ISSN 1021-4437, Russian Journal of Plant Physiology, 2017, Vol. 64, No. 4, pp. 576–587. © Pleiades Publishing, Ltd., 2017.

RESEARCH PAPERS

Molecular Characterization and Expression Profiling of the Phosphoenolpyruvate Carboxylase Genes in Peanut (Arachis hypogaea L.)1 L. Pana, b, J. Zhanga, b, N. Chena, M. Chena, M. Wanga, T. Wanga, X. Chia, M. Yuanc, Y. Wanb, S. Yua,*, and F. Liub,** a

Shandong Peanut Research Institute, Qingdao, 266100, China State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, College of Agronomic Sciences, Shandong Agricultural University, Tai’an, 271018, China c Xinjiang Agricultural University, Urumqi, 830052, China *e-mail: [email protected] **e-mail: [email protected]

b

Received April 13, 2016

Abstract⎯Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled enzyme located at the core of plant carbohydrate metabolism. Plant PEPCs belong to a small multigene family encoding several plant-type PEPC genes, along with at least one distantly related bacterial-type PEPC gene. The PEPC genes have been intensively studied in Arabidopsis, but not in peanut (Arachis hypogaea L.). Previously, we isolated five PEPC genes (AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4 and AhPEPC5) from peanut. Here, due to the sequencing of the peanut genome, we analyzed the complexity of its PEPC gene family, including phylogenetic relationships, gene structure and chromosome mapping. The results showed that AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4 encoded typical plant-type enzymes, while AhPEPC5 was a bacterial-type PEPC. The recombinant proteins of these genes were expressed in Escherichia coli, and the calculated molecular weights of the recombinant proteins were 110.8 kD (AhPEPC1), 110.7 kD (AhPEPC2), 110.3 kD (AhPEPC3), 110.8 kD (AhPEPC4), and 116.4 kD (AhPEPC5). The expression patterns of AhPEPC1-5 were analyzed under cold, salt and drought conditions. Our results indicated that the expression of AhPEPC3 was rapidly and substantially enhanced under abiotic stress, whereas the expression of AhPEPC1 and AhPEPC2 was slightly enhanced under certain stress conditions. Some genes were down-regulated in leaves under stress: AhPEPC1, AhPEPC4 and AhPEPC5 under salt stress and AhPEPC4 and AhPEPC5 under drought stress. These results suggest that peanut PEPC proteins may differ in their functions during acclimation to abiotic stresses. Keywords: Arachis hypogaea, AhPEPC, expression analysis, abiotic stress DOI: 10.1134/S1021443717040100

INTRODUCTION Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important enzyme at a crucial branch point in plant carbohydrate metabolism. It is widely distributed in all photosynthetic organisms, including vascular plants, algae, cyanobacteria and photosynthetic bacteria, as well as in non-photosynthetic bacteria and protozoa, but is absent from animals and fungi [1]. PEPC catalyzes the irreversible β-carboxylation of PEP in the presence of HCO3− to yield oxaloacetate and inorganic phosphate using 1 The article is published in the original.

Abbreviations: Ah—Arachis hypogaea; BTPC—bacterial-type phosphoenolpyruvate carboxylase; COS—castor oil seed; DAP—days after pegging; PTPC—plant-type phosphoenolpyruvate carboxylase; SDS-PAGE—sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Mg2+ as a cofactor [2, 3]. This reaction is the primary fixation step in photosynthetic CO2 assimilation in the leaves of C4 and Crassulacean acid metabolizing plants [1, 2]. However, PEPC also fulfils essential functions in non-photosynthetic tissues and photosynthetic cells of C3 plant leaves. It has an anaplerotic function, which replenishes the tricarboxylic acid cycle with intermediates that are consumed in a variety of biosynthetic pathways and through nitrogen assimilation [4–6]. Other physiological roles of PEPC in plants include maintaining cellular pH, supplying carbon to N2-fixing legume root nodules, absorbing and transporting cations in roots, control of stomatal movements, fruit maturation and seed germination [7–9]. Consistent with its wide range of activities, plant PEPCs belong to a small gene family encoding several plant-type PEPCs (PTPC), along with at least one

