The rate of CO(2) assimilation controls the expression and activity of glutamine synthetase through sugar formation in sunflower (Helianthus annuus L) leaves

The expression and activity of glutamine synthetase (GS, EC 6.3.1.2) were examined in relation to the rate of CO2 assimilation in sunflower (Helianthus annuus L.) leaves. Intact plants were kept in the dark for 72 h and subsequently exposed to light under different atmospheric CO2 concentrations (100, 400 and 1200 microl l-1) for 6 h. The in vivo rates of net CO2 assimilation correlated with atmospheric CO2 concentrations. Stomatal conductances and transpiration rates remained largely unaffected by CO2 levels. Exposure of the plants to increasing CO2 concentrations in the light caused concomitant increases in the contents of starch and soluble sugars and a decrease in the nitrate content in leaves. Both cytosolic and chloroplastic (GS2) GS activities were higher at elevated CO2. A greater accumulation of GS2 mRNA was also observed under high CO2. Exogenous supply of sucrose to detached leaves greatly increased the levels of GS enzyme activity and of mRNA for chloroplastic GS in the dark. These results indicate that GS expression and activity in sunflower leaves are modulated by the rate of CO2 assimilation, and that photosynthesized sugars are presumably involved as regulatory metabolites.     

温室CO2施肥对黄瓜幼苗根系生长及分泌物和伤流液组成的影响

为探讨温室蔬菜CO2施肥的根际效应,以黄瓜幼苗为试材,研究了CO2施肥(上午施肥/上、下午施肥;施肥浓度/对照浓度(950±50)/(350±50)μmol/molCO2)对根系生长及分泌物和伤流液组成的影响。结果表明,CO2施肥明显促进黄瓜幼苗根系发育,根系生物量显著增加;单株根系分泌物中氨基酸、糖、有机酸和酚酸总量增加,但单位鲜重根系分泌量却呈现增幅减少、无变化甚至降低趋势,说明单株分泌量增加主要由根系生长量的增加所引起。CO2施肥促进幼苗对养分的吸收,伤流液中矿质元素、ZT浓度增加,但GA、ABA和IAA浓度降低;与上午CO2施肥相比,上、下午均CO2施肥的效果更明显。CO2施肥促进了黄瓜幼苗根系发育及其代谢活性,为地上部的旺盛生长创造了条件。     

Impact of atmospheric CO2 on growth, photosynthesis and nitrogen metabolism in cucumber (Cucumis sativus L.) plants

Expression and activity of nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2) were analysed in relation to the rate of CO(2) assimilation in cucumber (Cucumis sativus L.) leaves. Intact plants were exposed to different atmospheric CO(2) concentrations (100, 400 and 1200microLL(-1)) for 14 days. A correlation between the in vivo rates of net CO(2) assimilation and the atmospheric CO(2) concentrations was observed. Transpiration rate and stomatal conductance remained unaffected by CO(2) levels. The exposure of the cucumber plants to rising CO(2) concentrations led to a concomitant increase in the contents of starch and soluble sugars, and a decrease in the nitrate content in leaves. At very low CO(2), NR and GS expression decreased, in spite of high nitrate contents, whereas at normal and elevated CO(2) expression and activity were high although the nitrate content was very low. Thus, in cucumber, NR and GS expression appear to be dominated by sugar levels, rather than by nitrate contents.     

Lateral root frequency decreases when nitrate accumulates in tobacco transformants with low nitrate reductase activity: consequences for the regulation of biomass partitioning between shoots and root1

