ABA activation of an MBP kinase in Pisum sativum epidermal peels correlates with stomatal responses to ABA
ABA activation of an MBP kinase in Pisum sativum epidermal peels correlates with stomatal responses to ABA
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DOI：
10.1093/jexbot/51.343.197
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发表时间：
2000-02-01
影响因子：
6.9
通讯作者：
Neill, SJ
中科院分区：
文献类型：
--
作者：
Burnett, EC;Desikan, R;Neill, SJ
用髓鞘碱性蛋白（MBP）作为底物的凝胶内蛋白激酶测定表明，脱落酸（ABA）激活了豌豆突变体叶片表皮皮中的一个MBP激酶（AMBP激酶）。AMBP激酶具有丝裂原活化蛋白激酶（MAPK）的特征。它优先利用MBP作为人工底物，它是快速和短暂激活的，它的大小合适（分子量约45 kDa），需要酪氨酸磷酸化才能发挥活性，并且在激活时酪氨酸被磷酸化。利用逆转录- pcr技术从保护细胞、表皮和叶肉细胞中克隆出一个MARK，并利用一种针对哺乳动物MAPK的抗体进行免疫印迹检测，在表皮表皮、叶肉和保护细胞中检测到MARK相关蛋白，包括45 kDa的一个。MARK激酶特异性抑制剂PD98059抑制AMBP激酶的激活，进而抑制MAPK的激活，与PD98059抑制ABA诱导的气孔关闭和脱氢基因表达相关，这表明ABA对豌豆表皮剥皮的作用需要MAPK的激活。经酶处理分离的保护细胞AMBP激酶未被ABA激活。然而，在分离的保护细胞中，c. 43 kDa的蛋白激酶被ABA激活，而在叶肉和表皮组织中则没有。
In-gel protein kinase assays using myelin basic protein (MBP) as substrate have been used to demonstrate that abscisic acid (ABA) activates an MBP kinase (AMBP kinase) in epidermal peels prepared from leaves of the Argenteum mutant of pea, Pisum sativum L. AMBP kinase has the characteristics of a mitogen-activated protein kinase (MAPK): it utilizes MBP preferentially as an artificial substrate, it is rapidly and transiently activated, it is of the appropriate size (molecular weight c. 45 kDa), requires tyrosine phosphorylation for activity and is tyrosine phosphorylated upon activation. Reverse transcription-PCR was used to generate a previously-cloned MARK from guard cells, epidermis and mesophyll and immunoblotting using an antibody raised against a mammalian MAPK detected MARK-related proteins, including one of 45 kDa, in epidermal peels, mesophyll and guard cells. Inhibition of AMBP kinase activation by PD98059, a specific inhibitor of MARK kinase, and thus MAPK activation, correlated with PD98059-inhibition of ABA-induced stomatal closure and dehydrin gene expression, suggesting that ABA effects in pea epidermal peels require MAPK activation. AMBP kinase was not activated by ABA in guard cells isolated by enzyme treatment. However, a protein kinase of c. 43 kDa was activated by ABA in isolated guard cells, but not in mesophyll or epidermal tissue.