Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.

The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.

We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.

Electron transfer from cytochrome f to photosystem I in green algae.

The time course of P700(+) reduction and cytochrome f oxidation following a single-turnover flash excitation of photosystem I was measured under various conditions in different strains of green algae. P700(+) was reduced with a half-time of 4 µs. The rate of cytochrome f oxidation was found to depend widely on physiological factors. Reversible transitions are described from a 'slow-oxidation' state (t 1/2=500 µs) to a 'fast-oxidation' state (t 1/2=80 µs). The addition of ionophore strongly favours and stabilizes the 'fast-oxidation' state. We suggest that these transitions reflect either reversible association between the cytochrome bf complex and the reaction center of photosystem I or changes in the mobility of oxidized plastocyanin. The transitions might be under the control of the membrane potential or the intracellular ATP content. The relation of these reversible transitions with the 'light state' transitions, and their possible involvement in a switch from linear to cyclic electron transfer, are discussed.

Turnover kinetics of photosystem I measured by the electrochromic effect in Chlorella.

The rise kinetics of the absorption changes induced at 515 nm and 480 nm by a flash were studied using two types of xenon flashes of different durations. The 'slow' rise of the absorption change (t 1/2 = 15--20 microseconds) observed by Cox and Delosme (1978 C.R. Acad. Sci. (Paris) Sér. D 282, 775--778) and Joliot was found to be due to double hits occurring in the reaction centers of System I during the flash. The turnover kinetics of the reaction centers of System I after a short flash were studied by a double flash method. They are in agreement with a second order reaction between P+-700 and its electron donor.

515 nm Absorption changes in Chlorella at short times (4--100 mus) after a flash.

Using Chlorella, three types of absorption changes at 515 nm have been studied in the 4-100 mus time range following a flash. (1) The absorption change observed when both photoreactions are blocked, probably due to the formation of the triplet state of a carotenoid, is show to depend on Photosystem II excitation only. (2) The absorption increase induced by photoreaction I is biphasic; first phase, complete in less than 4 mus, followed by a slower phase with a half-rise time of 15-20 mus. (3) On the other hand, photoreaction II induces only a fast absorption increase (lessthan 4 mus). The time course of the biphasic 515 nm absorption increase induced by photoreaction I is similar to the biphasic absorption decrease previously observed at 480 nm by Cox and Delosme (1976, C.R. Acad. Sci. Paris 282D, 775-778). No significant absorption change is observed at 490 nm. These results suggest that the transmembrane electric field induced by photoreaction I rises to its maximum value in at least two phases within 100 mus following flash excitation.