Lee Sun-Ji, Lee Mi Hun, Kim Jeong-Il, Kim Soo Young
Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, South Korea.
Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, South Korea
Plant Cell Physiol. 2015 Jan;56(1):84-97. doi: 10.1093/pcp/pcu148. Epub 2014 Oct 15.
We isolated an Arabidopsis ABA-insensitive mutant, ais143, by activation tagging screen. T-DNA was found to be located in the coding region of a putative mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) gene, Raf10, thereby abolishing its expression in the mutant. ais143 exhibited reduced seed dormancy as well as reduced ABA sensitivity. The phenotypes were complemented by the wild-type Raf10 gene, and the overexpression (OX) of Raf10 resulted in delayed seed germination and enhanced ABA sensitivity. Raf10 has high sequence identity to another MAP3K, Raf11. Parallel analysis of Raf11 knockout (KO) and OX lines showed that their phenotypes were similar to those of Raf10 KO and OX lines. An ais143 raf11 double mutant exhibited stronger phenotypes than single mutants, indicating the functional redundancy between Raf10 and Raf11. Transcript analysis revealed that the expression of many ABA-associated genes, including the key regulatory genes ABI3 and ABI5, was altered in the Raf10 and Raf11 OX lines. Recombinant Raf10 and Raf11 proteins exhibited kinase activity, which was inhibited by the MAP3K inhibitor BAY 43-9006 but not by the MAP2K inhibitor U0126. Collectively, our data indicate that Raf10 and Raf11 kinases are important regulators of seed dormancy and ABA response and that they affect the expression of ABI3, ABI5 and other ABA-regulated genes.
我们通过激活标签筛选分离出拟南芥脱落酸不敏感突变体ais143。发现T-DNA位于一个假定的丝裂原活化蛋白（MAP）激酶激酶激酶（MAP3K）基因Raf10的编码区，从而导致其在突变体中表达缺失。ais143表现出种子休眠降低以及脱落酸敏感性降低。野生型Raf10基因可互补这些表型，Raf10的过表达（OX）导致种子萌发延迟并增强了脱落酸敏感性。Raf10与另一个MAP3K，即Raf11具有高度的序列同一性。对Raf11敲除（KO）和过表达系的平行分析表明，它们的表型与Raf10 KO和OX系相似。ais143 raf11双突变体表现出比单突变体更强的表型，表明Raf10和Raf11之间存在功能冗余。转录分析显示，包括关键调控基因ABI3和ABI5在内的许多脱落酸相关基因的表达在Raf10和Raf11过表达系中发生了改变。重组Raf10和Raf11蛋白表现出激酶活性，该活性被MAP3K抑制剂BAY 43-9006抑制，但不被MAP2K抑制剂U0126抑制。总的来说，我们的数据表明Raf10和Raf11激酶是种子休眠和脱落酸反应的重要调节因子，并且它们影响ABI3、ABI5和其他脱落酸调控基因的表达。