Accumulation of nitrate in the shoot of low-nitrate reductase tobacco transformants leads to an increase of the shoot:root ratio to higher values than in nitrogen-sufficient wild-type plants, even though the transformants are severely deficient in organic nitrogen. In the present paper, wild-type plants and low- nitrate reductase transformants were grown on vertical agar plates to investigate whether this inhibition of root growth by internal nitrate (i) can be reversed by adding sugars to the roots and (ii) is due to slower growth of the main roots or to a decreased number of lateral roots. When grown with a low nitrate supply, the transformants resembled wild-type plants with respect to amino acid and protein levels, shoot-root allocation, lateral root frequency, and rates of growth. When the transformants were grown with a high nitrate supply in the absence of sucrose they grew more slowly and had lower levels of amino acids and protein than wild-type plants, but accumulated more nitrate and developed a high shoot:root ratio. Root length was not affected, but the number of lateral roots per plant decreased. The slower root growth was accompanied by an increase of the concentration of sugars in the roots. Addition of 2% sucrose to the medium partially reversed the high shoot:root ratio in the transformants, but did not increase the frequency of lateral roots. It is concluded that nitrate accumulation in the plant leads to decreased root growth via (i) changes in carbon allocation leading to decreased allocation of sugars to root growth, and (ii) a decrease in the number of lateral roots and a shift in the sensitivity with which root growth responds to the sugar supply. This revised version was published online in June 2006 with corrections to the Cover Date.     

Diurnal variations in growth rate and growth substrate levels of spinach (Spinacia oleracea L.) under nitrogen-limiting conditions

Spinach plants were grown in hydroponic culture provided with variable limiting amounts of N. During a complete diurnal cycle, growth of the root and shoot parts, as well as levels of soluble and insoluble sugars and of free amino acids, were monitored. No clear relationship could be detected between the level of N feeding and the levels of free sugars and amino acids. Analysis of variance revealed that the variances in the relative growth rates of plant root and shoot could be correlated with the levels of sugars and amino acids. Root amino acid concentration could be correlated with shoot amino acid concentration and root sugar concentration. No relationship was found between the variances in root and shoot free sugar concentrations.     

Accumulation of nitrate in the shoot acts as a signal to regulate shoot-root allocation in tobacco†

Mutants and transformants of tobacco (Nicotiania tabacum L. cv Gatersleben 1) with decreased expression of nitrate reductase have been used to investigate whether nitrate accumulation in the shoot acts as a signal to alter allocation between shoot and root growth. (a) Transformants with very low (1–3% of wild-type levels) nitrate reductase activity had growth rates, and protein, amino acid and glutamine levels similar to or slightly lower than a nitrate-limited wild-type, but accumulated large amounts of nitrate. These plants should resemble a nitrate-limited wild-type, except in responses where nitrate acts as a signal. (b) Whereas the shoot:root ratio decreases from about 3.5 in a well-fertilized wild-type to about 2 in a nitrate-limited wild-type, the transformants had a very high shoot:root ratio (8–10) when they were grown on high nitrate. When they were grown on lower nitrate concentrations their shoot:root ratio declined progressively to a value similar to that in nitrate-limited wild-types. Mutants with a moderate (30–50%) decrease of nitrate reductase also had a small but highly significant increase of their shoot:root ratio, compared to the wild-type. The increased shoot:root ratio in the mutants and transformants was due to a stimulation of shoot growth and an inhibition of root growth. (c) There was a highly significant correlation between leaf nitrate content and the shoot:root ratio for eight genotypes growing at a wide range of nitrate supply. (d) A similar increase of the shoot:root ratio in nitrate reductase-deficient plants, and correlation between leaf nitrate content and the shoot:root ratio, was found in plants growing on ammonium nitrate. (f) Split-root experiments, in which the transformants were grown with part of their root system in high nitrate and the other part in low nitrate, showed that root growth is inhibited by the accumulation of nitrate in the shoot. High concentrations of nitrate in the rooting medium actually stimulate local root growth. (g) The inhibition of root growth in the transformants was relieved when the transformants were grown on limiting phosphate, even though the nitrate content of the root remained high. This shows that the nitrate-dependent changes in allocation can be overridden by other signals that increase allocation to root growth. (h) The reasons for the changed allocation were investigated in transformants growing normally, and in split-root culture. Accumulation of nitrate in the shoot did not lead to decreased levels of amino acids or protein in the roots. However, it did lead to a strong inhibition of starch synthesis and turnover in the leaves, and to decreased levels of sugars in the root. The rate of root growth was correlated with the root sugar content. It is concluded that these changes of carbon allocation could contribute to the changes in shoot and root growth.     

