水稻種子發芽及幼苗期胚中澱粉累積及蔗糖轉運之研究

Abstract

第一部份： 水稻種子發芽及幼苗生長時，發育中的莖葉及根組織主要之能量來源為胚乳澱粉分解生成之醣類：葡萄糖及麥芽糖。種子發芽時，葡萄糖及麥芽糖由胚乳運入胚中並可進一步被轉變為蔗糖以運至胚中其他部位，或由維管束組織運至生長中的莖葉或根組織，此為其生長發育之主要能量來源；運至胚中之醣類亦會重新合成澱粉並暫時儲存於胚中，於種子發芽初期短暫累積於胚盤組織，之後胚中澱粉主要累積部位則轉移至胚軸維管束組織周圍。由去除莖葉組織造成胚中澱粉過量累積或將植株移至全黑暗處理抑制光合作用造成胚中澱粉減少之現象，推測莖葉等積儲組織對碳素的需求會影響胚中澱粉之代謝。並且，由黑暗處理後胚中澱粉代謝酵素α-amylase之活性並未受到影響，而澱粉合成酵素granule-bound starch synthase (GBSS)Ⅰ, Ⅱ及starch branching enzyme(SBE) Ⅰ, Ⅱ, Ⅳ基因表現受到抑制，推測澱粉合成酵素扮演黑暗處理下胚中澱粉代謝之調控關鍵角色，並由黑暗處理後胚中水溶性醣類含量下降及黑暗處理同時外加葡萄糖後SBEIII表現相較黑暗處理有升高之現象，推測黑暗處理下水溶性醣類含量亦為胚中澱粉之代謝調控之間接調控因子。 第二部份： 蔗糖轉運蛋白(sucrose transporter; SUT)為負責運輸蔗糖進行跨膜運輸之轉運蛋白，經由水稻基因組序列分析，目前已知水稻具有五個SUT基因，分別命名為OsSUT1, 2, 3, 4, 5。本研究之目的為探討葡萄糖分子對水稻胚發芽時，胚中OsSUT1及OsSUT2基因表現之影響及其調控機制，首先藉由水稻分離胚培養之實驗，發現於培養液中加入葡萄糖，其於短時間(一天)培養後，OsSUT1之基因表現會被抑制，但在長時間(五天)於含葡萄之培養液中培養的胚組織中，OsSUT1之表現則會明顯的升高，由乾燥或是浸潤後四天之水稻胚處理葡萄糖一天後OsSUT1表現皆會受到抑制，得知此葡萄糖短時間與長時間效應對於OsSUT1之不同影響主要為葡萄糖處理時間長短所造成；另一方面，外加葡萄糖於培養液中，不論於短時間或長時間培養皆會促進OsSUT2表現，且其促進之效果隨培養時間增長而更為明顯。藉由葡萄糖衍生物3-O-Methylglucose以及六碳糖激酶(hexokinase)抑制物N-acetyl glucosamine對胚中SUT1, 2之影響得知葡萄糖對於OsSUT1基因之表現調控，不論於短時間或長時間之影響皆為Hexokinase-dependent pathway，而對OsSUT2基因之表現調控則為Hexokinase-independent pathway。

Part1: Endosperm starch would be hydrolyzed to glucose and maltose and further being sent to the newly synthesized shoot and root through embryo during rice (Oryza sativa L.) seed germination and seedling establishment. In embryos, the soluble sugar from endosperm could be converted to starch and transiently accumulated in scutellums for few days. Following, the starch resynthesis was began to be presented in the cells surrounding the vascular bundles of embryos. Since the hyperaccumulation of starch in embryo tissues was observed in shoot-removing seedlings and low levels of starch content were found in low-photosynthetic efficiency seedling embryos in dark conditions, it was suggested that the amount of starch biosynthesis in embryo tissues were depended on the sink tissue demand. Accroding to the expressions of granule-bound starch synthase (GBSS)Ⅰ, Ⅱ, and starch branching enzyme (SBE) Ⅰ, Ⅱ, Ⅳ responsive to dark treatment, it indicated that the starch level in embryo tissues of growing seedlings in dark was mainly controlled by starch synthetic efficiency. Moreover, SBEIII expression in dark was recovered when glucose was supplied into medium. It was suggested that sugar may function as one of the factors to regulate SBEⅢ gene expression in embryos of young seedlings. Part 2: Sucrose transporters are transmembrane proteins in charge of transporting sucrose across cell membrane. There are five sucrose transporter isogenes in rice (Oryza sativa L.) and named OsSUT1, 2, 3, 4, 5. During embryo germination, effect of glucose on OsSUT1 expression in embryos presented a bi-phase process. OsSUT1 expression was repressed in embryos when germinated in glucose-containing medium after 1-d treatment; however, it was enhanced after 5-d treatment. It was proposed that the differential effects of glucose on OsSUT1 expression were mainly affected by glucose treatment period. Besides, OsSUT2 expression was enhanced in embryos when germinated in glucose-containing medium after 1 and 5-d treatment. According to the effects of sugar analog, 3-O-Methylglucose, and hexokinase inhibitor, N-acetyl glucosamine, on OsSUT1 expressions, it was suggested that the signal transduction for regulating glucose-responsive OsSUT1 expressions in embryos were mediated by hexokinase-dependent pathway. On the other hand, glucose affected OsSUT2 expression was through a hexokinase-independent.