Distribution of two isoforms of glutamine synthetase in bundle sheath and mesophyll cells of corn leaves

Localization of two isoforms of glutamine synthetase (GS; EC 6.3.1.2) was investigated in different cell types, mesophyll cells and bundle sheath cells, of corn ( Zea mays L. var. W64A × W182E) leaves by using ion exchange chrotnatography. In whole leaf extracts, relative activities of GS1 (cytosolic GS) and GS2 (chloroplastic GS) were almost equal. Purified mesophyll protoplasts and bundle sheath strands also showed similar proportions of GS1 and GS2. Methionine sulfoximine (1 mM ) enhanced the accumulation of ammonia when mesophyll protoplasts were incubated with nitrite or when bundle sheath strands were incubated with glycine. This clearly indicates a spatial separation of metabolism of NH⁺₄ derived from photorespiration and from reduction of NOJ.     

Effect of nitrogen starvation on ammonium assimilation by Chlamydomonas reinhardtii

In nitrogen-starved Chlamydomonas reinhardtii , wild type, strain 21 gr cells, consumption of nitrate, nitrite and ammonium may occur in the dark in the absence of an added carbon source. Consumption of ammonium in the dark was about 25% higher than in the light, while consumption of nitrate or nitrite in the dark was lower than in the light.
N starvation produced a linear increase with time in the intracellular level of glutamine synthetase (GS, EC 6.3.2.1) and glutamate synthase (NADH-GOGAT, EC 1.4.1.14 and ferredoxin-GOGAT, EC 1.4.7.1) activities in C. reinhardtii . The effect on GS₁ (3-fold) and NADH-GOGAT (4.5-fold) was higher than that on GS₂ (1.5-fold) and ferredoxin-GOGAT (1.5-fold).
Experiments with methylammonium, L-methionine-D, L-sulfoximine (MSX) and azaserine suggest that: 1) Ammonium itself decreases the intracellular levels of glutamine synthetase and ferredoxin-glutamate synthase activities; and 2) a metabolite resulting from ammonium assimilation by the alga may be a negative modulator of NADH-glutamate synthase activity.     

Effects of tabtoxin on nitrogen metabolism

Tabtoxin is a chlorosis-inducing toxin produced by the plant pathogenic bacterium Pseudomonas syringae pv. tabaci. Previous studies have indicated that tabtoxin inhibits glutamine synthetase (EC 6.3.1.2) in vitro. We report here that tabtoxin also inhibits glutamine synthetase in vivo. The main evidence was that assimilation of exogenous ¹⁵NH₃ into Asparagus sprengeri protein was rapidly inhibited in isolated cells exposed to tabtoxin. This was associated with an equivalent decline in glutamine synthetase activity in extracts of these cells and the accumulation of extracellular ammonia. Glutamine synthetase was also inhibited in leaves of Nicotiana tabacum L. cv. White Burley treated with tabtoxin and the affected tissue accumulated ammonia and became chlorotic. However, the development of symptoms and accumulation of ammonia was suppressed when the leaves were held in air containing 1% CO₂ to reduce photorespiration. This indicates that the chlorotic symptom did not result from the inhibition of nitrogen assimilation but was a consequence of the interruption of the photorespiratory nitrogen cycle.     

Ammonium assimilation in bryophytes. l-glutamine synthetase from Sphagnum fallax

Cytosolic and plastidic l -glutamine synthetase (EC 6.3.1.2) isoenzymes from Sphagnum fallax Klinggr. (Klinggr. clone 1) were separated by size-exclusion and ion exchange chromatography. The cytosolic enzyme (GS₁) was purified to apparent electrophoretic homogeneity. The native enzyme had a molecular mass of 390 ± 20 kDa as estimated by gel filtration and was apparently composed of 8 subunits with molecular masses of 48 kDa. GS₁ activity could be measured from pH 6.8 to 8.6 in 50 m M imidazole buffer, with a broad optimum between pH 7.2 and 8.0. The K_m values were 2.5 m M , 0.5 m M and 0.5 m M for l -glutamate, ammonium and ATP, respectively. The enzyme was inhibited by more than 10 m M ammonium or glutamate. The incorporation of ¹⁵NH₄⁺ into amino acids was observed in vivo using ¹⁵ NMR. Label from ammonium was first detected in the amide N of glutamine, and only subsequently in the amino N of glutamate. Moreover, no assimilation was detected in the presence of the specific GS inhibitor methionine sulfoximine. These observations are consistent with a dominant role for GS in the assimilation of ammonium in Sphagnum .     

