The role of isoprenylcysteine methylation and metabolism in phytohormone signaling

David H Huizinga, Purdue University

Abstract

Abscisic acid (ABA) is the phytohormone responsible for mediating the response to long term stresses such as drought and salinity; and for preventing inappropriate seed germination and promotion of stomatal closure. Isoprenylated proteins have been shown to be negative regulators of ABA signaling. These proteins are modified by the attachment of a 15 or 20 carbon isoprenoid at a carboxyl terminal CaaX sequence, followed by aaX proteolysis and methylation of the carboxyl terminal isoprenylcysteine by isoprenylcysteine methyltransferase (ICMT). The methyl group can be subsequently removed from isoprenylated proteins by isoprenylcysteine methylesterase (ICME). In order to test the hypothesis that the methylation status of isoprenylated proteins has an effect on ABA signaling in plants, we analyzed a variety of transgenic Arabidopsis thaliana plants. Over-expression of ICMT in Arabidopsis caused an ABA insensitive phenotype, indicating that ICMT is a negative regulator of ABA signaling. In addition, the over-expression of ICME in Arabidopsis led to ABA hypersensitivity and drought tolerance in Arabidopsis, demonstrating that ICME is a positive regulator of ABA signaling. Interestingly, ICME was induced by ABA, indicating that ABA signaling is under positive feedback control, a result that makes sense considering the long term nature of ABA responses. Moreover, a T-DNA insertion near the carboxyl terminus of ICME induced increased ICME mRNA, and the 3' untranslated region of ICME caused decreased GUS expression in epidermal cells, and increased GUS expression in stomatal guard cells, particularly under drought conditions. Farnesylcysteine (FC), a product of the degradation of farnesylated proteins, is a known inhibitor of ICMT. We detected a lyase activity in Arabidopsis membranes that is specific to FC, and identified a gene that codes for the corresponding enzyme, farnesylcysteine lyase ( FCLY). T-DNA insertions in this gene caused reduced FCLY activity, and led to the accumulation of FC and an ABA hypersensitive phenotype. However, over-expression of ICMT in fcly plants suppressed or reversed the ABA hypersensitivity of fcly plants. These results are consistent with the hypothesis that the ABA hypersensitive phenotype of fcly plants is due to FC inhibition of ICMT. The results described here firmly establish that the methylation status of isoprenylated proteins determines their ability to negatively regulate ABA signaling and suggest possible approaches for genetically engineering drought tolerant crop species that would increase agricultural yields in arid regions or in times of drought.

Degree

Ph.D.

Advisors

Staiger, Purdue University.

Subject Area

Molecular biology|Cellular biology|Biochemistry

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