Genome-wide Analysis of Phosphoenolpyruvate Carboxylase Gene Family and Their Response to Abiotic Stresses in Soybean

Abstract

Phosphoenolpyruvate carboxylase (PEPC) plays an important role in assimilating atmospheric CO₂ during C₄ and crassulacean acid metabolism photosynthesis, and also participates in various non-photosynthetic processes, including fruit ripening, stomatal opening, supporting carbon-nitrogen interactions, seed formation and germination, and regulation of plant tolerance to stresses. However, a comprehensive analysis of PEPC family in Glycine max has not been reported. Here, a total of ten PEPC genes were identified in soybean and denominated as GmPEPC1-GmPEPC10. Based on the phylogenetic analysis of the PEPC proteins from 13 higher plant species including soybean, PEPC family could be classified into two subfamilies, which was further supported by analyses of their conserved motifs and gene structures. Nineteen cis-regulatory elements related to phytohormones, abiotic and biotic stresses were identified in the promoter regions of GmPEPC genes, indicating their roles in soybean development and stress responses. GmPEPC genes were expressed in various soybean tissues and most of them responded to the exogenously applied phytohormones. GmPEPC6, GmPEPC8 and GmPEPC9 were significantly induced by aluminum toxicity, cold, osmotic and salt stresses. In addition, the enzyme activities of soybean PEPCs were also up-regulated by these treatments, suggesting their potential roles in soybean response to abiotic stresses.

Figure 1. Phylogenetic analysis of PEPC proteins in soybean and other plant species.

The full-length amino acid sequences of 75 PEPC proteins from 13 different plant species were used to construct the neighbor-joining tree using ClustalX 2.0 and MEGA 6.0 with 1000 bootstrap replicates. Branches with less than 50% bootstrap support were collapsed. The PEPCs were classified into two subfamilies (PTPC and BTPC). Subfamily PTPC is further separated into five clades from PTPC I to PTPC V, whereas subfamily BTPC is separated into two clades including BTPC I and BTPC II. Different plant species were distinguished by different colors.

Figure 2. The phylogenetic relationship and exon-intron structures of PEPC family in soybean.

Exon-intron structure was analyzed by online tool Gene Structure Display Server (GSDS). Lengths of exons and introns of GmPEPC genes were exhibited proportionally as indicated by the scale on the bottom. The classification of soybean PEPCs was indicated by the phylogenetic relationship on the left.

Figure 3. Multiple alignment of Soybean PEPC amino acid sequences.

Black, gray, and light shading indicate 100%, 75%, and 50% similarities, respectively. Spots represent gaps. Amino acid residues of experimentally proven function are indicated by *above the alignment.

Figure 4. Conservation and diversity of the motifs in PEPC proteins.

The schematic representation of ten motifs in PEPC family is elucidated by MEME. Amino acid residues of experimentally proven function are indicated by black arrows.

Figure 5. Predicted cis-elements in the promoter regions of GmPEPC genes.

The 1500 bp promoter regions of 10 GmPEPC genes were analyzed to predict the cis-elements, which were presented as colored ellipses: ABA responsive element (ABRE), anaerobic responsive element (ARE), auxin responsive element (TGA-element, AuxRR-core), light-responsive element (G-box), gibberellin responsive element (GARE-motif), ethylene responsive element (ERE, GCC-box), heat stress responsive element (HSE), low-temperature-responsive element (LTR), MYB binding site (MBS), pathogen-related cis-element (S-box), defense and stress-responsive element (TC-rich repeats), salicylic acid responsive element (TCA-element), jasmonic acid responsive element (TGACG-motif, CGTCA-motif), wound-responsive element (WUN-motif), endosperm development (P-box), and WRKY binding site (W-box). The numbers on the top indicate the relative positions to the start codon.

Figure 6. Expression patterns of GmPEPC genes in different soybean tissues.

The expression patterns of 10 GmPEPC genes in 14 soybean tissues were investigated by qRT-PCR, using soybean GmRP15 gene as the internal control. Root, stem, leaf, flower, and seeds from different development stages were subjected to analysis. DAF: day after flowering. The experiments were repeated three times, log 2 based value was used to create the heat map with clustering of genes. The expression levels are shown from the lowest (blue) to highest (yellow) in heat colors as indicated by the scale on the top.

Figure 7

Expression profiles of PEPC genes in leaves (A) and roots (B) of soybean in response to ABA, ACC, GA, JA, Al, cold, salt (NaCl) and osmotic (PEG) treatments. The relative expression levels of the 10 PEPC genes were quantified by qRT-PCR, using soybean GmRP15 gene as the internal control. Leaves and roots of 14-d soybean seedlings are used to investigate the changes in PEPC expression under different treatments, including 100 μM ABA (Abscisic acid), 100 μM ACC (aminocyclopropane carboxylatesythase), 100 μM GA (Gibberellin), 100 μM JA (Jasmonic acid), 25 μM AlCl₃ (pH 4.3), 4 °C cold, 200 mM NaCl, and 20% PEG6000. The experiments were repeated three times, log2 based value (fold change) was used to create the heat map with clustering of genes. All data were normalized to the expression level of control (0 h). The scale represents the relative expression levels from low (blue) to high (red).

Figure 8. PEPC activities in soybean leaves and roots subjected to different abiotic and phytohormone treatments.

Leaves and roots of 14-d soybean seedlings are used to investigate the changes in PEPC activities under different treatments, including 100 μM ABA (Abscisic acid), 100 μM ACC (aminocyclopropane carboxylatesythase), 100 μM GA (Gibberellin), 100 μM JA (Jasmonic acid), 25 μM AlCl₃ (pH 4.3), 4 °C cold, 200 mM NaCl, and 20% PEG6000. Data shown are means ± SD of three independent experiments. Statistical significance of differences between control and treated groups was analyzed using Student’s t-test (*indicates P < 0.05).