Enhancing spinach (Spinacia oleracea L.) resilience in pesticide-contaminated soil: Role of pesticide-tolerant Ciceribacter azotifigens and Serratia marcescens in root architecture, leaf gas exchange attributes and antioxidant response restoration.

This study unveils the detoxification potential of insecticide-tolerant plant beneficial bacteria (PBB), i.e., Ciceribacter azotifigens SF1 and Serratia marcescens SRB1, in spinach treated with fipronil (FIP), profenofos (PF) and chlorantraniliprole (CLP) insecticides. Increasing insecticide doses (25-400 µg kg-1 soil) significantly curtailed germination attributes and growth of spinach cultivated at both bench-scale and in greenhouse experiments. Profenofos at 400 µg kg-1 exhibited maximum inhibitory effects and reduced germination by 55%; root and shoot length by 78% and 81%, respectively; dry matter accumulation in roots and shoots by 79% and 62%, respectively; leaf number by 87% and leaf area by 56%. Insecticide application caused morphological distortion in root tips/surfaces, increased levels of oxidative stress, and cell death in spinach. Application of insecticide-tolerant SF1 and SRB1 strains relieved insecticide pressure resulting in overall improvement in growth and physiology of spinach grown under insecticide stress. Ciceribacter azotifigens improved germination rate (10%); root biomass (53%); shoot biomass (25%); leaf area (10%); Chl-a (45%), Chl-b (36%) and carotenoid (48%) contents of spinach at 25 µg CLP kg-1 soil. PBB inoculation reinvigorated the stressed spinach and modulated the synthesis of phytochemicals, proline, malondialdehyde (MDA), superoxide anions (O2•-), and hydrogen peroxide (H2O2). Scanning electron microscopy (SEM) revealed recovery in root tip morphology and stomatal openings on abaxial leaf surfaces of PBB-inoculated spinach grown with insecticides. Ciceribacter azotifigens inoculation significantly increased intrinsic water use efficiency, transpiration rate, vapor pressure deficit, intracellular CO2 concentration, photosynthetic rate, and stomatal conductance in spinach exposed to 25 µg FIP kg-1. Also, C. azotifigens and S. marcescens modulated the antioxidant defense systems of insecticide-treated spinach. Bacterial strains were strongly colonized to root surfaces of insecticide-stressed spinach seedlings as revealed under SEM. The identification of insecticide-tolerant PBBs such as C. azotifigens and S. marcescens hold the potential for alleviating abiotic stress to spinach, thereby fostering enhanced and safe production within polluted agroecosystems.

Ca2SiO4 chemigation reduces cadmium localization in the subcellular leaf fractions of spinach (Spinacia oleracea L.) under cadmium stress.

Heavy metal like cadmium (Cd) is inessential and highly toxic and is posing serious environmental problems for agriculture worldwide. Presence of Cd gives rise to several physiological and structural disorders that leads to reduction in growth and performance of agricultural plants. Evidence related to subcellular distribution and accumulation of Cd is still enigmatic. Experiment was conducted using hydroponic culture to examine the subcellular accumulation of Cd in Spinacia oleracea L. leaves under Cd stress (50 µM and 100 µM); moreover, the Cd toxicity alleviation using 5 mM silicon (Si) was investigated. Our findings suggest that fresh and dry biomass, shoot and root length, leaf area and length of leaf declined when exposed to Cd stress (50 µM and 100 µM); however, an increase was noticed when Cd treated plants were supplied with Si (5 mM). The content of Ca2+, Mg2+ and Fe2+ in apoplastic washing fluid and symplasm were found to be lower in plants treated with alone Cd, when compared to control. Higher Cd2+:Ca2+, Cd2+:Fe2+ and Cd2+:Mg2+ ratios were detected under cadmium stress in both apoplast and symplast of leaves which were lowered by the addition of 5 mM Si. The novelty of the current study is the detection of increased apoplastic and symplastic Cd concentration in aerial part (i.e., spinach leaves) under alone Cd treatment which was considerably reduced when supplied with Si. Moreover, a noticeable increase in spinach growth and beneficial ionic concentrations suggest that Si can ameliorate the Cd stress in crop plants.

