Light-harvesting Chlorophyll-protein Research Articles

The emission spectra of thermoluminescence from the photosynthetic apparatus

Emission spectra of two thermoluminescence (TL) components emitted from the photosynthetic apparatus were determined by the use of an imaging photon detector system. The following results were obtained: (i) The TL B-band emitted from whole thylakoids and PS II core complex showed the same emission spectrum peaking at about 690 nm (uncorrected for wavelength dependence of the photocathode) with a broad tailing in far-red region. This spectrum agrees with the reported emission spectrum of delayed luminescence, consistent with the view that the TL B-band arises from thermally stimulated recombination of charge pairs in PS II. (ii) The emission spectrum of TL Z-band was variant, depending on the PS I/PS II ratio in the sample. This was because the Z-band consists of two spectral components emitting at about 740 nm and 690 nm (also uncorrected for the wavelength dependence of the photocathode); the former originates exclusively from PS I and the latter from PS II. (iii) Light-harvesting chlorophyll protein complexes (LHCI and LHCII) emit the respective spectral components of Z-band more strongly than do their respective core complexes, indicating that reaction center photochemistry is not involved in energy storage for the Z-band. (iv) A methanol extract of thylakoids emitted only a weak Z-band at 690 nm, but the formation of a chlorophyll aggregate markedly enhanced the Z-band intensity, concomitant with a shift in emission maximum to 740 nm. The mechanism of energy storage for Z-band is discussed in relation to the local chlorophyll concentration.

Characterization of a cDNA encoding for the 28.5-kDa LHCII apoprotein from the unicellular marine chlorophyte, Dunaliella tertiolecta

Three distinct clones, encoding light-harvesting chlorophyll (LHCII) proteins associated with photosystem II were isolated from a cDNA library of the unicellular marine chlorophyte, Dunaliella tertiolecta. We determined the nucleotide sequence of one of these clones, as well as the N-terminal amino acid (aa) sequences of the four mature LHCII apoproteins (24.5, 28.5, 30 and 31 kDa). The sequenced cDNA clone encoded the precursor of the 28.5-kDa apoprotein. We deduced that the transit peptide is 30 aa long and the mature protein is processed between A and V within the peptide RAAVEFYGP. Southern blots of D. tertiolecta genomic DNA indicated the presence of three to five genes. The algal aa sequence differs from higher plants mainly in the transit peptide and the N terminus. Several aa residues, highly conserved in higher plants, which are believed to play a role in chlorophyll binding, are not conserved in the chlorophytes.

Photoregulation of the Light-Harvesting Chlorophyll Protein Complex Associated with Photosystem II in Dunaliella tertiolecta

The marine chlorophyte Dunaliella tertiolecta Butcher responds to a one-step transition from a high growth irradiance level (700 micromoles quanta per square meter per second) to a low growth irradiance level (70 micromoles quanta per square meter per second) by increasing the total amount of light-harvesting chlorophyll (Chl) a/b binding protein associated with photosystem II (LHC II), and by modifying the relative abundance of individual LHC II apoproteins. When high light-adapted cells were incubated with gabaculine, which inhibits Chl synthesis, and transferred to low light, the LHC II apoproteins were still synthesized and the (35)S-labeled LHC II apoproteins remained stable after a 24 hour chase. These results suggest that Chl synthesis is not required for stability of the LHC II apoproteins in this alga. However, when the control cells are transferred from high light to low light, the amount of the four LHC II apoproteins per cell increases, whereas it does not in the presence of gabaculine. These results suggest that Chl synthesis is required for a photoadaptive increase in the cellular level of LHC II.

Emerson enhancement, photosynthetic control and protein phosphorylation in isolated maize mesophyll chloroplasts; dependence upon carbon metabolism

Intact chloroplasts isolated from maize mesophyll have been used to investigate the relationship between the metabolic demand for ATP and the control of light-harvesting and electron transport. It was found that the rate of reduction of glycerate 3-phosphate can be either inhibited or stimulated by the addition of pyruvate, depending on their relative concentrations, pointing to optimisation of the ΔpH and the rate of electron transport. Pyruvate addition was associated with a stimulation of the phosphorylation of the light-harvesting chlorophyll protein complex, even when O 2 evolution was stimulated. Under a variety of conditions, the level of phosphorylation was inversely correlated with the ΔpH. Emerson enhancement of the reduction of glycerate 3-phosphate is increased when pyruvate is added; the increased rate of O 2 evolution brought about by far-red light is associated with an increase in the ΔpH. It is suggested that the ΔpH transmits the metabolic demand for ATP to the thylakoids, which respond by altering the relative rates of excitation of Photosystems I and II.

