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[学术文献 ] A dominant negative mutation of GhMYB25-like alters cotton fiber initiation, reducing lint and fuzz 进入全文
PLANT CELL
Cotton (Gossypium hirsutum) fibers, vital natural textile materials, are single-cell trichomes that differentiate from the ovule epidermis. These fibers are categorized as lint (longer fibers useful for spinning) or fuzz (shorter, less useful fibers). Currently, developing cotton varieties with high lint yield but without fuzz remains challenging due to our limited knowledge of the molecular mechanisms underlying fiber initiation. This study presents the identification and characterization of a naturally occurring dominant negative mutation GhMYB25-like_AthapT, which results in a reduced lint and fuzzless phenotype. The GhMYB25-like_AthapT protein exerts its dominant negative effect by suppressing the activity of GhMYB25-like during lint and fuzz initiation. Intriguingly, the negative effect of GhMYB25-like_AthapT could be alleviated by high expression levels of GhMYB25-like. We also uncovered the role of GhMYB25-like in regulating the expression of key genes such as GhPDF2 (PROTODERMAL FACTOR 2), CYCD3; 1 (CYCLIN D3; 1), and PLD (Phospholipase D), establishing its significance as a pivotal transcription factor in fiber initiation. We identified other genes within this regulatory network, expanding our understanding of the determinants of fiber cell fate. These findings offer valuable insights for cotton breeding and contribute to our fundamental understanding of fiber development. A dominant negative mutation of GhMYB25-like inhibits fuzz initiation but only slightly reduces lint initiation by compensating with higher wild-type protein level during lint cell differentiation.
[学术文献 ] Gland-specific GhVQ22 negatively regulates gland size and affects secondary metabolic accumulation in cotton 进入全文
PLANT BIOTECHNOLOGY JOURNAL
Cotton (Gossypium spp.) has evolved pigment glands (PGs) that accumulate toxic terpenoids, such as gossypol, which serve as a defence mechanism against pests (Gadelha et al., 2014). Laboratory experiments and field trials have confirmed that PGs are essential for tolerance to chewing pests in cotton (Benedict et al., 1977; Mao et al., 2007). Altering the ability of PGs to synthesize and accumulate secondary metabolites is a promising strategy for pest resistance. The discovery of PG development-related genes, such as Gl2/Gl3, CGF1 and CGF2, has preliminarily revealed the genetic mechanisms involved in PG biogenesis and the gossypol synthesis pathway (Janga et al., 2019; Ma et al., 2016). However, few studies have focused on the regulation of PG size. Here, we isolated a PG-specific valine glutamine (VQ) gene, GhVQ22 (GH_A12G0470/GH_D12G0482), regulates PG size and affects the composition and content of secondary metabolites in PGs. GhVQ22 has potential applications in novel anti-pest strategies for cotton. Comparative transcriptome analyses between the PG tissues (PGT) and the PG-adjacent tissues (PGAT) in embryos at 18 days post-anthesis (DPA) were performed through laser-capture microdissection (Figure S1). We detected 506 differentially expressed genes (DEGs) in PGT compared with PGAT (Figure 1a). The 20 DEGs with the highest fold changes were selected for preliminary gland phenotype screening by virus-induced gene silencing (Table S1). GhVQ22-silenced plants (TRV2:GhVQ22) showed significantly enlarged PGs compared to the wild-type (WT) (Figure S2a). GhVQ22 expression was barely detected in PGAT (Figure 1b) and glandless cotton Z12YW (gl2gl2gl3gl3) (Figure 1c), β-Glucuronidase (GUS) reporter expression driven by the GhVQ22 promoter was limited to PGs in stable transgenic G. hirsutum (Figure 1d). These results confirmed that GhVQ22 expression is PG-specific. The Ghvq22 mutant exhibited significant increase in PG size across most tissues (Figure 1e), and PG diameter was approximately 2.7 times larger as compared to WT (Figure S3a). In true leaves of the Ghvq22 mutant, the PG size was significantly larger than that of the WT across the pseudo-developmental trajectory (Figure 1f, Figure S3b). In Ghvq22 mutant, mature PGs exhibited 3–5 sheath cell layers, whereas in WT, there were only 1–3 sheath cell layers (Figure 1g, Figure S3c). Similarly, the cavity diameter was correspondingly increased (Figure 1h, Figure S3c), meanwhile its PG density was half as compared to WT (Figure 1i). But