- 學位論文
阿拉伯芥FIN219在遠紅光與茉莉酸訊息傳遞間交互作用之功能性研究
Functional Studies of FIN219 in the Integration of Far-Red Light and Jasmonate Signaling in Arabidopsis
摘要
Chapter 1 FAR-RED INSENSITIVE 219 (FIN219)參與在阿拉伯芥之遠紅光訊息傳遞中，扮演正向調控者。先前研究得知於黑暗下，FIN219是具有抑制CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)之功能，然而，其中之調控機制尚未明瞭。我們利用大量表達不同FIN219片段之轉殖株探討其調控關係，並建構大量表現GUS-FIN219-N53 (NCox)及GUS-FIN219-N300 (NTox)的建構體，其分別含有FIN219的N端coiled-coil結構之53及300個胺基酸，將其轉入野生型。僅於遠紅光下，NCox 及 NTox皆呈現dominant-negative長下胚軸外表型；並亦於相同處理下發現，突變株與轉殖株皆累積較多的COP1蛋白質，而HY5蛋白質卻明顯降低，此現象並未發現在其他光源下。為了釐清NCox及NTox呈現遠紅光不敏感之原因，進一步利用yeast two-hybrid (Y2H)、in vitro pull-down及bimolecular fluorescence complementation (BiFC)等分析，發現FIN219可以藉由N端的coiled-coil結構與另一個FIN219蛋白質的C端coiled-coil結構產生蛋白質交互作用，且此作用是發生在細胞質中。利用Y2H、pull-down、in vivo coimmunoprecipitation及BiFC分析，得知FIN219的C端coiled-coil結構可與COP1的WD40 repeats結構發生蛋白質交互作用，此亦發生在細胞質中。於fin219-2/cop1-6雙突變株發現並無改變cop1-6突變株在黑暗下之外表型，亦得知HY5蛋白質的穩定需要FIN219存在。於不同強度遠紅光實驗發現，不同強度的遠紅光可藉由phyA調控FIN219蛋白質含量，進而影響COP1的功能。綜合以上結果，推測NCox及NTox蛋白質與COP1蛋白質同時競爭內生型FIN219蛋白質之C端coiled-coil結構，當野生型阿拉伯芥大量表現NCox及NTox蛋白質會導致COP1不易被內生型FIN219蛋白質篏制於細胞質中，反而進入細胞核降解HY5，因此呈現出遠紅光不敏感外表型。 Chapter 2 植物光形態發育同時需要光訊息及植物荷爾蒙的協同作用，以適應植物於光照下之生存。先前研究發現FIN219具有調控光訊息傳遞與合成茉莉酸－異亮胺酸之雙功角色。因此，探討光與荷爾蒙訊息交互作用是提供研究植物光形態發育之重要資訊。coi1-16突變株於不同光源下呈現長下胚軸外表型；於遠紅光下，FIN219蛋白質受COI1調控，且外加coronatine處理，導致回饋抑制之調控現象。另外，利用GUS-COP1大量表現的轉殖株於MeJA或coronatine處理下，發現內生型COP1蛋白質量呈現下降趨勢，此結果亦呈現在野生型及fin219-2突變株中；另外，GUS-COP1轉基因蛋白質亦於MeJA或coronatine處理下皆明顯降低。此外，茉莉酸引起阿拉伯芥大量累積花青素主要取決於HY5蛋白質累積量。綜合以上結果可推斷COP1可能扮演在光與茉莉酸訊息傳遞下之另外一個調控者，可提供探討兩者訊息傳遞交互作用之方向。 Chapter 3 番茄青枯病是一種非常普遍和廣泛的疾病，主要是由青枯菌引起，特別是在炎熱和潮濕的環境下更為嚴重；於自然環境中，因為寄主抗性的不穩定，以及青枯菌種類與變異性極大，至目前為止，並無良好的防治方式；因此，番茄抵禦青枯菌的分子機制的瞭解仍然有限。本篇報導由番茄釣取到番茄SlJAR1同源性基因－SlJRL1，並且建立可誘導SlJRL1基因表現的番茄轉殖株。比對兩基因之蛋白質序列竟然高達99 %相同性；先前發現SlJAR1於地下根部之基因表現量最高，相反地，SlJRL1於地下部表現量最低，反而是地上部有較高的基因表現量；進一步利用南方氏墨點法分析，發現於CL5915番茄品系之基因體含有多個SlJRL1基因。進一步建構誘導啟動子驅動SlJRL1之載體，將其轉殖進番茄基因體中，利用DEX處理轉殖株，其誘導SlJRL1表現量於第二天即達到高峰；並利用此誘導方式，進行青枯菌感染，經DEX誘導之轉殖番茄可延緩萎凋病徵發生；並分析體內之青枯菌族群，發現轉殖番茄經由DEX誘導後，亦可減緩於莖頂處青枯菌累積之數量；由此可知，植物茉莉酸荷爾蒙具有參與番茄防禦青枯菌感染之功能。因此，本篇SlJRL1轉殖番茄之結果，可提供抵抗番茄青枯病之分子機制的瞭解。
並列摘要
Chapter 1 FAR-RED INSENSITIVE 219 (FIN219) in Arabidopsis is involved in phytochrome A-mediated far-red (FR) light signaling. Previous genetic studies revealed that FIN219 acts as an extragenic suppressor of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). However, the molecular mechanism underlying the suppression of COP1 remains unknown. Here, we used a transgenic approach to study the regulation of COP1 by FIN219. Transgenic seedlings containing ectopic expression of the FIN219 N-terminal domain in wild-type Columbia (named NCox for the expression of the N-terminal coiled-coil domain and NTox for the N-terminal 300 amino-acid region) exhibited a dominant-negative long-hypocotyl phenotype under FR, reflected as reduced photomorphogenic responses and altered levels of COP1 and HY5. