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To investigate the role of stress in nitrogen management in plants, the effect of pathogen attack, elicitors, and phytohormone application on the expression of the two senescence-related markers GS1 (cytosolic glutamine synthetase EC 6.3.1.2) and GDH (glutamate dehydrogenase, EC 1.4.1.2) involved in nitrogen mobilization in senescing leaves of tobacco (Nicotiana tabacum L.) plants, was studied. The expression of genes involved in primary nitrogen assimilation such as GS2 (chloroplastic glutamine synthetase) and Nia (nitrate reductase, EC 1.6.1.1) was also analysed. The Glubas gene, coding a beta-1,3-glucanase, was used as a plant-defence gene control. As during natural senescence, the expression of GS2 and Nia was repressed under almost all stress conditions. By contrast, GS1 and GDH mRNA accumulation was increased. However, GS1 and GDH showed differential patterns of expression depending on the stress applied. The expression of GS1 appeared more selective than GDH. Results indicate that the GDH and GS1 genes involved in leaf senescence are also a component of the plant defence response during plant-pathogen interaction. The links between natural plant senescence and stress-induced senescence are discussed, as well as the potential role of GS1 and GDH in a metabolic safeguard process.

Nitrogen is known to modulate plant development and resistance to pathogens. Four selected lines (Alg, NS1, NR1 and NR2) of chicory (Cichorium intybus L.) were grown on low (0.6 mM) and high (3 mM) NO(-)(3) nutrition in order to study the effect of N on the expression of three traits, namely, shoot/root ratio, chicon morphology and resistance to soft rot caused by Erwinia sp. For all genotypes, increasing N supply led to a higher shoot/root ratio, resulting from an increased shoot biomass but with no effect on root growth. In contrast, the effect of N on chicon morphology and resistance to bacteria was genotype-dependent and we distinguished two groups of lines according to their phenotypic characteristics. In the group consisting of NR1 and NR2, increasing NO(-)(3) supply during the vegetative phase made the chicon morphology switch from an opened to a closed type while resistance to bacteria was not affected by N supply. In the NS1 and Alg group, the effect of N on chicon morphology was the opposite to that observed in the NR1-NR2 group while NS1 and Alg exhibited a partial resistance to Erwinia sp. , only expressing soft-rot disease when the N supply reached 3 mM. Characterization by DNA amplification fingerprinting (DAF) allowed the generation of 110 polymorphic bands and confirmed that the lines NR1 and NR2, on the one hand, and NS1 and Alg, on the other hand, belong to two distinct genetic groups. The DAF results indicate that chicon morphology and partial resistance to Erwinia sp. are complex traits which would be amenable to quantitative trait loci analysis. The split growth phase of chicory means that any changes in chicon related to N supply during vegetative growth were mediated by a root-originating signal. No variation in root carbon content among genotypes and NO(-)(3) treatments was observed. In contrast, differences in root N content revealed the same grouping of the chicory lines, NR1 and NR2 being systematically richer in amino acids and NO(-)(3) than NS1 and Alg. However, no correlation existed between N compounds and chicon morphology or pathology if all genotypes were considered together. Thus, the effect of N on plant development and pathology as well as putative identified signals might be specific for a genotype. Our study indicates that it is necessary to consider the genetic variability within a species in any signalling-pathway research.

The activity of nitrate reductase (NR) in leaves is regulated by light and photosynthesis at transcriptional and posttranscriptional levels. To understand the physiological role of these controls, we have investigated the effects of light and CO2 on in vivo NO3- reduction in transgenic plants of Nicotiana plumbaginifolia lacking either transcriptional regulation alone or transcriptional and posttranscriptional regulation of NR. The abolition of both levels of NR regulation did not modify the light/dark changes in exogenous 15NO3- reduction in either intact plants or detached leaves. The same result was obtained for 15N incorporation into free amino acids in leaves after 15NO3- was supplied to the roots, and for reduction of endogenous NO3- after transfer of the plants to an N-deprived solution. In the light, however, deregulation of NR at the posttranscriptional level partially prevented the inhibition of leaf 15NO3- reduction resulting from the removal of CO2 from the atmosphere We concluded from these observations that in our conditions deregulation of NR in the transformants investigated had little impact on the adverse effect of darkness on leaf NO3- reduction, and that posttranscriptional regulation of NR is one of the mechanisms responsible for the short-term coupling between photosynthesis and leaf NO3- reduction in the light.

In chicory, we examined how NO3 − supply affected NO3 − uptake, N partitioning between shoot and root and N accumulation in the tuberized root throughout the vegetative period. Plants were grown at two NO3 − concentrations: 0.6 and 3 mM. We used 15N-labelling/chase experiments for the quantification of N fluxes between shoot and root and for determining whether N stored in the tuberized root originates from N remobilized from the shoot or from recently absorbed NO3 −. The rate of 15NO3 − uptake was decreased by low NO3 − availability at all stages of growth. In young plants (10–55 days after sowing; DAS), in both NO3 − treatments the leaves were the strongest sink for 15N. In mature (tuberizing) plants, (55–115 DAS), the rate of 15NO3 − uptake increased as well as the amount of exogenous N allocated to the root. In N-limited plants, N allocation to the tuberized root relied essentially on recent N absorption, while in N-replete plants, N remobilized from the shoot contributed more to N-reserve accumulation in the root. In senescing plants (115–170 DAS) the rate of 15NO3 − uptake decreased mainly in N-replete plants whereas it remained almost unchanged in N-limited plants. In both NO3 − treatments the tuberized root was the strongest sink for recently absorbed N. Remobilization of previously absorbed N from shoot to tuberized root increased greatly in N-limited plants, whereas it increased slightly in N-replete plants. As a consequence, accumulation of the N-storage compounds vegetative storage protein (VSP) and arginine was delayed until later in the vegetative period in N-limited plants. Our results show that although the dynamics of N storage was affected by NO3 − supply, the final content of total N, VSP and arginine in roots was almost the same in N-limited and N-replete plants. This indicates that chicory is able to build up a store of available N-reserves, even when plants are grown on low N. We also suggest that in tuberized roots there is a maximal capacity for N accumulation, which was reached earlier (soon after 100 DAS) in N-replete plants. This hypothesis is supported by the fact that in N-replete plants despite NO3 − availability, N accumulation ceased and significant amounts of N were lost due to N efflux.

