Michael T. Black

Regulation of phosphorylation of chloroplast membrane polypeptides by the redox state of plastoquinone

Higher plant chloroplasts possess a light-activated protein kinase that catalyses phosphorylation of several thylakoid proteins including LHCP [l-5]. A decrease in the yield of chlorophyll fluorescence from PSI1 at room temperature was caused by addition of ATP under conditions necessary for kinase activity. Under the same conditions there is an increase in the relative fluorescence emission from PSI at -196°C. These observations support the proposal that phosphorylation of LHCP controls the distribution of quanta between PSI1 and PSI [6,7]. On the basis of the ability of various partial reactions of photosynthetic electron transport to promote kinase activity, it was proposed that the redox state of plastoquinone controls protein phosphorylation and hence also the distribution of quanta [8]. A similar suggestion was made to explain ATP-induced fluorescence quenching [7] and potentiometric redox titration indeed showed the involvement of a 2 electron carrier with E,, 7.8 -Ok+50 mv [9]. However, it remained to be shlwn that: (i) Phosphorylation was responsible for the fluorescence changes, (ii) Plastoquinone in pea chloroplasts titrates with this midpoint potential. Here, the crucial links are established between the redox state of plastoquinone, the activation of protein kinase and changes in chlorophyll fluorescence. A model by which the redox state of plastoquinone can control the relative rates of excitation of PSI1 and PSI is presented.

Activation of adenosine 5′ triphosphate-induced quenching of chlorophyll fluorescence by reduced plastoquinone: The basis of state I–state II transitions in chloroplasts

The distribution of absorbed radiation arriving at the reaction centres of PSI1 and PSI in chloroplasts is subject to regulation [ 1,2]; this phenomenon was first defined in terms of the state I-state II transitions, whereby light absorbed by PSI1 causes a change that enhances the efficiency of PSI and light absorbed by PSI induces a reversal of this change [3]. Experimentally, this process has been studied by observing the slow quenching of chlorophyll fluorescence that occurs during a period of several minutes following illumination [4-61. At least part of this quenching is due to the establishment of a transmembrane proton gradient [4-81 but it has been shown that such quenching does not elicit a change in exciton distribution between PSI1 and PSI [8,9]. In isolated chloroplast membranes addition of ATP causes a 25-30% quenching of chlorophyll fluorescence [9,10] after 10 min incubation. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU. Fluorescence emission spectra at -196’C indicate an increase in the fraction of energy transferred to PSI after ATP treatment. It was suggested therefore that this effect could be the basis for physiological regulation of exciton distribution. The molecular mechanism for the ATP-induced quenching is the presence in

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5′-triphosphate

Addition of ATP to chloroplasts causes a reversible 25-30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at -196 degrees C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F0) and variable (Fv) fluorescence such that the ratio of Fv to the maximum (Fm) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of Fv against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.

Heterogeneity in chloroplast photosystem II.

Photosystem-two (PSII) in the chloroplasts of higher plants and green algae is not homogeneous. A review of PSII heterogeneity is presented and a model is proposed which is consistent with much of the data presented in the literature. It is proposed that the non-quinone electron acceptor of PSII is preferentially associated with the sub-population of PSII known as PSIIß.

On the nature of the fluorescence decrease due to phosphorylation of chloroplast membrane proteins

Abstract 1. Phosphorylation of chloroplast membranes by illumination in the presence of ATP results in a 15–20% increase in the rate of Photosystem I electron transfer at low light intensity. 2. Phosphorylated membranes when depleted of Mg2+ and resuspended in a low salt medium still show a 17% lower yield of Photosystem II fluorescence than do unphosphorylated membranes. A 31% difference is seen after restoration of the maximal yield by addition of Mg2+. 3. The concentration of Mg2+ required to induce a half-maximal increase in fluorescence is 0.9 mM for control and 1.8 mM for phosphorylated chloroplasts. Phosphorylation at 1 mM Mg2+ can therefore cause more than double the amount of decrease in fluorescence yield from Photosystem II compared to phosphorylation at 5 mM. 4. The above results are discussed in terms of the mechanism of the ATP-induced fluorescence changes and a suggestion is made that the apparent interaction between phosphorylation and Mg2+ concentration may be a physiologically important phenomenon.

