Evolution of C4 Photosynthesis in the GenusFlaveria: How Many and Which Genes Does It Take to Make C4?

Abstract

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.

Introgression and repeated co-option facilitated the recurrent emergence of C4photosynthesis among close relatives

“…3), the carbon shuttle of A. cimicina relies on enzymes and transporters associated with the most common form of C 4 photosynthesis (NADP‐malic enzyme type; Gowik et al. 2011; Bräutigam et al. 2014; Mallman et al.…”

Understanding metabolite transport and metabolism in C4 plants through RNA-seq

“…NADP-ME based decarboxylation occurs in the chloroplasts and requires the import of malate and the export of pyruvate (Figure 1d). Transcripts of NADP-ME are reliably of higher abundance in F. bidentis (Table 1) [6,7] as well as in Z. mays, Setaria viridis, and Echinochloa glabrescens [8,9 ,10]. The transport protein catalyzing the import of malate and/or the export of pyruvate is unknown at the molecular level ( Table 1).…”

“…The dicarboxylate transporter DiT2 (alternate name DCT) was considered a candidate transporter for malate in maize [35], however, the Flaveria trinervia DiT2 is unable to catalyze the reaction based on in vitro analysis of the transport substrates [36]. DiT2 is moderately upregulated in conjunction with NADP-ME [6][7][8] and DiT2 is strongly expressed in maize bundle sheath cells (Table 1) and S. bicolor bundle sheath cells [25]. Mutant analysis of maize showed symptoms consistent with a role in malate import in the C 4 cycle [37 ] raising the question whether the original assay was unsuitable for detecting the exchange [36] or whether Flaveria and maize may use different transporters (Figure 1d).…”

“…Alternatively, the OAA is converted to aspartate (Asp) in chloroplasts by plastidic aspartate transaminase (AspAT) [12,46] after import by DiT2 [36] in exchange for aspartate. Both enzymes and transporters are moderately upregulated [7]; in maize, AspAT, MDH, and DiT1 are mesophyll specific while DiT2 is bundle sheath specific ( Table 1). The transamination reaction requires an amino group donor in the chloroplast which may come directly from the N assimilation or from a plastidic AlaAT reaction realizing the regeneration of pyruvate.…”

“…This indicates that the complex trait C 4 photosynthesis is traceable at the transcript abundance level [5 ]. After the initial comparison in the genus Cleomaceae [5 ], the C 4 trait has also been traced in the Flaveriaceae [6,7], the (X = 9) Panicoideae [8], and in comparison of grass species with wider phylogenetic spacing, rice (Ehrhartoideae) vs Echinocloa (X = 9 Panicoideae) [9 ] and rice (Ehrhartoideae) vs maize (Andropogonae) [10]. C 4 photosynthesis is a complex trait which consists of changes in cellular and leaf tissue architecture, changes in regulation, and changes in metabolism.…”