The effect of nitrogen supply to chicory plants on carbon partitioning between shoot, root and tuberized root was studied at different stages of vegetative growth, using long-term 13 CO 2 labelling-chase experiments. This approach was complemented by measurement of storage carbohydrates and activities of enzymes involved in root sucrose metabolism (sucrose-sucrose fructosyl transferase (SST), sucrose synthase, invertase). In both young and mature plants, low NO 3 - resulted in a 30-35% decrease in 13 C assimilation. However, the partitioning of 13 C between shoot and root was affected differently at different stages of development. In young plants, in which carbohydrates were being used for structural root and shoot growth, neither 13 C shoot/ 13 C root ratio nor root activities of the above enzymes were modified by NO 3 - supply. In contrast, in mature plants storing large amounts of carbohydrates as fructan in the tuberized root, low NO 3 - caused the ratio to decrease from 0.6 to 0.2, despite unchanged net flux of 13 C from shoot to root. The extractable activity of SST was elevated in mature plants, compared to young plants, at both low and high NO 3 - , consistent with its role in fructan synthesis. However, mature plants grown at low NO 3 - exhibited SST activity double that of plants grown at high NO 3 - . From these results, it is concluded that the observed decrease in shoot/root dry weight ratio at low NO 3 - supply is caused by increased utilization of carbohydrates for storage due to elevated root SST activity.

In this paper we discuss the ways in which our understanding of the nature of the molecular controls of nitrogen assimilation has been increased by the use of non-leguminous and leguminous plants with genetically-altered capacities for ammonia assimilation. Using tobacco or Lotus as model plants, Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities have been altered by stimulating or inhibiting in an organ- or tissue-specific manner the expression of the corresponding genes. In a few selected examples, the physiological impact of these genetic manipulations has been studied on plants grown under different nitrogen regimes. The use of such genetically-modified plants will allow us to better understand the molecular control of this metabolic pathway. It is also potentially of great importance in agriculture if such internal and stable modifications are beneficial in terms of nitrogen use efficiency, thus avoiding an excessive utilization of fertilizers or herbicides (GS inhibitors). Our current knowledge and prospects for future development are explored. Manipulation der NH4-Assimilation in transgenen Nicht-Leguminosen und Leguminosen Das Verstandnis der molekularen Steuerungsmechanismen der Stickstoffassimilation ist durch die Verwendung von Pflanzen mit genetisch veranderter Kapazitat der NH4+-Assimilation betrachtlich gewachsen. Bei den Modellpflanzen Tabak (fur Nicht-Leguminosen) und Lotus corniculatus (fur Leguminosen) lassen sich die Aktivitaten der Glutaminsynthetase (GS) und der Glutamatsynthase (GOGAT) auf Organ-und Gewebsebene durch Stimulierung oder Hemmung der Expression der entsprechenden Gene verandern. Die physiologischen Konsequenzen dieser Genmanipulation wurden an Pflanzen unterschiedlicher N-Versorgung untersucht. Sollten sich solche internen und stabilen Modifikationen der Pflanzen als gunstig im Hinblick auf eine Verbesserung der N-Dungereffizienz erweisen, konnte zukunftig der Aufwand fur N-Dunger und (uber die GS-Inhibierung wirkende) Herbizide reduziert werden. Die Chancen hierfur bedurfen jedoch noch weiterer Forschung.

The responses of photosynthesis to high light and low temperature were studied in vines cultivated in the greenhouse in low light. Exposure to high light (1000 /umol m−2 s−1) or low temperature (5 °C) alone had no measurable effect on the photosynthetic processes, but the combination of high light and low temperature caused rapid loss of photosynthetic capacity and a decrease in the efficiency of photosynthetic energy conversion. After a 15 h exposure to 5°C at high light, the Fv/sb/Fmratio had decreased by 80% and the apparent quantum yield by 75%. Nevertheless, when the leaves were returned to low light at 22°C, these parameters recovered rapidly. The foliar pools of ascorbate and glutathione decreased in the first hours of photoinhibitory treatment while the zeaxanthin content increased from negligible levels to about 50% of the total foliar xanthophyll pool. There was a clear correlation between the zeaxanthin content of the leaves and their Fv/Fm ratio during both photoinhibition and recovery. However, there was also a good correlation between the decrease in theFv Fm ratio and the measured decrease in the total foliar levels of the antioxidants ascorbate and glutathione. The amount of D, protein diminished over the same period as the zeaxanthin levels were increasing. This approach, involving simultaneous measurements of several parameters considered to influence photosystemy II activity, clearly demonstrates that measured decreases in Fv/Fm may not simply be related to zeaxanthin levels or to amounts of D1 protein alone but result from multifactoral influences.

