黑穗醋栗果实生长发育过程中抗坏血酸含量及相关酶活性的变化

The Changes in the Contents of Ascorbic Acid and the Activities of Related Enzymes in Black Currant Fruits During the Process of Its Growth and Development

【目的】研究不同品种不同生长时期黑穗醋栗果实内抗坏血酸(AsA)含量及其合成代谢过程中相关酶活性的变化,以明确果实生长发育过程中AsA含量与代谢合成相关酶之间的关系,为全面揭示黑穗醋栗果实AsA积累规律提供理论依据。【方法】以3个不同黑穗醋栗品种(‘亚德’‘布劳德’和‘黑丰’)为试材,测定果实在幼果期、膨大期、半转色期、转色期和成熟期时还原型抗坏血酸(AsA)、氧化型抗坏血酸(DHA)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量以及AsA合成与代谢相关酶活性。【结果】不同品种黑穗醋栗果实大小、AsA含量以及AsA相关代谢物水平存在明显的多样性。其中‘亚德’单果重最大,为1.97 g。果实生长发育过程中,总抗坏血酸(T-AsA)和AsA含量在3个品种中变化趋势一致,均在幼果期含量最高,其中‘亚德’幼果期果实中AsA含量最高,为83.17μmol·g^(-1) FW,随着果实的生长迅速下降,在成熟期降至21.28μmol·g^(-1) FW;3个品种果实中GSH和T-GSH含量随着果实发育呈升高趋势,但不同品种升高时期或增加幅度不同;GSSG含量在不同品种间存在较大差异,成熟果中‘黑丰’含量最低,为0.008μmol·g^(-1) FW,仅为‘亚德’的10.2%。AsA-GSH循环再生代谢中,脱氢抗坏血酸还原酶(DHAR)和单脱氢抗坏血酸还原酶(MDHAR)活性在果实膨大期达到最高,成熟期降至最低,其中‘布劳德’果实中DHAR和MDHAR活性略高于‘亚德’和‘黑丰’;谷胱甘肽还原酶(GR)活性在幼果期最高,‘亚德’幼果期果实中GR活性最高(0.06μmol·min^(-1)·g^(-1) FW),之后随着果实的生长发育不断下降,抗坏血酸过氧化物酶(APX)活性变化与之相似;L-半乳糖途径的关键酶L-半乳糖^(-1),4-内酯脱氢酶(GalLDH)活性随着果实生长发育的变化趋势与AsA含量变化相一致,且‘亚德’果实中GalLDH活性在幼果期和成熟期均高于其他两个品种。通过相关性分析发现,GalLDH与T-AsA、AsA、DHA、DHAR和MDHAR呈现极显著正相关关系,相关系数可达0.91以上,即果实中GalLDH活性越高,果实中AsA含量也越高;DHAR和MDHAR与T-AsA、AsA间也存在极显著正相关关系,而APX与T-GSH、GSH间相关性较强。【结论】黑穗醋栗幼果期果实中AsA含量最高,且品种间差异显著;GalLDH、MDHAR和DHAR可能是黑穗醋栗果实中AsA合成代谢的关键酶,黑穗醋栗果实中AsA含量积累主要取决于GalLDH活性,说明合成途径起着更关键的作用,而AsA-GSH循环再生途径相关酶对AsA合成也有一定的贡献,黑穗醋栗高AsA含量的积累是由合成途径与循环途径共同作用的结果。

【Objective】 The changes in the ascorbic acid(AsA) contents and the enzymatic activities during the anabolic process of different cultivars and growth stages of black currant fruits were studied to determine the relationship between AsA contents and anabolic enzymes during the growth and development of fruits, so as to provide a theoretical basis for comprehensively revealing the accumulation rule of AsA in black currant fruits. 【Method】 Three different cultivars of black currant fruits(Adelinia, Brodtrop and Heifeng) were studied and determined the contents of reduced AsA, oxidized ascorbic acid(DHA), reduced glutathione(GSH) and oxidized glutathione(GSSG) and anabolic enzymatic activities of young, expansion, half-veraison, veraison and maturity stages. 【Result】 There were significant diversities in fruit sizes, AsA contents and AsA metabolites of different cultivars of black currant fruits. The Adelinia had the largest weight of single fruit(1.97 g). During the growth and development process of fruits, the changes in the total ascorbic acid(T-AsA) and AsA contents of fruits were consistent among the three cultivars, and the young fruits had the highest contents. The AsA content of young Adelinia fruit was the highest(83.17 μmol·g^-1FW) and then sharply decreased rapidly to the maturity stage with the growth of the fruit, which decreased to 21.28 μmol·g^-1FW at maturity stage. The contents of GSH and T-GSH in the three cultivars increased with the development of fruits, but the different cultivars increased in different stages and degrees. The content of GSSG was quite different among different cultivars. For the mature fruits, the GSSG content of Heifeng was the lowest, which was 0.008 μmol·g^-1FW and only accounted for 10.2% of Adelinia. In AsA-GSH recycling regeneration metabolism, the activities of dehydroascorbate reductase(DHAR) and monodehydroascorbate reductase(MDHAR) showed the highest level at expansion period, and finally decreased to the lowest level at maturity stage. The DHAR and MDHAR activities of Brodtrop fruits showed slightly higher than those of Adelinia and Heifeng fruits. The activity of glutathione reductase(GR) was the highest level at young stage. The GR activity of Adelinia young fruits was the highest(0.06 μmol·min-1·g^-1FW), and then decreased with the growth of fruits. The changes in the activities of ascorbate peroxidase(APX) were similar to the changes in the activities of GR. The changes in the activities of L-galactose-1,4-lactone dehydrogenase(GalLDH), a key enzyme of L-galactose pathway, were consistent with the changes in AsA contents. The GalLDH activity of Adelinia young and mature fruits showed higher than that of Heifeng and Brodtrop young and mature fruits, respectively. According to the correlation analysis, the GalLDH activity showed a highly significant positive correlationship with T-AsA, AsA, DHA, DHAR and MDHAR. The correlation coefficient was above 0.91. The higher GalLDH activity was found in the fruits, the higher AsA contents of fruits also was found. There was a highly significant positive correlationship between DHAR and MDHAR, T-AsA and AsA. The APX had a high correlation with T-GSH and GSH. 【Conclusion】 The AsA content of black currant young fruits was the highest and there were significant differences among the three cultivars. The GalLDH, MDHAR and DHAR might be the key enzymes for AsA anabolism in black currant fruits. The accumulation of AsA content of black currant fruits resulted from the activity of GalLDH, which indicated that the anabolic pathway played a more important role and were found to be a dominant position. The related enzymes of AsA-GSH recycling regeneration pathway also contributed to the AsA anabolism. The accumulation of high AsA content in black currant fruits resulted from the combined effects of anabolic and recycling pathways.