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Analysis of gene expression and protein accumulation of an E3 ligase mcCPN1 in ice plant
|關鍵字:||冰花;Salt stress;mcCPN1;ubiquitination;mcSKD1;cell growth||出版社:||生命科學系所||引用:||Abas L, Benjamins R, Malenica N, Paciorek T, Wiśniewska J, Moulinier-Anzola JC, Sieberer T, Friml J and Luschnig C (2006) Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol 8: 249-56 Adams P, Nelson D, Yamada S, Chmara W, Jensen RG, Bohnert HJ and Griffiths H (1998) Growth and development of Mesembryanthemum crystallinum (Aizoaceae). New Phytol 138: 171-90 Apse MP, Aharon GS, Snedden WA and Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285: 1256-8 Aravind L and Koonin EV (2000) The U box is a modified RING finger - a common domain in ubiquitination. Curr Biol 10: R132-4 Ardley HC and Robinson PA (2005) E3 ubiquitin ligases. 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McCOPINE1 (mcCPN1)是在耐鹽植物冰花中鑑定到的一個copine蛋白，與鹽逆境相關蛋白mcSKD1 (suppressor of K+ transport defect 1)及mcSNF1 (sucrose non-fermenting 1)具有蛋白質交互作用。已知鹽誘導蛋白mcSKD1參與鹽逆境下intracellular protein trafficking之過程，而蛋白激酶mcSNF1則與鹽逆境訊息傳遞路徑有關。由於mcCPN1蛋白上具有兩個保留性區位，分別為N端copine A domain及C端RING-finger domain，將其歸類為RING-type copine。已知大部分含有RING-finger domain 的蛋白皆具有E3 ubiquitin ligase的活性，在ubiquitination過程中的功能為辨識並標定受質蛋白。本論文之目的在於分析鹽逆境下mcCPN1的基因表現及蛋白累積的情形，並進一步偵測其E3 ligase的活性，以期對mcCPN1蛋白的作用有更深入的了解。
為了解mcCPN1與鹽逆境的相關性，分別偵測鹽處理的細胞及植株中mcCPN1基因及蛋白累積的情況。此部分的分析是以RT-PCR及北方墨點法偵測mcCPN1基因表現量，並以西方墨點法偵測mcCPN1蛋白的累積量。在細胞層次上，鹽處理後的冰花培養細胞中mcCPN1基因皆呈現穩定的表現。然而在蛋白的累積量上，鹽處理七天後，mcCPN1在細胞中的累積量會隨著鹽濃度增加而增加，反之，鹽處理三天卻會減緩mcCPN1的累積量，進一步以0 mM及200 mM鹽處理冰花培養細胞一周後，分析不同時間點收集的細胞中mcCPN1蛋白累積量，發現mcCPN1蛋白的累積在細胞層次與細胞的增生具有較大的相關性。在冰花植株中，mcCPN1基因在根部與葉部中亦呈現穩定表現，然而200 mM鹽處理後，mcCPN1蛋白累積量均上升，顯示mcCPN1蛋白在冰花植株中可能參與鹽逆境的反應。經由上述結果推測冰花mcCPN1蛋白參與了細胞增生及鹽逆境的反應。
由於mcCPN1蛋白累積量在鹽誘導下及細胞增生過程中產生變化，進一步針對其蛋白活性做測試，首先利用in vitro ubiquitination assay證實mcCPN1確實具有E3 ligase的活性，並嚐試以mcSKD1及mcSNF1蛋白當作mcCPN1的受質蛋白，結果顯示mcSKD1可被mcCPN1蛋白辨識並標定ubiquitin，証實mcSKD1為mcCPN1的受質蛋白之一。此外，in vitro下mcSNF1會對減緩mcSKD1被mcCPN1標定ubiquitin的程度。由以上結果可知mcCPN1為一E3 ubiquitin ligase且mcSKD1為其受質蛋白，且mcSNF1可能在mcCPN1主導的mcSKD1 ubiquitination過程中扮演調控的角色。
綜合本論文之結果，E3 ligase mcCPN1可能參與冰花的細胞增生和鹽逆境下的反應，且其蛋白累積受到轉譯或後轉譯層次的調控。在鹽逆境下，mcCPN1可能會藉由控制鹽逆境相關蛋白mcSKD1之protein ubiquitination進而參與冰花鹽逆境下之反應機制。
McCOPINE1 (mcCPN1), a RING-type copine, was identified from halophyte ice plant (Mesembryanthemum crystallinum L.) and known to interact with salt stress-induced proteins mcSKD1 (suppressor of K+ transport defect 1) and mcSNF1 (sucrose non-fermenting 1). McSKD1 is a salt-induced gene and involves in intracellular protein trafficking, whereas mcSNF1 is predicted to be a protein kinase and possibly involves in salt-stress signaling. Our previous study showed that mcCPN1 contains two conserved domains, N-terminal copine A domain and C-terminal RING (really interesting new gene)-finger domain. RING-finger proteins usually possess E3 ligase catalytic activity executing the last step of ubiquitination in recognizing and ubiquitinating substrate proteins. Thus, the main focus of this thesis is to examine the expression and accumulation of mcCPN1 under salt stress, along with the analysis of its E3 ligase activity in order to access its function in salt-tolerant mechanism of ice plant.
To understand the role of mcCPN1 in salt-tolerant mechanism, gene expression and protein accumulation of mcCPN1 were examined at both cellular and intact plant levels. RT-PCR and Northern blotting were used to analyze mcCPN1 expression and anti-mcCPN1 antibody was used to detect mcCPN1 accumulation. At cellular level, gene expression of mcCPN1 was constitutive after addition of salt, whereas a significant change was found in the accumulation of mcCPN1. Long-term salt stress induced mcCPN1 accumulation; however, short-term salt stress suppressed it. Temperal accumulation of mcCPN1 indicated that cell proliferation has a profound influence on mcCPN1 accumulation. At intact plant level, constitutive expression of mcCPN1 was found in salt-stressed roots and leaves, whereas protein accumulation of mcCPN1 was salt-induced suggesting that mcCPN1 might participate in the salt stress response of ice plant.
Due to the reason that the changes in the accumulation mcCPN1 were found at cellular and intact plant level, the catalytic activity of mcCPN1 was further examined. The activity of mcCPN1 was demonstrated by the in vitro ubiquitination assay. When using mcCPN1 interacting protein mcSKD1 or mcSNF1 as the in vitro substrate, mcSKD1 can be ubiquitinated but mcSNF1 can not. Furthermore, mcSNF1 reduced the amount of ubiquitinated mcSKD1 in the in vitro system. These results suggested that mcSKD1 is one of the substrate proteins for E3 ligase mcCPN1 and mcSNF1 might play a regulatory role in mcCPN1-mediated mcSKD1 ubiquitination.
Taken together, these results indicated that the E3 ligase mcCPN1 participates in the cell proliferation and salt stress response in ice plant and the accumulation of mcCPN1 was regulated at translational and/or post-translational level. The involvement of mcCPN1 in salt-tolerant mechanism of ice plant is possibly by way of recognizing and ubiquitinated salt stress-related protein mcSKD1.
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