An artificial ecosystem was developed with the aim of associating fish production with a vegetable crop purifying the fish water, in a closed system. The nitrogenous compounds excreted in dissolved form by the fish, and transformed by the bacteria, provide nitrogen for the plants. Previous studies have focused on the system's design and the management of the plant compartment in order to achieve a state of equilibrium. The present paper deals with the performance of the ecosystem over a 2 year period in different conditions of fish and plant culture. Yield measurement of the biological production and the dynamics of the nitrogen transfers in the ecosystem made it possible to define for each trial the efficiency of the system both in terms of productivity and of waste nitrogen recovery. In the spring-summer trial, 80 tomatoes in staggered crops were associated with 216 growing fingerlings. Fish growth paralleled that obtained in a traditional breeding tank and tomato yield reached 70% of the conventional hydroponic culture but with a drastic saving in fertilizers. The recirculating water was well purified as its nitrogen content remained low at 1 mmol l −1 . About 60 % of the nitrogen supplied was recovered from fish (31%) and plants (28%). In the winter trial, 176 lettuce cultivars were associated with 65 adult fish. The plant production varied with the lettuce genotype used, with only butterhead lettuces reaching the same yield as in conventional hydroponics. The size of the plant compartment was too small in relation to the fish compartment so only 29 % of the nitrogen supplied was recovered (fish 21 %, plants 7.5 %). Therefore, the water nitrogen content increased to 15% of the nitrogen supplied. When the main conditions to ensure equilibrium of the ecosystem are satisfactory, size relationship between the three interacting compartments, dynamic management of the fish compartment (growing fingerlings) and the plant compartment (staggered crops, application of a mineral complement), biological production can reach a high level with low fresh water input and low pollution of the environment. The nitrogen balance highlighted the great efficiency of the purifying plant compartment (nitrogen assimilation by plants was high and nitrogen remained low in water) which has been not observed in earlier experiments with artificial ecosystems.

To address the questions of whether allocation of carbohydrates to roots is influenced by ionic form of nitrogen absorbed and whether allocation of carbohydrates to roots in turn influences proportionality between NH4+ and NO3- uptake from mixed sources, NH4+ and NO3- were supplied separately to halves of a split-root hydroponic system and were supplied in combination to a whole-root system. Dry matter accumulation in the split-root system was 18% less in the NH4(+)-fed axis than in the NO3(-)-fed axis. This, however, does not indicate that partitioning of carbohydrate between the two axes was different. Most of the reduction in dry matter accumulation in the NH4(+)-fed axis can be accounted for by the retransport of CH2O equivalents from the root back to the shoot with amino acids produced by NH4+ assimilation. Uptake of NH4+ or NO3- by the respective halves of the split-root system was proportional to the estimated allocation of carbohydrate to that half. When NH4+ and NO3- were supplied to separate halves of the split-root system, the cumulative NH4+ to NO3- uptake ratio was 0.81. When supplied in combination to the whole-root system, the cumulative NH4+ to NO3- uptake ratio was 1.67. Thus, while the shoot may affect total nitrogen uptake through the export of carbohydrates to roots, the shoot (common for halves of the split-root system) apparently does not exert a direct effect on proportionality of NH4+ and NO3- uptake by roots. For whole roots supplied with both NH4+ and NO3-, the restriction in uptake of NO3- may involve a stimulation of NO3- efflux rather than an inhibition of NO3- influx. While only the net uptake of NH4+ and NO3- was measured by ion chromatography, monitoring at approximately hourly intervals during the first 3 days of treatment revealed irregularly occurring intervals of both depletion (net influx) and enrichment (net efflux) in solutions. In the case of NH4+, numbers of net efflux events were similar (21 to 24 out of 65 sequential sampling intervals) whether NH4+ was supplied with NO3- to whole-root systems or separately to an axis of the split-root system. In the case of NO3-, however, the number of net efflux events increased from 8 when NO3- was supplied to a separate axis of the split-root system to between 19 and 24 when NO3- was supplied with NH4+ to whole-root systems.

In Vitis vinifera L. cv. Chardonnay maintained in a greenhouse, the maximum rate of photosynthesis, the measured rates of de novo sucrose and starch synthesis and the total leaf sucrose and starch contents were relatively constant throughout the period from April to July although the partitioning of newly fixed carbon was modified in favour of sucrose synthesis half-way through the growing period. In these experimental conditions, no significant differences in these parameters were observed in plants from which the fruit had been removed in comparison to the controls

Transformed Nicotiana plumbaginifolia plants, constitutively expressing a chimaeric nitrate reductase gene (nia2 gene transcript fused to the constitutive CaMV 35S promoter), were cultivated in greenhouses at INRA, Versailles (from October to December 1991). They were supplied with either 12 mM or 1 mM NO 3 - , and given either a 12 h or a 24 h photoperiod. These plants exhibited a nitrate reductase activity in the leaves 25-150% higher than that of the wild-type plants. The physiological consequences of this deregulated expression were further characterized

An investigation to determine the magnitude of the back reactions which occur during net ammonium uptake by roots and during net ammonium assimilation within roots was undertaken with maize (Zea mays L.). Ten-day-old seedlings, which had been grown on 250 mmol m~3 ammonium at pH 4 or 6, were pretreated for 3 h in the absence or presence of 500 mmol m3 MSX (methionine-DL-sulphoximine), an inhibitor of the glutamine synthetase-catalysed pathway of ammonium assimilation. They were then exposed for 2 h to 99 A% 15N-ammonium + MSX. Substantial ammonium cycling occurred during net ammonium uptake. Efflux was enhanced by MSX treatment, reflecting a 2- to 3-fold accumulation of ammonium in the root tissue. Influx of ammonium was also increased by treatment with MSX, indicating that influx was enhanced when products of ammonium assimilation were dissipated. The decline in root 14N-ammonium accounted for only a small fraction of the 14N-ammonium recovered in the ambient 15N-ammonium solution, revealing a substantial generation of endogenous 14N-ammonium during the 2 h exposure. The net quantity of ammonium generated was increased appreciably when assimilation of ammonium was restricted by MSX and it was estimated to occur at least 50% faster than net ammonium uptake. Presence of MSX severely decreased translocation of 15N to shoots but had a smaller influence on incorporation of 15N into macromolecules of the root tissue. The various ammonium flux rates were not greatly affected by growth at pH 4 0, implying a considerable resistance of ammonium assimilation processes in these maize roots to the high ambient acidity commonly induced by exposure to ammonium.

