Jean-François Morot-Gaudry

Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence

Abstract. 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.

Characterization of Markers to Determine the Extent and Variability of Leaf Senescence in Arabidopsis. A Metabolic Profiling Approach

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.

Adaptations of Photosynthetic Electron Transport, Carbon Assimilation, and Carbon Partitioning in Transgenic Nicotiana plumbaginifolia Plants to Changes in Nitrate Reductase Activity.

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.

The absence of photoinhibition during the mid-morning depression of photosynthesis in Vitis vinifera grown in semi-arid and temperate climates

Summary 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 CO 2 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 F v /F m 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 CO 2 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.

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

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 μmol photons·m−2 ·s−1). This relationship disappeared at higher irradiance (450 μmol 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 μmol photons·m−2·s−1 than in those grown at 170 μmol 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.

Cellular and subcellular localisation of glutamine synthetase and glutamate dehydrogenase in grapes gives new insights on the regulation of carbon and nitrogen metabolism

Abstract. 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 (15NO3) limitation affects nitrogen partitioning between metabolic and storage sinks and nitrogen reserve accumulation in chicory (Cichorium intybus L.)

Abstract. 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.

Evidence for an action of fusicoccin on the vacuolar pH of acer pseudoplatanus cells in suspension culture

Abstract 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.

Effect of exogenous nitrogen (15NO3) on utilization of vegetative storage proteins (VSP) during regrowth in chicory (Cichorium intybus)

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.

Nitrogen-induced changes in morphological development and bacterial susceptibility of Belgian endive (Cichorium intybus L.) are genotype-dependent

Abstract. 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.