阿拉伯芥熱休克因子HsfA7a/b在非生物逆境反應之功能性研究

Abstract

氣候變化導致自然災害頻發，如高溫、洪水、土壤鹽鹼化和長期乾旱，給全球各地帶來了巨大挑戰。非生物逆境，包括乾旱、高鹽、極端溫度及水淹等因素，已成為全球農業生產的主要問題，影響植物生長和作物產量，並對糧食安全構成威脅。植物在非生物逆境的應對過程中，需要啟動調控機制來維持細胞穩定性。熱休克轉錄因子 (HSFs)，在植物對多種非生物逆境反應中扮演重要角色。除了在高溫逆境中表現突出外，HSFs也廣泛參與植物應對乾旱、鹽鹼及低溫等多重逆境條件的反應，負責啟動下游逆境防禦基因的表達，促進植物的適應能力。為了更加了解HSFA7a/b在非生物逆境下扮演的角色，我們利用CRISPR-Cas9技術建立HSFA7a 與 HSFA7b的雙突變株。結果表明，HSFA7a 和 HSFA7b 在不同的非生物逆境條件下均被顯著誘導表達，顯示出它們可能參與植物多種非生物逆境反應。透過耐熱性、離層酸處理後的發芽狀況以及鹽處理後子葉綠化狀況試驗，證實 HSFA7a 負向調控逆境反應，而HSFA7b 扮演正向調控的角色。值得注意的是，兩者的單突變體呈現出相反的表現型，反映了這兩者在逆境反應中的功能差異。為了進一步解析HSFA7a/b的功能，我們透過雙分子螢光互補實驗以及冷光素酶互補成像分析確認了HSFA7a/b與 HSFA2 之間的蛋白質-蛋白質相互作用，並透過雙螢光素酶報導基因分析證實了它們在APX2基因表達調控中的協同作用。這些結果顯示，HSFA7a/b與HSFA2共同形成一個協同調控網路，幫助植物在非生物逆境中保持穩定，增強其適應能力。最後，RNA-seq分析中差異表達基因 (DEGs) 的重疊顯示，HSFA7a/b可能與植物清除活性氧反應有著密切相關。總結來說，我們的研究探討HSFA7a/b在植物應對多種非生物逆境中的調控機制，並識別出潛在的下游基因。

Climate change has resulted in more natural disasters, including higher temperatures, floods, soil salinization, and droughts, creating significant challenges for global communities. Abiotic stresses such as drought, high salinity, and extreme temperatures have a severe impact on agriculture and food security. Heat shock transcription factors (HSFs) are crucial in helping plants respond to these stresses by regulating defense gene expression. In our study, we used CRISPR-Cas9 to create double mutants of the HSFA7a and HSFA7b genes to explore their roles in abiotic stress responses. Our findings revealed that both genes are activated by heat, abscisic acid (ABA), salt stress, and cold stress. Specifically, HSFA7a negatively regulates responses to abiotic stress, while HSFA7b acts as a positive regulator. Protein interactions between HSFA7a/b and HSFA2 were confirmed using BiFC and LCI assays, and dual-luciferase reporter assays showed their involvement in regulating the APX2 gene. Additionally, RNA-seq analysis indicated their role in reactive oxygen species (ROS) scavenging. Overall, our research emphasizes the roles of HSFA7a/b in plant responses to abiotic stresses and identifies potential downstream genes involved.