The temperature-dependent accumulation of Mg-protoporphyrin IX and reactive oxygen species in Chlorella vulgaris

Photosynthetic organisms undergo photoacclimation in response to changes in environmental conditions to maximize energy production and at the same time protect the light-sensitive pigments and proteins from excess light. Low temperature and high irradiance both cause the electron transport chain to become more reduced which can result in the production of damaging reactive oxygen species. In the unicellular green alga Chlorella vulgaris Beij., light and temperature regulate light-harvesting protein accumulation via the redox state of the plastoquinone pool. To investigate temperature-dependent factor (s) regulating light-harvesting protein accumulation we measured the abundance of chlorophyll biosynthetic precursors and reactive oxygen species production in C. vulgaris cells acclimated to a series of growth conditions. We observed that Mg-protoporphyrin accumulates in response to low temperature, but its abundance does not correlate with light-harvesting protein levels. Reactive oxygen levels measured under the same growth conditions strongly correlated with light-harvesting protein levels. Therefore, we suggest that reactive oxygen species may act as part of both a temperature- and irradiance-dependent signalling mechanism in the regulation of light-harvesting protein accumulation in response to growth conditions.     

The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

A cytochrome b ₆ f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a ‘high light’ acclimation state. The cytochrome b ₆ f complex may be involved indirectly in the regulation of photoacclimation via 1) regulation of the plastoquinone redox state; 2) regulation of the redox-controlled thylakoid protein kinase allowing exposure of the dephosphorylated LHC II to acclimative proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Coregulation of light-harvesting complex II phosphorylation and lhcb mRNA accumulation in winter rye

Winter rye plants grown under contrasting environmental conditions or just transiently shifted to varying light and temperature conditions, were studied to elucidate the chloroplast signal involved in regulation of photosynthesis genes in the nucleus. Photosystem II excitation pressure, reflecting the plastoquinone redox state, and the phosphorylation level of thylakoid light-harvesting proteins (LHCII and CP29) were monitored together with changes occurring in the accumulation of lhcb, rbcS and psbA mRNAs. Short-term shifts of plants to changed conditions, from 1 h to 2 d, were postulated to reveal signals crucial for the initiation of the acclimation process. Comparison of these results with those obtained from plants acclimated during several weeks' growth at contrasting temperature and in different light regimes, allow us to make the following conclusions: (1) LHCII protein phosphoylation is a sensitive tool to monitor redox changes in chloroplasts; (2) LHCII protein phosphorylation and lhcb mRNA accumulation occur under similar conditions and are possibly coregulated via an activation state of the same kinase (the LHCII kinase); (3) Maximal accumulation of lhcb mRNA during the diurnal light phase seems to require an active LHCII kinase whereas inactivation of the kinase is accompanied by dampening of the circadian oscillation in the amount of lhcb mRNA; (4) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead (5) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for the regulation of lhcb gene expression in the nucleus.     

Temperature-induced greening of<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis>. The role of the cellular energy balance and zeaxanthin-dependent nonphotochemical quenching

When cells of the green alga Chlorella vulgaris Beij. are transferred from growth at 5 degrees C and an irradiance of 150 micromol photons m(-2) s(-1) to 27 degrees C and the same irradiance, they undergo what is normally considered a high-light to low-light phenotypic change. This involves a 3-fold increase in cellular chlorophyll content with a concomitant increase in light-harvesting complex polypeptide levels. This process appears to occur in response to the cellular capacity to utilize the products of photosynthesis, with the redox state of the plastoquinone pool sensing the cellular energy balance. The phenotypic adjustment can be enhanced or blocked using chemical inhibitors that modulate the redox state of the plastoquinone pool. The functional changes in the photosynthetic apparatus that occurred during the high-light to low-light acclimation were examined with special consideration paid to the paradox that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated cells, with non-functional photosystem II (PSII), accumulate light-harvesting polypeptides. At the structural and basic functional levels, the light-harvesting complex of the cells treated with DCMU was indistinguishable from that of the untreated, control cells. To examine how PSII was protected in the DCMU-treated cells, we measured the content of xanthophyll-cycle pigments. It appeared that a zeaxanthin-dependent nonphotochemical quenching process was involved in PSII protection during greening in the presence of DCMU. Metabolic inhibitors of mitochondrial respiration were used to examine how the change in cellular energy balance regulates the greening process. Apparently, the mitochondrion acts to supply energy to the chloroplast during greening, and inhibition of mitochondrial respiration diminishes chlorophyll accumulation apparently through an increase in the redox state of the plastoquinone pool.     

Thylakoid protein phosphorylation and the thiol redox state

Illumination of thylakoid membranes leads to the phosphorylation of a number of photosystem II-related proteins, including the reaction center proteins D1 and D2 as well as the light-harvesting complex (LHCII). Regulation of light-activated thylakoid protein phosphorylation has mainly been ascribed to the redox state of the electron carrier plastoquinone. In this work, we show that this phosphorylation in vitro is also strongly influenced by the thiol disulfide redox state. Phosphorylation of the light-harvesting complex of photosystem II was found to be favored by thiol-oxidizing conditions and strongly downregulated at moderately thiol-reducing conditions. In contrast, phosphorylation of the photosystem II reaction center proteins D1 and D2 as well as that of other photosystem II subunits was found to be stimulated up to 2-fold by moderately thiol-reducing conditions and kept at a high level also at highly reducing conditions. These responses of the level of thylakoid protein phosphorylation to changes in the thiol disulfide redox state are reminiscent of those observed in vivo in response to changes in the light intensity and point to the possibility of a second loop of redox regulation of thylakoid protein phosphorylation via the ferredoxin-thioredoxin system.     

Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase.

Higher plants acclimate to various light environments by changing the antenna size of a light-harvesting photosystem. The antenna size of a photosystem is partly determined by the amount of chlorophyll b in the light-harvesting complexes. Chlorophyllide a oxygenase (CAO) converts chlorophyll a to chlorophyll b in a two-step oxygenation reaction. In our previous study, we demonstrated that the cellular level of the CAO protein controls accumulation of chlorophyll b. We found that the amino acids sequences of CAO in higher plants consist of three domains (A, B, and C domains). The C domain exhibits a catalytic function, and we demonstrated that the combination of the A and B domains regulates the cellular level of CAO. However, the individual function of each of A and B domain has not been determined yet. Therefore, in the present study we constructed a series of deleted CAO sequences that were fused with green fluorescent protein and overexpressed in a chlorophyll b-less mutant of Arabidopsis thaliana, ch1-1, to further dissect functions of A and B domains. Subsequent comparative analyses of the transgenic plants overexpressing B domain containing proteins and those lacking the B domain determined that there was no significant difference in CAO protein levels. These results indicate that the B domain is not involved in the regulation of the CAO protein levels. Taken together, we concluded that the A domain alone is involved in the regulatory mechanism of the CAO protein levels.     

Role of the low-molecular-weight subunits PetL, PetG, and PetN in assembly, stability, and dimerization of the cytochrome b6f complex in tobacco

The cytochrome b(6)f (Cyt b(6)f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum 'Petit Havana') to analyze and compare their roles in assembly and stability of the complex. Deletion of petG or petN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b(6)f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast, DeltapetL plants accumulate about 50% of other Cyt b(6)f subunits, appear green, and grow photoautotrophically. However, DeltapetL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b(6)f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in DeltapetL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b(6)f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent plastoquinone reduction, which functions in cyclic electron transport around PSI in vivo, was not impaired in DeltapetL.     

High-intensity-light-dependent and transient expression of new genes encoding distant relatives of light-harvesting chlorophyll-a/b proteins in Chlamydomonas reinhardtii

We identified four Lhc-like genes (Lhl) encoding proteins that are distant relatives of light-harvesting chlorophyll a/b-binding (LHC) proteins in the green alga Chlamydomonas reinhardtii. Their mRNA levels after transfer from low-intensity light to high-intensity light (HL) were examined and compared with those of Lhc genes encoding LHC proteins of PSII. The transfer caused a decrease in the mRNA level of Lhl3, a homolog of Arabidopsis thaliana Lil3, within 2 h, followed by gradual restoration depending on the intensity of HL. The response was similar to that of Lhc genes. In contrast, the mRNA levels of Lhl1, Lhl2 and Lhl4 significantly increased, reached a maximum within 1 h after the transfer and then rapidly returned to a low level. The intensity of HL little influenced the response of these genes. While the Lhl1 and Lhl2 proteins were homologs of early light-inducible protein (ELIP) and high-light-inducible protein (HLIP), respectively, Lhl4 encoded a novel protein. The HL-induced expression of Lhl4 was most prominent among the genes tested. Studies using various inhibitors indicate that the HL response is not mediated by the redox state of plastoquinone pool or reactive oxygen species, but required de novo protein synthesis in the cytosol.     

Correlation between changes in light energy distribution and changes in thylakoid membrane polypeptide phosphorylation in Chlamydomonas reinhardtii

We have used a new method to extensively modify the redox state of the plastoquinone pool in Chlamydomonas reinhardtii intact cells. This was achieved by an anaerobic treatment that inhibits the chlororespiratory pathway recently described by P. Bennoun (Proc. Natl. Acad. Sci. USA, 1982, 79:4352-4356). A state I (plus 3,4-dichlorophenyl-1,1- dimethylurea) leads to anaerobic state transition induced a decrease in the maximal fluorescence yield at room temperature and in the FPSII/FPSI ratio at 77 degrees K, which was three times larger than in a classical state I leads to state II transition. The fluorescence changes observed in vivo were similar in amplitude to those observed in vitro upon transfer to the light of dark-adapted, broken chloroplasts incubated in the presence of ATP. We then compared the phosphorylation pattern of thylakoid polypeptides in C. reinhardtii in vitro and in vivo using gamma-[32P]ATP and [32P]orthophosphate labeling, respectively. The same set of polypeptides, mainly light-harvesting complex polypeptides, was phosphorylated in both cases. We observed that this phosphorylation process is reversible and is mediated by the redox state of the plastoquinone pool in vivo as well as in vitro. Similar changes of even larger amplitude were observed with the F34 mutant intact cells lacking in photosystem II centers. The presence of the photosystem II centers is then not required for the occurrence of the plastoquinone-mediated phosphorylation of light-harvesting complex polypeptides.