María José Quiles

Chlororespiration and tolerance to drought, heat and high illumination

Sun (Chrysanthemum morifolium) and shade (Spathiphyllum wallisii) plants were used to study the effects of drought, heat and high illumination. The stress conditions caused a greater accumulation of hydrogen peroxide in Chrysanthemum morifolium than in Spathiphyllum wallisii leaves. They also resulted in down-regulation of linear electron transport in the leaves of both species, as indicated by a gradual reduction in the photochemistry efficiency of PS II, which was associated with an increase in the non-photochemical quenching of fluorescence. Only a slight decrease in F(v)/F(m) was observed under stress conditions in either plant species, suggesting that the chloroplast is protected by mechanisms that dissipate excess excitation energy to prevent damage to the photosynthetic apparatus. In addition to the effects on photosynthetic activity, changes were also observed by immunoblot analysis in the plastidial NADH DH complex, PTOX and PGR5. The quantities of the PTOX and NDH-H subunit of the thylakoidal NADH DH complex, and the NADH DH activity in the thylakoid membranes were similar in control plants of both species and increased in stressed plants, particularly in Spathiphyllum wallisii. The level of PGR5 polypeptide was higher in Chrysanthemum morifolium than in Spathiphyllum wallisii control plants, while after stress, the quantity of PGR5 increased significantly in Chrysanthemum morifolium and remained constant in Spathiphyllum wallisii. These results indicate that the relative importance of chlororespiration and the cyclic electron pathways in the tolerance to drought, heat and high illumination differs in sun and shade plants, indicating different adaptive mechanisms to the environment. In the conditions studied, the PGR5-dependent cyclic pathway is more active in Chrysanthemum morifolium, a sun species, whereas in Spathiphyllum wallisii, a shade species, other ways involving the NADH DH complex and PTOX are stimulated in response to stress, which results in lower levels of ROS accumulation in the leaves.

Comparison of the thylakoidal NAD(P)H dehydrogenase complex and the mitochondrial complex I separated from barley leaves by blue-native PAGE

Plastids contain an NAD(P)H dehydrogenase (NAD(P)H DH) complex preferentially located in the stroma thylakoids, which is homologous to the mitochondrial complex I (EC 1.6.5.3). However, until now, the two complexes have not been simultaneously studied in the same plant species. In this work we initiate a comparative study between both complexes from Hordeum vulgare L. The mitochondrial complex I and the stroma thylakoid NAD(P)H DH complex from barley, when separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by NADH nitroblue tetrazolium staining, showed different sizes and electrophoretic mobilities. Complex I had an apparent molecular mass of 740 kDa and the thylakoidal NAD(P)H DH complex a mass of 690 kDa. The two complexes were identified by immunoblotting assays, since antibodies raised against the NdhA, NdhH and NdhK thylakoidal polypeptides recognized three polypeptides of the barley thylakoidal complex, and antibodies raised against the B12 and B15 bovine complex I subunits reacted with two polypeptides of the barley mitochondrial complex I. Additional immunoblotting revealed that the thylakoidal NAD(P)H DH complex contains the three polypeptides (24, 56, 76 kDa) from the NADH-oxidizing unit of the complex, similar in size and antigenicity to the mitochondrial subunits. Both complexes have at least one more homologous subunit (29 kDa) of the complex minimal structure, which was recognized by the anti-TYKY antibody.

Stimulation of chlororespiration by drought under heat and high illumination in Rosa meillandina.

Rosa meillandina plants were used to study the effects of water deficit on photosynthesis and chlororespiration. Plants showed high tolerance to heat and high illumination in controlled conditions that ensured that there was no water deficit. However, when heat and high illumination were accompanied by low watering photosynthetic linear electron transport was down regulated, as indicated by the reduced photochemistry efficiency of PS II, which was associated with an increase in the non-photochemical quenching of fluorescence. In addition to the effects on the photosynthetic activity, changes were also observed in the plastidial NDH complex, PTOX and PGR5. In plants exposed to heat and high illumination without water deficit, the activities and amounts of the chlororespiration enzymes, NDH complex and PTOX, remained similar to the control and only increased in response to drought, high light and heat stress, applied together. In contrast, both the PS I activity and the amount of PGR5 polypeptide were higher in plants exposed to heat and high illumination without water deficit than in those with water deficit. The results indicated that in the conditions studied, the contribution of chlororespiration to regulating photosynthetic electron flow is not relevant when there is no water deficit, and another pathway, such as cyclic electron flow involving PGR5 polypeptide, may be more important. However, when PS II activity is inhibited by drought, chlororespiration, together with other routes of electron input to the electron transfer chain, is probably essential.

