Systems analysis of cis-regulatory motifs in C4 photosynthesis genes using maize and rice leaf transcriptomic data during a process of de-etiolation

Abstract

Identification of potential cis-regulatory motifs controlling the development of C4 photosynthesis is a major focus of current research. In this study, we used time-series RNA-seq data collected from etiolated maize and rice leaf tissues sampled during a de-etiolation process to systematically characterize the expression patterns of C4-related genes and to further identify potential cis elements in five different genomic regions (i.e. promoter, 5'UTR, 3'UTR, intron, and coding sequence) of C4 orthologous genes. The results demonstrate that although most of the C4 genes show similar expression patterns, a number of them, including chloroplast dicarboxylate transporter 1, aspartate aminotransferase, and triose phosphate transporter, show shifted expression patterns compared with their C3 counterparts. A number of conserved short DNA motifs between maize C4 genes and their rice orthologous genes were identified not only in the promoter, 5'UTR, 3'UTR, and coding sequences, but also in the introns of core C4 genes. We also identified cis-regulatory motifs that exist in maize C4 genes and also in genes showing similar expression patterns as maize C4 genes but that do not exist in rice C3 orthologs, suggesting a possible recruitment of pre-existing cis-elements from genes unrelated to C4 photosynthesis into C4 photosynthesis genes during C4 evolution.

Keywords: C4 photosynthesis; cell specificity; cis element; etiolation; evolution; systems biology..

Fig 1.

Pathway-level gene expression of maize and rice during the de-etiolation process. The dot size represents the gene expression level across the whole genome. The color code indicates the relative gene expression level within a given pathway, from low (yellow) to high (red). (A) Photosynthetic pathways, and (B) non-photosynthesis related pathways. CCM, CO₂ concentration mechanism.

Fig 2.

Expression curves of C₄ gene families. The x-axis represents different time points and the y-axis represents the RPKM value. Expression curves were 3^rd-order polynomial regressed, whilst points indicate actual RPKM values.

Fig 3.

Expression patterns of C₄ orthologous gene pairs between maize (red) and rice (blue). RPKM values were normalized after 3^rd-order polynomial regression.

Fig 4.

Diagram showing numbers and species of conserved DNA motifs between maize and rice. Conserved DNA motifs identified with the k80 appraoch are marked with color as indicated in the keys, and the number of mapped sites are shown. Overlapping results between the k80 and k30 approaches are marked in bold in the keys.

Fig 5.

The number of recruited motif sites in different segments of C₄ genes. The total number of mapped sites for potential recruited motifs in maize identified using the k80 approach are given in the corresponding genomic segments, and the number of overlapping motifs is indicated in brackets.