阿拉伯芥熱逆境反應基因於生殖生長期之功能分析

Abstract

發生在作物生殖生長期的高溫逆境往往直接導致作物減產。因此，植物在生殖生長時期的高溫逆境反應是一個重要的研究課題。然而，過去在這方面的研究卻很少。本文以模式植物—阿拉伯芥( Arabidopsis thaliana )花及花粉進行耐熱性試驗，探討生殖生長期植物的耐熱機制。利用受熱處理後之果莢以及種子發育和花粉發芽率及花粉管長度分別建立花和花粉的耐熱量化指標，可發現熱馴化處理使剛展開的花和離體的花粉明顯產生後天耐熱性。利用此方法分析幼苗期重要的耐熱基因在生殖生長期的角色，發現 hsp101、hsa32、以及轉錄因子HSFA1的三重突變體eTK，其剛展開的花在熱馴化處理後無法產生後天耐熱性。在後天耐熱試驗處理後，這些突變體的果莢顯著比野生型短，種子的數目也顯著降低。表示HSA32、 HSP101及轉錄因子HSFA1a/b/d 為花的後天耐熱性機制所必需。西方墨點法檢測熱馴化處理後HSP101與HSA32的表現，結果與幼苗期一致，在花朵中HSP101與HSA32能夠相互正回饋調節，使其產生長期後天耐熱性。然而hsp101與hsa32突變體的成熟花粉後天耐熱性並不受到該基因缺失的影響，表示這兩個蛋白質對於花粉後天耐熱性機制不扮演重要功能。檢測熱休克轉錄因子HSFA1a/b/d/e 的T-DNA三重突變體花粉耐熱性，結果顯示除了HSFA1a以外，HSFA1e為啓動花粉後天耐熱性的重要轉錄因子。然而HSFA1e對幼苗和整朵花的後天耐熱性不具有重要功能，故HSFA1e在花粉耐熱試驗結果與在幼苗的結論相反。綜合以上結果，植物生殖生長期的花器與花粉，可以透過熱馴化產生後天耐熱性，幫助植物避免第二次更高溫逆境的傷害。但成熟花粉的後天耐熱性機制顯然與其它組織有所不同。

Heat stress response has been investigated intensively in vegetative tissues of plants, but not in reproductive tissues, which are directly relevant to productivity in agriculture. In this study, we established biological assays for Arabidopsis flowers and pollen to identify important components for acquired thermotolerance (AT) at reproductive stage. Like seedlings, newly opened flowers could endure harsh heat if primed with milder heat, manifesting by elongation of siliques and seed setting following the heat treatment. Pollen also acquire enhanced thermotolerance after heat acclimation, which was demonstrated by measuring in vitro pollen germination rate and pollen tube length. We then used these assays to show whether previously identified components for AT in seedling stage are also required at reproductive stage. The T-DNA knockout mutants of HSP101 and HSA32 showed significantly retarded growth of silique and decreased seed number after treatments, suggesting HSP101 and HSA32 play important roles in heat acclimation in flowers. The pollens of HSP101 and HSA32 mutants did not showed significant defect in AT, indicating that these thermotolerance components have differential functions in different organs. Furthermore, we evaluated the individual role of the four HSFA1 genes, HSFA1a/b/d/e, in pollen AT. HSFA1a, A1b, and A1d were shown to be the redundant master regulators of heat stress response in vegetative tissues. Intriguingly, HSFA1a and A1e, but not HSFA1b and A1d, play major role in pollen AT, suggesting the subfunctionalization of HSFA1 genes in Arabidopsis. Taken together, we suggest that flowers and pollens have the capacity of acquiring thermotolerance by acclimation, which protects them from damage caused by severely high temperature. Our data also indicate that common and distinct thermotolerance components are employed in different organs.