Detection of singlet oxygen and superoxide with fluorescent sensors in leaves under stress by photoinhibition or UV radiation

In order to understand the physiological functions of reactive oxygen species (ROS) generated in leaves, their direct measurement in vivo is of special importance. Here we report experiments with two dansyl-based ROS sensors, the singlet oxygen specific DanePy and HO-1889NH, which is reactive to both singlet oxygen and superoxide radicals. Here we report in vivo detection of (1)O(2) and O(2)(-*) by fluorescence quenching of two dansyl-based ROS sensors, the (1)O(2) specific DanePy and HO-1889NH, which was reactive with both (1)O(2) and O(2)(-*). The ROS sensors were administered to spinach leaves through a pinhole, and then the leaves were exposed to either excess photosynthetically active radiation or UV (280-360 nm) radiation. Microlocalization of the sensors' fluorescence and its ROS-induced quenching was followed with confocal laser scanning microscopy and with fluorescence imaging. These sensors were specifically localized in chloroplasts. Quenching analysis indicated that the leaves exposed to strong light produced (1)O(2), but hardly any O(2)(-*). On the other hand, the dominant ROS in UV-irradiated leaves was O(2)(-*), while (1)O(2) was minor.     

Influence of ascorbate and the Mehler peroxidase reaction on non-photochemical quenching of chlorophyll fluorescence in maize mesophyll chloroplasts

 Non-photochemical quenching of chlorophyll fluorescence (NPQ) and quantum yield of photosystem II (PSII) were studied with intact mesophyll chloroplasts of maize (Zea mays L.) during the initial minutes of illumination using the pulse-modulated chlorophyll fluorescence technique. Non-photochemical quenching was rapidly reversible in the dark at any point during illumination, which is indicative of energy-dependent dissipation of energy (mediated via thylakoid ΔpH changes and ascorbate-dependent synthesis of zeaxanthin). In chloroplasts suspensions including 15 mM ascorbate in the medium, with addition of oxaloacetate and pyruvate, the PSII yield, rate of reduction of oxaloacetate and phosphorylation of pyruvate reached a maximum after approximately 2 min of illumination. Under these conditions, which promote phosphorylation and a decreased ΔpH across the thylakoid membrane, NPQ rose to a maximum after 2–3 min of illumination, dropped to a minimum after about 6 min, and then increased to a steady-state level. A rather similar pattern was observed when leaves were illuminated following a 30-min dark period. Providing chloroplasts with higher levels of ascorbate (60 mM), prevented the transient drop in NPQ. Anaerobic conditions or addition of potassium cyanide caused a decrease in PSII yield, providing evidence for operation of the ascorbate-dependent Mehler-peroxidase reaction. These conditions also strongly suppressed the transient drop in NPQ. Dithiothreitol, an inhibitor of violaxanthin de-epoxidase, caused a large drop in NPQ even in the presence of high levels of ascorbate. The results suggest that the decline of NPQ occurs in response to an increase in lumen pH after initiation of phosphorylation, that this decline can be suppressed by conditions where ascorbate is not limiting for violaxanthin de-epoxidase, and that the increase of NPQ after such a decline is the result of development of energy dissipation in PSII reaction centers. Received: 13 August 1999 / Accepted: 17 September 1999     

Membrane barriers and Mehler-peroxidase reaction limit the ascorbate available for violaxanthin de-epoxidase activity in intact chloroplasts

