Antisense-mediated suppression of tomato zeaxanthin epoxidase alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated and antisense transgenic tomato plants were produced. Northern, southern, and western blot analyses demonstrated that antisense LeZE was transferred into the tomato genome and the expression of LeZE was inhibited. The ratio of (A+Z)/(V+A+Z) in antisense transgenic plants was maintained at a higher level than in the wild type (WT) plants under high light and chilling stress with low irradiance. The value of non-photochemical quenching (NPQ) in WT and transgenic plants was not affected during the stresses. The oxidizable P700 and the maximal photochemical efficiency of PSII (F_v/F_m) in transgenic plants decreased more slowly at chilling temperature under low irradiance. These results suggested that suppression of LeZE caused zeaxanthin accumulation, which was helpful in alleviating photoinhibition of PSI and PSII in tomato plants under chilling stress.     

The increase in unsaturation of fatty acids of phosphatidylglycerol in thylakoid membrane enhanced salt tolerance in tomato

Overexpression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) in tomato increased cis-unsaturated fatty acid content in phosphatidylglycerol (PG) of thylakoid membrane. By contrast, suppressing the expression of LeGPAT decreased the content of cis-unsaturated fatty acid in PG. Under salt stress, sense transgenic plants exhibited higher activities of chloroplastic antioxidant enzymes, lower content of reactive oxygen species (ROS) and less ion leakage compared with the wild type (WT) plants. The net photosynthetic rate (P _N) and the maximal photochemical efficiency (F_v/F_m) of photosystem II (PSII) decreased more slightly in sense lines but more markedly in the antisense ones, compared to WT. D1 protein, located in the reactive center of the PSII, is the primary target of photodamage and has the highest turnover rate in the chloroplast. Under salt stress, compared with WT, the content of D1 protein decreased slightly in sense lines and significantly in the antisense ones. In the presence of streptomycin (SM), the net degradation of the damaged D1 protein was faster in sense lines than in other plants. These results suggested that, under salt-stress conditions, increasing cis-unsaturated fatty acids in PG by overexpression of LeGPAT can alleviate PSII photoinhibition by accelerating the repair of D1 protein and improving the activity of antioxidant enzymes in chloroplasts.     

Relationships between phosphatidylglycerol molecular species of thylakoid membrane lipids and sensitivities to chilling-induced photoinhibition in rice

In an attempt to explore the relationships between phosphatidylglycerol （PG） molecular species of thylakoid membrane lipids and sensitivities to chilling-induced photoinhibition, PG molecular species, D1 protein, electron transport activities of thylakoid membrane and the potential quantum yield （FvlFm） in rice treated under middle and low photon flux density （PFD） at 11℃ were analyzed by high performance liquid chromatography, enzyme hydrolysis, gas phase chromatography （GC） and so on. Results showed that the major molecular species of PGs in rice thylakoid membrane were 18：3/16：0, 18：3/16：1（3t）, 18：2/16：0, 18：2/16：1（3t）, 18：1/16：0, 18：1/16：1（3t）, 16：0/16：0, 16：0/16：1（3t）. There were large differences in the contents of unsaturated PG molecular species such as 18：1-3/16：0-16：1（3t） and saturated PG molecular species like 16：0/16：0-16：1（3t） among japonica cv 9516 0-9516）, japonica-indica hybrid F1 j-9516/i-SY63 （ji-95SY） and indica cv Shanyou 63 （i-SY63）. J-9516 containing higher contents of unsaturated PG molecular species was manifest in stable D1 protein contents under chill and tolerant to chill-induced photoinhibition. In contrast to j-9516, i-SY63 with lower contents of unsaturated PG molecular species, exhibited unstable D1 protein contents under chill and was sensitive to chill-induced photoinhibition, ji-95SY containing middle contents of unsaturated PG molecular species between those of j-9516 and i-SY63, exhibited mid extent of sensitivity to chill-induced photoinhibition. The losses in D1 protein also account for the inhibition in electron transport activity of thylakoid membrane and the observed decline in FvlFm. The PG molecular species that is efficient in raising chilling-resistant capacity were those containing unsaturated fatty acids, namely, unsaturated PG molecular species. These results implied that the substrate selectivity of the glycerol-3-phosphate acyltransferase in chloroplasts towards 16：0 or 18：1 displayed greatly the difference between japonica and indica rice. Itwas possible to enhance the capacity of resistance to chilling-induced photoinhibition by improving or modifying the GPAT gene.     

