The Effect of Sulfur Re-Addition on H<Subscript>2</Subscript> Photoproduction by Sulfur-Deprived Green Algae

Sulfur deprivation of algal cultures selectively and partially inactivates photosystem II (PSII)-catalyzed O₂ evolution, induces anaerobiosis and hydrogenase expression, and results in sustained H₂ photoproduction for several days. We show that re-addition of limiting amounts of sulfate (1–10 μM final concentration) to the cultures during the H₂-production phase temporarily reactivates PSII photochemical and O₂-evolution activity and re-establishes higher rates of electron transport through the photosynthetic electron transport chain. The reactivation of PSII occurs by de novo D1 protein synthesis, but does not result in the re-establishment of aerobic conditions in the reactor, detectable by dissolved-O₂ sensors. However, concomitant H₂ photoproduction is inhibited, possibly due to excessive intra-cellular levels of photosynthetically-evolved O₂. The partial recovery of electron transport rates correlates with the re-oxidation of the plastoquinone (PQ) pool, as observed by pulse-amplitude modulated (PAM) and fluorescence-induction measurements. These results show that the presence of a more oxidized PQ pool releases some of the down-regulation of electron transport caused by the anaerobic conditions.     

Hydrogen production by <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: an elaborate interplay of electron sources and sinks

The unicellular green alga Chlamydomonas reinhardtii possesses a [FeFe]-hydrogenase HydA1 (EC 1.12.7.2), which is coupled to the photosynthetic electron transport chain. Large amounts of H₂ are produced in a light-dependent reaction for several days when C. reinhardtii cells are deprived of sulfur. Under these conditions, the cells drastically change their physiology from aerobic photosynthetic growth to an anaerobic resting state. The understanding of the underlying physiological processes is not only important for getting further insights into the adaptability of photosynthesis, but will help to optimize the biotechnological application of algae as H₂ producers. Two of the still most disputed questions regarding H₂ generation by C. reinhardtii concern the electron source for H₂ evolution and the competition of the hydrogenase with alternative electron sinks. We analyzed the H₂ metabolism of S-depleted C. reinhardtii cultures utilizing a special mass spectrometer setup and investigated the influence of photosystem II (PSII)- or ribulosebisphosphate-carboxylase/oxygenase (Rubisco)-deficiency. We show that electrons for H₂-production are provided both by PSII activity and by a non-photochemical plastoquinone reduction pathway, which is dependent on previous PSII activity. In a Rubisco-deficient strain, which produces H₂ also in the presence of sulfur, H₂ generation seems to be the only significant electron sink for PSII activity and rescues this strain at least partially from a light-sensitive phenotype. The latter indicates that the down-regulation of assimilatory pathways in S-deprived C. reinhardtii cells is one of the important prerequisites for a sustained H₂ evolution.     

Autotrophic and Mixotrophic Hydrogen Photoproduction in Sulfur-Deprived Chlamydomonas Cells

In Chlamydomonas reinhardtii cells, H₂ photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H₂ production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H₂ photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H₂ production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H₂ photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H₂ production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H₂ production was measured. H₂ production was enhanced in autotrophic conditions by removing O₂ using N₂ bubbling, thereby showing that substantial H₂ production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H₂ photoproduction.     

The Role of Various Domains of the Iron–Sulfur Protein in the Assembly and Activity of the Cytochromme bc 1 Complex of Yeast Mitochondria

Assembly studies in vitro of deletion mutants of the iron–sulfur protein into the cytochromebc ₁ complex revealed that mutants localized in the extramembranous regions of the proteinwere not assembled into the complex in contrast to the efficient assembly of mutants in themembrane-spanning region. Charged amino acids located in the extramembranous 1-4 loopand the 1 helix were mutated and expressed in yeast cells lacking the gene for the iron–sulfurprotein. Mutating the charged amino acid residues H124, E125, R146, K148, and D149 aswell as V132 and W152 resulted in loss of enzymatic activity due to the loss of iron–sulfurprotein suggesting that these amino acids are required to maintain protein stability. By contrast,no loss of iron–sulfur protein accompanied the 30–50% loss of bc ₁ complex activity in mutantsof three conserved alanine residues, A86, A90, and A92, suggesting that these residues maybe involved in the proposed movement of the flexible tether of the iron–sulfur proteinduring catalysis.     

