Eliane Deléens

Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development

Abstract. A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L. plants. Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots. To eliminate any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants and progeny devoid of TL- and TR-DNA sequences were chosen for further analyses. These plants had a 50–80% increase in total leaf GS activity. Plants were grown under different nitrogen regimes (4 or 12 mM NH4+) and aspects of carbon and nitrogen metabolism were examined. In roots, an increase in free amino acids and ammonium was accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH4+ in comparison to the wild type grown under the same conditions. Labelling experiments using 15NH4+ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids. This experiment showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants overexpressing GS. It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots was the result of shoot protein degradation. Moreover, following three weeks of hydroponic culture early floral development was observed in the transformed plants. As all these properties are characteristic of senescent plants, these findings suggest that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature flowering when plants are grown on an ammonium-rich medium.

Does root glutamine synthetase control plant biomass production in Lotus japonicus L.

Abstract. To investigate the contribution of root cytosolic glutamine synthetase (GS) activity in plant biomass production, two different approaches were conducted using the model legume Lotus japonicus. In the first series of experiments, it was found that overexpressing GS activity in roots of transgenic plants leads to a decrease in plant biomass production. Using 15N labelling it was shown that this decrease is likely to be due to a lower nitrate uptake accompanied by a redistribution to the shoots of the newly absorbed nitrogen which cannot be reduced due to the lack of nitrate reductase activity in this organ. In the second series of experiments, the relationship between plant growth and root GS activity was analysed using a series of recombinant inbred lines issued from the crossing of two different Lotus ecotypes, Gifu and Funakura. It was confirmed that a negative relationship exists between root GS expression and plant biomass production in both the two parental lines and their progeny. Statistical analysis allowed it to be estimated that at least 13% of plant growth variation can be accounted for by variation in GS activity.

Carbon isotope composition of biochemical fractions isolated from leaves of Bryophyllum daigremontianum berger, a plant with crassulacean acid metabolism: Some physiological aspects related to CO2 dark fixation.

Isotope analysis of the biochemical fractions isolated quantitatively from young and mature leaves of Bryophyllum daigremontianum Berger have been carried out before and after a dark period of accumulation of organic acids. The mature leaf is enriched in 13C compared to the young leaf. The δ13C values of the different leaf constituents vary between the δ13C values of C4 plants (-11‰) and those of C3 plants (-27‰). During the dark period, the two types of leaves store organic acids with δ13C values of ≃-15‰ and lose insoluble sugars, including starch with a δ13C value of ≃-12‰. Furthermore, young leaves store phosphorylated compounds with δ13C values of ≃-11‰ and lose weakly polymerised sugars with δ13C values of ≃-18‰. These results led to the formulation of a hypothesis of the origin of the two substrates of β-carboxylation: phosphoenolpyruvate arises from the glycolytic breakdown of the insoluble sugars rich in 13C, and the major portion of the CO2 is the result of the complete breakdown (respiration) of the soluble sugars rich in 12C. The existence of two independent sugar pools leads to the assumption that there are two separate glycolytic pathways. The 13C enrichment of the stored products of the young leaves in the day seems to be the result of a weak discrimination for 13C by ribulose diphosphate carboxylase, which reassimilates to a great extent the CO2 released from malate accumulated in the night.

Relationships between flag leaf carbon isotope discrimination and several morpho-physiological traits in durum wheat genotypes under Mediterranean conditions

Relationships between flag leaf carbon isotope discrimination (Delta), water status parameters, residual transpiration (RT) and stomatal density (SD) were examined on a collection of 144 durum wheat accessions. Associations between Delta, grain yield (GY) and harvest index (HI) were also studied. The field trial was conducted under Mediterranean conditions. The crop cycle was characterised by a period of drought from February until maturity. A broad range of values we obtained for Delta (16.5-19.9 per thousand) and other physiological traits. Flag leaf Delta was positively and significantly correlated with both HI and GY. Delta was better correlated with HI than with GY, which suggests that higher Delta values indicate higher efficiency of carbon partitioning to the kernel, leading to higher GY. Delta was found positively related with RT and negatively related with SD. This relationship may indicate a possible SD component of RT due to the association between conductance and SD. Strong positive correlations were found between Delta and water status parameters, suggesting that Delta may provide a good indication of plant water status in durum wheat under rainfed Mediterranean conditions.