Cyclic electron flow within PSII protects PSII from its photoinhibition in thylakoid membranes from spinach chloroplasts

Cyclic electron flow within PSII (CEF-PSII) was proven to alleviate the photoinhibition of PSII. We set the conditions where CEF-PSII functioned or did not, by adding nigericin to the reaction mixture for the dissipation of DeltapH across thylakoid membranes, and then the thylakoids were illuminated. When CEF-PSII did not function and the activity of linear electron flow (LEF) was low, light-treated thylakoid membranes largely lost the activity of LEF. The inactivation of LEF was due to the loss of the activity of PSII, but not that of PSI. The inactivation of PSII was suppressed, when CEF-PSII functioned or LEF was enhanced. These results imply that CEF-PSII contributes to the protection of PSII from its photoinhibition with LEF, as an electron sink.     

Overexpression of zeaxanthin epoxidase gene enhances the sensitivity of tomato PSII photoinhibition to high light and chilling stress

A tomato ( Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene ( LeZE ) was isolated. The deduced amino acid sequence of LeZE showed high identities with zeaxanthin epoxidase in other plant species. Northern blot analysis showed that the mRNA accumulation of LeZE in the wild-type (WT) was not induced by light and temperature but regulated by the diurnal rhythm. The sense transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analysis confirmed that sense LeZE was transferred into the tomato genome and overexpressed. The ratio of (A + Z)/(V + A + Z) and the values of non-photochemical quenching were lower in transgenic plants than in WT plants under high light and chilling stress with low irradiance. The O₂ evolution rate and the maximal photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more quickly during both stresses and recovered slower than that in WT under optimal conditions. These results suggested that overexpression of LeZE impaired the function of the xanthophyll cycle and aggravated PSII photoinhibition in tomato under high light and chilling stress.     

低温胁迫下外源褪黑素对番茄幼苗光抑制的缓解效应

为了探究外源褪黑素对低温逆境条件下番茄光合系统破坏的缓解机制,设置常温+水(NW)、常温+褪黑素(NM)、低温+水(CW)、低温+褪黑素(CM)4个处理,对番茄幼苗光合与叶绿素荧光参数、叶绿体抗坏血酸-谷胱甘肽(AsA-GSH)循环效率进行分析。结果表明: 与NW相比,CW的光合速率下降了50.3%～72.6%,叶绿体中的丙二醛含量升高了17.5%～132.7%,超氧阴离子产生速率、H₂O₂含量分别升高了86.5%～235.9%、96.6%～208.4%;而低温下施用褪黑素显著缓解了这一趋势,CM处理光合速率比CW提升了22.7%～24.7%,丙二醛含量、超氧阴离子产生速率、H₂O₂含量分别降低了16.6%～29.0%、14.9%～22.7%、10.7%～27.1%。与CW相比,CM处理光系统Ⅱ光化学能转化系数和调节性能量耗损系数分别升高了15.8%和7.2%,非调节性能量耗损系数下降了24.7%,AsA-GSH循环中关键代谢酶活性均有不同程度的增强。综上,低温下施用外源褪黑素可以平衡光系统Ⅱ的能量分布,增强叶绿体中AsA-GSH循环的活性氧清除效率,进而缓解低温引起的光抑制。     

Overexpression of tomato chloroplast omega-3 fatty acid desaturase gene alleviates the photoinhibition of photosystems 2 and 1 under chilling stress

In transgenic (TG) tomato (Lycopersicon esculentum Mill.) overexpressed ω-3 fatty acid desaturase gene (LeFAD7) was identified, which was controlled by the cauliflower mosaic virus 35S promoter and induced increased contents of unsaturated fatty acids in thylakoid membrane. Under chilling stress at low irradiance (4 °C, 100 μmol m⁻² s⁻¹) TG plants with higher linolenic acids (18: 3) content maintained a higher O₂ evolution rate, oxidizable P700 content, and maximal photochemical efficiency (F_v/F_m) than wild type (WT) plants. Low temperature treatment for 6 h resulted in extensive changes of chloroplast ultrastructure: in WT plants most chloroplasts became circular, the number of amyloids increased, appressed granum stacks were dissolved, grana disappeared, and the number of grana decreased, while only a few grana were found in leaves of TG plants. Hence the overexpression of LeFAD7 could increase the content of 18: 3 in thylakoid membrane, and this increase alleviated the photoinhibition of photosystem (PS) 1 and PS2 under chilling at low irradiance.     