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distantly related bacterial-type PEPC (BTPC) [10– 14]. PTPC genes encode closely related 100–110 kD polypeptides with a conserved N-terminal seryl-phosphorylation domain and a distinguishing C-terminal tetrapeptide QNTG signature that typically exist as homotetrameric Class-1 PEPCs [15, 16]. PTPC activity is tightly controlled by a combination of allosteric effectors and reversible phosphorylation at the conserved N-terminal seryl-residue catalyzed by a dedicated Ca2+-independent PTPC protein kinase and protein phosphatase type-2A [1, 2, 17]. Plant BTPC genes encode 116–118 kD polypeptides exhibiting low (<40%) sequence identities with PTPCs that are more closely related to prokaryotic PEPCs than to PTPCs [6]. Compared with PTPC, BTPC lacks the N-terminal phosphorylatable Ser residue and contains a prokaryotic-like (R/K)NTG tetrapeptide at its C-terminus [18, 19]. BTPC genes and transcripts have been well documented in vascular plants and green algae [10–13, 20]. Although PEPC genes and proteins have been extensively studied in many organisms [14, 20], little has been reported on the characterization of the PEPC gene family in peanut. Previously, we isolated five PEPC genes from peanut. Recently, the peanut genome was sequenced, and its gene prediction and annotation are publicly available (http://peanutbase. org/home). Therefore, in this study, using the peanut genome sequence, we analyzed the complexity of the PEPC gene family, including phylogenetic relations, gene structures and chromosome location. We expressed the recombinant proteins of these genes in Escherichia coli. The expression patterns of these genes were investigated in different tissues and at different stages of peanut seed development. Additionally, the expression patterns of these genes were analyzed under cold, salt and drought conditions. These findings expanded our knowledge of PEPC genes in peanut and provided a theoretical basis for the study of abiotic stress tolerance in peanut. MATERIALS AND METHODS Plant materials. Peanut plants (Arachis hypogaea L. cultivar E11) were grown in a growth chamber with a 16 h light/8 h dark photoperiod at 28/23°C day/night temperatures. Leaves, stems and roots were sampled from the seedlings at the trefoil leaf stage. Seeds were sampled at 10, 20, 30, 40, 50 and 60 days after pegging (DAP). For the cold treatment, seedlings in the soil at the trefoil leaf stage were kept at 4°C, and leaves were sampled separately either before cold treatment (0 h) or after continuous exposure to 4°C for 4, 8, 12, 24 or 72 h. For stress treatments, the roots of seedlings grown in soil were flushed carefully with tap water to remove all soil, and then submerged in solutions of 200 mM NaCl or 20% polyethylene glycol (PEG) 6000. Leaves were sampled separately after treatment for 0, 4, 8, 12, 24 or 72 h. All samples were immediately RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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frozen in liquid nitrogen and stored at –80°C until required. Sequence analysis. The open reading frames and encoded amino acid sequences of all genes were deduced using the BioXM 2.6 software (http://zhanglab. njau.edu.cn/hj.htm). Physicochemical properties of the deduced proteins were predicted using Protparam (http://www.expasy.ch/tools/protparam.html). The active sites of the protein sequences were analyzed by comparisons with the PROSITE database (http:// prosite.expasy.org/). A multiple sequence alignment was performed with DNAMAN version 5.2 (http:// www.lynnon.com/dnaman.html). Phylogenetic analysis. Homologs of each member of the Arabidopsis PEPC family were identified by BLASTP searches with datasets from Phytozome v10.2 (http://phytozome.jgi.doe.gov/pz/portal.html). Only the sequences with an e-value below e–50 were considered the members of the PEPC family. Supplementary Table 1 provides a detailed description of the proteins used and the corresponding accession numbers. Amino acid sequences were aligned using the ClustalX program with the implanted BioEdit [21]. The neighbor-joining method in MEGA4 [22] was used to construct the phylogenetic tree. Bootstrapping with 1000 replicates and default program parameters was used to establish the confidence limits of the tree branches. Gene structure prediction and chromosome mapping. Cultivated peanut (A. hypogaea) is an allotetraploid of recent origin, with an AABB genome. The most probable wild ancestors of cultivated peanut are Arachis duranensis and Arachis ipaensis whose genomes merged several thousand years ago, in a rare genetic event. A. duranensis (2n = 2x = 20) most likely contributed the A genome. A. ipaensis (2n = 2x = 20) probably contributed the B genome. These two “simpler” genomes have first been sequenced toward achieving its ultimate goal: the complete genomic sequence for cultivated peanut [23]. The gene structure displayed by the server program [24] was used to illustrate the exon/intron organization of individual PEPC genes by comparing the cDNAs with their corresponding genomic DNA sequences. PEPC genes were mapped on chromosomes by determining their chromosomal positions in PeanutBase (http://peanutbase.org/). Construction of expression plasmids. The AhPEPCs cDNA fragments were amplified by PCR with the LA PCR system (TaKaRa, China). Five pairs of genespecific primers carrying the specific restriction sites (Table 1) were designed analyzing the target gene sequences. The LA PCR system was comprised of 2.5 μL of 10×PCR buffer with MgCl2, 1 μL of each primer (10 μM), 4.0 μL of 10 mM dNTPs, 1 μL of cDNA sample, 0.5 μL of LA Taq™ DNA polymerase, and 16 μL of doubledistilled water. The PCR products were separated by electrophoresis through a 1% agaNo. 4

