The effect of nitrate assimilation deficiency on the carbon and nitrogen status of Arabidopsis thaliana plants

Carbon (C) and nitrogen (N) metabolism are integrated processes that modulate many aspects of plant growth, development, and defense. Although plants with deficient N metabolism have been largely used for the elucidation of the complex network that coordinates the C and N status in leaves, studies at the whole-plant level are still lacking. Here, the content of amino acids, organic acids, total soluble sugars, starch, and phenylpropanoids in the leaves, roots, and floral buds of a nitrate reductase (NR) double-deficient mutant of Arabidopsis thaliana (nia1 nia2) were compared to those of wild-type plants. Foliar C and N primary metabolism was affected by NR deficiency, as evidenced by decreased levels of most amino acids and organic acids and total soluble sugars and starch in the nia1 nia2 leaves. However, no difference was detected in the content of the analyzed metabolites in the nia1 nia2 roots and floral buds in comparison to wild type. Similarly, phenylpropanoid metabolism was affected in the nia1 nia2 leaves; however, the high content of flavonol glycosides in the floral buds was not altered in the NR-deficient plants. Altogether, these results suggest that, even under conditions of deficient nitrate assimilation, A. thaliana plants are capable of remobilizing their metabolites from source leaves and maintaining the C–N status in roots and developing flowers.     

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide and O-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO(2) from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5'-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO(2) led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO(2) on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO(2), APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO(2) also recovered both enzyme activities, with OAS again influenced only APR. (35)SO(4)(2-) feeding showed that treatment in air without CO(2) severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of (35)S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of (35)S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.     

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.     

Effects of cotyledons and nitrate on the nitrogen assimilation ofPhaseolus vulgaris

Summary In an experiment performed under greenhouse conditions usingPhaseolus vulgaris cv. Carioca inoculated withRhizobium strain CO5, effects of cotyledons and mineral nitrogen on the initial process of nitrogen assimilation were evaluated. Plants were maintained intact or had either both or half of both cotyledons removed six days after planting. Levels of mineral nitrogen corresponded to the addition of 0 or 1.5 mg N/plant/day three days before each of the four harvests (8, 10, 12 and 14 days after planting). Cotyledon removal generally decreased nodule number and dry weight and total nitrogenase activity, although there was no effect on specific nodule efficiency, but the nitrate reductase activity was increased in both shoots and roots. Mineral nitrogen decreased nodulation and nitrogenase activity when applied 9 and 11 days after planting, but increased shoot and root nitrate reductase activity and total nitrogen incorporation, indicating that plants could be nitrogen limited during the initial period of growth.

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Resumen En un experimento realizado en invernadero conPhaseolus vulgaris, cv. Carioca, inoculado conRhizobium (cepa CO5) fue evaluado los efectos de los cotiledones y del nitrógeno mineral en el proceso inicial de la assimilación de N. Las plantas fueron mantenidas intactas o con la mitad o todos los cotiledones retirados a los 6 dias despues de la siembra (DDS). El N mineral fue aplicado en la dosis de 0 ó 1.5 mg N/planta/dia, 3 dias antes de cada una de los 4 casechas realizadas (8, 10, 12 y 14 DDS). La retirada de los cotiledones generalmente disminuyó el número y el peso seco de los nódulos y la actividad de la nitrogenase, mas não hubo influencia en la eficiencia específica de los nódulos. La actividad de la nitrato reductasa aumentó tanto en la parte aérea como en las raíces. El nitrógeno mineral disminuyó la nodulación y la actividad de la nitrogenasa cuando la aplicación ocurrió a los 9 y 11 DDS, mas aumentó la actividad de la nitrato reductasa de las raíces y de la parte aérea, asi como el contenido de N en la planta, indicando que estas podrian estar limitadas por nitrógeno en la fase inicial de crescimento.

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Résumé Dans une expérimentation réalisée dans les conditions de serre au moyen dePhaseolus vulgaris cv Carioca inoculée avec la souche CO5 deRhizobium, on a évalué les effets de cotylédons et de l'azote minéral sur le processus initial de l'assimilation azotée. Les plants ont été soit maintenus intacts soit amputés des deux ou de la moitié des deux cotylédons, 6 jours après la plantation. Les teneurs en azote minéral correspondaient à l'ajout de 0 ou 1.5 mg d'azote par plant et par jour, trois jours avant chacune des 4 récoltes (8, 10, 12 et 14 jours après la plantation). L'amputation de cotyledon diminue d'une manière générale le nombre de nodules, le poids sec et l'activité totale de nitrogénase, bien qu'il n'y ait aucun effet sur l'efficience spécifique de nodule mais l'activité de nitrate réductase était augmentée tant dans les pousses que dans les racines. L'azote minéral a déterminé la nodulation et l'activité de nitrogénase lorsque l'application avait lieu les 9ème et 11ème jours après la plantation, mais elle a augmenté l'activité de nitrate réductase chez les pousses et les racines ainsi que l'incorporation totale d'azote, indiquant par là que la plante pouvait être limitée en azote durant la période initiale de croissance.

