Molecular Mechanism Of Formation And Regulation Of Red Leaf Traits In Cotton(Gossypium Hirsutum L.)

Objective: Anthocyanins are naturally water-soluble pigments.The accumulation and distribution of anthocyanins not only provide flowers or fruits with colorful colors and rich patterns to attract pollinators and seed dispersers,and protect plants from abiotic stresses such as ultraviolet,cold,salt and drought,as well as biological stresses of bacteria,insects and herbivores.In addition,anthocyanins are widely used in the food and medical industries for their antioxidant and anti-mutagenic properties.To data,biosynthesis and regulation of anthocyanins have been widely studied in model plants and horticultural plants,while less research has been reported in cotton.Our research group discovered a red-leaf germplasm resource of Upland cotton(named HY)during the introduction of cotton germplasm resources in the early stage.The preliminary study found that the formation of red leaf phenotype of this germplasm was caused by anthocyanin accumulation and was induced by light.Therefore,this study was conducted to investigate the mechanism of anthocyanin synthesis and regulation in Upland cotton based on HY material,and to provide a theoretical basis for cotton stress resistance research and natural coloured cotton breeding.Methods: Identification of candidate genes responsible for the red leaf phenotype of HY using bulked segregant analysis(BSA-seq),gene function annotation and clonal sequencing.Candidate genes were validated by VIGS,transgenic overexpression and gene editing techniques.A comparative transcriptome(RNA-seq)approach to analyse the effects of different light qualities on anthocyanin biosynthesis and regulatory genes in HY plants,and to reveal the regulatory network of anthocyanin biosynthesis in Upland cotton by GO,KEGG and GSEA analysis.We performed genome-wide identification and bioinformatics analysis of CRY,HY5 and GST gene families in Upland cotton,and CRY,HY5 and GST genes involved in anthocyanin accumulation and regulation were validated by VIGS,or dual luciferase,GUS tissue staining,transient overexpression and Arabidopsis mutant complementation assays.Results and conclusions:(1)The red-leaf phenotype of HY was caused by anthocyanin accumulation;the candidate gene were located on chromosome D07 using bulked segregant analysis(BSA-seq)with a candidate interval size of 8.12 Mb.A MYB113 transcription factor(Gh RLC1/GH＿D07G0852)was selected as the main candidate gene of red-leaf germplasm(HY)in cotton by gene annotation.Further sequencing revealed a 227 bp tandem repeat and a 1 bp insertion in the promoter of Gh RLC1,which is tentatively believed to be the main cause of the red leaf phenotype.The promoter activity assay showed that the minimum active fragment of the gene promoter was 455 bp.Both transient and stable overexpression of Gh RLC1 in green-leaf cotton can upregulated the structural genes involved in anthocyanin biosynthesis,causing green tissues to accumulate anthocyanins and turn red.The knockdown of Gh RLC1 expression by VIGS and gene editing techniques was followed by a decrease in the expression of structural genes in the anthocyanin biosynthesis pathway as well as anthocyanin content in HY leaves.Furthermore,hybridization experiments of knockout lines and overexpression lines confirmed that Gh RLC1 regulates anthocyanin biosynthesis of cotton in a dose-dependent manner.Taken together,these results suggest that the repetitive segment(227 bp duplication and 1 bp insertion)on the Gh RLC1 promoter is responsible for the overexpression of the gene,the activation of the anthocyanin synthesis pathway,and the emergence of the anthocyanin accumulation phenotype.(2)GO,KEGG and GSEA analyses showed that 6838 non-redundant differentially expressed genes(DEGs)from the three comparison groups were mainly distributed in the "circadian rhythm","phenylpropanoid biosynthesis","anthocyanin biosynthesis" and "flavonoid and flavonol biosynthesis" pathways.Genes involved in light signal sensing and transduction(PHY,CRY,HY5),early anthocyanin biosynthesis(PAL,C4 H,4CL,CHI,CHS,F3 H,F3’H,F3’5’H),late anthocyanin biosynthesis(DFR,ANS,UFGT)and anthocyanin transport(GST,ABC,MATE)were induced by red or blue light,increasing the HY plant’s anthocyanin content.Although the induction of anthocyanin biosynthesis and regulatory genes was significantly stronger with red light than with blue light,more anthocyanins could be accumulated under the induction of blue light,while the effect of red light on the accumulation of proanthocyanidins was more significant.(3)A total of 10 Gh CRY genes were identified by genome-wide analysis of Upland cotton.They were divided into three categories: Gh CRY1,Gh CRY2 and Gh CRY-DASH,based on their conserved domains and phylogenetic tree analysis.Promoter analysis showed that the most abundant cis-acting elements among the 10 Gh CRY genes was light-responsive motifs,followed by MYB-binding,MYC-binding,hormone-responsive and stress-responsive motifs.In addition,expression analysis showed that Gh CRY1,Gh CRY2 and Gh CRY-DASH were highly expressed in stems,sepals and leaves,respectively,and were induced by blue light.The results of VIGS experiment showed that silencing of Gh CRY1 a and Gh CRY1 b resulted in a significant decrease in anthocyanin biosynthesis-related genes and anthocyanin content in HY plants.In addition,a gene annotated as HY5 was identified from the "circadian rhythm-plants" pathway in the KEGG enrichment analysis,which showed a down-regulation trend in the Gh CRY1-silenced plants.The phylogenetic tree and transcriptional profile tentatively identified this gene as being involved in the regulation of anthocyanin biosynthesis.After the Gh HY5 transcription level was reduced by VIGS technique,the blue light induced anthocyanin accumulation phenotype was lost in HY plants.GUS and dual-luciferase assay showed that Gh HY5 could bind to the G-box motif of the Gh RLC1 promoter,thereby driving its expression.These results suggest that Gh HY5 regulates anthocyanin accumulation in cotton by activating the expression of Gh RLC1.(4)A number of GST genes induced by red and blue light were identified in the transcriptome data.Further research on GST genes of Upland cotton found that,except for A06,a total of 100 Gh GST genes were randomly distributed on all chromosomes.Phylogenetic analysis revealed that 100 Gh GST could be divided into 10 subfamilies,among which tau(U)and Phi(F)subfamilies had the largest number of members,which was consistent with reports from other plants.Phylogenetic and transcriptional analysis of F subfamily members revealed that Gh GSTF12(GH＿A07G0814 and GH＿D07G0816)genes were involved in anthocyanin accumulation.The ectopic expression of Gh GSTF12 was found to complement leaf colour-deficient phenotype of Arabidopsis tt19 mutant.In HY plants,virus-induced silencing of Gh GSTF12 resulted in the loss of the red phenotype due to anthocyanin accumulation and did not affect the expression of structural and regulatory genes for anthocyanin biosynthesis.Transient overexpression of Gh GSTF12 only accelerated anthocyanin accumulation in HY plants under light,but had no effect on green plants.These results suggest that Gh GSTF12 is involved in anthocyanin accumulation in cotton.