John Bennett

Biosynthesis of the Light-Harvesting Chlorophyll a/b Protein

1. When etiolated pea seedlings were exposed to continuous light for 24 h and then returned to darkness, 38% of the chlorophyll a, 74% of the chlorophyll b and 84% of the light-harvesting chlorophyll a/b protein that had accumulated under illumination proved to be unstable in darkness. The unstable chlorophyll displayed a half-life of about 90 min. In contrast, alpha and beta subunits of the chloroplast coupling factor and the large and small subunits of ribulose 1,5-biphosphate carboxylase continued to accumulate in darkness, although at a slower rate than in plants maintained under light. 2. Short-term labelling in vivo with L-[35S]methionine showed that leaves continued to synthesize the light-harvesting protein and the small subunit of ribulose 1,5-biphosphate carboxylase for up to 48 h after transfer of plants from light and darkness. However, after long-term labelling (16 h), the light-harvesting chlorophyll a/b protein was found to be labelled to high specific activity only in illuminated leaves. 3. I conclude that the light-harvesting chlorophyll a/b protein is subject to turnover after transfer of plants from light to darkness. The site of breakdown appears to be the photosynthetic membrane. I suggest that turnover of the protein is part of the normal physiological mechanism for co-ordinating the accumulation of the pigment and protein components of the light-harvesting chlorophyll a/b complex.

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.

Chloroplast phosphoproteins. The protein kinase of thylakoid membranes is light-dependent

Isolated intact pea cHoroplasts incorporate ~‘*P]~rthophosphate into several polypeptides in the 7000-79 000 mol. wt range [ I

Thylakoid protein phosphorylation during State 1—State 2 transitions in osmotically shocked pea chloroplasts

The three most Lonspicuous of the phosphoproteins are bound to the thylakoid membrane and comprise a 9000 Mr polypeptide and two polypeptides of about 26 OOOIU~ whicl~ form a closely-spaced doublet on SDS-palyacrylamide gels. The doublet is derived from the light-harvesting chlorophyll a/b binding protein complex of the thylakoid [2]. Labelling of the phosphoproteins with [32P]ortl~ophosphate is lightdependent in intact cldoroplasts [ 1 I_ In this paper I show that the protein kinase responsible for the

Photosynthetic protein phosphorylation in intact chloroplasts: Inhibition by DCMU and by the onset of CO2 fixation

o:;phorylation is also bound to the thylakoids and is itself light-dependent, Protein kinases generaily utilize [y-32P]ATP as phosphoryl group donor in in vitro assays. An obvious Ii@-dependent step in chloroplast phosphoprotein label.!ing from [32P]ortlloy)llospl~ate is the generation of [y-32P]ATP by photophoi,phorylation within the intact 0rgAnelles. -The fact that the uncoupler CCCB inhibits such phosphoprotein labelling f l] supports the notion that photophosp!~orylation is involved. However, when protein kinm activity is assayed in osmotically-shocked chloroplasts or in Isolated thylakoids, using exogenous [y-32P]ATP as phosphoryl group donor, protein phosphorylation is insensitive to CCCP but still light-dependent.

Research lettersPhosphorylation of thylakoid proteins and synthetic peptide analogs: Differential sensitivity to inhibition by a plastoquinone antagonist

Abstract In osmotically shocked pea chloroplasts illuminated with modulated blue-green light (light 2), phosphorylation of the light-harvesting chlorophyll a b- protein complex (LHCP) accompanies the slow decrease in modulated fluorescence that indicates adaptation to light absorbed predominantly by Photosystem II (State 2). On subsequent additional illumination with continuous far-red light (absorbed predominantly by Photosystem I; light 1) both effects are reversed: modulated chlorophyll fluorescence emission increases (indicating adaptation towards State 1) and LHCP is dephosphorylated. Net phosphorylation and dephosphorylation of LHCP induced by light 2 and excess light 1, respectively, occur on the same time scale as the ATP-dependent chlorophyll fluorescence changes indicative of State 2 and State 1 transitions. The phosphatase inhibitor NaF (10 mM), stimulates the effect of blue-green light on fluorescence and prevents the effect of far-red light. These results provide a demonstration that light of different wavelengths can control excitation energy distribution between the two photosystems via the plastoquinol-activated LHCP phosphorylation mechanism suggested previously (Allen, J.F., Bennett, J., Steinback, K.E. and Arntzen, C.J. (1981) Nature 291, 25–29; and Horton, P. and Black, M.T. (1980) FEBS Lett. 119, 141–144).