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Table 1. DNA sequences of oligonucleotide primers used in this study Name

Oligonucleotide sequence 5'–3'

Construction of expression plasmids yPEPC1-F GACACCATATGATGGCTTCAATTGATGCTCAG yPEPC1-R GTGTCCTCGAGTTAACCTGTGTTTTGCATGC yPEPC2-F GACACGAATTCATGGCAAAGAAGTTGGAAAAGATG yPEPC2-R GTGTCCTCGAGTTAACCAGTGTTCTGCATGCCAG yPEPC3-F GACACCATATGATGGCTACTAAGAAAGTTGAG yPEPC3-R GTGTCGAATTCTTAACCAGTGTTTTGCATGCCAG yPEPC4-F GACACCATATGATGGCGAATAGGAATTTGGAG yPEPC4-R GTGTCGAGCTCTTAACCAGTGTTCTGCATTCCG yPEPC5-F GACACCATATGATGACTGATACTACTGATGATATTG yPEPC5-R GTGTCGGATCCTCAACCCGTGTTCTTCATCCCAG Real-time RT-PCR β-actin-F TTGGAAT GGGTCAGAAGGATGC β-actin-R AGTGGTGCCTCAGTAAGAAGC qPEPC1-F GGCAAACGGTTCAAGATTGT qPEPC1-R CCTGTGGGGACTTCTTCAG qPEPC2-F CGGGGAAGCATTTAGACATG qPEPC2-R GCAGGAGTTTCTTGGTTTCG qPEPC3-F TATGGCTTGGCTTTGGGAAG qPEPC3-R CGCTCACCAAACAGCCATAG qPEPC4-F TCGCTGCTCTGAATGATAGG qPEPC4-R ACTTGTTGGGTTCAGCGAGA qPEPC5-F TTGTGCTGGTGATTAGTGGC qPEPC5-R GATCTTTCGGTTGTCGTCCT

rose gel, and purified using a Gel Extraction Kit (TaKaRa) according to the manufacturer’s protocol. The purified products were then cloned into the pMD18-T simple vector (TaKaRa) and sequenced (Shangon, Shanghai, China). The generated recombinant plasmids AhPEPC/pMD18-T were digested, and the inserts were cloned into restriction sites (AhPEPC1: NdeI/ XhoI, AhPEPC2: EcoRI/ XhoI, AhPEPC3: NdeI/ EcoRI, AhPEPC4: NdeI/ SacI, AhPEPC5: NdeI/ BamHI) of the expression vector pET-28b (Novagen, United States). The resulting expression plasmids AhPEPC/pET-28b were finally transformed into E. coli BL21 (DE3) (Tiangen Biotech, China) for PEPC expression. Inducible expression of recombinant proteins. The E. coli BL21 (DE3) strain was transformed with the identified plasmids. Five single bacterial colonies were selected from the transformants. The selected single bacterial colonies were inoculated into 4 mL of LuriaBertani (LB) medium containing 30 μg/mL kanamycin and incubated at 37°C for 12 h. Then, 1 mL of the cultures (1 mL) were inoculated into 100 mL LB (containing 30 μg/mL kanamycin) and cultured at 37°C until the absorbance at 600 nm reached 0.6. To induce

