Identification and expression analysis of a gene encoding a bacterial-type phosphoenolpyruvate carboxylase from Arabidopsis and rice

Abstract

Phosphoenolpyruvate carboxylase (PEPC) is distributed in plants and bacteria but is not found in fungi and animal cells. Important motifs for enzyme activity and structure are conserved in plant and bacterial PEPCs, with the exception of a phosphorylation domain present at the N terminus of all plant PEPCs reported so far, which is absent in the bacterial enzymes. Here, we describe a gene from Arabidopsis, stated as Atppc4, encoding a PEPC, which shows more similarity to Escherichia coli than to plant PEPCs. Interestingly, this enzyme lacks the phosphorylation domain, hence indicating that it is a bacterial-type PEPC. Three additional PEPC genes are present in Arabidopsis, stated as Atppc1, Atppc2, and Atppc3, encoding typical plant-type enzymes. As most plant PEPC genes, Atppc1, Atppc2, and Atppc3 are formed by 10 exons interrupted by nine introns. In contrast, Atppc4 gene has an unusual structure formed by 20 exons. A bacterial-type PEPC gene was also identified in rice (Oryza sativa), stated as Osppc-b, therefore showing the presence of this type of PEPC in monocots. The phylogenetic analysis suggests that both plant-type and bacterial-type PEPCs diverged early during the evolution of plants from a common ancestor, probably the PEPC from gamma-proteobacteria. The diversity of plant-type PEPCs in C3, C4, and Crassulacean acid metabolism plants is indicative of the evolutionary success of the regulation by phosphorylation of this enzyme. Although at a low level, the bacterial-type PEPC genes are expressed in Arabidopsis and rice.

Figure 1.

Sequence alignment of PEPC from different sources. The protein sequences deduced from Atppc1, Atppc2, Atppc3, Atppc4, and Osppc-b genes were compared with the E. coli and Synechocystis sp. PCC 6803 enzymes using the program BioEdit sequence alignment editor (Hall, 1999). The accession numbers of these sequences are described in ”Materials and Methods.” The asterisk marks the phosphorylable Ser residue at the phosphorylation domain (underlined). Boxes I to V mark domains important for enzyme catalysis.

Figure 2.

Structure of the Arabidopsis PEPC gene family and Osppc-b gene from rice. Black rectangles, exons; lines, introns. The numbers indicate intron size in nucleotides. Exons and introns in the Arabidopsis genes were deduced by comparing the corresponding cDNA with the gene sequence obtained from the Arabidopsis database (http://www.Arabidopsis.org). The structure of Osppc-b was deduced with the GENSCAN program (http://genes.mit.edu/GENSCAN.html).

Figure 3.

Phylogenetic tree of PEPCs from different sources. The phylogenetic tree was constructed with full-length PEPC amino acid sequences using the neighbor-joining method of the ClustalX version 1.8 program (Thompson et al., 1997). Bootstrap analysis was computed with 1,000 replicates. Numbers in branches indicate bootstrap values (percent). The accession numbers are described in ”Materials and Methods.”

Figure 4.

Differential expression of PEPC genes in Arabidopsis. DNA-free total RNA (0.8 μg) isolated from different Arabidopsis tissues were analyzed by relative quantitative RT-PCR using gene-specific primers in the presence of 18S rRNA primers and competimers. Experiments were repeated at least three times obtaining similar results. The expected sizes of the bands corresponding to the different transcripts are: Atppc1, 197 bp; Atppc2, 100 bp; Atppc3, 170 bp; Atppc4, 195 bp and rRNA, 315 bp, as indicated on the left. Ethidium bromide-stained bands for each gene were quantified with the ScionImage software and compared with the corresponding rRNA band. The highest value in each analysis was arbitrarily considered as 100%. R, Root; Rl, rosette leaves; St, stem; Sl, stem leaves; Sq, silique; F, flower.

Figure 5.

Differential expression of PEPC genes in rice seeds and seedlings. Rice seeds were allowed to germinate for 5 d and aleurone layer (A) and scutellum (Sc) were dissected. Roots (R) and shoots (Sh) were dissected from 5-day-old seedlings. Total RNA was extracted from these tissues and from mature seeds (MS) and analyzed by relative quantitative RT-PCR using gene-specific primers in the presence of 18S rRNA primers and competimers. Experiments were repeated at least three times obtaining similar results. The expected sizes of the bands corresponding to the different transcripts are: Osppc1, 196 bp; Osppc2, 121 bp; Osppc-b, 165 bp; and rRNA, 315 bp, as indicated on the left. Ethidium bromide-stained bands were quantified with the ScionImage software and compared with the corresponding rRNA band. The highest value in each analysis was arbitrarily considered as 100%.