Heavy metals-resistant bacteria (HM-RB): Potential bioremediators of heavy metals-stressed Spinacia oleracea plant.

Microorganism technologies can provide a potential alternative to traditional methods of removing heavy metals to conserve agricultural soils. This study aimed to identify and characterize heavy metals-resistant bacteria (HM-RB) isolated from industry-affected soil and their desired impact as bioremediators of heavy metals-stressed spinach plants. Three of 135 isolates were selected based on a high level of resistance to heavy metals. Based on morphological and biochemical characteristics, the selected isolates were identified as Bacillus subtilis subsp. spizizenii DSM 15029 T DSM (MA3), Paenibacillus jamilae DSM 13815 T DSM (LA22), or Pseudomonas aeruginosa DSM 1117 DSM (SN36). Experiments were implemented to investigate the three isolated HM-RB ability on improving attributes of growth, physio-biochemistry, and components of the antioxidant defense system of spinach plant exposed to the stress of cadmium (Cd2+; 2 mM), lead (Pb2+; 2 mM) or 2 mM Cd2++2 mM Pb2+. Compared to control, Cd2+ or Pb2+ stress markedly lowered plant fresh and dry weights, leaf contents of chlorophylls and carotenoids, rates of transpiration (Tr), net photosynthesis (Pn) and stomatal conductance (gs), relative water content (RWC), and membrane stability index (MSI). In contrast, contents of α.tochopherol (α.TOC), ascorbic acid (AsA), glutathione (GSH), proline, soluble sugars, Cd2+, and Pb2+, as well as activities of enzymatic and non-enzymatic antioxidants were markedly elevated. The application of HM-RB promoted the tolerance to heavy metal stress in spinach plants by improving Tr, Pn, gs, RWC, and MSI, while activities of enzymatic and non-enzymatic antioxidants were suppressed. These results reflected positively in promoting plant growth under heavy metal stress. Therefore, the application of HM-RB as potential bioremediators may be a promising strategy for promoting plant growth and productivity under heavy metal stress.

Glutathione biosynthesis plays an important role in microcystin-LR depuration in lettuce and spinach.

Irrigation of crop plants with microcystins (MCs) contaminated water could be a threat to human health via bioaccumulation. Despite the fact MCs bioaccumulation in crop plants is well documented, MCs depuration, as well as the mechanism involved remains unclear. The objectives of the present study were to investigate the bioaccumulation and depuration of microcystin-LR (MC-LR) in lettuce (Lactuca sativa L.) and spinach (Spinacia oleracea L.), as well as to explore the role of glutathione (GSH) biosynthesis in MC-LR depuration. The tested plants were irrigated with deionized water containing 10 µg L-1 MC-LR for 12 days (bioaccumulation), and subsequently, with either deionized water only or deionized water containing 0.5 mM buthionine sulfoximine (BSO, a specific inhibitor of GSH biosynthesis) for 12 days (depuration). After bioaccumulation period, highest concentrations of MC-LR found in lettuce and spinach were 114.4 and 138.5 µg kg-1 dry weight (DW) respectively. Depuration rates of MC-LR in lettuce and spinach were 9.5 and 8.1 µg kg-1 DW d-1, which deceased to 3.7 and 4.6 µg kg-1 DW d-1 in treatments with BSO application. GSH content in both lettuce and spinach were not significantly affected during depuration without BSO; whereas after treatment with BSO, GSH content significantly decreased by 36.0% and 24.7% in lettuce and spinach on 15 d, and the decrease remained on 18 d and 21 d in lettuce. Moreover, during the bioaccumulation period, activities of glutathione reductase (GR) and glutathione S-transferase (GST) were enhanced in both plants. Our results suggested that GSH biosynthesis played an important role in MC-LR depuration in the tested plants. Concerning human health risk, most of the estimated daily intake (EDI) values during the bioaccumulation period exceeded the tolerable daily intake (TDI) guideline. However, the risk could be alleviated by irrigating with MCs-free water for a certain amount of time before harvest.

