RNA-Seq based phylogeny recapitulates previous phylogeny of the genus Flaveria (Asteraceae) with some modifications.

BACKGROUND: The genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the genus Flaveria contains 21 of the 23 known Flaveria species and has been previously constructed using a combination of morphological data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnL-F). RESULTS: Here we developed a new strategy to update the phylogenetic tree of 16 Flaveria species based on RNA-Seq data. The updated phylogeny is largely congruent with the previously published tree but with some modifications. We propose that the data collection method provided in this study can be used as a generic method for phylogenetic tree reconstruction if the target species has no genomic information. We also showed that a "F. pringlei" genotype recently used in a number of labs may be a hybrid between F. pringlei (C3) and F. angustifolia (C3-C4). CONCLUSIONS: We propose that the new strategy of obtaining phylogenetic sequences outlined in this study can be used to construct robust trees in a larger number of taxa. The updated Flaveria phylogenetic tree also supports a hypothesis of stepwise and parallel evolution of C4 photosynthesis in the Flavaria clade.

Initial events during the evolution of C4 photosynthesis in C3 species of Flaveria.

The evolution of C4 photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO2 pump, termed C2 photosynthesis. How C3 species evolved C2 metabolism is critical to understanding the initial phases of C4 plant evolution. To evaluate early events in C4 evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C3 and C2 species of Flaveria, a model genus for C4 evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C3 species that are most closely related to the C2 Flaveria species, would show rudimentary characteristics of C2 physiology. Compared with less-related C3 species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C2 species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO2 compensation point of photosynthesis was slightly lower than in the less-related C3 species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO2 by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO2 within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C2 photosynthesis.

Evolution of C4 photosynthesis in the genus flaveria: establishment of a photorespiratory CO2 pump.

C4 photosynthesis is nature's most efficient answer to the dual activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the resulting loss of CO(2) by photorespiration. Gly decarboxylase (GDC) is the key component of photorespiratory CO(2) release in plants and is active in all photosynthetic tissues of C(3) plants, but only in the bundle sheath cells of C(4) plants. The restriction of GDC to the bundle sheath is assumed to be an essential and early step in the evolution of C(4) photosynthesis, leading to a photorespiratory CO(2) concentrating mechanism. In this study, we analyzed how the P-protein of GDC (GLDP) became restricted to the bundle sheath during the transition from C(3) to C(4) photosynthesis in the genus Flaveria. We found that C(3) Flaveria species already contain a bundle sheath-expressed GLDP gene in addition to a ubiquitously expressed second gene, which became a pseudogene in C(4) Flaveria species. Analyses of C(3)-C(4) intermediate Flaveria species revealed that the photorespiratory CO(2) pump was not established in one single step, but gradually. The knowledge gained by this study sheds light on the early steps in C(4) evolution.

Evolution of C4 photosynthesis in the genus Flaveria: how many and which genes does it take to make C4?

Selective pressure exerted by a massive decline in atmospheric CO(2) levels 55 to 40 million years ago promoted the evolution of a novel, highly efficient mode of photosynthetic carbon assimilation known as C(4) photosynthesis. C(4) species have concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex C(4) trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of related C(3) and C(4) species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C(3) to C(3)-C(4) intermediate to C(4) species, to quantify the differences in the transcriptomes of closely related plant species with varying degrees of C(4)-associated characteristics. Single gene analysis defines the C(4) cycle enzymes and transporters more precisely and provides new candidates for yet unknown functions as well as identifies C(4) associated pathways. Molecular evidence for a photorespiratory CO(2) pump prior to the establishment of the C(4) cycle-based CO(2) pump is provided. Cluster analysis defines the upper limit of C(4)-related gene expression changes in mature leaves of Flaveria as 3582 alterations.

The future of C4 research--maize, Flaveria or Cleome?

C4 photosynthesis has evolved multiple times among the angiosperms: the spatial rearrangement of the photosynthetic apparatus, combined with alterations to the leaf structure, allows CO2 to be concentrated around Rubisco. Higher CO2 concentrations at Rubisco decrease the rate of oxygenation and therefore reduce the amount of energy lost through photorespiration. C4 plants are particularly prevalent in tropical and subtropical regions because they can sustain higher rates of net photosynthesis; they also represent some of our most productive crops. To date, most progress in identifying genes crucial for C4 photosynthesis has been made using maize and Flaveria. We propose that Cleome, the most closely related genus containing C4 species to the C3 model Arabidopsis, be used together with Arabidopsis resources to accelerate our progress in elucidating the genetic basis of C4 photosynthesis.