The Role of Acyl Lipids in Reconstitution of Lipid-Depleted Light-Harvesting Complex II from Cold-Hardened and Nonhardened Rye

The role of acyl lipids in the in vitro stabilization of the oligomeric form of light-harvesting complex II of winter rye (Secale cereale L. cv Muskateer) grown at 5 or 20°C was investigated. Purified light-harvesting complex II was enzymically delipidated to various extents by treatment with the following lipolytic enzymes: phospholipase C, phospholipase A₂, and galactolipase. Complete removal of phosphatidylcholine had no effect on the stability of the oligomeric form, whereas the removal of phosphatidylcholine plus phosphatidylglycerol caused a decrease in the ratio of oligomeric:monomeric forms from 1.86 ± 0.17 to 0.85 ± 0.17 and 3.51 ± 0.82 to 0.81 ± 0.29 for purified cold-hardened and nonhardened light-harvesting complex II, respectively, with no change in free pigment content. Incubation of delipidated cold-hardened or nonhardened light-harvesting complex with purified thylakoid phosphatidylglycerol containing trans-Δ³-hexadecenoic acid resulted in 48% reconstitution of the oligomeric form on a total chlorophyll basis with an oligomer:monomer of about 1.90. Incubation in the presence of di- 16:0 or di- 18:1 phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglyceride, or digalactosyldiacylglyceride caused no oligomerization, but rather a further destabilization of the monomeric form. These lipid-dependent structural changes were correlated with significant changes in the 77K fluorescence emission spectra for purified light-harvesting complex II. We conclude that the stabilization of the supramolecular organization of light-harvesting complex II from rye is specifically dependent upon molecular species of phosphatidylglycerol containing trans-Δ³-hexadecenoic acid.     

Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii

Photosynthetic organisms developed multiple strategies for balancing light-harvesting versus intracellular energy utilization to survive ever-changing environmental conditions. The light-harvesting complex (LHC) protein family is of paramount importance for this function and can form light-harvesting pigment protein complexes. In this work, we describe detailed analyses of the photosystem II (PSII) LHC protein LHCBM9 of the microalga Chlamydomonas reinhardtii in terms of expression kinetics, localization, and function. In contrast to most LHC members described before, LHCBM9 expression was determined to be very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g., when nutrient availability was limited. LHCBM9 was localized as part of PSII supercomplexes but was not found in association with photosystem I complexes. Knockdown cell lines with 50 to 70% reduced amounts of LHCBM9 showed reduced photosynthetic activity upon illumination and severe perturbation of hydrogen production activity. Functional analysis, performed on isolated PSII supercomplexes and recombinant LHCBM9 proteins, demonstrated that presence of LHCBM9 resulted in faster chlorophyll fluorescence decay and reduced production of singlet oxygen, indicating upgraded photoprotection. We conclude that LHCBM9 has a special role within the family of LHCII proteins and serves an important protective function during stress conditions by promoting efficient light energy dissipation and stabilizing PSII supercomplexes.     

Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (II. Adjustment of Photosynthetic Capacity in Winter Wheat and Winter Rye)

Winter wheat (Triticum aestivum L. cv Monopol), spring wheat (Triticum aestivum L. cv Katepwa), and winter rye (Secale cereale L. cv Musketeer) grown at 5[deg]C and moderate irradiance (250 [mu]mol m-2 s-1) (5/250) exhibit an increased tolerance to photoinhibition at low temperature in comparison to plants grown at 20[deg]C and 250 [mu]mol m-2 s-1 (20/250). However, 5/250 plants exhibited a higher photosystem II (PSII) excitation pressure (0.32-0.63) than 20/250 plants (0.18-0.21), measured as 1 - qP, the coefficient of photochemical quenching. Plants grown at 20[deg]C and a high irradiance (800 [mu]mol m-2 s-1) (20/800) also exhibited a high PSII excitation pressure (0.32-0.48). Similarly, plants grown at 20/800 exhibited a comparable tolerance to photoinhibition relative to plants grown at 5/250. In contrast to a recent report for Chlorella vulgaris (D.P. Maxwell, S. Falk, N.P.A. Huner [1995] Plant Physiol 107: 687-694), this tolerance to photoinhibition occurs in winter rye with minimal adjustment to polypeptides of the PSII light-harvesting complex, chlorophyll a/b ratios, or xanthophyll cycle carotenoids. However, Monopol winter wheat exhibited a 2.5-fold stimulation of sucrosephosphate synthase activity upon growth at 5/250, in comparison to Katepwa spring wheat. We demonstrate that low-temperature-induced tolerance to photoinhibition is not a low-temperature-growth effect per se but, instead, reflects increased photosynthetic capacity in response to elevated PSII excitation pressure, which may be modulated by either temperature or irradiance.     

