植物CONSTANS (CO)及CONSTANS-Like (COL)基因對於整合來自於環境中光與溫度變化訊號具有極重要的地位，但是對於這類基因如何在個體接收環境變化訊息後反應在生長變化上的調節機制尚未明瞭。本篇研究分析來自於台灣原生種白花蝴蝶蘭台灣阿嬤CONSTANS-Like 1 (Phalaenopsis aphrodite CONSTANS-Like 1, PaCOL1) 基因生理調節機制，並進一步探究其同源性基因在其他原生種蝴蝶蘭之種間差異性。PaCOL1為B-box蛋白質家族成員之一，分布於細胞質與細胞核執行其生理功能，基因表現分析顯示PaCOL1在白花蝴蝶蘭中主要分布於葉部組織，累積量會因溫度變動而改變；PaCOL1蛋白質具有日夜差異性累積分布，自傍晚至午夜為累積高峰時段。大量表現PaCOL1基因之阿拉伯芥轉殖植株於短日照 (short-day conditions: 8小時光照、23℃/16小時黑暗、23℃)環境下與野生型 (WT)植株相較有提早開花現象；但在長日照 (long-day conditions: 16小時光照、23℃/8小時黑暗、23℃) 環境栽培中的PaCOL1轉殖株與野生植株其開花時間則無顯著差異。核糖核酸測序分析 (RNA transcriptome sequencing, RNAseq) 結果顯示在短日照環境種植之PaCOL1阿拉伯芥轉殖株，其差異性表現基因數據裡與光訊息傳遞路徑上已知可促進開花相關部分基因呈現正向調控情形。酵母菌雜交實驗(Yeast two-hybrid, Y2H)與雙分子螢光互補試驗 (Bimolecular Fluorescence Complementation, BiFC)顯示PaCOL1蛋白質具有與其他蛋白質鍵結能力，可與阿拉伯芥生理時鐘調節蛋白質AtCCA1、開花抑制因子AtFLC產生蛋白質互動反應。阿拉伯芥cca1.lhy雙基因突變株導入PaCOL1基因轉殖株之基因互補實驗，觀察到cca1與lhy雙基因突變株失去基因而產生的光無感反應可因PaCOL1基因表達而部份恢復其對光反應機制，造成植株形態發育與開花時間改變，此結果證實PaCOL1為一參與光週期調節相關基因。本篇研究亦發現PaCOL1同源基因存在於姬蝴蝶蘭 (P. equestris)、大葉蝴蝶蘭 (P. bellina)及螢光蝴蝶蘭 (P. violacea)，且PaCOL1蛋白質可與蝴蝶蘭中過氧化物 (ROS) 清除者PaAPX及開花素PaFT等蛋白質進行交互作用，顯示PaCOL1可能也參與逆境訊息之整合而影響蘭花的生理調節機制。;CONSTANS (CO) and CONSTANS-Like (COL) genes play important roles in coalescing signals from photoperiod and temperature pathways. However, the mechanism of CO and COLs involved in regulating the developmental stage transition and photoperiod/temperature senescing remains unclear. In this study, we identified a COL ortholog gene from the Taiwan native orchid Phalaenopsis aphrodite (P. aphrodite). The P. aphrodite CONSTANS-Like 1 (PaCOL1) belongs to the B-box protein family and functions in the nucleus and cytosol. Expression profile analysis revealed that PaCOL1 was significantly expressed in leaves, but its accumulation was over-repressed during environmental temperature shift. We found a differential profile for PaCOL1 accumulation, with peak accumulation at late afternoon and at the middle of the night. Arabidopsis with PaCOL1 overexpression showed earlier flowering under short-day (SD) conditions (8-h/23℃ light and 16-h/23℃ dark) but no difference under long-day (LD) conditions (16-h/23℃ light and 8-h/23℃ dark). Transcriptome sequencing revealed several genes involved in flowering regulation of the photoperiod pathway were upregulated in PaCOL1-overexpressing Arabidopsis plants. Yeast two-hybrid (Y2H) analysis and bimolecular fluorescence complementation (BiFC) analysis revealed that PaCOL1 could interact with a crucial clock-associated regulator, AtCCA1, and a flowering repressor, AtFLC. Expressing PaCOL1 in cca1.lhy double mutant partially reversed the mutant flowering time under photoperiod treatment, which confirms the role of PaCOL1 function in the rhythmic associated factors for modulating flowering. We found PaCOL1 homologs existed in other 3 Phalaenopsis plants: P. equestris, P. bellina and P. violacea. PaCOL1 showed the potential interaction with two Phalaenopsis native proteins, the florigen PaFT and a ROS scavenger PaAPX. These evidences shedding the light on PaCOL1 may function in photoperiodical and stress signaling modulation of orchid physiological regulatory networks.