The xanthophyll cycle affects reversible interactions between PsbS and light-harvesting complex II to control non-photochemical quenching.

To maintain high photosynthetic rates, plants must adapt to their light environment on a timescale of seconds to minutes. Therefore, the light-harvesting antenna system of photosystem II in thylakoid membranes, light-harvesting complex II (LHCII), has a feedback mechanism, which determines the proportion of absorbed energy dissipated as heat: non-photochemical chlorophyll fluorescence quenching (NPQ). This is crucial to prevent photo-oxidative damage to photosystem II (PSII) and is controlled by the transmembrane pH differences (ΔpH). High ΔpH activates NPQ by protonation of the protein PsbS and the enzymatic de-epoxidation of LHCII-bound violaxanthin to zeaxanthin. But the precise role of PsbS and its interactions with different LHCII complexes remain uncertain. We have investigated PsbS-LHCII interactions in native thylakoid membranes using magnetic-bead-linked antibody pull-downs. The interaction of PsbS with the antenna system is affected by both ΔpH and the level of zeaxanthin. In the presence of ΔpH alone, PsbS is found to be mainly associated with the trimeric LHCII protein polypeptides, Lhcb1, Lhcb2 and Lhcb3. However, a combination of ΔpH and zeaxanthin increases the proportion of PsbS bound to the minor LHCII antenna complex proteins Lhcb4, Lhcb5 and Lhcb6. This pattern of interaction is not influenced by the presence of PSII reactions centres. Similar to LHCII particles in the photosynthetic membrane, PsbS protein forms clusters in the NPQ state. NPQ recovery in the dark requires uncoupling of PsbS. We suggest that PsbS acts as a 'seeding' centre for the LHCII antenna rearrangement that is involved in NPQ.

Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics.

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors-single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal-embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm-1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

Effects of high voltage nanosecond pulsed plasma and micro DBD plasma on seed germination, growth development and physiological activities in spinach.

In this study, we analyzed seed germination, seedling growth, and physiological aspects after treatment with high voltage nanosecond pulsed plasma and micro DBD plasma in spinach (Spinacia oleracea L.), a green leafy vegetable known to have low germination rate. Both germination and dry weight of seedlings increased after high voltage pulse shots were applied to spinach seeds. However seeds treated with many shots (10 shots) showed a decrease in germination rate and seedling growth. Seeds treated with air DBD plasma exhibited slightly higher germination and subsequent seedling growth than those treated with N2 plasma. Seed surface was degenerated after treated with high voltage pulsed plasma and micro DBD plasma but no significant difference in the degree of degeneration was observed among micro DBD plasma treatment time. Level of GA3 hormone and mRNA expression of an amylolytic enzyme-related gene in seeds were elevated 1 day after treatment with high voltage pulsed plasma. The relative amount of chlorophyll and total polyphenols in spinach seedlings grown from seeds treated with air DBD plasma was increased in 30 s, 1 min, and 3 min treatments. Taken together, our results suggest a possibility that plasma can enhance seed germination by triggering biochemical processes in seeds.

The effect of medium viscosity on kinetics of ATP hydrolysis by the chloroplast coupling factor CF1.

The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca(2+)- and Mg(2+)-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca(2+)-dependent ATPase without changing the apparent Michaelis constant (К m), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg(2+)-dependent reaction, the presence of sucrose increased К m without changing the maximal rate that is many-fold as high as that of Ca(2+)-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg(2+)-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water.

Molecular cloning and characterization of pathogenesis-related protein family 10 gene from spinach (SoPR10).

