Analysis Of Key Metabolites And Regulatory Pathways For Inner Quality Formation In Sweet Cherry Fruit Development

Sweet cherry（Prunus avium L.）belongs to the plum tree of the rose family,also known as the big cherry.It has a unique flavor and rich nutritional value and is widely popular in China as an important economic fruit tree.In order to cultivate sweet cherry varieties with high quality,it is very important to study the dynamic changes in fruit quality during fruit development.At the same time,since sweet cherry is a typical self-incompatibility species,exogenous application of gibberellin is often used in production to improve its fruit setting rate.Therefore,this study measured and analyzed the fruit quality of sweet cherry in 12periods,explored the regulatory mechanism of exogenous GA₃-mediated changes in sweet cherry endogenous hormones,established the synthetic regulatory network of fruit quality and5 kinds of endogenous hormones such as abscisic acid and gibberellin,and systematically identified and analyzed the WRKY gene family of sweet cherry fruits for the first time.To provide a theoretical basis for improving sweet cherry fruit quality and efficiency.The main research results are as follows:1.The dynamics of substance accumulation in the growth and development of sweet cherry fruits were analyzed.Through the determination of soluble sugar,titratable acid,anthocyanin,and other contents of sweet cherry fruits in 12 periods and the observation of fruit traits,we analyzed the external morphological and physiological quality characteristics during fruit development and explored the changing pattern and the correlation between hormones and quality indexes.According to the fruit development characteristics,the sweet cherry fruit development was divided into three periods:green ripening period,color conversion period,and full red period.Meanwhile,the fruit soluble sugar,anthocyanin,and soluble solids indexes increased continuously with fruit maturity and reached the peak at the ripening stage.The analysis of the fractions of soluble sugars,organic acids,and anthocyanins by high-performance liquid chromatography（HPLC）revealed that glucose,malic acid,and centaureidin-3-O-rutinoside were the major components,accounting for 41.59%,93.53%,and72.37%,respectively.In the hormone change spectrum,the endogenous hormones JA and GA₃showed a decreasing trend,and IAA,ABA,and GA₄showed a decreasing and then increasing trend.Among them,ABA content was the most abundant and changed most drastically,increasing from 32.91 ng/g FW to 119.05 ng/g FW,which may play the most important role in fruit growth and development.By correlating the physiological indexes with the five hormones,the results showed that there was some correlation between hormone content and quality indexes,but the correlation of the same hormone at different growth stages also differed,indicating that each hormone played an important inhibitory or promoting role at different growth stages,respectively.2.Transcriptome and metabolome were combined to analyze the regulatory mechanism of fruit quality formation.In the metabolome,various glycans such as D-Glucose,Sucrose,Trehalose,Mannose,Dihydroquercetin,Dihydromyricetin,Dephinidin-3-O-glucoside,Cyanidin-3-O-rutincoside,and many other flavonoids.Among them,D-Glucose and Cyanidin-3-O-rutinoside were among the most important components of sweet cherry soluble sugars and anthocyanins,respectively,which were consistent with the HPLC findings.Further transcriptional analysis was performed to map the synthesis network of soluble sugars,organic acids,and anthocyanins and identify the key structural genes involved in the synthesis pathway.q RT-PCR further verified the expression of key structural genes in the synthesis pathway,showing similar expression abundance to that in the transcriptome.This study established a precise regulatory network for quality changes and laid the foundation for fruit quality and efficiency improvement.3.Study on the regulation of endogenous hormones in sweet cherry fruits by exogenous GA₃.We treated with exogenous GA₃at the flowering stage,analyzed the change patterns of endogenous hormones,constructed the anabolic regulatory network of each endogenous hormone,and screened key genes of endogenous hormone anabolism that responded to exogenous GA₃regulation.In the GA pathway,exogenous GA₃significantly suppressed the expression of three GA2ox genes regulating GA₄catabolism at fruit ripening,but significantly up-regulated the expression of the CYP701 gene,indicating that GA2ox and CYP701cooperated to jointly promote the massive accumulation of endogenous GA₇and GA₃.The expression of NCED2,a major regulator gene of ABA synthesis,was significantly increased after the application of exogenous GA₃significantly increased,contributing to a 7.42-fold increase in ABA content at maturity.The results indicated that exogenous GA₃mainly acted by influencing the anabolism of endogenous ABA and GA₃in fruits,which further clarified that ABA plays an important role in fruit quality regulation during sweet cherry fruit development.Also,in conducting transcription factor analysis,it was found that a variety of transcription factors play a role in hormone regulation,among which the WRKY gene family showed the highest percentage of variability after GA₃application,indicating that it is more sensitive to GA₃application.4.Identification and analysis of WRKY gene families in sweet cherry fruit.Fifty-eight WRKY gene families,named Pa WRKY1-Pa WRKY58,were identified from the sweet cherry genome database and divided into three subfamilies,Group I,Group II,and Group III,based on phylogenetic analysis,with Group II divided into five subgroups.The promoter region of Pa WRKYs genes is rich in cis-acting elements related to growth and development and stress response,and each gene contains at least one hormone-acting element,of which ABA is more abundant than other hormones.Protein interactions were predicted to show that WRKY can interact with multiple proteins.Transcriptome data showed that sweet cherry WRKY gene family transcription factors are strongly influenced by exogenous GA₃with a high percentage of differences,especially genes belonging to Group III（Pa WRKY16,Pa WRKY21,Pa WRKY38,Pa WRKY52,and Pa WRKY53）,which are potential regulators of sweet cherry fruit growth.These transcription factors can bind to genes such as NCED and YUCCA to regulate endogenous hormone content.These findings lay the foundation for the preliminary determination of the mechanism of GA₃effect on endogenous hormones in sweet cherry and the biological function study of WRKY transcription factors.