The Regulation of Phosphoenolpyruvate Carboxylase in Stomatal Guard Cells of Commelina communis

Nelson, John Paul S (1994) The Regulation of Phosphoenolpyruvate Carboxylase in Stomatal Guard Cells of Commelina communis. PhD thesis, University of Glasgow.

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A great deal of work has been done recently to investigate mechanisms which control plant metabolism. In the CAM and C4 systems, it was found that phosphoenolpyruvate carboxylase which catalyses the primary fixation of CO2 in these plants is controlled by a reversible phosphorylation mechanism, with the enzyme being more active in the phosphorylated form than the nonphosphorylated form. Stomatal guard cells exhibit a form of malate metabolism very similar to CAM, and require the pathway of malate synthesis to be active during stomatal opening, and inactive during closure. This pathway involves flux through phosphoenolpyruvate carboxylase. The main aim of the work described in this thesis was to investigate the regulation of phosphoenolpyruvate carboxylase in guard cells. In addition, signal transduction in stomatal guard cells has recently been investigated by many workers, and this system is often now regarded as a model for signal transduction in plants. An additional aim of this project, therefore, was to study aspects of the signalling process involved in regulation of guard cell phosphoenolpyruvate carboxylase. The first part of the work involved the definition of conditions that would reproducibly cause stomatal movements. Stomata in epidermal strips from the C3 plant Commelina communis were found to open when incubated in the light on a solution containing 25mM K2SO4 and which had air with a reduced concentradon of CO2 bubbled through it. When the epidermis was placed into darkness and normal air was bubbled through the medium, the stomata closed. Stomata were also found to open in response to the fungal toxin fusicoccin when incubated on 25mM K2SO4 in the dark. The possibility that guard cell phosphoenolpyruvate carboxylase is regulated by phosphorylation was investigated in several ways. The incubation of extracts of guard cell protoplasts with [gamma-32P] ATP, Mg2+ and cyclic AMP dependent protein kinase catalytic subunits showed that guard cell phosphoenolpyruvate carboxylase became phosphorylated in vitro. In the absence of added kinase, no phosphorylation of the enzyme was observed, even in the presence of 1mM free Ca2+. Incubation of intact guard cell protoplasts with [32P]-orthophosphate showed that the enzyme was also phosphorylated in vivo. The phosphorylation state of guard cell phosphoenolpyruvate carboxylase was increased when guard cell protoplasts were incubated with 50mM K+ in the light or with fusicoccin. The phosphorylation state of the enzyme did not increase when protoplasts were incubated in the presence of 50mM K+ in darkness or with either abscisic acid or okadaic acid, an inhibitor of protein phosphatases. Light stimulated phosphorylation of the enzyme was reversed when protoplasts were placed in darkness. Protein phosphatase 2A activity, known to be responsible for the dephosphorylation of phosphoenolpyruvate carboxylase in the CAM plant Bryophyllum fedtschenkoi, was detected in extracts of guard cell protoplasts. Fusicoccin-stimulated phosphorylation was prevented by the protein synthesis inhibitor cycloheximide, a phenomenon similar to observations made in the CAM and C4 systems. It was also found that cycloheximide appeared to retard the stomatal opening that occors in response to both light and fusicoccin. Assays were carried out to measure the activity and some of the kinetic parameters of guard cell phosphoenolpyruvate carboxylase. Enzyme activity was almost five fold higher at pH 8.0 than at pH 7.2, but the enzyme was relatively insensitive to the feedback inhibitor malate at higher pH. When measured at pH 7.8, increasing concentration of the inhibitor malate increased both apparent Km and apparent Vmax values and at malate concentrations higher than 5mM, the enzyme appeared to depart from Michaelis-Menten kinetics. When guard cell protoplasts were incubated in the presence of K2SO4 in the light and dark or with and without fusicoccin, no significant differences in Ki (malate) from protoplasts in different conditions were observed. Further attempts to change Ki (malate) by the incubation of guard cell extracts with protein phosphatases and kinases also produced no significant change. When enzyme assays were carried out at pH 7.0 in the presence of glucose 6- phosphate, the enzyme extracted from guard cell protoplasts which had been incubated in the light had a Km (PEP) of 4.7muM, whilst that from those incubated in the dark had a Km (PEP) of 10.7muM, with a probability of identity between the two of less than 7%. Extracts of fusicoccin-stimulated protoplasts contained phosphoenolpyruvate carboxylase with a Km (PEP) of 6.7muM, with a less than 18% probability of identity with the dark control. Ki (malate) was not found to change significantly when measured under these conditions, although Vmax was decreased in protoplasts which were incubated in the light or with fusicoccin compared to the dark control. These results provide strong evidence to suggest that guard cell phosphoenolpyruvate carboxylase is controlled by reversible phosphorylation, in a manner analagous to the CAM and C4 enzymes. In addition, consideration of these results leads to the suggestion that cytoplasmic K+ concentration is an important signalling factor in guard cells.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: H G Nimmo
Keywords: Biochemistry, Cellular biology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75639
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:02
Last Modified: 19 Nov 2019 19:02

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