Insights Into the Regulation of the Expression Pattern of Calvin-Benson-Bassham Cycle Enzymes in C3 and C4 Grasses

Abstract

C₄ photosynthesis is characterized by the compartmentalization of the processes of atmospheric uptake of CO₂ and its conversion into carbohydrate between mesophyll and bundle-sheath cells. As a result, most of the enzymes participating in the Calvin-Benson-Bassham (CBB) cycle, including RubisCO, are highly expressed in bundle-sheath cells. There is evidence that changes in the regulatory sequences of RubisCO contribute to its bundle-sheath-specific expression, however, little is known about how the spatial-expression pattern of other CBB cycle enzymes is regulated. In this study, we use a computational approach to scan for transcription factor binding sites in the regulatory regions of the genes encoding CBB cycle enzymes, SBPase, FBPase, PRK, and GAPDH-B, of C₃ and C₄ grasses. We identified potential cis-regulatory elements present in each of the genes studied here, regardless of the photosynthetic path used by the plant. The trans-acting factors that bind these elements have been validated in A. thaliana and might regulate the expression of the genes encoding CBB cycle enzymes. In addition, we also found C₄-specific transcription factor binding sites in the genes encoding CBB cycle enzymes that could potentially contribute to the pathway-specific regulation of gene expression. These results provide a foundation for the functional analysis of the differences in regulation of genes encoding CBB cycle enzymes between C₃ and C₄ grasses.

Keywords: C4 photosynthesis; Calvin-Benson-Bassham cycle; cis-regulatory elements; gene expression regulation; transcription factor binding sites.

FIGURE 1

SBPase coding sequence is highly conserved among C₃ and C₄ grasses in comparison to putative regulatory regions. (A,C) mVISTA plots of Brachypodium distachyon (A) and Sorghum bicolor (C) SBPase aligned to SBPase orthologs in C₃ and C₄ grasses. Genomic region includes approximately 2 kb upstream from the transcription start site and 1 kb after the end of the 3′ untranslated region (UTR). UTRs, exons, and introns are annotated. The vertical line with a small perpendicular arrow indicates the transcription start site and the arrowhead the orientation of the gene. The graph shows sequences with 50–100% identity and regions with > 70% identity within 50 base pairs are highlighted in purple if they are located in exons, in cyan if they are located in UTRs, or in pink if they are located outside exons or UTRs. Boxes highlight conserved non-coding sequences (CNSs), and the predicted position weight matrix for each conserved sequence is included (B). On the left side, the phylogenetic relationship between C₃ (in green) and C₄ (in brown) grasses is shown. Common ancestor of BOP clade and PACMAD clade species are shown as a red and as a blue dot, respectively. Note that Dichanthelium oligosanthes is a C₃ species within the PACMAD.

FIGURE 2

Arabidopsis thaliana transcription factor binding sites (TFBS) identified in the potential regulatory sequences of genes encoding C₃ and C₄ Calvin-Benson-Bassham (CBB) cycle enzymes. (A) Upset plot showing the identified A. thaliana TFBS and in which orthologous genes they are found. Horizontal bars represent the number of common motifs identified within orthologs, vertical bars represent the motifs shared between different orthologous genes, as indicated by the dots below. The name of the A. thaliana TFBS is included inside the vertical bars, and the number of motifs as well as the name of the gene group are indicated above. There is only one common motif shared across all orthologs (common_CBB: VRN1, gray bar), and most of the common motifs identified in GAPDHB are not common in the genes encoding other enzymes (common_GADPHB). In addition, many motifs are shared between GAPDHB, FBPase, and SBPase (common_SFG), whereas most PRK motifs are not shared with the genes encoding other enzymes (common_PRK). (B) Localization of each gene group in the genomic region around genes encoding CBB cycle enzymes in Brachypodium distachyon and Sorghum bicolor. The x-axis corresponds to the genomic coordinates with the start codon corresponding to the + 1 position. The colored arrow represents the gene structure with UTRs in blue, exons in red, and potential promoter and terminator as a black line. The dots represent the genomic coordinates of each of the motifs within each gene group. Different gene groups are separated along the y-axis. Despite being comprise by the same TFBS, the distribution of the dots changes between species. Notably, TFBS can be found at multiple coordinates in the same gene. Most of the trans-acting factors binding to the identified A. thaliana TFBS belong to the same family, and often bind to similar sequences. In fact, TFBS tend to cluster in discrete regions that might play a role in the regulation of the expression of the corresponding gene.