Assessment of Photosynthesis Tolerance to Herbicides, Heat and High Illumination by Fluorescence Imaging

Fluorescence imaging represents a non-invasive tool for revealing and understanding spatial heterogeneity in leaf performance caused by external factors, such as abiotic stress. Sun (Rosa meillandina and Chrysanthemum morifo- lium) and shade (Spathiphyllum wallisii) plants were used to study their tolerance to heat and high illumination. Fluores- cence yield, effective PSII quantum yield and non-photochemical quenching were analysed in leaves attached to plants by fluorescence imaging. The control plants of all species showed homogeneous images of the fluorescence parameters throughout the leaf. The fluorescence yield (F) was 0.1 or less, the effective PSII quantum yield (Y(II)) around 0.75 and non-photochemical quenching (NPQ) less than 0.3. The two sun plants showed higher tolerance to stress conditions. Few variations were observed in F and Y(II) images after stress photoperiods and some leaf regions showed an increase in NPQ, indicating more thermal energy dissipation in these zones than in other leaf regions. The images of the fluorescence parameters were similar to those of control plants after one recovery photoperiod without stress conditions. Shade plant showed lower tolerance and irreversible damage was observed after the first photoperiod, particularly at the base of the leaf and in the areas adjacent to the ribs. The centre and top of the leaf were less damaged, and effective PSII quantum yield remained high because the leaf curved to reduce the incident radiation. Incubation with the herbicides DCMU and paraquat led to differences in the fluorescence parameter images. The effect of DCMU (0.1 mM) was visible after 30 min incubation, beginning at the ribs and adjacent areas of the leaf. The three species studied showed different degree of sensi- tivity to paraquat (0.2 mM), and the effective quantum yield in each species was affected at different incubation times.

The Effects of Cold Stress on Photosynthesis in Hibiscus Plants

The present work studies the effects of cold on photosynthesis, as well as the involvement in the chilling stress of chlororespiratory enzymes and ferredoxin-mediated cyclic electron flow, in illuminated plants of Hibiscus rosa-sinensis. Plants were sensitive to cold stress, as indicated by a reduction in the photochemistry efficiency of PSII and in the capacity for electron transport. However, the susceptibility of leaves to cold may be modified by root temperature. When the stem, but not roots, was chilled, the quantum yield of PSII and the relative electron transport rates were much lower than when the whole plant, root and stem, was chilled at 10°C. Additionally, when the whole plant was cooled, both the activity of electron donation by NADPH and ferredoxin to plastoquinone and the amount of PGR5 polypeptide, an essential component of the cyclic electron flow around PSI, increased, suggesting that in these conditions cyclic electron flow helps protect photosystems. However, when the stem, but not the root, was cooled cyclic electron flow did not increase and PSII was damaged as a result of insufficient dissipation of the excess light energy. In contrast, the chlororespiratory enzymes (NDH complex and PTOX) remained similar to control when the whole plant was cooled, but increased when only the stem was cooled, suggesting the involvement of chlororespiration in the response to chilling stress when other pathways, such as cyclic electron flow around PSI, are insufficient to protect PSII.

Water deficit and heat affect the tolerance to high illumination in hibiscus plants.

This work studies the effects of water deficit and heat, as well as the involvement of chlororespiration and the ferredoxin-mediated cyclic pathway, on the tolerance of photosynthesis to high light intensity in Hibiscus rosa-sinensis plants. Drought and heat resulted in the down–regulation of photosynthetic linear electron transport in the leaves, although only a slight decrease in variable fluorescence (Fv)/maximal fluorescence (Fm) was observed, indicating that the chloroplast was protected by mechanisms that dissipate excess excitation energy to prevent damage to the photosynthetic apparatus. The incubation of leaves from unstressed plants under high light intensity resulted in an increase of the activity of electron donation by nicotinamide adenine dinucleotide phosphate (NADPH) and ferredoxin to plastoquinone, but no increase was observed in plants exposed to water deficit, suggesting that cyclic electron transport was stimulated by high light only in control plants. In contrast, the activities of the chlororespiration enzymes (NADH dehydrogenase (NDH) complex and plastid terminal oxidase (PTOX)) increased after incubation under high light intensity in leaves of the water deficit plants, but not in control plants, suggesting that chlororespiration was stimulated in stressed plants. The results indicate that the relative importance of chlororespiration and the cyclic electron pathway in the tolerance of photosynthesis to high illumination differs under stress conditions. When plants were not subjected to stress, the contribution of chlororespiration to photosynthetic electron flow regulation was not relevant, and another pathway, such as the ferredoxin-mediated cyclic pathway, was more important. However, when plants were subjected to water deficit and heat, chlororespiration was probably essential.