Genetic decrease in fatty acid unsaturation of phosphatidylglycerol increased photoinhibition of photosystem I at low temperature in tobacco leaves

Leaves of transgenic tobacco plants with decreased levels of fatty acid unsaturation in phosphatidylglycerol (PG) exhibited a slightly lower level of the steady state oxidation of the photosystem I (PSI) reaction center P700 (P700(+)) than wild-type plants. The PSI photochemistry of wild-type plants was only marginally affected by high light treatments. Surprisingly, all plants of transgenic lines exhibited much higher susceptibility to photoinhibition of PSI than wild-type plants. This was accompanied by a 2.5-fold faster re-reduction rate of P700(+) in the dark, indicating a higher capacity for cyclic electron flow around PSI in high light treated transgenic leaves. This was associated with a much higher intersystem electron pool size suggesting over-reduction of the PQ pool in tobacco transgenic lines with altered PG unsaturation compared to wild-type plants. The physiological role of PG unsaturation in PSI down-regulation and modulation of the capacity of PSI-dependent cyclic electron flows and distribution of excitation light energy in tobacco plants under photoinhibitory conditions at low temperatures is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Mechanism of copper-enhanced photoinhibition in thylakoid membranes

The effect of copper on photoinhibition of photosystem II (PSII) in vitro was studied in bean ( Phaseolus vulgaris L. cv. Dufrix) and pumpkin ( Cucurbita pepo L.) thylakoids. The thylakoids were illuminated at 200–2 000 μmol photons m⁻² s⁻¹ in the presence of 70–1 830 added Cu²⁺ ions per PSII. Three lines of evidence show that the irreversible damage of PSII caused by illumination of thylakoids in the presence of Cu²⁺ was mainly due to donor-side photoinhibition resulting from inhibition of the PSII donor side by Cu²⁺. First, addition of an artificial electron donor partially restored PSII activity of thylakoids that had been illuminated in the presence of Cu²⁺. Second, already moderate light was enough to cause rapid inhibition of PSII, and the inhibition could be saturated by light. Third, the extrinsic polypeptides of the oxygen-evolving complex were found to become oxidized by the combined effect of Cu²⁺ and light. The presence of oxygen was not necessary for the copper-induced enhancement of photoinhibition of PSII. When the illumination was prolonged, copper caused a gradual collapse of the thylakoid structure by increasing degradation of thylakoid proteins.     

Glycine betaine improves thylakoid membrane function of tobacco leaves under low-temperature stress

Glycine betaine (GB) is an effective compatible solute that improves the tolerance in plants to various stresses. We investigated the effects of 2 mM GB applied to the roots of a tobacco (Nicotiana tabacum L.) cultivar on enhancing photosynthesis under low-temperature (LT) stress (5/5 °C, 12/12 h, 300 μmol m⁻² s⁻¹) and in the subsequent recovery (25/18 °C) from the stress. The net photosynthetic rate, intrinsic efficiency measured as the ratio of variable to maximum fluorescence, and actual efficiency of the photochemistry of photosystem 2 as well as the ATPase activity in the thylakoid membrane decreased, and a distinct K step in the fluorescence transient O-J-I-P appeared under cold stress. Exogenous GB alleviated the decrease in all these parameters. The LT-stress induced the accumulation of 33–66 kDa polypeptides and decreased the proportion of unsaturated fatty acids in the thylakoid membrane. In plants subjected to LT-stress, GB protected these polypeptides from damage and enhanced the proportion of unsaturated fatty acids. An increase in non-radiative energy dissipation (NPQ) may be involved in the improvement of the function of the thylakoid membrane by GB since exogenous GB protected violaxanthin de-epoxidase and enhanced NPQ.     

Photoinhibition in mutants of Arabidopsis deficient in thylakoid unsaturation

Thylakoid lipid composition in higher plants is characterized by a high level of fatty acid unsaturation. We have screened four mutants of Arabidopsis that have reduced levels of fatty acid unsaturation. Three of the mutant lines tested, fad5, fad6, and the fad3-2 fad7-2 fad8 triple mutant, were more susceptible to photoinhibition than wild-type Arabidopsis, whereas one mutant, fab1, was indistinguishable from wild type. The fad3-2 fad7-2 fad8 triple mutant, which contains no trienoic fatty acids in its thylakoid membranes, was most susceptible to photoinhibition. Detailed investigation of photoinhibition in the triple mutant revealed that the rate of photoinactivation of PSII was the same in wild-type and mutant plants. However, the recovery of photoinactivated PSII was slower in fad3-2 fad7-2 fad8, relative to wild type, at all temperatures below 27 degrees C. These results indicate that trienoic fatty acids of thylakoid membrane lipids are required for low-temperature recovery from photoinhibition in Arabidopsis.     

