Comprehensive identification, evolutionary patterns and the divergent response of PRX genes in Phaseolus vulgaris under biotic and abiotic interactions

Abstract

Peroxiredoxins (Prxs) are novel cysteine-based peroxidases which are involved in protecting cells from oxidative damage by catalyzing the reduction of different peroxides. The present study addressed, for the first time, genome-wide identification, evolutionary patterns and expression dynamics of Phaseolus vulgaris Prx gene family (PvPrx). Nine Prx proteins were identified in P. vulgaris based on homology searches. The phylogeny analysis of Prxs from seven plant species revealed that Prx proteins can be clustered into four groups (1C-Prx, 2C-Prxs, PrxQ and type II Prxs). Both tandem and segmental duplication contributed to PvPrx gene family expansion. Intragenic reorganizations including gain/loss of exon/intron and insertions/deletions have also contributed to PvPrx gene diversification. The collinearity analysis revealed the presence of some orthologous Prx gene pairs between A. thaliana and P. vulgaris genomes. The Ka/Ks ratio indicated that two of the three PvPrx duplicated gene pairs have undergone a purifying selection. Redundant stress-related cis-acting elements were also found in the promoters of most PvPrx genes. RT q-PCR analysis revealed an upregulation of key PvPrx members in response to symbiosis and different abiotic factors. The upregulation of targeted PvPrx members, particularly in leaves exposed to salinity or drought, was accompanied by an accumulation of hydrogen peroxide (H₂O₂). When exogenously applied, H₂O₂ modulated almost all PvPrx genes, suggesting a potential H₂O₂-scavenging role for these proteins. Collectively, our analysis provided valuable information for further functional analysis of key PvPrx members to improve common bean stress tolerance and/or its symbiotic performance.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03246-8.

Keywords: Abiotic stress; Common bean; Peroxiredoxins; Redox regulation; Reverse-transcription q-PCR; Symbiosis.

Fig. 1

Phylogenetic analysis of Prx proteins in seven different plant species. The multiple alignment of 59 Prx protein sequences including 10 of Arabidopsis thaliana (At1C-Prx, At1g48130; At2C-PrxA, At3g11630; At2C-PrxB, At5g06290; AtPrxQ, At3g26060; AtPRXII-A, At1g65990; AtPRXII-B, At1g65980; AtPRXII-C, At1g65970; AtPRXII-D, At1g60740; AtPRXII-E, At3g52960; AtPRXII-F, At3g06050), 10 Orysa sativa (Os1C-PrxA, LOC_Os07g44430; Os1C-PrxB, LOC_Os07g44440; Os2C-PrxA, LOC_Os04g33970; Os2C-PrxB, LOC_Os02g33450; OsPrxQ, LOC_Os06g09610; OsPRXII-B, LOC_Os01g24740; OsPRXII-C, LOC_Os01g48420; OsPRXII-D, LOC_Os02g09940; OsPRXII-E, LOC_Os06g42000; OsPRXII-F, LOC_Os01g16152), 8 Populus trichocarpa (Pt1C-Prx, POPTR_0008s09930; Pt2C-PrxA, POPTR_0016s07280; Pt2C-PrxB, POPTR_0006s22130; PtPrxQ, POPTR_0006s13980; PtPRXII-B, POPTR_0001s44990; PtPRXII-C, POPTR_0018s09030; PtPRXII-E, POPTR_0013s10250; PtPRXII-F, POPTR_0019s04070), 7 of Medicago trancatula (Mt1C-Prx, Medtr4g094720.1; Mt2C-PrxA, Medtr1g105090.1; Mt2C-PrxB, Medtr7g105830.1; MtPrxQ, Medtr4g124790.1; MtPrxII-A, Medtr6g087990.1; MtPrxII-F, Medtr2g022660.1; MtPrxII-E,Medtr7g103610.1), 7 of Lotus japonicus (Lj1C-Prx, Lj4g0025954.1; Lj2C-PrxA, Lj5g0012215.1; Lj2C-PrxA, Lj1g0014032.1; Lj4g0020079.1, LjPrxQ; Lj2g0015698.1, LjPrxII-A; Lj6g0011312.1, LjPrxII-F; Lj1g0019128.1, LjPrxII-E), 8 of Brachypodium distachyon (Bd1C-Prx, Bradi1g20040.1.p; Bd2C-PrxA, Bradi3g45140.1.p; Bd2C-PrxB, Bradi5g09650.1.p; BdPrxQ, Bradi1g46622.1.p; BdPRXII-B, Bradi2g46380.2.p; BdPRXII-C, Bradi3g06750.1.p; BdPRXII-E, Bradi1g35660.1.p; BdPRXII-F, Bradi2g09910.1.p) and 9 of P. vulgaris (accession numbers of the 9 PvPrx sequences are indicated in Table S1) was performed by ClustalW software and the phylogenetic tree was constructed by the neighbor-joining (NJ) method. Numbers on branches indicate bootstrap values for 1000 replicates. The Prx sequences from A. thaliana, P. vulgaris, P. trichocarpa, M. trancatula, L. japonicus (Dicots), O. sativa and B. distachyon (Monocots) are preceded by At, Pv, Pt, Mt, Lj, Os and Bd, respectively. PvPrxs are mentioned in red. The name of each Prx group is indicated next to the corresponding group

Fig. 2

Motifs and structures of PvPrxs. A A phylogenetic tree of PvPrxs constructed by the neighbor-joining (NJ) method after performing a multiple sequence alignment using ClustalW software. B Conserved motifs in PvPrx proteins obtained by MEME suite program. C Gene structure of Prxs in P. vulgaris as revealed by the GSDS2.0 software

