PpWRKY4 regulates carotenoids accumulation in peach fruit by affecting the expression of PpCCD4

Abstract:【Objective】Peach (Prunus persica) is one of the most important economic fruit tree in the world. The color of fruit flesh is a notable feature for consumers and one of the important breeding objectives. The flesh color of white/yellow is a typical Mendelian trait controlled by a single locus (Y), and white phenotype (YY or Yy) is fully dominant over the yellow one (yy). The yellow- flesh peach mainly depends on the accumulation of carotenoids in chromoplasts. The carotenoids is considered as an indispensable part for human diet health and nutrition. The previous studies indicated that three types of the PpCCD4 mutation caused no expression or extremely lower expression of the PpCCD4 in yellowflesh peach, suggesting that the carotenoids would be negatively correlated with the expression of thePpCCD4. Although the PpCCD4 is considered as the key gene controlling the white/yellow flesh trait of peach, the regulatory mechanism of carotenoids degradation remains unclear. The PpWRKY4, a WRKY transcription factors associated with carotenoids degradation, was isolated from a white- flesh peach exhibiting lower carotenoids content in this study and the regulatory mechanism of the PpWRKY4 was explored in carotenoid metabolism of peach fruit.【Methods】The carotenoids content and the relative expression of the PpCCD4 at S1- S4 stages of the fruits of Zhongyoutao 14 (CN14) were analyzed, respectively. The PpWRKY4 (the homology of OfWRKY3) was cloned by PCR. PpWRKY4 and other three homologous proteins were analyzed for domains using MEGA 11. The expression pattern of the PpWRKY4 and PpCCD4 at S1-S4 periods of the fruits of CN14 were obtained by qRT-PCR. The cis-elements of the PpCCD4 promoter were analyzed using PlantRegMap and a binding site map was drawn by GSDS. The CDS of the PpWRKY4 was inserted into pSAK277-GFP vector, and then transformed into GV3101 and Marker mixed annotated Nicotiana benthamiana. The fluorescence in tobacco leaf cells was observed using laser scanning confocal microscopy after 36- 48 h. The PpWRKY4 was cloned and inserted into pGADT7 and transfected into Y1HGold with PpAbAiPpCCD4-promoter for Y1H assay. The CDS of the PpWRKY4 was cloned into the pSAK277 vector to create the effector. The promoter sequence of the PpCCD4 (-2 Kb upstream of ATG) was cloned into the dual reporter vector pGreenII-0800. The Agrobacterium cells containing pSAK277:PpWRKY4 and pGreenⅡ-0800:PpCCD4 were co-infiltrated into the tobacco leaves, and the leaves infiltrated with the empty vector pSAK277 and pGreenII-0800:PpCCD4 were used as controls. 48 h after infiltration, the luciferase (LUC) and Renilla luciferase (REN) activities were measured using a dual-luciferase assay kit.【Results】The expression pattern of the PpCCD4 and the carotenoids content during the S1-S4 periods of the fruits of CN14 showed opposite tendency. The relative expression of the PpCCD4 increased gradually, while the carotenoids content of CN14 fruits significantly decreased, ranged from 1.97 μg·g-1 at S1 stage to 0.68 μg · g- 1 at S4 stage. Several cis-elements of the PpCCD4 promoter were observed, mainly including Dof, MADS, MYB, bHLH, WRKY etc. The PpWRKY4 contained 1764 bp of ORF (open reading frame) and encoded 587 amino acids, which contained two WRKY conserved domains, as the character of a group I of WRKY gene subfamily. The sequence alignment results indicated that PpWRKY4 showed the highest expected value with OfWRKY3 which is a key transcription activator of the OfCCD4 gene participating in biosynthesis of carotenoids. qRT-PCR analysis of S1-S4 periods of the fruits of CN14 showed that the PpWRKY4 showed the opposite expression pattern of the PpCCD4. The expression level of the PpWRKY4 showed higher at S1 stage and then sharply decreased at S2-S4 stages. Based on the expression patterns of the PpWRKY4 and PpCCD4, the PpWRKY4 might negatively regulate the PpCCD4. The subcellular localization analysis demonstrated that the PpWRKY4 would be a nucleus-localized transcription factor. The Y1H results showed that the yeast cells containing the PpWRKY4 and PpCCD4 were able to grow well in SD-Leu/AbA200, but the yeast containing the empty AD vector and the PpCCD4 did not, indicating that the PpWRKY4 was bound to the PpCCD4. The results of dual-luciferase assays using the transiently transformed tobacco leaves indicated that the activity of the PpCCD4 promoter greatly decreased after co-infiltration of the promoter reporter construct with a construct expressing PpWRKY4. These results showed that the PpWRKY4 would be bound to the integral PpCCD4 and repressed its transcription.【Conclusion】A homology of OfWRKY3 denoted as the PpWRKY4 was cloned in CN14 peach. The PpWRKY4 is a typical group I WRKY transcription factor with two WRKY domains. The PpWRKY4 was expressed higher at S1 stage of CN14 fruits than that at S2-S4 stages, showing opposite expression pattern with the PpCCD4. The PpWRKY4 could di- rectly bind to the PpCCD4 and inhibited its expression. The PpWRKY4 was likely a transcription inhibitor of the PpCCD4 gene, involved in regulating carotenoids accumulation.