Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of <Emphasis Type="Italic">Brassica napus</Emphasis> leaf protoplasts

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H₂O₂ and enzymes involved in H₂O₂ generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H₂O₂ and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H₂O₂ was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H₂O₂ in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H₂O₂ generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.     

Biochemical and molecular analysis of a temperature-sensitive albino mutant in kale named “White Dove”

“White Dove” is a mutant in kale (Brassica oleracea var. acephala f. tricolor), which exhibits a mutant albino phenotype in the interior of the plant under low temperature conditions. Chlorophyll content in “White Dove” was dramatically reduced under low temperature conditions, while the content in “Green Dove” decreased slightly under the same conditions. The levels of five chlorophyll precursors suggested that chlorophyll biosynthesis in white kale was inhibited by low temperature stress at the step of Pchlide. However, Mg-Proto IX was not inhibited in white kale grown under low temperature conditions. The results of quantitative RT-PCR illustrated that the chlorophyll biosynthetic genes in the white cultivar were dramatically down-regulated by low temperature stress from the step of POR, while CISC and DBB1B in the white cultivar were dramatically induced under low temperature conditions. The results of transmission electron microscopy analysis showed that there were normal chloroplasts in the young leaves of white kale grown at 20 °C, whereas proplastids were observed in white kale grown at 5 °C. These results strongly suggested that low-temperature stress significantly inhibited plastid development in the young leaves of white kale, and repressed chlorophyll biosynthesis at the step of Pchlide by down-regulating the expression of downstream chlorophyll biosynthetic genes, resulting in undifferentiated proplastids and the albino phenotype observed in young leaves. Several genes associated with chlorophyll accumulation were also affected by low temperature conditions in white kale, especially CISC and DBB1B.     

Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus

To establish a cytoplasmic male-sterile/restored fertility (cms-Rf) system for F₁ seed production in Brassica napus, we transferred a gene from fertillity restored radish to B. napus by protoplast fusion. X-irradiated protoplasts, isolated from shoots of Raphanus sativus cv Kosena (Rf line), were fused with iodoacetamide-treated protoplasts of a B. napus cms cybrid. Among 300 regenerated plants, six were male-fertile. The fertile plants were characterized for petal color, chromosome number and the percentage of viable pollen grains. Three fertile plants had aneuploid chromosome numbers and white or cream petals, which is a dominant marker in radish. Of these three plants, one which had 2n = 47 chromosomes and white petals was used for further backcrosses. After two backcrosses, chromosome number and petal color became identical to that of B. napus. No female sterility was observed in the BC₃ generations.     

Nontranslation of foreign genetic information for fraction 1 protein under circumstances favorable for direct transfer ofNicotiana gossei isolated chloroplasts intoN. Tabacum protoplasts

Summary The nuclei and cytoplasm ofN. gossei andN. tabacum are compatible to the extent that reciprocal, interspecific F₁ hybrids can be produced by conventional breeding techniques. Conditions were established in which manyN. gossei isolated chloroplasts could be seen by phase and fluorescence microscopy to adhere to 40% of the population of protoplasts obtained from white tissue of variegatedN. tabacum plants and to remain attached after washing the protoplasts. Chloroplasts also could be seen to enter the interior of the protoplasts. After treating albino protoplasts withN. gossei chloroplasts, the protoplasts were subjected to further conditions whereby 65 calluses containing shoots developed. TwentyN. tabacum protoplasts not treated with foreign chloroplasts also produced calluses with shoots to serve as a control. All calluses developed chlorophyll irrespective of whether or not the albino protoplasts had been treated with isolatedN. gossei chloroplasts. The Fraction 1 protein ofN. tabacum has a different electrophoretic mobility from the protein ofN. gossei or anN. gossei xN. tabacum F₁ hybrid. The Fraction 1 protein large subunit is coded by chloroplast DNA, whereas the small subunit is coded by nuclear DNA. Fraction 1 protein was isolated from the variegated shoots of the 65 calluses obtained after treating albino protoplasts with foreign chloroplasts. Immunoelectrophoresis demonstrated the protein from each callus to have a mobility identical toN. tabacum protein. Therefore, under circumstances highly favorable for the direct transfer ofN. gossei isolated chloroplasts (and possibly nuclei also) intoN. tabacum protoplasts, no evidence was obtained to suggest that genetic information contained in the isolated foreign organelles was being translated into the polypeptides of either the large or small subunits of Fraction 1 protein contained in newly differentiated leaves derived from the protoplasts. Supported by Research Grant PCM-75-07368 from the National Science Foundation.     