protein expression 0.5 mM isopropyl β-D-1-thiogalactopyranoside was added. Uninduced vector control groups were established in parallel. After induction, cells were harvested by centrifugation at 4000 rpm for 10 min and then resuspended using 5 mL phosphate buffer saline (PBS). Samples were analyzed by 5% SDS-PAGE. Quantitative real-time RT-PCR. A real-time RTPCR analysis was performed using a LightCycler 2.0 instrument system (Roche, Germany). The amplification of β-actin was used as an internal control to normalize all data. Six pairs of gene-specific primers (Table 1) were designed after an analysis of the target gene sequences. The real-time RT-PCR reactions were performed using the SYBR Premix Ex Taq polymerase (TaKaRa) according to the manufacturer’s instructions. Each 20 μL reaction was comprised of 2 μL template, 10 μL 2× SYBR Premix, and 0.4 μL (200 nM) each primer. The reactions were subjected to an initial denaturation step of 95°C × 10 s, followed by 40 cycles of 95°C × 5 s, 60°C × 30 s and 72°C × 10 s. A melting curve analysis was performed at the end of the PCR run over the range 60–95°C, increasing the temperature stepwise by 0.5°C every 10 s. The baseline

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Table 2. Phosphoenolpyruvate carboxylase genes in A. hypogaea Gene symbol AhPEPC1 AhPEPC2 AhPEPC3 AhPEPC4 AhPEPC5

NCBI Genbank accession number EU391629 FJ222240 FJ222826 FJ222827 FJ222828

Gene length, bp

ORF, bp

4.974 6.515 5.897 4.645 6.334

2.907 2.901 2.901 2.910 3.111

Protein length, aa 968 966 966 969 1.036

Molecular weight, kD

pI

110.8 110.7 110.3 110.8 116.4

5.95 5.94 5.65 6.05 6.14

and quantification cycle were automatically determined using the Light Cycler Software. No template controls were included for each primer pair, and each PCR reaction was carried out in triplicate. The relative quantification method (delta-delta Cp) was used to evaluate quantitative variation [25].

genes shared a high degree of identity at the amino acid level, however AhPEPC5 shared only 36.7%– 37.9% identity with AhPEPC1–4. These results indicate that the AhPEPC1-4 genes encode the plant-type PEPCs and the AhPEPC5 gene encodes a bacterialtype PEPC.

RESULTS

Phylogenetic Analysis To establish the phylogenetic relationships among different sources of PEPC genes, sequences from representative eukaryotic species belonging to plant eudicots (A. thaliana, G. raimondii, L. usitatissimum, R. communis, P. trichocarpa, M. truncatula, G. max, Arachis hypogaea, P. vulgaris, B. rapa and M. esculenta), monocotyledons (O. sativa, Z. mays, B. distachyon, S. bicolor, S. italica and P. virgatum), fern (S. moellendorffii), moss (P. patens) and algae (C. reinhardtii, C. subellipsoidea, V. carteri, O. lucimarinus and M. pusilla CCMP1545), were selected to construct the phylogenetic tree using the neighbor-joining method. This tree (Fig. 1) shows that all PEPC protein family members clustered into two major clades: the plant-type PEPC clade and the bacterial-type PEPC clade. The plant-type PEPC clade was divided into two distinct subfamilies. The AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4 proteins were grouped with PEPC enzymes from eudicots and were separate from those of monocotyledons, moss and fern. The AhPEPC5 protein, which is similar to AtPPC4, was clearly distinguished from the plant-type PEPCs and is more closely related to PEPCs from algae. Apparently both types of plant PEPCs diverged early during the evolution of plants.

