Environmentally driven evolution of Rubisco and improved photosynthesis and growth within the C3 genus Limonium (Plumbaginaceae).

Carbon assimilation by most ecosystems requires ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Its kinetic parameters are likely to have evolved in parallel with intracellular CO2 availability, with the result that faster forms of Rubisco occur in species with CO2 -concentrating mechanisms. The Rubisco catalytic properties were determined and evaluated in relation to growth and carbon assimilation capacity in Mediterranean Limonium species, inhabiting severe stress environments. Significant kinetic differences between closely related species depended on two amino acid substitutions at functionally important residues 309 and 328 within the Rubisco large subunit. The Rubisco of species facing the largest CO2 restrictions during drought had relatively high affinity for CO2 (low Michaelis-Menten constant for CO2 Kc) but low maximum rates of carboxylation (kcatc), while the opposite was found for species that maintained higher CO2 concentrations under similar conditions. Rubisco kinetic characteristics were correlated with photosynthetic rate in both well-watered and drought-stressed plants. Moreover, the drought-mediated decrease in plant biomass accumulation was consistently lower in species with higher Rubisco carboxylase catalytic efficiency (kcatc/Kc). The present study is the first demonstration of Rubisco adaptation during species diversification within closely related C3 plants, revealing a direct relationship between Rubisco molecular evolution and the biomass accumulation of closely related species subjected to unfavourable conditions.

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.

Photosynthetic assimilation of ¹4C into amino acids in potato (Solanum tuberosum) and asparagine in the tubers.

Asparagine is the predominant free amino acid in potato tubers and the present study aimed to establish whether it is imported from the leaves or synthesised in situ. Free amino acid concentrations are important quality determinants for potato tubers because they react with reducing sugars at high temperatures in the Maillard reaction. This reaction produces melanoidin pigments and a host of aroma and flavour volatiles, but if free asparagine participates in the final stages, it results in the production of acrylamide, an undesirable contaminant. ¹4CO2 was supplied to a leaf or leaves of potato plants (cv. Saturna) in the light and radioactivity incorporated into amino acids was determined in the leaves, stems, stolons and tubers. Radioactivity was found in free amino acids, including asparagine, in all tissues, but the amount incorporated in asparagine transported to the tubers and stolons was much less than that in glutamate, glutamine, serine and alanine. The study showed that free asparagine does not play an important role in the transport of nitrogen from leaf to tuber in potato, and that the high concentrations of free asparagine that accumulate in potato tubers arise from synthesis in situ. This indicates that genetic interventions to reduce free asparagine concentration in potato tubers will have to target asparagine metabolism in the tuber.

An engineered pathway for glyoxylate metabolism in tobacco plants aimed to avoid the release of ammonia in photorespiration.

BACKGROUND: The photorespiratory nitrogen cycle in C3 plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO2. Because of the loss of CO2 and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C3 plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle. RESULTS: When grown in ambient air, but not in elevated CO2, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation. CONCLUSIONS: Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.

Rubisco activities, properties, and regulation in three different C4 grasses under drought.

In C4 plants, water deficit may decrease photosynthetic CO2 assimilation independently of changes in stomatal conductance, suggesting decreased turnover by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The activity and biochemistry of Rubisco was studied in three different C4 grasses: Paspalum dilatatum, Cynodon dactylon, and Zoysia japonica. The objectives were to characterize the C4 Rubisco in these species and to identify factors associated with decreased photosynthetic rates caused by drought. Rubisco isolated from each of the three C4 grasses was characterized by smaller specificity factors (SC/O), larger Michaelis-Menten constants for CO2 (Kc) and O2 (Ko), and larger maximum carboxylation velocities (Vc) than Rubisco from wheat, which can be rationalized in terms of the CO2-rich environment of C4 Rubisco in the bundle sheath. During leaf dehydration the quantity and maximum activity of Rubisco remained unchanged but the initial and total activities declined slightly, possibly due to increased inhibition. Tight-binding inhibitors were present in the light but were more abundant in the dark, especially in Z. japonica, and increased in quantity with drought stress. The inhibitor from darkened leaves of Z. japonica was identified as 2-carboxyarabinitol-1-phosphate (CA1P). Consistent with the presence of CA1P, the total activity of Rubisco was decreased after 12 h darkness in Z. japonica. Ribulose-1,5-bisphosphate (RuBP) in the leaves decreased with drought stress, to quantities approximating those of Rubisco catalytic sites. The magnitude of the decrease in RuBP suggested that, at least in C. dactylon and Z. japonica, it could contribute to the drought-induced decrease in photosynthesis.

