Date Approved

2008

Degree Type

Open Access Senior Honors Thesis

Department

Biology

First Advisor

Dr. Marianne Laporte

Second Advisor

Dr. James VandenBosch

Abstract

Plants that reduce water loss by transpiration present less agricultural stress to the environment. Transpiration is limited by the size of pores, or stomata, on the surfaces of leaves. Stomatal aperture is related to the ion concentration in surrounding guard cells, which varies in response to fluctuating concentrations of potassium ions, the principle counter-ion chloride, and the organic counter-ion malate. Levels of malate in guard cells may be altered by increased activity of NADP-Malic Enzyme (NADP-ME) expression. The goal of our research is to create transgenic Arabidopsis thaliana plants with increased NADP-ME expression in the guard cells, limiting transpiration through decreased stomatal aperture size. Such plants are expected to have less open stomata, be sufficient in yield, and retain the ability to respond to changing environmental conditions.

Expression of NADP-ME in Arabidopsis thaliana required the isolation of a strong, guard-cell specific promoter. A promoter of a potassium channel gene, KAT1, is strongly expressed in guard cells. Analysis of the Arabidopsis genome was done to locate KAT1. The KAT1 promoter was amplified and isolated through Polymerase Chain Reaction (PCR). Sequence analysis confirmed isolation of KAT1 with minor mutations. Visualization of KAT1 expression was confirmed through the analysis of transgenic plants with the KAT1 promoter fused to the GUS reporter gene. Significant expression of KAT1 was detected exclusively in guard cells of 9-day-old seedlings. The KAT1 promoter will be inserted into a binary vector and then Agrobacterium to transform Arabidopsis thaliana and create transgenic plants. Expression of KAT1 with NADP-ME in Arabidopsis thaliana should result in lower concentrations of malate in guard cells, decreased aperture size of stomata, and a decrease in transpiration rate during gas exchange.

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