Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Identification of the Product of ndhA Gene as a Thylakoid Protein Synthesized in Response to Photooxidative Treatment

A 76 amino acid sequence of NDH-A (the protein encoded by plastidndhA gene) from barley (Hordeum vulgare L.) was expressed asa fusion protein with rß-galactosidase in E. coli.The corresponding antibody generated in rabbits was used toinvestigate localization, expression and synthesis in vitroof NDH-A. NDH-A was identified as a 35 kDa polypeptide localizedin thylakoid membrane. Western blots shows a large increasein NDH-A levels when barley leaves were incubated under photooxidativeconditions, which was more pronounced in mature-senescent leavesthan in young leaves. Immunoprecipitation of the [³⁵S]methioninelabelled proteins, synthesized in vitro by isolated chloroplasts,demonstrated the synthesis in chloroplasts of the NDH-A 35 kDapolypeptide when barley leaves had been incubated under photooxidativeconditions. The results indicate that ndh genes may be involvedin the protection of chloroplasts against photooxidative stress,particularly in mature-senescent leaves. (Received November 13, 1995; Accepted February 5, 1996)     

Regulation of Mo-cofactor, NADH- and NAD(P)H-specific nitrate reductase activities in the wild type and two nar-mutant lines of barley (Hordeum vulgare L.)

Seedlings of three genotypes of barley, Hordeum vulgare L.,cv. Winer, were grown in nutrient solutions for 12 d: (a) Wt,the wild type; (b) Chlo19 and (c) Chlo29, two nitrate reductase(NR) deficient nar-mutants. Nar-mutant plants grown in nitratedeveloped about 5–24% of NADH-NR (EC 1.6.6.1 [EC] .) activitylevel characteristic of the Wt. The NR in vitro assays in whichNADH or NADPH were used as electron donors showed that the twomutant lines contained a mixture of NADH-specific and NAD(P)H-bispecific(EC 1.6.6.2 [EC] .) NRs. Chlo19 had a very low level of MoCo activityas compared to Chlo29 and Wt. Chlo19 appeared to be mutatedin a MoCo gene rather than in the genes coding for the nitrateNR apoenzyme. NAD(P)H-NR was found in the shoots and roots of both mutantsbut only in the roots of Wt. Several aspects of the regulationof NADH and NAD(P)H specific NRs in plants of the barley cv.Winer genotypes are discussed. MoCo was a strong limiting factorfor NR biosynthesis in nitrate-fed plants of Chlo19, but lesslimited in N-starved and ammonium-fed plants. Biomass productionby the three genotypes was similar during first 12 d after germination,regardless of the level of NR detected in vitro. Mutant plantsmay be able to supply the nitrogen required for growth withonly 5–24% of the NR level of the WT. Key words: Hordeum vulgare, mutants, nitrate, nitrate reductase, molybdenum cofactor     

DNA Levels in Dividing and Developing Plastids in Expanding Primary Leaves of Avena sativa

The amounts of plastid DNA in the primary leaves of 4-d-oldlight- and dark-grown seedlings of Avena sativa were measuredby microspectrofluorometry using the DNA-fluorochrome DAPI (4',6-diamidino-2-phenylindole). In the light-grown primary leaves (40–45 mm long) therewas a marked increase in DNA level per plastid from 10.2 to18.5 ? 10^–15 g between 2.0 mm and 10 mm from the leafbase, resulting from the rate of plastid DNA synthesis beinghigher than the rate of plastid division. Beyond 30 mm the plastidDNA level was reduced to 14 ? 10^–15g due to chloroplastdivision rates being higher than the rate of plastid DNA synthesis,while from 20 mm plastid DNA levels were constant at 2.2 ? 10^–12g per cell, which corresponds to 16000 plastome copies per cell. Observations of dark-grown leaves establish that, in Avena,light is not necessary for plastid division and the dark-grownleaf cells accumulate higher amounts of plastid DNA than light-grownleaf cells. Plastid nucleoids showed a change of distribution after completionof plastid DNA synthesis in light-grown leaves. A change inthe distribution of plastid nucleoids was also observed duringthe greening of etioplasts of dark-grown leaves while plastidDNA level remained constant. Such changes in plastid nucleoiddistribution appear to be independent of plastid DNA synthesisand correlate with the formation of grana stacks. Key words: Avena sativa, microspectrofluorometry, plastid DNA     

