Nitrogen (N) plays the most important role of all soil mineral nutrients in plant growth and development. In grapevine, nitrogen is most likely to be defi cient although it is the main fertilizer commonly applied to vineyards to increase productivity (Keller et al. 1998) and to influence grape juice composition (Ough and Bell 1980). Considerable new information has been obtained over the past twenty years on the uptake, translocation, distribution, partitioning, and storage of nitrogenous compounds in grapevines as well as new insights toward a better understanding on the regulation of the synthesis and degradation of amino acids and other nitrogenous compounds, and of the enzymes associated with these re actions.

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.

Fernao Pires and Pinot noir grapevines were grown in field-sites in Lisbon and Versailles respectively. The grapevines at the Versailles site were grown in pots in artificial media while the vines in Lisbon were grown in soil in the field. A substantial inhibition of net CO2 assimilation from mid-morning onwards was observed at both sites. Prior to the period of measurement the Lisbon site had received no rain for 45 days but the Versailles vines were watered twice a day. In neither case was there any evidence to suggest that substantial photoinhibition was occurring during the photoperiod. The Fv/Fm ratio was relatively constant from dawn until dusk. The foliar zeaxanthin content showed a marked diurnal variation with maximum values obtained at midday and at minimum values at dawn and dusk. Interestingly, the total xanthophyll pool was greater in the leaves of Versailles vines than the Lisbon site. The maximum zeaxanthin levels of the former at midday were three times those of the latter. The foliar pools of ascorbate and glutathione either remained constant or increased slighdy from dawn to dusk. The pools of glycine and serine in the leaves declined from mid-morning onwards at both sites but the ratio of 3-PGA/triosephosphates remained constant throughout the photoperiod. This implies that energy supply and utilisation in the leaves were well-balanced throughout the photoperiod despite the decrease in CO2 assimilation. We conclude that similar strategies for the regulation of the photosynthetic apparatus occurred in both cases. This afforded effective protection against photoinhibition. Even though the vines grown in Lisbon were subjected to a significant period of drought they exhibited lower levels of stress-indicating substances particularly proline and zeaxanthin than the vine leaves grown in Versailles. We are drawn to the conclusion that vines grown in pots with the resultant restriction on root growth suffer greater stress than the field-grown vines. The cooler growth conditions encountered in Versailles may be suboptimal for grapevines and, hence, add additional stress factors.

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 interval 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.

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 (CO2)-C-13 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 NO3- resulted in a 30-35% decrease in C-13 assimilation. However, the partitioning of C-13 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 C-13 shoot/C-13 root ratio nor root activities of the above enzymes were modified by NO3- supply. In contrast, in mature plants storing large amounts of carbohydrates as fructan in the tuberized root, low NO3- caused the ratio to decrease from 0.6 to 0.2, despite unchanged net flux of C-13 from shoot to root. The extractable activity of SST was elevated in mature plants, compared to young plants, at both low and high NO3-, consistent with its role in fructan synthesis. However, mature plants grown at low NO3- exhibited SST activity double that of plants grown at high NO3-. From these results, it is concluded that the observed decrease in shoot/root dry weight ratio at low NO3- supply is caused by increased utilization of carbohydrates for storage due to elevated root SST activity.

In the present work we evaluate the accumulation and further remobilization of vegetative storage proteins (VSP) in chicory. A protein with molecular weight of 17 kDa, 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 (NO3)-N-15 (1.79 % atom excess N-15) or with demineralized water (control). We determined the cycling of endogenous nitrogen (N-14), protein (VSP) and amino acids, and the movement of concurrently absorbed nitrogen (N-15). Soluble proteins were remobilized at the onset of forcing as a primary response of nitrogen cycling in chicory root. Amino add remobilization took place only when the chicon growth began with arginine remobilized first. Although N-14 aux into the chicon was similar in all three treatments, indicating that NO3 supply did not effect endogenous N remobilization, VSP use was effected by NO3 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 NO3 supply; with higher NO3 availability, there was lower VSP remobilization. Furthermore, neo-synthesis of VSP could occur during the forcing process, The finding that N-15 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 NO3 supply, N-15 was translocated to the chicon almost without mixing with the bulk nitrogen of the root.

ABSTRACT 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 F v /sb/F m ratio 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 F v /F m ratio during both photoinhibition and recovery. However, there was also a good correlation between the decrease in theF v F m 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 F v /F m may not simply be related to zeaxanthin levels or to amounts of D 1 protein alone but result from multifactoral influences.

Chicory plants (Cichorium intybus), which produce a tuberized root containing high concentrations of fructans, were grown with nitrate at either optimal (4 mol m(-3)) or suboptimal (0.6 mol m(-3)) concentration. At 120 BAS (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 (CO2)-C-14 showed increased allocation of carbon to the root and decreased partitioning 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 particularly 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, suggested that SST plays a role in determining the sink strength, Possible interactions between N status and SST activity/sink strength are discussed.

