Two different modes of early development and nitrogen assimilation in gymnosperm seedlings†

Light-independent chloroplast development and expression of genes encoding chloroplast proteins occur in many but not all species of gymnosperms. Early development in maritime pine (Pinus pinaster) seedlings was strongly light-independent, whereas Ginkgo biloba seedlings exhibited a typical angiosperm-like morphogenesis with differentiated patterns in light and dark. In pine, chloroplast polypeptides were undetectable in the seed embryo and accumulated in cotyledons of both light- and dark-grown plants in good correlation with light-independent chlorophyll synthesis. In contrast, chlorophyll and chloroplast proteins were only detected in light-grown ginkgo. Pine cytosolic glutamine synthetase (GS) and ferredoxin glutamate synthase (Fd-GOGAT) were present at low levels in the seeds and accumulated at comparable amounts in light- and dark-grown seedlings. Fd-GOGAT was also barely detectable in the seeds of ginkgo and only accumulated in green plants with mature chloroplasts. In G. biloba seeds and etiolated plants only cytosolic GS was identified, while in light-grown seedlings this molecular form was present at low abundance and choroplastic GS was the predominant isoenzyme. The above results have been confirmed by immunolocalization of GS protein in pine and ginkgo plantlets. In pine, GS was present in the peripheral cytoplasm of mesophyll cells and also in the phloem region of the vascular bundle. Immunocytochemical analysis showed that the labelling of mesophyll and phloem cells was only cytoplasmic. In developing ginkgo, GS antigens were present in the chloroplasts of mesophyll parenchyma cells of leaflets and green cotyledons. In contrast, a weak labelling of GS was observed in the parenchyma and phloem cells of non-green cotyledons enclosed in the seed coat. Taking all this into account, our data indicate the existence of two different modes of GS and GOGAT regulation in gymnosperms in close correlation with the differential response of plants to light. Furthermore, the results suggest that glutamine and glutamate biosynthesis is confined to the chloroplast of mesophyll cells in species with light-dependent chloroplast, development whereas compartmentation would be required in species with light-independent plastid development.     

Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.

Summary Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.Abbreviations BSA bovine serum albumin - EM electron microscope - GOGAT glutamate synthase - GS glutamine synthetase - GUS -glucuronidase - IgG immunoglobulin - PBS phosphate buffer saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Coordinated accumulation of the chloroplastic and cytosolic pyruvate,Pi dikinases with enhanced expression of CAM in Kalanchoë blossfeldiana

Recent studies reveal that the intracellular localization of pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) in mesophyll cells of malic enzyme (ME)-dependent Crassulacean acid metabolism (CAM) plants varies among species, occurring not only in the chloroplasts but also in the cytosol in some species. The facultative CAM plant Kalanchoë blossfeldiana accumulates PPDK in both compartments of the mesophyll cells. In this study, the patterns of accumulation of the chloroplastic and cytosolic PPDKs were investigated for K. blossfeldiana plants with different CAM activities by immunogold labeling and electron microscopy. Greater CAM activity was found in plants grown under drought conditions with short days than under well-watered conditions with long days, and in lower leaves than in higher leaves. There was a trend that plants and leaves with greater CAM activity show denser labeling for PPDK in both the cytosol and chloroplasts. However, the ratio of the density of PPDK labeling in the cytosol to that in the chloroplasts was almost constant (2.4–3.0). Higher labeling for phosphoenolpyruvate carboxylase (EC 4.1.1.31) in the cytosol was also correlated with higher CAM activity but there was almost no difference in the density of labeling for ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in the chloroplasts. These results indicate that the increase in accumulation of cytosolic PPDK is closely associated with the increase of chloroplastic PPDK during enhanced CAM expression. This suggests that both PPDKs are involved in CAM function.     

Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.     

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.     