Sequence Analysis of the Peanut PEPC Gene Family We previously isolated five PEPC genes (AhPEPCs) from peanut. The sequence information for the five genes was submitted to GenBank, with the accession numbers EU391629, FJ222240, FJ222826, FJ222827 and FJ222828 (Table 2). As shown in Table 2, AhPEPC1-4 genes encode 968, 966, 966, 969 amino acids, respectively. However, the AhPEPC5 gene encodes 1.036 amino acids. An analysis of the deduced amino acid sequences of the AhPEPCs revealed further differences between the plant-type and bacterialtype PEPCs as shown in Supplementary Fig. 1. Because the Arabidopsis PEPC gene family has been extensively studied, we included the amino acid sequence of PEPCs from Arabidopsis into the Supplementary Fig. 1. One important and invariable property of plant-type PEPCs is their regulation by the reversible phosphorylation of a serine residue, which is part of the sequence acid-base-XX-SIDAQLR in an extended stretch at the N termini of these enzymes [20]. The phosphorylation domain was found in the polypeptides AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4 (underlined in Supplementary Fig. 1). However, no phosphorylation domain was found in the polypeptide deduced from the AhPEPC5 gene. The plant-type PEPC also contains a characteristic Cterminal tetrapeptide QNTG signature, and we found it in AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4. Compared with plant-type PEPCs, bacterial-type PEPCs lack the N-terminal phosphorylatable serine reside and contain a prokaryotic-like (R/K)NTG tetrapeptide at their C-termini [18, 19]. In AhPEPC5, the C terminus was formed by the residues KNTG, while AtPPC4 contains a RNTG tetrapeptide at its C-terminus (Supplementary Fig. 1). In addition, the multiple sequence alignment showed that AhPEPC1-4 RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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Gene Structures and Chromosome Mapping Genes in the same clade are closer in their exon/intron structures when compared to the genes in the other clades (Fig. 2). Almost all of the plant-type PEPC genes have a conserved structure composed of 10 exons interrupted by nine introns [2, 20]. AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4 genes, encoding plant-type PEPCs from peanut, also showed such conserved gene structure (Fig. 2). However, the AhPEPC5 gene encoding a bacterial-type PEPC was No. 4

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Fig. 1. Phylogenetic tree of PEPCs from different sources. Colored branches indicate different groups of proteins. Purple: bacterial-type PEPCs; red: plant-type PEPCs from eudicots; and green: plant-type PEPCs from monocotyledons, moss, and fern. The blue underlines indicate the five AhPEPCs. In each tree, gene sequences other than peanut PEPCs were displayed using the nomenclature with the following abbreviations: At—Arabidopsis thaliana TAIR10; Glyma—Glycine max Wm82.a2.v1; Bradi— Brachypodium distachyon v2.1; Brara—Brassica rapa FPsc v1.3; Medtr—Medicago truncatula Mt4.0v1; Lus—Linum usitatissimum v1.0; Mes—Manihot esculenta v4.1; Gorai—Gossypium raimondii v2.1; Os—O. sativa v7.0; Zm—Zea mays 6a; Sobic—Sorghum bicolor v2.1; Si—Setaria italica v2.1; Pavir—Panicum virgatum v1.1; Phvul—Phaseolus vulgaris v1.0; Rc—R. communis v0.1; Potri—Populus trichocarpa v3.0; Sm—Selaginella moellendorffii v1.0; Cre—Chlamydomonas reinhardtii v5.5; Csu—Coccomyxa subellipsoidea C-169 v2.0; Mpu—Micromonas pusilla CCMP1545 v3.0; Olu—Ostreococcus lucimarinus v2.0; Phpat—Physcomitrella patens v3.0; Vocar—Volvox carteri v2.0.

composed of 20 exons, which was similar to the AtPPC4 and GmPEPC17 genes (Fig. 2) [10, 20]. The bacterialtype PEPC genes found in plants showed a different gene structure than the plant-type genes, which further supported the independent evolution of both types of PEPC genes in plants. Cultivated peanut (A. hypogaea) is an allotetraploid of recent origin, with an AABB genome. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaensis with genome types AA

and BB, respectively, which were used in the Peanut Genome Project. Most pseudomolecules showed a one-to-one correspondence between A. duranensis and A. ipaensis: pairs 02, 03, 04 and 10 were collinear; pairs 05, 06 and 09 were each differentiated by a large inversion in one arm of one of the pseudomolecules; and the pseudomolecules in pair 01 were differentiated by large inversions of both arms. In contrast, A07 has only one normal (upper) euchromatic arm and A08 is abnormally small, with low repetitive content [23].

