| 摘要: | 為瞭解植物耐受高溫逆境之機轉，進而以遺傳工程改良作物耐熱力，以因應全球暖化之威脅，本實驗室以前向式遺傳學的策略，自ethyl methanesulfonate (EMS)處理過之阿拉伯芥M2子代中，篩選出一對持續性高溫逆境 (37°C培養4天)失去耐受性之突變植株，命名為heat-intolerant 5-1 (hit5-1)。後續分析發現，hit5-1雖對持續性高溫敏感，但對熱休克(44°C 培養40分鐘) 則比野生型更具耐受力。經基因定位、互補實驗及序列比對，確認hit5-1突變點落在編號At5g40280 之 ERA1 基因上。ERA1 所編碼的蛋白質產物，為蛋白質法尼酯化酵素(protein farnesyltransferase, PFT) 的β 次單元。法尼酯化(farnesylation)是蛋白質後轉譯修飾的一種方式。目前已知，ERA1 突變株會有種籽延遲萌芽、氣孔關閉、耐旱等ABA誘導的性狀。一般認定，植物細胞內，至少有一個ABA負調節因子於常態下須被法尼酯化。至於法尼酯化對植物熱逆境反應的調節，及熱逆境下存活力的影響，則未被報導過。hit5-1 的篩選及初步分析，無疑提供了最新穎及最直接的證據，指出法尼酯化在植物熱逆境反應中，扮演了必要性的角色。也據此，幾個重要的疑題亟須釐清。第一、hit5-1 突變所造成的高溫誘導性狀，是否有ABA訊息的介入？第二、hit5-1 對長期溫合高溫及短期熱休克有不同的反應。PFT的下游是否還有訊息傳遞的分歧，以區隔此二種不同的反應？如果是，有否可能以遺傳學的方式，進一步將這兩個分歧途徑區別出來？意即以hit5-1 為親代，篩出長期溫合高溫逆境「或」短期熱休克逆境下，性狀回復成野生型的雙突變植株？第三、已知熱休克蛋白質(heat shock proteins, HSPs) 的累積與否，直接影響植物熱休克的耐受能力。如是觀之，hit5-1 突變是否改變了HSP 基因的表現？hit5-1耐受熱休克性狀是否來自此一改變？本研究計畫的目的，就是設計並進行實驗，解釋闡說此三疑題。本研究計畫的結果，除了可以讓吾人對植物耐熱機轉有創新的瞭解，也將揭露更多法尼酯化調節細胞生理生化功能的面貌。 ;Scientific consensus suggests that global warming induced heat stress, which results when the ambient temperature is elevated above the normal optimum, will impact agriculture and food production worldwide. Thus, elucidating how plants respond to heat stress is essential for developing heat-tolerant crops for meeting the future food needs. For this and to identify genetic determinants that induce such responses, we used a forward genetics approach to screen ethyl methane sulfonate-mutagenized Arabidopsis mutants that were more thermosensitive than the wild-type plants. One of these mutants, hit5 (for heat-intolerant 5), was isolated because prolonged incubation at 37°C for 4 days was determined to be lethal for the mutant, but not for the wild-type seedlings. However, sudden heat shock treatment at 44°C for 40 min was found to be lethal for the wild-type plants but not for the hit5 seedlings. The mutated locus was mapped to the ENHANCED RESPONS TO ABA 1 (ERA1) gene, which encodes the β-subunit of the protein farnesyl transferase (PFT) that is responsible for the covalent linkage of a 15-carbon farnesyl moiety to target proteins. Farnesylation is a type of post-translational modification. As the name implies, mutations in ERA1 gene are known to confer enhanced response to abscisic acid (ABA) phenotypes. This indicated that at least one of the PFT-targeting proteins is a negative regulator of ABA signaling. However, the effects of protein farnesylation on plant survival under high temperature conditions have not yet been investigated. Thus, the isolation and initial characterization of hit5/era1 mutants provided novel and direct evidences of farnesylation being an essential part of heat stress response in plants. Nevertheless, several questions need to be clarified. First, is the change in thermosensitivity in hit5/era1 involves ABA signaling? Second, depending on the form of heat stress, hit5/era1 exhibit either heat-sensitive or heat-tolerant phenotypes. Hence, how are PFT-mediated heat stress response divided? Can this division be further distinguished genetically? That is, can a revertant, in which only one of the hit5 temperature-dependent phenotype is reverted to the wild type, be isolated? Third, the accumulation of heat shock proteins (HSPs) is known to contribute to the ability of plants to survive heat shock. Does hit5 mutation affect HSP gene expression? If so, is this directly involved in the hit5-mediated heat shock tolerance phenotype? In this research proposal, we designed specific experiments to address these substantial and important questions. Results from this study might not only provide novel insights on plant HSRs, but also reveal the versatile roles of protein farnesylation in the regulation of plant cell functions. |