Osmotic water permeability of isolated protoplasts. Modifications during development

A transference chamber was developed to measure the osmotic water permeability coefficient (P_os) in protoplasts 40 to 120 μm in diameter. The protoplast was held by a micropipette and submitted to a steep osmotic gradient created in the transference chamber. P_os was derived from the changes in protoplast dimensions, as measured using a light microscope. Permeabilities were in the range 1 to 1000 μm s⁻¹ for the various types of protoplasts tested. The precision for P_os was ≤40%, and within this limit, no asymmetry in the water fluxes was observed. Measurements on protoplasts isolated from 2- to 5-d-old roots revealed a dramatic increase in P_os during root development. A shift in P_os from 10 to 500 μm s⁻¹ occurred within less than 48 h. This phenomenon was found in maize (Zea mays), wheat (Triticum aestivum), and rape (Brassica napus) roots. These results show that early developmental processes modify water-transport properties of the plasma membrane, and that the transference chamber is adapted to the study of water-transport mechanisms in native membranes.     

Compartmentation and export of 14CO2 fixation products in mesophyll protoplasts from the C4-plant Digitaria sanguinalis

Isolated mesophyll protoplasts, and protoplast extracts containing intact chloroplasts, from the C₄ species Digitaria sanguinalis have been used to study Compartmentation and export of C₄ acids, using different C₃ precursors as substrate for ¹⁴CO₂ fixation. Mg²⁺ was necessary for maximum ¹⁴CO₂ fixation rates with both protoplasts and protoplast extracts, whereas Mg²⁺ was inhibitory for oxaloacetate and phosphoglycerate reduction. This inhibition could be overcome by preincubating the materials in the light with excess of EDTA before addition of Mg²⁺. Under these conditions pyruvate as substrate for ¹⁴CO₂ fixation induced mainly malate formation, whereas phosphoglycerate as substrate induced oxaloacetate formation, indicating competition for available NADPH between oxaloacetate and phosphoglycerate reduction. Oxaloacetate could be exported from the protoplasts at rates comparable to the rates of ¹⁴CO₂ fixation in intact leaves (200 μmol/mg Chl × h). This product probably passed the plasma membrane by simple diffusion, whereas the export of malate and aspartate seemed to be regulated, with the size of the intraprotoplast pool being relatively independent of the export rate. It is concluded that transport via the plasma membrane-cell wall path may play a role in metabolite flow during photosynthesis in C₄ plants.     

Formation kinetics and H2O2 distribution in chloroplasts and protoplasts of photosynthetic leaf cells of higher plants under illumination

The dye H₂DCF-DA, which forms the fluorescent molecule DCF in the reaction with hydrogen peroxide, H₂O₂, was used to study light-induced H₂O₂ production in isolated intact chloroplasts and in protoplasts of mesophyll cells of Arabidopsis, pea, and maize. A technique to follow the kinetics of light-induced H₂O₂ production in the photosynthesizing cells using this dye has been developed. Distribution of DCF fluorescence in these cells in the light has been investigated. It was found that for the first minutes of illumination the intensity of DCF fluorescence increases linearly after a small lag both in isolated chloroplasts and in chloroplasts inside protoplast. In protoplasts of Arabidopsis mutant vtc2-2 with disturbed biosynthesis of ascorbate, the rate of increase in DCF fluorescence intensity in chloroplasts was considerably higher than in protoplasts of the wild type plant. Illumination of protoplasts also led to an increase in DCF fluorescence intensity in mitochondria. Intensity of DCF fluorescence in chloroplasts increased much more rapidly than in cytoplasm. The cessation of cytoplasmic movement under illumination lowered the rate of DCF fluorescence intensity increase in chloroplasts and sharply accelerated it in the cytoplasm. It was revealed that in response to switching off the light, the intensity of fluorescence of both DCF and fluorescent dye FDA increases in the cytoplasm in the vicinity of chloroplasts, while it decreases in the chloroplasts; the opposite changes occur in response to switching on the light again. It was established that these phenomena are connected with proton transport from chloroplasts in the light. In the presence of nigericin, which prevents the establishment of transmembrane proton gradients, the level of DCF fluorescence in cytoplasm was higher and increased more rapidly than in the chloroplasts from the very beginning of illumination. These results imply the presence of H₂O₂ export from chloroplasts to cytoplasm in photosynthesizing cells in the light; the increase in this export falls in the same time interval as does the cessation of cytoplasmic movement.     