Effect of nitrate on water transfer across roots of nitrogen pre-starved maize seedlings

The addition of 10 mM KNO3 to the solution bathing the roots of young nitrogen-starved seedlings of Zea mays L. enhanced root water transfer within 15 h, compared with 10 mM KCl addition. The free exudation flux was 2.2–3.9 times higher in excised KNO3-treated roots than in KCl-treated ones. Cryo-osmometry data for xylem sap suggested that, compared with chloride, nitrate treatment increased the steady solute flux into the xylem, but did not modify the osmotic concentration of sap. Root growth was not significantly modified by nitrate within 15 h. Root hydraulic conductances were measured by using either hydrostatic-pressure or osmotic-gradient methods. During hydrostatic experiments, the conductance (kp), which is thought to refer mainly to the apoplasmic pathway, was 1.6 times larger in KNO3-than in KCl-treated plants. From experiments in which polyethylene glycol (PEG) 8000 was used as external osmolyte, osmotic conductances (ks) were found to be smaller by 5–20 times than kp for the two kinds of plants. The KCl-treated roots were characterized by a low ks which was the same for influx or efflux of water. By contrast, KNO3-treated roots exhibited two distinct conductances ks1 and ks2, indicating that influx of water was easier than efflux when the water flow was driven by the osmotic pressure gradient. Infiltration of roots with KNO3 solution supported the idea that nitrate might enhance the efficiency of the cell-to-cell pathway. The low ks value of KCl-treated roots and the existence of two contrasting ks values (ks1 and ks2) for KNO3-treated roots are discussed in terms of reversible closing of water channels.

Mobilisation and distribution of starch and total N in two grapevine cultivars differing in their susceptibility to shedding

As a part of a project aimed at elucidating the causal relationship between reserve mobilisation and the extent of shedding in Vitis vinifera L., we compared storage and fate of carbon (C) and nitrogen (N) reserves in two varieties differing in their susceptibility to fruitlet abscission. Merlot (susceptible) and Pinot Noir (P. Noir, not susceptible) vines were grown in trenches under semi-controlled conditions over a 3-y period after planting. Mobilisation of stored C and N, distribution of reserve materials within the vines and 15N uptake were followed particularly during the spring growth flush and floral development in the third year. At dormancy, starch levels in the perennial tissues (roots, trunk, canes) were higher in Merlot than in P. Noir. During the spring growth flush, starch level decreased markedly in the roots of both cultivars until early bloom. At that time, starch started to accumulate in P. Noir but not in Merlot. Similar variations were found with total N. Accordingly, 15N analysis showed that translocation of storage N to the annual tissues was nearly achieved at early bloom in P. Noir while it continued until pea berry size in Merlot. In parallel, N uptake increased during the spring growth flush, and it was higher in P. Noir than in Merlot. These results indicate that transition between heterotrophic (root) and autotrophic (leaf) mode of nutrient allocation towards the developing inflorescences occurs earlier in P. Noir. Possible consequences are discussed in relation to the susceptibility of each cultivar to shedding.

Relationships between carbon isotope discrimination, dry matter production, and harvest index in durum wheat

Summary Carbon isotope discrimination has been proposed as a criterion for the indirect selection to improve transpiration efficiency and grain yield in bread wheat and barley. Less attention has been devoted to durumwheat ( Triticum durum Desf.) despite its economic importance in the Mediterranean basin. The Δ genetic variation and its relationships to dry matter production and harvest index in durum wheat were investigated in this study. For this purpose, field experiments were conducted on 144 durum wheat accessions under Mediterranean conditions (South of France) during three consecutive years with contrasting climatic conditions. Grain yield, above-ground biomass, harvest index, and carbon isotope discrimination of flag leaf and kernel were measured. Differences between years, noted for both leaf and kernel carbon isotope discrimination, were probably related to the variation in water availability from year to year. A large genotypic variation was also noticed for both leaf and kernel carbon isotope discrimination. The two traits were found to be positively correlated with grain yield within and across years, which confirms the interest in carbon isotope discrimination for selection for grain yield improvement under Mediterranean conditions. Both kernel and leaf carbon isotope discrimination correlated better with harvest index than with grain yield, suggesting that carbon isotope discrimination could reflect the efficiency of carbon partitioning to the kernel. The lack of correlation between leaf carbon isotope discrimination and both harvest index and grain yield in favourable water conditions (1996) was probably due to the difference in water availability between the period until flag leaves sampling (favourable conditions) and the strong water stress which accompanied the grain filling. Kernel carbon isotope discrimination correlated better with both harvest index and grain yield than did leaf carbon isotope discrimination. Moreover, a higher broad-sense heritability was obtained for kernel carbon isotope discrimination than for leaf carbon isotope discrimination. As a result, kernel carbon isotope discrimination appeared to be a better predictive criterion for efficiency of the carbon partitioning to the kernel (harvest index), and hence for grain yield, than did flag leaf carbon isotope discrimination.

