The aba Mutant of Arabidopsis thaliana (L.) Heynh. Has Reduced Chlorophyll Fluorescence Yields and Reduced Thylakoid Stacking

It has been shown that the aba mutant of Arabidopsis thaliana (L.) Heynh. is impaired in epoxy-carotenoid biosynthesis and accumulates the epoxy-carotenoid precursor, zeaxanthin (C.D. Rock, J.A.D. Zeevaart [1991] Proc Natl Acad Sci USA 88: 7496-7499). In addition to providing conclusive evidence for the indirect pathway of abscisic acid biosynthesis from epoxy-carotenoids, the aba mutation offers a powerful means to study the function of xanthophylls (oxygenated carotenoids) in photosynthesis. We measured in vivo the chlorophyll (Chl) fluorescence parameters F_o (initial), F_m (maximum), F_v (variable = F_m − F_o), and t_½ (half-rise time of fluorescence induction) of wild-type (WT) and three allelic aba mutants. The mutant genotypes had significantly lower F_o and F_m values relative to those of WT. The F_v/F_m ratio and t_½, which are parameters affected by photochemical efficiency, photosystem II (PSII), and plastoquinone pool sizes, were similar in the aba alleles and WT. Because the aba genotypes accumulate high levels of zeaxanthin, which is involved in nonphotochemical quenching of Chl fluorescence, we propose that the reduced fluorescence yields in the aba genotypes are a consequence of the accumulated zeaxanthin. Measurement of PSII oxygen evolution rates in isolated thylakoid membranes of WT and aba-4 confirmed that quantum efficiency was not altered in aba-4 but indicated that the mutant had reduced PSII activity in vitro. Electron microscopy revealed an abnormal chloroplast ultrastructure in the aba plants: the mutants had significantly fewer thylakoid lamellae per granum stack but significantly more grana per chloroplast, as well as more chloroplasts per cell than WT. Immunoblot analysis established that aba-4 had normal levels of the Chl a/b-binding core polypeptide of PSII (CP29) and the PSII light-harvesting Chl a/b-binding complex. These results provide evidence for the role of zeaxanthin in nonphotochemical fluorescence quenching and suggest involvement of epoxy-carotenoids and/or zeaxanthin in thylakoid stacking and PSII activity.     

Some effects of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785) on the development of chloroplast thylakoid membranes in Hordeum vulgare L.

Chloroplast ultrastructural and photochemical features were examined in 6-d-old barley (Hordeum vulgare L. cv. Sundance) plants which had developed in the presence of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785). In spite of a substantial modification of the fatty-acid composition of thylakoid lipids there were no gross abnormalities in chloroplast morphology, and normal amounts of membrane and chlorophyll were present. Fluorescence kinetics at 77K demonstrated considerable energetic interaction of photosystem (PS)I and PSII chlorophylls within the altered lipid environment. An interference with electron transport was indicated from altered room-temperature fluorescence kinetics at 20°C. Subtle changes in the arrangements of chloroplast membranes were consistently evident and the overall effects of these changes was to increase the proportion of appressed to nonappressed membranes. This correlated with a lower chlorophyll a/b ratio, an increase in the amount of light-harvesting chlorophylls as determined by gel electrophoresis and fluorescence emission spectra, and an increase in excitation-energy transfer from PSII to PSI, as predicted from current ideas on the organisation of photosystems in appressed and non-appressed thylakoid membranes.Abbreviations CP1 P700-chlorophyll a protein - F_o, F_m, F_v minimal, maximal and variable fluorescence yield - LHCP light-harvesting chlorophyll-protein complex - PSI, PSII photosystem I, II - San 9785 4-chloro-5(dimethylamino)-2-phenyl-3(2H)-pyridazinone     

Alteration in chloroplast structure and thylakoid membrane composition due to in vivo heat treatment of rice seedlings: correlation with the functional changes