A comparison between cation and protein phosphorylation effects on the fluorescence induction curve in chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea

Abstract Fluorescence induction curves in chloroplasts phosphorylated by the thylakoid protein kinase activated at low light intensity and high chlorophyll concentration have been measured. At 5 mM Mg2+, phosphorylation did not preferentially quench variable fluorescence. At 1 mM, preferential quenching of variable fluorescence was observed, indicating a second effect of phosphorylation at low Mg2+ (Horton, P. and Black, M.T. (1982) Biochim. Biophys. Acta 680, 22–27). Comparison of the extent of fluorescence decrease and the resulting ratio of variable to maximum fluorescence after phosphorylation and after lowering Mg2+ concentration demonstrated a difference between these two mechanisms of lowering of fluorescence. The significance of these results in terms of how phosphorylation may alter membrane organization is discussed.

An investigation into the ATP requirement for phosphorylation of thylakoid proteins and for the ATP-induced decrease in the yield of chlorophyll fluorescence in chloroplasts at different stages of development

Abstract The phosphorylation of thylakoid membrane polypeptides has been investigated in chloroplasts prepared from peas that had been grown under intermittent light and then exposed to between 4 and 48 h of continuous light. At 4 h, when the ratio of the total amount of labelling of a 9 kDa-polypeptide relative to light-harvesting chlorophyll protein (LHCP) polypeptides was much greater than 1, the affinity for ATP was found to be the same ( S 0.5 , approx. 100 μM) for both polypeptides. In contrast, in fully greened chloroplasts, when labelling of LHCP was much greater than that of the 9 kDa-polypeptide, the S 0.5 for ATP was 40 μM for LHCP and 500 μM for the 9 kDa-polypeptide. A correlation was observed during development between the affinity for ATP of the 9 kDa-species and its abundance relative to LHCP. It is suggested that these polypeptides compete for phosphorylation by the same protein kinase. Simultaneous assay of the ATP-induced fluorescence decrease at different ATP concentrations revealed a close correlation with LHCP labelling but not with labelling of the 9 kDa-polypeptide. This correlation held irrespective of which polypeptide was the major phosphoprotein.

An investigation of the mechanistic aspects of excitation energy redistribution following thylakoid membrane protein phosphorylation

Abstract Accompanying thylakoid membrane protein phosphorylation is a redistribution of energy between PS II and PS I; mechanistic aspects of this redistribution have been investigated in both a mature and a developing chloroplast system. Data are presented which suggest that the mechanism of these changes is dependent upon the developmental status and/or morphological characteristics of the chloroplast.

Changes in topography and function of thylakoid membranes following membrane protein phosphorylation.

Changes in topography and function of pea (Pisum sativum L.) thylakoid membrane fractions following membrane protein phosphorylation have been studied. After protein phosphorylation the stromal membrane fraction had a higher chlorophyll a/b ratio, an increased content of light-harvesting chlorophyll protein and a higher ratio of chlorophyll to cytochrome f. This indicates that a pool of light-harvesting chlorophyll protein migrates from the photosystem II-enriched grana regions to the photosystem I-enriched stroma lamellae, in agreement with Kyle et al. (1984, Biochim. Biophys. Acta 765, 89–96) and Larsson et al. (1983, Eur. J. Biochem. 136, 25–29). Phosphorylation caused a stimulation in the rate of light-limited photosystem-I electron transfer in the unappressed membrane fraction, indicating that the translocated LHC-II becomes functionally associated with photosystem I.

Effects of Protein Phosphorylation on the Properties of Thylakoid Membranes

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.