ABA activation of an MBP kinase in Pisum sativum epidermal peels correlates with stomatal responses to ABA

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.