A. Keys

Rubisco regulation: a role for inhibitors

In photosynthesis Rubisco catalyses the assimilation of CO(2) by the carboxylation of ribulose-1,5-bisphosphate. However, the catalytic properties of Rubisco are not optimal for current or projected environments and limit the efficiency of photosynthesis. Rubisco activity is highly regulated in response to short-term fluctuations in the environment, although such regulation may not be optimally poised for crop productivity. The regulation of Rubisco activity in higher plants is reviewed here, including the role of Rubisco activase, tight binding inhibitors, and the impact of abiotic stress upon them.

The isolation and characterisation of a catalase-deficient mutant of barley (Hordeum vulgare L.)

A mutant line of barley, R(othamsted)-Pr 79/4, has been isolated which grows poorly in natural air, but normally in air enriched to 0.2% CO2. Analysis of the products of 14CO2 fixation showed that there was no major block in photosynthetic or photorespiratory carbon metabolism in the mutant and that rates of CO2 fixation were only slightly lower than those measured in the wild type (c.v. Maris Mink). Leaves of the mutant line contained only 10% of the catalase (EC 1.11.1.6) activity found in the wild type; and the two major bands of catalase activity detected after starch-gel electrophoresis of extracts of normal leaves were missing from similar extracts of RPr 79/4. Peroxisomes isolated from mutant leaves contained negligible catalase activity, but normal levels of other enzymes involved in photorespiration. Genetic analysis has shown that the mutation is recessive and that both air-sensitivity and catalase-deficiency segregate together in F2 plants derived from a cross between the mutant and the cultivar Golden Promise. [1-14C]Glycollate was not converted to 14CO2 faster in the mutant leaves than in the normal leaves. Thus there was no evidence that photorespiratory CO2 may be obtained by the chemical action of H2O2 on glyoxylate or hydroxypyruvate.

Increased levels of glutathione in a catalase-deficient mutant of barley (Hordeum vulgare L.)

Abstract The total glutathione content of leaves of the catalase deficient mutant barley RPr 79/4 increased 5- to 10-fold to a maximum of 3 μmol g −1 fresh wt. after growth in air. The proportion of reduced glutathione (GSH) fell from 86% to 42% during this period. Similar results were obtained when wild-type barley leaves were treated with aminotriazole, a catalase inhibitor. It is proposed that the excess H 2 O 2 synthesised during photorespiration causes the formation of oxidised glutathione (GSSG).

The regulation of the biosynthesis of glutathione in leaves of barley (Hordeum vulgare L.)

Abstract Between 50 and 65% of the glutathione in barley leaves was present in the chloroplasts depending upon the light regime. However, only 66–76% of the chloroplast glutathione was present in the reduced state (GSH) as opposed to 97–98% of that in the cytoplasm. In shoots treated with the catalase inhibitor aminotriazole and in shoots of the catalase deficient barley mutant RPr 79/4 exposed to air, the glutathione level increased 3-fold in 8 h in the light. The increase was accounted for by a rise in both the chloroplast and cytoplasm level of oxidised glutathione (GSSG), the GSH concentration remained relatively constant in both compartments. Only 2–3% of applied 35 SO 4 was metabolised to glutathione by wild-type shoots. In aminotriazole-treated plants this value rose to 17.9% and in the mutant RPr 79/4 exposed to air to 32%.

Increased capacity for photosynthesis in wheat grown at elevated CO2:the relationship between electron transport and carbon metabolism

Spring wheat (Triticum aestivum L.) was grown under optimal nutrition for six weeks at 700 and 350 μmol·mol−1 CO2 and simultaneous measurements of photosystem-II (PSII) chlorophyll fluorescence and gas exchange were conducted on intact attached leaves. Plants grown at elevated CO2 had double the concentration of CO2 at the carboxylation site (Cc) despite a lowered stomatal (gs) and mesophyll (gm) conductance compared with ambient-grown plants. Plants grown at elevated CO2 had a higher relative quantum yield of PSII electron transport (ΦPSII) and a higher relative quantum yield of CO2 fixation (ΦCO2). The higher ΦPSII was due to a larger proportion of open PSII centres, estimated by the coefficient of photochemical quenching of fluorescence (qp), with no change in the efficiency of light harvesting and energy transduction by open PSII centres (F′v/F′m). Analysis of the relationship between ΦPSII and ΦCO2 conducted under various CO2 and O2 concentrations showed that the higher ΦCO2 for a given ΦPSII in leaves developed under elevated CO2 was similar to that obtained in leaves upon a partial reduction in photorespiration. Calculation of the allocation of photosynthetic electron-transport products to CO2 and O2 showed that for leaves developed in elevated CO2, there was an increase in both total linear electron flow and electron flow to CO2 and a decrease in electron flow to O2. Plants developed under elevated CO2 showed positive acclimation manifested by a higher ΦCO2 when measured under ambient CO2 and higher assimilation rates in A/Ci curves. Initial and total activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco EC 4.1.1.39) measured in vitro increased by 16 and 15% respectively in leaves from plants grown in elevated CO2, which was in agreement with a 15% higher in vivo carboxylation efficiency. It is concluded that growth of spring wheat at elevated CO2 enhances photosynthesis due to a change in the balance of component processes manifested as an increased capacity for carbon fixation, total electron transport and Rubisco activity, and a concomitant partial reduction of photorespiration.