PR10 genes encode small, intracellular proteins that respond to biotic and abiotic stresses. In this study, a cDNA clone (designated as SoPR10, GenBank Accession No. KC142174) encoding a PR10 protein from spinach (Spinacia oleracea L.) was isolated and characterized. SoPR10 encoded a 161-amino acid polypeptide with a predicted molecular mass of 19.76 kDa and a pI of 4.61. Real-time quantitative analysis indicated that SoPR10 was constitutively expressed in root and shoot. The abundance of SoPR10 in salt-resistant cultivar (Chaoji) was generally greater than in salt-sensitive cultivar (Daye) under 160 mM L(-1) NO3(-) treatment for 0.5, 3, and 6 h. The expression of SoPR10 was also induced by other abiotic stresses including polyethylene glycol, NaCl, salicylic acid, and H2O2. Our results indicated that SoPR10 might play important roles under nitrate stress and other abiotic stresses.

Differential sensitivity of spinach and amaranthus to enhanced UV-B at varying soil nutrient levels: association with gas exchange, UV-B-absorbing compounds and membrane damage.

The metabolic reasons associated with differential sensitivity of C3 and C4 plant species to enhanced UV-B under varying soil nutrient levels are not well understood. In the present study, spinach (Spinacia oleracea L. var All Green), a C3 and amaranthus (Amaranthus tricolor L. var Pusa Badi Chaulai), a C4 plant were subjected to enhanced UV-B (280-315 nm; 7.2 kJ m(-2) day(-1)) over ambient under varying soil nutrient levels. The nutrient amendments were recommended Nitrogen (N), Phosphorus (P), Potassium (K), 1.5× recommended NPK, 1.5× recommended N and 1.5× recommended K. Enhanced UV-B negatively affected both the species at all nutrient levels, but the reductions varied with nutrient concentration and combinations. Reductions in photosynthetic rate, stomatal conductance and chlorophyll content were significantly more in spinach compared with amaranthus. The reduction in photosynthetic rate was maximum at 1.5× recommended K and minimum in 1.5× NPK amended plants. The oxidative damage to membranes measured in terms of malondialdehyde content was significantly higher in spinach compared with amaranthus. Enhanced UV-B reduced SOD activity in both the plants except in amaranthus at 1.5× recommended K. POX activity increased under enhanced UV-B at all nutrient levels in amaranthus, but only at 1.5× K in spinach. Amaranthus had significantly higher UV-B-absorbing compounds than spinach even under UV-B stress. Lowest reductions in yield and total biomass under enhanced UV-B compared with ambient were observed in amaranthus grown at 1.5× recommended NPK. Enhanced UV-B did not significantly change the nitrogen use efficiency in amaranthus at all NPK levels, but reduced in spinach except at 1.5× K. These findings suggest that the differential sensitivity of the test species under enhanced UV-B at varying nutrient levels is due to varying antioxidative and UV-B screening capacity, and their ability to utilize nutrients. Amaranthus tolerated enhanced UV-B stress more than spinach at all nutrient levels and 1.5× recommended NPK lowered the sensitivity maximally to enhanced UV-B with respect to photosynthesis, biomass and yield. PCA score has also confirmed the lower sensitivity of amaranthus compared with spinach with respect to the measured physiological and biochemical parameters.

Inhibition of CO2 fixation by iodoacetamide stimulates cyclic electron flow and non-photochemical quenching upon far-red illumination.