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MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING AhPEPC1 AhPEPC2 AhPEPC3 AhPEPC4 AhPEPC5 AtPPC1 AtPPC2 AtPPC3 AtPPC4 GmPEPC1 GmPEPC4 GmPEPC7 GmPEPC15 GmPEPC17 5'

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Fig. 2. Gene structures of the peanut, Arabidopsis and soybean PEPCs. The green-boxes represent exons; the lines represent introns; and the thick lines in black represent untranslated region. The intron phases between exon-intron junctions are presented as 0, 1 and 2. The sizes of exons and introns were estimated using the scale at the bottom. GenBank accession numbers are as follows: Arachis hypogaea (AhPEPC1, EU391629; AhPEPC2, FJ222240; AhPEPC3, FJ222826; AhPEPC4, FJ222827; and AhPEPC5, FJ222828), Arabidopsis thaliana (AtPPC1, AJ532901; AtPPC2, AJ532902; AtPPC3, AF071788; and AtPPC4, AJ532903) and Glycine max (GmPEPC1, XM_006591827; GmPEPC4, NM_001250465; GmPEPC7, AB008540; GmPEPC15, AB097087; and GmPEPC17, AY563043).

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150 Fig. 3. Chromosomal locations of peanut PEPC genes. Genes were plotted according to their sequence positions along the chromosomes. The sizes of the chromosomes were estimated using the scale on the left.

A search of the peanut genome database identified the distribution of the PEPC genes on different peanut chromosomes (Fig. 3). For Arabidopsis thaliana, four PEPC genes were found distributed on three chromosomes. AtPPC1 and AtPPC4 were located on chromosome 1, AtPPC2 on chromosome 2, and AtPPC3 on chromosome 3 [20]. As shown in Fig. 3, the five peanut PEPC genes were distributed on eight chromosomes. AhPEPC1 was located on chromosomes A8 and B2; AhPEPC2 and AhPEPC3 were located on chromosomes A3 and B3, respectively; AhPEPC4 was located on chromosomes A7 and B8; and AhPEPC5 was located on chromosomes A9 and B9. The five peanut PEPC genes, except AhPEPC2 and AhPEPC3, were distributed in different chromosomal regions. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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This indicated that the PEPC genes were distributed widely in the peanut genome and that they might originate from different ancestors. Expression of AhPEPCs in E. coli Cells As shown in Supplementary Fig. 2, our data revealed that AhPEPC1-4 genes were highly expressed after a 2–10 h induction at 37°C. However, the expression of AhPEPC5 achieved the highest level after a 2 h induction and had a downward trend thereafter (Supplementary Fig. 2). Apparently different expression of both types of PEPC genes in E. coli cells may result from their structural differences. The five AhPEPC genes were expressed in the cell inclusion bodies, and No. 4

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Fig. 4. Expression analyses of five AhPEPC genes in four peanut tissues (a) and at six stages of seed development (b). 1⎯AhPEPC1; 2⎯AhPEPC2; 3⎯AhPEPC3; 4⎯ AhPEPC4; 5—AhPEPC5. β-actin was used as an internal control. The bars represent SD of three repetitions.

the calculated molecular weights of the recombinant proteins were ~110.8 kD (AhPEPC1), 110.7 kD (AhPEPC2), 110.3 kD (AhPEPC3), 110.8 kD (AhPEPC4), and 116.4 kD (AhPEPC5) (Supplementary Fig. 2). These results were in accordance with the theoretical values. The molecular weight of AhPEPC5 was greater than those of the other four AhPEPCs. Tissue-Specific Expression Patterns Quantitative real-time RT-PCR was employed to confirm the expression patterns of the five PEPC genes in four peanut tissues and at different stages of seed development. As shown in Fig. 4, five genes displayed specific temporal and spatial expression patterns across different tissues and developmental stages. AhPEPC1 showed highest expression in root, leaf and seed, with the lowest level in stem (Fig. 4a). AhPEPC2 was preferentially expressed in leaves, with lower expression in all other tissues. The highest accumulation of AhPEPC3 transcript was found in roots followed by seeds. AhPEPC4 showed highest expression in leaves followed by seeds. AhPEPC5 transcript abundance was higher in roots and leaves than in any of the other tissues tested. The expression patterns of these genes across six developmental stages of seeds are shown in Fig. 4b. High expression levels of the AhPEPC1 gene were