Expanding knowledge of the Rubisco kinetics variability in plant species: environmental and evolutionary trends

The present study characterizes the kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from 28 terrestrial plant species, representing different phylogenetic lineages, environmental adaptations and photosynthetic mechanisms. Our findings confirm that past atmospheric CO(2)/O(2) ratio changes and present environmental pressures have influenced Rubisco kinetics. One evolutionary adaptation to a decreasing atmospheric CO(2)/O(2) ratio has been an increase in the affinity of Rubisco for CO(2) (Kc falling), and a consequent decrease in the velocity of carboxylation (kcat (c)), which in turn has been ameliorated by an increase in the proportion of leaf protein accounted by Rubisco. The trade-off between K(c) and k(cat)(c) was not universal among the species studied and deviations from this relationship occur in extant forms of Rubisco. In species adapted to particular environments, including carnivorous plants, crassulacean acid metabolism species and C(3) plants from aquatic and arid habitats, Rubisco has evolved towards increased efficiency, as demonstrated by a higher k(cat)(c)/K(c) ratio. This variability in kinetics was related to the amino acid sequence of the Rubisco large subunit. Phylogenetic analysis identified 13 residues under positive selection during evolution towards specific Rubisco kinetic parameters. This crucial information provides candidate amino acid replacements, which could be implemented to optimize crop photosynthesis under a range of environmental conditions.

Photorespiration in C4 grasses remains slow under drought conditions

The CO(2)-concentrating mechanism present in C(4) plants decreases the oxygenase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and, consequently, photorespiratory rates in air. Under drought conditions, the intercellular CO(2) concentration may decrease and cause photorespiration to increase. The C(4) grasses Paspalum dilatatum Poiret, Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel were grown in soil and drought was imposed by ceasing to provide water. Net CO(2) assimilation (A) and stomatal conductance to water vapour decreased with leaf dehydration. Decreased carbon and increased oxygen isotope composition were also observed under drought. The response of A to CO(2) suggested that the compensation point was zero in all species irrespective of the extent of drought stress. A slight decrease of A as O(2) concentration increased above 10% provided evidence for slow photorespiratory gas exchanges. Analysis of amino acids contained in the leaves, particularly the decrease of glycine after 30 s in darkness, supported the presence of slow photorespiration rates, but these were slightly faster in Cynodon dactylon than in Paspalum dilatatum and Zoysia japonica. Although the contents of glycine and serine increased with dehydration and mechanistic modelling of C(4) photosynthesis suggested slightly increased photorespiration rates in proportion to photosynthesis, the results provide evidence that photorespiration remained slow under drought conditions.

A comparison between the coupled spectrophotometric and uncoupled radiometric assays for RuBP carboxylase.

The coupled spectrophotometric assay for RuBP carboxylase was compared with the conventional radiometric assay to assess the validity of its use in the measurement of initial and total activities in crude leaf extracts. At high magnesium concentrations both assays gave the same absolute values of initial and total activities, and resolved similarly the changes of total activity and activation state (ratio of initial to total activity) which occurred when the water status and light environment of leaves was altered prior to sampling. Although the magnesium concentration supporting the maximum rate of initial activity in soybean extracts was similar in the two assays, substantial differences of initial activity were observed at sub-optimal concentrations of magnesium. At low magnesium concentrations reaction rates in the spectrophotometric assay exhibited an initial phase of non-linearity which subsequently gave way to a linear rate. In contrast, reaction rates at low magnesium were linear from the time of initiation in the radiometric assay. Inclusion of EDTA in the reaction medium did, however, induce non-linear rates in the radiometric assay. The pre-addition of RUBP to extract immediately prior to dilution into the reaction medium did not eliminate the non-linearity in either assay system. The significance of these observations is discussed briefly in relation to the use of the spectrophotometric assay.

The re-assimilation of ammonia produced by photorespiration and the nitrogen economy of C3 higher plants

Photorespiration involves the conversion of glycine to serine with the release of ammonia and CO2. In C3 terrestrial higher plants the flux through glycine and serine is so large that it results in the production of ammonia at a rate far exceeding that from reduction of new nitrogen entering the plant. The photorespiratory nitrogen cycle re-assimilates this ammonia using the enzymes glutamine synthetase and glutamine:2-oxoglutarateaminotransferase.

Photosynthetic responses of three C4 grasses of different metabolic subtypes to water deficit

C4 plants are considered to be less sensitive to drought than C3 plants because of their CO2 concentrating mechanism. The C4 grasses, Paspalum dilatatum Poiret (NADP-ME), Cynodon dactylon (L.) Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK) were compared in their response to water deficit imposed by the addition of polyethylene glycol to the nutrient solution in which they were grown. The effects of drought on leaf relative water content (RWC), net photosynthesis, stomatal conductance, carboxylating enzyme activities and chlorophyll a fluorescence were investigated. In C. dactylon the RWC was more sensitive, but the photosynthetic activity was less sensitive, to water deficit than in P. dilatatum and Z. japonica. The decrease of photosynthesis in P. dilatatum under water deficit was not closely related to the activities of the carboxylating enzymes or to chlorophyll a fluorescence. However, decreased activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase, in addition to decreased stomatal conductance, may have contributed to the decrease of photosynthesis with drought in C. dactylon and Z. japonica. The different responses to water deficit are discussed in relation to the natural habitats of C4 grasses.