Okadaic Acid, a Protein Phosphatase Inhibitor, Blocks Calcium Changes, Gene Expression, and Cell Death Induced by Gibberellin in Wheat Aleurone Cells

Abstract

The cereal aleurone functions during germination by secreting hydrolases, mainly alpha-amylase, into the starchy endosperm. Multiple signal transduction pathways exist in cereal aleurone cells that enable them to modulate hydrolase production in response to both hormonal and environmental stimuli. Gibberellic acid (GA) promotes hydrolase production, whereas abscisic acid (ABA), hypoxia, and osmotic stress reduce amylase production. In an effort to identify the components of transduction pathways in aleurone cells, we have investigated the effect of okadaic acid (OA), a protein phosphatase inhibitor, on stimulus-response coupling for GA, ABA, and hypoxia. We found that OA (100 nM) completely inhibited all the GA responses that we measured, from rapid changes in cytosolic Ca2+ through changes in gene expression and accelerated cell death. OA (100 nM) partially inhibited ABA responses, as measured by changes in the level of PHAV1, a cDNA for an ABA-induced mRNA in barley. In contrast, OA had no effect on the response to hypoxia, as measured by changes in cytosolic Ca2+ and by changes in enzyme activity and RNA levels of alcohol dehydrogenase. Our data indicate that OA-sensitive protein phosphatases act early in the transduction pathway of GA but are not involved in the response to hypoxia. These data provide a basis for a model of multiple transduction pathways in which the level of cytosolic Ca2+ is a key point of convergence controlling changes in stimulus-response coupling.

Signal Transduction in Barley Aleurone Protoplasts Is Calcium Dependent and Independent.

“…Also, okadaic acid, an inhibitor of serinehhreonine protein phosphatases, has been found to inhibit the GA and ABA response of wheat aleurone, perhaps highlighting a role for changes in protein phosphorylation in the regulatory systems in these cells (Kuo et al, 1996). The observation that an ABA-insensitive mutant of Arabidopsis (ABi-1) is defective in a gene with homology to Ca2+-dependent protein phosphatases (Leung et al, 1994) further suggests that Ca2+-dependent protein phosphorylation events may be a fruitful avenue for research into the ABA signal transduction pathway.…”

Programmed cell death during vascular system formation

“…In contrast, the tonoplast disruption is a critical factor of the death of endocarp cells during ovary senescence of pea (Vercher et al, 1987). Aleurone cells die in response to GA (Kuo et al, 1996). This cell death process involves vacuolar enlargement and then the disruption of the tonoplast and plasma membrane, although it is unknown whether the vacuolar collapse precedes that of the plasma membrane.…”

“…Phytohormones sometimes function in the induction of developmentally programmed cell death. For example, the death of aleurone cells is induced by gibberellic acid (GA) and suppressed by abscisic acid (ABA) (Kuo et al, 1996;Wang et al, 1996b). In contrast, GA inhibits the cell death process in pea ovary senescence (Vercher et al, 1987).…”

Gibberellins: regulating genes and germination

“…For example, okadaic acid, a protein phosphatase inhibitor, has been found to affect both the GA and ABA pathways, but not hypoxic responses in the wheat aleurone (Kuo et al, 1996). Okadaic acid inhibited the increase in expression of high-pI amylase, the increase in cytoplasmic [Ca# + ] and the cell death normally induced by GA, as well as antagonizing the induction of the PHVA1 gene by ABA (Kuo et al, 1996).…”

“…These toxins have been applied to oat and barley aleurone and found to affect the inhibitory action of ABA on GA-induced amylase production Ritchie & Gilroy, unpublished), although Kuo et al (1996) reported no effect on wheat aleurone function. Further indications of G-protein action arise from the observation that application of mastoparan, a small peptide that mimics activated G-proteincoupled receptors, mimics GA action in oat aleurone cells , although again wheat aleurone was unresponsive (Kuo et al, 1996). In oat, a non-hydrolysable GTP analogue also blocked GA action, consistent with a G-protein transducing the GA signal in this tissue.…”