阿拉伯芥核蛋白CIA2和CIL調控葉綠體發育機制研究

Abstract

CHLOROPLAST IMPORT APPARATUS 2 (CIA2)與CIA2-LIKE (CIL)隸屬於一羣植物特有、會參與開花時間或蓋日韻律調節的CCT [CONSTANS (CO), CO-LIKE (COL), TIMING OF CAB EXPRESSION1 (TOC1)] 轉錄因子。有別於其他CCT蛋白，CIA2會提高一些葉綠體蛋白合成基因的表現量，進而確保葉綠體的正常發育。 CIL與CIA2的胺基酸序列具有65％的相似性；CIL在葉子和花苞中表現，其表現型態與CIA2相似，這表明CIL和CIA2在阿拉伯芥中是同源的。CIL在cia2突變株中表現量增加，而cia2 /cil雙突變株的葉色比cia2突變株更為淺綠。對cia2 /cil雙突變株的微陣列分析(Microarray analysis) 顯示，與葉綠體發育有關的細胞核表現基因，包括與光合作用和葉綠素生合成相關的基因，表現量明顯降低，顯示CIA2和CIL共同調節了GOLDEN2-LIKE 1和葉綠體發育相關基因的表現。微觀結構觀察(Microstructure observation) 顯示10天齡的cia2 /cil雙突變株具有特定的發育異常。 CIA2是細胞核轉錄蛋白，包含位於氨基酸62-65和291-308的兩個細胞核導引訊息(nuclear localization signal, NLS)。 CIL也是細胞核轉錄蛋白，其NLS位於胺基酸 47-50。 CIA2和CIL的CCT結構不具有核定位信號功能。酵母雙雜交（yeast two-hybrid, Y2H）篩選確認了與CIA2 相互作用的蛋白。除了自身和CIL外，還確認了諸如CO、NUCLEAR FACTOR Y B1 (NF-YB1)、NF-YC1、NF-YC9 和ABSCISIC ACID-INSENSITIVE 3 等蛋白與葉綠體功能和開花時間的調節有關。Y2H進一步確認CIA2和CIL的N 和C 端區域對於與其他蛋白的交互作用很重要。儘管CIA2 和CIL 的CCT 結構是兩個蛋白質之間的主要相互作用片段，但CIA2和CIL N 端的CIA2 and CIL conserved 1 (CC1) 結構使上述CIA2、CIL和開花時間調節蛋白之間能夠交互作用。 本研究中顯示阿拉伯芥CIA2 和CIL 與CO 和NF-Y複合體(complex) 交互作用，並參與CO相關的開花機制調控。CIA2 和CIL 的N端CC1結構與CO 和NF-Ys (B1、C1和C9) 相互作用形成更高階的複合體，並且CC1 結構中的胺基酸序列與NF-Ys中NF-YA1 結構的序列相似。其中，NF-YAs 蛋白是利用NF-YA1結構與NF-YB / NF-YC複合體交互作用。最後，本研究的結果顯示CIA2 和CIL 參與葉綠體發育和CO 相關開花機制的調控。Chloroplast import apparatus 2 (CIA2) and CIA2-like (CIL) are classified to plant-specific, CCT [CONSTANS (CO), CO-LIKE (COL), TIMING OF CAB EXPRESSION1 (TOC1)] motif-containing transcription factors involving in regulation of flowering time or circadian rhythm. In contrast to other CCT proteins, CIA2 is able to increase the expression yields of genes encoding chloroplast proteins, and therefore to ensure the proper development of chloroplast. CIL shares 65% similarity of amino acid sequence with CIA2; CIL is expressed in leaves and young flower buds, and its expression pattern is similar to that of CIA2, suggesting that CIL and CIA2 are homologous in Arabidopsis. CIL is overexpressed in cia2 plants, and the pale-green phenotype of cia2/cil is more severe than that of cia2. Microarray analysis of cia2/cil double mutants revealed evidently decreased expression of nuclear genes involved in chloroplast development, including genes associated with photosynthesis and chlorophyll biosynthesis, indicating that CIA2 and CIL co-regulate the expression of GLK1 and chloroplast development-related genes. Microstructure observations revealed a specific developmental abnormality of chloroplasts in the 10-day-old cia2/cil double mutants. CIA2 is a nuclear protein containing two nuclear localization signals (NLSs) located at amino acid (aa) positions 62-65 and 291-308. CIL is also a nuclear protein, with an NLS located at 47-50 aa. The CCT motifs of CIA2 and CIL do not function as an NLS. CIA2-interacting candidates were identified by using yeast two-hybrid (Y2H) screening. In addition to CIA2 and CIL, CIA2-interacting proteins identified from Y2H such as CO, NUCLEAR FACTOR Y B1 (NF-YB1), NF-YC1,NF-YC9, and ABSCISIC ACID-INSENSITIVE 3 were speculated to be involved in the regulation of chloroplast function and flowering time. Y2H experiments revealed that the N- and C-terminal regions of CIA2 and CIL are important for interactions with other candidate proteins. Although the CCT motifs of CIA2 and CIL are the major interacting fragments between the two protein, the N-terminal CIA2 and CIL conserved 1 (CC1) motif enables the interactions among CIA2, CIL, and the flowering time regulatory proteins mentioned above. This study proposes that the Arabidopsis CIA2 and CIL interact with the CO and NF-Y complex and participate in CO-related flowering regulation. Moreover, the N-terminal CC1 motifs of CIA2 and CIL interact with CO and NF-Ys (B1, C1, and C9) to form a high-order complex, and the residues in the CC1 motif are similar to those in the NF-YA1 subdomain of NF-YAs, which interact with the NF-YB/NF-YC complexes. Finally, the results of this study suggest that CIA2 and CIL co-regulate the expression of genes involved in chloroplast development and CO-related flowering regulation.