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標題: 阿拉伯芥atSKD1基因參與auxin運輸的功能分析
Functional analyses of atSKD1 in auxin transport mechanism of Arabidopsis
作者: 楊婷婷
Yang, Ting-Ting
關鍵字: atSKD1
endosome trafficking
polar auxin transport
出版社: 生命科學系所
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摘要: Auxin負責調節植物生長發育及適應環境,是重要的訊息傳遞分子,在原生質膜上有許多auxin運輸蛋白,其中pin-formed (PIN)蛋白會在原生質膜上呈極性分布以調控auxin的極性運輸。然而PIN不會一直位於原生質膜上,其可經由endocytosis進入內膜系統並在不同的endosome間進行傳遞,當傳遞至late endosome/multivesicular body (MVB)時,MVB膜上的retromer complex可使PIN蛋白返回early endosome,再重返回到原生質膜以維持PIN在原生質膜上的極性分布。MVB除了參與PIN蛋白極性分布外,還可透過endosomal sorting complex required for transport (ESCRT)蛋白複合體進行MVB sorting,以便MVB與液胞融合時液胞蛋白得以釋放至液胞內,完成液胞蛋白的運輸。Suppressor of K+ transport growth defect 1 (SKD1)是作用於MVB的ATPase,當ESCRT蛋白複合體完成MVB sorting,atSKD1會利用ATP水解釋能使其瓦解,以便進行下一次MVB sorting所需。本論文藉由觀察反向atSKD1轉殖株之auxin極性運輸能力,探討atSKD1以至於ESCRT蛋白複合體參與auxin極性運輸的機制。 偵測反向atSKD1轉殖株T5子代之內生性atSKD1表現量,發現在各轉殖系間表現量下降程度不同。首先觀察內生性atSKD1表現量下降50%之7.3.6植株的側根密度,與wild-type比較下,發現未添加auxin時,7.3.6植株之側根密度高出104%,當NAA濃度達0.05 μM,7.3.6植株之側根密度則低約39%,除了側根密度變化外,7.3.6植株之根毛長度多出了39%,且根毛密度亦高出33%,顯示atSKD1參與調節auxin的極性運輸,進而維持側根及根毛發育。進一步偵測7.3.6植株中auxin極性運輸相關蛋白基因表現量,與wild-type比較下,發現AUX1、LAX3、PIN1、PIN2、PIN3、PIN4、PIN5、PIN7、PGP1、PGP19及ACL5之表現量呈不等程度下降,而PGP4表現量則是大幅提升,顯示當auxin的極性運輸途徑受到改變時,可能會影響auxin極性運輸相關蛋白基因的表現量。接著觀察7.3.6植株根部auxin分布情形,與wild-type比較,發現auxin在根尖以上之中柱部分的累積情形消失,顯示atSKD1會影響auxin的極性運輸。進一步偵測全株auxin的累積量,發現7.3.6植株不論在有無添加auxin環境下,auxin累積量皆高於wild-type,顯示atSKD1的確參與auxin的極性運輸,促使auxin大量累積在植株中。 接著觀察內生性atSKD1表現量下降程度無顯著差異之7.1.6及7.2.9植株,與wild-type比較下,當NAA濃度為0.2 μM,7.1.6植株之側根密度高出約38%,而在7.2.9植株中之側根密度變化則無顯著差異。進一步偵測7.1.6及7.2.9植株中auxin極性運輸相關蛋白基因表現量,與wild-type比較下,在7.1.6植株中發現PIN1、PIN2及PIN5之表現量下降,而PIN7、PGP1及PGP19之表現量則會提升,接著在7.2.9植株中發現AUX1、LAX3、PIN1、PIN2、PIN5、PIN7、PGP19及ACL5之表現量會下降,上述基因表現量變化幅度不大,顯示雖然atSKD1表現量無明顯下降,還是會造成植株對auxin反應的改變。 綜合以上結果推論,透過atSKD1維持ESCRT蛋白複合體的正常運作,使位於MVB上的retromer complex得以調節auxin運輸蛋白在原生質膜上的分布,維持auxin極性運輸使植株正常生長。
The plant hormone auxin is a small molecule regulates growth and development in response to environmental signals. There are many auxin-transport proteins localized to the plasma membrane. The polar localization of pin-formed (PIN) on plasma membrane affects the direction of polar auxin transport, while PINs are not always localized to the plasma membrane. PINs pass different endosomes after entering endosomal system via endocytosis. When PINs target to the late endosome/multivesicular body (MVB), they may be sent back to the early endosome by the retromer complex on MVB and then recycled to the plasma membrane. As the results, the recycling pathway maintains the polar localization of PINs on plasma membrane. The sorting of vacuolar proteins by the endosomal sorting complex required for transport (ESCRT) machinery is another important sorting process taking place in MVB. Fusion of MVB with the vacuole leads to the release of vacuolar proteins into the lumen of the vacuole. AtSKD1 has recently been shown to localize on MVB and catalyze the ATP hydrolysis. Once MVB sorting is completed, atSKD1 is required for releasing the subunits of ESCRT complex from MVB by ATP hydrolysis. In this study, examining the change of polar auxin transport in antisense atSKD1 mutants, I want to comprehend the exact role of atSKD1 in polar auxin transport. First, examining the lateral root density of 7.3.6 line, which is a line of mutant having 50% reduced expression of endogenous atSKD1, I found it is higher than wild-type to 104% without NAA treatment and lower than wild-type to 39% when treated with 0.05 μM NAA. Besides, the root hair length of 7.3.6 line was higher than wild-type to 39% and the root hair density of 7.3.6 line was higher than wild-type to 33%. The results suggested atSKD1 is involved in polar auxin transport and normal development of roots. Furthermore, after examining the expressions of auxin transport genes in 7.3.6 line, I found the expressions of several genes such as AUX1, LAX3, PIN1, PIN2, PIN3, PIN4, PIN5, PIN7, PGP1, PGP19, and ACL5 were lower than wild-type in different degrees. The results suggested atSKD1 is involved in polar auxin transport. Distinct auxin distributions between wild-type and 7.3.6 line root were observed. In 7.3.6 line, auxin did not accumulate in the stele but accumulate high amounts in root tip. Then, examining the amount of auxin in 7.3.6 line, no matter treated with NAA or not, I found the amount of auxin in 7.3.6 line was higher than that of wild-type. The results clearly indicated the participation of atSKD1 in polar auxin transport. Next, I examined the lateral root density of 7.1.6 and 7.2.9 lines which the expression of endogenous atSKD1 was not different from wild-type. Examining the lateral root density of 7.1.6 line, it was higher than wild-type to 38% when treated with 0.2 μM NAA. However, examining the lateral root density of 7.2.9 line, it was no significant difference from wild-type. Furthermore, examining the expressions of auxin transport genes in 7.1.6 line, I found the expressions of several genes such as PIN1, PIN2, and PIN5 were lower than wild-type and the expressions of several genes such as PIN7, PGP1, and PGP19 were higher than wild-type in different degrees. Then, examining the expressions of auxin transport genes in 7.2.9 line, I found the expressions of several genes such as AUX1, LAX3, PIN1, PIN2, PIN5, PIN7, PGP19, and ACL5 were lower than wild-type in different degrees. The results showed certain changes in auxin response were detected even when the mutants expressed atSKD1 at substantial levels. In conclusion, atSKD1 maintains the normal function of ESCRT complex and leads the retromer complex on MVB to regulate the polar localization of PINs and other auxin transport-related proteins. Therefore, the polar auxin transport continues to carry on, and the plant grows in normal circumstance.
其他識別: U0005-1908201113351400
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