Identification And Functional Analysis Of Light-induced Genes Involved In Anthocyanin Biosynthesis In Purple Grain Wheat

Anthocyanins are members of the flavonoid class of secondary metabolites and broadly accumulate in land plants.Due to their potent antioxidant and free-radical-scavenging properties,anthocyanins are believed to provide protection against damage caused by biotic and abiotic stressors in plants.Furthermore,they are beneficial for human health.Purplegrained wheat varieties accumulate anthocyanins in the pericarp,and thus they are considered as important germplasm sources in crop breeding.Previous studies have shown that light plays an essential role in anthocyanin biosynthesis in purple-grained wheat.However,the regulatory mechanisms underlying light-induced anthocyanin accumulation in the pericarps of purplegrained wheat varieties are still unknown.Therefore,it is important to identify the genes involved in light-induced anthocyanin biosynthesis in the pericarps of purple-grained wheat and characterize their functions.ELONGATED HYPOCOTYL5(HY5),a basic leucine zipper(b ZIP)protein,is a master regulator of photomorphogenesis and plays a key role in the connection between light and anthocyanin biosynthesis.The direct interaction between HY5 and the promoters of many genes involved in anthocyanin biosynthesis,particularly R2R3-MYB genes,has been reported in many plants.However,HY5 proteins usually lack a trans-activation domain and they may require additional cofactors to fine-tune their activity.Among these cofactors,the B-BOX(BBX)proteins are the most well-known.In Arabidopsis,members of the IV subfamily of BBX are closely related to anthocyanin biosynthesis.However,their functions and regulatory mechanisms in the light-induced anthocyanin biosynthesis in the pericarps of purple-grained wheat are still unclear.In this paper,the purple-grained wheat variety ‘Heixiaomai 76’ was used as the experimental material.First,the regulatory factors affecting the biosynthesis of anthocyanins in the pericarps were analyzed through direct exposure and shading treatment of the seeds.Then,based on transcriptome data from pericarps treated with different light conditions,key pathways and candidate genes involved in light-induced anthocyanin biosynthesis in the pericarps were identified,and the candidate HY5 and BBX genes were cloned and functionally characterized.The main results are as follows:1.Shading treatment of H76 spikes from 10 days after pollination(DAP)showed that there was only a small amount of anthocyanin accumulation in the late stage of grain development(26 DAP).Direct exposure of H76 with glumes and lemmas removal at 16 DAP showed that the grains of H76 were gradually turned purple after 6 h(L-6h),while the control remained light green(0h and UL-6h).These results indicate that light rapidly induces anthocyanin biosynthesis and accumulation in the pericarps of purple-grained wheat,and that the biosynthesis of anthocyanins mainly depends on light.A purple pericarp sample(L-6h)and two light green pericarp samples(0h and UL-6h)were subjected to transcriptome analysis.The results showed that the pathways with the most enriched differentially expressed genes were phenylpropanoid biosynthesis,flavonoid biosynthesis,carbon metabolism,plant hormone signal transduction,starch and sucrose metabolism,and MAPK signaling pathway.These pathways may be involved in the light-induced anthocyanin biosynthesis in the pericarps of purple-grained wheat.2.Based on transcriptome data and phylogenetic analysis,six HY5 genes(Ta HY5-6A,Ta HY5-6B,Ta HY5-6D,Ta HY5-7A,Ta HY5-7B,and Ta HY5-7D)and three BBX genes(Ta BBX-3A,Ta BBX-3B,and Ta BBX-3D)were isolated.q RT-PCR analysis showed that the expression of all these genes was significantly induced by light,consistent with the trend of anthocyanin accumulation induced by light in the pericarps of purple-grained wheat.Among them,Ta HY5-7A and Ta BBX-3B genes had the highest expression levels among their respective gene families.Furthermore,cis-element analysis showed that the HY5 promoter sequence contained multiple light-responsive cis-elements,such as the G-box and TCT-motif,which was consistent with the q RT-PCR results.3.Although single nucleotide polymorphisms(SNPs)exist in the nucleic acid sequences of Ta HY5-6A,Ta HY5-6B,and Ta HY5-6D,they share the same amino acid sequence(156amino acids).Ta HY5-7A,Ta HY5-7B,and Ta HY5-7D encode 164,166,and 164 amino acids,respectively,and share ～96% amino acid sequence identity.All four HY5 proteins possess a conserved b ZIP domain at the C-terminus and conserved amino acid motifs to the casein kinase II(CKII)phosphorylation site and the COP1 interaction site at the N-terminus.Ta BBX-3A,Ta BBX-3B,and Ta BBX-3D genes all encode 349 amino acids and share high sequence similarity(～98% identity).Structural analysis showed that they contain two conserved B-box domains(B-box I and B-box II)at their N-terminus,which is characteristic of the IV subfamily of BBX proteins.4.Subcellular localization results indicate that all the HY5 and BBX proteins localize in the nucleus.Overexpression of each of the four HY5 genes in Arabidopsis hy5 mutants resulted in partial restoration of the hy5 mutant phenotype,and overexpression of each of the three BBX genes in Arabidopsis Wassilewskija ecotype increased anthocyanin content in Arabidopsis Wassilewskija ecotype,indicating that they have a positive regulatory function in anthocyanin biosynthesis.5.Yeast self-activation assays showed that Ta HY5-6A,Ta HY5-7A,Ta HY5-7B,and Ta HY5-7D lack a trans-activation domain.Moreover,dual-luciferase assays showed that Ta HY5-7A alone is not sufficient to activate the expression of Ta Ppm1,an important R2R3-MYB regulator of anthocyanin biosynthesis in the pericarps of purple-grained wheat.These results indicate that HY5 proteins require the presence of co-factors to carry out their functions.6.Yeast two-hybrid and bimolecular fluorescence complementation assays showed that Ta HY5-6A and Ta HY5-7D can interact with all three BBX proteins,while Ta HY5-7A can interact with Ta BBX-3B and Ta BBX-3D,and Ta HY5-7B can only interact with Ta BBX-3B.These results suggest that Ta BBX-3A,Ta BBX-3B,and Ta BBX-3D may act as cofactors for HY5 proteins to function.Dual-luciferase assays using Ta HY5-7A and Ta BBX-3B as representatives showed that Ta HY5-7A and Ta BBX-3B can collaboratively activate Ta Ppm1 expression,thereby participating in the regulation of anthocyanin biosynthesis in the pericarps of purple-grained wheat.7.Transgenic Arabidopsis seedlings overexpressing BBX genes not only had increased anthocyanin content,but also exhibited a dwarf phenotype.Randomly selected Ta BBX-3A transgenic seedlings were subjected to transcriptome sequencing,and it was found that Ta BBX-3A regulates plant development by modulating the expression of genes related to plant hormone signaling pathways.Similarly,overexpression of Ta BBX-3A in the rice cultivar Nipponbare resulted in transgenic plants with a dwarf phenotype.These results suggest that BBX genes not only participate in the regulation of anthocyanin biosynthesis,but also participate in the regulation of plant height through hormone signaling pathways.