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays revealed that FIN219 could interact with the WD-40 domain of COP1 and with its N-terminal coiled-coil domain through its C-terminal domain. Further in vivo coimmunoprecipitation study confirms that FIN219 interacts with COP1 under continuous FR light. Studies of the double mutant fin219-2/cop1-6 indicated that HY5 stability requires FIN219 under darkness and FR light. Moreover, FIN219 levels positively regulated by phytochrome A can modulate the subcellular location of COP1 and are differentially regulated by various fluence rates of FR light. We concluded that the dominant-negative long-hypocotyl phenotype conferred by NCox and NTox in a wild-type background was caused by the misregulation of COP1 binding with the C terminus of FIN219. Our data provide a critical mechanism controlling the key repressor COP1 in response to FR light. Chapter 2 Photomorphogenic development requires the synergistic integration of light signaling and phytohormones to confer plants with the ability to perceive light energy and signals for survival in environments. Previous report has suggested that FIN219 plays a dual role with enzymatic activities and a regulatory interacting protein involved in JA and FR light signaling, respectively. Here, we showed that the coi1-16 mutant exhibited a long-hypocotyl phenotype in various light conditions and influenced the expression of light-regulated genes under FR. The levels of FIN219 were dependent on COI1 and negatively feedback regulated by coronatine treatment under FR. Furthermore, GUS-COP1 overexpressed transgenic seedlings (GUS-COP1ox) were used for studying the regulation of COP1 protein in MeJA and coronatine treatments. Under FR, the COP1 levels in wild-type Col, fin219-2, and GUS-COP1ox were reduced by MeJA and coronatine treatments; surprisingly, GUS-COP1 proteins were greatly decreased in MeJA and coronatine treatments. Besides, exogenous jasmonates highly induced anthocyanin accumulation, which was dependent on HY5 protein levels. Thus, we propose that COP1 may play a vital role in the modulation of the integration between light and JA signaling pathways. Chapter 3 Bacterial wilt in tomato caused by Ralstonia solanacearum infection is a common and widespread disease, especially in hot and humid environments. Combating the disease is difficult because of unstable host resistance and the variation and diversity of the bacterial strains. Thus, the molecular mechanisms underlying tomato resistance against Ralstonia solanacearum remain unknown. Here, we isolated a homolog of tomato Solanum lycopersicum JASMONATE RESISTANT 1 (SlJAR1), named SIJAR1-like 1 (SlJRL1) and generated transgenic tomato lines harboring an inducible promoter-driven SlJRL1 construct. SlJRL1 shares 99% amino acid identity with SlJAR1. Intriguingly, SlJRL1 showed preferential expression in aerial parts and SlJAR1 in roots. DNA gel blot analysis revealed multiple copies of SlJRL1 in the tomato genome. Transgenic tomato containing a single copy of the transgene SlJRL1 exhibited high levels of SlJRL1 expression at 2 days after dexamethasone (DEX) induction. Moreover, induction of SlJRL1 expression by DEX could delay the symptoms of tomato bacterial wilt, and efficiently reduce the amount of Ralstonia in stems, so the phytohormone jasmonic acid may participate in the resistant responses of tomato to bacterial wilt caused by Ralstonia. Therefore, transgenic tomato containing inducible expression of SlJRL1 may help further understand the molecular mechanism of tomato resistance against bacterial wilt.