Exposure of 25 °C-grown, seven-day-old rice seedlings to mild heat stress of 40 °C for 24 h in dark did not cause any change in protein or pigment content of the thylakoids, but produced major disorganization of chloroplast ultrastructure. This heat induced disorganization of thylakoid structure/organization caused significant (65 percnt;) loss in PSII activity, slight loss in PSI activity, and brought about a decrease in relative quantum efficiency of PSII. The herbicide ¹⁴C atrazine binding assay revealed a decreased number of binding sites of the herbicide and altered the herbicide dissociation constant, suggesting that the heat induced disorganization of the thylakoids affects the acceptor side of PSII. Cation induced Chla fluorescence analyses at room temperature and low temperature indicated thatin vivo heat exposure of rice seedlings altered the extent of energy transfer in favor of PSI. Immunoblotting analysis of several PSII polypeptides such as D1/D2 reaction dimer and Cyt b₅₅₉ showed no major changes due to mild heat exposure except for the PSII core antenna polypeptide (CP43), which could reflect the reduction in PSII activity observed in light saturation studies. Similarly, haeme staining did not indicate any change in other cytochrome related polypeptides. Our results therefore clearly suggest thatin vivo exposure of rice seedlings to elevated (40 °C) temperature caused thylakoid structural disorganization, and this disorganization of some of the thylakoid complexes resulted in a loss in thylakoid photochemical function.     

Pigment composition and functional state of the thylakoid membranes during preparation of samples for pigment-protein complexes separation by nondenaturing gel electrophoresis

The present study was conducted to examine changes in photosynthetic pigment composition and functional state of the thylakoid membranes during the individual steps of preparation of samples that are intended for a separation of pigmentprotein complexes by nondenaturing polyacrylamide gel electrophoresis. The thylakoid membranes were isolated from barley leaves (Hordeum vulgare L.) grown under low irradiance (50 μmol m⁻² s⁻¹). Functional state of the thylakoid membrane preparations was evaluated by determination of the maximal photochemical efficiency of photosystem (PS) II (F_V/F_M) and by analysis of excitation and emission spectra of chlorophyll a (Chl a) fluorescence at 77 K. All measurements were done at three phases of preparation of the samples: (1) in the suspensions of osmotically-shocked broken chloroplasts, (2) thylakoid membranes in extraction buffer containing Tris, glycine, and glycerol and (3) thylakoid membranes solubilized with a detergent decyl-β-D-maltosid. F_V/F_M was reduced from 0.815 in the first step to 0.723 in the second step and to values close to zero in solubilized membranes. Pigment composition was not pronouncedly changed during preparation of the thylakoid membrane samples. Isolation of thylakoid membranes affected the efficiency of excitation energy transfer within PSII complexes only slightly. Emission and excitation fluorescence spectra of the solubilized membranes resemble spectra of trimers of PSII light-harvesting complexes (LHCII). Despite a disrupted excitation energy transfer from LHCII to PSII antenna core in solubilized membranes, energy transfer from Chl b and carotenoids to emission forms of Chl a within LHCII trimers remained effective.     

Changes in D1-protein turnover and recovery of photosystem II activity precede accumulation of chlorophyll in plants after release from mineral stress

After seven weeks of a combined magnesium and sulphur deficiency, spinach (Spinacea oleracea L.) plants showed a substantial accumulation of inactivated photosystem II (PSII) centres as indicated by a 40% decrease of the chlorophyll (Chl) fluorescence parameter F_v/F_m (F_v being the yield of variable fluorescence and F_m the yield of maximal fluorescence when all reaction centres are closed) together with a severe loss of leaf Chl content of 75%. The responses of the photosynthetic apparatus were examined when the deficient plants were transferred back to a rich nutrient medium. During the first 24 h of the recovery phase, thylakoid protein synthesis measured as incorporation of [¹⁴C]leucine per unit of Chl increased substantially. The synthesis rate of the D1 reaction-centre polypeptide of PSII, which in the deficient plants was reduced to 50% of the non-deficient control, was stimulated eight- to ninefold. D1-protein content, which in the deficient plants was reduced to 40% of the non-deficient control, started to increase 2 d later. Thus, D1-protein degradation was also enhanced. The increased D1-protein turnover led to a rapid repair of the existing PSII centres as indicated by the rise of F_v/F_m. It was completed at day 7 of the recovery phase. At day 2 of the recovery phase, the synthesis of other thylakoid proteins such as the D2 protein, cytochrome b ₅₅₉, CP 47 and the 33-kDa polypeptide of the water-splitting system, became stimulated. This process resulted in an accumulation of new PSII centres. During the first week, formation of new PSII centres was not associated with an increase in leaf Chl content. The Chl content of the recovering leaves only started to increase when the ratio of PSII polypeptides versus LHCII (light-harvesting complex of PSII), which was substantially diminished in the deficient plants, became comparable to that of the control. The recovery process was accompanied by substantial changes in thylakoid protein phosphorylation. Their relevance to thylakoid protein turnover and stability is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - F_o yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed (after a saturating flash) under steady-state conditions - F_v yield of variable fluorescence, (difference between F_oand F_m) - F yield of variable fluorescence under steady state conditions - LHC light-harvesting complex - PQ plastoquinone - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_P photochemical quenching - q_n non-photochemical quenching The authors like to thank Dipl. Biol. Britta Untereiser for determining the chlorophyll fluorescence quenching factors. This work was supported by grants from the Bundesminister für Forschung und Technologie, the Project Europäisches Forschungszentrum and the German Israeli Foundation in cooperation with Prof. I. Ohad, Hebrew University, Jerusalem, Israel.     

