阿拉伯芥中非生物逆境誘導之轉錄因子AtERF53的功能分析

Functional Characterization of an Abiotic Stress-Inducible Transcription Factor AtERF53 in Arabidopsis thaliana

謝恩蓉(En-Jung Hsieh)

指導教授 ： 林讚標

國立臺灣大學/生命科學院/植物科學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.03121

摘要

AP2/ERF蛋白在植物生長發育和生物及非生物逆境反應中都扮演著重要的角色。Ethylene response factor 53 (AtERF53)蛋白為阿拉伯芥中的轉錄因子，在ERF家族中屬於第一群，其基因在乾旱及鹽逆境下很早就會被誘導表現，乾旱逆境下更是會被大量誘導表現。由於將AtERF53大量表現在阿拉伯芥的轉殖株時，皆測不到其蛋白質表現，造成AtERF53基因的功能不容易被研究。之前的研究發現AtERF53會和RING domain ligase2 (RGLG2)在細胞核中互相影響而導致ERF53蛋白被降解。將AtERF53大量表現在RING domain ligase1/ RING domain ligase2 (rglg1rglg2)雙基因突變株時，可以使AtERF53蛋白穩定的表現，此發現進而提供了研究AtERF53生理功能的機會。此篇研究中證實了在野生型阿拉伯芥中AtERF53的表現亦會被熱和離層酸(ABA)處理誘導其表現。組織學分析中(histochemical GUS analysis)將AtERF53啟動子接上GUS後，可以發現GUS活性表現主要位在根、莖、葉，特別在維管束組織中表現最為顯著，而保衛細胞以及花序中沒有測到活性表現。利用阿拉伯芥的原生質體(protoplast)進行暫時性表現質體(transient expression assay, cotransfection)的實驗中可判斷AtERF53具有正向活化下游基因的活性。當AtERF53大量表現在rglg1rglg2雙基因突變株時，其轉殖株具有較佳的耐熱性而且其ABA和脯胺酸(proline)含量相較於rglg1rglg2雙基因突變株都有增加。ABA誘導氣孔關閉的實驗中可觀察到AtERF53大量表現在rglg1rglg2雙基因突變株的轉殖株之氣孔孔徑較rglg1rglg2雙基因突變株小，顯示AtERF53參與調節氣孔之運動。利用微陣列技術(microarray assay)將大量表現之轉殖株和rglg1rglg2雙基因突變株的基因表現進行扣減後，找到許多和逆境相關的基因如DREB1A、COR15A、COR15B、PLC、cpHSC70s以及proline和ABA代謝相關的基因表現量都被調控。接著進一步利用染色體免疫沉澱研究法(chromatin immunoprecipitation assay)來鑑定由microarray assay找出的基因中哪些為AtERF53的直接下游基因。綜上所述，根據遺傳、分生、生化方面的實驗結果可以推斷AtERF53蛋白在阿拉伯芥中扮演一個轉錄因子的角色，進而提升植物對非生物逆境的耐受性。

關鍵字

AP2/ERF轉錄因子 ； AtERF53 ； 非生物逆境

並列摘要

AP2/ERF proteins play crucial roles in plant growth and development and in responses to biotic and abiotic stresses. ETHYLENE RESPONSE FACTOR 53 (AtERF53) is a transcription factor and belongs to group 1 in the ERF family which was induced in the early stage of dehydration and salt treatment, especially was highly induced by dehydration. The functional study of AtERF53 is hampered because its protein expression in Arabidopsis is vulnerable to degradation in overexpressed transgenic lines. In a previous study, we found that RING domain ligase2 (RGLG2) interacted with AtERF53 in the nucleus, and mediated AtERF53 ubiquitination for proteasome degradation. Taking advantage of the RING domain ligase1/ RING domain ligase2 (rglg1rglg2) double mutant in which AtERF53 can express stably, we investigated the physiological function of AtERF53. In this study, we demonstrate that expression of AtERF53 in wild-type Arabidopsis was responsive to heat and abscisic acid (ABA) treatment. Histological analysis showed that AtERF53 was expressed in stems, leaves, roots, and especially in the vasculature, but not in guard cells or inflorescence. From results of the cotransfection experiment, we concluded that AtERF53 has positive transactivation activity. Overexpression of AtERF53 in the rglg1rglg2 double mutant conferred better heat-stress tolerance and had resulted in higher endogenous ABA and proline levels compared to rglg1rglg2 double mutants. AtERF53 also has a function to regulate guard-cell movement because the stomatal apertutre of AtERF53 overexpressed in rglg1rglg2 double mutant was smaller than that in the rglg1rglg2 double mutant under ABA treatment. In a global gene expression study, we found higher expression of many stress-related genes, such as DREB1A, COR15A, COR15B , PLC, P5CS1, cpHSC70s, and proline and ABA metabolism-related genes. Furthermore, we identified several downstream target genes of AtERF53 by chromatin immunoprecipitation assay. In conclusion, these genetic, molecular and biochemical studies may explain how the AtERF53 transcription factor contributes to abiotic stress tolerance in Arabidopsis.

並列關鍵字

AP2/ERF transcription factor ； AtERF53 ； abiotic stress

參考文獻

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