Zea mays L. (cv Dea) plants grown to the stage of stalk elongation, were allowed to assimilate (13)CO(2) and (15)N-nitrates from 45 to 53 days after sowing. Isotopic abundances in labeled nutrients were slightly enriched compared to natural abundances. The new C in plant was acropetally distributed and the new N was preferentially accumulated in the sheath and stalk in the medium region. C input was 25-fold higher than N input. The new C in total plant C was 20%, whereas it was 10% for N. The stalk acted as a major sink because it accumulated, respectively, 27.5 and 47.5% of the C and N inputs. The new C in soluble carbohydrates was 76% in growing organs (upper stalk) and only 39% in source leaves, whereas it was 43% and 13% in starch, respectively. New N in nitrates+amino-acids spanned in the range from 20% (leaf) to 50% (stalk). New C and N in soluble proteins were, respectively, 13.4 and 3.8% in leaves, 8.8 and 9.6% in stalk, and 8.7 and 14.3% in roots. In the middle stalk and leaves, the proteins and carbohydrates represent an equivalent C and N source for remobilization.

For the first time in Arabidopsis thaliana, this work proposes the identification of quantitative trait loci (QTLs) associated with leaf senescence and stress response symptoms such as yellowing and anthocyanin-associated redness. When Arabidopsis plants were cultivated under low nitrogen conditions, we observed that both yellowing of the old leaves of the rosette and whole rosette redness were promoted. Leaf yellowing is a senescence symptom related to chlorophyll breakdown. Redness is a symptom of anthocyanin accumulation related to whole plant ageing and nutrient limitation. In this work, Arabidopsis is used as a model system to dissect the genetic variation of these parameters by QTL mapping in the 415 recombinant inbred lines of the Bay-0×Shahdara population. Fifteen new QTLs and two epistatic interactions were described in this study. The yellowing of the rosette, estimated by visual notation and image processing, was controlled by four and five QTLs, respectively. The visual estimation of redness allowed us to detect six QTLs among which the major one explained 33% of the total variation. Two main QTLs were confirmed in near-isogenic lines (heterogenous inbred family; HIF), thus confirming the relevance of the visual notation of these traits. Co-localizations between QTLs for leaf yellowing, redness and nitrogen use efficiency described in a previous publication indicate complex interconnected pathways involved in both nitrogen management and senescence- and stress-related processes. No co-localization between QTLs for leaf yellowing and redness has been found, suggesting that the two characters are genetically independent.

CABI:20073191056; International audience; Brassica napus (winter oilseed rape) is an important agricultural crop cultivated for oil, which can be used as an edible product or for industrial application, bioester for example. Despite the very high capacity of oilseed rape to take up nitrate, many authors have reported a very low recovery of nitrogen in field-grown crops whatever the level of N fertilizer applied. In this manuscript we describe the main biochemical and molecular mechanisms involved in nitrate uptake, reduction, assimilation and N recycling during the reproductive period to gain sufficient knowledge to determine the relative importance of environmental and genetic factors determining N management in plants. This understanding will provide the necessary background for improvement of oilseed rape varieties.

Dans certaines conditions environnementales, les plantes produisent des radicaux libres, formes tres reactives de l'oxygene capables de detruire les structures cellulaires. Les mecanismes de defense enzymatique dont disposent les vegetaux pour assurer le « nettoyage cellulairede ces radicaux libres ne sont pas toujours suffisants. Les premiers essais de transgenese, ayant pour but d'ameliorer l'efficacite des enzymes impliquees dans ces defenses, sont encourageants.

La photorespiration conduit-elle vraiment a une deperdition de matiere organique chez les plantes ? C'est, en tous cas, ce qu'ont pense les scientifiques pendant plus de vingt ans. Alors, pour tenter de reduire ce phenomene et augmenter le rendement des cultures, ils ont eu recours aux avancees des biotechnologies. En vain. Devant l'echec de leurs tentatives, ils ont donc cherche a savoir quel pouvait etre le role exact de cette photorespiration embarrassante.

Comparison of the extent of leaf senescence depending on the genetic background of different recombinant inbred lines (RILs) of Arabidopsis (Arabidopsis thaliana) is described. Five RILs of the Bay-0 × Shahdara population showing differential leaf senescence phenotypes (from early senescing to late senescing) were selected to determine metabolic markers to discriminate Arabidopsis lines on the basis of senescence-dependent changes in metabolism. The proportion of γ-aminobutyric acid, leucine, isoleucine, aspartate, and glutamate correlated with (1) the age and (2) the senescence phenotype of the RILs. Differences were observed in the glycine/serine ratio even before any senescence symptoms could be detected in the rosettes. This could be used as predictive indicator for plant senescence behavior. Surprisingly, late-senescing lines appeared to mobilize glutamine, asparagine, and sulfate more efficiently than early-senescing lines. The physiological basis of the relationship between leaf senescence and flowering time was analyzed.

An antisense nitrite reductase (NiR, EC 1.7.7.1) tobacco ( Nicotiana tabacum L.) transformant (clone 271) was used to gain insight into a possible correlation between nitrate reductase (NR, EC 1.6.6.1)-dependent nitrite accumulation and nitric oxide (NO(.)) production, and to assess the regulation of signal transduction in response to stress conditions. Nitrite concentrations of clone 271 leaves were 10-fold, and NO(.) emission rates were 100-fold higher than in wild type leaves. Increased protein tyrosine nitration in clone 271 suggests that high NO(.) production resulted in increased peroxynitrite (ONOO(-)) formation. Tyrosine nitration was also observed in vitro by adding peroxynitrite to leaf extracts. As in mammalian cells, NO(.) and derivatives also increased synthesis of proteins like 14-3-3 and cyclophilins, which are both involved in regulation of activity and stability of enzymes.