IMMUNOLOCALIZATION OF NITRATE REDUCTASE IN THE MARINE DINOFLAGELLATE GONYAULAX POLYEDRA (PYRROPHYTA)1

A polyclonal antibody, raised against nitrate reductase (NR) purified from the photosynthetic dinoflagellate Gonyaulax polyedra Stein, was used as a probe in immunogold-labeling experiments on thin sections prepared from cells harvested both during day and night phases. Previous experiments have shown that both NR activity and the amount of immunoreactive NR in cell extracts is greater when day-phase cells are examined, and this property was exploited as an internal control for the cytochemical labeling. We observed that in day-phase cells, chloroplasts contained approximately three times more gold particles than night-phase cells (highly significant difference; P < 0.0001), whereas cytoplasmic labeling levels remained relatively level between day and night. We conclude from the diurnal difference in labeling that our antibody faithfully reflects the distribution of NR in Gonyaulax cells. Thus, as in to some other higher plants and green algae, Gonyaulax compartmentalizes active NR in its chloroplasts.     

Photorespiration and light act in concert to regulate the expression of the nuclear gene for chloroplast glutamine synthetase.

In Pisum sativum, distinct chloroplast and cytosolic forms of glutamine synthetase (GS) are encoded by homologous nuclear genes that are differentially expressed in vivo (Tingey, S. V., Tsai, F.-Y., Edwards, J. W., Walker, E. L., and Coruzzi, G. M. [1988]. J. Biol. Chem. 263, 9651-9657). In leaves, light selectively affects the expression of the nuclear gene for chloroplast GS2. Differences in the maximal levels of GS2 mRNA in etiolated plants treated with red or white light indicate that only part of the white-light-induced accumulation of GS2 mRNA is due to a phytochrome-mediated response. The kinetics of GS2 mRNA accumulation in response to white-light illumination of etiolated or dark-adapted green plants indicates that GS2 mRNA accumulates more rapidly in plants containing mature, photosynthetically competent chloroplasts. Other evidence that GS2 mRNA levels are affected by the metabolic status of chloroplasts concerns the selective induction of GS2 mRNA in plants grown under conditions that result in the production of photorespiratory ammonia. These results indicate that the light-induced accumulation of GS2 mRNA in leaves results from the action of phytochrome as well as light-induced changes in chloroplast metabolism.     

Four isoforms of glutamine synthetase in light-grown soybeans

Glutamine synthetase (GS) activity recovered from linear sucrose gradients was associated with the cytosol of cells isolated from etiolated soybean hypocotyls whereas light-grown tissue contained increased GS activity localized in both the cytosol and chloroplasts. DEAE-cellulose chromatography indicated two GS isoforms in etiolated hypocotyls whereas light-grown hypocotyls and primary leaves contained four isoforms. Only one GS isoform was recovered from both etiolated and light-grown cotyledons.     

Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts

In higher plants, photorespiratory Gly oxidation in leaf mitochondria yields ammonium in large amounts. Mitochondrial ammonium must somehow be recovered as glutamate in chloroplasts. As the first step in that recovery, we report glutamine synthetase (GS) activity in highly purified Arabidopsis thaliana mitochondria isolated from light-adapted leaf tissue. Leaf mitochondrial GS activity is further induced in response to either physiological CO(2) limitation or transient darkness. Historically, whether mitochondria are fully competent for oxidative phosphorylation in actively photorespiring leaves has remained uncertain. Here, we report that light-adapted, intact, leaf mitochondria supplied with Gly as sole energy source are fully competent for oxidative phosphorylation. Purified intact mitochondria efficiently use Gly oxidation (as sole energy, NH(3), and CO(2) source) to drive conversion of l-Orn to l-citrulline, an ATP-dependent process. An A. thaliana genome-wide search for nuclear gene(s) encoding mitochondrial GS activity yielded a single candidate, GLN2. Stably transgenic A. thaliana ecotype Columbia plants expressing a p35S::GLN2::green fluorescent protein (GFP) chimeric reporter were constructed. When observed by laser scanning confocal microscopy, leaf mesophyll and epidermal tissue of transgenic plants showed punctate GFP fluorescence that colocalized with mitochondria. In immunoblot experiments, a 41-kD chimeric GLN2::GFP protein was present in both leaf mitochondria and chloroplasts of these stably transgenic plants. Therefore, the GLN2 gene product, heretofore labeled plastidic GS-2, functions in both leaf mitochondria and chloroplasts to faciliate ammonium recovery during photorespiration.     

