Showing papers by "Christine H. Foyer published in 1984"

Compartmentation and fluxes of inorganic phosphate in photosynthetic cells.

Abstract: An analysis of the compartmentation and fluxes of inorganic phosphate in isolated cladophyll cells from Asparagus officinalis was made in parallel with an ultrastructural study. The elution pattern of labelled inorganic phosphate (which indicates that the asparagus cells are behaving as a system of three compartments in series) was used to quantify the fluxes between the vacuole, cytoplasm and free space. A relaxation time of 198 min was calculated for inorganic phosphate exchange between the vacuole and cytoplasm. It is, therefore, suggested that the vacuole serves to buffer the cytoplasmic inorganic phosphate concentration in the long term. However, in the short term, exchange with the vacuole will not appreciably affect the cytoplasmic inorganic phosphate concentration and thus the partitioning of photosynthetically fixed carbon.

Phosphorylation of a stromal enzyme protein in maize (Zea mays) mesophyll chloroplasts

Abstract: When intact maize (Zea mays) mesophyll chloroplasts were illuminated in the presence of [32P]orthophosphate and subsequently subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a major polypeptide species of Mr 100000 was found to be heavily labelled. This polypeptide was not found in maize mesophyll thylakoid or cytoplasmic fractions, but was localized solely in the chloroplast stroma. No phosphorylation of polypeptides in the 100000-Mr region was observed in the mesophyll chloroplasts from C3 species (where the primary product of CO2 fixation is a 3-carbon compound), suggesting that this polypeptide arises from a protein associated with C4 metabolism (where the first product of CO2 fixation is a 4-carbon compound). The 100kDa polypeptide was major component of the maize mesophyll chloroplast, comprising 10-15% of the total protein, which banded in an identical position to the apoprotein of the enzyme pyruvate, orthophosphate dikinase, which catalyses a reaction of the C4 cycle [Edwards & Walker (1983) C3, C4: Mechanisms, and Cellular and Environmental Regulation, of Photosynthesis, Blackwell Scientific Publications, Oxford and London]. Phosphorylation in the 100kDa species was prohibited by treatment of lysed chloroplasts with antibody to pyruvate, orthophosphate dikinase (EC 2.7.9.1). These data suggest that the phosphorylated polypeptide observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is the monomeric form of this enzyme. The 100kDa polypeptide was partially phosphorylated in darkness, but a significant increase in the degree of phosphorylation was found on illumination. This polypeptide was found to be dephosphorylated only slowly when the chloroplasts were returned to darkness. Maximum phosphorylation was observed in the presence of pyruvate or dihydroxyacetone phosphate, which also caused maximum activation of pyruvate, orthophosphate dikinase. Phosphorylation of the 100kDa polypeptide did not coincide with deactivation of pyruvate, orthophosphate dikinase, but maximum phosphorylation occurred under conditions that promoted maximum activity of the enzyme, at which time one phosphate group was associated with each enzyme molecule. Protein phosphorylation did not appear to arise from the reaction mechanism of the enzyme.

Regulation of Light Harvesting Chlorophyll a/b Binding Protein (LHCP) Phosphorylation in Intact Maize Mesophyll Chloroplasts

Abstract: The thylakoid membranes of higher plant chloroplasts show ATP-dependent protein kinase activity which results in phosphorylation of a number of membrane polypeptides including those which comprise LHCP (Horton, 1983a). The physiological role of this protein phosphorylation is to exert control over the rates of photon delivery to the two photosystems. Experiments using isolated pea thylakoids clearly demonstrated that the kinase activity was controlled by the redox state of the plastoquinone pool. More recently, it has been shown that ADP can act as a kinase inhibitor and subsequently that kinase activity is dependent on energy charge (Hartwell et al 1982). In the latter work it was suggested that redox transitions merely act as an ‘on/off’ switch rather than a continuous modulator, the major physiological control being attributed to energy charge. In order to fully understand the role of protein phosphorylation in vivo it is necessary to understand which of these regulators is a major control factor. Experiments using the manipulation of carbon metabolism in isolated intact mesophyll chloroplasts from maize, provides an ideal opportunity to distinguish between the regulation of kinase by plastoquinone redox state and by adenylate status. The maize mesophyll chloroplast contains the enzymes of the reductive phase of the Calvin Cycle, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase and the C4 enzymes, pyruvate Pi-dikinase and NADP-malate dehyrogenase. Addition of pyruvate stimulates ATP utilisation, the addition of oxaloacetate (OAA) causes the utilization of reducing equivalents without ATP consumption, while the addition of PGA requires that both ATP and NADPH are consumed. In this paper we describe simultaneous assays of redox state, ΔpH, ATP/ADP ratio, O2 evolution and protein kinase activity in these chloroplasts.

Effects of Protein Phosphorylation on the Properties of Thylakoid Membranes

Abstract: The State 1 to State 2 transition in higher plant chloroplasts involves the reversible phosphorylation of thylakoid membrane proteins (Horton 1983). This phosphorylation can alter the distribution of energy between PSII and PSI by either, or both, an alteration in the absorption cross-section of PSI (α) or by a change in spillover. An indication of mechanism is given by room temperature fluorescence induction curves of phosphorylated and non-phosphorylated thylakoids in the presence of DCMU (Fig.1). A lowering of fluorescence such that the Fv/Fm ratio is unchanged indicates a decrease in α whereas a preferential quenching of Fv (i.e. a decrease in the Fv/Fm ratio) indicates an increase in spillover of energy from PSII to PSI. We have previously shown (Horton, Black 1983) that the predominance of either factor depends upon the ionic composition of the suspension medium with phosphorylation promoting an increase in spillover only at low Mg2+ concentrations.