Evolution and function of a cis-regulatory module for mesophyll-specific gene expression in the C4 dicot Flaveria trinervia

Abstract

C(4) photosynthesis presents a sophisticated integration of two complementary cell types, mesophyll and bundle sheath cells. It relies on the differential expression of the genes encoding the component enzymes and transporters of this pathway. The entry enzyme of C(4) photosynthesis, phosphoenolpyruvate carboxylase (PEPC), is found exclusively in mesophyll cells, and the expression of the corresponding gene is regulated at the transcriptional level. In the C(4) dicot Flaveria trinervia, the mesophyll-specific expression of the C(4) PEPC gene (ppcA) depends on a 41-bp segment in the distal promoter region referred to as MEM1 (for mesophyll expression module1). Here, we show that a MEM1 sequence found in the orthologous ppcA gene from the C(3) species Flaveria pringlei is not able to direct mesophyll-specific gene expression. The two orthologous MEM1 sequences of F. pringlei and F. trinervia differ at two positions, a G-to-A exchange and the insertion of the tetranucleotide CACT. Changes at these two positions in the C(3) MEM1 sequence were necessary and sufficient to create a mesophyll-specificity element during C(4) evolution. The MEM1 of F. trinervia enhances mesophyll expression and concomitantly represses expression in bundle sheath cells and vascular bundles.

Figure 1.

Analysis of the ppcA GUS Reporter Gene Constructs FtPR, FtM-FtPR, and FbM-FtPR in Transgenic F. bidentis. (A) Schematic presentation of the ppcA GUS chimerical genes. Nucleotide numbers refer to the translation initiation codon. The Ft MEM1 region is indicated by light green boxes, and the proximal region (PR) is indicated by dark green boxes. The state of the C₃/C₄-associated polymorphisms in the A submodule (G or A) and the B submodule (presence or absence of CACT) of MEM1 are indicated. (B) GUS activities in leaves of transgenic F. bidentis plants. The median values (black bars) of the GUS activities are expressed in nanomoles of the reaction product 4-methylumbelliferone (MU) generated per milligram of protein per minute. The numbers of independent transgenic plants investigated (N) are indicated at the top of each column. (C) to (E) Histochemical localization of GUS activity in leaf sections of transgenic F. bidentis plants transformed with the FtPR (C), FtM-FtPR (D), or FbM-FtPR (E) construct. Incubation times were 25 h (C), 12 h (D), and 24 h (E).

Figure 2.

Analysis of the ppcA GUS Reporter Gene Constructs ppcAFp, FpM-FpPR, FpM-FtPR, FtM/A_ΔCACT-FtPR, and FpM/G_+CACT-FtPR in Transgenic F. bidentis. (A) Schematic presentation of the ppcA GUS chimerical genes. Nucleotide numbers refer to the translation initiation codon. The Ft MEM1 region is indicated by light green boxes, and the Fp MEM1 region is indicated by light yellow boxes. The Ft proximal region (PR) is indicated by dark green boxes, and the Fp PR is indicated by dark yellow boxes. The state of the C₃/C₄-associated polymorphisms in the A submodule (G or A) and the B submodule (presence or absence of CACT) of MEM1 are indicated. (B) GUS activities in leaves of transgenic F. bidentis plants. The numbers of independent transgenic plants investigated (N) are indicated at the top of each column. Median values (black bars) are shown. MU, 4-methylumbelliferone. (C) to (G) Histochemical localization of GUS activity in leaf sections of transgenic F. bidentis plants transformed with ppcAFp (C), FpM-FpPR (D), FpM-FtPR (E), FtM/A_ΔCACT-FtPR (F), and FpM/G_+CACT-FtPR (G). Incubation times were 2 h (C), 3.5 h (D), 18 h (E), 17 h (F), and 3 h (G).

Figure 3.

Analysis of the ppcA GUS Reporter Gene Constructs FtM-FtPR/Δnt12-41, FtM-FtPR/Δnt1-11, FtM/A-FtPR, and FtM/ΔCACT-FtPR in Transgenic F. bidentis. (A) Schematic presentation of the ppcA GUS chimerical genes. Nucleotide numbers refer to the translation initiation codon. The Ft MEM1 region is indicated by light green boxes, and the Ft proximal region (PR) is indicated by dark green boxes. The state of the C₃/C₄-associated polymorphisms in the A submodule (G or A) and the B submodule (presence or absence of CACT) of MEM1 are indicated. (B) GUS activities in leaves of transgenic F. bidentis plants. The numbers of independent transgenic plants investigated (N) are indicated at the top of each column. Median values (black bars) are shown. MU, 4-methylumbelliferone. (C) to (F) Histochemical localization of GUS activity in leaf sections of transgenic F. bidentis plants transformed with FtM/Δnt12-41-FtPR (C), FtM/Δnt1-11-FtPR (D), FtM/A-FtPR (E), and FtM/ΔCACT-FtPR (F). Incubation times were 21 h ([C], [D], and [F]) and 24 h (E). Note that some FtM/ΔCACT-FtPR plants were analyzed previously by Gowik et al. (2004). In the course of this study, they were reanalyzed by extending the staining period to 2 d in order to increase the sensitivity of detection. In addition, new plants were generated.

Figure 4.

Comparisons of MEM1 and MEM1-Like Sequences. (A) MEM1 sequences of the ppcA promoters from C₄, C₄-like, C₃-C₄ intermediate, and C₃ Flaveria species. Ft, F. trinervia; Fb, F. bidentis; Fpa, F. palmerii; Fv, F. vaginata; Fbr, F. brownii; Fpu, F. pubescens; Fc, F. cronquistii; Fp, F. pringlei. (B) Comparison of MEM1 sequences from the ppcA promoters of F. trinervia (Ft) and F. pringlei (Fp) with their MEM1-like counterparts from the ppcB genes. The MEM1 A and B submodules are highlighted by boxes. Asterisks label identical nucleotides in the A or B submodule of all promoters. Gray bars indicate the single nucleotide difference in the A submodule and the insertion/deletion of the CACT tetranucleotide in the B submodule. For a comparison of the whole promoters, see Supplemental Figure 1 online.

Figure 5.

Analysis of the ppcA GUS Reporter Gene Construct FtppcA in Transgenic Arabidopsis. (A) Schematic presentation of the ppcA GUS chimerical gene. Nucleotide numbers refer to the translation initiation codon. The Ft MEM1 region and the proximal region (PR) are indicated by dark green boxes. (B) GUS staining of a transgenic Arabidopsis seedling. Incubation time was 2.5 h. (C) Histochemical localization of GUS activity in a leaf section of a transgenic Arabidopsis plant transformed with FtppcA. Incubation time was 2.5 h. BSC, bundle sheath cells; PP, palisade parenchyma; SP, sponge parenchyma; X, xylem.

Figure 6.

MEM1 Evolution in the Genus Flaveria. (A) Phylogenetic tree of the ppcA, ppcB, and ppcC PEPC promoter sequences of Flaveria. The sequence alignment was constructed with Dialign (Morgenstern et al., 1998) and corrected by hand. The tree was generated by the distance method as implemented in PAUP 4.0b10 (Swofford, 2002), where uninformative characteristics were excluded. Bootstrap analysis was performed with 1000 replications, and the obtained bootstrap values are indicated. (B) Model of MEM1 evolution in the genus Flaveria. The model relies on the phylogeny of the genus Flaveria, which is based on morphological and molecular data sets (McKown et al., 2005).