Improvement of element uptake and antioxidative defense in Brassica napus under lead stress by application of hydrogen sulfide

Heavy metal pollution is one of the major constraints in oilseed rape (Brassica napus L.) production. In this study, protective role of hydrogen sulfide (H₂S) on plant growth under lead (Pb) stress was studied in B. napus. Plants were grown hydroponically in greenhouse conditions under three levels (0, 100, and 400 μM) of Pb and three levels (0, 100 and 200 μM) of H₂S donor sodium hydrosulfide. Outcomes demonstrated that Pb stress significantly reduced the plant biomass, leaf chlorophyll contents, nutrients uptake in the leaves and roots of B. napus plants. Exogenous application of H₂S significantly improved the plant biomass, chlorophyll contents and concentration of macro- and micronutrients in the leaves and roots of B. napus plants under Pb-toxicity conditions. The data indicated that application of Pb alone significantly increased the reactive oxygen species (ROS) as well as malondialdehyde (MDA) in the leaves and roots of plants. Meanwhile, application of H₂S decreased the production of MDA and ROS in the leaves and roots by increasing antioxidant activities under Pb stress. Moreover, this study also revealed that plants treated with H₂S at different concentrations enhanced the contents of total glutathione and glutathione reduced/glutathione oxidized ratio in leaves and roots under different levels of Pb. The results depicted that H₂S improved the plant biomass, uptake of nutrients in the leaves and roots of B. napus plants and enhanced the performance of antioxidant defense system due to its ameliorative potential under Pb stress conditions.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Subcellular localization of spermidine synthase in the protoplasts of chinese cabbage leaves

Previous studies on the presence of spermidine synthase (EC 2.5.1.16) in the protoplasts of Chinese cabbage (Brassica pekinensis var Pak Choy) leaves had detected a small but significant fraction of the enzyme in a crude chloroplast fraction (Cohen, Balint, Sindhu 1981 Plant Physiol 68: 1150-1155). To establish whether this enzyme is truly a chloroplast component, we have isolated purified intact chloroplasts from protoplasts by density gradient centrifugation in silica sols (Ludox AM). Such chloroplasts contained all of the diaminopimelate decarboxylase (EC 4.1.1.20) of the protoplasts, but were essentially devoid of spermidine synthase. Control experiments showed that the latter had not been inactivated under conditions of isolation, purification, and assay of the intact chloroplasts. Isolation and assay of protoplast vacuoles in a further examination of the supernatant fluid containing the enzyme revealed a significant fraction of the enzyme in the vacuole fraction. However this fraction was found to contain similar proportions of a soluble enzyme, glucose 6-phosphate dehydrogenase. It has been concluded that vacuolar fractions are difficultly separable from soluble cytoplasmic material, which is probably the only compartment containing spermidine synthase.     

Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O₂ evolution of about 100 to 150 micromoles of O₂ per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO₃ with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O₂ per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.

Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.

Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO₃ and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

     

Analysis of plants regenerated from protoplast fusions between Brassica napus and Eruca sativa

Summary Protoplasts from etiolated hypocotyls of Brassica napus stained with carboxyfluorescein were fused with mesophyll protoplasts from Eruca sativa. Hybrid cells could be identified under the light microscope by (1) fully developed chloroplasts derived from E. sativa and (2) the cytoplasmic strands of the B. napus hypocotyl protoplasts, or (3) by the presence of both red and green fluorescence when investigated under UV light. The heterokaryons were selected using either a micro-manipulator or a flow sorter. On average, 5.4% of the calli obtained after selection differentiated into shoots. Regenerated shoots were subjected to isozyme analysis for verification of their hybrid character. Of the 23 hybrids successfully transferred to the greenhouse, 11 were asymmetric according to isozyme analysis. The nuclear DNA content of the hybrids was determined by flow cytometry, which gives an estimate of chromosome number. Most of the hybrids had a DNA content, and thus a chromosome number, that deviated from the expected sum of the parents. Almost all of the hybrids had some degree of fertility and produced seeds. Seed set, expressed as seeds per pollinated flower, was on average 7% of that of B. napus in the case of self-pollination and 26% of that of B. napus when backcrossed to B. napus. The chloroplast genotype was investigated in 13 hybrids. Of these, 11 had chloroplasts derived from B. napus, while only 2 had chloroplasts of E. sativa origin.     

Transfer and segregation of triazine tolerant chloroplasts in Brassica napus L.

Summary Hypocotyl protoplasts of 45 different genotypes of German winter oilseed rape Brassica napus L. (double zero quality: high in yield, seeds low in erucic acid and glucosinolate content) were regenerated to plants. Triazine/triazinone (tri)-tolerant chloroplasts of the Canadian spring oilseed rape variety OAC Triton were introduced into some winter oilseed rapes by means of protoplast fusion. X-ray irradiation was used to limit the transfer of nuclear DNA of Triton protoplasts and to promote the selective transfer of tri-tolerant chloroplasts. Regenerated cybrid plants survived a treatment rate of 1000 g/ha metribuzin. The presence and segregation of the tri-tolerant chloroplasts in winter oilseed rape plants, regenerated from fusion products and their progeny, was investigated by restriction fragment length polymorphism (RFLP). Our results indicate that chloroplast segregation was not completed in plants regnerated from fusion products derived from X-irradiated OAC Triton mesophyll protoplasts and German winter oilseed rape hypocotyl protoplasts. In regenerants and their progeny both chloroplast types can still be present. Chloroplasts derived from wintertype protoplasts can outcompete tritolerant chloroplasts during plant development. In some instances, even progeny plants not kept under selective conditions (metribuzin) lost tri-tolerant chloroplasts. A homogenous population of tri-tolerant chloroplasts was necessary to obtain stable tri-tolerant winter oilseed rape plants.     

Endogenous nitric oxide generation in protoplast chloroplasts

Key message

NO generation is studied in the protoplast chloroplasts. NO, ONOO ^? and ROS (O ₂ ^? and H ₂ O ₂ ) are generated in chloroplasts. Nitric oxide synthase-like protein appears to be involved in NO generation.

Abstract

Nitric oxide stimulates chlorophyll biosynthesis and chloroplast differentiation. The present study was conducted to better understand the process of NO generation in the leaf chloroplasts and protoplasts. NO, peroxynitrite and superoxide anion were investigated in the protoplasts and isolated chloroplasts using specific dyes, confocal laser scanning and light microscopy. The level of NO was highest after protoplast isolation and subsequently decreased during culture. Suppression of NO signal in the presence of PTIO, suggests that diaminofluorescein-2 diacetate (DAF-2DA) detected NO. Detection of peroxynitrite, a reaction product of NO and superoxide anion, further suggests NO generation. Moreover, generation of NO and peroxynitrite in the chloroplasts of wild-type Arabidopsis and their absence or weak signals in the leaf-derived protoplasts of Atnoa1 mutants confirmed the reactivity of DAF-2DA and aminophenyl fluorescein to NO and peroxynitrite, respectively. Isolated chloroplasts also showed signal of NO. Suppression of NO signal in the presence of 100 μM nitric oxide synthase inhibitors [l-NNA, Nω-nitro-l-arginine and PBIT, S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea] revealed that nitric oxide synthase-like system is involved in NO synthesis. Suppression of NO signal in the protoplasts isolated in the presence of cycloheximide suggests de novo synthesis of NO generating protein during the process of protoplast isolation. Furthermore, the lack of inhibition of NO production by sodium tungstate (250 μM) and inhibition by l-NNA, and PBIT suggest involvement NOS-like protein, but not nitrate reductase, in NO generation in the leaf chloroplasts and protoplasts.     