Increased Turnover of a Polypeptide Associated with the Lightharvesting Chlorophyll-protein Complex II in Partially Bleached Euglena gracilis

Summary The levels of a 24,500 Mr polypeptide of the light-harvesting chlorophyll-protein complex associated with photosystem II decrease in plastids of growing photoheterotrophic cultures of Euglena gracilis during heat-bleaching at 33 °C. The polypeptide, however, continues to be synthesized, imported and incorporated into thylakoids despite reduced levels of accumulation. The failure of bleaching plastids to maintain normal levels of this polypeptide could be explained by an increase in turnover of the newly synthesized polypeptide at the elevated temperature. In this report, I show that the polypeptide is rapidly degraded when isolated plastids from [ 35 S]labelled partially bleached Euglena are incubated at 33 °C. Pulse-chase experiments carried out in vivo with partially bleached cell cultures essentially confirm the results obtained with isolated plastids. The results presented suggest that post-translational events regulate the levels of the 24,500 Mr polypeptide in bleaching Euglena.

Movement of newly imported light-harvesting chlorophyll-binding protein from unstacked to stacked thylakoid membranes is not affected by light treatment or absence of amino-terminal threonines.

In higher plants and algae, the transduction of captured light energy is highly regulated as excess excitation of photosystem II (PSII) reaction centers can be redirected to photosystem I (PSI) reaction centers. Models that attempt to explain this phenomenon involve light-harvesting chlorophyll-protein complexes (LHCII) that capture light energy and migrate between PSII and PSI. This report shows that in pea chloroplasts, the major protein component of LHCII, light-harvesting chlorophyll-binding protein (LHCP), can indeed migrate within the thylakoid membrane. We show, however, that although newly imported LHCP inserts into both stacked and unstacked thylakoid membranes, it then moves only from the unstacked, PSI-rich membranes to the stacked, PSII-rich membranes. The observed migration is not affected by light treatment that induces a redistribution of captured light energy (state I-state II transition) that previously was thought to induce LHCP to migrate in the opposite direction, from stacked to unstacked membranes. A mutation that removes the site of LHCP phosphorylation, the proposed trigger of state transitions, also has no effect on the integration and movement of LHCP, but does render LHCP more susceptible to proteolytic degradation. These results are not consistent with current models that deal with the short-term change in the distribution of light energy.

Inhibition of energy transfer from light harvesting chlorophyll-protein complex to photosystem II by oxyfluorfen

The effect of magnesium on the photochemical activity and fluorescence yield of chlorophyll a in vivo of oxyfluorfen-treated chloroplasts was studied. The magnesium-induced increase in Photosystem II activity at low light intensity was abolished by incubation of chloroplasts with oxyfluorfen. Photosystem I activity in the presence of Mg 2+ was higher with than without oxyfluorfen. The magnesium-induced increase in fluorescence yield was progressively lowered with increasing concentration of oxyfluorfen. DCMU did not increase the fluorescence yield of high salt chloroplasts incubated with oxyfluorfen. These results suggest that oxyfluorfen inhibits energy transfer from LHC II to PS II and increases spill-over of energy from PS II to PS I.

The light-harvesting system of Euglena gracilis during the cell cycle

The apoproteins of the light-harvesting chlorophyll-protein complexes LHCI and CP29 (apparent molecular weights of 27 kDa and 29 kDa, respectively) of Euglena gracilis were identified immunologically. Both complexes are present in the thylakoids of autotrophically cultured Euglena cells during the whole cell cycle. The relative amount of each apoprotein tends to increase towards the end of the cell cycle. The light-harvesting chlorophyll-protein complex of photosystem II, LHCII, of E. gracilis contains chlorophyll a, chlorophyll b, neoxanthin, diadinoxanthin and beta-carotene. Its chlorophyll a/b ratio is about 1.7 during the whole cell cycle. About 9 h after cell division the ratio of diadinoxanthin to chlorophyll a is doubled for a time of 3-4 h. The relevance of this increase during one developmental stage is discussed in relation to the insertion and-or assembly of newly synthesized LHCII.