observed at 30 and 40 DAP and much lower levels during the other stages. The AhPEPC3 transcript remained at the relatively low level during the initial stage of seed development, however, the expression increased gradually during later stages, peaking at 40 DAP and dramatically decreased thereafter until 60 DAP. The expression patterns of AhPEPC2, AhPEPC4 and AhPEPC5 were similar over the course of seed development. The expression levels of AhPEPC2, AhPEPC4 and AhPEPC5 were highest at 30 DAP but decreased gradually thereafter. These results suggest that the AhPEPC genes may play important roles in the seed development process. AhPEPC Expression Patterns in Peanut under Abiotic Stress To confirm the expression patterns of AhPEPC genes under stress, we monitored the changes in these transcripts in peanut leaves. The level of AhPEPC1 transcripts increased under cold stress, with a peak level of about 3.5-fold observed at 8 h, and then it decreased gradually (Fig. 5). The expression levels of AhPEPC2 and AhPEPC4 were slightly increased under cold stress, with a peak level at 24 h, and then they decreased gradually (Fig. 5). The transcript levels of AhPEPC3 increased obviously under cold stress, with a peak ~10-fold increase at 24 h (Fig. 5). Transcript

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Fig. 5. Expression analyses of five AhPEPC genes in peanut leaves during cold stress using quantitative real-time RT-PCR method. β-actin was used as an internal control. The bars represent SD of three repetitions.

levels of AhPEPC5 exhibited no obvious changes in the leaves during cold treatment (Fig. 5). Except for the AhPEPC5 gene, the AhPEPC genes were induced at varying degrees under cold stress, especially the AhPEPC3 gene. Our results indicated that these genes may play important roles in enhancing peanut resistance to cold stress. The expression patterns of AhPEPCs in peanut leaves after treatment with 200 mM NaCl were also monitored (Fig. 6). There was no obvious change in the expression of AhPEPC1 in peanut leaves after 4 h of salt treatment (Fig. 6). However, the AhPEPC1 gene was down-regulated from 8 to 72 h of salt treatment. The levels of AhPEPC2 and AhPEPC3 transcripts gradually accumulated between 4 h and 12 h after salt stress, and then decreased (Fig. 6). However, the transcript levels of AhPEPC3 increased obviously under salt stress, with a peak ~7-fold increase at 12 h. The expression level of AhPEPC4 decreased under salt stress, with the lowest level being detected at 24 h (Fig. 6). The transcript level of AhPEPC5 in leaves decreased between 4 h and 12 h after salt stress, and then returned to the untreated level (Fig. 6). The results suggested that AhPEPC3 maybe played a signifRUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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icant positive role in the salt tolerance of peanut. However, AhPEPC1, AhPEPC4 and AhPEPC5 probably had negative functions in the regulation of salt stress. 20% PEG-6000 solution was used to mimic drought stress when monitoring the expression patterns of AhPEPCs in peanut leaves (Fig. 7). There was no obvious change in the expression of AhPEPC1 in peanut leaves under drought treatment. Within 12 h of treatment, the AhPEPC2 gene was slightly up-regulated in the leaves of peanut seedlings subjected to drought stress, and then decreased. The transcript level of AhPEPC3 slightly increased 4 h after treatment, with a peak ~2.2-fold increase at 8 h. The expression of AhPEPC4 exhibited no obvious pattern, with distinct down-regulation at 24 h in the leaves. The level of the AhPEPC5 transcript decreased gradually under drought stress. DISCUSSION PEPCs are enzymes of great importance in plant carbohydrate metabolism. To date, there are two types of PEPC having significant differences in gene No. 4

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Fig. 6. Expression analyses of five AhPEPC genes in peanut leaves during salt stress using quantitative real-time RT-PCR method. β-actin was used as an internal control. The bars represent SD of three repetitions.