Examination of chlorophyll fluorescence in sulfur-deprived cells of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Pulse amplitude modulation fluorimetry was used to assess chlorophyll fluorescence parameters in Chlamydomonas reinhardtii cells during sulfur deprivation. A significant (fourfold) increase in the chlorophyll fluorescence yield (parameters F ₀ and F _m) normalized to the chlorophyll concentration was shown for deprived cells. The chlorophyll content did not change during the deprivation experiments. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in photosystem II (PSII) of sulfur-deprived cells. For example, starved cells exhibited a less pronounced pH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in the reaction centers of PSII. It was also shown that the photosynthetic apparatus of starved cells is primarily in state 2 and that back transition to state 1 is suppressed. However, these changes cannot cause the discovered elevation of chlorophyll fluorescence intensity (F ₀ and F _m) in the cells under sulfur limitation. The observed increase in the chlorophyll fluorescence intensity under sulfur deprivation may be due to partial dissociation of peripheral light-harvesting complexes from the reaction centers of PSII or a malfunction of the dissipative cycle in PSII, involving cytochrome b ₅₅₉.     

Photosynthetic H<Subscript>2</Subscript> metabolism in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> (unicellular green algae)

Unicellular green algae have the ability to operate in two distinctly different environments (aerobic and anaerobic), and to photosynthetically generate molecular hydrogen (H₂). A recently developed metabolic protocol in the green alga Chlamydomonas reinhardtii permitted separation of photosynthetic O₂-evolution and carbon accumulation from anaerobic consumption of cellular metabolites and concomitant photosynthetic H₂-evolution. The H₂ evolution process was induced upon sulfate nutrient deprivation of the cells, which reversibly inhibits photosystem-II and O₂-evolution in their chloroplast. In the absence of O₂, and in order to generate ATP, green algae resorted to anaerobic photosynthetic metabolism, evolved H₂ in the light and consumed endogenous substrate. This study summarizes recent advances on green algal hydrogen metabolism and discusses avenues of research for the further development of this method. Included is the mechanism of a substantial tenfold starch accumulation in the cells, observed promptly upon S-deprivation, and the regulated starch and protein catabolism during the subsequent H₂-evolution. Also discussed is the function of a chloroplast envelope-localized sulfate permease, and the photosynthesis–respiration relationship in green algae as potential tools by which to stabilize and enhance H₂ metabolism. In addition to potential practical applications of H₂, approaches discussed in this work are beginning to address the biochemistry of anaerobic H₂ photoproduction, its genes, proteins, regulation, and communication with other metabolic pathways in microalgae. Photosynthetic H₂ production by green algae may hold the promise of generating a renewable fuel from nature’s most plentiful resources, sunlight and water. The process potentially concerns global warming and the question of energy supply and demand.     

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.     

The effect of sulfur compounds on H<Subscript>2</Subscript> evolution/consumption reactions,mediated by various hydrogenases,in the purple sulfur bacterium, <Emphasis Type="Italic">Thiocapsa roseopersicina</Emphasis>

The influence of reduced sulfur compounds (including stored S⁰) on H₂ evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S₂O₃²⁻, SO₃²⁻, S²⁻ and S⁰ as electron donors for light-dependent H₂ production. Dark H₂ evolution from organic substrates via Hox hydrogenase was inhibited by S⁰. Under light conditions, endogenous H₂ uptake by Hox or Hup hydrogenases was suppressed by S compounds. СО₂-dependent H₂ uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process. Dark H₂ consumption via Hyn hydrogenase was connected to utilization of S⁰ as an electron acceptor and resulted in the accumulation of H₂S. In wild type BBS, with high levels of stored S⁰, dark H₂ production from organic substrates was significantly lower, but H₂S accumulation significantly higher, than in the mutant GB1121(Hox⁺). There is a possibility that H₂ produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S⁰.     

Influence of Histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the P_D1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P_680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA₁ and psbA₂ genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT^?) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA₃ gene. The O₂-evolving activity of His-tagged PSII isolated from WT^? was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA₁ is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT^?. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P₆₈₀, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to P_D1 is not an important determinant of the redox and spectroscopic properties of P₆₈₀ in T. elongatus.     

