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Characterization of Arabidopsis carbonic anhydrase over- expression lines and novel methods development for Brassica napus large scale guard cell isolations

Abstract

In Arabidopsis thaliana, the carbon dioxide and abscisic acid signaling pathways are known to diverge early on, and [beta]-carbonic anhydrases ([beta]CAs) have been found to be upstream mediators of the carbon dioxide pathway. Recently, we reported that guard cell-targeted over- expression of [beta]CA1 and [beta]CA4 in wild-type plants displayed a reduced stomatal conductance at all CO₂ concentrations³⁵. The differences in drought tolerance of [beta]CA1 and [beta]CA4 over-expression plants may be explained by the sub-cellular localization of these proteins. Imaging of fluorescently labeled β-carbonic anhydrases showed that [beta]CA1 localizes to both the chloroplasts and the plasma membrane, while [beta]CA4 localizes to the plasma membrane. We propose that the beta]CA1 over-expression lines show more long-term drought -tolerance, because photosynthesis and energy production take place in the chloroplast. In contrast, [beta]CA4 over -expression lines were found to be more resilient to short -term osmotic stress, because the gene localizes to the plasma membrane Brassica napus is an edible oilseed crop species that is closely related to Arabidopsis thaliana, which indicate that B. napus may be useful for comparative genomic analyses³⁷⁻³⁸. A novel method for large scale B. napus guard cell protoplast isolation was developed for transcriptomic analysis of guard cell responses to abscisic acid (ABA) and drought. The optimimum B. napus growth conditions were similar to Arabidopsis thaliana growth conditions. By understanding the mechanisms regulating stomatal responses, we can develop strategies for improving drought tolerance of B. napus and, potentially, other crop plants

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