the PG diameter and density were significantly reduced in gl2gl2Gl3Gl3 and Gl2Gl2gl3gl3 mutants with lower GhVQ22 expression (Figure S4b–d). The total secondary metabolites extracted from Ghvq22 leaves were significantly different from the WT (Figure S5). Liquid chromatography–mass spectrometry revealed that 2193 and 1879 metabolites were up-regulated and down-regulated, respectively, in Ghvq22 mutant compared with the WT (Figure 1k). The gossypol content was decrease approximately 50% as compared to WT (Figure 1j). The kaempferol and catechin content was significantly increased and decreased compared with the WT, respectively (Figure 1l,m, Figures S6 and S7). These results suggested that GhVQ22 might regulate secondary metabolite synthesis in PGs. The regulatory relationship between GhVQ22 and PG development-related genes was investigated to determine how GhVQ22 regulates PG development. As PG development progressed with embryo development from 13 to 30 DPA, GhVQ22 expression lagged behind that of Gl2/Gl3 (Figure 1n). Gl2 and Gl3, the core factors that redundantly regulate PG development, activate target gene expression by binding to the G-box (5′-CACGTG-3′) cis-regulatory element (Lin et al., 2023). The GhVQ22 promoter contains a G-box at −234 to −229 bp (Figure S8). Through conducting an electrophoretic mobility shift assay (EMSA) (Figure 1o) and yeast one-hybrid analysis (Figure S9), we confirmed that Gl2 can bind to the GhVQ22 promoter. In addition, Gl2 could activate its transcriptional activity as indicated by a dual-luciferase reporter assay in leaves of Nicotiana benthamiana (Figure 1p). The comparative transcriptome of 18 DPA embryos revealed that 1464 and 1370 genes were up-regulated and down-regulated, respectively, in Ghvq22 (Figure S10). Gene Ontology (GO) enrichment analysis demonstrated that the up-regulated DEGs enriched in cell division, secondary metabolite and flavonoid biosynthesis (Figure 1q). The expression level of PG development-related genes, such as Gl2/Gl3, CGF1, CGF2, JUB1 and ERF105, was significantly enhanced in Ghvq22 mutant (Figures S11 and S12a). These results suggested that GhVQ22 regulation of PG development might depend on the genetic networks of Gl2/Gl3 and are involved in cell division and secondary metabolite pathway. Our hypothesis suggests that Gl2/Gl3 not only activates the expression of genes involved in PG development but also triggers the expression of the negative regulator GhVQ22 (Figure 1r). These two opposing mechanisms form a delicate balance in regulating PG development and secondary metabolite synthesis. Our study revealed that GhVQ22 as a downstream target of Gl2/Gl3, negatively regulates PG size and affects secondary metabolic accumulation. We speculate that the significant changes in PG development-related genes and gland morphogenesis might affect secondary metabolic synthesis. We also observed the stem trichome density of the Ghvq22 mutant was significantly lower than that of the WT (Figure S13b), suggesting that the PGT and PGAT might influence each other by the signal molecular communications. In general, the present results lay a foundation for further research on the regulation of PG development.
[学术文献 ] iJAZ-based approach to engineer lepidopteran pest resistance in multiple crop species 进入全文
NATURE PLANTS
The fall armyworm (FAW) poses a significant threat to global crop production. Here we showed that overexpression of jasmonate ZIM-domain (JAZ) protein GhJAZ24 confers resistance to cotton bollworm and FAW, while also causing sterility in transgenic cotton by recruiting TOPLESS and histone deacetylase 6. We identified the NGR motif of GhJAZ24 that recognizes and binds the aminopeptidase N receptor, enabling GhJAZ24 to enter cells and disrupt histone deacetylase 3, leading to cell death. To overcome plant sterility associated with GhJAZ24 overexpression, we developed iJAZ (i, induced), an approach involving damage-induced expression and a switch from intracellular to extracellular localization of GhJAZ24. iJAZ transgenic cotton maintained fertility and showed insecticidal activity against cotton bollworm and FAW. In addition, iJAZ transgenic rice, maize and tobacco plants showed insecticidal activity against their lepidopteran pests, resulting in an iJAZ-based approach for generating alternative insecticidal proteins with distinctive mechanisms of action, thus holding immense potential for future crop engineering.