The unsaturation of phosphatidylglycerol in thylakoid membrane alleviates PSII photoinhibition under chilling stress

Over-expression of chloroplast glycerol-3-phosphate acyltransferase gene (LeGPAT) in tomato increased cis-unsaturated fatty acid content in phosphatidylglycerol (PG) of the thylakoid membrane. Under chilling stress, the oxygen evolving activity, the maximal photochemical efficiency of PSII (F _v/F _m), and superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities decreased less in sense lines than in antisense lines compared to wild-type (WT) plants. Consistently, the relative electric conductivity, \textO₂ ^{. -} {\text{O}}_{2} ^{{. - }} and H₂O₂ contents in sense lines were lower than those of WT and antisense lines. The antisense lines with low level of unsaturated fatty acids in PG were extremely susceptible to photoinhibition of PSII and had a significant reduction in the D1 protein content of PSII reaction center under chilling stress. However, in the presence of streptomycin (SM), the degradation of D1 protein was faster in sense lines than in WT and antisense plants. These results suggested that, under chilling stress conditions, increasing cis-unsaturated fatty acids in PG through over-expression of LeGPAT can alleviate PSII photoinhibition by accelerating the repair of D1 protein and improving the activities of antioxidant enzymes in chloroplasts.     

Stress-induced chlorosis and increase in D1-protein turnover precede photoinhibition in spinach suffering under magnesium/sulphur deficiency

To test wether chlorosis is induced by photoinhibitory damage to photosystem II (PSII), onset of chlorosis and loss of PSII function were compared in young spinach (Spinaciae oleracea L.) plants suffering under a combined magnesium and sulphur deficiency. Loss of chlorophyll already occurred after the first week of deficiency and preceded any permanent functional inhibition of the photosynthetic apparatus. Permanent disturbancies of photosynthetic electron transport measured in isolated thylakoids and of PSII function, determined via the ratio of variable fluorescence to maximal fluorescence, F_v/F_m, could be detected only after the second week of deficiency. After the third week, the plants had lost about 60% of their chlorophyll; even so, fluorescence data indicated that 85% of the existing PSII was still capable of initiating photosynthetic electron transport. However, quenching analysis of steady-state fluorescence showed an early increase in non-photochemical quenching and in down-regulated PSII centres with low steady-state quantum efficiency. Together with the down-regulation of PSII centres, a 1.4-fold increase in D1-protein synthesis, measured as incorporation of [¹⁴C]leucine, could be observed at the end of the first week before any loss of D1 protein, chlorophyll or photosynthetic activity could be detected. Immunological determiation by Western-blotting did not show a change in D1-protein content; thus, at this time, D1 protein was not only faster synthesised but was also faster degraded than before the imposition of mineral deficiency. The increased turnover was high enough to prevent any loss or functional inhibition of PSII. After 3 weeks, D1-protein synthesis on a chlorophyll basis was further stimulated by a factor of 2. However, this was not enough to prevent a net loss of D1 protein of about 70%, showing that the D1-protein was now degraded faster than it was synthesised. Immunological determination and electron-transport measurements showed that together with the loss of D1 protein the other polypetides of PSII were also degraded, resulting in a specific loss of PSII centres. The degradation of PSII centres prevented a large accumulation of damaged PSII centres. We assume that the decrease in PSII centres initiates the breakdown of the other thylakoid proteins.Abbreviations Fo yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed under steady-state conditions - F_v yield of variable fluorescence, (difference between F_o and F_m) - F yield of variable fluorescence under steady-state conditions, difference between F_m and F_t, the fluorescence yield under steady-state conditions - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_p photochemical quenching - q_n non-photochemical quenching This work was supported by grants from the Bundesminister für Forschung und Technologie and the German Israeli Foundation. The authors thank Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) for fruitful discussions.     

