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Thèse de doctorat : Biologie Végétale et Forestière : Nancy 1 : 2008. / Titre provenant de l'écran-titre. Bibliogr.

The relationship between carbon flux pathways in the plant must be explained. Fixation of carbon by the plant is the first step in dry matter yield production. Photorespiration (PR) appears to depress dry matter yield potential through its release of carbon, potentially fixed by photosynthesis, in the light. If PR rate could be reduced genetically in the plant, net carbon gain might be increased. A population of alfalfa (Medicago sativa L.) was screened for low PR potential per unit of leaf area. 'Hayden' alfalfa, grown under competitive stand field conditions, was investigated for five harvests for carbon flux relationships. Field-grown shoots of the plants were analyzed in the laboratory for per unit leaf area total carbon uptake (TCU), PR, apparent photosynthesis (AP), and dark respiration (DR) rates. An exceptionally low PR plant, designated ∅5, was isolated. Nine randomly selected Hayden plants were crossed on to the ∅5 and seed collected from the latter. Forty F₁ progeny, grown under space planted field conditions, were analyzed by the same means for carbon flux. The 40 genotypes approached a normal distribution for PR, TCU, AP, and DR rates. Significant differences were found among genotypes for the pathways sufficient variability was present for selection of low PR rate plants. Clone cuttings of a high (number 40) and a low (number 12) PR F₁ selection, plus an ∅5 parent clone, were grown in the greenhouse for carbon flux measurements and analyzed as described previously. No significant differences were found for PR, TCU, AP, or DR between genotypes. In the F₁ population, 12 and 40 were concomitant extremes for PR and TCU rates. Correlation between PR and TCU rates in the population was positive but moderate with an r = 0.53. Genotypic expression of PR was observed in the field plants, but only trends toward these differences were expressed in the greenhouse. Environmental factors in the greenhouse masked the genotypic expression of PR, TCU, AP, and DR. This indicates that greenhouse selection of PR levels may not be possible. The selection of low PR plants under Arizona field conditions may be a feasible operation, if significance between genotypes and ranking persistence is maintained over seasons.

Bibliography: leaves [i]-xvi.

Most plants produce leaves with stomata on either both surfaces (amphistomatous) or on the lower surface only (hypostomatous). The importance of stomata to plant survival suggests that these two stomatal distribution patterns may be adaptive, and this problem is explored. It is concluded that amphistomaty is an adaptation to produce a high conductance to CO₂ diffusion into the leaf. As such it is advantageous to plants with high photosynthetic capacity leaves in high light environments, experiencing rapidly fluctuating or continuously available soil water. Plants meeting these criteria are found to be almost exclusively amphistomatous; those not meeting the criteria are mostly hypostomatous. Also investigated is the adaptive significance of differences in stomatal conductances and conductance responses to environmental factors between the two surfaces of amphistomatous leaves. Although differences in stomatal conductance are found between the two surfaces in sunflower, differences in conductance response to light intensity and water vapor pressure difference across the stomatal pore were neglible. Water stress relieved one day prior to experiments caused upper stomatal conductance to be reduced more than lower, but responses to light and water vapor pressure difference remained essentially parallel for the two surfaces. For these differences in conductance to be adaptive differences in photosynthetic characteristics between the two surfaces. In addition, estimation of the resistance to diffusion of CO₂ across the mesophyll yields values low enough to preclude steep gradients in CO₂ partial pressure in the mesophyll. In the absence of CO₂ gradients within the leaf, differences in photosynthetic characteristics between the two surfaces cannot exist. It is concluded that differences in stomatal conductance between the two surfaces of amphistomatous leaves are not adaptations to differences in CO₂ uptake characteristics.

