Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H₂ production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over‐reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress‐related genes, down‐regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H₂ production because it supplies electrons but the evolved O₂ inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50‐fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn‐cluster of PSII, and afterwards, it can donate electrons to tyrozin Z⁺ at a slow rate. This stage is followed by donor‐side‐induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H₂ production. We also show that ascorbate influenced H₂ evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.     

Comparative analysis of photosensitivity in photosystem I donor and acceptor side mutants of Chlamydomonas reinhardtii

Electron input from plastocyanin into photosystem I (PSI) is slowed down in the Chlamydomonas reinhardtii mutants affected at the donor side (PsaF or PsaB, lumenal loop j) of PSI. In contrast, electron exit from PSI to ferredoxin is diminished in the PSI acceptor side PsaC mutants K35E and FB₁. Although, the electron transfer reactions are diminished to a similar extent in both type of mutants, the PsaC mutants K35E and FB₁ are more light‐sensitive than the PsaF‐deficient strain 3bF or the PsaB mutants E613N and W627F. To assess the differential photosensitivity of donor and acceptor side mutants fluorescence transients, gross oxygen evolution and uptake, PSII photo‐inhibition and rate of recovery were measured as well as NADP⁺ photoreduction. The NADP⁺ photoreduction measurements indicated that the donor side is limiting the reduction rate. In contrast, measurements of gross oxygen evolution and uptake showed that the reducing side limits linear electron transfer. However, under high light, donor and acceptor side mutations lead to PSII photo‐inhibition and to a diminished rate of PSII recovery, cause lipid peroxidation and result in a decrease in the levels of PSI and PSII. The wild type is not affected under the same conditions. These responses are most pronounced in the PsaC‐K35E and PsaB‐W627F mutants, and they correlate with the light sensitivity of these strains. The correlation between limitation of electron transfer through PSI and the formation of reactive oxygen species as a cause for the light‐sensitivity is discussed.     

Induction of tolerance to oxidative stress in the green alga, Chlamydomonas reinhardtii, by abscisic acid

The effect of abscisic acid (ABA) on the tolerance to oxidative stress in a freshwater green alga, Chlamydomonas reinhardtii, was investigated. Exogenously added ABA enhanced the growth of this alga, which was observed under continuous illumination but not in the dark. The cells treated with ABA for 24 h showed tolerance to oxidative stress caused by exposure to paraquat or hydrogen peroxide. In the ABA‐treated cells, the activities of two antioxidant enzymes, catalase (CAT) and ascorbate peroxidase (APX), were significantly higher than those in the untreated control. The result suggests that ABA plays a role in the enhancement of tolerance to oxidative stress by increasing the activity of antioxidant enzymes.     

INORGANIC CARBON REPLETION CONSTRAINS STEADY‐STATE LIGHT ACCLIMATION IN THE CYANOBACTERIUM SYNECHOCOCCUS ELONGATUS1

Cyanobacteria show high metabolic plasticity by re‐allocating macromolecular resources in response to variations in both environmental inorganic carbon (Ci) and light. We grew cultures of the picoplanktonic cyanobacterium Synechococcus elongatus Nägeli across a 50‐fold range of growth irradiance at either a dissolved [Ci] <0.1 mM, sufficient to induce strongly the carbon‐concentrating mechanism (CCM) or a dissolved [Ci] of ～4 mM, sufficient to strongly induce the CCM to basal constitutive activity. There was no detectable growth cost of acclimation to low Ci across the entire range of irradiance and growth was nearly light saturated at 50 l mol photons·m^?2·s^?1. Cells acclimated to low Ci significantly re‐allocated macromolecular resources to support their CCM, while maintaining near homeostatis of metabolic flux per unit photosynthetic complex. Changing growth irradiance also drove re‐organization of the photosynthetic machinery to balance excitation flux and metabolic demands, but flux per complex varied widely across the range of tolerable growth irradiances. Across the range of growth irradiance, low Ci cells had significantly less phycocyanin than high Ci cells, which corresponded to a lower PSII absorbance capacity. Furthermore, low Ci cells maintained more PSI per cell^?1 than high Ci cells under high growth irradiance. Low Ci cells could therefore maintain more of their PSII reaction centers open at high growth irradiance than could high Ci cells, which experienced a significant PSII closure. Thus, acclimation to growth under high available Ci actually constrained acclimation to high light by restricting electron transport downstream from PSII in S. elongatus.     