The Benson-Calvin cycle enzymes are activated in vivo when disulfide bonds are opened by reduction via the ferredoxin-thioredoxin system in chloroplasts. Iodoacetamide reacts irreversibly with free -SH groups of cysteine residues and inhibits the enzymes responsible for CO2 fixation. Here, we investigate the effect of iodoacetamide on electron transport, when infiltrated into spinach leaves. Using fluorescence and absorption spectroscopy, we show that (i) iodoacetamide very efficiently blocks linear electron flow upon illumination of both photosystems (decrease in the photochemical yield of photosystem II) and (ii) iodoacetamide favors cyclic electron flow upon light excitation specific to PSI. These effects account for an NPQ formation even faster in iodoacetamide under far-red illumination than in the control under saturating light. Such an increase in NPQ is dependent upon the proton gradient across the thylakoid membrane (uncoupled by nigericin addition) and PGR5 (absent in Arabidopsis pgr5 mutant). Iodoacetamide very tightly insulates the electron current at the level of the thylakoid membrane from any electron leaks toward carbon metabolism, therefore, providing choice conditions for the study of cyclic electron flow around PSI.

Evidence that putrescine modulates the higher plant photosynthetic proton circuit.

The light reactions of photosynthesis store energy in the form of an electrochemical gradient of protons, or proton motive force (pmf), comprised of electrical (Δψ) and osmotic (ΔpH) components. Both components can drive the synthesis of ATP at the chloroplast ATP synthase, but the ΔpH component also plays a key role in regulating photosynthesis, down-regulating the efficiency of light capture by photosynthetic antennae via the q(E) mechanism, and governing electron transfer at the cytochrome b(6)f complex. Differential partitioning of pmf into ΔpH and Δψ has been observed under environmental stresses and proposed as a mechanism for fine-tuning photosynthetic regulation, but the mechanism of this tuning is unknown. We show here that putrescine can alter the partitioning of pmf both in vivo (in Arabidopsis mutant lines and in Nicotiana wild type) and in vitro, suggesting that the endogenous titer of weak bases such as putrescine represents an unrecognized mechanism for regulating photosynthetic responses to the environment.

N-formylkynurenine as a marker of high light stress in photosynthesis.

Photosystem II (PSII) is the membrane protein complex that catalyzes the photo-induced oxidation of water at a manganese-calcium active site. Light-dependent damage and repair occur in PSII under conditions of high light stress. The core reaction center complex is composed of the D1, D2, CP43, and CP47 intrinsic polypeptides. In this study, a new chromophore formed from the oxidative post-translational modification of tryptophan is identified in the CP43 subunit. Tandem mass spectrometry peptide sequencing is consistent with the oxidation of the CP43 tryptophan side chain, Trp-365, to produce N-formylkynurenine (NFK). Characterization with ultraviolet visible absorption and ultraviolet resonance Raman spectroscopy supports this assignment. An optical assay suggests that the yield of NFK increases 2-fold (2.2 ± 0.5) under high light illumination. A concomitant 2.4 ± 0.5-fold decrease is observed in the steady-state rate of oxygen evolution under the high light conditions. NFK is the product formed from reaction of tryptophan with singlet oxygen, which can be produced under high light stress in PSII. Reactive oxygen species reactions lead to oxidative damage of the reaction center, D1 protein turnover, and inhibition of electron transfer. Our results are consistent with a role for the CP43 NFK modification in photoinhibition.

Dynamics of the antioxidant system during seed osmopriming, post-priming germination, and seedling establishment in Spinach (Spinacia oleracea).

Osmopriming is a pre-sowing treatment that improves seed germination performance and stress tolerance. To understand osmopriming physiology, and its association with post-priming stress tolerance, we investigated the antioxidant system dynamics during three stages: during osmopriming, post-priming germination, and seedling establishment. Spinach seeds (Spinacia oleracea L. cv. Bloomsdale) were primed with -0.6 MPa PEG at 15°C for 8 d, and dried at room temperature for 2 d. Unprimed and primed germinating seeds/seedlings were subjected to a chilling and desiccation stresses. Seed/seedling samples were collected for antioxidant assays and germination performance and stress tolerance were evaluated. Our data indicate that: (1) during osmopriming the transition of seeds from dry to germinating state represses the antioxidant pathways (residing in dry seeds) that involve CAT and SOD enzymes but stimulates another pathway (only detectable in imbibed seeds) involving APX; (2) a renewal of antioxidant system, possibly required by seedling establishment, occurs after roughly 5 d of germination; (3) osmopriming strengthens the antioxidant system and increases seed germination potential, resulting in an increased stress tolerance in germinating seeds. Osmopriming-mediated promotive effect on stress tolerance, however, may diminish in relatively older (e.g. ~5-week) seedlings.