Reflected Far-Red Light Effects on Chlorophyll and Light-Harvesting Chlorophyll Protein (LHC-II) Contents under Field Conditions

The influence of various colors of soil cover (mulch) on the farred/red (FR/R) ratio in upwardly reflected light and on concentrations of chlorophyll (Chl) and light-harvesting Chl protein (LHC-II) were measured under field conditions. The FR/R ratios above green surfaces were higher than over white surfaces. Even though plants (Gossyplum hirsutum L. cv PD-1) were grown in full sunlight, those that received higher FR/R ratios in upwardly reflected light were taller and had thinner leaves with higher concentrations of Chl and LHC-II. A controlled environment experiment showed FR/R control of Chl and LHC-II concentrations. The results illustrate the importance of spectral distribution of reflected light on plant growth and a potential means of altering the chemistry of leaf crops under field conditions.

Recovery from photoinhibition: effect of light and inhibition of protein synthesis of 32-kD chloroplast protein

Recovery from photoinhibition of photosynthesis in intact Lemna gibba was studied in presence of the protein synthesis inhibitors chloramphenicol and cycloheximide. Exposure to an irradiance of 1000 μmol m(-2)s(-1) in N2 for 90 min induced 80% photoinhibition. The plants recovered photosynthesis when transfered to normal irradiances (210 μmol m(-2)s(-1)) and air. Chloramphenicol added to the medium was taken up by the plant and reduced photosynthesis slightly. Recovery from photoinhibition was more inhibited than photosynthesis. Cycloheximide was also taken up by the plants and reduced synthesis of light harvesting chlorophyll protein: however, neither photosynthesis nor recovery were much affected. Synthesis of 32-kD chloroplast protein during recovery was inhibited by chloramphenicol, but not by cycloheximide. Synthesis of 32-kD protein was enhanced by 20-210 μmol m(-2)s(-1) light. The results support the hypothesis that synthesis of 32-kD protein is important for recovery of photosynthesis after photoinhibition.

Gibberellic Acid Effects on Greening in Pea Seedlings

The effect of gibberellic acid (GA) on light-induced greening of etiolated pea plants (Pisum sativum [L.] cultivars Alaska and Progress) was characterized. Progress, a GA-deficient dwarf of Alaska, was found to accumulate chlorophyll and light harvesting chlorophyll protein associated with photosystem II (LHC-II) more rapidly than Alaska, Alaska treated with GA, or Progress treated with GA. A slightly lower chlorophyll content was noted after 24 hours of light induced greening for Alaska treated with GA relative to untreated Alaska. GA-treated Progress, Alaska, and GA-treated Alaska all gave essentially identical patterns for LHC-II accumulation. Similar patterns of LHC-II mRNA induction were found in all four treatments indicating that differences in mRNA induction did not cause differences in LHC-II accumulation. Chlorophyll and LHC-II accumulation in each treatment followed the same patterns of accumulation and a significant correlation (at the 0.01 level of significance) was found between chlorophyll and LHC-II content. Since Progress treated with GA accumulated LHC-II and chlorophyll in a manner similar to that of Alaska, it is clear that GA alters the process of greening either directly or indirectly.

Energy transfer in the light-harvesting complex II of Dunaliella tertiolecta is unusually sensitive to Triton X-100

Triton X-100, a detergent commonly used to solubilize higher plant thylakoid membranes, was found to be deleterious to Dunaliella LHC II. It disrupted the transfer of excitation energy from chlorophyll b to chlorophyll a. Based on analysis of pigments and immunoassays of LHC II apoproteins from sucrose density gradient fractions, Triton X-100 caused aggregation of the complex, but apparently did not remove chlorophyll b from the apoprotein. Following solubilization with Triton X-100 only CPI could be resolved by electrophoresis. In contrast, solubilization of Dunaliella thylakoids with octyl-β-D-glucopyranoside preserved energy transfer from chlorophyll b to chlorophyll a. This detergent also effectively prevented aggregation on sucrose gradients and preserved CPI oligomers, as well as LHCP1 and LHCP3 on non-denaturing gels. Solubilization with Deriphat gave similar results. We propose that room temperature fluorescence excitation and emission spectroscopy be used in conjunction with other biophysical and biochemical probes to establish the effects of detergents on the integrity of light harvesting chlorophyll protein complexes. Methods used here may be applicable to other chlorophytes which prove refractory to protocols developed for higher plants.