The presence of an acidic lumen and the xanthophylls, zeaxanthin and antheraxanthin, are minimal requirements for induction of non-radiative dissipation of energy in the pigment bed of Photosystem II. We recently reported that ascorbate, which is required for formation for these xanthophylls, also can mediate the needed lumen acidity through the Mehler-peroxidase reaction [Neubauer and Yamamoto (1992) Plant Physiol 99: 1354–1361]. It is demonstrated that in non-CO₂-fixing intact chloroplasts and thylakoids of Lactuca sativa, L. c.v. Romaine, the ascorbate available to support de-epoxidase activity is influenced by membrane barriers and the ascorbate-consuming Mehler-peroxidase reaction. In intact chloroplasts, this results in biphasic kinetic behavior for light-induced de-epoxidation. The initial relatively high activity is due to ascorbate preloaded into the thylakoid before light-induction and the terminal low activity due to limiting ascorbate from the effects of chloroplast membranes barriers and a light-dependent process. A five-fold difference between the initial and final activities was observed for light-induced de-epoxidation in chloroplasts pre-incubated with 120 mM ascorbate for 40 min. The light-dependent activity is ascribed to the competitive use of ascorbic acid by ascorbate peroxidase in the Mehler-peroxidase reaction. Thus, stimulating ascorbic peroxidase with H₂O₂ transiently inhibited de-epoxidase activity and concomitantly increased photochemical quenching. Also, the effects inhibiting ascorbate peroxidase with KCN, and the K_M values for ascorbate peroxidase and violaxanthin de-epoxidase of 0.36 and 3.1 mM, respectively, support this conclusion. These results indicate that regulation of xanthophyll-dependent non-radiative energy dissipation in the pigment bed of Photosystem II is modulated not only by lumen acidification but also by ascorbate availability.Abbreviations APO ascorbate peroxidase - MP Mehler ascorbate-peroxidase - NIG nigericin - NPQ non-photochemical quenching - F_o dark fluorescence - F fluorescence at any time - F_M maximal fluorescence of the (dark) non-energized state - F_M maximal fluorescence of the energized state - q_P coefficient for photochemical fluorescence quenching - VDE violaxanthin de-epoxidase - k first-order rate constant for violaxanthin de-epoxidase activity     

Photosynthesis, respiration and antioxidant enzymes in pepper leaves under drought and heat stresses

Leaf gas exchange, chlorophyll fluorescence and activities of antioxidant enzymes were studied in two pepper (Capsicum annuum L.) cultivars grown under drought (D) and heat (H), as well as under drought and heat in combination (HD). The drought-tolerant cv. Zhengjiao 13 exhibited greater net photosynthetic rate (P_N) and cytochrome respiratory pathway activity (R_SHAM), and lower contents of superoxide radical and hydrogen peroxide, as compared to the drought-sensitive cv. Longkouzaojiao. The P_N and R_SHAM decreased and ROS production increased under D and HD in both cultivars. As compared to the Longkouzaojiao, Zhengjiao 13 retained higher non-photochemical quenching (NPQ), photorespiration rate (R_L), and alternative respiratory pathways (R_KCN) under D and HD. Drought increased the superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities in the cytosol, chloroplasts and mitochondria in the two cultivars. Although SOD and APX activities decreased in Longkouzaojiao under HD, SOD activity increased in Zhengjiao 13. There was no H-induced reactive oxygen species production due to increase of R_L, NPQ, R_SHAM, R_KCN and activities of SOD and APX. However, H slightly decreased the P_N. The results indicated that HD was more detrimental than either stress alone.     

The causes of altered chlorophyll fluorescence quenching induction in the Arabidopsis mutant lacking all minor antenna complexes

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is the process by which excess light energy is harmlessly dissipated within the photosynthetic membrane. The fastest component of NPQ, known as energy-dependent quenching (qE), occurs within minutes, but the site and mechanism of qE remain of great debate. Here, the chlorophyll fluorescence of Arabidopsis thaliana wild type (WT) plants was compared to mutants lacking all minor antenna complexes (NoM). Upon illumination, NoM exhibits altered chlorophyll fluorescence quenching induction (i.e. from the dark-adapted state) characterised by three different stages: (i) a fast quenching component, (ii) transient fluorescence recovery and (iii) a second quenching component. The initial fast quenching component originates in light harvesting complex II (LHCII) trimers and is dependent upon PsbS and the formation of a proton gradient across the thylakoid membrane (ΔpH). Transient fluorescence recovery is likely to occur in both WT and NoM plants, but it cannot be overcome in NoM due to impaired ΔpH formation and a reduced zeaxanthin synthesis rate. Moreover, an enhanced fluorescence emission peak at ~679?nm in NoM plants indicates detachment of LHCII trimers from the bulk antenna system, which could also contribute to the transient fluorescence recovery. Finally, the second quenching component is triggered by both ΔpH and PsbS and enhanced by zeaxanthin synthesis. This study indicates that minor antenna complexes are not essential for qE, but reveals their importance in electron stransport, ΔpH formation and zeaxanthin synthesis.     

