications in Populus R2R3-MYBs and the roles of these six genes in secondary wall formation and flowering control.