釐清CIA2及CIL是否透過GLKs及GATA轉錄因子促進阿拉伯芥葉綠體發育與開花

Abstract

CIA2 (CHLOROPLAST IMPORT APPARATUS 2)與CIL (CIA2-LIKE)為阿拉伯芥中具有65 %序列相同的CCT [CONSTANS (CO), CO-LIKE, and TIMING OF CAB1 (TOC1)]家族同源蛋白。CIA2是上調TOC33 (TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS 33)及TOC75基因表現的轉錄因子，進而協調蛋白質運送到葉綠體，並促進葉綠體的發育；CIL則可能對上述功能扮演輔助角色並促進開花時間。為了確認CIA2及CIL調控葉綠體發育以及開花時間的下游機制，我以染色質免疫沉澱實驗證明CIA2直接與葉綠體發育相關的轉錄因子GLK1 (GOLDEN 2-LIKE 1)、GLK2及GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM-INVOLVED )的基因序列進行交互作用，而CIL則作用在GLK2及GNC的基因序列上。即時反轉錄PCR結果顯示CIA2促進GLK1，並與CIL共同促進GLK2但抑制GNC的基因表現。進一步分析cia2、cil、glk1、glk2及不同組合的雜交突變植株，發現相比於Col-0，cia2的葉綠素及類胡蘿蔔素含量減少約35%，cia2/cil及glk1/glk2約下降50%，而cia2/cil/glk1/glk2則只剩20%以下。缺失CIA2之葉綠體大小為Col-0的60%，同時缺失CIA2/CIL或GLK1/2下降至40%，四突變株則小於Col-0的四分之一，顯示CIA2與GLK1/2共同調控葉綠體發育。在CIA2缺失下單一細胞所含的葉綠體數量上升，則可能是因GNC基因表現量上升促進葉綠體分裂的結果。開花時間檢測指出，含cil之突變株延遲開花時間但只稍微影響花器形成；而CIA2或GLKs的缺失不影響開花時間，但明顯呈現花器數量減少與花序變短的表現型。綜上所述，CIA2可能藉由GLK1、GLK2及GNC協調阿拉伯芥的葉綠體發育與分裂，CIL則輔助其功能；此外，CIA2與GLKs共同正調控花器與花序的發育，CIL則促進植物開花。Arabidopsis CIA2 (CHLOROPLAST IMPORT APPARATUS 2) and CIL (CIA2-LIKE) are homologous CCT [CONSTANS (CO), CO-LIKE, and TIMING OF CAB1 (TOC1)] proteins sharing 65% sequence identity. CIA2 is a transcription factor up-regulating the expression of TOC33 (TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS 33) and TOC75 to modulate protein import into chloroplasts, and therefore promotes chloroplast development. In addition to play an assistant role to CIA2, CIL also promotes flowering. To determine the downstream candidates regulated by CIA2 and CIL, ChIP (chromatin immunoprecipition) assay was performed and identified that CIA2 interacts with the gene sequences of GLK1 (GOLDEN 2-LIKE 1), GLK2 and GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM-INVOLVED); whereas CIL interacts with the last two gene’s sequences. Based on RT-qPCR (quantitative reverse transcription polymerase chain reaction) results, CIA2 particularly up-regulates GLK1. However, CIA2 and CIL both up-regulate and down-regulate GLK2 and GNC, respectively. Comparison of cia2, cil, glk1, glk2 and their double, triple, quadruple mutant plants, the contents of chlorophylls and carotenoids were reduced approximately 35%, 50 %, 50 %, and more than 80 % in cia2, glk1/glk2 and cia2-related double and quadruple mutants, respectively, relative to Col-0. The chloroplast size in cia2, cia2-related and glk-related double, triple and quadruple mutants were about 60 %, 40 %, 40 % and less than 25%, respectively, of Col-0 chloroplast size, suggesting that CIA2 and GLK1/2 enhance chloroplast development. The chloroplast number increased in cia2 that may result from the higher expression of GNC, which advancing chloroplast division. Furthermore, delayed flowering time was observed in cil-related single, double, triple and quadruple mutants; but CIL had little effect on the development of reproductive organ. Lack of CIA2 and GLKs significantly attenuate the height and flower number of primary inflorescences, but do not affect flowering time. In summary, CIA2 may coordinate chloroplast development and division through GLK1, GLK2 and GNC, whereas CIL is supplementary to CIA2’s function; CIA2 and GLKs co-regulate inflorescence and flower organ development, but CIL promotes flowering time.