Desiccation enhances phosphorylation of PSII and affects the distribution of protein complexes in the thylakoid membrane

Desiccation has significant effects on photosynthetic processes in intertidal macro‐algae. We studied an intertidal macro‐alga, Ulva sp., which can tolerate desiccation, to investigate changes in photosynthetic performance and the components and structure of thylakoid membrane proteins in response to desiccation. Our results demonstrate that photosystem II (PSII) is more sensitive to desiccation than photosystem I (PSI) in Ulva sp. Comparative proteomics of the thylakoid membrane proteins at different levels of desiccation suggested that there were few changes in the content of proteins involved in photosynthesis during desiccation. Interestingly, we found that both the PSII subunit, PsbS (Photosystem II S subunit) (a four‐helix protein in the LHC superfamily), and light‐harvesting complex stress‐related (LHCSR) proteins, which are required for non‐photochemical quenching in land plants and algae, respectively, were present under both normal and desiccation conditions and both increased slightly during desiccation. In addition, the results of immunoblot analysis suggested that the phosphorylation of PSII and LHCII increases during desiccation. To investigate further, we separated out a supercomplex formed during desiccation by blue native‐polyacrylamide gel electrophoresis and identified the components by mass spectrometry analysis. Our results show that phosphorylation of the complex increases slightly with decreased water content. All the results suggest that during the course of desiccation, few changes occur in the content of thylakoid membrane proteins, but a rearrangement of the protein complex occurs in the intertidal macro‐alga Ulva sp.     

Cyclic flow of electrons within PSII in thylakoid membranes

In photosynthesis, the electrons released from PSII are considered to be shared mainly by carbon metabolism and the water-water cycle. We demonstrated previously that some electrons are utilized in a CO2- and O2-independent manner in leaves of wild watermelon [Miyake and Yokota (2000) Plant Cell Physiol: 41: 335]. In the present study, we examined the mechanism of this alternative flow of electrons in thylakoid membranes, isolated from fresh spinach leaves, by simultaneously measuring the quantum yield of PSII and the flux of the linear flow of electrons. In the presence of the protonophore nigericin, which eliminates the pH gradient across thylakoid membranes, the quantum yield and the flux of the linear flow of electrons were directly proportional to one another. The quantum yield at a given linear flux of electrons was much higher in the absence of nigericin than in its presence, indicating that an additional or alternative flow of electrons can occur independently of the linear flow in the absence of nigericin. In the presence of nigericin, the alternative flux of electrons increased with decreasing pH and with increasing reduction of the plastoquinone pool. Cyclic flow of electrons in PSII appears to be the most plausible candidate for the alternative flow of electrons. The flux reached 280 micromol x e(-) (mg Chl)(-1) x h(-1) and was similar to that of the CO2- and O2-independent alternative flow of electrons that we found in leaves of wild watermelon. The cyclic, alternative flow of electrons in PSII provides a possible explanation for the alternative flow of electrons observed in vivo.     

Sidedness studies of thylakoid phosphatidylglycerol in higher plants.

The transmembrane distribution of phosphatidylglycerol was determined in thylakoids from barley (Hordeum vulgare), lettuce (Lactuca sativa) and pea (Pisum sativum) chloroplasts. Phospholipase A2 and phospholipase D digestion and chemical-labelling methods were used. Phosphatidylglycerol was preferentially localized in the outer (stromal) leaflet. The proportion of the phospholipid in this leaflet ranged from about 66% in pea to about 75% for barley and lettuce thylakoids. One of the main fatty acids, trans-delta 3-hexadecenoic acid, was exclusively located in the outer leaflet in all three plant types. The data are discussed in relation to suggested roles for phosphatidylglycerol in thylakoid function.     

