Calcium-Independent Steps in Guard Cell Regulation by Abscisic Acid: The Kinase Connection

Project: Research project

Project Details

Description

The epidermes of aerial plant organs contain microscopic pores called stomata through which gas exchange with the environment occurs. Stomatal apertures are regulated by pairs of guard cells that border and define the stomatal pores. Guard cells regulate stomatal apertures by osmotic swelling and shrinking, driven in large part by uptake or loss of potassium ions and anions. The plant hormone abscisic acid (ABA) inhibits stomatal opening and promotes closure when plants experience drought or are otherwise stressed. ABA effects on guard-cell ion-transport pathways include inhibition of potassium uptake channels and activation of channels mediating potassium and anion efflux. One pathway of ABA action appears to utilize elevation of cytosolic calcium levels as a signaling step, and experimental elevation of cytosolic calcium concentration can mimic ABA inhibition of potassium uptake channels and activation of anion channels. However, other data point to calcium independent modes of ABA action as well, which forms the focus of this research. Utilizing simultaneous whole-cell patch clamp analysis of potassium currents and confocal ratiometric cytosolic calcium imaging with Indo-1 dye under conditions where ABA does not evoke an increase in cytosolic calcium levels, it has been observed that ABA still inhibits the potassium uptake channels. Electrophysiological and imaging experiments are designed to test whether alterations in cytosolic pH play a signaling role in this pathway, and whether this pathway is linked to an intracellular ABA receptor. In addition, the role of phosphorylation/dephosphorylation in the pathway will be evaluated, because of evidence recently obtained for an ABA-activated, calcium independent kinase (ABA-activated protein kinase; AAPK) in guard cells of Vicia faba. This serine/threonine kinase is activated within one minute by physiological levels of ABA, and is detected in guard cells, but not epidermal or mesophyll cells. Utilizing probes based on AAPK peptide sequence recently obtained, AAPK cDNA and genomic clones will be identified in Vicia faba and Arabidopsis thaliana. Immunolocalization will be utilized to pinpoint the subcellular localization of AAPK. T-DNA insertion lines will be evaluated for 'knockouts' in AAPK; in addition, transgenic Arabidopsis plants over- or under-expressing AAPK will be produced. These plants will be evaluated under a battery of different growth conditions for phenotype effects of altered AAPK levels. In addition, guard cells from these plants will be subjected to a detailed patch clamp analysis to determine whether ABA regulation of channel activity is altered.

Guard cells are a vital control point in the regulation of photosynthetic carbon dioxide uptake and transpirational water loss, and, accordingly, represent potential targets for biotechnological manipulations to alter plant water use efficiency. It has been known for some time that alteration of other genes in the ABA-response pathways of guard cells, namely ABI1 and ABI2 in Arabidopsis (which encodes phosphatases), results in wilty plants, but these genes are also involved in developmental pathways in other cell types. The results of this research should allow the specific manipulation of a guard-cell ABA-response gene (namely AAPK), with potentially useful effects on plant function. For example, one could hypothesize that overexpression of AAPK would result in more drought-tolerant plants. Such hypotheses are testable.

StatusFinished
Effective start/end date3/1/995/31/03

Funding

  • National Science Foundation: $509,845.00

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