Functional characterization of sweet cherry PaPME1 and PaPME2 during fruit ripening and softening

Abstract:【Objective】Pectin methyl esterases (PMEs) are key enzymes in the demethylation of pectin, which participates in the degradation of pectin during fruit softening. However little has been stud- ied about the functional characteristics of PME genes during fruit ripening, In order to provide a theoretical basis for determining the function of pectin methyl esterase genes (PMEs) during fruit ripening and softening, we characterized the biological function of PaPME1 and PaPME2 genes in sweet cherry.【Methods】7-year-old trees of sweet cherry (P. avium) cultivar‘Zaohongzhu’were selected grown in the resource collections of the National Fruit Tree Germplasm Repository, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences (Zhengzhou, China). To examine the expression pattern of PaPME1 and PaPME2 at different growth and developmental stages of sweet cherry fruits, quantitative real-time PCR (qRT-PCR) was implemented using total RNA from different fruit developmental stages. In addition, we used TRV-mediated virus-induced gene silencing (VIGS) to further evaluate the function of PaPME1 and PaPME2 during the course of sweet cherry fruit ripening and softening. Mean- while, The fruit hardness, soluble solids, soluble pectin and PME activity of the TRV::PaPME1- andPaPME2 infected fruit were detected.【Results】Gene expression analysis showed that the expression level of PaPME2 was low during early fruit growth and development. Subsequently, PaPME2 expres- sion was sequentially and significantly upregulated during fruit ripening and softening, suggesting that the expression pattern was consistent with the ripening and softening period of sweet cherry fruit. The expression of PaPME1 gene was low in the early stage of fruit development, and was up-regulated with the development of fruit until the fruit ripened and softened. In addition, the expression of PaPME1gene showed two peaks on 28 days and 56 days after flowering, but the expression of PaPME1 gene showed a trend of up-regulation and down-regulation during fruit maturation and development. The tobacco rattle virus-induced gene silencing (TRV-VIGS) technique was used to knock down expression of the PaPME1 and PaPME2 genes in the sweet cherry‘Zaohongzhu’. RT-PCR was performed using cD-NA samples from infiltrated fruit at 15 dpi to verify if the PaPME1 and PaPME2 genes in‘Zaohongzhu’were effectively silenced. This showed that the expression of PaPME1 and PaPME2 genes were markedly reduced in the TRV::PaPME1- and PaPME2 infected fruit compared with the TRV::00-infect- ed fruit, suggesting that each gene was effectively silenced. Furthermore, silencing of PaPME2 of sweet cherry fruit delayed fruit ripening and softening in comparison with TRV::00-infected control fruit on 21 days post- inoculation (dpi). The fruit firmness, soluble solid concentration, water soluble pectin (WSP) content, CDTA soluble pectin (CSP) content, Na2CO3 soluble pectin (NSP) content, PME activity, cellulose content, and hemicelluloses content of the TRV::PaPME1- and TRV::PaPME2- infected fruits were analyzed. The results showed fruit firmness, WSP content, and PME activity of the TRV:: PaPME2-infected fruit was significantly reduced compared with that of the TRV::00-infected fruit on 15 dpi. And in PaPME2-silenced fruits, CSP content and NSP content were significantly increased compared with those of the TRV::00-infected fruits on 15 dpi. However, soluble solid concentration, cellu- lose content, and hemicelluloses content of TRV::PaPME2- infected fruit did not differ significantly from that of TRV::00-infected fruit on 15 dpi. Compared with TRV::00-infected fruit, TRV::PaPME2-in-fected sweet cherry fruits had no significant differences in fruit firmness, soluble solid concentration,WSP content, CSP content, NSP content, PME activity, cellulose content, and hemicelluloses content on 15 dpi. Together, our findings indicate that PaPME2 is involved in sweet cherry fruit ripening and soft- ening.【Conclusion】In conclusion, this study highlighted an important role for PaPME2 that would most likely be a key gene regulating fruit softening during sweet cherry fruit ripening and softening.These findings would provide novel insights into understanding the molecular mechanisms underlyingfruit softening determination during fruit ripening and softening in fruit trees and might have potential value for improving the postharvest quality and shelf life of fruits in sweet cherry.