A small heat-shock protein confers stress tolerance and stabilizes thylakoid membrane proteins in cyanobacteria under oxidative stress

Small heat-shock proteins are molecular chaperones that bind and prevent aggregation of nonnative proteins. They also associate with membranes. In this study, we show that the small heat-shock protein HspA plays a protective role under oxidative stress in the cyanobacterium Synechococcus elongatus strain ECT16-1, which constitutively expresses HspA. Compared with the reference strain ECT, ECT16-1 showed much better growth and viability in the presence of hydrogen peroxide. Under the peroxide stress, pigments in thylakoid membrane, chlorophyll, carotenoids, and phycocyanins, were continuously reduced in ECT, but in ECT16-1 they decreased only during the first 24 h of stress; thereafter no further reduction was observed. For comparison, we analyzed a wild type and an hspA deletion strain from Synechocystis sp. PCC 6803 and found that lack of hspA significantly affected the viability of the cell and the pigment content in the presence of methyl viologen, suggesting that HspA stabilizes membrane proteins such as the photosystems and phycobilisomes from oxidative damage. In vitro pull down assays showed a direct interaction of HspA with components of phycobilisomes. These results show that HspA and small heat-shock proteins in general play an important role in the acclimation to oxidative stress in cyanobacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Grafting onto Cucurbita moschata rootstock alleviates salt stress in cucumber plants by delaying photoinhibition

To determine how the use of a given rootstock can influence the functioning of the photosynthetic apparatus of the scion under salt stress, the growth, gas exchange, photosystem II (PSII) efficiency, xanthophyll cycle, and chloroplast ultrastructure of nongrafted, self-grafted, and pumpkin-grafted (hereafter referred to as rootstock-grafted) cucumber (Cucumis sativus L.) plants were investigated at day 15 after being treated with 90 mM NaCl. The reductions in plant growth of the rootstock-grafted plants were lower than those of the nongrafted and self-grafted plants under 90 mM NaCl. The net photosynthetic rate, stomatal conductance, maximal and effective quantum yield of PSII photochemistry, photochemical quenching coefficient, and effective quantum-use efficiency of PSII in the light-adapted state of the nongrafted and self-grafted plants were significantly decreased under 90 mM NaCl. However, these reductions were alleviated when the cucumber plants were grafted onto the pumpkin (Cucurbita moschata Duch.) rootstock. The intercellular CO₂ concentrations were significantly increased in the nongrafted and self-grafted plants under 90 mM NaCl, whereas it was decreased in the rootstock-grafted plants. Nonphotochemical quenching (NPQ) and the deepoxidation state of the xanthophyll cycle were significantly increased under 90 mM NaCl, particularly in the rootstockgrafted plants, suggesting the rootstock-grafted plants had higher potential to dissipate excess excitation energy and reduce the probability of photodamage to PSII. Under 90 mM NaCl, the number of grana was reduced, the thylakoids were swollen, and starch granules accumulated in all plants. However, the damage of chloroplast ultrastructure was alleviated in the rootstock-grafted plants. Taken together, the use of C. moschata rootstock alleviated salt stress in cucumber plants by delaying photoinhibition, probably due to a lower incidence of both stomatal and nonstomatal factors limiting photosynthesis.     

Molecular changes of Arabidopsis thaliana plastoglobules facilitate thylakoid membrane remodeling under high light stress

Plants require rapid responses to adapt to environmental stresses. This includes dramatic changes in the size and number of plastoglobule lipid droplets within chloroplasts. Although the morphological changes of plastoglobules are well documented, little is known about the corresponding molecular changes. To address this gap, we have compared the quantitative proteome, oligomeric state, prenyl-lipid content and kinase activities of Arabidopsis thaliana plastoglobules under unstressed and 5-day light-stressed conditions. Our results show a specific recruitment of proteins related to leaf senescence and jasmonic acid biosynthesis under light stress, and identify nearly half of the plastoglobule proteins in high native molecular weight masses. Additionally, a specific increase in plastoglobule carotenoid abundance under the light stress was consistent with enhanced thylakoid disassembly and leaf senescence, supporting a specific role for plastoglobules in senescence and thylakoid remodeling as an intermediate storage site for photosynthetic pigments. In vitro kinase assays of isolated plastoglobules demonstrated kinase activity towards multiple target proteins, which was more pronounced in the plastoglobules of unstressed than light-stressed leaf tissue, and which was diminished in plastoglobules of the abc1k1/abc1k3 double-mutant. These results strongly suggest that plastoglobule-localized ABC1 kinases hold endogenous kinase activity, as these were the only known or putative kinases identified in the isolated plastoglobules by deep bottom-up proteomics. Collectively, our study reveals targeted changes to the protein and prenyl-lipid composition of plastoglobules under light stress that present strategies by which plastoglobules appear to facilitate stress adaptation within chloroplasts.