Quantifying and monitoring functional photosystem II and the stoichiometry of the two photosystems in leaf segments: approaches and approximations.

Given its unique function in light-induced water oxidation and its susceptibility to photoinactivation during photosynthesis, photosystem II (PS II) is often the focus of studies of photosynthetic structure and function, particularly in environmental stress conditions. Here we review four approaches for quantifying or monitoring PS II functionality or the stoichiometry of the two photosystems in leaf segments, scrutinizing the approximations in each approach. (1) Chlorophyll fluorescence parameters are convenient to derive, but the information-rich signal suffers from the localized nature of its detection in leaf tissue. (2) The gross O(2) yield per single-turnover flash in CO(2)-enriched air is a more direct measurement of the functional content, assuming that each functional PS II evolves one O(2) molecule after four flashes. However, the gross O(2) yield per single-turnover flash (multiplied by four) could over-estimate the content of functional PS II if mitochondrial respiration is lower in flash illumination than in darkness. (3) The cumulative delivery of electrons from PS II to P700(+) (oxidized primary donor in PS I) after a flash is added to steady background far-red light is a whole-tissue measurement, such that a single linear correlation with functional PS II applies to leaves of all plant species investigated so far. However, the magnitude obtained in a simple analysis (with the signal normalized to the maximum photo-oxidizable P700 signal), which should equal the ratio of PS II to PS I centers, was too small to match the independently-obtained photosystem stoichiometry. Further, an under-estimation of functional PS II content could occur if some electrons were intercepted before reaching PS I. (4) The electrochromic signal from leaf segments appears to reliably quantify the photosystem stoichiometry, either by progressively photoinactivating PS II or suppressing PS I via photo-oxidation of a known fraction of the P700 with steady far-red light. Together, these approaches have the potential for quantitatively probing PS II in vivo in leaf segments, with prospects for application of the latter two approaches in the field.

Changes in cyclic and respiratory electron transport by the movement of phycobilisomes in the cyanobacterium Synechocystis sp. strain PCC 6803.

Phycobilisomes (PBS) are the major accessory light-harvesting complexes in cyanobacteria and their mobility affects the light energy distribution between the two photosystems. We investigated the effect of PBS mobility on state transitions, photosynthetic and respiratory electron transport, and various fluorescence parameters in Synechocystis sp. strain PCC 6803, using glycinebetaine to immobilize and couple PBS to photosystem II (PSII) or photosystem I (PSI) by applying under far-red or green light, respectively. The immobilization of PBS at PSII inhibited the increase in cyclic electron flow, photochemical and non-photochemical quenching, and decrease in respiration that occurred during the movement of PBS from PSII to PSI. In contrast, the immobilization of PBS at PSI inhibited the increase in respiration and photochemical quenching and decrease in cyclic electron flow and non-photochemical quenching that occurred when PBS moved from PSI to PSII. Linear electron transport did not change during PBS movement but increased or decreased significantly during longer illumination with far-red or green light, respectively. This implies that PBS movement is completed in a short time but it takes longer for the overall photosynthetic reactions to be tuned to a new state.

[The progress in research on the reduced nicotinamide adenine di(tri)-nucleotide phosophate [NAD(P)H] dehydrogenase complex and chlororespiration].

Besides the non-cyclic electron transport driven by the two photosystems (PSII and PSI), the cyclic electron transport pathways around PSI are also essential for efficient photosynthesis. As one of these pathways, the NAD(P)H dehydrogenase complex (NDH complex) mediated cyclic electron transport has been well studied. Along with the identification of the plastid terminal oxydase (PTOX), the functions of NDH-mediated cyclic and chlororepiratory electron transport in energy supply for photosynthesis as well as in the resistance to photooxidative stress have increasingly been brought to the researchers' attention. In the present paper, the structural characteristics of NDH complex, the regulatory mechanism, and the physiological significance of NDH mediated cyclic electron transport and chlororespiration are reviewed.

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes.

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress.

After incubation at 42 degrees C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (deltandhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of deltandhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42 degrees C for 6 h, the photochemical activity declined more markedly in deltandhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (deltapH), while the consumption of deltapH was highly inhibited in deltandhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO2 assimilation by providing extra deltapH, thus reducing the photooxidative damage.

[State transition of the photosynthetic apparatus in plant].

State transition of the photosynthetic apparatus in plants is a short-term adaptation mediated mainly by the reversible phosphorylation of the main light-harvesting complex protein (LHCII) and its migration between photosystem I (PSI) and photosystem II (PSII). In higher plants and Chlamydomonas, LHCII phosphorylation is mainly controlled by the redox state of plastoquinone pool and cytochrome b(6)f complex, while salt could induce a redox-independent LHCII phosphorylation via transient changes in ion concentrations in Dunaliella. State transition can balance the distribution of excitation energy between PSII and PSI by changes in light absorption cross section and excitation energy spillover between the two photosystems. The preliminary results got in the studies of green algae reveal that state transition can also balance the ATP supply and demand.

[Changes in trans-thylakoid membrane proton motive force induced by treatments with red and far-red light in Dunaliella salina].

