阿拉伯芥種子萌發及後萌發時期RNA解旋酶突變株呈現對葡萄糖和離層酸高度敏感之特性

Abstract

為了適應多變環境，高等植物會感受逆境並調節其生長和發育。在植物的發育過程，葡萄糖除了提供養份之外，也扮演十分重要的訊息傳遞角色。本實驗室利用 3% 葡萄糖生長環境篩選數千個阿拉伯芥 T-DNA 插入突變株，得到數個突變株具有對葡萄糖高敏感之特性。本研究針對其中一個對葡萄糖高敏感之 rh57-1 突變株進行研究。AtRH57 的 cDNA 全長為 2049 bp，可轉譯出 541 個胺基酸，預測分子量為 60.9 kDa。蛋白質序列比對結果顯示指出，AtRH57 為第二類的 DEAD-box RNA 解旋酶，具兩個特性：一為演化樹分析屬於獨立分支ㄧ群，與其他 DEAD-box RNA 解旋酶蛋白質序列無全部或部份相同；二為與其他 DEAD-box RNA 解旋酶基因至少催化功能區中有一段相同序列之前含內含子。由反轉錄聚合酶鏈鎖反應 (RT-PCR) 分析，發現 AtRH57 在阿拉伯芥的每個器官皆有表現，其中以根的表現量最多，以腋生葉和果莢的 AtRH57 表現量較低。在 3% 與 4.5% 葡萄糖逆境下生長九天，野生株 (Col-0) 和 rh57-1 突變株的種子萌發率並無明顯差異。但相較於野生株，rh57-1 突變株有較低子葉綠化和子葉擴張率，且 rh57-1 突變株之胚軸與根的長度均明顯被抑制，顯示 rh57-1 對於葡萄糖具有高敏感性，且不是高濃度下的葡萄糖滲透壓造成。兩個不同插入位點 T-DNA 之 rh57-2 和 rh57-3 突變株，也與 rh57-1 突變株在葡萄糖逆境下的外表型相符合，證明 AtRH57 基因的確受到破壞而導致 rh57-1 突變株對葡萄糖具高敏感性。即時定量反轉錄聚合酶鏈鎖反應 (real-time PCR) 分析 4.5% 葡萄糖逆境中，rh57-1 突變株的醣類調節相關基因表現，發現與野生株相比確實有明顯改變。此外，rh57-1 的種子萌發和早期植株發育也對離層酸具高度敏感性。添加離層酸抑制劑 fluridone 到不同濃度葡萄糖培養基中，發現 rh57-1 子葉擴張皆有明顯恢復。高濃度葡萄糖逆境下，rh57-1 突變株離層酸含量變化的確比野生株明顯高出兩倍。且 rh57-1 突變株內之離層酸訊息傳遞相關基因 ABI3、ABI4 和 ABI5 明顯受到誘導。最後，利用阿拉伯芥原生質體進行 AtRH57 蛋白質定位分析，證實 EGFP-AtRH57 以及 AtRH57- EGFP 的融合蛋白皆位於細胞核內。

To cope with changing environment, higher plants can sense sugar levels and accordingly adjust their growth and development. In addition to supply nutrients, glucose plays an important signaling role in different developmental processes in plants and other organisms. Thousands of Arabidopsis T-DNA insertion mutants grown at 3% glucose condition have been screened. Subsequently, several mutants exhibiting glucose hypersensitivity were obtianed. One of glucose hypersensitive muntants, rh57-1, was selected for further investigations. The full length AtRH57 cDNA contains 2049 bp encoding a polypeptide of 541 amino acids with a calculated molecular mass of 60.9 kDa. Sequence alignment revealed that AtRH57 is a member of Class II DEAD-box RNA helicase family because this class exhibits (1) Phylogenetic tree analysis indicated that completely identical or partially identical protein sequence with other DEAD-box protein;(2) AtRH57 does not share at least one intron at an identical position with one other gene of the class. Reverse transcription (RT-PCR) analysis showed that AtRH57 mRNA was present in all the organ, and the expression was highest in root and lower in cauline leaf and silique. When grown in the presence of either 3% or 4.5% glucose concentration for 9 d, no seed germination rate of the rh57-1 mutant did not change when compared with wild-type (WT; Col-0) plants. However, the rh57-1 mutant exhibited impaired cotyledon greening and expansion. Moreover, the hypocotyl and root of rh57-1 mutant are singnificantly inhibited. No developmental difference between wild-type and rh57-1 mutant plants was observed when grown at the same concentration mannitol, indicating that the growth impairment of rh57-1 exhibits glucose hypersensitivity, but the impairment is not caused by osmotic stress. The rh57-2 and rh57-3 seedlings exhibited phenotypes similar to rh57-1 on the medium supplemented with 4.5% glucose, further confirm the disruption of the AtRH57 gene resulting in the rh57-1 mutant hypersensitive to glucose. Real-time PCR analysis indicated that some of these glucose-responsive genes were singnificantly altered in rh57-1 mutant seedlings in the presence of 4.5% glucose conditions. The rh57-1 seed germination and early seedling development also exhibited sensitivity to ABA. The glucose inhibition of cotyledon expansion of rh57-1 was markedly rescued when various concentration of glucose supplymented with 1 μM fluridone. In the presence of 4.5% glucose, variations of ABA content in the rh57-1 seedling increased 2-fold more than that in WT. Real-time PCR analysis revealed several key genes involved in the ABA signaling genes were induced by glucose in the rh57-1 mutant, such as ABI3, ABI4 and ABI5. Finally, subcellular localization of the AtRH57 in Arabidopsis protoplast analysis showed that the EGFP-AtRH57 and AtRH57-EGFP fusion protein were localized to the nucleus.