Escherichia coli expressing chloroplast chaperones as a proxy to test heterologous Rubisco production in leaves.

Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimize, and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome-transformed tobacco, we show that the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal that heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low-cost screening tool that can accurately emulate chloroplast expression.

Predicting plant Rubisco kinetics from RbcL sequence data using machine learning.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is responsible for the conversion of atmospheric CO2 to organic carbon during photosynthesis, and often acts as a rate limiting step in the later process. Screening the natural diversity of Rubisco kinetics is the main strategy used to find better Rubisco enzymes for crop engineering efforts. Here, we demonstrate the use of Gaussian processes (GPs), a family of Bayesian models, coupled with protein encoding schemes, for predicting Rubisco kinetics from Rubisco large subunit (RbcL) sequence data. GPs trained on published experimentally obtained Rubisco kinetic datasets were applied to over 9000 sequences encoding RbcL to predict Rubisco kinetic parameters. Notably, our predicted kinetic values were in agreement with known trends, e.g. higher carboxylation turnover rates (Kcat) for Rubisco enzymes from C4 or crassulacean acid metabolism (CAM) species, compared with those found in C3 species. This is the first study demonstrating machine learning approaches as a tool for screening and predicting Rubisco kinetics, which could be applied to other enzymes.

Rubisco substitutions predicted to enhance crop performance through carbon uptake modelling.

Improving the performance of the CO2-fixing enzyme Rubisco is among the targets for increasing crop yields. Here, Earth system model (ESM) representations of canopy C3 and C4 photosynthesis were combined with species-specific Rubisco parameters to quantify the consequences of bioengineering foreign Rubiscos into C3 and C4 crops under field conditions. The 'two big leaf' (sunlit/shaded) model for canopy photosynthesis was used together with species-specific Rubisco kinetic parameters including maximum rate (Kcat), Michaelis-Menten constant for CO2 at ambient atmospheric O2 (Kc21%O2), specificity for CO2 to O2 (Sc/o), and associated heat activation (Ha) values. Canopy-scale consequences of replacing native Rubiscos in wheat, maize, and sugar beet with foreign enzymes from 27 species were modelled using data from Ameriflux and Fluxnet databases. Variation among the included Rubisco kinetics differentially affected modelled carbon uptake rates, and Rubiscos from several species of C4 grasses showed the greatest potential of >50% carbon uptake improvement in wheat, and >25% improvement in sugar beet and maize. This study also reaffirms the need for data on fully characterized Rubiscos from more species, and for better parameterization of 'Vcmax' and temperature response of 'Jmax' in ESMs.

Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop an RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a rbcS gene whose codon use and 5' untranslated-region matched rbcL Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pST-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pST However, the ßA-ßB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15%, respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.

Exploring molecular evolution of Rubisco in C3 and CAM Orchidaceae and Bromeliaceae.

BACKGROUND: The CO2-concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO2 availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C3 and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence. RESULTS: We identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C3 orchids and between strong CAM and C3 bromeliads suggested Rubisco had evolved in response to differing CO2 concentration. CONCLUSIONS: The results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ13C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.

Correction: Positively selected amino acid replacements within the RuBisCO enzyme of oak trees are associated with ecological adaptations.

[This corrects the article DOI: 10.1371/journal.pone.0183970.].

Positively selected amino acid replacements within the RuBisCO enzyme of oak trees are associated with ecological adaptations.

Phylogenetic analysis by maximum likelihood (PAML) has become the standard approach to study positive selection at the molecular level, but other methods may provide complementary ways to identify amino acid replacements associated with particular conditions. Here, we compare results of the decision tree (DT) model method with ones of PAML using the key photosynthetic enzyme RuBisCO as a model system to study molecular adaptation to particular ecological conditions in oaks (Quercus). We sequenced the chloroplast rbcL gene encoding RuBisCO large subunit in 158 Quercus species, covering about a third of the global genus diversity. It has been hypothesized that RuBisCO has evolved differentially depending on the environmental conditions and leaf traits governing internal gas diffusion patterns. Here, we show, using PAML, that amino acid replacements at the residue positions 95, 145, 251, 262 and 328 of the RuBisCO large subunit have been the subject of positive selection along particular Quercus lineages associated with the leaf traits and climate characteristics. In parallel, the DT model identified amino acid replacements at sites 95, 219, 262 and 328 being associated with the leaf traits and climate characteristics, exhibiting partial overlap with the results obtained using PAML.

Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C3 photosynthesis.

