Poster: Water relations

Abs # 158: Expression and Mutagenesis of NADP⁺ Malic Enzyme in Guard Cells

Presenter:	Coliadis, Jason , jcoliadis@emich.edu
Authors	Laporte, Marianne M. (A)   Coliadis, Jason  (A)   Muniz, Jennifer  (A)
Affiliations:	(A): Eastern Michigan University

NADP⁺ malic enzyme (NADP-ME) has been implicated in the stomatal conductance mechanism of C₃ plants and tobacco plants that have been genetically modified to express high levels of NADP-ME have been shown to consume less water per unit mass gained than wild-type plants. Therefore, this protein has potential for altering water use in crop plants. Our focus is to characterize the expression and role of NADP-ME activity in stomatal conductance. Here, we present stomatal expression patterns of the six isoforms of NADP-ME in the Arabidopsis thaliana genome. RNA was extracted from Arabidopsis guard cell protoplasts, as well as from leaf, stem, and root tissue. Expression patterns were determined using RT-PCR with gene specific primers. At5g11670 was the only isoform consistently expressed at high levels in guard cells, indicating that it is the major form of NADP-ME in Arabidopsis guard cells. Expression of all six NADP-ME loci was observed in root, leaf, and tissue samples. We hypothesize that the mechanism through which malic enzyme influences stomatal conductance is a result of its effect on malate content in guard cells, which is integral to the enzyme’s catalytic function. To test this hypothesis, we have created site-directed mutants of NADP-ME that maintain ion-binding capacity but lack malate catalysis. The mutant cDNAs have been inserted into expression vectors and their enzymatic activity has been assessed in E. coli. We plan experiments to asses the effect of mutagenized NADP-ME on transgenic Arabidopsis and tobacco plants. This strategy will allow us to characterize the role of NADP-ME in stomatal conductance, and may lead to new strategies in engineering plant water use.