甘藷MYC型IbbHLH3轉錄因子利用蛋白質多重交互作用調控受傷反應途徑

Abstract

過去研究已知，甘藷在受到昆蟲嚙食時，IbNAC1轉錄因子會調控sporamin基因表達以活化抗蟲防禦系統。而IbNAC1的基因表達是由MYC型的IbbHLH3及IbbHLH4兩個轉錄因子相互作用以調控其表達量，當IbbHLH3-IbbHLH3 homodimer形成時可啟動IbNAC1表達；反之，IbbHLH3-IbbHLH4 heterodimer則抑制IbNAC1表達。本研究顯示，IbbHLH3蛋白質的bHLH-domain 末端處有一特異區域，透過此區域與IbbHLH4結合以抑制IbNAC1基因表現。其他IbNAC1之抑制子 (repressor) 如IbJAZs及IbEIL1亦能夠與IbbHLH3結合並 抑制IbNAC1之表現。當葉片受傷時IbbHLH3的基因被誘導大量表現並產生IbbHLH3-IbbHLH3複合蛋白進而正向調控IbNAC1的轉錄以對抗昆蟲，而此時IbJAZ2 與IbEIL1的基因表現會減少。到了受傷晚期，IbJAZ1開始大量表現，藉由形成IbbHLH3-IbJAZ1複合蛋白以抑制IbNAC1表現。為進一步研究IbbHLH3與作用蛋白質結合後的功能，將這些基因分別以35S 啟動子驅動並過量表現於阿拉伯芥。這些轉殖株當中，過量表現IbJAZ2的植株在經JA處理後其表型與野生型相比無明顯差別；而過量表現IbJAZ1的植株中其生長卻不受JA抑制，且呈現花青素含量降低及JA防禦相關基因VSP2、LOX2、COI1表現下降等JA不敏感表型之特徵，此表示IbJAZ1的功能為JA訊息路徑中的抑制者。而在IbEIL1過量表現的轉殖株中亦出現對JA不敏感型的現象。綜合以上結果可知，甘藷中有許多蛋白質能與IbbHLH3產生交互作用，然而這些不同的蛋白質在面對受傷誘導的JA反應時分別扮演其特定的角色，共同協調受傷逆境生理反應。

Sweet potato IbNAC1 is known that activates the defense programs against herbivory. Previous study reported that two bHLH transcription factors, IbbHLH3 and IbbHLH4, dynamically coordinate the expression of IbNAC1 during wounding. In this study, we evidenced that IbbHLH4 interacts with IbbHLH3 by binding the end of HLH-domain of IbbHLH3 which is higher diversity compared to other bHLH homologous. Several proteins, including IbJAZs and IbEIL1, were found that interact with IbbHLH3 to regulate wounding response. When sweet potato leaves are exposed to wounding, the transcriptions of IbJAZ2 and IbEIL1 are immediately repressed, while the expression of IbbHLH3 is induced. The IbbHLH3-IbbHLH3 protein complex activates IbNAC1 against herbivory. Until late wounding, the expression of IbJAZ1 is elevated that repressed the activation function of IbbHLH3 by forming IbbHLH3-IbJAZ1 protein complex as IbbHLH3-IbbHLH4 complex. To characterize the biological functions of these IbbHLH3-intereacting proteins, the genes were overexpressed into Arabidopsis by 35S promoter. Among these transformants, the phenotype of IbJAZ2-overexpressing plants had no significant difference under JA treatment compared with WT. On the other hand, IbJAZ1-overexpressing plants showed insensitity to JA-inhibited anthocyanin accumulation, JA-inhibited root formation, and decrease of JA-regulated defence gene VSP2, LOX2, and COI1. Moreover, overexprssion of IbEIL1 also exhibited JA-insensitive response as IbJAZ1. These results suggested that the IbbHLH3-interacting proteins are multifunctional in response to wound-induced JA responses in sweet potato.