Date Approved

2004

Degree Type

Open Access Senior Honors Thesis

Department

Biology

First Advisor

Dr. Marianne Laporte

Second Advisor

Dr. Jim Vandenbosch

Abstract

A reliable supply of water is required to maintain virtually all forms of life. Plants, which lack the ability to migrate away from harsh environments, have evolved numerous mechanisms to cope with natural fluctuations of water availability. One type of modification is known as stomata, taken from the Greek word for “mouth”. Stomata resemble tiny mouths on the underside of leaves, opening and closing to regulate cellular water loss. Research has shown that over expression of NADP Malic Enzyme in transgenic plants causes the stomata to remain more closed. This decreases the amount of organismal water loss via transpiration, ultimately resulting in plants that consume less water.

Little is known about the link between NADP-ME and stomatal conductance. In order further characterize this relationship, we have compared enzyme expression between the guard cells and various other tissues of Arabidopsis thaliana. Using gene-specific primers for each of the six isoforms of Arabidopsis malic enzyme, RT-PCR was performed on RNA extracted from isolated guard cell protoplasts. The expression pattern of malic enzyme was then analyzed using gel electrophoresis. It was discovered that At5g11670 was the most highly expressed isoform in Arabidopsis guard cells, with significant expression of At5g25860 and At2g13566. Expression of all six isoforms was observed in root, stem, and leaf tissues.

We have developed a strategy to more accurately determine the mechanism by which NADP ME over-expression induces a decrease in stomatal conductance. Both the catalytic function and ion-binding activity of NADP-ME have the potential to affect guard cell activity of these transgenic plants, but it is not known which actually plays the significant role. To explore this, we have started work to create a deactivated form of maize NADP Malic Enzyme that retains ion-binding capabilities without catalyzing the malate to pyruvate reaction. The incapacitated enzyme will be introduced into an expression vector and expressed first in E. coli., and then in Arabidopsis thaliana. Stomatal conductance will be measured, answering questions regarding the role of the enzyme in guard cell activity. We hope that these findings may be used in engineering plant water use, further contributing to the success of world-wide agriculture.

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