Fig. 3

CAREs detection and PvPrx gene ontology analysis. A The cis-acting regulatory element (CARE) analysis using the online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/search_CARE.html). B Gene ontology terms identified for the PvPrx genes on the expression atlas PvGEA (www.plantgrn.noble.org/PvGEA) database (O'Rourke et al. 2014)

Fig. 4

PvPrx gene mapping, dupplication events and collineraity analysis. A Chromosomal distribution of Prx genes in P. vulgaris. The chromosome numbers are indicated above each vertical bar. B PvPrx gene dupplication events. Circos visualization of both tandem and segmental duplicated PvPrx genes shown on the chromosome maps (yellow). C Syntenic relationship assessment. Collinear analysis between A. thaliana (orange) and P. vulgaris (green) genomes was performed by MCScanX. Chr1–Chr5, five chromosomes of A. thaliana. Chr1-Chr11, 11 chromosomes of common bean. Red lines depict Prx homologous gene pairs between Common bean and A. thaliana chromosomes, while gray lines indicate all syntenic blocks between Common bean and A. thaliana genomes

Fig. 5

RT-qPCR analysis of PvPrx gene expression during nodule development and according to the N₂-fixing performance of the Rhizobial strain. A RT-qPCR analysis of PvPrx gene expression during P. vulgaris nodule development. RNA was isolated from common bean nodules at different developmental stages including PN: nodule primordium (15 dpi), MN: mature nodules (49 dpi), and SN: senescent nodules (69 dpi) and used for RNA extraction and RT-qPCR analysis. Data are expressed relative to the amount of mRNA transcripts of PvPrxF.1 in the PN (calibrator). B Expression profiles of PvPrx genes in nodules under efficient and less-efficient Rhizobial strains. RNA was isolated from common bean nodules induced either with efficient (Eff, R. gallicum 8a3) or less-efficient (L-Eff, R. etli Ma1A32) strains at the same above mentioned developmental stages (NP, MN and SN) and used for PvPrx gene expression by RT-qPCR. Data are expressed relative to the amount of mRNA transcripts of PvPrxF.1 in the PN (calibrator). The data represent means of three independent biological experiments. For each nodule developmental stage, eight plants were used per treatment per replicate by pooling similar samples together. Two technical replicates were done for each Prx gene. Log₂ transformed values were used for the heat map construction using the TBTools software. The colors indicate expression intensity (red, high expression; blue, low expression). C Nitrogen fixing capacity estimated by the acetylene-reducing activity (ARA µmol h⁻¹plant⁻¹) in mature nodules under efficient and less-efficient Rhizobial strains. D leghemoglobin content (mg g⁻¹ fw) in mature nodules under efficient and less-efficient Rhizobial strains. Values in each column with identical letters are not significantly different (p < 0.05)

Fig. 6

Modulation of PvPrx genes in response to symbiosis. RNA was isolated from roots of fertilized (control) and symbiotic N₂-fixing common bean plants (inoculated with 8a3 strain) within 15 dpi and used for RT-qPCR analysis. In order to calculate the relative expression, expression value of each PvPrx gene in roots of fertilized (control) plants was set as 1. The PvUbq gene was used as an internal control. Nine plants were used per treatment per replicate. Error bars indicate standard deviations of three biological replicates. Different letters marked on the same bar chart indicate significant differences at the 0.05 level

Fig. 7

Prx gene expression changes in response to salinity in P. vulgaris. Plant roots and leaves were harvested within 0, 6 and 24 h post-treatment with NaCl (100 mM) for RNA extraction and RT-qPCR analysis. The 2^–ΔΔCt method was used to calculate relative gene expression. The default expression value for each gene was 1 at 0 h before treatment (control). The PvUbq gene was used as an internal control. Error bars show the standard deviation of three biological and two technical replicates. Different letters marked on the same bar chart indicate significant differences at the 0.05 level. A Genes upregulated in both leaves and roots; B genes upregulated in leaves and downregulated in roots; C genes either unchanged or downregulated in both leaves and roots

Fig. 8

Hydrogen peroxide levels in common bean leaves and roots under salt and drought stresses. Leaves (third position) and roots were harvested within 6 and 24 hpt with 100 mM NaCl or 20% PEG-6000 for H₂O₂ measurements. Three independent biological replicates were done and nine plants were used per treatment per replicate. Values in each column with identical letters are not significantly different (p < 0.05)

Fig. 9

Modulation of PvPrx gene expression under drought. Plant roots and leaves were harvested within 0, 6 and 24 h post-treatment with PEG 6000 (20%) for RNA extraction and RT-qPCR analysis. The 2^–ΔΔCt method was used to calculate relative gene expression. The default expression value for each gene was 1 at 0 h before treatment (conrol). The PvUbq gene was used as an internal control. Error bars show the standard deviation of three biological and two technical replicates. Different letters marked on the same bar chart indicate significant differences at the 0.05 level as follows: A Genes upregulated in both leaves and roots; B genes downregulated in leaves and unchanged in roots; C genes unchanged in both leaves and roots

Fig. 10

Exogenous application of hydrogen peroxide differentially modulated PvPrx genes. Roots and leaves of common bean plants either treated or not with 10 mM H₂O₂ were harvested within 6 h post-treatment (hpt) for RNA extraction and RT-qPCR analysis. The 2^–ΔΔCt method was used to calculate relative gene expression. The amount of each PvPrx gene transcripts in control samples was set as 1. The PvUbq gene was used as an internal control. The data represent means of three independent experiments. Nine common bean plants were used per treatment per replicate. a Relative expression patterns of PvPrx genes in leaves. b Relative expression patterns of PvPrx genes in roots