The mechanism of photosystem‐II inactivation during sulphur deprivation‐induced H2 production in Chlamydomonas reinhardtii

Sulphur limitation may restrain cell growth and viability. In the green alga Chlamydomonas reinhardtii, sulphur limitation may induce H₂ production lasting for several days, which can be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystem II (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has long been assumed to be caused by the sulphur‐limited turnover of its reaction center protein PsbA. Here we reinvestigated this issue in detail and show that: (i) upon transferring Chlamydomonas cells to sulphur‐free media, the cellular sulphur content decreases only by about 25%; (ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover, and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP‐L‐galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen‐evolving complex and provide electrons to PSII, albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers are inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation.     

Bicarbonate protects the water-oxidizing complex of photosystem II against thermoinactivation in intact <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Currently available data about bicarbonate (BC) action on the Mn-containing water-oxidizing complex (WOC) of the photosystem II (PSII) were obtained almost solely in vitro, e.g. on subchloroplast membrane fragments enriched with PSII. To investigate the in vivo BC effect on the PSII donor side, we used the method of dark thermoinactivation of intact Chlamydomonas reinhardtii cells. Photosynthetic activity of PSII was measured as photoinduced changes in the PSII chlorophyll fluorescence yield and as the rate of photosynthetic oxygen evolution. To exclude a “direct” effect of the absence of BC on the PSII activity, before measurements of the photosynthetic activity, the concentration of BC in all samples was equalized by addition of NaHCO₃ to each of them (except for those that contained 5 mM of NaHCO₃ during thermoinactivation) to reach the final concentration of 5 mM. This allowed registering only so-called “irreversible” (i.e., not reversible by subsequent addition of BC) effect of the absence of BC during thermoinactivation. It was shown that, if 5 mM NaHCO₃ was added to the medium before thermoinactivation, the rate of inactivation of the PSII donor side was lower than in BC-depleted medium 1.5-to 2-fold. The obtained results are interpreted as an indication that BC protects the donor side of PSII against thermoinactivation in vivo, in intact C. reinhardtii cells. This proves the correctness of the earlier proposition that BC is an integral constituent of the Mn-containing water-oxidizing complex of PSII. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 342–349. The article was translated by the authors.     

Measurement of OH radical CT for inactivating Cryptosporidium parvum using photo/ferrioxalate and photo/TiO2 systems

Aim: This study investigates the inactivation of Cryptosporidium parvum using the OH radical and reports the OH radical CT (OH radical concentration × contact time) values for C. parvum inactivation. Methods and Results: Although a wealth of information has demonstrated the efficacy of the microbial inactivation activity of the OH radical, no studies have performed a quantitative estimation of the OH radical for C. parvum inactivation. The CT value of the OH radical required for 2 log C. parvum inactivation was measured with two OH radical‐generating systems, photo/ferrioxalate and photo/TiO₂. The OH radical was approx. 10⁴–10⁷‐fold more effective for microbial inactivation than other popular chemical disinfectants such as ozone, chlorine dioxide and free chlorine. Conclusions: The OH radical appears to be suitable for microbial inactivation with a calculated CT value required for 2 log C. parvum inactivation of 9·3 × 10^?5 mg min l^?1. Significance and Impact of the Study: This study is the first report of an investigation on the role of the OH radical in the photo/ferrioxalate and photo/TiO₂ systems and on the OH radical CT required for C. parvum inactivation.     

Carbon dioxide enrichment alleviates heat stress by improving cellular redox homeostasis through an ABA‐independent process in tomato plants

Plant responses to elevated CO₂ and high temperature are critically regulated through a complex network of phytohormones and redox homeostasis. However, the involvement of abscisic acid (ABA) in plant adaptation to heat stress under elevated CO₂ conditions has not been thoroughly studied. This study investigated the interactive effects of elevated CO₂ (800 μmol·mol^?1) and heat stress (42 °C for 24 h) on the endogenous level of ABA and the cellular redox state of two genotypes of tomato with different ABA biosynthesis capacities. Heat stress significantly decreased maximum photochemical efficiency of PSII (Fv/Fm) and leaf water potential, but also increased levels of malondialdehyde (MDA) and electrolyte leakage (EL) in both genotypes. Heat‐induced damage was more severe in the ABA‐deficient mutant notabilis (not) than in its parental cultivar Ailsa Craig (Ailsa), suggesting that a certain level of endogenous ABA is required to minimise the heat‐induced oxidative damage to the photosynthetic apparatus. Irrespective of genotype, the enrichment of CO₂ remarkably stimulated Fv/Fm, MDA and EL in heat‐stressed plants towards enhanced tolerance. In addition, elevated CO₂ significantly strengthened the antioxidant capacity of heat‐stressed tomato seedlings towards a reduced cellular redox state for a prolonged period, thereby mitigating oxidative stress. However, elevated CO₂ and heat stress did not alter the endogenous level of ABA or the expression of its biosynthetic gene NCED2 in either genotype, indicating that ABA is not involved in elevated CO₂‐induced heat stress alleviation. The results of this study suggest that elevated CO₂ alleviated heat stress through efficient regulation of the cellular redox poise in an ABA‐independent manner in tomato plants.     