Identification and immunolocalization of a 65 kDa drought induced protein in cultivated tomatoLycopersicon esculentum

Summary A 65 kDa protein with a pI value of 5.2 accumulated gradually in tomato leaves during water stress. Protein levels returned to those of the control upon rehydration of the plants. Antiserum raised against the protein, purified from two dimensional electrophoresis gels, was obtained and used as a probe to localize the protein in tomato leaves by immunofluorescence and immunogold labeling. The protein was found to be mainly localized in different areas of nuclei (peripheral chromatin masses, nucleoli and nucleoplasm), chloroplasts, and some leaf cell cytoplasmic regions. Quantification of the gold labeling clearly demonstrated that the amount of the protein increased significantly in nuclei and chloroplasts of cells in drought-stressed plants. Cytological changes occurring in leaf tissues during water stress are also reported.     

Evidence for an operative glutamine translocator in chloroplasts from maritime pine (Pinus pinaster Ait.) cotyledons

In higher plants, ammonium is assimilated into amino acids through the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. This metabolic cycle is distributed in different cellular compartments in conifer seedlings: glutamine synthesis occurs in the cytosol and glutamate synthesis within the chloroplast. A method for preparing intact chloroplasts of pine cotyledons is presented with the aim of identifying a glutamine–glutamate translocator. Glutamine–glutamate exchange has been studied using the double silicone layer system, suggesting the existence of a translocator that imports glutamine into the chloroplast and exports glutamate to the cytoplasm. The translocator identified is specific for glutamine and glutamate, and the kinetic constants for both substrates indicate that it is unsaturated at intracellular concentrations. Thus, the experimental evidence obtained supports the model of the GS/GOGAT cycle in developing pine seedlings that accounts for the stoichiometric balance of metabolites. As a result, the efficient assimilation of free ammonia produced by photorespiration, nitrate reduction, storage protein mobilisation, phenylpropanoid pathway or S‐adenosylmethionine synthesis is guaranteed.     

Changes in the activities of chloroplast and cytosolic isoenzymes of glutamine synthetase during normal leaf growth and plastid development in wheat

Soluble protein extracts and chloroplasts from a serial sequence of transverse sections of a 7-d-old wheat leaf (Triticum aestivum cv. Maris Huntsman) were used to study changes in the activity of glutamine synthetase (GS; EC 6.3.1.2) during cell and chloroplast development. Glutamine synthetase activity increased more than 50-fold per cell from the base to the tip of the wheat leaf. Two isoenzymes of GS were separated using fast protein liquid chromatography (FPLC). Glutamine synthetase localized in the cytoplasm (GS1) eluted at about 0.21 M NaCl, and the isoenzyme localized in the chloroplast (GS2) eluted at about 0.33 M NaCl. The increase in GS activity during leaf development was found to be caused primarily by an increase in the activity of the chloroplast GS2. The activity of the cytoplasmic GS1 remained constant as the cells were displaced from the base to the tip of the leaf, whereas GS2 activity increased within the chloroplast throughout development. At the base of the leaf, 26% of total GS activity was cytoplasmic; the remaining 74% was in the chloroplast. At 10 cm from the base, only 4% of the activity was cytoplasmic, and 96% was in the chloroplast. The results indicate that the chloroplast GS2 is probably responsible for most of the ammonia assimilation in the mature wheat leaf, whereas cytoplasmic GS1 may serve a role in immature developing leaf cells.Abbreviations FPLC fast protein liquid chromatography - GS glutamine synthetase - GS1 cytoplasmic glutamine synthetase - GS2 chloroplast glutamine synthetase     

Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L

Glutamine synthetase was localized in nodules, roots, stems, and leaves of red kidney bean (Phaseolus vulgaris L.) by immunocytochemistry. Affinity purified antibodies reactive with glutamine synthetase were prepared using purified nodule-enhanced glutamine synthetase. Immunogold labeling was observed in the cell cytoplasm in each plant organ. In nodules, the labeling was more intense in the infected cells than in the uninfected cells. No labeling was observed in nodule bacteroids, peribacteroid spaces, or in peribacteroid membranes, while previous reports of glutamine synthetase immunolabeling of legume nodules showed labeling in the bacteroid fraction. Significant labeling was observed in nodule proplastids which contained starch granules. Substantial labeling was also observed in leaf chloroplasts. No labeling was observed in other organelles including mitochondria, peroxisomes, and endoplasmic reticulum. Preimmune IgGs did not bind to any structure in the tissues examined.     

Immunocytochemical determination of the subcellular distribution of ascorbate in plants

Ascorbate is an important antioxidant in plants and fulfills many functions related to plant defense, redox signaling and modulation of gene expression. We have analyzed the subcellular distribution of reduced and oxidized ascorbate in leaf cells of Arabidopsis thaliana and Nicotiana tabacum by high-resolution immuno electron microscopy. The accuracy and specificity of the applied method is supported by several observations. First, preadsorption of the ascorbate antisera with ascorbic acid or dehydroascorbic acid resulted in the reduction of the labeling to background levels. Second, the overall labeling density was reduced between 50 and 61% in the ascorbate-deficient Arabidopsis mutants vtc1-2 and vtc2-1, which correlated well with biochemical measurements. The highest ascorbate-specific labeling was detected in nuclei and the cytosol whereas the lowest levels were found in vacuoles. Intermediate labeling was observed in chloroplasts, mitochondria and peroxisomes. This method was used to determine the subcellular ascorbate distribution in leaf cells of plants exposed to high light intensity, a stress factor that is well known to cause an increase in cellular ascorbate concentration. High light intensities resulted in a strong increase in overall labeling density. Interestingly, the strongest compartment-specific increase was found in vacuoles (fourfold) and in plastids (twofold). Ascorbate-specific labeling was restricted to the matrix of mitochondria and to the stroma of chloroplasts in control plants but was also detected in the lumen of thylakoids after high light exposure. In summary, this study reveals an improved insight into the subcellular distribution of ascorbate in plants and the method can now be applied to determine compartment-specific changes in ascorbate in response to various stress situations.     

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.     

Proof of C4 photosynthesis without Kranz anatomy in Bienertia cycloptera (Chenopodiaceae)

Kranz anatomy, with its separation of elements of the C4 pathway between two cells, has been an accepted criterion for function of C4 photosynthesis in terrestrial plants. However, Bienertia cycloptera (Chenopodiaceae), which grows in salty depressions of Central Asian semi-deserts, has unusual chlorenchyma, lacks Kranz anatomy, but has photosynthetic features of C4 plants. Its photosynthetic response to varying CO2 and O2 is typical of C4 plants having Kranz anatomy. Lack of night-time CO2 fixation indicates it is not acquiring carbon by Crassulacean acid metabolism. This species exhibits an independent, novel solution to function of the C4 mechanism through spatial compartmentation of dimorphic chloroplasts, other organelles and photosynthetic enzymes in distinct positions within a single chlorenchyma cell. The chlorenchyma cells have a large, spherical central cytoplasmic compartment interconnected by cytoplasmic channels through the vacuole to the peripheral cytoplasm. This compartment is filled with mitochondria and granal chloroplasts, while the peripheral cytoplasm apparently lacks mitochondria and has grana-deficient chloroplasts. Immunolocalization studies show enzymes compartmentalized selectively in the CC compartment, including Rubisco in chloroplasts, and NAD-malic enzyme and glycine decarboxylase in mitochondria, whereas pyruvate, Pi dikinase of the C4 cycle is localized selectively in peripheral chloroplasts. Phosphoenolpyruvate carboxylase, a cytosolic C4 cycle enzyme, is enriched in the peripheral cytoplasm. Our results show Bienertia utilizes strict compartmentation of organelles and enzymes within a single cell to effectively mimic the spatial separation of Kranz anatomy, allowing it to function as a C4 plant having suppressed photorespiration; this raises interesting questions about evolution of C4 mechanisms.     

