Evolution of C4 photosynthesis in the genus Flaveria: 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.

Figure 1.

The Genus Flaveria as a Model Organism to Study C₄ Evolution. (A) Schematic view of the NADP-ME type C₄ pathway as it can be found in C₄ Flaveria species modified from Gowik and Westhoff (2011). See the text for abbreviations and a detailed description of the pathway. (B) Phylogeny of the genus Flaveria according to McKown et al. (2005).

Figure 2.

The Quantitative Patterns of Transcript Accumulation in C₃ and C₄ Flaverias Are Distinct. (A) Comparison of F. trinervia (Ft, C₄) and F. robusta (Fro, C₃). (B) Comparison of F. bidentis (Fb, C₄) and F. pringlei (Fp, C₃). Shown are the percentages of genes with significantly higher abundance of transcripts in the C₄ species (green bars), percentages of genes unchanged (gray bars, including genes not detected), and percentages of genes with significantly lower abundance of transcripts in C₄ species (yellow bars). Percentages are based on the total number of genes in each annotation class (values in parentheses on the y axis). TCA, tricarboxylic acid.

Figure 3.

Overrepresentation Analyses of Up- and Downregulated Genes within Functional Gene Classes Defined by MapMan Bins. Fisher’s exact test followed by the Bonferroni correction was used to identify functional categories enriched in up- or downregulated genes when transcript abundances in F. trinervia (Ft, C₄) and F. robusta (Fro, C₃), F. bidentis (Fb, C₄) and F. pringlei (Fp, C₃), or F. ramosissima (Fra, C₃-C₄) and F. robusta (Fro, C₃) were compared. Blue, up- or downregulated genes are significantly overrepresented; red, up- or downregulated genes are significantly underrepresented. aa, amino acid; LHC, light-harvesting complex; PS, photosynthesis.

Figure 4.

Differences in C₄ Pathway Gene Expression for F. trinervia (C₄), F. ramosissima (C₃-C₄), F. robusta (C₃), F. bidentis (C₄), and F. pringlei (C₃). (A) Schematic view of the NADP-ME type C₄ pathway. Relative transcript abundances are given in small inset boxes. The transcript levels for F. trinervia, F. ramosissima, and F. robusta were normalized by setting the F. robusta transcript level to one, and the F. bidentis and F. pringlei transcript levels were normalized by setting the F. pringlei transcript level to one for each gene. (B) Activity of NAD-ME in the extractable enzyme fractions of leaves from all five species (+ se; n = 3). FW, fresh weight. (C) Ala and Asp amounts in the leaves of all five species (+ se; n = 3).

Figure 5.

Photorespiration Is Altered between F. trinervia (C₄), F. ramosissima (C₃-C₄), F. robusta (C₃), F. bidentis (C₄), and F. pringlei (C₃). (A) Schematic view of the photorespiratory pathway. Relative transcript abundances are given in small inset boxes. The transcript levels for F. trinervia, F. ramosissima, and F. robusta were normalized by setting the F. robusta transcript level to one, and the F. bidentis and F. pringlei transcript levels were normalized by setting the F. pringlei transcript level to one for each gene. (B) Amounts of important photorespiratory metabolites in the leaves of all five species (6 se; n = 3).

Figure 6.

Cluster Analysis of Transcript Abundances in F. bidentis (C₄), F. trinervia (C₄), F. ramosissima (C₃-C₄), F. robusta (C₃), and F. pringlei (C₃). (A) Hierarchical sample clustering of all expressed transcripts. The tree was calculated with the MEV program using the HCL module with the Euclidean distance criterion and the average linkage method. According to their transcript profiles, the two C₄ species are more closely related to each other than to the other three Flaveria species. (B) C₄-related clusters. K-means analysis was used to define 26 clusters identifying different expression profiles. The six clusters with a C₄-related pattern are shown. All 26 clusters can be found in Supplemental Figure 2 online. (C) Functional category (MapMan bins) enrichment among the six C₄-related clusters. Enrichment of genes belonging to distinct functional categories was analyzed with the Wilcoxon statistic followed by the Benjamini-Hochberg correction. Blue, significantly overrepresented; red, significantly underrepresented. The complete enrichment analysis for all 26 clusters is shown in Supplemental Figure 4 online. aa, amino acid; CHO, carbohydrate; PS, photosynthesis; TCA, tricarboxylic acid cycle.

Figure 7.

Schematic of the CO₂ Concentrating and Photorespiratory Pathways in the C₃-C₄ Intermediate Species F. ramosissima. Three distinct CO₂ concentrating mechanisms, the NADP-ME type (green), the NAD-ME type (blue) C₄ pathway, and the photorespiratory Gly shuttle (orange), operate in parallel in this C₃-C₄ intermediate. F. ramosissima, with a working C₄ cycle, can compensate for the massive ammonia imbalance introduced by the photorespiratory CO₂ pump, by adjusting the ratios of the transport metabolites Ala/pyruvate and Asp/malate.