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.     

Xanthophyll Cycle and Inactivation of Photosystem Ⅱ Reaction Centers Alleviating Reducing Pressure to Photosystem Ⅰ in Morning Glory Leaves under Short-term High Irradiance

Under 30-min high irradiance （1500μmol m^-2 s^-1）, the roles of the xanthophyll cycle and D1 protein turnover were investigated through chlorophyll fluorescence parameters in morning glory （Ipomoea setosa） leaves, which were dipped into water, dithiothreitol （DTT） and lincomycin （LM）, respectively. During the stress, both the xanthophyll cycle and D1 protein turnover could protect PSI from photoinhibition. In DTT leaves, non-photochemical quenching （NPQ） was inhibited greatly and the oxidation level of P700 （P700^＋） was the lowest one. However, the maximal photochemical efficiency of PSII （Fv/Fm） in DTT leaves was higher than that of LM leaves and was lower than that of control leaves. These results suggested that PSI was more sensitive to the loss of the xanthophyll cycle than PSII under high irradiance. In LM leaves, NPQ was partly inhibited, Fv/Fm was the lowest one among three treatments under high irradiance and P700^＋ was at a similar level as that of control leaves. These results implied that inactivation of PSII reaction centers could protect PSI from further photoinhibition. Additionally, the lowest of the number of active reaction centers to one inactive reaction center for a PSII cross-section （RC/CSo）, maximal trapping rate in a PSll cross-section （TRo/CSo）, electron transport in a PSll cross-section （ETo/CSo） and the highest of 1-qP in LM leaves further indicated that severe photoinhibition of PSII in LM leaves was mainly induced by inactivation of PSII reaction centers, which limited electrons transporting to PSh However, relative to the LM leaves the higher level of RC/CSo, TRo/CSo, Fv/Fm and the lower level of 1-qP in DTT leaves indicated that PSI photoinhibition was mainly induced by the electron accumulation at the PSI acceptor side, which induced the decrease of P700^＋ under high irradiance.     

Membrane stabilization by abscisic acid under cold aids proline in alleviating chilling injury in maize (Zea mays L.) cultured cells

Previous studies of maize suspension‐cultured cells showed that abscisic acid (ABA) treatment at warm temperatures improved the tolerance of cells to subsequent chilling. In the present study, it is shown that both ABA‐treated and untreated maize cells accumulated proline in response to chilling. However, ABA‐treated cells displayed less lipid peroxidation during chilling, and thus, unlike untreated cells, were able to retain the accumulated proline intracellularly. Proline application experiments indicate that an intracellular proline level higher than 2 µmole (g FW)^?1 prior to chilling was needed to meaningfully reduce chilling‐enhanced lipid peroxidation and significantly improve chilling tolerance. The results suggest that total proline accumulation in ABA‐treated as well as untreated cells during chilling was enough to potentially improve chilling tolerance, but proline leakage rendered the control cells unable to benefit from the endogenous synthesis of proline in relation to the alleviation of chilling injury. Proline participated in chilling tolerance improvement in ABA‐treated maize cells, as evidenced by: (1) the inhibition of proline accumulation by l ‐methionine‐d , l ‐sulphoximine (MSO), an inhibitor of glutamine synthetase, reduced ABA‐improved chilling tolerance, and (2) the addition of glutamine into the medium prevented the MSO‐induced reduction in chilling tolerance. The revised relationship between proline accumulation and membrane stability at cold is discussed in the light of these current findings.     

Directed inactivation of the psbl gene does not affect Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants. To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and determined its nucleotide sequence. Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein. Analysis of these mutants revealed that absence of the PsbI protein results in a 25–30% loss of PSII activity. However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred. These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80–90%) titer of the PSII complex. In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity.     

