A comparative analysis of the differences in starch degradation and soluble sugar accumulation between 'Zhongjiao No.9' and 'Baxijiao' during fruit ripening

Abstract: 【Objective】‘Zhongjiao No. 9'is a new cavendish banana variety bred by the Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences. The genetic relationship between‘Zhongjiao No. 9'and‘Baxijiao'is close. Both are AAA genotypes and have similar fruit shape and ripening process. However, there are significant differences in the taste and sweetness between them,which makes them excellent materials for studying starch degradation and soluble sugar accumulation.In this study, the differences in fruit quality and sugar metabolism-related genes between‘Zhongjiao No. 9'and‘Baxijiao'were comparatively analyzed. Our goal is to clarify the reasons for the differences in taste and sweetness between the two varieties from the perspectives of starch degradation and soluble sugar accumulation during fruit ripening process.【Methods】Green mature fruits of‘Zhongjiao No.9'and‘Baxijiao'were socked in 1 000 mg · L-1 ethephon aqueous solution for 1.5 min, and then transferred in a storage room with a temperature of 20 ℃ and a relative humidity of 80%-90%. A total of five samples were collected for each cultivar, including 1, 2, 3, 4 and 5 d after ethylene treatment. Fruit firmness was determined using a digital fruit firmness tester(GY-4, China). Total soluble solids were measured with a hand-held digital refractometer(PAL-1, UNI-IT Inc., Tokyo, Japan). The content of total starch was determined using a starch assay kit(K-TSTA-100 A), and the contents of amylose and amylopectin were measured using an amylose/amylopectin assay kit(K-AMYL). The contents of sucrose,glucose and fructose were determined with high performance liquid chromatography(HPLC). The full length of 23 sugars metabolism-related gene sequences were obtained by searching the NCBI(National Center for Biotechnology Information) database and banana genome database. Specific primers were designed by using Primer Express 5.0(Applied Biosystems, Foster City, CA, USA). Quantitative real time polymerase chain reaction(qPCR) was performed to analyze the differences in gene expression. The activities of the enzymes involved in sugar metabolism were determined by using enzyme labeling instrument.【Results】The contents of amylose and total starch were higher in green-mature‘Zhongjiao No.9'fruit than in‘Baxijiao'fruit. After ethylene treatment, the fruit firmness of both cultivars reached the lowest level at d3, and there was no significant change in fruit firmness later on. The contents of total soluble solids, glucose and fructose in the yellow-mature‘Zhongjiao No. 9'fruit were higher than those in the yellow-mature‘Baxijiao'fruit, while the content of sucrose was the opposite. Starch degradation rate of‘Zhongjiao No. 9'fruit was significantly faster than that of‘Baxijiao'. Meanwhile, the gene expression levels of six starch degradation-related genes encoding two α-amylases(AMY), two isoamylase(ISA), starch phosphorylase(SPh) and glucan water dikinase(GWD) in‘Zhongjiao No. 9'fruit were significantly higher than those in‘Baxijiao'. The enzyme activity of α-amylase in‘Zhongjiao No. 9'fruit was also significantly higher than that in‘Baxijiao'fruit. The increase rates of fructose and glucose contents in‘Zhongjiao No. 9'fruit were significantly higher than in‘Baxijiao'fruit, while the increase rate of sucrose content in‘Zhongjiao No. 9'fruit was significantly higher than in‘Baxijiao'fruit. The gene expression levels of five soluble sugars metabolism-related genes encoding two UDP-glucose pyrophosphorylases(UDPase), fructokinase(FK), hexokinase(HXK) and phosphoglucomutase(PGM) in‘Zhongjiao No. 9'fruit were higher in‘Baxijiao'fruit. The acid invertase(AI) and sucrose synthase(SS) activities in‘Zhongjiao No. 9'fruit were also significantly higher than those of‘Baxijiao'fruit. In addition, correlation analysis was used to identify genes and enzymes that were functionally related or co-regulated with sugar characters in both cultivars. Results showed that the accumulation of soluble sugars in both‘Zhongjiao No. 9'and‘Baxijiao'fruit was negatively correlated with starch content, fruit firmness and sucrose synthase activity, while positively with amylase activity and the expression of starch degradation-and sugar metabolism-related genes. Interestingly, there were significant positive correlations between soluble sugar contents and the expressions of BMY, ISA, SPS,PGM and FK in‘Zhongjiao No. 9'fruit, while the accumulation of soluble sugars were significantly positively correlated with the gene expressions of AMY, GWD, ISA and HXK in‘Baxijiao'fruit, indicating that the two banana cultivars showed significant preference in the expression of genes related to starch degradation and sugar metabolism. Partial least squares discrimination analysis(PLS-DA) was used to explore the preference of the two banana cultivars. In the first two days after ethylene treatment,no significant separation was observed between‘Zhongjiao No. 9'and‘Baxijiao'fruit samples. The samples of the two banana cultivars could be separated from 3 d after ethylene treatment, and the degree of separation reached the maximum at 5 d, indicating that there were significant differences in sugar metabolism between the two banana cultivars. The percentage of amylose in starch and SS activity contributed significantly to the sample groups of two banana cultivars at 1 and 2 d after ethylene treatment. Six starch degradation-and soluble sugars metabolism-related genes contributed more to the sample groups of‘Zhongjiao No. 9'. Five starch degradation-and soluble sugars metabolism-related genes contributed more to the sample groups of‘Baxijiao'.【Conclusion】The expression levels of six starch degradation-related genes and five soluble sugars metabolism-related genes in‘Zhongjiao No. 9'fruit were significantly higher than those in‘Baxijiao', resulting in higher contents of glucose and fructose in the yellow-mature‘Zhongjiao No. 9'fruit than in the yellow-mature‘Baxijiao'fruit, while the content of sucrose in the yellow-mature‘Zhongjiao No. 9'fruit was lower than in the yellow-mature‘Baxijiao'fruit.