The subcellular localisation of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in grapevine (Vitis vinifera L.) leaves and flowers was investigated using immunogold-labelling experiments. In mature leaf tissue or fully developed flowers, GS was visualised both in the cytosol and in the chloroplasts, a high proportion of the protein being present in the phloem companion cells. GDH was preferentially located in the mitochondria of the phloem companion cells in both leaves and flowers. This observation suggests that, in conjunction with GS, GDH plays a major role in controlling the translocation of organic carbon and nitrogen metabolites in both vegetative and reproductive organs. Significant amounts of GDH protein were also visualised in multivesicular bodies within the flower receptacle. Although the function of such organelles is still unknown, its is possible that the presence of GDH in such cellular structures is important for the recycling of carbon and nitrogen molecules in senescing tissues in which the enzyme is generally induced.

Nitrate Assimilation: Historical Resume ASSIMILATION OF NITRATE AND AMMONIUM: Sources of Nitrogen, Nitrogen Cycle, Root Structure, and Nitrogen Assimilation Transport of Nitrate by Roots Nitrate Assimilation: Nitrate and Nitrite Reductases Ammoniacal Nutrition of Plants Ammonium Assimilation Integration of Nitrate Uptake in the Whole Plant SYMBIOTIC ASSIMILATION OF NITROGEN: Symbiotic Nitrogen Fixation Symbiotic Signals in Rhizobium Nitrogenase: Biochemical, Molecular, and Genetic Aspects Nitrogen Assimilation by Ectomycorrhizal Symbiosis Plant Growth Enhancement by Rhizobacteria Rice Field Cyanobacteria: Ecology Contribution to Soil Fertility and Practical Utilization SYNTHESIS AND TRANSPORT OF AMINO ACIDS AND CARBON/NITROGEN RELATIONSHIPS: Synthesis of Amino Acids Transport of Amino Acids in Plants Co-ordination between Nitrogen Photosynthetic and Respiratory Metabolism Nitrogen (NO3) Nutrition and Distribution of Carbon in the Plant DISTRIBUTION OF NITROGEN DURING PLANT GROWTH AND DEVELOPMENT: 15N Methodology Mobilization of Nitrogen Reserve in the Herbaceous Plants Management of Nitrogen in Ligneous Species Protein Synthesis in Grains and Seeds Proteolysis in Higher Plants: Nature, Function, and Regulation NITROGEN:ECOPHYSIOLOGY AND AGRONOMY: Nitrogen Nutrition and Growth of Cultivated Swards Nitrogen: Crops Production and Environment Nodular Diagnosis: Agronomic Basis for an Integrated Improvement of Symbiotic Nitrogen Fixation and the N2-dependent Yield of Grain-legumes Nitrogen and Pollution

The metabolic, biochemical and molecular events occurring during tobacco (Nicotiana tabacum) leaf ageing are presented, with a particular emphasis on nitrogen metabolism. An integrated model describing the source/sink relationship existing between leaves of different developmental stages along the main plant axis is proposed. The results of our study show that a tobacco plant can be divided into two main sections with regards to sink/source relationships. Sink-to-source transition occurs at a particular leaf stage in which a breakpoint corresponding to an accumulation of carbohydrates and a depletion of both organic and inorganic nitrogen is observed. The sink/source transition is also marked by the appearence of endoproteolytic activities and the induction of both cytosolic glutamine synthetase and NAD(H)-dependent glutamate dehydrogenase transcripts, proteins and activities. The role of the newly induced enzymes and the nature of the potential metabolic and developmental signals involved in the regulation of their expression during leaf senescence are discussed.

Summary In the present work we evaluate the accumulation and further remobilization of vegetative storage proteins (VSP) in chicory. A protein with molecular weight of 17kDa, corresponding to 7 isoforms with pi ranging between 5 and 7, accumulated dramatically over the vegetative phase from spring to autumn and was extensively depleted during the flowering period in the following summer, a pattern typical for a VSP. When mature tuberized roots of chicory are harvested in autumn and forced in darkness, an etiolated bud (chicon) grows: this is the salad known as Belgian endive. In our experiments plants were fed, during the forcing process, nutrient solutions containing 1.5 or 18 mmol/L 15 NO 3 (1.79 % atom excess 15 N) or with demineralized water (control). We determined the cycling of endogenous nitrogen ( 14 N), protein (VSP) and amino acids, and the movement of concurrently absorbed nitrogen ( 15 N). Soluble proteins were remobilized at the onset of forcing as a primary response of nitrogen cycling in chicory root. Amino acid remobilization took place only when the chicon growth began with arginine remobilized first. Although 14 N flux into the chicon was similar in all three treatments, indicating that NO 3 supply did not effect endogenous N remobilization, VSP use was effected by NO 3 supply. SDS-PAGE and 2-D gel electrophoresis analyses showed an extensive depletion of VSP (especially five isoforms) only in the control. We suggested that extensive and specific depletion of VSP was delayed by NO 3 supply; with higher NO 3 availability, there was lower VSP remobilization. Furthermore, neo-synthesis of VSP could occur during the forcing process. The finding that 15 N was incorporated into the protein pool during this period supports this hypothesis. The chicon constituted a very strong sink for absorbed nitrogen. Either in high or low NO 3 supply, 15 N was translocated to the chicon almost without mixing with the bulk nitrogen of the root.