Light Intensity Regulates the Accumulation of the Major Light-Harvesting Chlorophyll-Protein in Greening Seedlings

Etiolated pea (Pisum sativum [L.] cv Progress 9) and barley (Hordeum vulgare [L.] cv Boone) seedlings greened under either low (40 microeinsteins per square meter per second) or high (550 microeinsteins per square meter per second) intensity light were analyzed for chlorophyll (Chl) content and the levels of mRNA and protein for the major light-harvesting chlorophyll (Chl)-protein of photosystem II (LHC-II). Low intensity plants accumulated Chl more rapidly than high intensity plants. Both single radial immunodiffusion analysis and mild sodium dodecyl sulfate-polyacrylamide gel electrophoresis green gels showed that low intensity plants also accumulated LHC-II protein more rapidly than high intensity plants, following a kinetic pattern similar to the total Chl data. In contrast, LHC-II mRNA levels appeared to be independent of LHC-II protein levels although pea and barley LHC-II mRNA exhibited different light intensity responses. The absence of coordination between LHC-II mRNA and protein levels suggested that the biosynthesis of LHC-II in greening seedlings is not limited by mRNA. A correlation (better than the 0.01 significance level) between LHC-II protein accumulation and Chl accumulation was found for both pea and barley. The accumulation of LHC-II protein was not linked to the development of photosynthetic electron transport. These results and the similar effect of light intensity on Chl content and LHC-II protein levels suggested that the availability of Chl may limit LHC-II protein accumulation in greening seedlings.

Factors that Affect the Synthesis and Accumulation of a Polypeptide of the Light-Harvesting Chlorophyll-Protein Complex II in Euglena gracilis

Cultures of photoheterotrophically grown Euglena gracilis Z maintained at 33 ° experience a time-dependent loss of chlorophyll and the preferential loss of the 24,500Mr polypeptide associated with lightharvesting chlorophyll a/b-binding protein the complex of Photosystem II. It is, therefore, important to establish whether temperature or chlorophyll loss is the overriding factor affecting the ability of bleaching plastids to accumulate the 24,500 Mr species. In this study we show that treatment with levulinic acid, an inhibitor of chlorophyll biosynthesis, resulted in a complete loss of the 24,500 Mr species from thylakoids. Light-grown cells exposed to darkness for up to 15 hours, on the other hand, experienced a rapid loss of chlorophyll but little or no reduction in the levels of the 24,500 Mr component. These observations suggest that, in Euglena, loss of chlorophyll and loss of the 24,500 Mr polypeptide from thylakoids are not always directly correlated. We also show that despite the reduction in the levels of the 24,500 Mr species from bleaching thylakoids the polypeptide continues to be synthesized at 33 °C. The failure of bleaching thylakoids to accumulate this polypeptide can be explained by an increased turnover of the newly synthesized polypeptide.

500 MHz 1H NMR of chlorophylls in the major light-harvesting chlorophyll-protein complex of photosystem II

500 MHz 1H NMR spectra were obtained of solutions containing oligomeric and monomeric forms of Chl a/b-P2, the major light-harvesting chlorophyll a/b-protein complex of photosystem II, isolated from thylakoid membranes of barley (Hordeum vulgare). Oligomers showed only a broad unresolved spectrum, but for monomers several downfield-shifted chlorophyll proton resonances were observed, assigned to the alpha and beta methine protons and the formyl proton of Chl-b. Identifying the observed shifts as ring-current shifts, these NMR data can be matched with previously obtained optical data confirming the trimeric arrangement of Chl-b in Chl a/b-P2 protein, with a distance between the chromophore centers of approximately 12 A.

