Physiological and molecular mechanism of Ca+GA₃ treatment alleviating fruit cracking incidence in ponkan

Abstract:【Objective】Fruit cracking is a physiological disease, which seriously affects the appearance and quality of fruit. Ponkan fruit cracking rate can be as high as 70% in serious cases, which has become a major problem in the development of the ponkan production. Fruit cracking is regulated by multiple cell physiological and biochemical metabolisms during peel development. Therefore, the physiological and molecular mechanisms of ponkan fruit cracking was investigated in order to provide theoretical basis for the prevention and alleviation of the fruit cracking in production.【Methods】In this study, 10-year-old early-maturing ponkan fruit (cracking-susceptible cultivar) in Tianma Citrus Farm in Yong-chun County, Fujian, China, were used as the experimental material. Eighteen trees with relatively consistent nutrition level and growth state under conventional cultivation management were selected for the experiment. At the expansion period of ponkan fruit in June and July, the fruit sprayed with 0.3 g · L- 1 chelated calcium and 10 mg·L-1 GA3 (Ca+GA3) were used as the treatment group, and the fruit sprayed with water as the control (CK). Ponkan fruit started to crack on August 15, 2022, and the fruit were collected at 15 d intervals (105, 120, 135, 150 and 165 d after full bloom). The fruit cracking rate and appearance quality were investigated, including transverse diameter, vertical diameter, fruit shape index, single fruit weight and peel thickness. In addition, H2O2 and MDA contents, and O2 production rate as well as antioxidant enzyme activities (SOD, CAT, POD, and PPO) in ponkan peel were determined. Furthermore, the cell wall metabolic enzyme activities (PME, PL, PG, CX, PAL, 4CL, and C4H) and the structural components (protopectin, soluble pectin, cellulose, and lignin) were measured. And the expression of the related genes of antioxidant enzymes and cell wall metabolic enzymes in the peel were analyzed.【Results】Ca + GA3 treatment significantly reduced the cracking rate of ponkan fruit. The transverse diameter and vertical diameter of the fruit increased, and fruit shape index, single fruit weight and top peel thickness in Ca+GA3 treatment were greater than those of CK. Abnormal metabolism of ROS is one of the most important causes of fruit cracking. During the period of 105-165 days after full bloom, the contents of H2O2, MDA and the rate of O2 production in ponkan peel showed a general trend of increasing and then decreasing and reached a peak at 135 d after full bloom. Ca+GA3 treatment reduced the degree of lipid peroxidation in cell membrane of the peel. The activities of antioxidant enzymes SOD and CAT in ponkan peel peaked at 135 d after full bloom, which was significantly higher, while POD and PPO activities significantly lower in Ca+GA3 treatment than in CK. The results indicated that Ca+GA3 treatment increased SOD and CAT activities, and decreased POD and PPO activities in ponkan peel. The higher activities of peel cell wall hydrolases, the faster the cell wall polysaccharide degradation, and the higher fruit cracking incidence. The Ca+GA3 treatment resulted in a consistently higher protopectin and lower soluble pectin contents in ponkan peel than those of CK. In addition, PME, PL and PG in ponkan peel were significantly reduced after Ca+GA3 treatment. The results indicated that Ca+GA3 treatment reduced the rate of degradation of protopectin into soluble pectin. The content of cellulose in ponkan peel decreased slowly during 105-165 days after full bloom. Ca+GA3 treatment effectively increased the content of cellulose in ponkan peel, while decreased CX activity. Lignin content in ponkan peel treated with Ca+GA3was lower than that of CK during the period of 105-165 d after full bloom, and the activities of PAL, 4CL, and C4H, key enzymes in lignin synthesis, decreased in the peel throughout the whole period of growth and development of ponkan fruit. These results indicate that Ca+GA3 treatment can effectively reduce the activities of key enzymes in lignin synthesis in the peel. Ca+GA3 treatment significantly increased the relative expression of antioxidant enzyme genes CrSOD and CrCAT, and decreased the relative expressions of CrPOD and CrPPO, as well as cell wall metabolism related genes CrPME, CrPL, CrPG, CrCX, CrPAL, Cr4CL, and CrC4H in ponkan peel. Ca+GA3 treatment improved the ductility and toughness of the peel and reduced the occurrence of the fruit cracking by influencing the ROS content, antioxidant capacities, cell wall metabolic enzyme activities and structural components in the peel.【Conclusion】In summary, Ca + GA3 treatment increased SOD and CAT activities, decreased O2 production rate and H2O2 and MDA contents, as well as POD and PPO activities in ponkan peel. Ca+GA3 treatment reduced PME, PL, PG, PAL, 4CL and C4H activities and lignin content, increased the protopectin and cellulose contents in ponkan peel. The expression levels of CrSOD, CrCAT, CrPOD, CrPPO, CrPME, CrPL, CrPG, CrCX, CrPAL, Cr4CL and CrC4H in the peelof ponkan fruit treated with Ca+GA3 were consistent with the activities of related enzymes and the accumulation of the metabolites. Ca + GA3 treatment is effective to reduce fruit cracking rate by affecting ROS metabolism and cell wall metabolism in fruit peel at both molecular and physiological levels.