17个建兰品种光合特性分析

以17个建兰(Cymbidium ensifolium)品种为材料,采用改良的丙酮法提取叶绿素,再通过Arnon丙酮法公式计算光合色素含量,利用捷克FluorCam开放式叶绿素荧光仪测定不同品种的叶绿素荧光参数。结果表明,17个建兰品种的光合色素和叶绿素荧光参数具有不同程度的差异,其中‘铁骨素’(C. ensifolium ‘Tiegusu’)、‘逸红双娇’(C. ensifolium ‘Yihongshuangjiao’)和‘闽南黄蝶’(C. ensifolium ‘Minnanhuangdie’)的光合色素含量高于其他品种,表明这3个品种具有良好的光合效率,吸收光能的能力较强;‘铁骨素’最大荧光产量(Fm)、Kautsky诱导效应最大荧光(Fp)、PS Ⅱ原初光能转化效率(Fv/Fm)和非光化荧光淬灭系数(NPQ)均为最高。综上可知,‘铁骨素’的光合生理特性优于其他品种,可作为优良建兰品种进行种植推广。     

The giant kelp Macrocystis pyrifera presents a different nonphotochemical quenching control than higher plants

Here the mechanisms involved in excitation energy dissipation of Macrocystis pyrifera were characterized to explain the high nonphotochemical quenching of chlorophyll a (Chla) fluorescence (NPQ) capacity of this alga. We performed a comparative analysis of NPQ and xanthophyll cycle (XC) activity in blades collected at different depths. The responses of the blades to dithiothreitol (DTT) and to the uncoupler NH4Cl were also assayed. The degree of NPQ induction was related to the amount of zeaxanthin synthesized in high light. The inhibition of zeaxanthin synthesis with DTT blocked NPQ induction. A slow NPQ relaxation upon the addition of NH4Cl, which disrupts the transthylakoid proton gradient, was detected. The slow NPQ relaxation took place only in the presence of de-epoxidated XC pigments and was related to the epoxidation of zeaxanthin. These results indicate that in M. pyrifera, in contrast to higher plants, the transthylakoid proton gradient alone does not induce NPQ. The role of this gradient seems to be related only to the activation of the violaxanthin de-epoxidase enzyme.     

Non-photochemical quenching kinetics during the dark to light transition in relation to the formation of antheraxanthin and zeaxanthin

Nonlinear regression analysis (NLR) is applied to quantify the dynamic response of non-photochemical fluorescence quenching (NPQ) of Trifolium repens cv. Regal upon dark to light transition. Commonly, only steady-state levels of NPQ are evaluated, ignoring transient kinetics. Experimental NPQ kinetics are fitted best with a sum of two functions: a sigmoidal Hill function plus a transient logarithmic normal function. It is shown that not only steady-state level of NPQ, but also the speed at which steady state is reached, increased with light intensity. The question is raised which biological processes cause the induction of the components of NPQ kinetics. The NPQ kinetics are found to resemble the kinetics of antheraxanthin and zeaxanthin formation during a dark to light transition. Furthermore, both molecules are known to induce NPQ. The hypothesis is put forward that a transient phase of NPQ (0-2 min after transition) is dependent upon concentrations of antheraxanthin, whereas the saturating phase corresponds with the production of zeaxanthin. A mathematical model, based on the presented hypothesis, predicts the effect of increasing light intensity on concentrations of antheraxanthin and zeaxanthin which correspond with experimental results. Implications of the hypothesis are discussed as well as the role of NLR in evaluating chlorophyll a fluorescence kinetics.     

Trapping of the quenched conformation associated with non-photochemical quenching of chlorophyll fluorescence at low temperature

The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures between 5 and 25°C and during rapid jumps of the leaf temperature. At 5°C, NPQ relaxed very slowly in the dark and was sustained for up to 30 min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to 25°C, the quenched state relaxed within 1 min, characteristic for qE, the energy-dependent component of NPQ. Measurements of the membrane permeability (ΔA₅₁₅) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending the model of Horton and co-workers (FEBS Lett 579:4201–4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

ABA Increases the Desiccation Tolerance of Photosynthesis in the Afromontane Understorey Moss Atrichum androgynum