Evaluation of the toxicity of stress-related aldehydes to photosynthesis in chloroplasts.

Aldehydes produced under various environmental stresses can cause cellular injury in plants, but their toxicology in photosynthesis has been scarcely investigated. We here evaluated their effects on photosynthetic reactions in chloroplasts isolated from Spinacia oleracea L. leaves. Aldehydes that are known to stem from lipid peroxides inactivated the CO(2) photoreduction to various extents, while their corresponding alcohols and carboxylic acids did not affect photosynthesis. alpha,beta-Unsaturated aldehydes (2-alkenals) showed greater inactivation than the saturated aliphatic aldehydes. The oxygenated short aldehydes malondialdehyde, methylglyoxal, glycolaldehyde and glyceraldehyde showed only weak toxicity to photosynthesis. Among tested 2-alkenals, 2-propenal (acrolein) was the most toxic, and then followed 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal. While the CO(2)-photoreduction was inactivated, envelope intactness and photosynthetic electron transport activity (H(2)O --> ferredoxin) were only slightly affected. In the acrolein-treated chloroplasts, the Calvin cycle enzymes phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphophatase, sedoheptulose-1,7-bisphosphatase, aldolase, and Rubisco were irreversibly inactivated. Acrolein treatment caused a rapid drop of the glutathione pool, prior to the inactivation of photosynthesis. GSH exogenously added to chloroplasts suppressed the acrolein-induced inactivation of photosynthesis, but ascorbic acid did not show such a protective effect. Thus, lipid peroxide-derived 2-alkenals can inhibit photosynthesis by depleting GSH in chloroplasts and then inactivating multiple enzymes in the Calvin cycle.

[Evolutonary modifications of molecular structure of ATP-synthase gamma-subunit].

The ATP-synthase gamma-subunit (FoF1) belongs to the rotor part of this oligomeric complex. Catalytic hydrolysis of adenosine triphosphate (ATP) is accompanied by rotation of gamma-polypeptide inside the sphere formed by six subunits (alphabeta)3 of the enzyme. The gamma-subunit regulates ATPase and ATP-synthase activities of the FoF1. In the present work, evolutionary and reverse changes of this regulatory polypeptide and their effect on properties of the enzyme are studied. It is suggested that elongation of the gamma-subunit globular part had resulted from the atpC intragene duplication in the process of adaptive evolution. The evolved fragment participates in light regulation of the chloroplast ATP-synthase.

The decay of the ATPase activity of light plus thiol-activated thylakoid membranes in the dark.

Oxidized ATP synthase of spinach thylakoid membranes catalyzes high rates of ATP synthesis in the light, but very low rates of ATP hydrolysis in the dark. Reduction of the disulfide bond in the gamma subunit of the ATP synthase in the light enhances the rate of Mg2+-ATP hydrolysis in the dark. The light plus thiol-activated state decays in a few minutes in the dark after illumination in Tris buffer, but not when Tricine was used in place of Tris. In this paper, it is shown that Tris in the assay mixture is an inhibitor of the light plus thiol-activated ATPase activity of thylakoids, but only after the activated membranes had incubated in the dark. Aminopropanediols and diethanolamine, also selectively inhibited ATPase activity of activated membranes after storage in the dark, whereas NH4Cl and imidazole inhibit the ATPase activity of activated thylakoids almost equally whether they are added directly after the illumination or several minutes later. The fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA) is quenched by the establishment of proton gradients by ATP-dependent proton uptake. Addition of ATP to activated membranes results in rapid quenching of ACMA fluorescence. If the activated membranes were incubated in the dark prior to ATP addition, a lag in the ATP-dependent ACMA fluorescence quenching as well as a similar lag in the rate ATP hydrolysis were seen. It is concluded that ADP rebinds to CF1 in the dark following illumination and inhibits the activity of the ATP synthase. Reactivation of the ATP synthase in the dark can occur by the slow generation of proton gradients by ATP hydrolysis in the dark. This reactivation takes place in Tricine buffer, but not in Tris because of its uncoupling action. Whether ADP binding plays a role in the regulation of the activity of the ATP synthase in situ remains to be established.