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.     

Xanthophyll cycle and light stress in nature: uniform response to excess direct sunlight among higher plant species

Photosystem II (PS II) efficiency, nonphotochemical fluorescence quenching, and xanthophyll cycle composition were determined in situ in the natural environment at midday in (i) a range of differently angled sun leaves ofEuonymus kiautschovicus Loesener and (ii) in sun leaves of a wide range of different plant species, including trees, shrubs, and herbs. Very different degrees of light stress were experienced by these leaves (i) in response to different levels of incident photon flux densities at similar photosynthetic capacities amongEuonymus leaves and (ii) as a result of very different photosynthetic capacities among species at similar incident photon flux densities (that were equivalent to full sunlight). ForEuonymus as well as the interspecific comparison all data fell on one single, close relationship for changes in intrinsic PSII efficiency, nonphotochemical fluorescence quenching, or the levels of zeaxanthin + antheraxanthin in leaves, respectively, as a function of the actual level of light stress. Thus, the same conversion state of the xanthophyll cycle and the same level of energy dissipation were observed for a given degree of light stress independent of species or conditions causing the light stress. Since all increases in thermal energy dissipation were associated with increases in the levels of zeaxanthin + antheraxanthin in these leaves, there was thus no indication of any form of xanthophyll cycle-independent energy dissipation in any of the twenty-four species or varieties of plants examined in their natural environment. It is also concluded that transient diurnal changes in intrinsic PSII efficiency in nature are caused by changes in the efficiency with which excitation energy is delivered from the antennae to PSII centers, and are thus likely to be purely photoprotective. Consequently, the possibility of quantifying the allocation of absorbed light into PSII photochemistry versus energy dissipation in the antennae from changes in intrinsic PSII efficiency is explored.Abbreviations A antheraxanthin - F actual level of fluorescence - F_a, F _o minimal fluorescence in the absence, presence of thylakoid energization - F_m, F _m maximal fluorescence in the absence, presence of thylakoid energization - F_m, - F)/F _m actual PSII efficiency ( = percent of absorbed light utilized in PSII photochemistry) - F_v/F_m, F _v /F_m/ PSII efficiency of open centers in the absence, presence of thylakoid energization - NPQ nonphotochemical fluorescence quenching - F_m/F _m - 1; q_p quenching coefficient for photochemical quenching - V violaxanthin - Z zeaxanthin     

Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress,and evidence for zeaxanthin-associated photoprotection

The function of photosystem (PS)II during desiccation and exposure to high photon flux density (PFD) was investigated via analysis of chlorophyll fluorescence in the desert resurrection plant Selaginella lepidophylla (Hook. and Grev.) Spring. Exposure of hydrated, physiologically competent stems to 2000 mol · m^–2 · s^–1 PFD caused significant reductions in both intrinsic fluorescence yield (F_O) and photochemical efficiency of PSII (F_V/F_M) but recovery to pre-exposure values was rapid under low PFD. Desiccation under low PFD also affected fluorescence characteristics. Both F_V/F_M and photochemical fluorescence quenching remained high until about 40% relative water content and both then decreased rapidly as plants approached 0% relative water content. In contrast, the maximum fluorescence yield (F_M) decreased and non-photochemical fluorescence quenching increased early during desiccation. In plants dried at high PFD, the decrease in F_V/F_M was accentuated and F_O was reduced, however, fluorescence characteristics returned to near pre-exposure values after 24-h of rehydration and recovery at low PFD. Pretreatment of stems with dithiothreitol, an inhibitor of zeaxanthin synthesis, accelerated the decline in F_V/F_M and significantly increased F_O relative to controls at 925 mol · m^–2 · s^–1 PFD, and the differences persisted over a 3-h low-PFD recovery period. Pretreatment with dithiothreitol also significantly decreased non-photochemical fluorescence quenching, increased the reduction state of Q_A, the primary electron acceptor of PSII, and prevented the synthesis of zeaxanthin relative to controls when stems were exposed to PFDs in excess of 250 mol · m^–2 · s^–1. These results indicate that a zeaxanthin-associated mechanism of photoprotection exists in this desert pteridophyte that may help to prevent photoinhibitory damage in the fully hydrated state and which may play an additional role in protecting PSII as thylakoid membranes undergo water loss.Abbreviations and Symbols DTT dithiothreitol - EPS epoxidation state - F_O yield of instantaneous fluorescence at open PSII centers - F_M maximum yield of fluorescence at closed PSII centers induced by saturating light - F_M F_M determined during actinic illumination - F_V yield of variable fluorescence (F_M-F_O) - F_V/F_M photochemical efficiency of PSII - q_P photochemical fluorescence quenching - q_NP non-photochemical fluorescence quenching of Schreiber et al. (1986) - NPQ non-photochemical fluorescence quenching from the Stern-Volmer equation - PFD photon flux density - RWC relative water content This paper is based on research done while W.G.E. was on leave of absence at Duke University during the fall of 1990. We would like to thank Dan Yakir, John Skillman, Steve Grace, and Suchandra Balachandran and many others at Duke University for their help and input with this research. Dr. Barbara Demmig-Adams provided zeaxanthin for standard-curve purposes.     

