Study On The Molecular Mechanism Of ABA In Regulating Chlorophyll Degradation And Anthocyanin Biosynthesis In Pericarp Of Litchi Chinensis

Litchi(Litchi chinensis Sonn.)is a particular subtropical fruit tree widely cultivated in southern China,and has plentiful germplasm resources which varied in fruit color phenotype.Color is an important part of litchi fruit appearance quality,during litchi fruit ripening,the change of pericarp color is green-yellow-red,following the degradation of chlorophyll and biosyntheiss of anthocyanins.Litchi fruit belongs to the non-climacteric fruit,and mainly regulated by ABA during ripening.Moreover,ABA modulated the coloration of litchi pericarp strongly.To study the molecular mechanism of ABA in regulating the coloration of litchi pericarp,uneven-red cv.'Feizixiao' and well-red cv.'Guwiei' were used.By means of RNA-seq,RT-qPCR,subcellular localization,transient expresion,dual luciferase assay and Yeast One-Hybrid system,the network of LcABFs transcription factors in regulating chlorophyll degradation and anthocyanin biosynthesis in pericarp was investigated.The results from this study filled the blank of molecular mechanism of ABA in regulating color development in litchi pericarp,and provided theoretical basises for quality modifying and fruit color regulation of litchi.The main results are as follows:1.'Feizixiao' fruit was used to analyze the different expressed genes(DEGs)between pericarp of 0 d,10 d,20 d after treatmented by exogenous ABA,CPPU and control by means of RNA-seq.It was found that a total of 2263 DEGs were identified in control during litchi pericarp pigmentation,and there were 1986 DEGs at the three stages after ABA treatment,2862 DEGs were indentified after CPPU treatment.579 genes with significantly different expression patterns were found in response to exogenous ABA treatment compared to control,and 827 DEGs were found in response to exogenous CPPU treatment compared to control.The most DEGs responsed to ABA were related to cellular process,single-organism process,metabllic process,catalytic activity,with the top three pathway groups were plant-pathogen interactions,plant hormone signal transduction,flavonoid biosynthesis.The most DEGs responsed to CPPU were related to single-organism process,cellular process,biological regulation,catalytic activity,with the top three pathway groups were carbon metabolism,plant-pathogen interactions and photosynthesis.After exogenous ABA treatment,chlorophyll biosynthesis-related genes were down-regulated,chlorophyll degradation-related and most of anthocyanin biosynthesis-related genes were up-regulated.However,chlorophyll biosynthesis-related genes were up-regulated,and chlorophyll degradation-related and most of anthocyanin biosynthesis-related genes were down-regulated after exogenous CPPU treatment.2.'Guiwei' fruit was used,and the changes of fruit shape,fruit color and endogenesis ABA content were tracked from fruit set to commercial maturation.As a result,'Guiwei' fruit belong to single sigmoid growth curve,and the turning point of ? stage to ? stage was 45~50 d after female flowers bloom.From the change of ABA content during fruit ripening,it can be demonstrated that ABA can regulate the turning of two stages.Three ABFs were isolated from 'Guiwei' pericarp,and named LcABF1,LcABF2 and LcABF3,respectively.From the phylogenetic analysis,LcABF1 and LcABF2 was more homology than LcABF3.From the expression analysis by RT-qPCR,LcABF1 mainly expressed in seed,LcABF2 expressed in all the six different tissues,and LcABF3 was highest expressed in seed and then in young leaf.According to the expression analysis in pericarp development stages,it was deduced that all of LcABF1,LcABF2 and LcABF3 were related to the rippening of litchi fruit.However,LcABF1 mostly participated in the degradation of chlorophyll,LcABF2 and LcABF3 induced the rippening of fruit,and LcABF3 mostly participated in the biosynthesis of anthocyanin.3.When fused with GFP,all of LcABF1,LcABF2 and LcABF3 were localized to the nucleus.By means of dual luciferase assay and Yeast One-Hybrid system,both of LcABF2 and LcABF3 could bind to the fragment of LcMYB1 promoter which containing ABRE element.With further investigation,LcABF1 and(or)LcABF2 were(was)able to activate the promoters of LcPAO and LcSGR,LcABF3 was able to activate the promoters of LcNYC and LcCLH.Moreover,LcABF2 and(or)LcABF3 could activate the expression of structure genes in anthocyanin biosynthesis,such as LcCHS,LcCHI,LcF3 H,LcF3'H,LcDFR,LcANS,and regulator genes LcbHLH1.By transient expression assays in Nicotiana benthamiana leaves,it was proved that LcABF1 and(or)LcABF2 were(was)able to accelerate the chlorophyll degradation with up-regulating the expression of NbNYC,NbCLH,NbPAO and NbSGR.Although LcABF3 could up-regulate the expression of NbNYC,NbCLH,there was no phenotype in the transformed leaves.There was no relationship between LcABF1 and anthocyanin biosynthesis-related genes.LcABF2 induced expression of NbDFR,NbANS,NbUFGT and NbAN1.And LcABF3 was able to induce more anthocyanin accumulation in transformed leaves with up-regulating the expression of NbANS and NbAN1.In summary,LcABF1 and LcABF2 mainly regulated the chlorophyll degradation,LcABFs and LcABF3 maily regulated the anthocyanin biosynthesis.