植物細胞依賴囊泡運輸系統(vesicle trafficking system)來進行胞器間的物質傳送，而該系統也多方面的影響了植物的生長與發育。例如，因逆境而受損的生物膜，需經該系統之運補而得以更新修復。此外，花粉管的延伸，也需要囊泡將細胞壁的組成分子傳送到生長尖端。顯然，囊泡運輸必須要有專一性，以確保各囊泡能將特定物質正確運至特定目的地。然而，與植物逆境耐受性及植物尖端生長有關的囊泡專一性因子，所知匱乏。進一步瞭解囊泡運輸對植物逆境耐受力及尖端生長的影響，將相當有助於改良具經濟價值之作物。本實驗室用功能性遺傳學的方法，從阿拉伯芥中篩選出若干對高溫逆境過度敏感之突變植株。其中的hit1-1 突變株 (as heat intolerant )，除了對高溫逆境失去正常之耐受性，對滲透逆境(osmotic stress)也過份敏感。經由基因定位及染色體行進的辨識，我們得知HIT1 cDNA 所對應之產物與酵母菌之Vps53p 具有相似性。Vps53p 在酵母菌裡，與Vps52p 及 Vps54p 共構成為囊泡繫鏈複合體 (Vesicle tethering complex)，調節與高基氏體有關的囊泡運送 (vesicle trafficking)。缺乏Vps53p 的酵母菌突變株，對熱逆境也同樣失去耐受性，卻可藉由HIT1 的表現得到補償。以HIT1 啟動子嵌合GUS 報導基因做實驗，結果顯示在花部組織中，HIT1 基因的表現只有在成熟的花粉粒及延伸的花粉管。此外，點突變的 hit1-1 會減緩花紛管的延伸。而帶有T-DNA 插入之hit1-2 及hit1-3，其花粉管的發育則完全受抑制。綜合論之，HIT1 是目前唯一經實驗證實，與植物逆境耐受度有關聯性的囊泡繫鏈同源蛋白質。而其對花粉管的伸長，也有決定性的角色。本研究計劃主要將針對以上發現，就HIT1 對植物囊泡運輸、尖端生長，及逆境耐受之影響做進一步的探究。研究策略包括利用pull down assay 及fluorophore-based protein interaction assay 等方法來判定 HIT1 是否會與阿拉伯芥同源於酵母菌Vps52p 及Vps54p 之蛋白質互動；利用生物膜染劑 FM4-64 及囊泡運送阻斷劑Brefeldin A 等藥物來追蹤HIT1 及 hit1-1 所參與之囊泡傳輸路徑；利用HIT1:GFP 或hit1-1:GFP 在花粉管的表現及分析HIT1 啟動子活性等方法來解析 HIT1 對植物尖端生長的調控; 利用熱引發葉綠素螢光之測量及氧化逆境誘導劑methyl viologen 之處理等方法來檢視hit1-1 對生物膜穩定性的影響。藉由以上之探討與確認，必能擴展學界對植物囊泡運輸的了解，進而提供改良作物適應逆境及提高受孕能力之途逕。本研究也會對實驗室所篩選出之其它hit 突變株，進行基因定位。The vesicle trafficking system is responsible for membrane transport and cargo delivery within the cell, and is crucial for plant growth and development at various aspects. For example, membrane rejuvenation through vesicle trafficking is important for plant to tolerate environmental stresses. Enhanced vesicle trafficking is also essential for pollen tube elongation as many molecules must be translocated to the growing tip. It is vital for these vesicles to maintain their identity so that each of them can find its unique target and deliver its cargo to the correct destination. Nevertheless, the genetic determinants that are required to achieve the necessary accuracy for plant tip growth and stress tolerance are largely unknown. Previously, we isolated an ethyl methanesulfate (EMS)-mutagenized Arabidopsis hit1-1 (heat-intolerant) mutant that is hypersensitive to both heat and osmotic stresses. Map-based cloning revealed that HIT1 encodes a homolog of the yeast Vps53p protein (Lee et al., 2006). The yeast Vps53p protein has been shown to be a tethering factor that associates with Vps52p and Vps54p in a complex formation to mediate the recognition between vesicles and the late Golgi. The growth of yeast vps53Δ null mutant also shows reduced thermotolerance, and expression of HIT1 in this mutant can partially complement the defect. Meanwhile, our HIT1 promoter-driven GUS reporter assay revealed that the expression of HIT1 in reproductive tissues is male-specific. Furthermore, while hit1-1 mutation results in reduced pollen tube length, disruption of HIT1 by T-DNA insertion completely inhibits the pollen tube growth. Collectively, HIT1 is the first and so far the only gene homologous to tethering factors linked to the tolerance of environmental stresses in higher eukaryotes, and the molecular event governed by HIT1 is also required for pollen tube elongation. More understanding of the HIT1 should definitely provide new insights into the importance of the vesicle tethering machinery in plants. Experiments are therefore proposed in this study with specific aims. The interaction of HIT1 with Arabidopsis homologs of yeast Vps52p and Vps54p will be examined by approaches such as pull-down assay and fluorophore-based protein interaction assay. The vesicle trafficking events mediated by HIT1 will be analyzed using methods such as tracing the movement of membrane-selective dye FM4-64 within HIT1 and hit1-1 cells. The roles of HIT1 in tip-growing cells will be studied with transgenic plants expressing HIT1 or hit1-1 fluorescent fusion protein and by HIT1 promoter analysis. The effects of HIT1 on plant stress tolerance will be investigated by analyzing membrane stability using methods such as methyl viologen treatment and heat-induced fluorescence measurement. In the meantime, screening for more hit mutants and mapping of new hit loci will be continued. 研究期間：9808 ~ 9907
