Lu Xin, Dun Hui, Lian Conglong, Zhang Xiaofei, Yin Weilun, Xia Xinli
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 10083, PR China.
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 10083, PR China.
Plant Physiol Biochem. 2017 Jun;115:418-438. doi: 10.1016/j.plaphy.2017.04.009. Epub 2017 Apr 8.
Plant miR164 family is highly conserved and miR164 members regulate conserved targets belonging to NAC transcription factors. Our previous studies have revealed that peu-miR164a-e and its target gene POPTR_0007s08420 participate in abiotic stress response in Populus euphratica according to deep sequencing and degradome sequencing. In this study, miR164 family comprises six members that generate two mature products (miR164a-e and miR164f) and target seven NAC genes in P. euphratica. Co-expression in Nicotiana benthamiana and 5' RACE confirmed that peu-miR164 directs PeNAC070, PeNAC012 and PeNAC028 mRNAs cleavage. Expression profiles of primary peu-miR164 a/b/c/d/e bear similarity to those of peu-miR164a-e, whereas PeNAC070 and PeNAC081 showed inverse expression patterns with peu-miR164a-e under abiotic stresses. Existence of cis-acting elements in PeNAC070 promoter (ABRE,MBs, Box-W1, GC-motif, and W-box) and in peu-MIR164b promoter (HSE) further confirmed different responses of peu-miR164 and PeNAC070 to abiotic stresses. Histochemical β-glucuronidase (GUS) staining revealed that GUS activities increased when Pro::GUS transgenic Arabidopsis plants were exposed to NaCl, mannitol and abscisic acid (ABA), whereas GUS activity of Pro::GUS plants decreased under ABA treatment. Subcellular localization and transactivation assays showed that PeNAC070 protein was localized to the nucleus and exhibited transactivation activity at the C-terminal. Overexpression of PeNAC070 in Arabidopsis promoted lateral root development, delayed stem elongation, and increased sensitivity of transgenic plants to drought and salt stresses. This study aids in understanding the adaptability of P. euphratica to extreme drought and salt environment by analysing tissue-specific expression patterns of miR164-regulated and specific promoter-regulated PeNAC genes.
植物miR164家族高度保守，miR164成员调控属于NAC转录因子的保守靶标。我们之前的研究通过深度测序和降解组测序揭示，胡杨peu-miR164a-e及其靶基因POPTR_0007s08420参与胡杨的非生物胁迫响应。在本研究中，miR164家族包含六个成员，可产生两种成熟产物（miR164a-e和miR164f），并靶向胡杨中的七个NAC基因。在本氏烟草中的共表达和5' RACE证实，peu-miR164指导PeNAC070、PeNAC012和PeNAC028 mRNA的切割。初级peu-miR164 a/b/c/d/e的表达谱与peu-miR164a-e的表达谱相似，而在非生物胁迫下，PeNAC070和PeNAC081与peu-miR164a-e呈现相反的表达模式。PeNAC070启动子（ABRE、MBs、Box-W1、GC-motif和W-box）和peu-MIR164b启动子（HSE）中顺式作用元件的存在进一步证实了peu-miR164和PeNAC070对非生物胁迫的不同响应。组织化学β-葡萄糖醛酸酶（GUS）染色显示，当Pro::GUS转基因拟南芥植株暴露于NaCl、甘露醇和脱落酸（ABA）时，GUS活性增加，而在ABA处理下，Pro::GUS植株的GUS活性降低。亚细胞定位和反式激活分析表明，PeNAC070蛋白定位于细胞核，并在C端表现出反式激活活性。在拟南芥中过表达PeNAC070促进了侧根发育，延迟了茎的伸长，并增加了转基因植物对干旱和盐胁迫的敏感性。本研究通过分析miR164调控的和特定启动子调控的PeNAC基因的组织特异性表达模式，有助于了解胡杨对极端干旱和盐环境的适应性。