Modification of the pII protein in response to carbon and nitrogen availability in filamentous heterocystous cyanobacteria

Abstract In the filamentous cyanobacterium Calothrix PCC 7504, which fixes N₂ aerobically, the modification state of the regulatory P_II protein (GlnB) was shown to depend on nitrogen and carbon availability, as observed in the unicellular non-fixing strain Synechococcus PCC 7942. However, the conditions for modifications, the time dependence of the process and the electrophoretic behavior of the native P_II isoforms differed somewhat between the two strains. In another strain, Calothrix PCC 7601, which has lost the capability to fix N₂, P_II was modified only if ammonia plus an inhibitor of glutamine synthetase were present. It is proposed that: (i) the behavior of the P_II proteins depends upon the physiological properties of the strains; and (ii) the modification system of P_II per se may differ between the two cyanobacterial genera.     

Localization of nitrite and sulfite reductase in bundle sheath and mesophyll cells of maize leaves

The distribution of nitrite reductase (EC 1.7.7.1) and sulfite reductase (EC 1.8.7.1) between mesophyll ceils and bundle sheath cells of maize ( Zea mays L. cv. Seneca 60) leaves was examined. This examination was complicated by the fact that both of these enzymes can reduce both NO-₂ and SO^2-₃ In crude extracts from whole leaves, nitrite reductase activity was 6 to 10 times higher than sulfite reductase activity. Heat treatment (10 min at 55°C) caused a 55% decrease in salfite reductase activity in extracts from bundle sheath cells and mesophyll cells, whereas the loss in nitrite reductase activity was 58 and 82% in bundle sheath cells and mesophyll cell extracts, respectively. This result was explained, together with results from the literature, by the hypothesis that sulfite reductase is present in both bundle sheath cells and mesophyll cells, and that nitrite reductase is restricted to the mesophyll cells. This hypothesis was tested i) by comparing the distribution of nitrite reductase activity and sulfite reductase activity between bundle sheath and mesophyll cells with the presence of the marker enzymes ribulose-l, 5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoe-nolpyruvate carboxylase (EC 4.1.1.32), ii) by examining the effect of cultivation of maize plants in the dark without a nitrogen source on nitrite reductase activity and sulfite reductase activity in the two types of cells, and iii) by studying the action of S^2-on the two enzyme activities in extracts from bundle sheath and mesophyll cells. The results from these experiments are consistent with the above hypothesis.     

An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli

The P_II protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that P_II, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain. We describe a new operon, glnK amtB , which encodes a homologue of P_II and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as P_II, reacted with polyclonal P_II antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli P_II. Like P_II, purified GlnK can activate the adenylylation of glutamine synthetase in vitro , and, in vivo , the GlnK protein is uridylylated in a glnD -dependent fashion. Unlike P_II, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a σ^N (σ⁵⁴) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two-component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on P_II. Possible regulatory implications of this conditional redundancy of P_II are discussed.     

Effect of applied benzyladenine on endogenous cytokinin content during the early stages of bud development of kiwifruit

The filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) was able to utilize glutamine as the sole nitrogen source. The addition to ammonium-grown cultures of the irreversible inhibitor of glutamine synthetase activity L-methionine-D, L-sulfoximine (MSX) inhibited cell growth. Supplying glutamine to the culture restored cell growth. This re-established growth was not due to interference by glutamine of MSX uptake by the cells, since glutamine synthetase (GS, EC 6.3.1.2) activity remained completely inhibited by MSX even when glutamine was simultaneously present. Both glutamine and ammonium exerted a negative effect on nitrate reductase (NR. EC 1.7.7.2) and nitrite reductase (NiR, EC 1.7.7.1) in vivo. This negative effect was reversed by MSX. When glutamine was added to MSX-treated cells, intracellular glutamine level was high, but the activity of both reductases remained at a high level. These results suggest that the presence of the active form of glutamine synthetase is required for the in vivo prevention of nitrate assimilation caused by ammonium and glutamine.     

Opinion: Stomatal responses to light and CO2 depend on the mesophyll

The mechanisms by which stomata respond to red light and CO₂ are unknown, but much of the current literature assumes that these mechanisms reside wholly within the guard cells. However, responses of guard cells in isolated epidermes are typically much smaller than those in leaves, and there are several lines of evidence in the literature suggesting that the mesophyll is necessary for these responses in leaves. This paper advances the opinion that although guard cells may have small direct responses to red light and CO₂, most of the stomatal response to these factors in leaves is caused by an unknown signal that originates in the mesophyll.     

Profiles of photosynthesis within red and green leaves of Quintinia serrata

We have measured photosynthesis at the cellular, tissue, and whole leaf levels to understand the role of anthocyanin pigments on patterns of light utilization. Profiles of chlorophyll fluorescence through sections of red and green leaves of Quintinia serrata showed that anthocyanins in the mesophyll restricted absorption of green light to the uppermost palisade mesophyll. The distribution was further restricted when anthocyanins were also present in the upper epidermis. Mesophyll cells located beneath a cyanic light-filter assumed the characteristic photosynthetic features of shade-adapted cells. As a result, red leaves showed a 23% reduction in CO₂ assimilation under light-saturating conditions, and a lower threshold irradiance for light-saturation, relative to those of green leaves. The photosynthetic characteristics of red leaves are comparable to those of shade-acclimated plants.     