International audience; Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO3- content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild-type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO3- content was observed in plants grown in nitrogen-replete conditions at low irradiance (170 mu mol photons . m(-2). s(-1)). This relationship disappeared at higher irradiance (450 mu mol photons . m(-2). s(-1)) because the maximal extractable NR activity in the leaves of the wild-type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO3- accumulated in the leaves of plants grown at 450 mu mol photons . m(-2). s(-1) than in those grown at 170 mu mol photons . m(-2). s(-1) in N-replete conditions. The foliar NO3- level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO3- was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO3- in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.

interest. During the vegetative phase of growth, it pro duces a tuberized root that operates as a sink for photo synthates, which are mainly converted into inulin ( j3-(2,1 ) linked fructan) and stored in the vacuole. Once harvested in autumn, these roots may be forced hydroponically in darkness to produce an etiolated bud, or chicon, which is used for human consumption. Alternatively, as fructans represent 80-85% of root dry weight (Limami and Fiala 1993), the tubers themselves may be used in the sugar industry for production either of inulin or of fructose syrup following inulin hydrolysis. Possible ways of aug menting root sink activity are, therefore, of great interest. One established and effective way of doing so is through manipulation of the nitrogen supply to the plant (Rufty et al., 1984). NOf supply is known to aifect not only N status and growth, but also dry matter partitioning between aerial and underground parts (Gasta 1 and Saugier, 1986; Smolders and Merckx, 1992; Limami et al., 1993). In particular, N deficiency reduces total biomass and leads to a decreased shoot/root ratio. Under these conditions, the strength of storage sinks relative to meta bolic sinks is increased, as shown by the enhanced alloca tion of C to the root, and accumulation of sugars there (Farrar, 1993). In chicory, such responses may allow production of tuberized roots in the absence of fertilizer NO3" concentrations damaging to the environment. Sink strength may be considered as the product of sink size and sink activity (Clifford, 1992). Little is known about the mechanisms by which root sink activity is modulated in chicory. Synthesis of fructan is thought to be the result of the sequential action of two enzymes, sucrose-sucrose fructosyl transferase (SST) and fructan fructan fructosyl transferase (Edelman and Jefford, 1968; Lusher et al., 1993). It is not yet known whether activities of these enzymes are the chief determinants of root sink strength, or whether other enzymes involved in sucrose Chicory plants (Cichorium intybus), which produce a tuberized root containing high concentrations of fruc tans, were grown with nitrate at either optimal (4 mol m"3) or suboptimal (0.6 mol m 3) concentration. At 120 DAS (days after sowing), shoot/root dry mass ratio in the plants grown at low N was less than one-third of that of plants grown at the higher concentration. This reflected differences in shoot dry mass, as root dry mass was similar in the two treatments. Pulse-chase experiments with 14C02 showed increased allocation of carbon to the root and decreased parti tioning to the growing shoot in the plants grown at low N. Root activities of three enzymes involved in sucrose and fructan metabolism were measured, in an attempt to identify the factors which determine sink strength. Whereas invertase activity showed little difference between the treatments, sucrose synthase and par ticularly sucrose-sucrose fructosyl transferase were higher in the roots of plants grown at low N. Activity of SST was also much greater in roots of young plants synthesizing large amounts of fructan than in mature roots no longer producing fructan. It is, therefore, sug gested that SST plays a role in determining the sink strength. Possible interactions between N status and SST activity/sink strength are discussed.

Transgenic Nicotiana plumbaginifolia plants that express either a 5-fold increase or a 20-fold decrease in nitrate reductase (NR) activity were used to study the relationships between carbon and nitrogen metabolism in leaves. Under saturating irradiance the maximum rate of photosynthesis, per unit surface area, was decreased in the low NR expressors but was relatively unchanged in the high NR expressors compared with the wild-type controls. However, when photosynthesis was expressed on a chlorophyll (Chl) basis the low NR plants had comparable or even higher values than the wild-type plants. Surprisingly, the high NR expressors showed very similar rates of photosynthesis and respiration to the wild-type plants and contained identical amounts of leaf Chl, carbohydrate, and protein. These plants were provided with a saturating supply of nitrate plus a basal level of ammonium during all phases of growth. Under these conditions overexpression of NR had little impact on leaf metabolism and did not stimulate growth or biomass production. Large differences in photochemical quenching and nonphotochemical quenching components of Chl a fluorescence, as well as the ratio of variable to maximum fluorescence, (FV/FM), were apparent in the low NR expressors in comparison with the wild-type controls. Light intensity-dependent increases in nonphotochemical quenching and decreases in FV/FM were greatest in the low NR expressors, whereas photochemical quenching decreased uniformly with increasing irradiance in all plant types. Nonphotochemical quenching was increased at all except the lowest irradiances in the low NR expressors, allowing photosystem II to remain oxidized on its acceptor side. The relative contributions of photochemical and nonphotochemical quenching of Chl a fluorescence with changing irradiance were virtually identical in the high NR expressors and the wild-type controls. Zeaxanthin was present in all leaves at high irradiances; however, at high irradiance leaves from the low NR expressors contained considerably more zeaxanthin and less violaxanthin than wild-type controls or high NR expressors. The leaves of the low NR expressors contained less Chl, protein, and amino acids than controls but retained more carbohydrate (starch and sucrose) than the wild type or high NR expressors. Sucrose phosphate synthase activities were remarkably similar in all plant types regardless of the NR activity. In contrast phosphoenolpyruvate carboxylase activities were increased on a Chl or protein basis in the low NR expressors compared with the wild-type controls or high NR expressors. We conclude that large decreases in NR have profound repercussions for photosynthesis and carbon partitioning within the leaf but that increases in NR have negligible effects.