Drought stress increases the production of 5-hydroxynorvaline in two C4 grasses.

Plants produce various compounds in response to water deficit. Here, the presence and identification of a drought-inducible non-protein amino acid in the leaves of two C(4) grasses is first reported. The soluble amino acids extracted from the leaves of three different species were measured by high-performance liquid chromatography of derivatives formed with o-phthaldialdehyde and beta-mercaptoethanol. One amino acid that increased in amount with drought stress had a retention time not corresponding to any common amino acid. Its identity was determined by metabolite profiling, using (1)H NMR and GC-MS. This unusual amino acid was present in the dehydrated leaves of Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel, but was absent from Paspalum dilatatum Poir. Its identity as 2-amino-5-hydroxypentanoic acid (5-hydroxynorvaline, 5-HNV) was confirmed by synthesis and co-chromatography of synthetic and naturally occurring compounds. The amount of 5-HNV in leaves of the more drought tolerant C(4) grasses, C. dactylon and Z. japonica, increased with increasing water deficit; therefore, any benefits from this unusual non-protein amino acid for drought resistance should be further explored.

Grasses of different C4 subtypes reveal leaf traits related to drought tolerance in their natural habitats: Changes in structure, water potential, and amino acid content.

Three grasses (Poaceae) of different C(4) subtypes, Paspalum dilatatum (NADP-malic enzyme [ME]), Cynodon dactylon (NAD-ME) and Zoysia japonica (phosphoenolpyruvate carboxykinase), occur in natural habitats that differ in annual rainfall. Their leaf characteristics were studied to identify traits related to drought tolerance. Plants were grown in pots, and water deficit was gradually induced by withholding water. Leaves of Z. japonica had the greatest and P. dilatatum the lowest relative dry matter content. Transverse sections of leaves that developed during the water deficit showed little change compared to control leaves, consistent with low phenotypic plasticity. Anatomical features distinguished the three species, with xeromorphic characteristics most strongly represented in Z. japonica. The leaf relative water content (RWC) decreased with the soil water content similarly for the three grasses. However, at 80% RWC, the leaf water potential was -3.1 MPa for Z. japonica and only -1.3 MPa for P. dilatatum and C. dactylon. Soluble amino acids, especially proline, increased as RWC decreased in leaves of C. dactylon and Z. japonica. Phenylalanine, valine, leucine, and isoleucine increased more in Z. japonica than in the other two species. The results provide evidence that C. dactylon and, especially, Z. japonica have evolved leaf traits better suited to arid habitats.

The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes.

The C4 photosynthetic pathway involves the assimilation of CO2 by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C4 acids. The enzymes of the CO2 concentrating mechanism could be affected under water deficit and limit C4 photosynthesis. Three different C4 grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C4 acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C4 enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of L-malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO2 by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.

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.

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.

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.

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 CO(2). In C(3) 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.

Evidence for recycling of inorganic phosphate by wheat chloroplasts during photosynthesis at air levels of CO2 and O2.