The influence of root assimilated inorganic carbon on nitrogen acquisition/assimilation and carbon partitioning

Understanding of the influences of root-zone CO2 concentration on nitrogen (N) metabolism is limited. The influences of root-zone CO2 concentration on growth, N uptake, N metabolism and the partitioning of root assimilated 14C were determined in tomato (Lycopersicon esculentum). Root, but not leaf, nitrate reductase activity was increased in plants supplied with increased root-zone CO2. Root phosphoenolpyruvate carboxylase activity was lower with NO3(-)- than with NH4(+)-nutrition, and in the latter, was also suppressed by increased root-zone CO2. Increased growth rate in NO3(-)-fed plants with elevated root-zone CO2 concentrations was associated with transfer of root-derived organic acids to the shoot and conversion to carbohydrates. With NH4(+)-fed plants, growth and total N were not altered by elevated root-zone CO2 concentrations, although 14C partitioning to amino acid synthesis was increased. Effects of root-zone CO2 concentration on N uptake and metabolism over longer periods (> 1 d) were probably limited by feedback inhibition. Root-derived organic acids contributed to the carbon budget of the leaves through decarboxylation of the organic acids and photosynthetic refixation of released CO2.     

Growth, photosynthesis, nitrogen partitioning and responses to CO2 enrichment in a barley mutant lacking NADH-dependent nitrate reductase activity

Plant growth, photosynthesis and leaf constituents were examined in the wild-type (WT) and mutant nar1 of barley (Hordeum vulgare L. cv. Steptoe) that contains a defective structural gene encoding NADH-dependent nitrate reductase (NADH-NAR). In controlled environment experiments, total biomass, rates of photosynthesis, stomatal conductance, intercellular CO(2) concentrations and foliar non-structural carbohydrate levels were unchanged or differed slightly in the mutant compared with the WT. Both genotypes displayed accelerated plant growth rates when the CO(2) partial pressure was increased from 36 to 98 Pa. Total NADH-NAR activity was 90% lower in the mutant than in the WT, and this was further decreased by CO(2) enrichment in both genotypes. Inorganic nitrate was greater in the mutant than in the WT, whereas in situ nitrate assimilation by excised leaves was two-fold greater for the WT than for the mutant. Foliar ammonia was 50% lower in the mutant than in the WT under ambient CO(2). Ammonia levels in the WT were decreased by about one-half by CO(2) enrichment, whereas ammonia was unaffected by elevated CO(2) in mutant leaves. Total soluble amino acid concentrations in WT and mutant plants grown in the ambient CO(2) treatment were 30.1 and 28.4 micromol g(-1) FW, respectively, when measured at the onset of the light period. Seven of the twelve individual amino acids reported here increased during the first 12 h of light in the ambient CO(2) treatment, leading to a doubling of total soluble amino acids in the WT. The most striking effect of the mutation was to eliminate increases of glutamine, aspartate and alanine during the latter half of the photoperiod in the ambient CO(2) treatment. Growth in elevated CO(2) decreased levels of total soluble amino acids on a diurnal basis in the WT but not in mutant barley leaves. The above results indicated that a defect in NADH-NAR primarily affected nitrogenous leaf constituents in barley. Also, we did not observe synergistic effects of CO(2) enrichment and decreased foliar NADH-NAR activity on most N-containing compounds.     

Stimulatory effects of certain amino acids,sugars, organic acids,nucleic acids and metals on nitrate utilization by alkalophilic Bacillus

The growth of alkalophilic Bacillus no. A-40-2 with nitrate as the nitrogen source was highly stimulated by the addition of 0.1% of certain amino acids, sugars, organic acids, nucleic acids, or Fe²⁺ or Mn²⁺ at concentrations of 10 mM or more to the medium, resulting in maximum growth after 24 h. Other alkalophilic Bacillus strains also showed the same results. A decrease in the amount of nitrate in the medium was observed. The optimum pH of nitrate reductase was 7.5.     

