| 摘要: | 在暗形態發育的期間，植物的幼苗呈現拉長的下胚軸及閉鎖的子葉。當光照時，幼苗會產生去白化現象，呈現光形態的發育，包括較短的下胚軸長度及子葉展開的情況。植物激素乙烯會抑制白化苗子葉的展開，但在光照後會促進子葉的綠化。先前研究得知ETHYLENE OVERPRODUCER1(ETO1)及ETO1-LIKE(EOL1/2)蛋白質為Cullin3 (CUL3)-based泛素連接酶，ETO1及EOL1/2藉由降解第2型ACS蛋白質，共同負向調控阿拉伯芥的乙烯生合成。在本研究中，我們發現在光暗轉換時，EOL1及EOL2在幼苗發育中扮演新穎的角色。在4天暗處理後，eol1及eol2單一或雙重突變株的白化苗呈現較高的子葉展開率，接著在轉移至2天全光照後，突變株幼苗則呈現較低的子葉綠化率。利用微矩陣法所進行的基因轉錄研究分析結果，我們發現在四天暗處裡下eto1、eol1及eol2白化苗中，數個參與光合作用的基因與野生種呈現顯著差異性的表現。其中，NADPH-原葉綠素酯氧化還原酶(PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, POR) PORA及PORB為促進葉綠素合成的重要酵素，催化原葉綠素(protochlorophyllide, Pchlide)轉化成脫植醇葉綠素(chlorophyllide, Chlide)。我們發現在eol1及eol2中PORA/B的基因表現為下降，而且脫植醇葉綠素的量在eol1及eol2中亦隨之減少，但在eto1與野生種控制組並沒有顯著差異。此結果表示在光暗轉換時EOL1及EOL2促進白化苗的綠化，在光形態發生中扮演著異於ETO1的角色。為了探討EOL蛋白質在幼苗發育的新功能，我們利用酵母菌雙雜交法，由3天大白化苗所製備而成的阿拉伯芥之cDNA庫，篩選出與EOL1有交互作用的蛋白質。在數個可能的候選蛋白中，我們發現JASMONATE ZIM DOMAIN9(JAZ9)與EOL1有專一性的交互作用，JAZ蛋白質為茉莉酸訊息傳遞路徑中的負調控因子。進一步的研究結果顯示，同屬為第五型JAZ蛋白的JAZ3及JAZ9，皆能夠與ETO1、EOL1及EOL2在酵母菌雙雜交系統中有交互作用，而位於JAZ9之羧基端的Jas結構域呈現與EOL1蛋白質有專一的交互作用。本研究結果揭露，EOL蛋白質和茉莉酸之間可能存在著共同調控幼苗光形態發育訊息的交互作用。最後，我們提出一個假設模型説明EOL1和EOL2在幼苗發育時對光反應的可能調節機制。;When exposed to light, plant seedlings undergo de-etiolation development, termed photomorphogenesis, which exhibits reduced hypocotyl elongation and cotyledon opening followed by greening prepared for photosynthesis. The plant gaseous hormone ethylene inhibits cotyledon opening and reduces hypocotyl elongation in the dark-grown (etiolated) seedlings but promotes cotyledon greening during photomorphogenesis. The Arabidopsis ETHYLENE OVERPRODUCER1 (ETO1) and ETO1-LIKE (EOL1 and EOL2) proteins were previously identified as components of cullin3(CUL3)-based ubiquitin E3 ligase to negatively regulate ethylene biosynthesis by promoting protein degradation of type 2 ACC synthases (ACS). In this study, we reveal a novel role of EOL1 and EOL2 in seedling development during dark-to-light transition. We found that the 4-d-old etiolated seedlings of eol1 and eol2 single and double mutants showed a higher percentage of cotyledon opening but a lower cotyledon greening rate after being exposed to white light for 2 days. Microarray analysis of 4-d-old etiolated eto1, eol1 and eol2 seedlings showed several genes involved in photosynthesis, including the key enzymes essential for chlorophyll synthesis, PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) PORA and PORB that catalyze the conversion of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), were repressed in eol1 and eol2 but not in eto1. In line with this finding, the accumulation of Chlide was reduced in the etiolated seedlings of eol1 and eol2 but not in eto1. Our data suggest that EOL1 and EOL2 may play a regulatory role in photomorphogenesis by facilitating greening of etiolated seedlings upon exposure to light. To investigate the functional role of EOL proteins, we sought to identify interacting proteins of EOL1 by screening an Arabidopsis cDNA library prepared from 3-d-old etiolated seedlings using the yeast two-hybrid (Y2H) system. Among several potential interacting proteins, we found that JASMONATE ZIM DOMAIN9 (JAZ9) interacts with EOL1. JAZ proteins act as negative regulators of JA signaling pathway in Arabidopsis. Further analysis indicated that the Jas domain at C terminus in JAZ9 is responsible for the specific interaction with EOL1 in yeast. In addition, JAZ3 and JAZ9, which belong to the group of type 5 JAZ proteins as JAZ9, can interact with ETO1, EOL1 and EOL2 by the Y2H assay. Our data suggest a potential relationship between EOL proteins and JA signaling pathway in seedling development responsive to light. Finally, we propose a hypothetical model to further study the underlying regulatory mechanisms of EOL1 and EOL2 in seedling development during photomorphogenesis. |