The effect of pretreatment with abscisic acid (ABA) on the physiologyof the moss Atrichum androgynum during a desiccation–rehydrationcycle was examined. During rehydration following desiccationfor 16 h, net CO₂fixation recovered much more slowly than photosystemII (PSII) activity, conditions conducive to the formation ofreactive oxygen species (ROS) in the photosynthetic apparatus.Pretreatment with ABA increased the rate of recovery of photosynthesisand PSII activity, and also doubled non-photochemical quenching(NPQ). Increased NPQ activity will reduce ROS formation, andmay explain in part how ABA hardens the moss to desiccation.In ABA-pretreated, but not untreated mosses, desiccation significantlyincreased the concentration of soluble sugars. Sugar accumulationmay promote vitrification of the cytoplasm and protect membranesduring desiccation. Starch concentrations in freshly collectedA. androgynum were only approx. 40 mg g^-1dry mass; they roseslightly during desiccation but were only slightly affectedby ABA pretreatment. ABA did not reduce chlorophyll breakdownduring desiccation. Copyright 2001 Annals of Botany Company Moss, desiccation, abscisic acid, photosynthesis, chlorophyll fluorescence     

The relations between antioxidant enzymes and chlorophyll fluorescence parameters in common bean cultivars differing in sensitivity to drought stress

Effects of the antioxidant system and chlorophyll fluorescence on drought tolerance of four common bean (Phaseolus vulgaris L.) cultivars were studied. The cultivars were positioned in the order of a decrease in their drought tolerance: Yakutiye, Pinto Villa, Ozayse, and Zulbiye on the basis of changes in the water potential, stomatal conductance, photosynthetic pigment content, and lipid peroxidation. Under drought conditions, the level of H₂O₂ was not changed in cv. Pinto Villa but decreased in other cultivars. Antioxidant enzymes (superothide dismutase (SOD), guaiacol peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR)) were generally activated in all cultivars. Interestingly, CAT, APX, and GR activities were not changed in cv. Pinto Villa, APX activity decreased in cv. Yakutiye, and CAT activity was not changed in cv. Zulbiye. The increases in SOD and GPX activities in cv. Ozayse were higher than in other cultivars. Drought stress reduced the effective quantum yield of PS2 (Φ_PS2) and the photochemical quenching (q_p), while it increased nonphotochemical quenching (NPQ) in all cultivars. The reduction or increase was more pronounced in cv. Zulbiye. There were generally significant correlations between q_p, NPQ, and ROS scavenging by SOD and APX. Also, there were significant correlations between SOD and q_p in tolerant cultivars and APX and q_p in sensitive ones. The results indicate that activation of SOD and APX was closely related to the efficiency of PS2 in common bean cultivars. This interaction was essential for protection of photosystems and plant survival under drought.     

Role of thylakoid Ndh complex and peroxidase in the protection against photo-oxidative stress: fluorescence and enzyme activities in wild-type and ndhF-deficient tobacco

An Ndh-deficient mutant of tobacco ( Nicotiana tabacum cv. Petit Havana) was prepared by disrupting the ndhF gene in a transplastomic approach. The mutant (Δ ndhF ) showed 10% of the Ndh complex activity (EC 1.6.5.3) and 8% of the NDH-F polypeptide of that of non-transformed plants. However, in Δ ndhF , NDH-A, another Ndh polypeptide, was still present at 50% of the level in non-transformed plants. Δ ndhF tobacco showed higher sensitivity than non-transformed plants to photo-oxidative stress (as judged by chlorophyll bleaching) caused by increased light intensity and paraquat applications. These photo-oxidative treatments increased the amount and activity of the Ndh complex, thylakoid peroxidase, post-illumination chlorophyll fluorescence and non-photochemical quenching (NPQ) of chlorophyll fluorescence in non-transformed but not in Δ ndhF tobacco. Highly stressed non-transformed plants showed a rapid post-rise decline of chlorophyll fluorescence, probably indicating a re-oxidation of reduced plastoquinone. The results indicate that, in normal plants, the Ndh complex and thylakoid peroxidase (EC 1.11.1.7) provide and remove electrons, respectively, to balance the redox level of the intermediates of cyclic electron transport. In this way, they optimize the generation of the transmembrane H⁺ gradient of thylakoids and, as a consequence, increase the NPQ and the protection against photo-oxidative stress.     

