阿拉伯芥heat intoleran 2（hit2）突變株一顆對高溫敏感。HIT2基因編碼的產物是核運輸受體EXPORTIN1A，其功能為辨識帶有NES的蛋白質，助其從細胞核輸出到細胞質。阿拉伯芥有兩個XPO1同源基因，分別為XPO1A及XPO1B。在先前的研究中發現，hit2/xpo1a突變株對熱休克、持續性高溫逆境和強光逆境敏感，而xpo1b突變株則無，這明確顯示出兩者在多種逆境耐受能力上，扮演著不同的角色。已知高溫逆境會刺激熱逆境轉錄因子heat stress transcription factors（HSFs）表現並進入細胞核啟動下游抵禦機制，例如熱休克蛋白。欲釐清HIT2/XPO1A的突變究竟困住哪些受質以致造成種種性狀，本研究嘗試從帶有NES的HSFs著手。為了找尋是否有HIT2/XPO1A專一辨認的受質（HSFs），我們將其接上Green fluorescent protein（GFP），產生融和蛋白，觀察他們在野生型（WT）、hit2 和xpo1b突變株的阿拉伯芥原生質體中，常溫下與高溫下細胞核質之分佈變化。我們找到AtHsfA6b是HIT2的專一受質。惟在高溫下觀察到AtHsfA6bΔNES/a6b-1與AtHsfA6b/a6b-1兩者對高溫都不敏感，顯示AtHsfA6b雖會被HIT2的專一調節，但HIT2之高溫敏感性狀並不源自於AtHsfA6b困之於細胞核內。本研究進而觀測，AtHsfA6b/a6b-1與AtHsfA6bΔNES/a6b-1對其他類型的環境逆境的耐受性，並與hit2突變株進行比對，發現AtHsfA6b之細胞核質分佈扮有其角色在植物耐受除高溫以外的環境逆境中。;Arabidopsis heat-intolerant 2（hit2）mutant was isolated because it displays heat and light sensitive phenotypes. The mutated locus was later mapped to be at the EXPORTIN1A（XPO1A）gene, which encodes a nuclear transport receptor whose function is to mediate transportation of nuclear export sequence（NES）-containing proteins from nucleus to cytoplasm. There are two XPO1 homologous genes, HIT2/XPO1A and XPO1B, in Arabidopsis genome, and they share ~87% amino acid sequence identity. However, only hit2/xpo1a but not xpo1b mutant plants exhibit heat- and high light-sensitivities phenotypes. This indicates that HIT2/XPO1Aand XPO1B are not functionally redundant, and implies the existence of HIT2/XPO1A -specific substrate. It is known that many heat stress transcription factors（HSFs）contain both nuclear localization sequence nuclear localization signal（NLS）and nuclear export signal（NES）, and are the major regulators for heat and high light stress responses in plant. To identify which of them might cause hit2/xpo1a phenotypes, we started by examining the intracellular localization of green fluorescent protein（GFP）-tagged HSFs in protoplasts prepared from WT, hit2/xpo1a and xpo1b leaves, at both normal（22°C）and high（37°C）temperature conditions. These results indicated that AtHSFA6b is HIT2/XPO1A-specific substrate. Both AtHsfA6b/a6b-1 and AtHsfA6bΔNES/a6b-1 are not sensitive to high-temperatures. These indicated that the retaintion of AtHsfA6b in the nucleous is not the cause resulting the hit2/xpo1a heat-intolerant phenotypes. I therefore examined the responses of AtHsfA6b/a6b-1 and AtHsfA6bΔNES/a6b-1 to other environmental stimuli and compared to that hit2/xpo1a. Results indicated that the Nucleo-cytoplasmic distribution of AtHsfA6b is necessary for plant to tolerate certain types of eveironmental stresses other than high temperatures. However, we found that hit2 and AtHsfA6bΔNES/a6b-1 has freezing-sensitive phynotype. AtHsfA6b/a6b-1 is not sensitive to freezing. At different degrees, how nucleo-cytiplasmic transport of AtHsfA6b cause the confining AtHsfA6b in nucleus of the different phenotype.