Photoregulation of the Light-Harvesting Chlorophyll Protein Complex Associated with Photosystem II in Dunaliella tertiolecta: Evidence that Apoprotein Abundance but Not Stability Requires Chlorophyll Synthesis

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 ³⁵S-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.     

Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (I. Light-Harvesting Complex II Abundance and Zeaxanthin Content in Chlorella vulgaris)

The basis of the increased resistance to photoinhibition upon growth at low temperature was investigated. Photosystem II (PSII) excitation pressure was estimated in vivo as 1 - qp (photochemical quenching). We established that Chlorella vulgaris exposed to either 5[deg]C/150 [mu]mol m-2 s-1 or 27[deg]C/2200 [mu]mol m-2 s-1 experienced a high PSII excitation pressure of 0.70 to 0.75. In contrast, Chlorella exposed to either 27[deg]C/150 [mu]mol m-2 s-1 or 5[deg]C/20 [mu]mol m-2 s-1 experienced a low PSII excitation pressure of 0.10 to 0.20. Chlorella grown under either regime at high PSII excitation pressure exhibited: (a) 3-fold higher light-saturated rates of O2 evolution; (b) the complete conversion of PSII[alpha] centers to PSII[beta] centers; (c) a 3-fold lower epoxidation state of the xanthophyll cycle intermediates; (d) a 2.4-fold higher ratio of chlorophyll a/b; and (e) a lower abundance of light-harvesting polypeptides than Chlorella grown at either regime at low PSII excitation pressure. In addition, cells grown at 5[deg]C/150 [mu]mol m-2 s-1 exhibited resistance to photoinhibition comparable to that of cells grown at 27[deg]C/2200 [mu]mol m-2 s-1 and were 3- to 4-fold more resistant to photoinhibition than cells grown at either regime at low excitation pressure. We conclude that increased resistance to photoinhibition upon growth at low temperature reflects photosynthetic adjustment to high excitation pressure, which results in an increased capacity for nonradiative dissipation of excess light through zeaxanthin coupled with a lower probability of light absorption due to reduced chlorophyll per cell and decreased abundance of light-harvesting polypeptides.     

Xanthophyll Cycle and Inactivation of Photosystem Ⅱ Reaction Centers Alleviating Reducing Pressure to Photosystem Ⅰ in Morning Glory Leaves under Short-term High Irradiance

Under 30-min high irradiance （1500μmol m^-2 s^-1）, the roles of the xanthophyll cycle and D1 protein turnover were investigated through chlorophyll fluorescence parameters in morning glory （Ipomoea setosa） leaves, which were dipped into water, dithiothreitol （DTT） and lincomycin （LM）, respectively. During the stress, both the xanthophyll cycle and D1 protein turnover could protect PSI from photoinhibition. In DTT leaves, non-photochemical quenching （NPQ） was inhibited greatly and the oxidation level of P700 （P700^＋） was the lowest one. However, the maximal photochemical efficiency of PSII （Fv/Fm） in DTT leaves was higher than that of LM leaves and was lower than that of control leaves. These results suggested that PSI was more sensitive to the loss of the xanthophyll cycle than PSII under high irradiance. In LM leaves, NPQ was partly inhibited, Fv/Fm was the lowest one among three treatments under high irradiance and P700^＋ was at a similar level as that of control leaves. These results implied that inactivation of PSII reaction centers could protect PSI from further photoinhibition. Additionally, the lowest of the number of active reaction centers to one inactive reaction center for a PSII cross-section （RC/CSo）, maximal trapping rate in a PSll cross-section （TRo/CSo）, electron transport in a PSll cross-section （ETo/CSo） and the highest of 1-qP in LM leaves further indicated that severe photoinhibition of PSII in LM leaves was mainly induced by inactivation of PSII reaction centers, which limited electrons transporting to PSh However, relative to the LM leaves the higher level of RC/CSo, TRo/CSo, Fv/Fm and the lower level of 1-qP in DTT leaves indicated that PSI photoinhibition was mainly induced by the electron accumulation at the PSI acceptor side, which induced the decrease of P700^＋ under high irradiance.     