The changes in trans-thylakoid membrane proton motive force caused by red light and caused by far-red light in the halotolerant green alga, Dunaliella salina are investigated. Irradiation with red light decreased the intensity of the fast phase of millisecond delayed light emission (ms-DLE) in D. salina, and far-red light led to the opposite effects. Under low temperature conditions (4 degrees C), red light still decreased ms-DLE fast phase intensity, however, far-red light did not enhance the ms-DLE fast phase intensity as it did at room temperature. In the presence of the uncoupler, nigericin, which eliminates the proton gradient across the thylakoid membrane, there was still a decrease in ms-DLE after red light irradiation, while far-red light had no stimulatory effects anymore. The far-red light-induced increase in ms-DLE fast phase is thus suggested to be due to the proton gradient formed by water oxidation in photosystem II. Previous studies with higher plants revealed that far red light increased ms-DLE fast phase intensity slightly, while red light caused a transient increase in ms-DLE fast phase intensity followed by a gradual decrease. Taken together, green algae differ from higher plants with respect to red light- and far red light-induced changes in ms-DLE. The possible reason is discussed.

NaCl-induced phosphorylation of light harvesting chlorophyll a/b proteins in thylakoid membranes from the halotolerant green alga, Dunaliella salina.

Light could induce phosphorylation of light harvesting chlorophyll a/b binding proteins (LHCII) in Dunaliella salina and spinach thylakoid membranes. We found that neither phosphorylation was affected by glycerol, whereas treatment with NaCl significantly enhanced light-induced LHCII phosphorylation in D. salina thylakoid membranes and inhibited that in spinach. Furthermore, even in the absence of light, NaCl and several other salts induced LHCII phosphorylation in D. salina thylakoid membranes, but not in spinach thylakoid membranes. In addition, hypertonic shock induced LHCII phosphorylation in intact D. salina under dark conditions and cells adapted to different NaCl concentrations exhibited similar LHCII phosphorylation levels. Taken together, these results show for the first time that while LHCII phosphorylation of D. salina thylakoid membranes resembles that of spinach thylakoid membranes in terms of light-mediated control, the two differ with respect to NaCl sensitivity under light and dark conditions.

Expression of the atpE Gene Coding for the epsilon Subunit of Maize Chloroplast Coupling Factor.

The entire atpE gene of the maize chloroplast coupling factor was inserted into the polylinker region of vectors pJLA505 and pWA to form recombinant plasmids pJLA505-atpE and pWA-atpE respectively. These expression plasmids were transformed into E. coli NM522 which induced at 42 degrees. By the analysis of SDS-PAGE, the expressed product of interest was observed to account fore more than 3o% of total E. coliproteins. The identification of the expressed product demonstrated that its immunological specificity was well retained. The antiserum cross-reacted with the expressed epsilon protein and CF(1)-epsilon protein of spinach and produced precipitin lines on Ouchterlony immunodiffusion test. The expressed product aggregated insolubly as the inclusion body and was purified to over 80% purity. The purified product had the same function as that of the native epsilon subunit.

[Separation of hydrophobic NAD(P)H dehydrogenase subcomplexes from cyanobacterium Synechocystis PCC6803].

Many efforts have been paid to the separation of an integrated NA(D)PH dehydrogenase (NDH) complex. Several hydrophilic subcomplexes of NDH have been purified from the cyanobacterium Synechocystis PCC6803. However, no hydrophobic NDH subcomplex has ever been separated from cyanobacteria yet. In this paper, two NDH subcomplexes were separated from n-dodecyl beta-D-maltoside(DM)-treated whole cell extracts of Synechocystis PCC6803 by anion exchange chromatography and gel filtration. Both subcomplexes contained the hydrophobic subunit NdhA, suggesting that they were hydrophobic NDH subcomplexes. Of the two subcomplexes, only one subcomplex contained NdhH. These subcomplexes showed NADPH-nitroblue tetrazolium (NBT) oxidoreductase activity and could specifically oxidize NADPH when several quinone analogues were used as electron acceptors, such as ferricyanide, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichlorophenol indophenol (DCPIP), duroquinone, ubiquinone-0 (UQ-0), etc.

The Photochemical Change in Photosynthetic Reaction Center of Purple Bacterium with Pheophytin Substitution.

The reaction centers are isolated from chromatophores of Rhodobacter sphaeroides 601 by detergent LDAO, and purified by chromatography on a DEAE-52 cellulose column. In the presence of acetone and an access of free pheophytins (Phes), bacteriopheophytins (Bphes) in reaction centers are replaced by pheophytins at sites H(A) and H(B) when incubated under high temperature. The substituting amounts are about 50% and 71% Bphes in reaction centers with incubation of fifteen and sixty minutes respectively. In the absorption spectra of reaction centers containing Phes (Phe RC), the Q(X) 537 nm and Q(Y) 758 nm bands of Bphe disappeared, three distinct bands assigned to the Q(X 509/542 nm and QY) 674 nm bands of phe appeared. Compared to reaction centers in control, the photochemical activities of Phe RCs, with incubating time of fifteen and sixty minutes, drop to 78 and 71% of that in control respectively.

