阿拉伯芥毛狀根形成相關之轉錄因子 NAC13 的功能性分析

Abstract

毛狀根是植物受到根毛農桿菌 (Agrobacterium rhizogenes) 感染所誘導產生的轉形根組織。先前研究中指出誘根質體 (Ri plasmid) 上的 rol 基因群在誘發毛狀根的過程中扮演重要的角色；其中 rolC 基因又被報導與毛狀根快速生長及側根發育有關。本實驗室先前研究顯示：缺失 rolC 的菸草毛狀根生長能力明顯下降，且 rolC 對毛狀根的發育及其快速生長的特性密切相關，但目前對於 rolC 如何調控毛狀根的形成與其特性仍屬未知。同時，阿拉伯芥中已有許多轉錄因子對於細胞、組織的生長有著全面性調控之報導。綜合以上，本研究針對阿拉伯芥中可能受到 rolC 調控的轉錄因子進行功能性探討，以推測 rolC 基因影響毛狀根組織生成及發育的機制。根據先前基因微陣列 (microarray) 分析結果，本研究以阿拉伯芥基因缺陷株的主根長度為參數，挑選出一個可能為轉錄因子的 NAC13 蛋白質。我們先在酵母菌的系統中，利用轉錄分析法 (transactivation assay) 證明 NAC13 具有轉錄下游基因的能力；再將 NAC13 與黃色螢光蛋白質融合連接後，利用共軛焦顯微鏡發現 NAC13 蛋白質在細胞內的表現位置是在細胞核中及其周圍。同時，藉由比較缺失 nac13 基因的阿拉伯芥毛狀根與野生形毛狀根的外觀、誘導率及粒線體含量，推測 rolC可能透過 nac13 來調控下游 aox1a 及 hre2 基因的表現，降低細胞中的活性含氧物質，以阻止粒線體的凋亡及維持毛狀根快速生長。

Hairy root is a specialized, fast-growing tissue that is induced by the infection of Agrobacterium rhizogenes. Root locus genes (rol genes) which encoded in root-inducing plasmid (Ri plasmid) of A. rhizogenes play key roles in hairy root formation. Among the rol genes, researches showed that fast and branched root growth required the presence of rolC gene. Our previous data showed that the viability of rolC-deficient hairy roots decreased significantly compared to wild-type hairy roots, hairy root formation and its fast growth were regulated by rolC with unknown mechanism. Previous researches also indicated that many transcription factors were thought to have large impacts on tissue characteristics in Arabidopsis. Took the things together, we focused on revealing the transcription factors which under the regulation of rolC, attempting to elucidate the mechanism of hairy root formation. Based on the previous microarray results, a putative transcription factor, NAC13, was identified according to its capacity for regulating the main root length in Arabidopsis mutants. The activation ability of NAC13 was demonstrated by using transactivation assay in yeast. By confocal microscopy observation, fusion proteins of NAC13 and yellow fluorescence protein (NAC13-YFP) were visualized in and around the nucleus. Moreover, comparing the morphology, induction efficiency and the mitochondria of nac13-deficient hairy roots and wild-type hairy roots showed that rolC might involve in the mitochondria stress signaling pathway through nac13 gene and its downstream genes, aox1a and hre2, to eliminate the excess reactive oxygen species for maintaining fast growth and preventing mitochondrial apoptosis in hairy roots.