Research On The Transcriptional Regulation Of Phytochrome Interacting Factor CsPIF3a On Key Genes Of Chlorophyll Biosynthesis In Camellia Sinensis

Tea plant [Camellia sinensis(L.)O.Kuntze] is an important perennial woody plant,which is rich in active ingredients beneficial to human health,and has special drinking and economic value.With the deciphering of the whole tea plant genome and the development of modern biotechnology,the high amino acid tea cultivars with different leaf colors have attracted extensive attention due to their unique colors and diverse health attributes.Currently,albino tea varieties have been widely studied because of their high amino acid content,while little research has been conducted on green-leaf high amino acid tea cultivars.Genes related to chlorophyll accumulation in tea leaves of different leaf colors have been reported,while the underlying transcriptional regulation of these genes remains unclear.In this study,one bud and two leaves of ‘Baojing Huangjincha 1’(BJ1),‘Huangjincha2’(HJC2),‘Baiye 1’(BY1),and ‘Huangjinya’(HJY),which are high amino acid tea cultivars with different leaf colors,were used as test materials,and the leaf phenotype,content of lipid-soluble pigments and biochemical component,leaf anatomical structure,and chloroplast ultrastructure were analyzed.Based on RNA-Seq data,key genes and transcription factors related to leaf color were screened,focusing on phytochrome interacting factors(PIFs).The bioinformatics and expression patterns of the identified Cs PIFs transcription factor were analyzed.The transcriptional regulation of Cs PIF3 a on the key genes in chlorophyll biosynthesis was deeply studied by molecular biological methods such as tobacco transient expression system,electrophoretic mobility shift assay(EMSA),and heterologous overexpression.The main findings are as follows:1.The leaf color of HJC2,BJ1,BY1,and HJY were dark-green(purplish),light-green,milky-white,and yellow,respectively.The content of chlorophyll from high to low was:HJC2>BJ1>BY1>HJY,and the content of carotenoids from high to low was:HJC2>BJ1>HJY>BY1.The ratio of carotenoids to total chlorophyll was significantly higher in albino tea cultivars(BY1 and HJY)than in green tea cultivars(BJ1 and HJC2),especially the highest in HJY.The contents of tea polyphenols and total catechins from high to low were as follows: BJ1>BY1>HJY>HJC2.The free amino acid content of HJC2 was the highest,followed by BY1,while the content of HJY was the highest at the early stage of shoot development and then decreased.The content of caffeine was lowest in BY1,while the content of soluble sugar was highest.The leaf,main vein,upper and lower epidermis,and palisade tissue of green-leaf tea cultivars(BJ1 and HJC2)were significantly thicker than those of albino tea cultivars(BY1 and HJY).Among them,the leaf and main veins of HJC2 were the thickest,and the palisade tissue cells were closely arranged.The chloroplast ultrastructure of BJ1 and HJC2 was normal,but the chloroplast ultrastructure of BY1 and HJY was defective,and chloroplast cavitation appeared.BY1 had significantly less granular lamellae,while HJY had almost no granular lamella stacking.2.The transcriptome libraries of BY1,HJY,BJ1,and HJC2 were constructed using RNA-Seq technology.Through counting the number of differentially expressed genes(DEGs),it was found that the number of DEGs in the comparative combinations of "HJY vs.HJC2" and "BY1 vs.HJC2" was much more than that in "BJ1 vs.HJC2".A total of 3246 common DEGs were obtained from the transcriptome data.The results of GO and KEGG enrichment analysis indicated that DEGs were mainly involved in amino acid biosynthesis,photosynthesis,and chlorophyll metabolism pathways.The expression pattern of DEGs showed that most genes involved in photosynthesis,photosynthetic carbon fixation and chlorophyll metabolism pathways were significantly higher in green tea leaves than in albino tea leaves,among which Cs HEMA and Cs POR were significantly differentially expressed key genes in chlorophyll metabolism pathways.The prediction results of protein-protein interaction network revealed that the chlorophyll biosynthesis-related proteins Cs HEMA(TEA026253),Cs HEMB(TEA023791),Cs HEMC(TEA022286)and Cs HEME(TEA016019)had strong interactions.Three types of transcription factors with more differential expression were screened in the comparative combinations of “HJY vs.HJC2”,“BY1 vs.HJC2” and “BJ1 vs.HJC2”: AP2,MYB,and b HLH,suggesting that these transcription factors were potential factors regulating chlorophyll metabolism.3.Combined with the tea plant genome and transcriptome data,a total of seven Cs PIFs genes were identified and divided into four groups,and their physiological and biochemical properties,amino acid sequences,evolutionary relationships,and molecular characteristics were further characterized.All Cs PIFs contain APB and HLH domains,only Cs PIF1,Cs PIF3 a and Cs PIF3 b contained APA domain.Cs PIFs within the same group share highly similar gene structures and conserved motifs.Phylogenetic analysis showed that Cs PIFs in tea plants were more closely related to homologous proteins in Populus tremula and Theobroma cacao.The expression profiles and correlation analysis of Cs PIFs and chlorophyll metabolism-related genes in tea cultivars with different leaf colors indicated that Cs PIFs were closely related to the chlorophyll content in tea leaves.The DNA-binding sites for Cs PIFs were abundant in promoter regions of structural genes related to chlorophyll metabolism.Protein-protein interaction networks showed that Cs PIFs could interact with phytochromes,PIF4,HY5,TOC1,COP1 and other proteins.These findings lay the foundation for better exploring the potential biological functions of Cs PIFs in tea plants.4.The full-length coding region of Cs PIF3 a and the promoter sequences of key chlorophyll biosynthesis genes Cs HEMA and Cs POR were successfully cloned from tea leaves.Subcellular localization revealed that Cs PIF3 a was a nuclear-localized protein.In vivo activation assays in yeast cells and tobacco leaves indicated that Cs PIF3 a possesses transcriptional activation.The CsPIF3a-GST fusion protein was obtained by prokaryotic expression and protein purification.The results of EMSA assay showed that Cs PIF3 a could bind to Cs HEMA-Probe and Cs POR-Probe in vitro.The dual-luciferase reporter gene assay system analysis showed that Cs PIF3 a could activate the transcriptional activities of Cs HEMA and Cs POR promoters in tobacco leaves.The p BWA(V)BS-Cs PIF3 a overexpression vector was successfully constructed and transformed into Arabidopsis thaliana to obtain the CsPIF3a-overexpression line.Compared with wild-type Arabidopsis thaliana,CsPIF3a-OE leaves had darker green color and higher chlorophyll content,and the At HEMA,At HEME,At CHLI,and At PORC genes involved in chlorophyll biosynthesis were significantly up-regulated.The above results indicate that Cs PIF3 a,as an important regulator of chlorophyll biosynthesis in tea leaves,can positively activate the expression of chlorophyll biosynthesis-related genes,thereby inducing the accumulation of chlorophyll in tea leaves.