The Regulatory Mechanisms Of High CO₂-induced Persimmon Fruit Softening

Persimmon（Diospyros kaki Thunb.）is a deciduous perennial fruit tree native to China.It can be simply divided into two types（astringent and non-astringent）according to whether the fruit has astringency at maturity.As astringent type is the main cultivated persimmon variety in our country,post-harvest deastringency technology is essential.However,most deastringency treatments,including high CO₂（95%CO₂）treatment,would lead to excessive softening of fruit,which reduced the commodity value of fruit and limited the development of industry.In this research,astringent persimmon fruit‘Jingmianshi’was selected as material,and the transcriptome,gene expression,regulatory effect,binding ability and protein interaction were analyzed.The mechanism of high CO₂-induced persimmon fruit softening by accumulating ethylene and abscisic acid（ABA）and accelerating cell wall degradation was elucidated.The main results are as follows:1.Identification of 227 candidate genes probably involved in astringent persimmon fruit softening by transcriptome analysis.The post-harvest deastringency treatment of high CO₂ caused excessive softening of fruit,while high CO₂ and1-methylcyclopropene mixed treatment could achieve deastringency effect and maintain fruit firmness.By transcriptome analysis and comparison between these treatments,227 differentially expressed genes were obtained as candidate genes involved in fruit deastringency softening.Further analysis showed that these genes contained 12 transcription factors,three transcription regulators besides transcription factor,three protein phosphorylation kinases and three phytohormone biosynthesis related genes.2.NAC-ERF complex was involved in fruit softening by synergistically regulating cell wall degradation.Dual-luciferase assay showed that,among 12transcription factors identified above,Dk NAC9 had a regulatory effect on Dk EGase1,a gene related to cellulose degradation that reported previously.Furthermore,EMSA indicated that Dk NAC9 specifically bind to the promoter of Dk EGase1.Meanwhile,Dk NAC9 could interact with Dk ERF8/16,two previously characterized transcription factors involved in persimmon fruit softening,and the combination of them resulted in synergistic activation effects on Dk EGase1 promoter.These results indicated that Dk NAC9-Dk ERF8/16 complexes synergistically regulated cellulose degradation to participate in fruit deastringency softening.3.High CO₂ treatment accumulated ethylene and ABA to accelerate fruit softening.The results showed that the contents of ethylene and ABA were higher in high CO₂-treated samples.Further analysis showed that the differentially expressed RNA binding protein Dk RBM24-1 could regulate the transcription levels of Dk ACS1and Dk NCED2+3’involved in ethylene and ABA biosynthesis,by specifically bind to the 3’UTR regions of m RNA.In addition,transcription factor Dkb HLH11 was identified with binding ability to the Dk NCED1+3’promoter.Since there was no significant difference in the expression of Dkb HLH11 between different treatments,the differentially expressed protein kinase Dk MAPKKK1 was found to interact with Dkb HLH11 protein,which increased the transcriptional activation activity of Dkb HLH11 on Dk NCED1+3’promoter.In conclusion,the synergistically regulating mechanism of NAC-ERF complex in cell wall degradation of astringent persimmon fruit was analyzed.Besides,high CO₂ was likely to induce ethylene and ABA accumulation by shared（Dk RBM24-1）and distinct（Dkb HLH11）regulators,leading to the deastringency softening of persimmon.These results can deepen our understanding of fruit ripening and the regulatory mechanisms of plant hormones,and provide theoretical basis and potential targets for fruit quality improvement by biotechnologies.