Abstract 【Objective】Peach (Prunus persica) is one of the most important economic fruit tree in the world. Fruit flesh color is a notable feature for consumers and an important breeding objective.
The flesh color of peach is mainly divided into white, yellow and red. 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 (yy). The yellow-flesh peach mainly depends on the accumulation of carotenoids in chromoplasts. Carotenoids is considered as an indispensable part for human diet health and nutrition. Previous studies indicated three types of PpCCD4 mutation caused no expression or extremely lower expression of PpCCD4 in yellow-flesh peach, suggesting carotenoids negatively correlated with the expression of PpCCD4. Although PpCCD4 is considered as the key gene controlled the white/yellow flesh trait of peach, the regulatory mechanism of carotenoids degradation is still unclear. PpWRKY4, a WRKY transcription factors associated with carotenoids degradation, was isolated from a white-flesh peach exhibiting lower carotenoids content. In this study, the regulatory mechanism of PpWRKY4 was explored in carotenoid metabolism of peach fruit. 【Methods】The carotenoids content and the relative expression of PpCCD4 in S1-S4 stages of fruit in Zhongyoutao 14 (CN14) were analyzed, respectively. 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 PpWRKY4 and PpCCD4 of S1-S4 periods of fruit in CN14 were obtained by qRT-PCR. The cis-elements of PpCCD4 promoter were analyzed using PlantRegMap and a binding site map was drawn by GSDS. The CDS of 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. PpWRKY4 was cloned and inserted into pGADT7 and transfected into Y1HGold with PpAbAi-PpCCD4-promoter for Y1H assay. The CDS of PpWRKY4 was cloned into the pSAK277 vector to create the effector. The promoter sequence of PpCCD4 (~2 Kb upstream of ATG) was cloned into the dual reporter vector pGreenII-0800. Agrobacterium cells containing pSAK277:PpWRKY4 and pGreenII-0800:PpCCD4 were co-infiltrated into tobacco leaves, and 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 PpCCD4 and the carotenoids content during the S1-S4 periods of fruit in CN14 showed opposite tendency. The relative expression of PpCCD4 increased gradually, while the carotenoids content of CN14 fruits significantly decreased, ranged from 1.97 μg·g-1 in S1 stage to 0.68 μg·g-1 in S4 stage. Several cis-elements of PpCCD4 promoter were observed, mainly including Dof, MADS, MYB, bHLH, WRKY etc. 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. 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 fruit in CN14 showed that PpWRKY4 showed the opposite expression pattern with PpCCD4. The expression level of PpWRKY4 showed higher in S1 stage and then sharply decreased in S2-S4 stages. Based on the expression patterns of PpWRKY4 and PpCCD4, it implied that PpWRKY4 might negatively regulated PpCCD4. Subcellular localization analysis demonstrated that PpWRKY4 is a nucleus-localized transcription factor. The Y1H results showed that yeast cells containing PpWRKY4 and PpCCD4 were able to grow well in SD-Leu/AbA200, but yeast containing the empty AD vector and PpCCD4 did not, indicating that PpWRKY4 was bound to PpCCD4. The results of dual-luciferase assays using 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 PpWRKY4 was bound to the integral p PpCCD4 and repressed its transcription. 【Conclusion】 A homology of OfWRKY3 denoted as PpWRKY4 was cloned in CN14 peach. PpWRKY4 is a typical group I WRKY transcription factor with two WRKY domains. PpWRKY4 was expressed higher in S1 stage of CN14 fruits than that in S2-S4 stages, showing opposite expression pattern with PpCCD4. YIH and dual-luciferase assays results indicated that PpWRKY4 could directly bind to PpCCD4 and inhibited its expression. These results indicated that PpWRKY4 was likely a transcription inhibitor of the PpCCD4 gene, involved in regulating carotenoids accumulation and provided the theoretical basis of regulation of flesh color in peach.
PDF （）