Productivity and carbon isotope discrimination in durum wheat organs under a Mediterranean climate

For durum wheat, we promote the use of carbon isotope discrimination (delta) as an indirect selection criterion for transpiration efficiency and grain yield (GY), and we identify the most effective organ for characterising delta genotypic variation. A field experiment was conducted in the South of France on 144 accessions, with a drought period occurring from February to June. Harvest index (HI), GY and delta (delta L, flag leaf; delta A, awn, delta G grain) were measured. Significant positive genetic correlations were noted between delta and both GY and HI. A larger genotypic variation and a higher broad-sense heritability were noted for delta G compared to delta L and delta A. delta G correlated better with GY and HI than delta L and delta A, showing that delta G could provide a better assessment of genotypic behaviours under drought during grain filling. Moreover, the indirect selection based on delta G (even when evaluated with one replicate) appeared more efficient than the direct selection for grain yield. This result emphasised the potential value of grain carbon isotope discrimination as a criterion for grain yield improvement under stressed Mediterranean conditions.

Carbon isotope composition of intermediates of the starch-malate sequence and level of the crassulacean acid metabolism in leaves of Kalanchoe blossfeldiana Tom Thumb.

Isotype analyses were performed on biochemical fractions isolated from leaves of Kalanchoe blossfeldiana Tom Thumb. during aging under long days or short days. Irrespective of the age or photoperiodic conditions, the intermediates of the starch-malate sequence (starch, phosphorylated compounds and organic acids) have a level of 13C higher than that of soluble sugars, cellulose and hemicellulose. In short days, the activity of the crassulacean acid metabolism pathway is predominant as compared to that of C3 pathway: leaves accumulate organic acids, rich in 13C. In long days, the activity of the crassulacean acid metabolism pathway increases as the leaves age, remaining, however, relatively low as compared to that of C3 pathway: leaves accumulate soluble sugars, poor in 13C. After photoperiodic change (long days→short days), isotopic modifications of starch and organic acids suggest evidence for a lag phase in the establishment of the crassulacean acid metabolism pathway specific to short days. The relative proportions of carbon from a C3-origin (RuBPC acitivity as strong discriminating step, isotope discrimination in vivo=20‰) or C4-origin (PEPC activity as weak discriminating step, isotope discrimination in vivo=4‰) present in the biochemical fractions were calculated from their δ13C values. Under long days, 30 to 70% versus 80 to 100% under short days, of the carbon of the intermediates linked to the starch-malate sequence, or CAM pathway (starch, phosphorylated compounds and organic acids), have a C4-origin. Products connected to the C3 pathway (free sugars, cellulose, hemicellulose) have 0 to 50% of their carbon, arising from reuptake of the C4 from malate, under long days versus 30 to 70% under short days.

Coefficient réel d'utilisation du 15Nurée pulvérisé sur des couverts de blé à floraison

Abstract The efficiency of recovery of N urea by plants or true recovery coefficient of N urea [TRC(N urea ) %] was determined after fertilization of wheat via the leaves. A chemical preparation containing 15 N urea (0.55 %) was sprayed on crops of two varieties at flowering. Two concentrations were given: 20 and 40 kg N urea . ha −1 . The crops were previously fertilized (NH4NO 3 ) at optimum or at sub optimum doses at 1-cm ear stage. Leaf damage due to urea spray remained in a low range. At flowering, the tips of the leaves already exhibited necroses (10 % of the total leaf area). These necrotic area proportions doubled at the largest urea dose 72 h after the spray. A hypothesis involving leaf urea metabolism is proposed. The true recovery coefficients of N urea [TRC(N urea ) %] were determined 6 h after spraying whole plants. They were found to be between 50 and 72 %, N urea being localized mainly in the ears and the leaves, and only slightly in the roots. The coefficients increased to 80–90 % at maturity. Thus, N urea assimilation was rapid and efficient. Eighty-six percent of the absorbed N urea was recovered in the seeds. The seed TRC(N urea ) values were between 65 and 81 %. The high and constant values of the TRC(N urea ) values described above contrasted with the low and variable data found in the literature. A metabolic compartmentation between N urea and the remobilized N from the other plant organs is proposed.