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

Abstract

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 MAPK from guard cells, epidermis and mesophyll and immunoblotting using an antibody raised against a mammalian MAPK detected MAPK-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 MAPK 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.

Mitogen-Activated Protein Kinases and Reactive Oxygen Species Signaling in Plants

“…Thus, NtMPK4 might function at early stages of ROS signaling by controlling the entrance gate for ozone uptake from the environment. A MAPK that is activated by ABA was also identified in pea (Pisum sativum) guard cells (Burnett et al, 2000), providing further evidence for a role of MAPKs in stomatal control. MAPK modules do not only affect the aperture but also the density of stomata.…”

Abscisic Acid Biosynthesis and Response

“…ABA may enhance either expression (Hwang and Goodman, 1995;Hong et al, 1997;Lee et al, 1998;Mikami et al, 1998;GomezCadenas et al, 1999;Piao et al, 1999) or activity (Li and Assmann, 1996;Burnett et al, 2000) of these kinases. Although initial functional studies with pharmacological inhibitors did not discriminate among the roles of individual family members, recent studies with dominant negative alleles have assayed the role of specific kinases (Sheen, 1996;Li et al, 2000).…”

MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling

“…Furthermore, a study with pea epidermal peels showed that the MAPKK inhibitor PD98059 inhibits ABA-induced stomatal closure and expression of an ABA-inducible dehydrin gene (16,17). Despite these studies indicating that MAPK cascades function in ABA signaling, it remains to be established which specific MAPKs, MAPKKs, and MAPKK kinases (MAPKKKs) form a complete cascade to mediate ABA signaling in guard cells.…”