Enhancing the catalytic properties of the CO2-fixing enzyme Rubisco is a target for improving agricultural crop productivity. Here, we reveal extensive diversity in the kinetic response between 10 and 37 °C by Rubisco from C3 and C4 species within the grass tribe Paniceae. The CO2 fixation rate (kcatc) for Rubisco from the C4 grasses with nicotinamide adenine dinucleotide (NAD) phosphate malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic pathways was twofold greater than the kcatc of Rubisco from NAD-ME species across all temperatures. The declining response of CO2/O2 specificity with increasing temperature was less pronounced for PCK and NADP-ME Rubisco, which would be advantageous in warmer climates relative to the NAD-ME grasses. Modelled variation in the temperature kinetics of Paniceae C3 Rubisco and PCK Rubisco differentially stimulated C3 photosynthesis relative to tobacco above and below 25 °C under current and elevated CO2. Amino acid substitutions in the large subunit that could account for the catalytic variation among Paniceae Rubisco are identified; however, incompatibilities with Paniceae Rubisco biogenesis in tobacco hindered their mutagenic testing by chloroplast transformation. Circumventing these bioengineering limitations is critical to tailoring the properties of crop Rubisco to suit future climates.

Rubisco Catalytic Properties and Temperature Response in Crops.

Rubisco catalytic traits and their thermal dependence are two major factors limiting the CO2 assimilation potential of plants. In this study, we present the profile of Rubisco kinetics for 20 crop species at three different temperatures. The results largely confirmed the existence of significant variation in the Rubisco kinetics among species. Although some of the species tended to present Rubisco with higher thermal sensitivity (e.g. Oryza sativa) than others (e.g. Lactuca sativa), interspecific differences depended on the kinetic parameter. Comparing the temperature response of the different kinetic parameters, the Rubisco Km for CO2 presented higher energy of activation than the maximum carboxylation rate and the CO2 compensation point in the absence of mitochondrial respiration. The analysis of the Rubisco large subunit sequence revealed the existence of some sites under adaptive evolution in branches with specific kinetic traits. Because Rubisco kinetics and their temperature dependency were species specific, they largely affected the assimilation potential of Rubisco from the different crops, especially under those conditions (i.e. low CO2 availability at the site of carboxylation and high temperature) inducing Rubisco-limited photosynthesis. As an example, at 25°C, Rubisco from Hordeum vulgare and Glycine max presented, respectively, the highest and lowest potential for CO2 assimilation at both high and low chloroplastic CO2 concentrations. In our opinion, this information is relevant to improve photosynthesis models and should be considered in future attempts to design more efficient Rubiscos.

Surveying Rubisco Diversity and Temperature Response to Improve Crop Photosynthetic Efficiency.

The threat to global food security of stagnating yields and population growth makes increasing crop productivity a critical goal over the coming decades. One key target for improving crop productivity and yields is increasing the efficiency of photosynthesis. Central to photosynthesis is Rubisco, which is a critical but often rate-limiting component. Here, we present full Rubisco catalytic properties measured at three temperatures for 75 plants species representing both crops and undomesticated plants from diverse climates. Some newly characterized Rubiscos were naturally "better" compared to crop enzymes and have the potential to improve crop photosynthetic efficiency. The temperature response of the various catalytic parameters was largely consistent across the diverse range of species, though absolute values showed significant variation in Rubisco catalysis, even between closely related species. An analysis of residue differences among the species characterized identified a number of candidate amino acid substitutions that will aid in advancing engineering of improved Rubisco in crop systems. This study provides new insights on the range of Rubisco catalysis and temperature response present in nature, and provides new information to include in models from leaf to canopy and ecosystem scale.

Kranz and single-cell forms of C4 plants in the subfamily Suaedoideae show kinetic C4 convergence for PEPC and Rubisco with divergent amino acid substitutions.

The two carboxylation reactions performed by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are vital in the fixation of inorganic carbon for C4 plants. The abundance of PEPC is substantially elevated in C4 leaves, while the location of Rubisco is restricted to one of two chloroplast types. These differences compared with C3 leaves have been shown to result in convergent enzyme optimization in some C4 species. Investigation into the kinetic properties of PEPC and Rubisco from Kranz C4, single cell C4, and C3 species in Chenopodiaceae s. s. subfamily Suaedoideae showed that these major carboxylases in C4 Suaedoideae species lack the same mutations found in other C4 systems which have been examined; but still have similar convergent kinetic properties. Positive selection analysis on the N-terminus of PEPC identified residues 364 and 368 to be under positive selection with a posterior probability >0.99 using Bayes empirical Bayes. Compared with previous analyses on other C4 species, PEPC from C4 Suaedoideae species have different convergent amino acids that result in a higher K m for PEP and malate tolerance compared with C3 species. Kinetic analysis of Rubisco showed that C4 species have a higher catalytic efficiency of Rubisco (k catc in mol CO2 mol(-1) Rubisco active sites s(-1)), despite lacking convergent substitutions in the rbcL gene. The importance of kinetic changes to the two-carboxylation reactions in C4 leaves related to amino acid selection is discussed.

Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone.

