Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase (A Cardinal Event Influencing the Photosynthesis Rate in Sorghum and Maize).

C4 leaf phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is subject to a day/night regulatory phosphorylation cycle. By using the cytoplasmic protein synthesis inhibitor cycloheximide (CHX), we previously reported that the reversible in vivo light activation of the C4 PEPC protein-serine kinase requires protein synthesis. In the present leaf gas-exchange study, we have examined how and to what extent the CHX-induced inhibition of PEPC protein kinase activity/PEPC phosphorylation in the light influences C4 photosynthesis. Detached Sorghum vulgare and maize (Zea mays) leaves fed 10 [mu]M CHX showed a gradual but marked decrease in photosynthetic CO2 assimilation capacity. A series of control experiments designed to assess deleterious secondary effects of the inhibitor established that this reduction in C4 leaf CO2 assimilation was not due to (a) an increased stomatal resistance to CO2 diffusion, (b) a decrease in the activation state of other photoactivated C4 cycle enzymes, and (c) a perturbation of the Benson-Calvin C3 cycle, as evidenced by the absence of an inhibitory effect of CHX on leaf photosynthesis by a C3 grass (Triticum aestivum). It is notable that the CHX-induced decrease in CO2 assimilation by illuminated Sorghum leaves was highly correlated with a decrease in the apparent phosphorylation status of PEPC and a concomitant change in carbon isotope discrimination consistent with a shift from a C4 to a C3 mode of leaf CO2 fixation. These collective findings indicate that the light-dependent activation of the PEPC protein-serine kinase and the resulting phosphorylation of serine-8 or serine-15 in Sorghum or maize PEPC, respectively, are fundamental regulatory events that influence leaf C4 photosynthesis in vivo.

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

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 (15)N 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.

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.

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

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 (13)C 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 (13)C. 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 (13)C. 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 δ(13)C 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.

Temperature Dependence of Carbon Isotope Fractionation in CAM Plants.

The carbon isotope fractionation associated with nocturnal malic acid synthesis in Kalanchoë daigremontiana and Bryophyllum tubiflorum was calculated from the isotopic composition of carbon-4 of malic acid, after appropriate corrections. In the lowest temperature treatment (17 degrees C nights, 23 degrees C days), the isotope fractionation for both plants is -4 per thousand (that is, malate is enriched in (13)C relative to the atmosphere). For K. daigremontiana, the isotope fractionation decreases with increasing temperature, becoming approximately 0 per thousand at 27 degrees C/33 degrees C. Detailed analysis of temperature effects on the isotope fractionation indicates that stomatal aperture decreases with increasing temperature and carboxylation capacity increases. For B. tubiflorum, the temperature dependence of the isotope fractionation is smaller and is principally attributed to the normal temperature dependences of the rates of diffusion and carboxylation steps. The small change in the isotopic composition of remaining malic acid in both species which is observed during deacidification indicates that malate release, rather than decarboxylation, is rate limiting in the deacidification process.

C and N Mobilization from Stalk and Leaves during Kernel Filling by C and N Tracing in Zea mays L.

The sink capacity of the stalk in Zea mays L. (cv DEA) during the elongation period was previously investigated with (13)C and (15)N tracing. The chase experiment described here demonstrates the different behavior of intermediary reserves for C and N remobilization until full maturity of the kernels. Carbon incorporated during stalk elongation participated mainly in cellulose formation in vegetative organs appearing after the labeling period; the remobilization to kernels was low (0.5%). Soluble carbohydrates and proteins were the main intermediary sink compounds, starch being little remobilized. N first incorporated in roots, sheaths, stalk, blades was translocated to the kernel; 42% of the labeled N were recovered in kernels where they represented 8% of the total N. Cob, husk, and shank acted first as N sinks and then as N sources during ear development. It appeared that aminoacids used for synthesis of kernel proteins have a common origin, except for glutelin G(3).

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.

Estimation of Carbon and Nitrogen Allocation during Stalk Elongation by C and N Tracing in Zea mays L.

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.

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.

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.