Miao Ying, Laun Thomas M, Smykowski Anja, Zentgraf Ulrike
Department of General Genetics, ZMBP (Centre of Molecular Biology of Plants), University of Tuebingen, Auf der Morgenstelle 28, Tuebingen, Germany.
Plant Mol Biol. 2007 Sep;65(1-2):63-76. doi: 10.1007/s11103-007-9198-z. Epub 2007 Jun 21.
Despite the importance of the senescence processes in plants, our knowledge on regulatory mechanisms of senescence is still poor. WRKY transcription factors have been shown to be involved in the regulation of leaf senescence. However, almost nothing is known about the upstream regulation of the senescence specific expression of WRKY factors. Therefore, we characterized proteins that bind and activate the promoter of WRKY53, which participates in leaf senescence in Arabidopsis thaliana. Surprisingly, a mitogen activated protein kinase kinase kinase (MEKK1) was identified as a DNA-binding protein. The binding motif for MEKK1 in the WRKY53 promoter could be characterized and promoter:GUS analyses revealed that this region is important for the switch of WRKY53 expression from a leaf age dependent to a systemic plant age dependent expression during bolting time. In addition to its promotor-binding activity, MEKK1 was also able to interact with the WRKY53 protein. Using bimolecular fluorescence complementation assays the complex formation of MEKK1 and WRKY53 could be localized predominately in the nucleus of Arabidopsis cells. MEKK1 could also phosphorylate WRKY53 in vitro and phosphorylation could increase DNA-binding activity of WRKY53 in vitro and transcription of a WRKY53 promoter driven reporter gene in vivo. These results suggest that MEKK1 is a bifunctional protein: it binds to the promoter of the WRKY53 gene regulating the switch from a leaf age dependent to a plant age dependent expression and it can phosopharylate WRKY53 in vitro increasing its DNA binding activity. Thus, MEKK1 might be able to take a very direct short cut in mitogen-activated protein kinase (MAPK) signalling by directly phosphorylating a transcription factor.
尽管衰老过程在植物中具有重要意义,但我们对衰老调控机制的了解仍然有限。WRKY转录因子已被证明参与叶片衰老的调控。然而,关于WRKY因子衰老特异性表达的上游调控几乎一无所知。因此,我们对与WRKY53启动子结合并激活该启动子的蛋白质进行了表征,WRKY53参与拟南芥的叶片衰老。令人惊讶的是,一种丝裂原活化蛋白激酶激酶激酶(MEKK1)被鉴定为一种DNA结合蛋白。可以对WRKY53启动子中MEKK1的结合基序进行表征,启动子:GUS分析表明,该区域对于抽薹期WRKY53表达从叶片年龄依赖性向全株年龄依赖性转变很重要。除了其启动子结合活性外,MEKK1还能够与WRKY53蛋白相互作用。使用双分子荧光互补分析,MEKK1和WRKY53的复合物形成主要定位于拟南芥细胞的细胞核中。MEKK1还可以在体外磷酸化WRKY53,磷酸化可以增加WRKY53在体外的DNA结合活性以及体内WRKY53启动子驱动的报告基因的转录活性。这些结果表明,MEKK1是一种双功能蛋白:它与WRKY53基因的启动子结合,调节从叶片年龄依赖性表达向全株年龄依赖性表达的转变,并且它可以在体外磷酸化WRKY53,增加其DNA结合活性。因此,MEKK1可能能够通过直接磷酸化转录因子在丝裂原活化蛋白激酶(MAPK)信号传导中采取非常直接的捷径。