Atrazine resistance entails a limited xanthophyll cycle activity,a lower PSII efficiency and an altered pattern of excess excitation dissipation

Atrazine-resistant (AR) weeds have a modified D1 protein structure, with a Ser₂₆₄→Gly mutation on the D1 protein, near the plastoquinone binding niche. The photosynthetic performance, the light response of the xanthophyll cycle and chlorophyll fluorescence quenching-related parameters were compared in attached leaves of susceptible (S) and AR biotypes of the C₃ dicot Chenopodium album L., Epilobium adenocaulon Hausskn., Erigeron canadensis L., Senecio vulgaris L. and Solanum nigrum L. and the C₄ dicot Amaranthus retroflexus L. grown under natural high-light conditions. No significant difference in CO₂ assimilation rate per leaf area unit was found between the S and AR biotypes of the investigated C₃ plants, whereas the AR biotype of A. retroflexus exhibited a relatively poor photosynthetic performance. The D1 protein mutant plants expressed a reduced activity of light-stimulated zeaxanthin formation. Neither the lower violaxanthin de-epoxidase activity nor the depletion of ascorbate seems to be the cause of the lower in vivo zeaxanthin formation in the AR plants. All the D1 mutant weeds had limited light-induced non-photochemical (NPQ) and photochemical (q_P) quenching capacities, and displayed a higher photosensitivity, as characterized by the ratio (1-q_P)/NPQ and a higher susceptibility to photoinhibition. Analysis of the chlorophyll fluorescence parameters showed that a lower proportion of excitation energy was allocated to PSII photochemistry, while a higher excess of excitation remained in the AR weeds relative to the S plants.     

硝基苯酚胁迫下外源褪黑素对水稻幼苗生长及生理特性的影响

采用液体培养实验方法,研究硝基苯酚胁迫对水稻(Oryza sativa L.)幼苗生长、抗氧化特性、光系统Ⅱ(PSⅡ)光合特性的影响,以及添加外源褪黑素对缓解硝基苯酚胁迫的作用。结果显示,随着硝基苯酚胁迫浓度的升高,水稻幼苗株高、根长、地下部干重、地上部干重、全株干重和叶片PSⅡ实际光化学效率[Y(Ⅱ)]、光化学淬灭系数(q P)、PSⅡ电子传递速率(ETR)、叶绿素含量均有所下降,而叶片非光化学淬灭系数(qN、NPQ)上升;同时,根系活性氧[过氧化氢(H_2O_2)和超氧阴离子(O·-2)]积累量、抗氧化酶[超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)]活性,以及渗透调节物质(可溶性蛋白和可溶性糖)含量呈先升高后降低的趋势。在非硝基苯酚胁迫下,与对照组相比,添加外源褪黑素显著提高了幼苗地下部干重、根系可溶性糖含量和SOD活性、叶片PSⅡ光化学效率和叶绿素含量。与单独添加硝基苯酚处理相比,硝基苯酚+褪黑素复合处理显著缓解了硝基苯酚胁迫对幼苗生长、叶片PSⅡ光化学效率和叶绿素合成的抑制作用;降低了根系活性氧水平、抗氧化酶活性和渗透调节物质含量。研究结果表明添加外源褪黑素能够显著缓解硝基苯酚胁迫对水稻幼苗生长、根系活性氧水平、抗氧化酶活性、叶片PSⅡ光化学效率及叶绿素合成的不良影响,提高水稻幼苗对硝基苯酚胁迫的适应性。     

Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo

As a response to high light, plants have evolved non-photochemical quenching (NPQ), mechanisms that lead to the dissipation of excess absorbed light energy as heat, thereby minimizing the formation of dangerous oxygen radicals. One component of NPQ is pH dependent and involves the formation of zeaxanthin from violaxanthin. The enzyme responsible for the conversion of violaxanthin to zeaxanthin is violaxanthin de-epoxidase, which is located in the thylakoid lumen, is activated by low pH, and has been shown to use ascorbate (vitamin C) as its reductant in vitro. To investigate the effect of low ascorbate levels on NPQ in vivo, we measured the induction of NPQ in a vitamin C-deficient mutant of Arabidopsis, vtc2-2. During exposure to high light (1,500 micromol photons m(-2) s(-1)), vtc2-2 plants initially grown in low light (150 micromol photons m(-2) s(-1)) showed lower NPQ than the wild type, but the same quantum efficiency of photosystem II. Crosses between vtc2-2 and Arabidopsis ecotype Columbia established that the ascorbate deficiency cosegregated with the NPQ phenotype. The conversion of violaxanthin to zeaxanthin induced by high light was slower in vtc2-2, and this conversion showed saturation below the wild-type level. Both the NPQ and the pigment phenotype of the mutant could be rescued by feeding ascorbate to leaves, establishing a direct link between ascorbate, zeaxanthin, and NPQ. These experiments suggest that ascorbate availability can limit violaxanthin de-epoxidase activity in vivo, leading to a lower NPQ. The results also demonstrate the interconnectedness of NPQ and antioxidants, both important protection mechanisms in plants.     

Higher plant photosystem II light-harvesting antenna, not the reaction center, determines the excited-state lifetime-both the maximum and the nonphotochemically quenched

The maximum chlorophyll fluorescence lifetime in isolated photosystem II (PSII) light-harvesting complex (LHCII) antenna is 4 ns; however, it is quenched to 2 ns in intact thylakoid membranes when PSII reaction centers (RCIIs) are closed (Fm). It has been proposed that the closed state of RCIIs is responsible for the quenching. We investigated this proposal using a new, to our knowledge, model system in which the concentration of RCIIs was highly reduced within the thylakoid membrane. The system was developed in Arabidopsis thaliana plants under long-term treatment with lincomycin, a chloroplast protein synthesis inhibitor. The treatment led to 1), a decreased concentration of RCIIs to 10% of the control level and, interestingly, an increased antenna component; 2), an average reduction in the yield of photochemistry to 0.2; and 3), an increased nonphotochemical chlorophyll fluorescence quenching (NPQ). Despite these changes, the average fluorescence lifetimes measured in Fm and Fm' (with NPQ) states were nearly identical to those obtained from the control. A 77 K fluorescence spectrum analysis of treated PSII membranes showed the typical features of preaggregation of LHCII, indicating that the state of LHCII antenna in the dark-adapted photosynthetic membrane is sufficient to determine the 2 ns Fm lifetime. Therefore, we conclude that the closed RCs do not cause quenching of excitation in the PSII antenna, and play no role in the formation of NPQ.     

The super-excess energy dissipation in diatom algae: comparative analysis with higher plants

When grown at intermittent light regime, diatom alga Phaeodactylum tricornutum is able to form photoprotective non-photochemical chlorophyll fluorescence quenching (NPQ) three to five times larger than that observed in the higher plants. This quenching is sustained in the dark for 5 to 10 min, reverses completely within approximately 1 h and seems to be very tightly related to the presence of the zeaxanthin analogue, diatoxanthin. Addition of the uncoupler NH4Cl before illumination can completely abolish formation of NPQ, revealing the ΔpH-dependency of the xanthophyll cycle activity. Once established, NPQ can also be almost completely reversed by the uncoupler. However, the higher NPQ is formed the more time is required for its reversal. At the point when the fluorescence was approximately 90% recovered the level of illumination-induced diatoxanthin was found to be only partially reduced. This indicates that the proton gradient is a key triggering factor of NPQ. It was also noticed that NPQ in Phaeodactylum cells was absent even when majority of reaction centers were closed and the plastoquinone pool was significantly reduced. The absence of NPQ at these conditions could be due to very low levels of ΔpH. It is likely that in diatoms alternative sources of protons such as the PS I cyclic electron transfer and/or chlororespiration are important in generating the proton gradient sufficient to trigger NPQ. Absorption changes associated with the xanthophyll cycle activity were found to be larger than those for higher plants. The position of the positive maximum in the difference spectrum illuminated-minus-dark was 512–514 nm in comparison to the 505–508 nm for leaves. The 535 nm band associated with NPQ in plants is absent in Phaeodactylum. An uncoupler-sensitive absorption change at 522 nm was discovered. Kinetics of NPQ showed linear correlation with the 522 nm absorption change. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in electron transport,superoxide dismutase and ascorbate peroxidase isoenzymes in chloroplasts and mitochondria of cucumber leaves as influenced by chilling