Comparison of ATP-induced and State 1/State 2-related changes in excitation energy distribution in Chlorella vulgaris

Chloroplasts contain a membrane-bound protein kinase which brings about phosphorylation of a number of thylakoid proteins [ 11, including the chlorophyll a/b binding protein of the light-harvesting chlorophyll-protein complex, LHCP [2]. Although ATP is the substrate for this protein phosphorylation, intact chloroplasts will incorporate [32P]orthophosphate into thylakoid proteins by virtue of their possession of an endogenous pool of adenine nucleotides and their capacity for photosynthetic phosphorylation of ADP. Protein phosphorylation in intact chloroplasts is light-dependent not only because of its dependence on photophosphorylation but also because the protein kinase is itself activated by illumination of the thylakoids to which it is bound [3]. Published studies of chloroplast protein phosphorylation have hitherto been carried out on thylakoids in the absence of added electron acceptors or on intact chloroplasts incapable of CO2 fixation. The investigation we describe in this letter was designed to establish which type of photosynthetic electron transport (cyclic or non-cyclic) activates protein phosphorylation in intact chloroplasts, and to see whether protein phosphorylation accompanies CO2 fixation in intact chloroplasts when they are supplied with CO2 to provide a physiological electron acceptor for non-cyclic electron transport.

The protein that harvests sunlight

Spinach thylakoids contain at least 8 proteins (8.3–58 kDa) whose phosphorylation is strongly inhibited by the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Reduction of DBMIB with ascorbate completely or partially relieves inhibition for all proteins other than the 27 kDa light-harvesting complex (LHC) II and a synthetic dodecapeptide (MRKSATTKKAVC). The peptide, an analog of the phosphorylation site of pea 27 kDa LHC II, is phosphorylated with the same site specificity, kinetics, redox control and sensitivity to DBMIB/ascorbate as the protein itself. The data indicate that synthetic peptides can be used to study the number, substrate specificity and redox regulation of thylakoid protein kinases.Spinach thylakoids contain at least 8 proteins (8.3–58 kDa) whose phosphorylation is strongly inhibited by the plastoquinone antagonist 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone (DBMIB). Reduction of DBMIB with ascorbate completely or partially relieves inhibition for all proteins other than the 27 kDa light‐harvesting complex (LHC) II and a synthetic dodecapeptide (MRKSATTKKAVC). The peptide, an analog of the phosphorylation site of pea 27 kDa LHC II, is phosphorylated with the same site specificity, kinetics, redox control and sensitivity to DBMIB/ascorbate as the protein itself. The data indicate that synthetic peptides can be used to study the number, substrate specificity and redox regulation of thylakoid protein kinases.

Inhibition of chloroplast development by tentoxin

Abstract The addition of ATP to thylakoids isolated from Chlorella vulgaris is shown to lead to a quenching of fluorescence originating from Photosystem II and phosphorylation of chlorophyll a chlorophyll b light-harvesting protein (LHCP) directly analogous to that reported for higher-plant chloroplasts. The time courses of these two processes are shown to be identical. Parallel measurements of ATP-induced changes in the fluorescence properties of isolated algal thylakoids and light-driven (State 1 / State 2 changes) in whole cells strongly support the idea that LHCP phosphorylation plays an important role in State 2 adaptation under in vivo conditions.

Protein Phosphorylation as an Adaptive Mechanism in Photosynthesis and Vision

Abstract The efficient harvesting of sunlight by chloroplasts depends on a chlorophyll-protein complex whose synthesis and phosphorylation are regulated by light.