Flow cytometric analysis of tobacco and cowpea protoplasts infected in vivo and in vitro with alfalfa mosaic virus

Immunofluorescence flow cytometry was used to study the distribution of viral antigen in protoplast populations. Protoplasts were isolated from healthy and alfalfa mosaic virus (AMV) infected tobacco leaves (designated in vivo infected). Furthermore isolated tobacco and cowpea protoplasts were infected in vitro with AMV. The FITC-conjugated antibodies could penetrate formaldehyde fixed protoplasts. The flow cytometric measurements were rapid and reproducible. Comparable immunofluorescence patterns were found for all infected samples (per sample 10⁴ protoplasts were measured). Infectious virus could only be detected in in vivo infected tobacco protoplasts and in in vitro infected cowpea protoplasts.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Modulation of photosynthesis and respiration in guard and mesophyll cell protoplasts by oxygen concentration

Stomatal movement is an energetic oxygen-requiring process. In the present study, the effect of oxygen concentration on mitochondrial respiratory activity and red-light-dependent photosynthetic oxygen evolution by Vicia faba and Brassica napus guard cell protoplasts was examined. Comparative measurements were made with mesophyll cell protoplasts isolated from the same species. At air saturated levels of dissolved oxygen in the protoplast suspension media, respiration rates by mesophyll protoplasts ranged from 6 to 10μmoles O₂ mg^?1 chl h^?1, while guard cell protoplasts respired at rates of 200–300 μmoles O₂ mg chl^?1 h^?1, depending on the species. Lowering the oxygen concentration below 50–60 mmol m^?3 resulted in a decrease in guard cell respiration rates, while rates by mesophyll cell protoplasts were reduced only at much lower concentrations of dissolved oxygen. Rates of photosynthesis in mesophyll cell protoplasts isolated from both species showed only a minor reduction in activity at low oxygen concentrations. In contrast, photosynthesis by guard cell protoplasts isolated from V. faba and B. napus decreased concomitantly with respiration. Oligomycin, an inhibitor of oxidative phos-phorylation, reduced photosynthesis in mesophyll cell protoplasts by 27–46% and in guard cell protoplasts by 51–58%. The reduction in both guard cell photosynthesis and respiration following exposure to low oxygen concentrations suggest close metabolic coupling between the two activities, possibly mediated by the availability of substrate for respiration associated with photosynthetic electron transport activity and subsequent export of redox equivalents.     

Effects of Hydrogen Sulfide on Growth,Antioxidative Capacity,and Ultrastructural Changes in Oilseed Rape Seedlings Under Aluminum Toxicity

The present study evaluates the beneficial effects of the hydrogen sulfide (H₂S) donor, sodium hydrosulfide (0 and 0.3 mM), on the growth of oilseed rape (Brassica napus L. cv. ZS 758) seedlings under aluminum (Al) stress (0, 0.1, and 0.3 mM). Results showed that Al stress decreased the seedling growth by reducing the shoot and root length, biomass, and antioxidant enzymes, which could be illustrated by increased levels of malondialdehyde (MDA), production of hydrogen peroxide (H₂O₂), and accumulation of Al in the shoots. Pretreatment with H₂S reduced MDA and H₂O₂ levels in the leaves and roots of B. napus seedlings. Moreover, activities of antioxidant enzymes (APX, CAT, APX, SOD, POD, and GR) were elevated significantly with the application of H₂S under Al stress. The microscopic examination confirmed that higher levels of Al completely impaired leaf mesophyll and root tip cells. Chloroplasts were spongy shaped with dissolved thylakoid membranes and more starch grains. Root tip cells showed visible symptoms under Al toxicity such as deposition of Al in vacuoles and disruption of whole cell organelles. Under pretreatment with exogenous H₂S, cell structures were improved and presented a clean mesophyll cell and chloroplast possessing well-developed thylakoid membranes as well as fewer starch grains. A number of modifications could be observed in root tip cells, that is, mature mitochondria, long endoplasmic reticulum as well as golgi bodies, under the combined application of H₂S and Al. On the basis of our results, we can conclude that H₂S has a promotive effect which could improve plant survival under Al stress.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.