Effective Absorption Cross-Sections in Porphyridium cruentum: Implications for Energy Transfer between Phycobilisomes and Photosystem II Reaction Centers

Effective absorption cross-sections for O₂ production by Porphyridium cruentum were measured at 546 and 596 nanometers. Although all photosystem II reaction centers are energetically coupled to phycobilisomes, any single phycobilisome acts as antenna for several photosystem II reaction centers. The cross-section measured in state I was 50% larger than that measured in state II.     

Novel Evidence for a Reversible Dissociation of Light-harvesting Complex II from Photosystem II Reaction Center Complex Induced by Saturating Light Illumination in Soybean Leaves

After saturating light illumination for 3 h the potential photochemical efficiency of photosystem Ⅱ （PSII） （FJF,, the ratio of variable to maximal fluorescence） decreased markedly and recovered basically to the level before saturating light illumination after dark recovery for 3 h in both soybean and wheat leaves, indicating that the decline in FJ/Fm is a reversible down-regulation. Also, the saturating light illumination led to significant decreases in the low temperature （77 K） chlorophyll fluorescence parameters F685 （chlorophyll a fluorescence peaked at 685 nm） and F685/F735 （F735, chlorophyll a fluorescence peaked at 735 nm） in soybean leaves but not in wheat leaves. Moreover, trypsin （a protease） treatment resulted in a remarkable decrease in the amounts of PsbS protein （a nuclear gene psbS-encoded 22 kDa protein） in the thylakoids from saturating light-illuminated （SI）, but not in those from darkadapted （DT） and dark-recovered （DRT） soybean leaves. However, the treatment did not cause such a decrease in amounts of the PsbS protein in the thylakoids from saturating light-illuminated wheat leaves. These results support the conclusion that saturating light illumination induces a reversible dissociation of some light-harvesting complex Ⅱ （LHClI） from PSII reaction center complex in soybean leaf but not in wheat leaf.     

The Relation Between Changes in Non-Photochemical Quenching,Low Temperature Fluorescence Emission,and Membrane Ultrastructure Upon Binding of Polyionic Compounds and Fragments of Light-Harvesting Complex 2

Experiments were performed to distinguish some of the proposed mechanisms by which thylakoid membranes regulate the performance of photosynthetic apparatus in relation to non-photochemical quenching, q_N. Aliphatic diamines were used as uncouplers of transmembrane H⁺ gradient as they can be transported across the membrane at the expense of hydrogen cations. Diamines did not induce changes in low-temperature fluorescence emission but induced different changes in membrane ultrastructure. Positively charged peptides did not affect membrane ultrastructure but blocked q_N. In addition, they caused an increase of low temperature fluorescence emission between 710 and 720 nm. For control peptide, the maximal fluorescence increase was found at 715 nm. Fragments of light-harvesting complex 2 in their phosphorylated and non-phosphorylated form shifted the position of this increase. We believe that peptides bind to membrane surface and reduce the mobility of membrane components whose migration is needed for observation of q_N. Phosphorylated and non-phosphophorylated LHC2 fragments bind to different binding sites for corresponding forms of the protein. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biosynthesis of Photosystem II Reaction Centers, Antenna and Plastoquinone Pool in Greening Cells of Cyanidium caldarium Mutant III-C

Dark-grown etiolated cells of Cyanidium caldarium mutant III-C lacking ≥99% of their normal chlorophyll content and inactive for photosynthesis were greened in continuous light. Measurements of oxygen evolution and fluorescence kinetics indicate that during greening: (a) the photosystem II (PSII) antenna containing between 30 and 40 chlorophyll a per center undergoes little change in size from 5% of the centers synthesized per cell to fully active cells; (b) energy transfer between PSII centers appears very early in the greening process; (c) the plastoquinone pool size per PSII center (about 14 equivalents) does not vary during greening and has already attained full size after synthesis of only 13% of the full complement of centers.     