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP+ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.

Fractionation of thylakoid membranes into grana and stroma thylakoids.

The chloroplasts contain an extensive system of internal membranes or thylakoids in which all the light-harvesting and energy-transducing processes of the photosynthesis are located. Thylakoids are differentiated into stacked membrane regions (or grana thylakoids) and nonstacked membranes (or stroma thylakoids), each with a specialized structure and function. Both kinds of thylakoids can be separated by detergent-based methods or mechanical fragmentation such as sonication. We describe the fractionation of thylakoid membranes into grana and stroma thylakoids by treatment with the detergent digitonin and successive ultracentrifugation of the resultant vesicles. After their separation, the thylakoid fractions retain electron transport and enzymatic activities and are characterized using various parameters. The stroma thylakoids have higher chlorophyll a/chlorophyll b and protein/total chlorophyll ratios, and greater photosystem I and NADH dehydrogenase activities than the grana thylakoids. In the conditions used and on a protein basis of total thylakoids, the yield of stroma thylakoids is 5%, which is considerable taking into account that the stroma thylakoids are a minor component of total thylakoids.

Localization of the chloroplast NAD(P)H dehydrogenase, complex in stroma thylakoids from barley

Abstract Plastids contain an NAD(P)H dehydrogenase (NAD(P)H DH) complex which is homologous to the mitochondrial complex I (EC 1.6.5.3). We describe the isolation and partial characterization of the chloroplast NAD(P)H DH complex of Hordeum vulgare L. When chloroplasts were fractionated the stromal thylakoids had a specific NADH-ferricyanide oxidoreductase (NADH-FeCNR) activity seven times higher than that of granal thylakoids and contained abundant polypeptide which reacted with the antibody raised against barley NdhA polypeptide. Native PAGE resolved three NADH-nitroblue tetrazolium (NBT)-staining enzyme bands from detergent-solubilized stroma thylakoids and one from the stroma fraction of chloroplasts. The two major thylakoid enzymes and the stromal enzyme showed NAD(P)H-FeCNR and ferredoxin-NADP oxidoreductase (FNR) (EC 1.18.1.2) activities, and the presence of FNR protein was confirmed by Western blotting. Only the major thylakoid enzyme of high molecular weight was found to contain the NdhA polypeptide, a core subunit of the NAD(P)H DH complex. Analysis by SDS–PAGE of the enzyme complex revealed nine polypeptides with molecular masses ranging from 18 to 63 kDa.

Differential effects of abscisic acid and methyl jasmonate on endoproteinases in senescing barley leaves

Endoproteinase activity was analyzed in chloroplasts isolated from barley leaf segments incubated in the dark with various hormonal senescence effectors. As a control, the endoproteinase activity of the supernatant fraction obtained during chloroplast preparation was also analyzed. Measured against azocaseine as substrate, the endoproteinase activity in chloroplasts increased 18 fold during the induction of senescence. This rise in activity was inhibited by kinetin (the activity increased only 10 fold) and very strongly stimulated by abscisic acid (ABA) (117 fold) and methyl jasmonate (Me-JA) (57 fold). Although less so, the endoproteinase activity of the supernatant fraction, mainly vacuolar and with acid pH optimum, was affected in the same way by all three effectors. Among the five endoproteinases (EC) found in chloroplasts, EC2 and EC4 were induced after incubation in water. ABA increased the levels of EC2 and EC4 (5 fold), and induced the development of EC3 and EC5, while Me-JA totally inhibited EC2 and EC4, and induced the development of EC1. At least one of the endoproteinases, EC2, is synthesized in chloroplasts. Among the six endoproteinases found in the supernatant fraction (E), E1, E2, E3 and E5, which are very probably extrachloroplastic endoproteinases, are stimulated by ABA to varying degrees. However, Me-JA stimulates E1 to a greater extent and totally inhibits E3. The differential effects of ABA and Me-JA on chloroplast and supernatant fraction endoproteinases suggest different action mechanisms for both senescence promotors.