The Involvement of Plastoquinone in the Pyocyanine-mediated Cyclic Electron Transport around Photosystem I.

2,5 dibromo-3-methyl-5-isopropyl-p-benzoquinone (DBMIB), an inhibitor of plastoquinone, inhibited photosystem I cyclic electron transport mediated by pyocyanine of low concentration, but had no effect on that mediated by phenazine methosulphate (PMS). In the presence of pyocyanine, the thylakoids displayed a transient post-illumination increase in chlorophyll fluorescence which resembled that displayed in leaves. The above results indicate the involvement of plastoquinone in the pyocyanine-mediated cyclic electron transport around photosystem I.

[Fourier transform infrared spectroscopy study on the protein secondary structure of photosystem II reaction center].

A successful study on the secondary structure of the isolated photosystem II (PSII) particles with the Fourier transform infrared spectroscopy is reported in this paper. The beta condensation effect is obviously characterized by infrared absorption spectra. The infrared spectra of both living protein and beta condensed protein samples are measured at room temperature. The amide I band in infrared spectrum is used to perform the quantitative analysis of the sample properties. The recorded spectra show the irreversible effect for the PSII particles after the 400 K heating. A rather strong change of the infrared spectra is observed due to the beta condensation of PSII protein. All the spectra are well fitted by 3-Lorentz-peak. The FTIR spectroscopy shows its effectiveness in studying the heating effect on the PSII particles.

Evaluation of the quality of deep frying oils with Fourier transform near-infrared and mid-infrared spectroscopy.

Deep frying oils are subjected to high temperature and prolonged heating that may lead to a series of quality and safety problems for fried foods. This study evaluated the quality of deep frying oils collected from a local college canteen (n = 132) with Fourier transform mid-infrared (FT-IR) and Fourier transform near-infrared (FT-NIR) spectroscopy. Partial least squares (PLS) regression was used to correlate spectral data with free fatty acids (FFA) and peroxide (PO) values of frying oils. The coefficient of determination (R(2)), standard error of prediction (SEP), and the RPD (ratio of the standard deviation of data set to the SEP) were used as indicators for the predictability of the PLS models. The FT-IR and FT-NIR methods exhibited similar predictability for the FFA values (FT-IR: R(2) = 0.954, SEP = 0.14, RPD = 4.48; FT-NIR: R(2) = 0.948, SEP = 0.14, RPD = 4.38). Although the predictability of the FT-IR method for the PO values was not as satisfactory as that of the FT-NIR method (FT-IR: R(2) = 0.893, SEP = 6.17, RPD = 2.93; FT-NIR: R(2) = 0.953, SEP = 4.15, RPD = 4.36), both FT-IR and FT-NIR methods could be used as simple and rapid approaches to determining the quality of deep frying oils.

Transient decrease of light-harvesting complex II phosphorylation level by hypoosmotic shock in dark-adapted Dunaliella salina.

This study investigated the regulation of major light harvesting chlorophyll a/b protein (LHCII) phosphorylation by hypoosmotic shock in dark-adapted Dunaliella salina cells. When the external NaCl concentration decreased in darkness, D. salina LHCII phosphorylation levels transiently dropped within 20 min and then restored gradually to basal levels. The transient decrease in LHCII phosphorylation levels was insensitive to NaF, a phosphatase inhibitor. Inhibition of intracellular ATP production by addition of an uncoupler or an ATP synthase inhibitor increased LHCII phosphorylation levels in D. salina cells exposed to hypoosmotic shock. Taken together, these results indicate that hypoosmotic shock inhibits the LHCII phosphorylation process. The related mechanism and physiological significance are discussed.

[Environmental regulation of Citrus photosynthesis].

Recent researches on the mechanisms of environmental regulation of citrus photosynthesis showed that the photoinhibition of photosynthesis induced by strong light or ultraviolet radiation was related to the inactiveness of PS II reaction center, and photorespiration and xanthophyll cycle played a pivotal role in protecting photosynthetic apparatus. Under temperature stress, lower CO2 assimilation was mainly due to the decrease of RuBPCase activity and the inactiveness of PS II reaction center, and species sensitivity existed. The decline of photosynthetic CO2 assimilation under high water stress was due to non-stomatal limitation, while that under low water stress was due to stomatal limitation. The citrus growth, yield and quality could be increased by increasing photosynthesis under elevated CO2 concentration. The mechanisms of photosynthesis regulation by minerals such as nitrogen, phosphorus, sulfur and iron, as well as the effects of salt stress on photosynthesis were discussed, and the directions for future research were suggested.

Effects of site-directed mutation on the function of the chloroplast ATP synthase epsilon subunit.

The previous work in our lab showed that the spinach chloroplast ATP synthase epsilon mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast epsilon subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast epsilon subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants epsilonL3I, epsilonL3F, epsilonL3T, epsilonL3R, epsilonL3E and epsilonL3P, respectively. These epsilon variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type epsilon subunit (epsilonWT). The abilities of mutants epsilonL3I and epsilonL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of epsilonWT, but the abilities of the other epsilon variants were lower than that of epsilonWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast epsilon subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.