The Effect of Low Growth Temperature on Hill Reaction and Photosystem 1 Activities and Pigment Contents in Maize Inbred Lines and Their F1 Hybrids

Young plants of maize inbred lines CE777, CE704, and CE810 and their F₁ hybrids displaying a positive heterotic effect in various photosynthetic characteristics were exposed to low temperature during their early growth developmental stage. The photochemical activity of isolated mesophyll chloroplasts and the contents of photosynthetic pigments in leaves of stressed and non-stressed plants were compared with the aim to find out the possible changes in the relationship between parents and hybrids, and to determine the genetic basis of heterosis in F₁ generation. Strong decrease in the content of chlorophylls was observed for all genotypes examined when plants were subjected to low growth temperature. Similar change was recorded for Hill reaction activity (HRA) of inbred lines but not of their F₁ hybrids, and no significant response at all was found for photosystem 1 (PS1) activity or the total carotenoids content. The intraspecific variation due to differences between genotypes was found for most of photosynthetic characteristics examined. This variation was caused by the additive and dominance genetic effects. Positive dominance was the main cause of positive heterosis in HRA and in the contents of photosynthetic pigments and was much more pronounced in the stressed plants compared to the non-stressed ones. The maternal additive effects participated in the inheritance of contents of photosynthetic pigments in plants exposed to low temperature, too. This revised version was published online in June 2006 with corrections to the Cover Date.     

Light Quality and Irradiance Level Interaction in the Control of Expression of Light-Harvesting Complex of Photosystem II: Pigments, Pigment-Proteins, and mRNA Accumulation

Effects of red and blue light at irradiances from 1.6 to 28.3 micromolar per square meter per second on chloroplast pigments, light-harvesting pigment-proteins associated with photosystem II, and the corresponding mRNA were evaluated in maize (Zea mays L.) plants (OP Golden Bantum) grown for 14 days under 14 hours light/10 hours dark cycles. Accumulation of pigments, pigment-proteins, and mRNA was less in blue than in red light of equal irradiance. The difference between blue and red light, however, varied as a function of irradiance level, and the pattern of this variation suggests irradiance-controlled activation/deactivation (switching) of blue-light receptor. The maximum reduction in blue light of mRNA and proteins associated with light-harvesting complex occurs at lower irradiance levels than the maximum reduction of chlorophylls a and b.     

Effects of Low Temperature on Growth and Nutrient Accumulation in Rye (Secale cereale) and Wheat (Triticum aestivum)

Rye (Secale cereale cv. Rheidol) and wheat (Triticum aestivumcv. Mardler) were grown at shoot/root temperatures of 20/20°C (warm grown, WG plants), 8/8 °C (cold grown, CG plants)and 20/8 °C (differential grown, DG plants). Plants fromcontrasting growth temperature regimes were standardized andcompared using a developmental timescale based on accumulatedthermal time (°C d) at the shoot meristem. Accumulationof dry matter, nitrogen and potassium were exponential overthe time period studied (150–550 °C d). In rye, therates of plant dry matter and f. wt accumulation were linearlyrelated to the temperature of the shoot meristem. However, inwheat, although the rates of plant dry matter and f. wt accumulationwere temperature dependent, the linear relationship with shootmeristem temperature was weaker than in rye. The shoot/rootratio of rye was stable irrespective of growth temperature treatment,but the shoot/root ratio of wheat varied with growth temperaturetreatment. The shoot/root ratio of DG wheat was 50% greaterthan WG wheat. In both cereals, nutrient concentrations anddry matter content tended to be greater in organs exposed directlyto low temperatures. The mean specific absorption rates of nutrientswere calculated for the whole period studied for each species/temperaturecombination and were positively correlated with both plant shoot/rootratio and relative growth rate. The data suggest that nutrientuptake rates were influenced primarily by plant demand, withno indication of specific nutrient limitations at low temperatures. Nutrient accumulation, relative growth rate (RGR), rye, Secale cereale cv. Rheidol, temperature, thermal time, Triticum aestivum cv. Mardler, wheat     

Stabilization of Isolated Photosystem II Reaction Center Complex in the Dark and in the Light Using Polyethylene Glycol and an Oxygen-Scrubbing System

The photosystem II reaction center as isolated (O Nanba, K Satoh [1987] Proc Natl Acad Sci USA 84: 109-112) is quite dilute and very unstable. Precipitating the complex with polyethylene glycol and resuspending it in buffer without detergent concentrates the reaction center and greatly improves its stability at 4°C in the dark as judged by light-induced electron transport activity. Furthermore, a procedure was developed to minimize photodestruction of polyethylene-glycol-concentrated material at room temperature in the light. The ability to stabilize the photosystem II reaction center should facilitate future photophysical, biochemical, and structural studies of the complex.     