[学术文献 ] Single-cell transcriptome atlas identified novel regulators for pigment gland morphogenesis in cotton 进入全文
PLANT BIOTECHNOLOGY JOURNAL
Cotton (Gossypium spp.) is a leading economic crop that is grown in more than 50 countries. The cottonseeds, once regarded as the by-product of fibre production, contain a rich supply of unsaturated fatty acids, proteins and vitamins. To date, the annual production of cottonseeds has the potential to meet the protein requirements for 550 million people globally, which shows great potential as a food resource amidst a growing food shortage (Janga et al., 2019). However, the utilization of cottonseed for food purposes is limited owing to the presence of ‘pigment glands’, which contains gossypol and its derivatives that are toxic to humans (Gao et al., 2020). To study how pigment gland cells differentiate and to reveal the gene regulatory network in gland morphogenesis, scRNA-Seq was performed using a pair of NILs (gland cotton ‘CCRI12’ and glandless cotton ‘CCRI12gl’). The 1-week-old cotyledons were enzymatically digested, and the purified protoplasts were labelled with a 10x genomics barcode for high-throughput sequencing (Figure 1a). A total of 9186 individual cells, including 4790 cells from ‘CCRI12’ and 4396 cells from ‘CCRI12gl’, were obtained after cell filtering process (Figure S1, Table S1) and were divided into 12 clusters based on highly variable genes (Figure 1b, Figure S2). To verify and correct cell group classifications, the expression profile of reported marker genes in 12 cell clusters of cotton cotyledons was studied. The clusters 0, 1 and 4 were identified as spongy mesophyll cells (SMC) due to the enrichment of a photosynthesis-related gene LHCB, and clusters 3 and 6 were identified as palisade mesophyll cells (PMC) due to the high expression of RBCS. The dominantly expressed GSTF9 marked cluster 5 as epidermal cells (EPC), and GSTL3 identified cluster 10 as the primordial cells (PRC) that could differentiate. In addition, cell type that specifically expressed MYB44, PXY, LTP and CYP82A3 marked the clusters 2, 7, 8 and 11 as guard cells (GC), xylem cells (XC), parenchyma cells (PAC) and phloem cells (PHC), respectively (Figures 1c,d, Table S2). No well-known marker gene for pigment gland cells has been reported to date. GoPGF is the key factor that controls the biogenesis of pigment glands (Ma et al., 2016). However, the expression of GoPGF in different cell types has not been studied. Cluster 9 was identified in the cotyledons of gland cotton ‘CCRI12’ but not glandless cotton ‘CCRI12gl’, and GoPGF was specifically detected in the cells of cluster 9. This led to the tentative annotation of cluster 9 of the cotyledon cells as pigment gland cells (PGC; Figure 1c). A pseudotime analysis was performed to uncover the differentiation relationships of cotyledon cell types. The study of the individual cell distribution and trajectory revealed that the PRCs originated earlier than the PGCs, suggesting that the PGCs could have differentiated from the PRCs (Figure 1e, Figure S3). In addition, four representative genes were selected to show their distribution and expression levels in PRCs and PGCs (Figure 1f). To explore the potential regulators of gland development of cotton, the highly expressed genes in each cell cluster were studied. A total of 9325 DEGs were obtained with 1430 DEGs preferentially expressed in PGC, while the other cell clusters contained a range from 572 to 1704 (Table S3). Other than GoPGF, the previously reported GhERF105 (Wu et al., 2021), which determines the biogenesis of pigment glands in cotton leaves, was also identified as PGC-specific gene in our scRNA-Seq data. These results suggested the reliability of scRNA-Seq analyses in pigment gland cells and confirmed the accuracy of our classification of cell types. To date, the regulators involving in pigment gland biogenesis that have been identified are all TFs, including CGF1, CGF2, GoPGF/CGF3, GaGRAS/GoSPGF and GhERF105. Therefore, this study focused on the TFs that were preferentially expressed in PGCs (Figure 1g, Table S4). qPCR revealed that most of the identified TFs were highly expressed in gland cells, while they were expressed at very low levels in the mesophyll cells (Figure S4). In addition, five candidate genes, including GoPGF, were selected for RNA in situ hybridization. These results showed that these genes have strong hybridization signals in the glandular structure (Figure 1h). A 1.5-kb promoter upstream of the GoPGF initiation codon was cloned to drive the expression of GUS in the cotton gland cultivar ‘Coker312’. The transgenic lines that expressed GUS were obtained and used for histochemical staining. As shown in Figure 1i, a strong and clear GUS staining was restricted to the pigment glands. To our knowledge, this study is the first to use GUS staining to demonstrate that the transcription of GoPGF is restricted to gland cells. Virus-induced gene silencing was utilized to quickly screen the candidate genes that controlled the formation of pigment glands. The results suggested that knock down of some candidate genes, including ERF13 and MYB14, mildly reduced the gland