The influence of leaf thickness on the CO2 transfer conductance and leaf stable carbon isotope ratio for some evergreen tree species in Japanese warm-temperate forests

1. The influence of leaf thickness on internal conductance for CO₂ transfer from substomatal cavity to chloroplast stroma ( g _i) and carbon isotope ratio (δ¹³C) of leaf dry matter was investigated for some evergreen tree species from Japanese temperate forests. g _i was estimated based on the combined measurements of gas exchange and concurrent carbon isotope discrimination.
2. Leaves with thicker mesophyll tended to have larger leaf dry mass per area (LMA), larger surface area of mesophyll cells exposed to intercellular air spaces per unit leaf area ( S _mes) and smaller volume ratio of intercellular spaces to the whole mesophyll (mesophyll porosity).
3. g _i of these leaves was correlated positively to S _mes but negatively to mesophyll porosity. The variation in g _i among these species would be therefore primarily determined by variation of the conductance in liquid phase rather than that in gas phase.
4. δ¹³C was positively correlated to mesophyll thickness and leaf nitrogen content on an area basis. However, g _i values did not correlate to δ¹³C. These results suggest that difference in δ¹³C among the species was not caused by the variation in g _i, but mainly by the difference in long-term photosynthetic capacity.
5. Comparison of our results with those of previous studies showed that the correlation between leaf thickness and g _i differed depending on leaf functional types (evergreen, deciduous or annual). Differences in leaf properties among these functional types were discussed.     

Impact of gaseous nitrogen deposition on plant functioning

Dry deposition of NH₃ and NO_x (NO and NO₂) can affect plant metabolism at the cellular and whole-plant level. Gaseous pollutants enter the plant mainly through the stomata, and once in the apoplast NH₃ dissolves to form NH₄⁺, whereas NO₂ dissolves to form NO₃⁻ and NO₂⁻. The latter compound can also be formed after exposure to NO. There is evidence that NH₃-N and NO_x-N can be reversibly stored in the apoplast. Temporary storage might affect processes such as absorption rate, assimilation and re-emission. Once formed, NO₃⁻ and NO₂⁻ can be reduced, and NH₄⁺ can be assimilated via the normal enzymatic pathways, nitrate reductase (NR), nitrite reductase and the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Fumigation with low concentrations of atmospheric NH₃ increases in vitro glutamine synthetase activity, but whether this involves both or only one of the GS isoforms is still an open question. There seems to be no correlation between fumigation with low concentrations of NH₃ and in vitro GDH activity. The contribution of atmospheric NH₃ and NO₂ deposition to the N budget of the whole plant has been calculated for various atmospheric pollutant concentrations and relative growth rates ( RGRs ). It is concluded that at current ambient atmospheric N concentrations the direct impact of gaseous N uptake by foliage on plant growth is generally small.     

LEAF INFECTION OF COTTON BY XANTHOMONAS MALVACEARUM (E.F.SM.) DOWSON

Xanthomonas malvacearum spread more rapidly along vascular tissue than into mesophyll when inoculated to the main veins of susceptible cotton leaves. The extent of spread varied with the concentrations of inocula, tissue age and cotton variety.
Increasing concentrations of inocula accelerated the initial spread of disease.
Bacteria spread more rapidly in young leaves than in old—increasing age greatly decreased disease in the mesophyll. The initial invasion was quicker in young leaves of young plants than in young leaves of old plants.
Three types of behaviour, according to the host's reaction, distinguish Knight's resistance factors: ( a ) where X. malvacearum spread extensively along veins and into mesophyll of plants containing factors B₃ and B₅; ( b ) where it was restricted to the point of inoculation in plants containing B ₄, B₉ and combinations with B _6m; and ( c ) where it spread along veins but not appreciably into mesophyll in varieties containing B ₂ and B ₂ B ₃.
From this and four other different types of tests, factors B ₂ and B ₃ seem to increase mesophyll resistance but only B ₂ gives appreciable vascular resistance. Further, the vascular bundles in varieties with B ₂ seem to be surrounded by an additional 'barrier' which resists X. malvacearum.     