Expression of NADH-Specific and NAD(P)H-Bispecific Nitrate Reductase Genes in Response to Nitrate in Barley

Barley (Hordeum vulgare L.) has two, differentially regulated, nitrate reductase (NR) genes, one encoding the NADH-specific NR (Nar1) and the other encoding the NAD(P)H-bispecific NR (Nar7). Regulation of the two NR genes by nitrate was investigated in wild-type Steptoe and in an NADH-specific NR structural gene mutant (Az12). Gene-specific probes were used to estimate NADH and NAD(P)H NR mRNAs. The kinetics of induction by nitrate were similar for the two NR genes; expression was generally below the limits of detection prior to induction, reached maximum levels after 1 to 2 h of induction in roots and 4 to 8 h of induction in leaves, and then declined to steady-state levels. Derepression of the NAD(P)H NR gene in leaves of the NADH-specific NR gene mutant Az12 did not appear to be associated with changes in nitrate assimilation products or nitrate flux. Nitrate deprivation resulted in rapid decreases in NADH and NAD(P)H NR mRNAs in seedling roots and leaves and equally rapid decreases in the concentration of nitrate in the xylem sap. These results indicate that factors affecting nitrate uptake and transport could have a direct influence on NR expression in barley leaves.     

Alternative pathway is involved in the tolerance of highland barley to the low-nitrogen stress by maintaining the cellular redox homeostasis

Key message

Alternative pathway (AP) is involved in the tolerance of highland barley seedlings to the low-nitrogen stress by dissipating excessive reducing equivalents generated by photosynthesis and maintaining the cellular redox homeostasis.

Abstract

Low nitrogen (N) is a major limiting factor for plant growth and crop productivity. In this study, we investigated the roles of the alternative pathway (AP) in the tolerance of two barley seedlings, highland barley (Kunlun12) and barley (Ganpi6), to low-N stress. The results showed that the chlorophyll content and the fresh weight decreased more in Ganpi6 than those in Kunlun12 under low-N stress, suggesting that Kunlun12 has higher tolerance to low-N stress than Ganpi6. AP capacity was markedly induced by low-N stress; and it was higher in Kunlun12 than in Ganpi6. Comparatively, the cytochrome pathway capacity was not affected under all conditions. Western-blot analysis showed that the protein level of the alternative oxidase (AOX) increased under low-N stress in Kunlun12 but not in Ganpi6. Under low-N stress, the NAD(P)H content and the NAD(P)H to NAD(P)⁺+NAD(P)H ratio in Ganpi6 increased more than those in Kunlun12. Furthermore, photosynthetic parameters (Fv/Fm, qP, ETR and Yield) decreased markedly and qN increased, indicating photoinhibition occurred in both barley seedlings, especially in Ganpi6. When AP was inhibited by salicylhydroxamic acid (SHAM), the NAD(P)H content and the NAD(P)H to NAD(P)⁺+NAD(P)H ratio dramatically increased under all conditions, resulting in the marked accumulation of H₂O₂ and malondialdehyde in leaves of both barley seedlings. Meanwhile, the malate–oxaloacetate shuttle activity and the photosynthetic efficiency were further inhibited. Taken together, AP is involved in the tolerance of highland barley seedlings to low-N stress by dissipating excess reducing equivalents and maintaining the cellular redox homeostasis.