Characteristics of the Photosynthetic Apparatus of Copper Non-tolerant Spinach Exposed to Excess Copper

Summary Individual spinach plants ( Spinacia oleracea L., c.v. Matador) sensitive to excess copper were grown on a nutrient medium containing toxic amounts of the metal, and the characteristics of its photosynthetic apparatus were examined. In chloroplasts isolated from Cu-treated plants the accumulation of plastid pigments and lipoquinones was lower than in controls and it decreased with the increase of copper toxicity. The loss of photochemical activity of photosystem I and photosystem II was observed at the late stage of leaf degradation (the leaves turned yellow). The loss of photosystem II activity was faster than that of photosystem I. Consequently, changes in chlorophyll distribution of light harvesting chlorophyll-protein complexes were observed. The CPa level, being the presumable reaction center of photosystem II, decreased significantly. The fine structure of chloroplasts revealed degradation of grana stacks and stroma lamellae depending on the stage of their degeneration, an increase in the number and size of plastoglobuli and intrathylakoidal inclusions. The results obtained have been discussed with regard to direct or indirect effects of copper in chloroplasts of plants sensitive to excess copper.

Abundance of the Major Chloroplast Polypeptides during Development and Ripening of Tomato Fruits

During maturation and ripening of tomato (Lycopersicon esculentum, cv Tamar) fruits, there are differential changes in the steady state levels of chloroplast proteins. Western blot analysis indicated that with the exception of the core polypeptide of photosystem I (PSI) (subunit I) the whole complex disappears during the transition of chloroplast to chromoplast. The amounts of the core polypeptide of photosystem II (PSII) (43 kilodaltons) and the light harvesting chlorophyll protein complex increase during maturation and decrease thereafter. In contrast, the 33 kilodalton subunit of PSII is found at the highest levels from the early recorded stages and decreases gradually until late stages of ripening. The level of cytochrome f decreases slowly during the maturation and ripening process, whereas the Rieske protein of the same complex disappears at a faster rate. There are also differential changes in the subunits of the chloroplast coupling factor.ATPase complex; alpha and beta subunits increase during maturation, whereas the level of the gamma subunit is already maximal at the earliest recorded stage of development and depleted thereafter. The two subunits of the ribulose-1,5 bisphosphate carboxylase increase in abundance during chloroplast maturation and gradually disappear after the transition from chloroplast to chromoplast. However, there are substantial differences in the rates of increase and disappearance of the large and small subunits of this enzyme. This imbalance is attributed to different regulation of nuclear and chloroplast gene expression. In addition, the steady state levels of chloroplastic superoxide dismutase and phosphoenolpyruvate carboxylase have been followed. Both enzymes reach their maxima at the final stages of ripening. This increase coincides with the climacteric rise of CO(2) release.

Influence of thylakoid protein phosphorylation on photosystem I photochemistry

The influence of the phosphorylation of thylakoid proteins on photosystem I photochemistry has been measured under conditions of linear dependence of the rate of electron transport to NADP on light intensity. It was found that the phosphorylation by ATP of light harvesting chlorophyll protein complex (LHC) II and other polypeptides stimulates the rate up to ∼ 40%; the stimulation is larger when the wavelength of actinic light corresponds to the main absorption of LHC II.

Photosensitization of triplet carotenoid in photosynthetic light-harvesting complex of photosystem II

A laser photolysis study, on the nanosecond time scale, has been carried out on the major light-harvesting chlorophyll-protein complex of photosystem II (LHC II..beta..). The transient triplet absorption of the isolated LHC II..beta.. has been compared to those of its constituent chromophores in dilute micellar aqueous solutions, namely, chlorophyll a, chlorophyll b, chlorophyll a + chlorophyll b, and chlorophyll a + ..beta..-carotene. The results indicate that the carotenoid in the LHC II..beta.. is photosensitized by triplet chlorophyll a and that the decay of the transient triplet absorption exhibits two transients, one in the approx. 50-ns range and the other in the approx. 10..mu..s time scales. The former transient is light-intensity dependent and is attributed to an annihilation process between two triplets of chlorophyll a, while the latter is due to the decay of the triplet carotenoid to its ground state.

Time resolution of a short-wavelength chloroplast fluorescence component at low temperature

Using a streak camera and interference filters we observe a 676-nm 9-ps chloroplast emission band. It is attributed to light harvesting chlorophyll proteins.