Gland-specific GhVQ22 negatively regulates gland size and affects secondary metabolic accumulation in cotton
棉花腺体特异性GhVQ22负调控腺体大小并影响次生代谢积累
- 关键词：
- 来源：
- PLANT BIOTECHNOLOGY JOURNAL
- 类型：
- 学术文献
- 语种：
- 英语
- 原文发布日期：
- 2024-05-10
- 摘要：
- 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.
- 所属专题：
- 171