Regulation of the anion channel of the chloroplast envelope from spinach.

Several anions such as Cl-, NO2(-), SO4(2-), and PO4(3-) are known to modulate the photosynthetic activity. Moreover, the chloroplast metabolism requires the exchange of both inorganic and organic (e.g., triose phosphate, dicarboxylic acid, ATP) anions between the cytoplasm and the stroma. A chloride channel form the chloroplast envelope was reconstituted in planar lipid bilayers. We show that the channel is active in conditions prevailing in the plant. The open probability increases with the ionic strength of the experimental solutions and is maximal at 0 mV. This suggests that the channel could play a role in the osmotic regulation of the chloroplast. Amino group reagents affect the channel activity in a way that demonstrated that lysine residues are important for channel gating but not for ATP binding. Together, our results provide new information on the functioning of this channel in the chloroplast envelope membranes. They indicate that the open probability of the channel is low (Po < or = 0.2) in vivo and that this channel can account for the chloride flux through the chloroplast envelope.

Short-term and long-term effects of low total pressure on gas exchange rates of spinach.

In this study, spinach plants were grown under atmospheric and low pressure conditions with constant O2 and CO2 partial pressures, and the effects of low total pressure on gas exchange rates were investigated. CO2 assimilation and transpiration rates of spinach grown under atmospheric pressure increased after short-term exposure to low total pressure due to the enhancement of leaf conductance. However, gas exchange rates of plants grown at 25 kPa total pressure were not greater than those grown at atmospheric pressure. Stomatal pore length and width were significantly smaller in leaves grown at low total pressure. This result suggested that gas exchange rates of plants grown under low total pressure were not stimulated even with the enhancement of gas diffusion because the stomatal size and stomatal aperture decreased.

Polyphenolic allelochemicals from the aquatic angiosperm Myriophyllum spicatum inhibit photosystem II.

Myriophyllum spicatum (Haloragaceae) is a highly competitive freshwater macrophyte that produces and releases algicidal and cyanobactericidal polyphenols. Among them, beta-1,2,3-tri-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-D-glucose (tellimagrandin II) is the major active substance and is an effective inhibitor of microalgal exoenzymes. However, this mode of action does not fully explain the strong allelopathic activity observed in bioassays. Lipophilic extracts of M. spicatum inhibit photosynthetic oxygen evolution of intact cyanobacteria and other photoautotrophs. Fractionation of the extract provided evidence for tellimagrandin II as the active compound. Separate measurements of photosystem I and II activity with spinach (Spinacia oleracea) thylakoid membranes indicated that the site of inhibition is located at photosystem II (PSII). In thermoluminescence measurements with thylakoid membranes and PSII-enriched membrane fragments M. spicatum extracts shifted the maximum temperature of the B-band (S(2)Q(B)(-) recombination) to higher temperatures. Purified tellimagrandin II in concentrations as low as 3 microM caused a comparable shift of the B-band. This demonstrates that the target site of this inhibitor is different from the Q(B)-binding site, a common target of commercial herbicides like 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Measurements with electron paramagnetic resonance spectroscopy suggest a higher redox midpoint potential for the non-heme iron, located between the primary and the secondary quinone electron acceptors, Q(A) and Q(B). Thus, tellimagrandin II has at least two modes of action, inhibition of exoenzymes and inhibition of PSII. Multiple target sites are a common characteristic of many potent allelochemicals.