     

Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO₂ assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH₄ ⁺ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu.     

Effects of nitrogen dioxide and nitrate nutrition on growth and nitrate assimilation in bean leaves

The influence of nutrient nitrate level (0-20 millimolar) on the effects of NO₂ (0-0.5 parts per million) on growth, K, photosynthetic pigment, N contents, and the activities of enzymes of N assimilation was studied in bean (Phaseolus vulgaris L. cv Kinghorn Wax) leaves. Exposing 7-day old bean seedlings for 5 days continuously to 0.02 to 0.5 parts per million NO₂ increased plant height, fresh weight, chlorophyll, carotenoid, organic N and nitrate contents, and nitrate reductase and glutamate synthase activities in the leaves of seedlings supplied with no external N. At 20 millimolar nitrate, most of the parameters examined were inhibited except for organic N and nitrate contents and glutamate synthase activity which increased in most cases. Generally, with an increase in NO₂ concentration, the stimulatory effect declined and/or the inhibitory effect increased. A 3-hour exposure of 12-day-old bean seedlings to 0.1 to 2.0 parts per million NO₂ increased nitrate content and nitrate reductase activity at each nutrient nitrate level except for a slight inhibition of enzyme activity during exposure to 2.0 parts per million NO₂ at 20 millimolar nitrate. The experiments demonstrated that the effect of NO₂ is strongly influenced by nutrient N level and that NO₂ is assimilated into organic nitrogenous compounds to serve as a source of N, only to a limited extent.     

Carbon and nitrogen metabolism in the seagrass, Zostera marina L.: Environmental control of enzymes involved in carbon allocation and nitrogen assimilation

This study experimentally examined influences of environmental variables on the activities of key enzymes involved in carbon and nitrogen metabolism of the submersed marine angiosperm, Zostera marina L. Nitrate reductase activity in leaf tissue was correlated with both water-column nitrate concentrations and leaf sucrose levels. Under elevated nitrate, shoot nitrate reductase activity increased in both light and dark periods if carbohydrate reserves were available. When water-column nitrate was low, glutamine synthetase activity in leaf tissue increased with environmental ammonium. In contrast, glutamine synthetase activity in belowground tissues was statistically related to both nitrate and temperature. At the optimal growth temperature for this species (ca. 25 °C), increased water-column nitrate promoted an increase in glutamine synthetase activity of belowground tissues. As temperatures diverged from the optimum, this nitrate effect on glutamine synthetase was no longer evident. Activities of both sucrose synthase and sucrose-P synthase were directly correlated with temperature. Sucrose-P synthase activity also was correlated with salinity, and sucrose synthase activity was statistically related to tissue ammonium. Overall, the enzymatic responses that were observed indicate a tight coupling between carbon and nitrogen metabolism that is strongly influenced by prevailing environmental conditions, especially temperature, salinity, and environmental nutrient levels.     

Seasonal nitrogen assimilation and carbon fixation in a fjordic sea loch

Carbon (C) fixation and nitrogen (N) assimilation rates havebeen estimated from ¹⁴C and ¹⁵N techniques for a 12 month periodin a Scottish sea loch. The maximum rate of nitrogen assimilated(29.92 mmol N m^–2 day^–1) was in April at the mostseaward station; similar high rates were experienced duringMay at the other stations. Carbon fixation rates were maximal(488–4047 mg C m^–2day^–1) at the time of highphytoplankton biomass (maximum 8.3 mg m^–3 chlorophylla) during May, whilst nitrate concentrations remained >0.7µ.mol l^–1. C:N assimilation ratios suggest nitrogenlimitation only during the peak of the spring bloom, althoughat times nitrogen (nitrate and ammonium) concentration fellto 0.2 µmol l^–1 in the following months. The verticalstability of the water column, influenced by tidal and riverineflushing, varied along the axis of the loch, resulting in markeddifferences between sampling stations. Although ammonium waspreferentially assimilated by phytoplankton, >50% of productionwas supported by nitrate uptake and only during the summer monthswas the assimilation of ammonium quantitatively important.     