In order to clarify the relationship between chill-induced disturbance in photosynthetic, respiratory electron transport and the metabolism of reactive oxygen species (ROS), leaf gas exchange, chlorophyll fluorescence quenching, respiration, and activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were investigated in chloroplasts and mitochondria of cucumber (Cucumis sativus) leaves subjected to a chill (8 °C) for 4 d. Chilling decreased net photosynthetic rate (P _N) and quantum efficiency of photosystem 2 (Φ_PS2), but increased the ratio of Φ_PS2 to the quantum efficiency of CO₂ fixation (ΦCO₂) and non-photochemical quenching (NPQ) in cucumber leaves. While chilling inhibited the activity of cytochrome respiration pathway, it induced an increase of alternative respiration pathway activity and the reduction level of Q-pool. Chilling also significantly increased O₂ ^• production rate, H₂O₂ content, and SOD and APX activities in chloroplasts and mitochondria. There was a more significant increase in SOD and APX activities in chloroplasts than in mitochondria with the increase of membrane-bound Fe-SOD and tAPX in chloroplasts being more significant than other isoenzymes. Taken together, chilling inhibited P _N and cytochrome respiratory pathway but enhanced the photosynthetic electron flux to O₂ and over-reduction of respiratory electron transport chain, resulting in ROS accumulation in cucumber leaves. Meanwhile, chilling resulted in an enhancement of the protective mechanisms such as thermal dissipation, alternative respiratory pathway, and ROS-scavenging mechanisms (SODs and APXs) in chloroplasts and mitochondria.     

Non-photochemical quenching in cryptophyte alga Rhodomonas salina is located in chlorophyll a/c antennae

Photosynthesis uses light as a source of energy but its excess can result in production of harmful oxygen radicals. To avoid any resulting damage, phototrophic organisms can employ a process known as non-photochemical quenching (NPQ), where excess light energy is safely dissipated as heat. The mechanism(s) of NPQ vary among different phototrophs. Here, we describe a new type of NPQ in the organism Rhodomonas salina, an alga belonging to the cryptophytes, part of the chromalveolate supergroup. Cryptophytes are exceptional among photosynthetic chromalveolates as they use both chlorophyll a/c proteins and phycobiliproteins for light harvesting. All our data demonstrates that NPQ in cryptophytes differs significantly from other chromalveolates - e.g. diatoms and it is also unique in comparison to NPQ in green algae and in higher plants: (1) there is no light induced xanthophyll cycle; (2) NPQ resembles the fast and flexible energetic quenching (qE) of higher plants, including its fast recovery; (3) a direct antennae protonation is involved in NPQ, similar to that found in higher plants. Further, fluorescence spectroscopy and biochemical characterization of isolated photosynthetic complexes suggest that NPQ in R. salina occurs in the chlorophyll a/c antennae but not in phycobiliproteins. All these results demonstrate that NPQ in cryptophytes represents a novel class of effective and flexible non-photochemical quenching.     

The Growth,physiological and biochemical response of foxtail millet to atrazine herbicide

Foxtail millet (Pennisetum glaucum L.) is a vital crop that is planted as food and fodder crop around the globe. There is only limited information is present for abiotic stresses on the physiological responses to atrazine. A field experiment was conducted to investigate the effects of different atrazine dosages on the growth, fluorescence and physiological parameters i.e., malonaldehyde (MDA) and reactive oxygen species (ROS) (H₂O₂ and O₂) in the leaves to know the extent of atrazine on oxidative damage of foxtail millet. Our experiment consisted of 0, 2.5, 12.5, 22.5 and 32.5 (mg/kg) of labeled atrazine doses on 2 foxtaill millet varieties. High doses of atrazine significantly enhanced ROS and MDA synthesis in the plant leaves. Enzymes activities like ascorbate peroxidase (APX) and peroxidase (POD) activities enhanced, while catalase (CAD) and superoxide dismutase (SOD) activities reduced with increasing atrazine concentrations. Finally atrazine doses at 32.5 mg/kg reduced chlorophyll contents, while chlorophyll (a/b) ratio also enhanced. Biomass, plant height, chlorophyll fluorescence parameters, minimal and maximal fluorescence (Fo, Fm), maximum and actual quantum yield, photochemical quenching coefficient, and electron transport rate are decreased with increasing atrazine doses.