In non-nodulated soybean [Glycine max (L.) Merrill cv. Ransom] plants that were subjected to 15 d of nitrogen deprivation in flowing hydroponic culture, concentrations of nitrogen declined to 1.0 and 1.4 mmol N g(-1) dry weight in shoots and roots, respectively, and the concentration of soluble amino acids (determined as primary amines) declined to 40 mu mol g(-1) dry weight in both shoots and roots. In one experiment, nitrogen was resupplied for 10 d to one set of nitrogen-depleted plants as 1.0 mol m(-3) NH4+ to the whole root system, to a second set as 0.5 mol m(-3) NH4+ plus 0.5 mol m(-3) NO3- to the whole root system, and to a third set as 1.0 mol m(-3) NH4+ to one-half of a split-root system and 1.0 mol m(-3) NO3- to the other half. In a second experiment, 1.0 mol m(-3) of nitrogen was resupplied for 4 d to whole root systems in NH4+:NO3- ratios of 1:0, 9:1, and 1:1. Nutrient solutions were maintained at pH 6.0.When NH4+ was resupplied in combination with NO3- to the whole root system in Experiment I, cumulative uptake of NH4+ for the 10 d of resupply was about twice as great as when NH4+ was resupplied alone. Also, about twice as much NH4+ as NO3- was taken up when both ions were resupplied to the whole root system. When NH4+ and NO3- were resupplied to separate halves of a split-root system, however, cumulative uptake of NH4+ was about half that of NO3-. The uptake of NH4-, which is inhibited in nitrogen-depleted plants, thus is facilitated by the presence of exogenous NO3-, and the stimulating effect of NO3- on uptake of NH4+ appears to be confined to processes within root tissues. In Experiment II, resupply of nitrogen as both NH4+ and NO3- in a ratio of either 1:1 or 9:1 enhanced the uptake of NH4+. The enhancement of NH4+ uptake was 1.8-fold greater when the NH4+:NO3--resupply ratio was 1:1 than when it was 9:1; however, only 1.3 times as much NO3- was taken up by plants resupplied with the 1:1 exogenous ratio. The effect of NO3- on enhancement of uptake of NH4+ apparently involves more than net uptake of NO3- itself and perhaps entails an effect of NO3- uptake on maintenance of K+ availability within the plant. The concentration of K+ in plants declined slightly during nitrogen deprivation and continued to decline following resupply of nitrogen. The greatest decline in K+ concentration occurred when nitrogen was resupplied as NH4+ alone. It is proposed that decreased availability of K+ within the NH4+-resupplied plants inhibited NH4+ uptake through restricted transfer of amino acids from the root symplasm into the xylem.

The temporal mobilization of nitrogen reserves and their relative contribution to whole plant growth, have been studied in grapevines ( Vitis vinifera L. cv Pinot noir). Four-year-old grapevines received in addition to nutrient medium the isotopic tracer 15 N (as 15 N-NO 3 K) as a ten fold increase of the natural abundance, for a three week period. Two periods of 15 N application were made either three weeks before leaf fall (in autumn) or three weeks preceding the beginning of shoot growth (at early spring). After collection of the spring xylem sap, at pruning-time, the same plants were sampled at bloom in order to determine both the amount and the redistribution of the isotopic tracer in the different parts of the plant. During the bleeding period, the autumnal N reserves represented 4% of the total pool of nitrogen present in the xylem sap, while 40% of the same pool originated from the spring supply. At flowering time, high amounts of "autumn" nitrogen remained stored in the perennial parts (30% of the total "autumn" storage), whereas the figure was only 17% in the case of spring 15 N feeding. Thus, a major part (83%) of the N assimilated at spring was used to ensure the new shoot growth, and almost 60% was accumulated in the subapical part of these shoots. The relative specific allocation (RSA) values for N compounds originating from autumn or spring assimilation periods were calculated in the different parts of the grapevine at flowering time. The highest values of RSA observed in the base and the clusters indicated that they were fed preferentially at their earlier stages of growth by a highly labeled xylem sap. The lower values of RSA evidenced in the subapical part of the shoot, which developed later, could be explained by an important dilution of spring or autumn labeled nitrogen by unlabeled N compounds recently synthetized in the course of shoot elongation. This experiment underlined the importance of the stored nitrogen, particularly "spring" nitrogen, to ensure the first steps of new shoot growth until the leaves of the shoot base have been fully expanded.

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.

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 NO3-, 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. A main consequence of this over-expression was a decrease in the foliar NO3- content (32-47%) and an increase in the glutamine (74-133%) and malate (25-96%) pool sizes compared to the wild-type controls. The higher glutamine content of the leaves of the transgenic plants resulted in an increase in the total amino acid pool compared to the wild-type even though the levels of other amino acids remained relatively stable. However, the total nitrogen content, protein content and dry matter production were not changed as a result of over-expression of nitrate reductase activity. These results suggest that it is possible to decrease the nitrate content of the leaves, and possibly other organs, in winter crops by this means.

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.