The relative contribution of advantageous and neutral mutations to the evolutionary process is a central problem in evolutionary biology. Current estimates suggest that whereas Drosophila, mice, and bacteria have undergone extensive adaptive evolution, hominids show little or no evidence of adaptive evolution in protein-coding sequences. This may be a consequence of differences in effective population size. To study the matter further, we have investigated whether plants show evidence of adaptive evolution using an extension of the McDonald-Kreitman test that explicitly models slightly deleterious mutations by estimating the distribution of fitness effects of new mutations. We apply this method to data from nine pairs of species. Altogether more than 2,400 loci with an average length of approximately 280 nucleotides were analyzed. We observe very similar results in all species; we find little evidence of adaptive amino acid substitution in any comparison except sunflowers. This may be because many plant species have modest effective population sizes.

Background: Rubisco enzyme catalyzes the first step in net photosynthetic CO 2 assimilation and photorespiratory carbon oxidation and is responsible for almost all carbon fixation on Earth. The large subunit of Rubisco is encoded by the chloroplast rbcL gene, which is widely used for reconstruction of plant phylogenies due to its conservative nature. Plant systematicists have mainly used rbcL paying little attention to its function, and the question whether it evolves under Darwinian selection has received little attention. The purpose of our study was to evaluate how common is positive selection in Rubisco among the phototrophs and where in the Rubisco structure does positive selection occur.

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.

19Enhancing the catalytic properties of the CO2-fixing enzyme Rubisco is a target for 20 improving agricultural crop productivity. Here we reveal high diversity in the kinetic 21 response between 10°C to 37°C by Rubisco from C3-and C4-species within the grass tribe 22Paniceae. The CO2-fixation rate (kcat C ) for Rubisco from the C4-grasses with NADP-malic 23 enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic 24 pathways was two-fold greater than the kcat C of Rubisco from NAD-ME species over all 25 temperatures. The decline in the response of CO2/O2 specificity with increasing 26 temperature was slower for PCK and NADP-ME Rubisco -a trait which would be 27 advantageous in the warmer climates they inhabit relative to the NAD-ME grasses. 28Variation in the temperatures kinetics of Paniceae C3-Rubisco and PCK-Rubisco were 29 modelled to differentially stimulate C3-photosynthesis above and below 25°C under current 30 and elevated CO2. Identified are large subunit amino acid substitutions that could account 31 for the catalytic variation among Paniceae Rubisco. Incompatibilities with Paniceae 32Rubisco biogenesis in tobacco however hindered their mutagenic testing by chloroplast 33 transformation. Circumventing these bioengineering limitations is critical to tailoring the 34 properties of crop Rubisco to suit future climates. 35Concerns about how escalating climate change will influence ecosystems are particularly 36 focused on the consequences to global agricultural productivity where increases are 37 paramount to meet the rising food and biofuel demands. Strategies to improve crop yield 38 by increasing photosynthesis have largely focused on overcoming the functional 39 inadequacies of the CO2-fixing enzyme Rubisco. A competing O2-fixing reaction by 40Rubisco produces a toxic product whose recycling by photorespiration consumes energy 41 and releases carbon. The frequency of the oxygenation reaction increases with temperature. 42To evade photorespiration many plants from hot, arid ecosystems have evolved C4 43 photosynthesis that concentrates CO2 around Rubisco that also facilitates improved plant 44water, light and nitrogen use. Here we show extensive catalytic variation in Rubisco from 45Paniceae grasses that align with the biochemistry and environmental origins of the different 46 C4 plant subtypes. We reveal opportunities for enhancing crop photosynthesis under 47 current and future CO2 levels at varied temperatures. 48The realization of the dire need to address global food security has heightened the need for 49 new solutions to increase crop yields 1 . Field tests and modelling analyses have highlighted 50 how photosynthetic carbon assimilation underpins the maximal yield potential of crops 2 . 51This has increased efforts to identify solutions to enhance photosynthetic efficiency and 52 hence plant productivity 3 . Particular attention is being paid to improving the rate at which 53 ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) can fix CO2 54 (refs...

Rubisco, the primary photosynthetic carboxylase, evolved 3-4 billion years ago in an anaerobic, high CO(2) atmosphere. The combined effect of low CO(2) and high O(2) levels in the modern atmosphere, and the inability of Rubisco to distinguish completely between CO(2) and O(2), leads to the occurrence of an oxygenation reaction that reduces the efficiency of photosynthesis. Among land plants, C(4) photosynthesis largely solves this problem by facilitating a high CO(2)/O(2) ratio at the site of Rubisco that resembles the atmosphere in which the ancestral enzyme evolved. The prediction that such conditions favor Rubiscos with higher kcat(CO2) and lower CO(2)/O(2) specificity (S(C/O)) is well supported, but the structural basis for the differences between C(3) and C(4) Rubiscos is not clear. Flaveria (Asteraceae) includes C(3), C(3)-C(4) intermediate, and C(4) species with kinetically distinct Rubiscos, providing a powerful system in which to study the biochemical transition of Rubisco during the evolution from C(3) to C(4) photosynthesis. We analyzed the molecular evolution of chloroplast rbcL and nuclear rbcS genes encoding the large subunit (LSu) and small subunit (SSu) of Rubisco from 15 Flaveria species. We demonstrate positive selection on both subunits, although selection is much stronger on the LSu. In Flaveria, two positively selected LSu amino acid substitutions, M309I and D149A, distinguish C(4) Rubiscos from the ancestral C(3) species and statistically account for much of the kinetic difference between the two groups. However, although Flaveria lacks a characteristic "C(4)" SSu, our data suggest that specific residue substitutions in the SSu are correlated with the kinetic properties of Rubisco in this genus.