EARLY TOXIC EFFECTS OF ZINC ON PSII OF SYNECHOCYSTIS AQUATILIS F. AQUATILIS (CYANOPHYCEAE)1

Zinc toxicity on photosynthetic activity in cells of Synechocystis aquatilis f. aquatilis Sauvageau was investigated by monitoring Hill activity and fluorescence. The oxygen‐evolving activity decreased to about 80% of the initial value after exposure to 0.1 mM ZnSO₄ for 1 h. The PSII activity was inhibited by 40% in the presence of zinc concentrations ranging from 0.5 to 5.0 mM, suggesting that the metal effect is limited by zinc uptake. The fluorescence capacity (F_max–F/F_max) decreased from 0.57 to 0.35 and 0.20 in Zn‐treated cells for 15 and 60 min, respectively, thus providing evidence for rapid inactivation of electron transport at PSII. Zinc treatment promoted a rapid increase in PSII fluorescence that was counteracted by addition of 1,4‐benzoquinone, indicating that electron transfer at the reducing side of the PSII reaction center is arrested by zinc. Furthermore, a decline in the fluorescence yield could be observed after 1 h of zinc treatment as well as when Zn‐treated cells were excited in presence of 3‐(3′,4′‐dichlorophenyl)‐1,1‐dimethylurea. Under these conditions, zinc did not affect energy transfer from phycobilisomes to PSII, and the gradual quenching of PSII fluorescence may be due to a decrease in electron flow on the donor side of PSII. However, the 20% increase in the minimal fluorescence intensity (F_o) in parallel to the absence of changes in the maximal fluorescence intensity (F_max), observed in the first hour of zinc treatment, could also suggest a metal‐induced decline in the energy transfer from PSII‐chl a antenna to the PSII reaction center.     

Effects of Nano-anatase on Spectral Characteristics and Distribution of LHCII on the Thylakoid Membranes of Spinach

In the article, we report that effects of nano-anatase on the spectral characteristics and content of light-harvesting complex II (LHCII) on the thylakoid membranes of spinach were investigated. The results showed that nano-anatase treatment could increase LHCII content on the thylakoid membranes of spinach and the trimer of LHCII; nano-anatase could enter the spinach chloroplasts and bind to PSII. Meanwhile, spectroscopy assays indicated that the absorption intensity of LHCII from nano-anatase-treated spinach was obviously increased in the red and the blue region, fluorescence quantum yield near 685 nm of LHCII was enhanced, the fluorescence excitation intensity near 440 and 480 nm of LHCII significantly rose and F ₄₈₀/F ₄₄₀ ratio was reduced. Oxygen evolution rate of PSII was greatly improved. Together, nano-anatase promoted energy transferring from chlorophyll (chl) b and carotenoid to chl a, and nano-anatase TiO₂ was photosensitized by chl of LHCII, which led to enhance the efficiency of absorbing, transferring, and converting light energy.     

Effect of protein modification by malondialdehyde on the interaction between the oxygen-evolving complex 33 kDa protein and photosystem II core proteins

Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSII∆OEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSII∆OEE were modified, but when only OEC33 or PSII∆OEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40°C but not at 25°C. In spinach leaves treated at 40°C under light, maximal efficiency of PSII photochemistry (F _v/F _m ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40°C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.     