     

Inheritance and expression of NAD(P)H nitrate reductase in barley

Summary NADH-specific and NAD(P)H bispecific nitrate reductases are present in barley (Hordeum vulgare L.). Wild-type leaves have only the NADH-specific enzyme while mutants with defects in the NADH nitrate reductase structural gene (nar1) have the NAD(P)H bispecific enzyme. A mutant deficient in the NAD(P)H nitrate reductase was isolated in a line (nar1a) deficient in the NADH nitrate reductase structural gene. The double mutant (nar1a;nar7w) lacks NAD(P)H nitrate reductase activity and has xanthine dehydrogenase and nitrite reductase activities similar to nar1a. NAD(P)H nitrate reductase activity in this mutant is controlled by a single codominant gene designated nar7. The nar7 locus appears to be the NAD(P)H nitrate reductase structural gene and is not closely linked to nar1. From segregating progeny of a cross between the wild type and nar1a;nar7w, a line was obtained which has the same NADH nitrate reductase activity as the wild type in both the roots and leaves but lacks NADPH nitrate reductase activity in the roots. This line is assumed to have the genotype Nar1Nar1nar7nar7. Roots of wild type seedlings have both nitrate reductases as shown by differential inactivation of the NADH and NAD(P)H nitrate reductases by a monospecific NADH-nitrate reductase antiserum. Thus, nar7 controls the NAD(P)H nitrate reductase in roots and in leaves of barley.Scientific Paper No. 7617, College of Agriculture Research Center and Home Economics, Washington State University, Pullman, WA, USA. Project Nos. 0233 and 0745     

Targeted inactivation of the plastid ndhB gene in tobacco results in an enhanced sensitivity of photosynthesis to moderate stomatal closure

The ndh genes encoding for the subunits of NAD(P)H dehydrogenase complex represent the largest family of plastid genes without a clearly defined function. Tobacco (Nicotiana tabacum) plastid transformants were produced in which the ndhB gene was inactivated by replacing it with a mutant version possessing translational stops in the coding region. Western-blot analysis indicated that no functional NAD(P)H dehydrogenase complex can be assembled in the plastid transformants. Chlorophyll fluorescence measurements showed that dark reduction of the plastoquinone pool by stromal reductants was impaired in ndhB-inactivated plants. Both the phenotype and photosynthetic performance of the plastid transformants was completely normal under favorable conditions. However, an enhanced growth retardation of ndhB-inactivated plants was revealed under humidity stress conditions causing a moderate decline in photosynthesis via stomatal closure. This distinctive phenotype was mimicked under normal humidity by spraying plants with abscisic acid. Measurements of CO(2) fixation demonstrated an enhanced decline in photosynthesis in the mutant plants under humidity stress, which could be restored to wild-type levels by elevating the external CO(2) concentration. These results suggest that the plastid NAD(P)H:plastoquinone oxidoreductase in tobacco performs a significant physiological role by facilitating photosynthesis at moderate CO(2) limitation.     

Cellular compartmentation of ammonium assimilation in rice and barley

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.     

Involvement of the thylakoidal NADH-plastoquinone-oxidoreductase complex in the early responses to ozone exposure of barley (Hordeum vulgare L.) seedlings

A possible implication of the plastid NADH-plastoquinone-oxidoreductase (Ndh) complex in the response against ozone-mediated oxidative stress in barley (Hordeum vulgare L.) leaves was investigated. After a 4 h treatment, exposure of barley seedlings to moderate ozone concentrations produced leaf-age-dependent increases in lipid peroxidation, peroxidase, and Ndh complex activities in the thylakoid membranes. A significant amount and activity of the Ndh complex were detected in mature barley leaves, but not in young barley leaves. In fact, young barley leaves behaved like ndh-deficient leaves in most of the aspects studied. When plants were exposed to photo-oxidative light after ozone fumigation, the recovery of Fv/Fm was lower in young leaves than in mature leaves. Ozone treatment significantly decreased non-photochemical quenching (qN) in young leaves, but not in mature leaves. Mature leaves showed higher levels of the energy (DeltamuH+) dependent (qE) component of qN. Treatment with antimycin A, an inhibitor of cyclic electron flow, increased the decay of qN produced by ozone in young leaves, but not in mature ones. The reduction state of plastoquinone increased after ozone treatment in mature dark-adapted leaves and was strongly quenched by far red light. It is proposed that the function of the Ndh complex helps the maintenance of qN, probably through the poising of the redox steady-state level of the intersystem carriers and then by optimizing the rate of cyclic electron flow. This should constitute an age-dependent early response in barley leaves, by contributing to minimize photoinhibition in the presence of ozone and high light.     