The profiles of nitrate reductase and carbonic anhydrase activity in batch cultivation of the marine microalgae Tetraselmis gracilis growing under different aeration conditions

Tetraselmis gracilis, a Prasinophycean alga found in estuaries and in the open ocean, was cultivated under different conditions of aeration, which resulted in variations of inorganic carbon in the medium. Relative growth rates, nitrate reductase and carbonic anhydrase activities were daily determined and correlated to the concentration of nitrate, nitrite, phosphate, inorganic and organic carbon in the media. Nitrate reductase catalyzes the reversible carbon dioxide hydration reaction. The activity profiles of both enzymes during 10 days of cultivation under aeration with air showed an inverse relationship: the maximum in the activity of nitrate reductase coincided with the minimum of carbonic anhydrase activity. An ionizable organic carbon species with pKa in the range of metabolites of the photorespiratory path was found parallel with the increase of carbonic anhydrase activity and the decrease of nitrate reductase activity. The onset of photorespiration is probably one of the factors involved in the simultaneous regulation of these enzymatic processes. Cultures aerated with air containing 5% CO2 showed different profiles for nitrate reductase activity and nitrate uptake.     

Influence of nitrate supply on concentrations and translocation of abscisic acid in barley (Hordeum vulgare)

Spring barley ( Hordeum vulgare L. cv. Golf) was grown at different nitrate supply rates, controlled by using the relative addition rate technique, in order to elucidate the relationship between nitrate-N supply and root and shoot levels of abscisic acid (ABA). The plants were maintained as (1) standard cultures where nitrate was supplied at relative addition rates (RAs) of 0.03, 0.09 and 0.18 day⁻¹, and (2) split-root cultures at RA 0.09 day⁻¹ but with the nitrate distributed between the two root parts in ratios of 100:0, 80:20 and 60:40. Time-dependent changes in root and shoot concentrations of ABA (determined by radioimmunoassay using a monoclonal antibody) were observed in both standard and split-root cultures during 12 days of acclimation to the different nitrate regimes. However, the ABA responses were similar at all nitrate supply rates. Further experiments were performed with split-root cultures where the distribution of nitrate between the two root parts was reversed from 80:20 to 20:80 so that short-term effects to local perturbations of nitrate supply could be studied without altering whole-plant N absorption. Transient increases in ABA concentrations (maximum of 25 to 40% after 3 to 4 h) were observed in both subroot parts, as well as in xylem sap and shoot tissue. By pruning the root system it was demonstrated that the change in ABA had its origin in the subroot part receiving the increased nitrate supply (i.e. switched from 20 to 80% of the total nitrate supply). The data indicate that ABA responses are easily transmitted between different organs, including transmission from one set of seminal roots to another via the shoot. The data do not provide any indication that long-term nitrate supplies or general nitrogen status of barley plants affect, or are otherwise related to, the average tissue ABA concentrations of roots and shoots.     

Compartmentation of labeled fixation products in intact mesophyll protoplasts from Avena sativa L. after in-situ inhibition of the chloroplast phosphate translocator