density (Figure S5). Notably, the GH_A05G3906 could modulate the contents of gossypol without changing the number of pigment glands, which suggested a possible biosynthetic pathway of sesquiterpene metabolism that is independent of pigment gland biogenesis (Figure S5). Among all the candidates, JUNGBRUNNEN 1 (GhJUB1) is of particular interest. Knock down of GhJUB1 inhibits gland biogenesis and the accumulation of gossypol. The GhJUB1-silenced plants (TRV:JUB1) exhibited dramatically reduced pigment glands in newly growing tissues (Figure 1j–l). In addition, GhJUB1-silenced cotton plants exhibited gossypol levels of 15% in the leaves and 18% in the stems compared with those of the control plants (Figure 1m). These results revealed that GhJUB1 regulates gland morphogenesis, which was similar to that of GoPGF. To study the relation between GhJUB1 and GoPGF, the expression of GhJUB1 was studied in the GoPGF-silenced cotton (TRV:PGF), and the results showed that the expression of GhJUB1 dramatically decreased to an undetectable level (Figure 1n), which suggests that GhJUB1 could be downstream of GoPGF to control the biogenesis of pigment glands. The pigment gland of cotton is a highly distinctive structure, which provides an ideal system to study cell differentiation and organogenesis. Our study indicates that the initiation of cell differentiation of pigment glands is highly correlated with the specific expression of key genes. One of the major constraints in the study of glandular development of cotton is the lack of natural glandless mutants. The scRNA-Seq data that we provide is invaluable for producing novel glandless mutants, which will greatly accelerate the breeding of commercially desired cotton varieties with glandless seeds.
[学术文献 ] VILLIN2 regulates cotton defense against Verticillium dahliae by modulating actin cytoskeleton remodeling 进入全文
PLANT PHYSIOLOGY
The active structural change of actin cytoskeleton is a general host response upon pathogen attack. This study characterized the function of the cotton (Gossypium hirsutum) actin-binding protein VILLIN2 (GhVLN2) in host defense against the soilborne fungus Verticillium dahliae. Biochemical analysis demonstrated that GhVLN2 possessed actin-binding, -bundling, and -severing activities. A low concentration of GhVLN2 could shift its activity from actin bundling to actin severing in the presence of Ca2+. Knockdown of GhVLN2 expression by virus-induced gene silencing reduced the extent of actin filament bundling and interfered with the growth of cotton plants, resulting in the formation of twisted organs and brittle stems with a decreased cellulose content of the cell wall. Upon V. dahliae infection, the expression of GhVLN2 was downregulated in root cells, and silencing of GhVLN2 enhanced the disease tolerance of cotton plants. The actin bundles were less abundant in root cells of GhVLN2-silenced plants than in control plants. However, upon infection by V. dahliae, the number of actin filaments and bundles in the cells of GhVLN2-silenced plants was raised to a comparable level as those in control plants, with the dynamic remodeling of the actin cytoskeleton appearing several hours in advance. GhVLN2-silenced plants exhibited a higher incidence of actin filament cleavage in the presence of Ca2+, suggesting that pathogen-responsive downregulation of GhVLN2 could activate its actin-severing activity. These data indicate that the regulated expression and functional shift of GhVLN2 contribute to modulating the dynamic remodeling of the actin cytoskeleton in host immune responses against V. dahliae. The regulated expression and subsequent functional shift of VILLIN2 from actin bundling to severing contribute to the modulation of cotton immune responses against Verticillium dahliae.
[学术文献 ] Attenuation of ethylene signaling increases cotton resistance to a defoliating strain of Verticillium dahliae 进入全文
CROP JOURNAL
The severity of Verticillium wilt on cotton caused by defoliating strains of Verticillium dahliae has gradu-ally increased and threatens production worldwide. Identification of the molecular components of leaf defoliation may increase cotton tolerance to V. dahliae. Ethylene, a major player in plant physiological processes, is often associated with senescence and defoliation of plants. We investigated the cotton-V. dahliae interaction with a focus on the role of ethylene in defoliation and defense against V. dahliae. Cotton plants inoculated with V. dahliae isolate V991, a defoliating strain, accumulated more ethylene and showed increased disease symptoms than those inoculated with a non-defoliating strain. In cotton with a transiently silenced ethylene synthesis gene (GhACOs) and signaling gene (GhEINs) during cot-ton-V. dahliae interaction, ethylene produced was derived from cotton and more ethylene increased cot-ton susceptibility and defoliation rate. Overexpression of AtCTR1, a negative regulator in ethylene signaling, in cotton reduced sensitivity to ethylene and increased plant resistance to V. dahliae. Collectively, the results indicated precise regulation of ethylene synthesis or signaling pathways improve cotton resistant to Verticillium wilt.