Bibliography: leaves [i]-xvi / xi, 132, xvi, [137] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1984

The gas exchange responses of two cultivars of Hordeum vulgare L., to light, temperature, CO₂ and water stress were investigated in the laboratory. The optimum temperature for net CO₂ assimilation was found to be 25°C and 22.5°C for cv. Clipper and cv. Dayan respectively. Net CO₂ assimilation was reduced at 30°C in cv. Dayan. At low light intensity the highest quantum yield efficiency was 0.051 mol.mol⁻¹ at 30°C for cv. Clipper, and 0.066 mol.mol⁻¹ at 20°C for cv. Dayan. At the same temperature, cv. Clipper had a higher water use efficiency than cv. Dayan, but stomatal conductance for cv. Dayan was higher than cv. Clipper. Stomatal limitation to CO₂ was lowest at the optimum temperature for CO₂ assimilation in both cultivars. Stomata limited CO₂ assimilation in cv. Clipper to a larger degree than in cv. Dayan. Relative stomatal limitation for cv. Clipper at 25°C was 0.280 ± 0.010, and for cv. Dayan at 22.5°C was 0.028 ± 0.011. Short-term exposure to elevated CO₂ concentrations increased CO₂ assimilation in both cultivars, but more so for cv. Clipper. Transpiration rate at elevated CO₂ partial pressures were higher in cv. Dayan than in cv. Clipper. At very high CO₂ (860 μmol.m⁻²s⁻¹) partial pressure water use efficiency in cv. Clipper was higher than cv. Dayan, but at low CO₂ partial pressures water use efficiency in cv. Dayan was higher than cv. Clipper. Water stress reduced the relative leaf water content and net CO₂ assimilation in both cultivars. Cultivar Dayan was more tolerant to water stress, and CO₂ assimilation in this cultivar was less affected by water stress. In both cultivars water stress increased the concentration of chlorophyll a, chlorophyll b, and chlorophyll a+b. The chlorophyll a:b ratio remained relatively constant throughout the stress period. No correlation between relative leaf water content and total carotenoid concentration was observed.

A procedure for the preparation of spinach leaf mitochondria was developed. The procedure combines differential centrifugation, partition in dextran- polyethyleneglycol two-phase system and Percoli density gradient centri- fugation. The different steps separate the material mainly according to size, surface properties and density, respectively. No chlorophyll was present in the final mitochondrial preparation and the mitochondria were also markedly enriched relative to peroxisomes and microsomes as esti­mated from the recovery of marker enzymes. The latency of enzyme activities was used to study the apparent intactness of the mitochondrial membranes. These measurements showed that both the inner and outer mitochondrial membranes were more than 90 % intact. The mitochondria were also functionally intact since the coupling between respiration and oxidative phosphorylation was retained. The purity of the preparation made it possible to study cytochromes from leaf mitochondria. The cytochrome content of stalk and leaf mitochondria was measured in order to compare mitochondria from photosynthesizing and non-photosynthesizing tissue. The measurements were performed by difference spectroscopy both at room temperature and at liquid nitrogen temperature. Qualitatively the cytochrome content in mitochondria from stalks and leaves was identical. Quantiatively leaf mitochondria contained,on a protein basis, only half the amount of the different cytochromes as compared to stalk mitochondria. The relative content of the different cytochromes was, however, similar suggesting that the composition of the respiratory chain was the same. The photorespiratory conversion of glycine to serine takes place in the mitochondria and involves oxidative decarboxylation of glycine. The ability to oxidize glycine via the respiratory chain was present in spinach leaf mitochondria, but absent in mitochondria prepared from roots, stalks and leaf veins from the same plants. This confirmed the specific localization of the glycine oxidizing activity to photosyntheticaliy active tissue, as suggested by studies with other plant material. The conversion of glycine to serine is a complex reaction depending on the combined action of two enzymes: glycine decarboxylase and serine hydroxymethyltransferase. The effect of inhibitors on the serine hydroxy­methyl transferase activity and the rate of the glycine bicarbonate exchange reaction associated with glycine decarboxylase was studied. These reactions represent partial steps in the conversion of glycine to serine and the aim was to investigate the site of inhibition for the different inhibitors, namely, isonicotinyl hydrazide (a pyridoxa!phosphate antagonist), amino- acetonitrile, glycinehydroxamate (glycine analogues) and cyanide. The results showed that these inhibitors had a complex pattern of inhibition. The same inhibitor affected more than one site and often with an apparently different mechanism. It was, however, found that aminoacetonitrile at low concentrations specifically inhibited glycine decarboxylase and that cyanide specifically inhibited serine hydroxymethyltransferase. / digitalisering@umu