Dirunal leaf movement, chlorophyll fluorescence and carbon assimilation in soybean grown under different nitrogen and water availabilities

Diurnal heliotropic leaf movements, photosynthetic gas exchange, and the ratio of variable fluorescence to maximum fluorescence (Fv/Fm) of unrestrained and horizontally restrained leaves from soybean (Glycine max cv. Cumberland) plants grown in two different water and two different nitrogen treatments were measured. Leaves of plants grown in low water or low nitrogen availability treatments displayed more pronounced diaheliotropism (solar tracking) in the afternoon and a longer period of paraheliotropism (light avoiding) at midday relative to those of well-watered, high-nitrogen-grown plants. Photosaturated photosynthetic rates and the photon flux required to saturate photosynthesis were reduced by water stress and nitrogen deficiency. Compared to horizontal leaves, irradiance on orienting leaves was nearer to the breakpoint of the photosynthetic light response curve, where photosynthesis is co-limited by ribulose biphosphate regeneration and carboxylation. This would increase the carbon return on investments of nitrogen into photosynthesis. A positive linear relationship between Fv/Fm and quantum yield of photosynthesis was measured. Leaves of low-nitrogen-grown plants had earlier and more prolonged reductions in Fv/Fm at midday compared to leaves of high nitrogen grown plants of the same water treatment. Within the same water and nitrogen treatment, horizontally restrained leaves had lower midday Fv/Fm in relation to orienting leaves. Nitrogen deficiency and water stress enhanced this difference such that horizontally restrained leaves of low water and low nitrogen grown plants had earlier and longer midday depressions in Fv/Fm.     

The effects of nitrate on the enzymes involved in nitrogen assimilation in nodulated pea roots

Pea Plants ( Pisum sativaum L. ev. Little Marvel) were grown in N-free medium and when well nodulated (28 days) were supplied for 8 days with nitrate or ammonium. Over the 8 days of nitrate treatment, total amino and amide N in sap declined, and the proportion of aspartate relative to the other amino acids increased. After 8 days of treatment, nitrogenase (EC 1.18.2.1) activity in nitrate-treated plants declined to about 30% of the activity in controls even though nodules were not directly in contact with nutrient solution. Nitrogenase activity was also decreased by the addition of ammonium chloride (10 m M ). With addition of nitrate or ammonium. clear signs of senescence began to show in the nodules after 4 days. Nitrate reductase (EC 1.6.6.1) activity was induced in roots by nitrate, but decreased sharply in nodules. In response to nitrate addition, newly formed root tissues showed 3- to 5-times higher glutamine synthetase (GS. EC 6.3.1.4) activity than newly formed tissues of control plants, expressed on a protein or weight basis. In complementary experiments, when ammonium salts were used instead of nitrates, the increase in GS activity was significantly lower. GS activity decreased in nodules of treated plants and total extractable protein was 3 times lower in nodules of nitrate-treated plants than in controls at day 8 of treatment.     

Developmental changes of enzymes of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation in peach leaves

The developmental profile of the activities of some enzymes involved in malate metabolism, namely phosphoenolpyruvate carboxylase (PEPC; EC 4. 1. 1. 31), NAD⁺-linked (EC 1. 1. 1. 37) and NADP⁺-linked (EC 1. 1. 1. 82) malate dehydrosenase (MDH), NAD⁺linked (EC 1. 1. 1. 39) and NADP⁺-linked (EC 1. 1. 1. 40) malic enzyme (ME), has been determined in leaves of peach [ Prunus persica (L.) Batsch cv. Maycrest], a woody C₃ species. In order to study the role of these enzymes, their activities were related to developmental changes of photosynthesis, respiration, and capacity for N assimilation. Activities of PEPC, NAD(P)⁺-MDH and NADP⁺-ME were high in young expanding leaves and decreased 2- to 3-fold in mature ones, suggesting that such enzymes play some role during the early stages of leaf expansion. In leaves of peach, such a role did not seem to be linked to C₃ photosynthesis or nitrate assimilation, in that photosynthetic O₂ evolution and activities of nitrate reductase (EC 1. 6. 6. 1) and glutamine synthetase (EC 6. 3. 1. 2) increased during leaf development. In contrast, leaf respiration strongly decreased with increasing leaf age. We suggest that in expanding leaves of this woody species the enzymes associated with malate metabolism have anaplerotic functions, and that PEPC may also contribute to the recapture of respiratory CO₂.     

