Abstract:

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α-Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α-amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple (Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α-amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular-localization studies via immunogold electron-microscopy technique showed that α-amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid-distributed pattern of α-amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α-amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α-amylase is involved in starch hydrolysis in plastids of the fruit cells.

苹果果实发育过程中α-淀粉酶的活性、数量变化和亚细胞定位

王永章  张大鹏^*

(中国农业大学果树分子发育实验室,植物生理学与生物化学国家重点实验室,北京 100094)

摘要：淀粉降解代谢与种子萌发、叶片光合作用、块根贮藏及肉质果实的发育密切相关.α-淀粉酶是催化淀粉水解的重要酶之一,然而由于它在生活细胞中经常定位于叶绿体或质体之外,与淀粉基质在亚细胞水平上相互隔离,所以该酶在植物活体内的生理功能至今不完全清楚.研究表明,在苹果(Malus domestica Borkh cv. Starkrimson)果实发育过程中,α-淀粉酶活性由低到高,与淀粉含量大致呈现互为消长的变化.Western blotting实验证明,在果实发育过程中,α-淀粉酶的表观数量也是由少到多,与活性的变化一致.利用胶体金免疫电镜定位技术证明,果实内α-淀粉酶主要定位于质体内,其他亚细胞区域内α-淀粉酶分布很少;尤其在果实发育中后期,围绕质体内淀粉粒有高密度的α-淀粉酶分布,说明该酶主要分布于细胞内功能区域.α-淀粉酶优先定位于质体内的亚细胞分布特点在果实整个生长发育期没有变化.随着果实发育的推进,质体内胶体金分布密度显著增加,此结果与Western blotting实验相互印证.推测α-淀粉酶参与了果实细胞内质体中淀粉的水解过程.

关键词 α-淀粉酶 亚细胞定位 苹果果实

*通讯作者 E-mail: zhangdp@public2.east.cn.net

Key words: α-amylase, subcellular localization, apple fruit