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob(AtL) plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob(AtL-R1) plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob(AtL-R1) and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8(A)S8(t) Rubisco [comprising AtL and tobacco small (S) subunits] in tob(AtL-R1) leaves compared with tob(AtL), despite >threefold lower steady-state Rubisco mRNA levels in tob(AtL-R1). Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tob(AtL-R1) lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.

A novel Botrytis species is associated with a newly emergent foliar disease in cultivated Hemerocallis.

Foliar tissue samples of cultivated daylilies (Hemerocallis hybrids) showing the symptoms of a newly emergent foliar disease known as 'spring sickness' were investigated for associated fungi. The cause(s) of this disease remain obscure. We isolated repeatedly a fungal species which proved to be member of the genus Botrytis, based on immunological tests. DNA sequence analysis of these isolates, using several different phyogenetically informative genes, indicated that they represent a new Botrytis species, most closely related to B. elliptica (lily blight, fire blight) which is a major pathogen of cultivated Lilium. The distinction of the isolates was confirmed by morphological analysis of asexual sporulating cultures. Pathogenicity tests on Hemerocallis tissues in vitro demonstrated that this new species was able to induce lesions and rapid tissue necrosis. Based on this data, we infer that this new species, described here as B. deweyae, is likely to be an important contributor to the development of 'spring sickness' symptoms. Pathogenesis may be promoted by developmental and environmental factors that favour assault by this necrotrophic pathogen. The emergence of this disease is suggested to have been triggered by breeding-related changes in cultivated hybrids, particularly the erosion of genetic diversity. Our investigation confirms that emergent plant diseases are important and deserve close monitoring, especially in intensively in-bred plants.

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.

Molecular adaptation during a rapid adaptive radiation.

"Explosive" adaptive radiations on islands remain one of the most puzzling evolutionary phenomena and the evolutionary genetic processes behind such radiations remain unclear. Rapid morphological and ecological evolution during island radiations suggests that many genes may be under fairly strong selection, although this remains untested. Here, we report that during a rapid recent diversification in the Hawaiian endemic plant genus Schiedea (Caryophyllaceae), 5 in 36 studied genes evolved under positive selection. Positively selected genes are involved in defence mechanisms, photosynthesis, and reproduction. Comparison with eight mainland plant groups demonstrates both the relaxation of purifying selection and more widespread positive selection in Hawaiian Schiedea. This provides compelling evidence that adaptive evolution of protein-coding genes may play a significant role during island adaptive radiations.

Rubisco evolution in C4 eudicots: an analysis of Amaranthaceae sensu lato.

BACKGROUND: Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the key reaction in the photosynthetic assimilation of CO2. In C4 plants CO2 is supplied to Rubisco by an auxiliary CO2-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C4 Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C3 enzyme. Specific amino-acids in Rubisco are associated with C4 photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots. METHODOLOGY/PRINCIPAL FINDINGS: We investigated Rubisco evolution in Amaranthaceae sensu lato (including Chenopodiaceae), the third-largest family of C4 plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C4 Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C4 plant groups, such as C4 monocots, illustrating a striking parallelism in molecular evolution. CONCLUSIONS/SIGNIFICANCE: Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco.

Coevolution of amino acid residues in the key photosynthetic enzyme Rubisco.

BACKGROUND: One of the key forces shaping proteins is coevolution of amino acid residues. Knowing which residues coevolve in a particular protein may facilitate our understanding of protein evolution, structure and function, and help to identify substitutions that may lead to desired changes in enzyme kinetics. Rubisco, the most abundant enzyme in biosphere, plays an essential role in the process of carbon fixation through photosynthesis, thus facilitating life on Earth. This makes Rubisco an important model system for studying the dynamics of protein fitness optimization on the evolutionary landscape. In this study we investigated the selective and coevolutionary forces acting on large subunit of land plants Rubisco using Markov models of codon substitution and clustering approaches applied to amino acid substitution histories. RESULTS: We found that both selection and coevolution shape Rubisco, and that positively selected and coevolving residues have their specifically favored amino acid composition and pairing preference. The mapping of these residues on the known Rubisco tertiary structures showed that the coevolving residues tend to be in closer proximity with each other compared to the background, while positively selected residues tend to be further away from each other. This study also reveals that the residues under positive selection or coevolutionary force are located within functionally important regions and that some residues are targets of both positive selection and coevolution at the same time. CONCLUSION: Our results demonstrate that coevolution of residues is common in Rubisco of land plants and that there is an overlap between coevolving and positively selected residues. Knowledge of which Rubisco residues are coevolving and positively selected could be used for further work on structural modeling and identification of substitutions that may be changed in order to improve efficiency of this important enzyme in crops.

Changes in Rubisco kinetics during the evolution of C4 photosynthesis in Flaveria (Asteraceae) are associated with positive selection on genes encoding the enzyme.

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.

Genome wide analyses reveal little evidence for adaptive evolution in many plant species.

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.