sequences and molecular structures in all plants. In this report, we comprehensively described five peanut PEPC genes, AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4 and AhPEPC5. Our data substantially increased the understanding of the bacterial- and plant-type PEPC genes in peanut and their differences. Despite the low similarity between the AhPEPC5 gene and other PEPC genes from peanut, the former gene undoubtedly encoded a PEPC as shown by the high conservation level of the residues important for PEPC activity and structure (Supplementary Fig. 1). The polypeptides deduced from the AhPEPC5 gene showed slightly higher identities with AtPPC4, a bacterial-type PEPC [20], than with the plant-type PEPCs and, most importantly, lacked the phosphorylation domain at the protein N terminus, a hallmark used to differentiate plant and bacterial PEPCs [26]. We therefore conclude that the AhPEPC5 gene encodes the bacterial-type PEPC. In addition, AhPEPC5 has a peculiar gene structure. Like most

plant PEPC genes, AhPEPC1-4 genes are composed of 10 exons, while AhPEPC5 has 20 exons similar to the AtPPC4 and GmPEPC17 (Fig. 2) [10, 20]. The phylogenetic analysis reported here also suggests that both types of plant PEPCs diverged early during plant evolution. Moreover, in our study, we successfully produced AhPEPC1–5 proteins in a prokaryotic expression system. The SDS-PAGE analysis showed that the five AhPEPC genes were expressed in the cell inclusion bodies and the molecular weight of AhPEPC5 was obviously larger than those of other four AhPEPCs (Supplementary Fig. 2). These findings further support the conclusion that AhPEPC5 gene encodes a bacterial-type PEPC. Despite the low transcription level, the bacterialtype PEPC may play important physiological roles in plants having PEPC activities [11, 27] or without PEPC activity [12]. The formation of class-2 PEPCs through the tight interactions between bacterial-type and plant-type PEPCs in developing castor oil seeds

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Fig. 7. Expression analyses of five AhPEPC genes in peanut leaves during drought stress using quantitative real-time RT-PCR method. β-actin was used as an internal control. The bars represent SD of three repetitions.

provided a working model of bacterial-type PEPCs [28, 29]. The bacterial-type PEPC is proposed to function as a catalytic and regulatory subunit within the class-2 PEPC complex [12]. In Arabidopsis, the bacterial-type PEPC gene, AtPPC4, may modulate the transcription of plant-type PEPC genes, indicating a transcriptional interaction between bacterialtype and plant-type PEPC genes in plants [30]. This study uncovered the interactions among bacterialtype and plant-type PEPCs. In our experiments, expression of the five AhPEPC genes were demonstrated in four peanut tissues and at different developmental stages of seeds. The PEPC genes were differentially expressed in peanut (Fig. 4a). These results presume that the AhPEPC genes may play different functions in the peanut respectively. However, the expression patterns of the five AhPEPC genes across six developmental seed stages were similar to each other: they were expressed abundantly at 30 and 40 DAP, and weakly at the other stages (Fig. 4b). These data suggest RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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that all the AhPEPC genes are important for seed development. Earlier reports showed that AtPPC1, AtPPC3 and AtPPC4 were induced by salt stress in roots [18, 30]. Our results indicated that the five PEPC genes in peanut displayed different expression patterns and transcription abundances during abiotic stresses. The AhPEPC3 transcript levels in peanut leaves were distinctly enhanced under abiotic stress. The transcription of AhPEPC2 slightly increased during cold, salt and drought stress. The expression of AhPEPC1 was slightly enhanced under certain stress conditions suggesting that these proteins, especially AhPEPC3, are important for enhancing peanut resistance to abiotic stress. Some genes, such as AhPEPC1, AhPEPC4 and AhPEPC5 in salt-stressed leaves and AhPEPC4 and AhPEPC5 in drought-stressed leaves, were obviously down-regulated during stress treatment; these data indicate that these genes mat negatively affect peanut abiotic stress regulation. No. 4

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A better understanding of the differences between bacterial-type and plant-type PEPC genes will help understand PEPC functions in peanut and provide a theoretical basis for the study of abiotic stress tolerance in this crop. A great deal of experimental work will be required to determine the specific biological function of each of PEPC genes. ACKNOWLEDGMENTS This study was supported by grants from the National Ten Thousand Youth Talents Plan of 2014, China Agriculture Research System (CARS-14), the National Natural Science Foundation of China (project nos. 31000728 and 31200211), the Natural Science Fund of Shandong Province (project nos. ZR2014YL011 and ZR2014YL012), the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences (project no. 2016YQN14).

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