Age Dependent Damage and Glutathione Metabolism in Ozone Fumigated Barley: A Leaf Section Approach

First, second, and third leaves of barley (Hordeum vulgare L.cv. Atem) seedlings fumigated with 200 nl 1^–1 ozone for5 d were divided into equal length quarter sections before analysisof glutathione metabolism. Since the first leaf is the oldestand the third leaf is the youngest and since monocotyledonousleaves grow from the base, sections were of a widely differingage. Lipid peroxidation rose moderately in all sections in all leaves.However, severe damage, detected visibly as chlorosis and analyticallyas loss of soluble protein, occurred only in the oldest tissue. The specific activity of glutathione reductase was slightlyelevated in old and middle-aged tissue whilst the specific activityof glutathione-S-transferase rose markedly in old, middle-aged,and young tissue following fumigation. This may reflect theimportance of glutathione-S-transferase in the protection ofplant cells from oxidative stress products formed in membranes. Fumigation with ozone also caused a loss of total glutathionein the tip sections of all leaves. Explanations for these results, including age dependent proteinloss, are discussed. Key words: Barley, ozone, glutathione, developmental age, glutathione-S-transferase     

Colorimetric Estimation of Glutamate Dehydrogenase in Leaf Tissue of Vigna mungo (L.) Hepper

Glutamate dehydrogenase (E.C. 1.4.1.2 [EC] ) is usually assayed bythe disappearance of NAD(P)H from the assay medium. A new technique,in which the enzyme level in leaf tissue of Vigna mungo (L.)Hepper is estimated by the disappearance of 2-oxoglutarate,is described. It provides a simple visible-range colorimetricassay for the enzyme. Vigna mungo, black gram, glutamate dehydrogenase, colorimetry     

Chloroplast Doublets and Differential Staining of the Cell Walls in Photosynthetic Tissues of Amaranthus dubius Mart

Ultrastructural studies of the leaves of Amaranthus dubius revealpresence of chloroplast doublets in mature leaf tissues. Theyalso highlight the difference in the structural organisationof the bundle sheath and the mesophyll cell walls, a featurewhich may have functional significance. Amaranthus dubius Mart., Calaloo, mesophyll, bundle sheath, chloroplast doublets, plastid fusion, plastid division, differential staining of the cell walls     

Intergeneric somatic hybridization of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) by protoplast fusion

An intergeneric somatic hybrid was obtained upon fusion of protoplasts of rice and barley. Protoplasts isolated from suspension cultures of rice cells were fused by electrofusion with protoplasts that had been isolated from young barley leaves. Some of the resultant calli formed green spots and shoots. Only one shoot formed roots, and it was subsequently successfully transferred to soil in a greenhouse. Its morphology closely resembled that of the parental rice plant. Cytological analysis indicated that the plant had both small chromosomes from rice and large chromosomes from barley. Southern hybridization analysis with a fragment of the tryptophan B (trpB) gene revealed both a rice-specific band and a barley-specific band. Mitochondrial (mt) and chloroplast (cp) DNAs were also analyzed using the same method. The plant was shown to contain novel mitochondrial and chloroplast sequence rearrangements that were not detected in either of the parents. Received: 5 March 1997 / Revision received: 4 September 1997 / Accepted: 13 September 1997