Studies On The Role Of PacMYBA In ABA-Regulated Anthocyainin Biosynthesis In Red-Colored Sweet Cherry Fruit

Anthocyanins are the major water-soluble pigments in higher plants and are responsible for the blue, purple, and red colors of many fruits and vegetables. The anthocyanin biosynthetic pathway is part of the flavonoid pathway, which is a branch of the phenylpropanoid pathway. Recent studies have shown that a kind of R2R3MYB transcription factors have significantly regulation effect on anthocyanin biosynthesis. The present study describes the identification and functional characterization of the sweet cherry R2R3MYB protein, PacMYBA and also first obtained five full-length genes of anthocyanin biosynthesis pathway. The present work also first systematically expounds the role of plant hormone ABA in the fruit development process of sweet cherry. The main results are follows:(1) In the present study we have studied the physiological and morphological changes during sweet cherry fruit development. The results showed that sweet cherry exhibited a biphasic growth pattern. The soluble sugars of sweet cherry including:glucose, fructose and sorbitol. No sucrose was detected in the sweet cherry. The levels of Chl declined continually after the SG stage, whereas the anthocyanin content increased rapidly after veraison.(2) We first obtained the anthocyanin biosynthesis pathway genes of sweet cherry by using RACE technology and named as PacCHS, PacCHI, PacF3H, PacDFR, PacANS, and PacUFGT. The expression of PacCHS, PacCHI and PacF3H were at high levels during the early stages, declined during veraison (i.e., the onset of ripening; DQ YW, IR), and increased again in the last two stages. In accordance with the pattern of anthocyanin accumulation during ripening, the expression of PacDFR, PacANS and PacUFGT were expressed at low levels during the early developmental stages, and expression rapidly increased during veraison and remained at high levels through to the final developmental stage.(3) We obtained a single putative R2R3MYB gene product, which we named PacMYBA, using RACE (rapid amplification of cDNA ends) PCR. Compared to apple MdMYB10, PacMYBA shared84%amino acid identity in the R2R3DNA-binding domain and68%identity over the whole protein. The nuclear localization of PacMYBA was consistent with its predicted function as a transcription factor and it is has no self-transcriptional activation activity. Tissue specific expression analysis showed that PacMYBA have the highest expression level in mature skin and flesh. The expression level of PacMYBA during sweet cherry fruit development is positive correlation with the anthocyanin biosynthesis of sweet cherry. We also found several cis-elements which related to plant growth and development response, stress response and plant hormone response in the promoter sequence of PacMYBA.(4) The immature seeds of transgenic plants displayed red pigmentation<10days after anthesis, whereas the seeds of wild-type plants (ecotype Columbia) had no pigmentation. These data show that PacMYBA plays a role in anthocyanin biosynthesis.Silencing of PacMYBA, using a Tobacco rattle virus-induced gene silencing technique, resulted in sweet cherry fruit that lacked red pigment. Those fruits which were infiltrated with TRV alone pigmentation normally. These results shown that PacMYBA involve in regulating anthocyanin biosynthesis of sweet cherry directly.(5) Abscisic acid (ABA) treatment significantly induced anthocyanin accumulation, while treatment with the ABA biosynthesis inhibitor nordihydroguaiaretic acid (NDGA) blocked anthocyanin production. The exoression level of PacMYBA increased quickly treated with30μmol/L ABA. After1h treatment with ABA the expression level of PacMYBA was significantly higher than control. This result suggested that PacMYBA might be a ABA-response gene.The uncolored phenotype was also observed in the fruits, silencing of PacNCED1, which encodes a key enzyme in the ABA biosynthesis pathway. The endogenous ABA content as well as the transcript levels of six structural genes and PacMYBA in PacNCED1-RNAi fruit was significantly lower than in the TRV vector control fruit. These results suggest that ABA is a signal molecule that promotes red-color sweet cherry fruit accumulating anthocyanin.