Effect of ATP on the Content of Inactive PSII Complexes in Chlorella

A prolonged (20 h) dark incubation of Chlorella pyrenoidosa algae at 37°C resulted in a twofold rise of the slowly rising phase (10–15 min), sF _v, in the kinetics of variable chlorophyll fluorescence, F _v (F _v = F _m – F ₀) in diuron-treated cells. This effect suggests the accumulation of inactive photosystem II (PSII) complexes with low efficiency of primary quinone acceptor of electron of PSII (Q_A) reduction. The presence of methylamine (MA), a thylakoid membrane uncoupler, or N, N-dicyclohexylcarbodiimide, an inhibitor of ATPase, precluded the accumulation of inactive PSII complexes. When salicylhydroxamate promoted the reduction of the plastoquinone (PQ) pool, exogenous ATP accelerated the accumulation of inactive complexes. Dark PQ oxidation in the presence of nonmetabolized glucose analog, 2-deoxy-D-glucose, lowered the content of inactive PSII complexes, and NaF, an inhibitor of chloroplast phosphatases, retarded this process. These data are considered as evidence for a mechanism regulating the content of inactive PSII complexes in the process of redox-dependent phosphorylation of D1- and/or D2-proteins of PSII.     

Chlorophyll fluorescence and leaf chlorophyll content of bean leaves injured by spider mites (Acari: Tetranychidae)

The use of chlorophyll fluorescence as a method for detecting and monitoring plant stress arising from Tetranychus urticae (Koch) feeding injury was investigated. The effect of mite density (1–32 mites per 1.5 cm² of leaf) and the duration of the feeding period (1–5 days) on the chlorophyll fluorescence parameters of bean (Phaseolus vulgaris) leaves were examined. Changes in chlorophyll fluorescence parameters were dependent both on mite density and duration of feeding. Decreases in F _o, the initial fluorescence and F _m, the maximum fluorescence led to a decrease in the ratio of variable to maximum fluorescence, F _v/F _m. The decrease in F _v/F _m is typical of the response of many plants to a wide range of environmental stresses and indicates a reduced efficiency of photosystem II (PSII) photochemistry. T _1/2, which is proportional to the pool size of electron acceptors on the reducing side of PSII, was also reduced in response to mite-feeding injury. The leaf chlorophyll content decreased with increasing mite density and duration of feeding but did not appear to contribute to the decrease in F _v/F _m. Chlorophyll fluorescence is an effective method for detecting and monitoring stress in T. urticae-injured bean leaves.     

Studies on the limitations to photosynthesis in leaves of the atrazine-resistant mutant ofSenecio vulgaris L.

In leaves of an atrazine-resistant mutant ofSenecio vulgaris the quantum efficiency of CO₂ assimilation was reduced by 21% compared to the atrazine-susceptible wild type, and at a light level twice that required to saturate photosynthesis in the wild type the CO₂ fixation rate in the mutant was decreased by 15%. In leaves at steady-state photosynthesis there was a measurable increase in the reduction state of the photosystem II (PSII) primary quinone acceptor,Q _A. Although this would lead to a decreased rate of PSII electron transport and may thus explain the decrease in quantum efficiency, this cannot account for the fall in the maximum rate of CO₂ fixation. The atrazine-resistant mutant showed an appreciably longer photosynthetic induction time which indicates an effect on carbon metabolism; however, the response of CO₂-fixation rate to intercellular CO₂ concentration revealed no differences in carboxylation efficiency. There were also no differences in the ability to perform a State 1–State 2 transition between the atrazine-resistant and susceptible biotypes and no difference in the profiles of phosphorylated thylakoid polypeptides. It is concluded that the alteration of the redox equilibrium between PSII quinone electron acceptors in the atrazine-resistant biotype limits appreciably the photosynthetic efficiency in non-saturating light. Additionally, there is a further, as yet unidentified, limitation which decreases photosynthesis in the resistant mutant under light-saturating conditions.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F _max maximum fluorescence emission - F _o2 minimal fluorescence emission upon exposure to saturating light flash - F _v variable fluorescence emission - F _v2 variable fluorescence emission upon exposure to saturating light flash - kDa kilodalton - PSI, II photosystems I, II - Q _A primary quinone acceptor of PSH - Q _B secondary quinone acceptor of PSII - RuBP ribulose-1,5-bisphosphate     