A new paradigm for producing astaxanthin from the unicellular green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis has been exploited as a cell factory to produce the high‐value antioxidant astaxanthin for over two decades, due to its superior ability to synthesize astaxanthin under adverse culture conditions. However, slow vegetative growth under favorable culture conditions and cell deterioration or death under stress conditions (e.g., high light, nitrogen starvation) has limited the astaxanthin production. In this study, a new paradigm that integrated heterotrophic cultivation, acclimation of heterotrophically grown cells to specific light/nutrient regimes, followed by induction of astaxanthin accumulation under photoautotrophic conditions was developed. First, the environmental conditions such as pH, carbon source, nitrogen regime, and light intensity, were optimized to induce astaxanthin accumulation in the dark‐grown cells. Although moderate astaxanthin content (e.g., 1% of dry weight) and astaxanthin productivity (2.5 mg L^?1 day^?1) were obtained under the optimized conditions, a considerable number of cells died off when subjected to stress for astaxanthin induction. To minimize the susceptibility of dark‐grown cells to light stress, the algal cells were acclimated, prior to light induction of astaxanthin biosynthesis, under moderate illumination in the presence of nitrogen. Introduction of this strategy significantly reduced the cell mortality rate under high‐light and resulted in increased cellular astaxanthin content and astaxanthin productivity. The productivity of astaxanthin was further improved to 10.5 mg L^?1 day^?1 by implementation of such a strategy in a bubbling column photobioreactor. Biochemical and physiological analyses suggested that rebuilding of photosynthetic apparatus including D1 protein and PsbO, and recovery of PSII activities, are essential for acclimation of dark‐grown cells under photo‐induction conditions. Biotechnol. Bioeng. 2016;113: 2088–2099. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

Characterization of the wave phenomenon of flash-induced chlorophyll fluorescence in Chlamydomonas reinhardtii

Flash-induced chlorophyll fluorescence relaxation is a powerful tool to monitor the reoxidation reactions of the reduced primary quinone acceptor, Q_A^? by Q_B and the plastoquinone (PQ) pool, as well as the charge recombination reactions between the donor and acceptor side components of Photosystem II (PSII). Under certain conditions, when the PQ pool is highly reduced (e.g. in microaerobic conditions), a wave phenomenon appears in the fluorescence relaxation kinetics, which reflects the transient reoxidation and re-reduction of Q_A^? by various electron transfer processes, which in cyanobacteria is mediated by NAD(P)H dehydrogenase (NDH-1). The wave phenomenon was also observed and assigned to the operation of type 2 NAD(P)H dehydrogenase (NDH-2) in the green alga Chlamydomonas reinhardtii under hydrogen-producing conditions, which required a long incubation of algae under sulphur deprivation (Krishna et al. J Exp Bot 70 (21):6321–6336, 2019). However, the conditions that induce the wave remained largely uncharacterized so far in microalgae. In this work, we investigated the wave phenomenon in Chlamydomonas reinhardtii under conditions that lead to a decrease of PSII activity by applying hydroxylamine treatment, which impacts the donor side of PSII in combination with a strongly reducing environment of the PQ pool (microaerobic conditions). A similar wave phenomenon could be induced by photoinhibitory conditions (illumination with strong light in the presence of the protein synthesis inhibitor lincomycin). These results indicate that the fluorescence wave phenomenon is activated in green algae when the PSII activity decreases relative to Photosystem I (PS I) activity and the PQ pool is strongly reduced. Therefore, the fluorescence wave could be used as a sensitive indicator of altered intersystem electron transfer processes, e.g. under stress conditions.

     

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.     

Dynamic changes of canopy-scale mesophyll conductance to CO₂ diffusion of sunflower as affected by CO₂ concentration and abscisic acid

Leaf‐level measurements have shown that mesophyll conductance (g_m) can vary rapidly in response to CO₂ and other environmental factors, but similar studies at the canopy‐scale are missing. Here, we report the effect of short‐term variation of CO₂ concentration on canopy‐scale g_m and other CO₂ exchange parameters of sunflower (Helianthus annuus L.) stands in the presence and absence of abscisic acid (ABA) in their nutrient solution. g_m was estimated from gas exchange and on‐line carbon isotope discrimination (Δ_obs) in a ¹³CO₂/¹²CO₂ gas exchange mesocosm. The isotopic contribution of (photo)respiration to stand‐scale Δ_obs was determined with the experimental approach of Tcherkez et al. Without ABA, short‐term exposures to different CO₂ concentrations (C_a 100 to 900 µmol mol^?1) had little effect on canopy‐scale g_m. But, addition of ABA strongly altered the CO₂‐response: g_m was high (approx. 0.5 mol CO₂ m^?2 s^?1) at C_a < 200 µmol mol^?1 and decreased to <0.1 mol CO₂ m^?2 s^?1 at C_a >400 µmol mol^?1. In the absence of ABA, the contribution of (photo)respiration to stand‐scale Δ_obs was high at low C_a (7.2‰) and decreased to <2‰ at C_a > 400 µmol mol^?1. Treatment with ABA halved this effect at all C_a.     