A la lumière, l’activité carboxylase de la RuBisCO permet de fixer le CO2 inorganique en matière organique, sous forme de 3-phosphoglycérate (3-PGA), qui sera utilisé pour la biosynthèse de sucres, d’acides organiques et aminés, de la paroi végétale etc. Cependant, elle possède aussi une activité oxygénase qui produit du 3-PGA et du 2-phosphoglycolate. Ce dernier composé étant toxique, il est métabolisé en 3-PGA par le cycle photorespiratoire qui se déroule dans le chloroplaste, le peroxysome et la mitochondrie. Malgré une perte partielle en carbone et en azote, l’importance de la photorespiration pour les plantes est illustré par les phénotypes néfastes que les mutants d’enzymes photorespiratoires présentent dans l’air (comme un retard de croissance, la chlorose, et de la létalité) et qui sont absents en fort CO2. Ceci pourrait refléter des interactions étroites entre la photorespiration et le métabolisme primaire des plantes. Afin de mieux comprendre ces interactions et la mise en place des phénotypes photorespiratoires, des mutants pour la glycolate oxydase (GOX) et la glutamate:glyoxylate aminotransférase ont été caractérisés à travers plusieurs analyses complémentaires: des échanges gazeux, de la fluorescence chlorophyllienne, du marquage des métabolites avec du 13C, des dosages de métabolites, de cofacteurs, et de la RuBisCO. Les résultats montrent que, suite à un transfert de fort CO2 dans l’air, l’inhibition de la photosynthèse observée chez nos mutants est principalement due à un défaut du recyclage du carbone photorespiratoire qui diminue l’activité de la RuBisCO. Cette inhibition photosynthétique a un impact négatif sur la quantité de RuBisCO dans les feuilles de ces mutants par rapport aux plantes contrôles. De plus, lorsque l’inhibition de la photosynthèse est trop importante chez nos mutants photorespiratoires, la carence en carbone déclenche de la sénescence dans leurs feuilles âgées. En parallèle, une comparaison des paramètres cinétiques de la GOX d’A. thaliana (plante en C3) et de Z. mays (plante en C4) associée à la mesure d’effets isotopiques 13C et 2H a révélé que ces enzymes partageaient des paramètres Michaéliens équivalents pour le glycolate, ainsi qu’un mécanisme réactionnel identique mettant en jeu un transfert d’hydrure. / In the light, the RuBisCO carboxylase activity assimilates inorganic CO2 into organic compounds, via the production of 3-phosphoglycerate (3-PGA) that is used for the biosynthesis of sugars, organic and amino acids, plant cell walls etc. However, it also has an oxygenase activity that makes 3-PGA and 2-phosphoglycolate (2-PG). The toxic 2-PG is metabolized to 3-PGA by the photorespiratory cycle, which takes place in chloroplasts, peroxisomes and mitochondria. Despite a partial loss of carbon and nitrogen, the importance of photorespiration for growth can be seen by the negative phenotypes exhibited by photorespiratory enzyme mutants in air (i.e. slow growth, leaf chlorosis, and sometimes lethality), which are not observed under high CO2 conditions. This may reflect the metabolic interactions between photorespiration and plant primary metabolism. To better understand such interactions and the development of photorespiratory phenotypes, mutants for glycolate oxidase (GOX) and glutamate:glyoxylate aminotransferase have been characterized by several complementary methods: analysis of gas exchanges, chlorophyll fluorescence,13C-labeling of metabolites, measurements of metabolites, cofactors and RuBisCO levels. The results show that, after a high CO2-to-air transfer, the inhibition of photosynthesis in the mutants is mainly due to a defect in photorespiratory carbon recycling leading to a decreased RuBisCO activity. The inhibition of carbon assimilation negatively impacts mutant leaf RuBisCO content when compared to wild-type plants. In the mutants, when photosynthetic inhibition is too high, the resulting carbon starvation triggers the onset of senescence in their old leaves. In parallel to this work, a comparison of the kinetic parameters of GOX from A. thaliana (C3 plant) and Z. mays (C4 plant) coupled to measurements of 13C and 2H kinetic isotopic effects showed that these enzymes share similar Michaelian parameters for glycolate, and a similar hydride transfer reaction mechanism.