Promotion of Energy Transfer and Oxygen Evolution in Spinach Photosystem II by Nano-anatase TiO<Subscript>2</Subscript>

Being a proven photocatalyst, nano-anatase is capable of undergoing electron transfer reactions under light. In previous studies we had proven that nano-anatase improved photosynthesis and greatly promoted spinach growth. The mechanisms by which nano-anatase promotes energy transfer and the conversion efficiency of the process are still not clearly understood. In the present paper, we report the results obtained with the photosystem II (PSII) isolated from spinach and treated by nano-anatase TiO₂ and studied the effect of nano-anatase TiO₂ on energy transfer in PSII by spectroscopy and on oxygen evolution. The results showed that nano-anatase TiO₂ treatment at a suitable concentration could significantly change PSII microenvironment and increase absorbance for visible light, improve energy transfer among amino acids within PSII protein complex, and accelerate energy transport from tyrosine residue to chlorophyll a. The photochemical activity of PSII (fluorescence quantum yield) and its oxygen-evolving rate were enhanced by nano-anatase TiO₂. This is viewed as evidence that nano-anatase TiO₂ can promote energy transfer and oxygen evolution in PSII of spinach.     

Photochemical activity of photosystem II and hydrogen photoproduction in sulfur-deprived Chlamydomonas reinhardtii mutants D1-R323D and D1-R323L

The role of photosystem II in hydrogen photoproduction by Chlamydomonas reinhardtii cells was studied in mutants with modified D1-protein. In D1-R323D and D1-R323L mutants, the replacement of arginine by aspartate or leucine, respectively, resulted in the disruption of electron transport at the donor side of photosystem II. The rate of oxygen evolution in D1-R323D decreased twice as compared to the pseudo-wild type (pWT), and in D1-R323L no oxygen evolution was detected. The latter mutant was not capable of photoautotrophical growth. The dynamics of changes in oxygen content, the reduction of photosystem II active reaction centers (deltaF/F(1)m), and hydrogen production rate in pWT were found to be similar to the wild type if cultivated under sulfur deprivation in a closed bioreactor. The observed gradual decrease in the deltaF/F(1)m value turned to a sharp drop almost to zero followed by a partial recovery during which the production of hydrogen set in. The transition to the anaerobic phase in D1-R323D cultured in a sulfur-deprived medium occurred earlier than it happened in pWt under the same conditions. However, the partial recovery of photosystem II activity and hydrogen production started at a later time, and the rate of hydrogen production was low. The D1-R323L mutant incapable of oxygen evolution entered the rapidly anaerobiosis but produced no hydrogen. The kinetics of photoinduced redox transitions in P700 was similar in all investigated strains and was not affected by diuron addition. This implies that the mutants had a pool of reducers, which could donate electrons through the quinone pool or cytochrome to photosystem I. However, in D1-R323L mutant lacking the active photosystem II, this condition was not sufficient to support hydrogenase activity.     

Genome analysis ofElymus angulatus andE. patagonicus (Poaceae) and their hybrids with N. and S. AmericanHordeum spp.

The results of genome analysis of five hybrids, viz.Elymus patagonicus ×Hordeum procerum, E. patagonicus ×H. tetraploidum, E. angulatus ×H. jubatum, E. angulatus ×H. lechleri, andE. angulatus ×H. parodii, are reported. The genomic constitution ofHordeum tetraploidum andH. jubatum is best given as H₁H₁H₂H₂, ofH. lechleri andH. parodii as H₁H₁H₂H₂H₄H₄, ofH. procerum as H₁H₁H₂H₂H₃H₃, and ofElymus patagonicus andE. angulatus as SSH₁H₁H₂H₂.     

Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga)

Sulfur deprivation in green algae causes reversible inhibition of photosynthetic activity. In the absence of S, rates of photosynthetic O2 evolution drop below those of O2 consumption by respiration. As a consequence, sealed cultures of the green alga Chlamydomonas reinhardtii become anaerobic in the light, induce the "Fe-hydrogenase" pathway of electron transport and photosynthetically produce H2 gas. In the course of such H2-gas production cells consume substantial amounts of internal starch and protein. Such catabolic reactions may sustain, directly or in directly, the H2-production process. Profile analysis of selected photosynthetic proteins showed a precipitous decline in the amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a function of time in S deprivation, a more gradual decline in the level of photosystem (PS) II and PSI proteins, and a change in the composition of the PSII light-harvesting complex (LHC-II). An increase in the level of the enzyme Fe-hydrogenase was noted during the initial stages of S deprivation (0-72 h) followed by a decline in the level of this enzyme during longer (t >72 h) S-deprivation times. Microscopic observations showed distinct morphological changes in C. reinhardtii during S deprivation and H2 production. Ellipsoid-shaped cells (normal photosynthesis) gave way to larger and spherical cell shapes in the initial stages of S deprivation and H2 production, followed by cell mass reductions after longer S-deprivation and H2-production times. It is suggested that, under S-deprivation conditions, electrons derived from a residual PSII H2O-oxidation activity feed into the hydrogenase pathway, thereby contributing to the H2-production process in Chlamydomonas reinhardtii. Interplay between oxygenic photosynthesis, mitochondrial respiration, catabolism of endogenous substrate, and electron transport via the hydrogenase pathway is essential for this light-mediated H2-production process.     

Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii

Treatment with NaHSO₃ induces a 10-fold increase in H₂ photoproduction in the filamentous N₂-fixing cyanobacterium Anabaena sp. strain PCC 7120. However, it is unclear whether this treatment also increases H₂ photoproduction in green alga. In this study, treatment with 13 mM NaHSO₃ resulted in about a 200-fold increase in H₂ production in Chlamydomonas reinhardtii, and this increase was most probably the result of reduced O₂ content and enhanced hydrogenase activity. Compared to the conventional strategy of sulfur deprivation, NaHSO₃ treatment results in a higher maximum rate of H₂ photoproduction, greater efficiency of conversion of light energy into H₂, shorter half-time to produce the maximum accumulated H₂ levels, and reduced costs because no centrifugation is involved. We therefore conclude that NaHSO₃ treatment is an efficient, rapid, and economic strategy for improving photobiological H₂ production in the green alga C. reinhardtii.     

The <Emphasis Type="Italic">b</Emphasis><Subscript>arg36</Subscript> contributes to efficient coupling in F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli, the F₁F_O ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b _arg36→ile and the b _arg36→glu mutations. The b _arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b _arg36→glu mutation was largely suppressed in the b _{arg36→glu,glu39→arg} double mutant. E. coli expressing the b _{arg36→glu,glu39→arg} subunit grew well on succinate-based medium. F₁F_O ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F₁F_O ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.     

Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived chlamydomonas cells

In Chlamydomonas reinhardtii cells, H2 photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H2 production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H2 photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H2 production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H2 photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H2 production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H2 production was measured. H2 production was enhanced in autotrophic conditions by removing O2 using N2 bubbling, thereby showing that substantial H2 production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H2 photoproduction.     

Rice <Emphasis Type="Italic">phot1a</Emphasis> mutation reduces plant growth by affecting photosynthetic responses to light during early seedling growth

The aim of this work was to characterize the phot1 mutant of rice during early seedling growth in various light conditions. We isolated the rice T-DNA insertion mutant phot1a-1 and compared it to the Tos17 insertion mutant phot1a-2. When phot1a mutants were grown under WL (100) and BL (40 μmol m⁻² s⁻¹), they demonstrated a considerable reduction in photosynthetic capacity, which included decreased leaf CO₂ uptake and plant growth. Pigment analysis showed no significant difference between wild-type and mutants in the Chl a:b ratios, whereas in the latter, total concentration was reduced (a 2-fold decrease). Carotenoid contents of the mutants were also decreased considerably, implying the involvement of phot1a in pigment degradation. Deletion of phot1a showed higher contents of H₂O₂ in leaves. Chloroplastic APX and SOD activities were lower in the mutants whereas the activities of cytosolic enzymes were increased. Immunoblotting indicated reduced accumulation of photosystem proteins (D1, D2, CP43, Lhca2, and PsaC) relative to the other light-harvesting complexes in the mutant. We conclude that the defect of Os Phot1a affects degradation of chlorophylls and carotenoids, and under photosynthetically active photon fluxes, mutation of phot1a results in loss of photosynthetic capacity owing to the damage of photosystems caused by elevated H₂O₂ accumulation, leading to a reduction in plant growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Hydrogen peroxide production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The presence of 2 mM bicarbonate in the incubation medium induced stomatal closure in abaxial epidermis of Arabidopsis. Exposure to 2 mM bicarbonate elevated the levels of H₂O₂ in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H₂DCF-DA). Bicarbonate-induced stomatal closure as well as H₂O₂ production were restricted by exogenous catalase or diphenylene iodonium (DPI, an inhibitor of NAD(P)H oxidase). The reduced sensitivity of stomata to bicarbonate and H₂O₂ production in homozygous atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase is involved during bicarbonate induced ROS production in guard cells. The production of H₂O₂ was quicker and greater with ABA than that with bicarbonate. Such pattern of H₂O₂ production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H₂O₂ is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.