Elevated CO2 alters tissue balance of nitrogen metabolism and downregulates nitrogen assimilation and signalling gene expression in wheat seedlings receiving high nitrate supply

Tissue and canopy-level evidence suggests that elevated carbon dioxide (EC) inhibits shoot nitrate assimilation in plants and thereby affects nitrogen (N) and protein content of the economic produce. It is speculated that species or genotypes relying more on root nitrate assimilation can adapt better under EC due to the improved/steady supply of reductants required for nitrate assimilation. A study was conducted to examine the effect of EC on N assimilation and associated gene expression in wheat seedlings. Wheat genotypes, BT-Schomburgk (BTS) with comparatively high leaf nitrate reductase (NR) activity and Gluyas Early (GE) with high root NR activity were grown in hydroponic culture for 30 days with two different nitrate levels (0.05 mM and 5 mM) in the climate controlled growth chambers maintained at either ambient (400 ± 10 μmol mol⁻¹) or EC (700 ± 10 μmol mol⁻¹) conditions. Exposure to EC downregulated the activity of enzyme NR and glutamate synthase (GOGAT) in leaf tissues, whereas in roots, activities of both the enzymes were upregulated by exposure to EC. In addition, EC downregulated N assimilation and signalling gene expression under high N availability. Root N assimilation was less affected in comparison with shoot N assimilation; thereby, the proportion of root contribution towards total assimilation was higher. The results suggest that EC could alter and re-programme N assimilation and signalling in wheat seedlings. The genotype and tissue-specific effects of EC on N assimilation also warrants the need for identification of suitable genotypes and revision of fertiliser regime for tapping the beneficial effects of EC conditions.

     

Effects of elevated ozone and nitrogen addition on leaf nitrogen metabolism in poplar

臭氧和氮添加对杨树叶片氮代谢的影响臭氧(O₃)污染和氮(N)沉降/施肥都能同时影响植物的生长。然而,几乎没有研究探究O₃和N添加对植物叶片N代谢过程的复合影响。本研究在开顶式气室(OTC)中对杨树进行了为期95 d的熏蒸实验,包括两个O₃水平(NF,环境O₃水平;NF60,NF + 60 ppb O₃)和4个N处理(N0,没有N添加;N50,N0 + 50 kg N ha⁻¹ yr⁻¹;N100,N0 + 100 kg N ha⁻¹ yr⁻¹;N200,N0 + 200 kg N ha⁻¹ yr⁻¹)。测定了与叶片N代谢相关的一些指标,包括叶片N代谢酶的活性、总叶片N浓度、NO₃⁻-N浓度、NH₄⁺-N浓度、总氨基酸浓度(TAA)、总可溶性糖的浓度(TSP)。研究结果表明,相对于NF,在8月份NF60处理显著刺激了硝酸还原酶(NR)的活性,使其升高了47.2%。当平均所有的N处理和两次取样时间时,NF60处理下谷氨酰胺酶(GS)的活性比NF处理下的高57.3%。但是O₃处理并没有显著影响TSP浓度,并且在8月也没有降低TAA的浓度。相对N0,高的N添加处理(N200)显著增加了杨树叶片的饱和光合速率(A_sat) 24%,并且分 别在8和9月增加了总叶片N浓度70.3%和43.3%。但是在8月份,N200处理下光合N利用效率比N0的低26.1%。这表明N添加导致的A_sat和叶片总的N浓度的升高是不匹配的,高N处理下,叶片中一些剩余的N没有被用于优化植物碳的同化。同时,也发现高N添加显著刺激了叶片N代谢过程,叶片中的NO₃⁻-N浓度、NH₄⁺-N浓度、TAA浓度、NR和GS活性都显著升高。然而,O₃和N添加对杨树叶片所有N代谢相关的指标都没有交互影响。这些结果将有助于更好地了解在高O₃污染和N沉降/施肥下植物的N代谢过程以及生物地球化学循环过程。     

The effect of nitrate feeding levels on the pathway of nitrogen incorporation into photosynthesizing leaf metabolism ofDatura stramonium L

Datura stramonium leaves fed K¹⁵NO₃ at 25 g N ml^–1 and 200 g N ml^–1 for 7 and 17 min show that at both time courses the main route of newly reduced nitrogen is to glutamate at the low N-feeding level, and to glutamine at the high N-feeding level. At the high N-feeding level the amido-N of glutamine shows higher¹⁵N enrichment than the amino-N, whereas at the low N-feeding level the opposite is true. Feeding of glutamine (¹⁵N amido labelled) at the 25 and 200 g N ml^–1 level produces¹⁵N enrichment of leaf amino acids, the prime routing being to glutamate. This indicates the operation of glutamate synthase at both feeding levels.It is possible that inDatura stramonium leaves both the glutamate dehydrogenase and glutamine synthetase/GOGAT pathways are simultaneously operative, the former route being favoured at low N-feeding levels, the latter at high N-feeding levels.