水稻HSA32之耐熱功能分析

Abstract

研究報告指出，全球暖化趨勢將增加熱浪頻率與強度，熱浪所造成的高溫逆境對水稻品質與產量皆會造成負面影響。廣泛地探討並深入暸解水稻之耐熱機制及其限度，應有助於提出耐熱品種改良的方向與策略。前人以模式植物阿拉伯芥探討植物耐熱機制之結果顯示，熱休克蛋白HSP101大量表現不僅能幫助植物度過熱逆境，亦可維持其產量。此外，本實驗室發現HSP101的降解受到另一個熱誘導蛋白HSA32的調控，且HSA32的含量亦受到HSP101的正向調控。根據上述結果推論，HSP101與HSA32蛋白之間存在回饋交互作用以延長植物對熱逆境之記憶。基於這些研究，我們想進一步瞭解HSP101與HSA32是否在水稻中也存在類似的交互作用，以及此交互作用如何幫助不同品種的水稻對抗熱逆境。首先，我們取得由跳躍子Tos17插入破壞表現的基因默化突變株osha32，接著探討突變株於不同熱逆境條件下之耐受性。在熱馴化後經歷較長恢復期再處以熱逆境後，oshsa32的生長遲滯現象較野生型來得嚴重；然而，如熱馴化後僅經歷短期恢復的處理則與野生型無顯著差異。經由分子層次分析結果指出，oshsa32種子熱敏感的性狀與OsHSP101蛋白基礎量偏低相關；此外，相較於野生型植株而言，oshsa32 突變株經熱馴化後累積的 OsHSP101 蛋白降解較快，進而造成 oshsa32 幼苗亦較不耐熱。我們歸結出OsHSA32應經由維持熱馴化後OsHSP101的表達量，進而幫助水稻對抗熱逆境，其結果與阿拉伯芥有一致性的發現。有趣的是，熱誘導之OsHSP101含量維持時間在稉稻與秈稻中不同，推測與其需適應不同環境相關。相較秈稻品系N22，熱馴化後OsHSA32和OsHSP101的表現在稉稻品系Nipponbare中維持較長的時間，因此Nipponbare對長恢復期後再處以熱逆境的耐受性較佳；反之，N22則具有較高的基礎耐熱性。本研究提供合適的耐熱性篩選平台，未來將可用於瞭解水稻各品種之耐熱特性，以及應用於育種材料之篩選。

A number of reports predict that the frequency and intensity of heat wave will increase in the near future due to the effect of global warming. Heat waves result in heat stress (HS) that reduces the quality and productivity of rice. Extensive investigation and in-depth understanding of the mechanism and limitation of heat stress response may help the formulation of breeding strategy for improving rice heat tolerance. According to previous studies, HSP101 was shown to protect fruit production under HS condition in Arabidopsis. The degradation rate of HSP101 following its induction is retarded by HSA32, whose post-stress accumulation is enhanced by HSP101. The interplay between HSP101 and HSA32 is considered to extend the memory of heat acclimation in Arabidopsis. With this information, I was interested to know whether there is a similar interplay between these two heat shock proteins (HSPs) in rice, and what are their roles within different rice types under various heat stress conditions. We first obtained and studied the knockout mutants of OsHSA32 in one of rice subspecies japonica cv. Nipponbare. Disruption of OsHSA32 by independent Tos17 insertions caused significant growth retardation under the assay condition for long-term acquired thermotolerance (LAT), but not for short-term acquired thermotolerance (SAT). In addition, we noticed that OsHSP101 expression was reduced in the seeds of null oshsa32 mutants, which may account for the heat-sensitive phenotype of oshsa32. Furthermore, degradation of OsHSP101 was faster in oshsa32 than that in WT during recovery after acclimation treating (42oC, 2hr). Our data showed that OsHSA32 plays an important role in rice basal thermotolerance (BT) and LAT through maintaining the level of the major chaperone protein OsHSP101, which were in agreement with the previous studies done in Arabidopsis. Interestingly, differential degradation rates of HSP101 were observed in indica and japonica rice types. Both duration of the heat induced OsHSP101 and OsHSA32 were longer in rice subspecies japonica cv. Nipponbare than in indica cv. N22, which might account for a better tolerance for LAT assay in Nipponbare than in N22. However, Nipponbare became inviable after BT assay while N22 could continue to grow under the same condition. These data suggested that among various rice cultivars there are differential responses against different types of heat stress conditions. Thus, the assays established in this study allow a better characterization of different thermotolerance traits among rice types, which would be useful for selecting materials for breeding in the future.