Phosphate recycling under photorespiratory conditions was investigated using intact wheat chloroplasts from Triticum aestivum (cv. Maris dove). A decline in the optimal Pi level needed to support steady-state photosynthesis was observed (a) as the bicarbonate supply became limiting, or (b) as oxygen concentrations were increased. Further, at subsaturating CO2 and elevated O2 (52%), photosynthetic induction periods were shortest in the absence of exogenous Pi, and severely extended by its addition. Thus, photosynthesis under low CO2 levels which favor ribulose 1,5 bisphosphate (RuBP) oxygenase activity and glycolate synthesis by chloroplasts decreases their dependency on exogenous Pi from the initial illumination of chloroplasts through to the attainment of steady state rates of O2 evolution. Uptake of phosphate (Pi) was directly measured at ambient O2 concentrations and showed the stoichiometry of O2 evolved to Pi consumed at 10 mmol/L bicarbonate (saturating) had a mean value of 3.0, and was increased to 5.4 at 2.5 mmol/L bicarbonate and to > 8.0 at 1.0 mmol/L bicarbonate. The observation is consistent with enhanced stromal recycling of Pi released during hydrolysis of phosphoglycolate produced in greater quantities as the ratio of RuBP carboxylase relative to oxygenase activities (vc/vo) declines. The theoretical relationship between vc/vo and O2/Pi stoichiometries was derived and compared favorably to experimental data obtained.

Rubisco activity: effects of drought stress.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity is modulated in vivo either by reaction with CO2 and Mg2+ to carbamylate a lysine residue in the catalytic site, or by the binding of inhibitors within the catalytic site. Binding of inhibitors blocks either activity or the carbamylation of the lysine residue that is essential for activity. At night, in many species, 2-carboxyarabinitol-1-phosphate (CA1P) is formed which binds tightly to Rubisco, inhibiting catalytic activity. Recent work has shown that tight-binding inhibitors can also decrease Rubisco activity in the light and contribute to the regulation of Rubisco activity. Here we determine the influence that such inhibitors of Rubisco exert on catalytic activity during drought stress. In tobacco plants, 'total Rubisco activity', i.e. the activity following pre-incubation with CO2 and Mg2+, was positively correlated with leaf relative water content. However, 'total Rubisco activity' in extracts from leaves with low water potential increased markedly when tightly bound inhibitors were removed, thus increasing the number of catalytic sites available. This suggests that in tobacco the decrease of Rubisco activity under drought stress is not primarily the result of changes in activation by CO2 and Mg2+ but due rather to the presence of tight-binding inhibitors. The amounts of inhibitor present in leaves of droughted tobacco based on the decrease in Rubisco activity per mg soluble protein were usually much greater than the amounts of the known inhibitors (CA1P and 'daytime inhibitor') that can be recovered in acid extracts. Alternative explanations for the difference between maximal and total activities are discussed.

Elucidating the biosynthesis of 2-carboxyarabinitol 1-phosphate through reduced expression of chloroplastic fructose 1,6-bisphosphate phosphatase and radiotracer studies with 14CO2.

2-carboxyarabinitol 1-phosphate limits photosynthetic CO2 assimilation at low light because it is a potent, naturally occurring inhibitor of ribulose 1,5-bisphosphate carboxylase/oxygenase. Evidence is presented that this inhibitor is derived from chloroplastic fructose 1,6-bisphosphate. First, transgenic plants containing decreased amounts of chloroplastic fructose 1,6-bisphosphate phosphatase contained increased amounts of fructose 1,6-bisphosphate and 2-carboxyarabinitol 1-phosphate and greatly increased amounts of the putative intermediates hamamelose and 2-carboxyarabinitol, which in some cases were as abundant as sucrose. Second, French bean leaves in the light were shown to incorporate 14C from 14CO2 sequentially into fructose 1,6-bisphosphate, hamamelose bisphosphate, hamamelose monophosphate, hamamelose, and 2-carboxyarabinitol. As shown previously, 14C assimilated by photosynthesis was also incorporated into 2-carboxyarabinitol 1-phosphate during subsequent darkness.