探討阿拉伯芥ETO1基因家族調控乙烯荷爾蒙生合成之研究;Genetic and mechanistic studies of ETO1 gene family in regulation of ethylene biosynthesis in Arabidopsis thaliana

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Title:	探討阿拉伯芥ETO1基因家族調控乙烯荷爾蒙生合成之研究;Genetic and mechanistic studies of ETO1 gene family in regulation of ethylene biosynthesis in Arabidopsis thaliana
Authors:	顧心元;Gu, Shin-Yuan
Contributors:	生命科學系
Keywords:	乙烯;蛋白質二聚化;化學遺傳學;阿拉伯芥;基因家族;白化苗;Ethylene;ETO1;protein dimerization;CTL1;acsinones;chemical genetics;etiolated seedlings;ETHYLENE OVERPRODUCER1;CHITINASE LIKE1;ACC oxidase;root elongation
Date:	2020-07-22
Issue Date:	2020-09-02 15:26:43 (UTC+8)
Publisher:	國立中央大學
Abstract:	乙烯為分子構造簡單的烴類氣體，參與許多植物的生理現象和發育過程。為了研究乙烯生合成的機制及新發現參與此路徑的基因，我們分析阿拉伯芥ETHYLENE OVERPRODUCER1（ETO1）家族的功能特性及利用化學遺傳學鑑定能回復到eto1特徵（ret）之阿拉伯芥突變株。ETO1、ETO1-LIKE1（EOL1）和EOL2是Broad-complex，Tramtrack，Bric-a-brac（BTB）蛋白家族，此家族成員共同調節阿拉伯芥中第2型1-氨基環丙烷-1-羧酸合酶之活性。儘管ETO1和EOL1 / EOL2基因製造出結構上相似的蛋白質，但是遺傳研究上發現它們具有相同及相異的功能。ETO1，EOL1和EOL2基因表達具有重疊但不完全相同的組織特異性。然而，在ETO1啟動子的控制下， EOL1和EOL2基因的表達均不能完全回復eto1表型，這說明ETO1和EOL1 / EOL2蛋白質的功能不盡相同。ETO1蛋白本身會形成同源二聚體，亦與EOL蛋白形成異源二聚體。此外，與EOL1 / EOL2蛋白相比，CULLIN3（CUL3）會偏好與ETO1蛋白交互作用。ETO1的BTB結構區域足以與CUL3蛋白交互作用，並且此區域是ETO1同源二聚化所必需的。然而，在阿拉伯芥基因轉殖植物中表現功能性區域互換之嵌合蛋白質，分析顯示ETO1的BTB結構區域是必要的、不可缺少的，但其不足以達到ETO1所有的功能。eto1-5為錯義突變，其異亮氨酸取代了苯丙氨酸，ETO1F466I蛋白削弱了其本身蛋白二聚作用及與EOL蛋白之相互作用，但不影響其與CUL3或ACS5蛋白的結合。我們的研究發現ETO1和EOL1 / EOL2之蛋白質-蛋白質交互作用，此機制在乙烯生合成中扮演至關重要的角色。 Acsinones為抑制乙烯生合成之化學小分子，ret9突變會降低eto1突變株白化苗對acsinones敏感性。經由圖譜定址選殖(map-based cloning)分析RET9，證實為CHITINASE LIKE1（CTL1）之點突變中，其145半胱氨酸被置換為酪氨酸。CTL1為植物細胞壁生合成、抵抗逆境之重要基因。ctl1ret9的白化苗呈現較短的下胚軸及根部。ctl1ret9幼苗經外生性ACC的處理，在抑制主根生長上較為敏感。經遺傳學分析，ctl1ret9在乙烯不敏感突變株組合下證實，完整的乙烯反應途徑對於ctl1ret9影響白化苗的根部型態改變是重要的。此外ctl1突變株的乙烯產量上升，是由於ACC氧化酶（ACO）基因表現量和酵素活性的增加。在ctl1ret9白化苗，與乙烯生物合成及訊息路徑相關的基因均表現量上升。以上結果證明，CTL1會參與調控ACO酵素活性及乙烯訊息路徑，此發現有助於我們理解，當植物細胞壁完整性被破壞時，乙烯影響根部生長的作用機制。 ;Ethylene is a simple hydrocarbon gas that regulates a number of physiological and developmental events in plants. To understand the mechanism and uncover new genes involved in the ethylene biosynthesis, we studied the functional properties of the Arabidopsis ETHYLENE OVERPRODUCER1 (ETO1) family and characterized the Arabidopsis revert to eto1 9 (ret9) mutants by use of a chemical genetics. ETO1, ETO1-LIKE1 (EOL1) and EOL2 are members of the Broad-complex, Tramtrack, Bric-a-brac (BTB) protein family that collectively regulate type-2 1-aminocyclopropane -1-carboxylic acid synthase (ACS) activity. Despite ETO1 and EOL1/EOL2 encode structurally related proteins, genetic studies suggest that they do have redundant and distinct functions. ETO1, EOL1 and EOL2 exhibit overlapping but distinct tissue-specific expression patterns. Nevertheless, neither EOL1 nor EOL2 can fully complement eto1 phenotype under the control of ETO1 promoter, which suggests differential functions of ETO1 and EOL1/EOL2. ETO1 forms homodimers and heterodimers with EOLs. Furthermore, CULLIN3 (CUL3) interacts preferentially with ETO1. The BTB domain of ETO1 is sufficient for interaction with CUL3 and required for homodimerization. However, domain-swapping analysis in transgenic Arabidopsis suggests that the BTB domain of ETO1 is essential but insufficient for ETO1 full function. The missense mutation in eto1-5 strongly impairs its dimerization and interaction with EOLs but does not affect binding to either CUL3 or ACS5. Our findings reveal the mechanistic role of protein-protein interactions of ETO1 and EOL1/EOL2 that is crucial for their biological function in ethylene biosynthesis. The ret9 mutant exhibited reduced sensitivity to acsinones in etiolated eto1 seedlings. Map-based cloning of RET9 revealed a cysteine 145 to tyrosine substitution in CHITINASE LIKE1 (CTL1), which is required for cell wall biogenesis and stress resistance in Arabidopsis. Etiolated seedlings of ctl1ret9 showed short hypocotyls and roots. The ctl1ret9 seedlings showed enhanced sensitivity to exogenous ACC to suppress primary root elongation. Genetic analysis of combinational ethylene-insensitive mutants indicated that an intact ethylene response pathway is essential for the alterations in root but not hypocotyl in etiolated ctl1ret9 seedlings. Furthermore, an increased ethylene level in ctl1 mutants was related to elevated activity of ACC oxidase (ACO). Moreover, genes associated with ethylene biosynthesis and response were upregulated in etiolated ctl1ret9 seedlings. These results imply that CTL1 negatively regulates ACO activity and the ethylene response, which thus contributes to understanding a role for ethylene in root elongation in response to perturbed cell wall integrity.
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