Yoo Jae Hyuk, Park Chan Young, Kim Jong Cheol, Heo Won Do, Cheong Mi Sun, Park Hyeong Cheol, Kim Min Chul, Moon Byeong Cheol, Choi Man Soo, Kang Yun Hwan, Lee Ju Huck, Kim Ho Soo, Lee Sang Min, Yoon Hae Won, Lim Chae Oh, Yun Dae-Jin, Lee Sang Yeol, Chung Woo Sik, Cho Moo Je
Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea.
J Biol Chem. 2005 Feb 4;280(5):3697-706. doi: 10.1074/jbc.M408237200. Epub 2004 Nov 29.
Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca2+-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis, a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Delta1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants.
钙调蛋白（CaM）是一种普遍存在的钙结合蛋白，通过调节多种酶和蛋白质的活性来调控多种细胞功能。植物表达众多的CaM异构体，这些异构体在体外对依赖CaM的酶表现出不同的激活和/或抑制作用。然而，植物CaM的具体生物学功能尚不清楚。在本研究中，我们分离出一个编码CaM结合转录因子MYB2的cDNA，该转录因子调节拟南芥中盐和脱水响应基因的表达。这是通过使用盐诱导的CaM异构体（GmCaM4）作为探针，从盐处理的拟南芥表达文库中实现的。通过结构域定位，我们在MYB2中鉴定出一个Ca2+依赖的CaM结合结构域。通过定点诱变、凝胶迁移率变动分析、分裂泛素分析以及使用Ca2+/CaM依赖酶的竞争分析，证实了CaM与CaM结合结构域的特异性结合。有趣的是，特定的CaM异构体GmCaM4增强了AtMYB2的DNA结合活性，而与之密切相关的CaM异构体（GmCaM1）则抑制了这种活性。在拟南芥中过表达Gm-CaM4上调了AtMYB2调控基因的转录速率，包括脯氨酸合成酶P5CS1（Δ1-吡咯啉-5-羧酸合成酶-1），该酶通过促进脯氨酸积累赋予耐盐性。因此，我们认为特定的CaM异构体通过激活MYB转录激活因子介导盐诱导的Ca2+信号传导，从而导致植物的耐盐性。