Optimal Thermal Environments for Plant Metabolic Processes (Cucumis sativus L.) (Light-Harvesting Chlorophyll a/b Pigment-Protein Complex of Photosystem II and Seedling Establishment in Cucumber)

Analysis of the temperatures providing maximal photosystem II fluorescence reappearance following illumination and thermal kinetic windows (TKWs), obtained from the temperature characteristics of enzyme apparent Km values, have been proposed as indicators of the bounds of thermal stress in plants. In this study, we have evaluated the temperature optimum for the accumulation of the chlorophyll a/b light-harvesting complex of photosystem II (LHCP II), its mRNA, and the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in cucumber (Cucumis sativus L. cv Ashley) as a broader measure of metabolism than that provided by either the fluorescence reappearance or TKWs. The TKW for cucumber is between 23.5 and 39[deg]C, with the minimum apparent Km occurring at 32.5[deg]C. The photosystem II variable fluorescence reappearance following illumination was maximal between 30 and 35[deg]C. Maximum synthesis of the LHCP II occurred at 30[deg] C. The light-induced accumulation of the LHCP II and the small subunit of Rubisco mRNAs showed similar temperature characteristics. Suboptimal temperatures delayed germination, altered cotyledonary soluble sugar content, and broadened the temperature range for chlorophyll accumulation. These results demonstrate an effect of seed reserve mobilization on the range of temperatures for chlorophyll accumulation, and suggest that metabolic temperature characteristics may be broadened by increasing available substrates for enzyme utilization. This study provides new information about the relationship between TKWs and cellular responses to temperature. In addition, the results suggest that the temperature range outside of which plants experience temperature stress is narrower than traditionally supposed.     

Changes of Photosystem Ⅱ Electron Transport in the ChlorophyⅡ-deficient Oilseed Rape Mutant Studied by ChlorophyⅡ Fluorescence and Thermoluminescence

The photosystem Ⅱ （PSII） complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has decreased the amount of light-harvesting complexes with an increased amount of some core polypeptldes of PSII, including CP43 and CP47. By means of chlorophyll fluorescence and thermolumlnescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type. In the chlorophyll lPdeflclent mutant, the decay of the primary electron acceptor quinones （QA-） reoxidation was decreased, measured by the fluorescence. Furthermore, the thermoluminescence studies in the chlorophyll bdeficient mutant showed that the B band （S2/S3QB-） decreased slightly and shifted up towards higher temperatures. In the presence of dlchlorophenyl-dlmethylurea, which is inhibited in the electron flow to the second electron acceptor quinines （QB） at the PSll acceptor side, the maximum of the Q band （S2QA-） was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyⅡ b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA-via forward electron flow and slower reduction of the oxidation S states.     

Superoxide,Hydrogen Peroxide and Hydroxyl Radical in D1/D2/cytochrome b-559 Photosystem II Reaction Center Complex

Spin-trapping electron spin resonance (ESR) was used to monitor the formation of superoxide and hydroxyl radicals in D1/D2/cytochrome b-559 Photosystem II reaction center (PS II RC) Complex. When the PS II RC complex was strongly illuminated, superoxide was detected in the presence of ubiquinone. SOD activity was detected in the PS II RC complex. A primary product of superoxide, hydrogen peroxide, resulted in the production of the most destructive reactive oxygen species, *OH, in illuminated PS II RC complex. The contributions of ubiquinone, SOD and H(2)O(2) to the photobleaching of pigments and protein photodamage in the PS II RC complex were further studied. Ubiquinone protected the PS II RC complex from photodamage and, interestingly, extrinsic SOD promoted this damage. All these results suggest that PS II RC is an active site for the generation of superoxide and its derivatives, and this process protects organisms during strong illumination, probably by inhibiting more harmful ROS, such as singlet oxygen.