Overexpression of chloroplastic monodehydroascorbate reductase enhanced tolerance to temperature and methyl viologen‐mediated oxidative stresses

A tomato (Lycopersicon esculentum Mill.) monodehydroascorbate reductase gene (LeMDAR) was isolated. The LeMDAR–green fluorescence protein (GFP) fusion protein was targeted to chloroplast in Arabidopsis mesophyll protoplast. RNA and protein gel blot analyses confirmed that the sense‐ and antisense‐ LeMDAR were integrated into the tomato genome. The MDAR activities and the levels of reduced ascorbate (AsA) were markedly increased in sense transgenic lines and decreased in antisense transgenic lines compared with wild‐type (WT) plants. Under low and high temperature stresses, the sense transgenic plants showed lower level of hydrogen peroxide (H₂O₂), lower thiobarbituric acid reactive substance (TBARS) content, higher net photosynthetic rate (P_n), higher maximal photochemical efficiency of PSII (F_v/F_m) and fresh weight compared with WT plants. The oxidizable P700 decreased more obviously in WT and antisense plants than that in sense plants at chilling temperature under low irradiance. Furthermore, the sense transgenic plants exhibited significantly lower H₂O₂ level, higher ascorbate peroxidase (APX) activity, greater P_n and F_v/F_m under methyl viologen (MV)‐mediated oxidative stresses. These results indicated that overexpression of chloroplastic MDAR played an important role in alleviating photoinhibition of PSI and PSII and enhancing the tolerance to various abiotic stresses by elevating AsA level.     

The psbO mutant of Chlamydomonas reinhardtii is capable of assembling stable, photochemically active reaction center of photosystem II

The functional status of photosystem II (PSII) complex in the dark-grown PsbO-deficient mutant of green alga Chlamydomonas reinhardtii was studied. It was found that ΔpsbO mutant cells of C. reinhardtii grown under heterotrophic conditions (dark + acetate) were capable of assembling stable, photochemically-competent reaction centers of PSII (as confirmed by immunological analysis of D1 protein level, pigments content and photoinduced changes of PSII chlorophyll fluorescence yield), while O₂-evolution activity was not revealed. The ratio F _v/F _m for the dark-grown ΔpsbO mutant C. reinhardtii was 0.37 and that for the dark-grown wild type cells was 0.56. Analysis of chlorophyll fluorescence induction curve indicated that the absence of oxygen-evolving activity could be due to some defects in the organization of the PSII catalytic manganese cluster. Decrease of the rate of the electron donation from water-oxidizing complex to the PSII reaction center as well as the appearance of an additional transient fluorescence peak during the dark relaxation of F _v testify to the damages to the PSII donor side. The data obtained suggest that the dark-grown PsbO-deficient cells of C. reinhardtii are able to form stable, photochemically active PSII reaction center, unable to oxidize water due to probable defects in the assembly of the manganese cluster.     

Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48℃ in the dark for 30 min or gradually elevated temperature from 30℃ to 80℃ at 0.5℃/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of Fo-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3℃ lower in the Chl b-less mutants (48℃) than in the wild type (51℃). At temperature up to about 45℃, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastruc-ture in the wild type showed no obvious alteration. After 55℃ exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chl _b-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a cer-tain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the in-ternal oxidative stress produced at severely high temperature.     

Growth,fluorescence, photosynthetic O<Subscript>2</Subscript> production and pigment content of salt adapted cultures of <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The effect of salt concentration (NaCl) on growth, fluorescence, photosynthetic activities and pigment content of the cyanobacterium Arthrospira platensis has been investigated over 15 days. It has been observed that high NaCl concentration induces an increase of the growth, photosynthetic efficiency (α), phycobilin/chlorophyll ratio and a slight decrease of dark respiration and compensation points. Moreover, high NaCl concentration enhances photosystem II (PSII) activity compared to photosystem I (PSI). Results show that the phycobilin-PSII energy transfer compared to the chlorophyll-PSII (F_695,600/F_695,440) increases. However, data obtained about the maximal efficiency of PSII photochemistry are controversial. Indeed, the Fv/Fm ratio decreases in salt adapted cultures, while at the same time the trapping flux per PSII reaction center (TR₀/RC) and the probability of electron transport beyond Q_A (₀) remain unchanged at the level of the donor and the acceptor sites of PSII. This effect can be attributed to the interference of phycobilin fluorescence with Chl a when performing polyphasic transient measurements.