Leaf mesophyll protoplasts of oat (Avena sativa L.) were allowed to fix ¹⁴C-labeled bicarbonate in the absence or presence of pyridoxal phosphate (PLP), a specific inhibitor of the phosphate translocator of the inner envelope membrane of chloroplasts. The incubation was terminated by a method of rapid integrated protoplast homogenization and fractionation, and compartmented levels of label contained in sugars, phosphate esters, amino acids and organic acids were determined. The results show that the addition of PLP to a suspension of intact protoplasts causes an accumulation of phosphate esters in the chloroplasts stroma for up to 2.5 min of incubation, with a corresponding decrease in the cytosol. Prolonged treatment of protoplasts with PLP in the light resulted in a decrease of starch-associated label, combined with higher levels of labeled sugars in the cytosol, indicating a switch from phosphorolytic to hydrolytic starch degradation. Together with the determination of pool sizes of triose phosphates and of inorganic phosphate, the results demonstrate that the method employed is an important tool in investigating processes of intracellular regulation. They are discussed with respect to the permeability and possible side reactions of PLP, as well as in the light of reports on PLP action on isolated chloroplasts.Abbreviations P_i orthophosphate - PLP pyridoxal 5-phosphate - TP triosephosphate     

Strain-specific salt tolerance and chemotaxis ofAzospirillum brasilense and their associative N-fixation with finger millet in saline calcareous soil

Three salt-tolerantAzospirillum brasilense strains were isolated from the roots of finger millet grown in saline calcareous soil and characterized. The effect of various salts on growth and N₂ase activity of these strains was tested and strain STR1 was found more tolerant at higher concentrations of Cl^-, SO₄ ² and HCO₃ ^-. Bicarbonate was found to be the most toxic. The content and concentrations of root exudates of finger millet genotypes were different and chemotaxis to sugars, amino acids, organic acids and root exudates was strain specific. Under salt stress, significant interactions between strains and genotypes of finger millet resulted in different responses of N₂ase activity, endo- and exorhizospheric population, dry weight of root, shoot and grain yield.     

The Role of Nitrate in the Osmoregulation of Lettuce (Lactuca sativa L.) Grown at Different Light Intensities

Blom-Zandstra, M. and Lampe, J. E. M., 1985. The role of nitratein the osmoregulation of lettuce (Lactuca sativa L.) grown atdifferent light intensities.—J. exp. Bot. 36: 1043–1052. The effect of different light intensities on the nitrate accumulationvis-à-vis the concentration of other solutes in plantsap expressed from lettuce leaves was studied. After growinglettuce plants under constant environmental conditions for 52d, they were transferred to different light intensities andharvested periodically. A quantitative analysis of componentsin solution in the expressed plant sap showed a decrease innitrate concentration and an increase in the organic acids (mainlymalate) and sugars (mainly glucose) with increasing light intensity.The light intensity only slightly increased the osmolarity ofthe expressed plant sap. The measured osmolarity correspondedvery well with the value estimated from the quantitative analysesimplying that all osmotically active compounds had been accountedfor. The decrease in nitrate concentration in the expressedplant sap was fully compensated for by an increase in the dissociatedorganic acids that partly dissociate twofold to sustain electroneutralityand by an increase in both organic acids and sugars to maintainthe osmolarity. The suggestion is supported that nitrate mayserve as osmoticum at low light conditions to compensate forthe shortage of carbohydrates resulting from suboptimal photosynthesis. Key words: Nitrate accumulation, osmoregulation, Lactuca saliva L.     

Change in sugar, sterol and fatty acid composition in banana meristems caused by sucrose-induced acclimation and its effects on cryopreservation