Staurosporine Induces Astrocytic Phenotypes and Differential Expression of Specific PKC Isoforms in C6 Glial Cells

Abstract: In this study we examined the effects of staurosporine, a potent inhibitor of protein kinase C (PKC), on the differentiation of C6 glial cells and on the expression and cellular distribution of specific PKC isoforms. Staurosporine reduced cell proliferation and induced distinctive changes in the morphological appearance of the cells to that characteristic of cells exhibiting astrocytic phenotypes. The differentiative effect of staurosporine was further indicated by the increased expression of two proteins related to astrocytic phenotypes, glial fibrillary acidic protein (GFAP) and glutamine synthetase. Thus, staurosporine induced a dose-dependent increase both in GFAP immunoreactivity and in the activity and protein levels of glutamine synthetase. Staurosporine also induced a decrease in the expression of PKC-β₂ and an increase in that of PKC-γ. In addition, it induced translocation of PKC-ε from the membrane to the cytosol, whereas no differences were observed in the distribution of the other PKC isoforms. The results of our study indicate that staurosporine induced astrocytic phenotypes in glial cells and that changes in the expression and cellular distribution of these PKC isoforms may be related to astrocytic differentiation.     

Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO₂-enriched atmosphere [0.7% (v/v) CO₂] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO₂ to air. F₁ plants of back crosses to wild type were viable in air and F₂ populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO₂ conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO₂ conditions.     

The Effect of Elevated [CO2] on Uptake and Allocation of 13C and 15N in Beech (Fagus sylvatica L.) during Leafing

Abstract: A continuous dual ¹³CO₂ and ¹⁵NH₄¹⁵NO₃ labelling experiment was undertaken to determine the effects of ambient (350μmol mol^-1) or elevated (700μmol mol^-1) atmospheric CO₂ concentrations on C and N uptake and allocation within 3-year-old beech ( Fagus sylvatica L.) during leafing. After six weeks of growth, total carbon uptake was increased by 63 % (calculated on total C content) under elevated CO₂ but the carbon partitioning was not altered. 56 % of the new carbon was found in the leaves. On a dry weight basis was the content of structural biomass in leaves 10 % lower and the lignin content remained unaffected under elevated as compared to ambient [CO₂]. Under ambient [CO₂] 37 %, and under elevated [CO₂] 51 %, of the lignin C of the leaves derived from new assimilates. For both treatments, internal N pools provided more than 90 % of the nitrogen used for leaf-growth and the partitioning of nitrogen was not altered under elevated [CO₂]. The C/N ratio was unaffected by elevated [CO₂] at the whole plant level, but the C/N ratio of the new C and N uptake was increased by 32 % under elevated [CO₂].     

Photosynthesis of two poplar clones under long‐term exposure to ozone

The photosynthetic response was studied in two clones ( Populus deltoides × maximowiczii Eridano and Populus × euramericana I‐214), known for their differential response to ozone (O₃) in terms of visible symptoms, when exposed to O₃ (60 nl l⁻¹ 5 h day⁻¹, 7 and 15 days). The photosynthetic ability was tested using gas exchange and chlorophyll fluorescence analysis. O₃ caused a decrease in the CO₂ assimilation rate at light saturation level in mature leaves of both clones. Alterations of Chl fluorescence parameters, in particular the F_v/F_m ratio and non‐photochemical quenching were also observed. The effects were similar for both clones and it could not be concluded that differential effects on electron transport capacity were responsible for the observed reduction in photosynthesis. The reduction of photosynthetic rate in Eridano was due mainly to a reduced mesophyll activity, as evidenced by the increase in intercellular CO₂ concentration and the minimal changes in stomatal conductance. In contrast, in I‐214, stomatal effects were primarily responsible, although effects on the mesophyll cannot be excluded. Data obtained indicate that the effects observed at the mesophyll level may be attributed to indirect effects caused by membrane disorders.     

Variations in the dorso-ventral organization of leaf structure and Kranz anatomy coordinate the control of photosynthesis and associated signalling at the whole leaf level in monocotyledonous species

Photosynthesis and associated signalling are influenced by the dorso-ventral properties of leaves. The degree of adaxial/abaxial symmetry in stomatal numbers, photosynthetic regulation with respect to light orientation and the total section areas of the bundle sheath (BS) cells and the surrounding mesophyll (M) cells on the adaxial and abaxial sides of the vascular bundles were compared in two C₄[ Zea mays (maize) and Paspalum dilatatum ] and one C₃[ Triticum turgidum (Durum wheat)] monocotyledonous species. The C₃ leaves had a higher degree of dorso-ventral symmetry than the C₄ leaves. Photosynthetic regulation was the same on each side of the wheat leaves, as were stomatal numbers and the section area of the BS relative to that of the M cells (BS/M section area ratio). In contrast, photosynthetic regulation in maize and P. dilatatum leaves showed a marked surface-specific response to light orientation. Compared to the adaxial sides of the C₄ monocotyledonous leaves, the abaxial surfaces had more stomata and the BS/M section area ratio was significantly higher. Differences in dorso-ventral structure, particularly in Kranz anatomy, serve not only to maximize photosynthetic capacity with respect light orientation in C₄ monocotyledonous leaves but also allow adaxial and abaxial-specific signalling from the respective M cells.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.