An artificial ecosystem integrating three biological compartments (fish, bacteria, plants) in a closed system was developed with the aim of associating fish production with a vegetable crop purifying the fish water. The nitrogenous compounds excreted in dissolved form by the fish, and transformed by the bacteria, provide nitrogen for the plants. This association has the double advantage of savings in water for fish culture and the recycling of fish excretion as the main source of minerals for producing edible plants. A pilot system of 2 m 3 was set up for intensive animal and plant production and installed in a greenhouse to enable continuous production throughout all seasons. The fish chosen were tilapias ( Oreochromis niloticus ) and the plants were tomatoes ( Lycopersicum esculentum ) grown according to the nutrient film technique in recirculating hydroponics. A granular filter bearing the nitrifying bacteria was inserted between the fish tank and the plants. The system's design was aimed at optimizing the functioning of the ecosystem, by the size of the different elements (fish tank, bacterial filter, hydroponic troughs) as well as by the choice of the recirculating water flow rate. During the first production cycle, we followed the evolution of the physico-chemical characteristics of the water and of the plant tissues, especially the nitrogen (NH 3 , NO 2 , NO 3 ) and mineral compounds (K, Ca, Mg, SO 4 , PO 4 ), in order to evaluate the functioning of the three compartments and to progressively develop the management of the plant compartment. The latter determined the overall equilibrium of the ecosystem by its capacity to absorb NO 3 and NH 4 in the recirculating water. The results were satisfactory as there was a stabilization of the nitrogenous compounds, in particular NO 3 , at a low level and a large production; in the first trial no attempt was made to improve the animal production. This trial highlighted the main conditions to ensure the equilibrium of the ecosystem: size relationship between the three interacting compartments, dynamic management of the plant compartment (staggered crops) and the application of a mineral complement to obtain optimum plant growth. The nature of the mineral complement will depend on the composition of the water available on the production site.

Changes in the activation level of rubisco from bean leaves were studied by measuring carboxylation rates after either a prolonged period in the dark after photoinhibitory treatment. Plants kept for 11 h in darkness were subjected to three stages of strong irradiance: stage I, photosynthesis in air; stage II, exposure in1% O 2 and CO 2 -free N 2 ; stage III, reintroduction of air. Decrease in the rate of CO 2 uptake at stage III are due to photoinhibition. From the end of stage I to stage III, initial carboxylase activity, measured in extracts, exhibited large variations whereas changes for total activity were much smaller. Initial activity was four times lower at stage II compared to stage I. It increased in several minutes to high values at stage III where rates of CO 2 uptake were attained more gradually. No large amount of ribulose 1–5 bisp phosphate (RuBP) appeared to be bound to inactive enzyme at stage II. When darkened leaves were illuminated (stage I) the increase of both initial and total activity was slow and parallel to disappeance of the inhibitor carboxyarabinitol-1-phosphate (CA1P). Conversely, increase of CO 2 uptake rate was more rapid.

The mechanisms priming the production, the movement, and the transient and final storage of the photoassimilated carbon in the maize plant were examined at the metabolic level during the formation of the seed, with the ultimate aim to identify metabolic steps restricting grain yield and explaining the delay of formation of the reserve molecules. Under normal field conditions, we show that maize directly supplies the developing seed with the photoassimilated carbon which undergoes numerous interconversions from the ear leaf to the grain. The proteins, either in the leaf or in the seed, are primarily synthesized from incoming amino acids. Nevertheless, a secondary in situ synthesis of amino acids provides the proteins with new amino acids. The amino acids of this second set, slowly synthesized in the seed from the photosynthetic carbon skeletons, are not detected in their free form but immediately and regularly incorporated into the seed proteins, in such a way that, after 4 days of chase, the proportion of the radioactive labeling of the amino acids of the different storage protein groups corresponds to their amino acid composition. In the leaf, the labeling of proteins also arises from different metabolisms, but mainly from the photosynthetic metabolism. Contrary to the seed proteins, the time course of the labeled leaf proteins implies a rapid turnover. The second labeling of starch and proteins in the ear leaf involves a reassimilation of CO(2), a process optimizing the carbon uptake in maize.

L’étude des réactions de carboxylations photosynthétiques chez les maïs « WH x WJ » et « INRA 508 » montre qu’une baisse de l’activité photosynthétique chez le maïs « WH x WJ », par rapport au maïs « INRA 508 », s’accompagne de déviations métaboliques au sein des premières réactions qui assurent le transfert du carbone du CO2 atmosphérique vers les composés du cycle de CALVIN-BENSON, via les acides malique et aspartique. Le même comportement métabolique en relation avec la photosynthèse avait déjà été observé antérieurement chez le maïs « W64A » mutant opaque-2 comparé au maïs « W64A » normal, plus productif., A compared study of the C4 photosynthetic pathway carried out on different maize genotypes (« WH x WJ » and « INRA 508 »), showed that in « WH x WJ» maize an important decrease of the photosynthetic rate was associated with marked metabolic deviations. From short pulse and chase experiments with 14CO2 under steady-state photosynthesis it was evidenced that the transfer of carbon from the dicarboxylic acids to CALVIN-BENSON cycle components was decelerated in maize « WH x WJ » as compared with « INRA 508 ». Aspartate was more heavily labeled in « WH x WJ » than in the « INRA 508 » leaves. Conversely the incorporations of 14C into malate and 3-phosphoglycerate were always higher in the « INRA 508 » than in the « WH x WJ » leaves. Previously the same pattern of the 14C distribution was observed in maize mutant « W64A » opaque-2 known to differ from maize « W64A » normal in CO2 uptake rate and dry matter production.

The pH, malic acid and K + contents of the cell sap extracted from frozen thawed Acer pseudoplatanus cells were measured. A fusicoccin (FC) treatment of the cells induced an increase pf the pH and malic acid and K + contents indicating that a vacuolar pH modification was probably involved in FC action. Even though the decrease of the pH of the culture medium was measureable after a short period of FC treatment (10–15 min), at least 1 h was necessary for an increase of sap pH to be significant. The assumption of a vacuolar pH modification is supported by the modification of the distribution of [ 14 C] nicotine molecules between the cells and their culture medium induced by the FC treatment. As this lipophilic base is absorbed by diffusion of the neutral form and concentrated as ions in the acidic compartments of the cells (i.e. mainly the vacuole) FC action on nicotine accumulation can be interpreted as evidence for a vacuolar pH modification. Possible mechanisms of this FC action are tentatively discussed.