To understand the mechanisms of sucrose‐induced acclimation in relation to plant cryopreservation, sugars, sterols, fatty acids of different lipid fractions (neutral lipids, glycolipids and sphingolipids and phospholipids), as well as free fatty acids were analyzed in proliferating meristem cultures of different banana varieties. The four banana varieties that were selected show different post‐thaw shoot regeneration rates (0–53.4%). All mentioned parameters were analyzed using (1) control meristems that were cultured on a normal sucrose concentration (0.09 M), which resulted in low survival after cryopreservation; and (2) 2‐week sucrose precultured meristems (0.4 M). This sucrose preculture, essential for regeneration after cryopreservation, resulted in a significant increase of each of seven sugars detected. The ratio of stigmasterol/sitosterol (St/Si) in sucrose‐pretreated meristems significantly increased. The sucrose pretreatment also resulted in a significant increase of total fatty acid content of the neutral lipid fraction and of the glycolipid and sphingolipid fraction, as well as the total free fatty acid content. The individual fatty acid content of the phospholipids was differently changed by the sucrose pretreatment for the given varieties studied. In most cases, sucrose pretreatment resulted in an increase of the double bond index (DBI) in the neutral lipids and a decrease of DBI in the glycolipids and sphingolipids, in phospholipids as well as in free fatty acids. Principal component analysis of all collected data revealed that (1) for the control material, sucrose and total sugar contents were closely linked to the post‐thaw shoot regeneration, suggesting that sucrose and total sugar may be main limiting factors to survive cryopreservation; (2) accumulation of large quantities of sugars (glucose, fructose, sucrose and total sugar) in sucrose‐pretreated material cannot explain the differences in survival after cryopreservation of the four banana varieties. We assume that a minimal amount of sugars is needed in meristem cultures to survive cryopreservation. Still, other limiting factors do influence the survival following the sucrose pretreatment. We observed that the parameters which are closely linked to the post‐thaw shoot regeneration are a minimal change in the ratios of St/Si, the minimal change of the DBI of phospholipids and free fatty acids, as well as linoleic acid content (C18:2); and (3) inositol, raffinose, myristic acid (C14:0) and oleic acid (C18:1) were present in small quantities; however, they could be correlated to survival after cryopreservation, suggesting that they may be also involved in cryopreservation process.     

Growth and shoot formation in protoplast-derived calli of Brassica oleracea ssp. acephala and ssp. capitata

The effect of media components and environmental factors on growth and organogenesis of protoplast-derived calli of curly kale and cabbage were tested. Optimal growth (fresh weight increase of calli, shoots and roots) was found at 60 mM sucrose. Lower sucrose concentrations (3–30 mM) were favourable for shoot formation. Nitrate concentrations from 23 to 100 mM in combination with 8 or 21 mM ammonium were optimal for shoot formation. However, growth was reduced by high (100 mM) nitrate concentration. The effects of various organic nitrogen compounds at 0.5 and 2 mM were tested. Glutamine did not influence shoot formation and barely growth. Proline at 0.5 mM stimulated growth of cabbage calli but decreased growth of curly kale calli, and at 2 mM, proline also inhibited shoot production. Adenine sulphate decreased growth of cabbage calli at 0.5 mM, and at 2 mM shoot production was also reduced. Spermidine and spermine inhibited both growth and differentiation. Putrescine resulted in about 50% higher fresh weights, and also increased the number of calli producing shoots by about 35%. More calli produced shoots in white light than in blue or red light or in darkness. The length of the photoperiod or intensity of light was not critical for shoot production.     

C14 distribution in photosynthate of tomato as influenced by substrate copper and molybdenum level and nitrogen source

Summary The influence of nitrogen source and micronutrient treatment on the apportionment of C¹⁴ in alcohol extracts was studied in tomato. Plants were exposed at several stages of maturity to C¹⁴O₂ and the ethanol-soluble photosynthate fractionated into sugars, organic acids, and amino acids.Ammonium-treated plants had low levels of organic acids at all stages of maturity, suggesting a rapid conversion to amino acid residues. However, the addition of copper in the absence of molybdenum resulted in increased labelling of the organic acid fraction under nitrate nutrition. The amino acid fraction was reduced at some stages of growth under these conditions. This may indicate that copper interferes with the role of molybdenum in the enzymatic reduction of nitrate.Measurement of the total quantity of some individual free amino acids indicated more efficient utilization of these for protein synthesis in the presence of copper. On the other hand, assay of the C¹⁴-activity of individual free amino acids showed that degree of label bore little relation to micro-nutrient treatment.Plant Science Department and Botany Department, University of Connecticut. Scientific Contribution No. 116, Agricultural Experiment Station, University of Connecticut, Storrs. This paper is based on a portion of the senior author's Ph. D. thesis (University of Connecticut).