In situ immunofluorescent localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in mesophyll of C4 dicotyledonous plants

The location of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in the leaf mesophyll of some dicotyledonous C4 plants was confirmed by immunofluorescent labelling. The anti-RuBPCO immune serum was obtained by inoculating a rabbit with commercially obtained RuBPCO. Specificity of these antibodies was tested by immunodiffusion, immunoelectrophoresis, and Western blotting. Fresh hand-cuts of leaves from dicotyledonous C4 plants, Amaranthus caudatus, A. dubius, Gomphrena globosa, and Portulaca oleracea, were incubated with the conjugated anti-RuBPCO immune serum and then with a commercial FITC-anti-rabbit IgG conjugate. Nerium oleander was used a control C3 plant pattern and Zea mays as a C4 plant pattern. The immunofluorescent label was distributed in both mesophyll and bundle sheath in all the C4 plants tested. It is an unequivocal proof that in the C4 dicotyledonous plants the RuBPCO is not only located in the chloroplasts of the bundle sheath cells but also in the chloroplasts of the mesophyll cells. In these plants therefore, the C4 pathway cannot exclusively be viewed as an intercellular level concentration mechanism. In the mesophyll cytoplasm, phosphoenolpyruvate carboxylase traps CO2, while in the mesophyll chloroplasts, RuBPCO operates with atmospheric CO2 and CO2 from the C4 decarboxylation step at an intracellular level, which could mean a significant energetic economy. The CO2 from photorespiration could be saved and reincorporated. Location of RuBPCO in the mesophyll and/or bundle sheath chloroplasts is a matter of inter- and intracellular compartmentation which makes another variation of C4 photosynthetic pathway possible. This revised version was published online in September 2006 with corrections to the Cover Date.     

Effect of water stress on cotton leaves : I. An electron microscopic stereological study of the palisade cells

Palisade cells from fully expanded leaves from irrigated and nonirrigated, field grown cotton (Gossypium hirsutum L. cv. Paymaster 266) were subjected to a microscopic examination to evaluate the effect of water stress on subcellular structures. The water potential difference between the two treatments was 13 bars at the time of sampling. The dimensions of the palisade cells and their density per unit leaf area were determined by light microscopy. Palisade cells from stressed plants had the same diameter, but were taller than their counterparts in irrigated plants. The density of the palisade cells was the same in both treatments as was the fractional volume of the intercellular space. It was concluded that the reduced leaf area observed in the stressed plants resulted primarily from a mitotic sensitivity to water stress. Further, expansion of palisade cells was not inhibited by the stress imposed in this study.

Morphometric analysis of electron micrographs was used to evaluate the subcellular structure of palisade cells from nonstressed and stressed plants. The fractional volumes of cell walls, total cytoplasm, chloroplasts, starch granules, intrachloroplast bodies, mitochondria, peroxisomes, and central vacuoles were determined. The surface densities of grana and stroma lamellae, outer chloroplast membranes, mitochondrial cristae, endoplasmic reticulum and Golgi cisternae were also measured. The number of chloroplasts, mitochondria, and peroxisomes were determined. These data were expressed as actual volumes, areas, and numbers per palisade cell for each treatment. Palisade cells from stressed plants had thinner cell walls, larger central vacuoles and approximately the same amount of cytoplasm compared to cells from nonstressed plants. Within the cytoplasm, stressed plants had more but smaller chloroplasts with increased grana and stroma lamellae surfaces, larger mithchondria with reduced cristae surfaces, smaller peroxisomes and reduced membrane surfaces of endoplasmic reticulum and Golgi cisternae.