To gain a better understanding of the biochemical basis for partitioning of photosynthetically fixed carbon between leaf and grain, a (14)CO(2) labeling study was conducted with field-grown maize plants 4 weeks after flowering. The carbon flow was monitored by separation and identification of (14)C assimilates and (14)C storage components within each tissue during the chase period (from 4 to 96 hours) following a 5 minute (14)CO(2) pulse. In the labeled ear leaf, the radioactivity strongly decreased to reach, at the end of the experiment, about 12% of the total incorporated radioactivity, mostly associated with sucrose and proteins. Nevertheless, an unexpected reincorporation of radioactivity was observed either in leaf starch or proteins, the day following the pulse. Conversely, the radioactivity in the grain increased to attain 66% of the total incorporated (14)C after a 96 hour chase. The photosynthates, mostly sucrose, organic and free amino acids, rapidly translocated towards the developing seeds, served as precursors for the synthesis of seed storage compounds, starch, and proteins. They accumulate in free form for 24 hours before being incorporated within polymerized storage components. This delay is interpreted as a necessary prerequisite for interconversions prior to the polycondensations. In the grain, the labeling of the storage molecules, either in starch or in storage protein groups (salt-soluble proteins, zein, and glutelin subgroups), was independent of their chemical nature but dependent on their pool size.

In previous studies we have reported that marked metabolic deviations in the first steps of the C4 photosynthetic pathway occur among inbred lines of maize with changes In the photosynthetic rate and dry matter production (Morot-Gaudry et al., 1979; Morot-Gaudry et al., 1981). These results led us to study in different varieties of maize the photosynthetic capability in relation to heterosis manifested by a greater vigor in height, leaf area, growth and dry matter accumulation. To examine whether heterosis is accompanied by varietal differences in net photosynthesis, carboxylation reactions and activities of photosynthetic enzymes, we have compared the physiological and biochemical differences between inbred parents and hybrids.

Whereas elementary processes of carbon photoassimilation, sucrose synthesis, phloem loading and unloading, have been well described, little information is available regarding protein synthesis from photosynthates in leaf and kernel during grain filling. To gain a better understanding of the biochemical basis for partitioning of photosynthetically fixed carbon between leaf and grain a 14CO2 labelling study was conducted with field-grown plants 30 days after flowering. Carbon flow was followed by separation and identifixation of 14C assimilates and 14C storage molecules within each tissue.

Operation of the glycolate pathway in isolated bundle sheath (BS) strands of two C4 species was demonstrated from 14C incorporation into two intermediates, glycine and serine, under conditions favourable for photorespiratory activity. Isolated BS strands fixing 14CO2 under light at physiological rates incorporate respectively 3% (Zea mays L., cv. INRA 258) and 7% (Panicum maximum Jacq.) of total 14C fixed into glycine + serine, at low bicarbonate levels (less than the Km for CO2 fixation, 0.8 mM). Higher bicarbonate concentrations depressed the percentage of incorporation into the two amino acids. No labelling was observed in the absence of added glutamate. Oxygen was required for glycine + serine labelling, since 14C incorporation into glycine was largely depressed by argon flushing, and labelling of the two amino acids was nearly suppressed by the addition of the strong reductant, dithionite, especially in maize. Two inhibitors of the glycolate pathway were tested. With α-hydroxypyridine-methanesulfonic acid, an inhibitor of glycolate oxidase, labelling of glycine and serine remained minimal whereas glycolate was accumulated. Isoniazid, an inhibitor of the transformation of glycine to serine induced a 50% increased labelling of glycine in maize BS, and a large decrease in serine labelling. In Panicum, the increase in [14C]-glycine was 90%. These results suggest that the pathway glycolate glycine serine operates in these plants. However, leakage of metabolites occurs in BS cells, especially in maize and a large part of newly formed glycolate, glycine and serine is exported out of the cells. Operation of ribulose-1,5-bisphosphate oxygenase activity in competition with ribulose-1,5-bisphosphate carboxylase is demonstrated by the lowering of total 14CO2 fixation when O2 is increased at low bicarbonate concentration. An interesting feature observed in maize BS, at low bicarbonate concentration, was an increase in ribulose-1,5-bisphosphate labelling when the O2 level was decreased. This was accompanied by an increase in CO2 fixation. This could indicate an increased rate in synthesis of ribulose-1,5-bisphosphate (which accumulated) due to a stimulation of ATP synthesis by cyclic photophosphorylation under anaerobic conditions.

Summary Caprylylglycine and copper caprylylglycinate are lipoamino acids resulting from condensation of glycine and caprylic acid by formation of peptide bonds. These molecules are known to have interesting biological properties and especially to improve the yield of food crops. Caprylylglycine and copper caprylylglycinate applied to maize seeds modify the growth and increase the dry matter production at maturity. This beneficial effect is accompanied by enhanced photosynthetic activity correlated with a better transfer of carbon from the air to the C 3 compartment through the C 4 compartment. Lipoamino acids do not affect the partition of carbon into aminoacids, starch, and proteins. The mode of penetration and the target of the action of lipoamino acids are not elucidated.

The aim of this work was to describe the incorporation of 14CO2 into maize at the late kernel fill under chilling and the subsequent movement of the photoassimilated 14C out the fed ear leaf. Cool temperatures were observed to decrease the photosynthetic rate and to alter the operation of the carbon assimilation pathway with 14C accumulation in alpha-alanine. They were shown also to affect the rate of photoassimilated carbon out of the fed area, and especially by delaying the seed import processes.

International audience