Energy Transfer from Phycobilisomes to Photosystems of Nostoc flagelliforme Born. et Flah. During the Rewetting Course and Its Physiological Significance

During the non-frost season, the condensation of dew makes Nostocflagelliforme Born. et Flah., a highly drought-tolerant terrestrial cyanobacterium, frequently undergo rehydration-dehydration.Rehydration begins in the dark at night. After rewetting in the dark, photochemical activity and the structure of photosystem (PS)II were not recovered at all; the structure of PSI, energy transfer in phycobilisomes, and energy transfer from phycobilisomes to PSI were recovered within 5 min, as in the light. The recovery of energy transfer from phycobilisomes to PSII was light dependent and energy transfer from phycobilisomes to PSII was only partially recovered in the dark. These results suggest that the two-trigger control (water and light) of photosynthetic recovery may make IV, flagelliforme avoid unnecessary energy consumption and, at the same time, the partial recovery of energy transfer from phycobilisomes to PSII in the dark could help N. flagelliforme accumulate more photosynthetic products during the transient period of rehydration-dehydration.     

Movement of Abscisic Acid Across the Plasmalemma and the Tonoplast of Guard Cells of Valerianella locusta

Uptake experiments and efflux compartmental analyses of abscisic acid (ABA) with acid treated epidermal peels of Valerianella locusta were performed to elucidate the mechanisms of transport of ABA across the plasmalemma and tonoplast of guard cells. ABA uptake across the plasmalemma is linearly correlated with external ABA concentration in the incubation medium. Under alkaline conditions ABA-uptake was not significantly above background, indicating that ABA uptake occurs mainly by diffusion of undissociated ABAH as the most permeable species, which is trapped afterwards in the alkaline cytosol as impermeable ABA^?. Efflux analysis of ABA revealed a saturable component of ABA transfer across the tonoplast. A Woolf-Augustinsson-Hofstee analysis suggested the existence of two transport systems for ABA at the tonoplast. The high affinity transport system had a K_M of 0.21 mol m^?3 and a V_max 85.8 amol ABA cell^?1 h^?1. Using the data of the uptake and efflux experiments we calculated the permeability coefficients of ABA for the plasmalemma and the tonoplast of guard cells, which are 2.46 10^?7 m s–¹ and 1.26 10^?8m s^?1, respectively. The distribution of the pH-probe (¹⁴C)-DMO between medium, cytosol and vacuole was investigated and used to calculate cytosolic and vacuolar pH. The vacuolar pH is too low to explain the high vacuolar ABA concentration by trapping of ABA^?, whereas the cytosol is sufficiently alkaline to act as an efficient anion trap. Therefore we conclude that ABA transport across the guard cell tonoplast is catalyzed by a saturable uptake component.     

A Psb27 homologue in Arabidopsis thaliana is required for efficient repair of photodamaged photosystem II

Psb27 has been identified as a lumenal protein associated with photosystem II (PSII). To gain insight into the function of Psb27, we isolated a mutant Arabidopsis plant with a loss of psb27 function. The quantity of PSII complexes and electron transfer within PSII remained largely unaffected in the psb27 mutant. Our results also showed that under high-light-illumination, PSII activity and the content of the PSII reaction center protein D1 decreased more significantly in the psb27 mutant than in wild-type (WT) plant. Treatment of leaves with a chloroplast protein synthesis inhibitor resulted in similar light-induced PSII inactivation levels and D1 protein degradation rates in the WT and psb27 mutant plants. Recovery of PSII activity after photoinhibition was delayed in the psb27 mutant, suggesting that Psb27 is required for efficient recovery of the photodamaged PSII complex. Overall, these results demonstrated that Psb27 in Arabidopsis is not essential for oxygenic photosynthesis and PSII formation. Instead, our results provide evidence for the involvement of this lumenal protein in the recovery process of PSII. Hua Chen and Dongyuan Zhang contribute equally to this work.     

BIOSYNTHESIS OF POLY‐3‐HYDROXYBUTYRATE (PHB) IN THE TRANSGENIC GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A cell‐wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC‐849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris‐acetate‐phosphate media containing 10 μg · mL^?1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly‐3‐hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min^?1 · mg protein^?1 to 126 nmol · min^?1 · mg protein^?1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation.