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

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.

Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium.

Acacia saligna is an invasive alien species that has the ability to establish symbiotic relationships with rhizobia. In the present study, genotypic and symbiotic diversity of native rhizobia associated with A. saligna in Tunisia were studied. A total of 100 bacterial strains were selected and three different ribotypes were identified based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages and a single strain to Sinorhizobium meliloti. Thirteen slow-growing isolates representing the most dominant IGS (intergenic spacer) profile clustered distinctly from known rhizobia species within Bradyrhizobium with the closest related species being Bradyrhizobium shewense and Bradyrhizobium niftali, which had 95.17% and 95.1% sequence identity, respectively. Two slow-growing isolates, 1AS28L and 5AS6L, had B. frederekii as their closest species with a sequence identity of 95.2%, an indication that these strains could constitute a new lineage. Strains 1AS14I, 1AS12I and 6AS6 clustered distinctly from known rhizobia species but within the Rhizobium leguminosarum complex (Rlc) with the most closely related species being Rhizobium indicum with 96.3% sequence identity. Similarly, the remaining 11 strains showed 96.9 % and 97.2% similarity values with R. changzhiense and R. indicum, respectively. Based on nodC and nodA phylogenies and cross inoculation tests, these 14 strains of Rlc species clearly diverged from strains of Sinorhizobium and Rlc symbiovars, and formed a new symbiovar for which the name sv. "salignae" is proposed. Bacterial strains isolated in this study that were taxonomically assigned to Bradyrhizobium harbored different symbiotic genes and the data suggested a new symbiovar, for which sv. "cyanophyllae" is proposed. Isolates formed effective nodules on A. saligna.

Genome-wide identification, characterization and expression analysis of glutaredoxin gene family (Grxs) in Phaseolus vulgaris.

Glutaredoxins (Grxs) are ubiquitous oxidoreductase proteins implicated in development and abiotic stress response mainly through maintaining redox homoeostasis. Here, we conducted the first systematic analysis of the Grx gene family (PvGrx) in the most popular legume Phaseolus vulgaris (common bean). A total of 50 PvGrx genes were identified, and divided into four classes (CC-type, CGFS-type, CPYC-type and Grl-type) based on the phylogenetic analysis. The different classes have different introns-exons structures and conserved motifs, indicating functional divergence in the PvGrx family. Both tandem and segmental duplications were found to be involved in the expansion of PvGrx family that underwent a purifying selection by excluding the deleterious loss-of-function mutations. Cis-acting regulatory elements and gene ontology analyses predicted their role of distinctive members in abiotic stress response and hormonal signalling. RNA-seq based expression analysis revealed their differential expression pattern during plant development. On the other hand, RT q-PCR analysis revealed that target PvGrx isoforms were associated with nodule organogenesis and symbiosis based on their expression profiles. In addition, a battery of PvGrx candidates were markedly upregulated by different abiotic stressors suggesting their broad spectrum of functions. These findings serve as a reference for functional analysis and genetic improvement in P. vulgaris and related legume species.

Phylogenetic study of rhizobia nodulating pea (Pisum sativum) isolated from different geographic locations in Tunisia.

Nodulated Pisum sativum plants showed the presence of native rhizobia in 16 out of 23 soil samples collected especially in northern and central Tunisia. A total of 130 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, dnaK and glnII) assigned 35 isolates to Rhizobium laguerreae, R. ruizarguesonis, Agrobacterium radiobacter, Ensifer meliloti and two putative genospecies. R. laguerreae was the most dominant species nodulating P. sativum with 63%. The isolates 21PS7 and 21PS15 were assigned to R. ruizarguesonis, and this is the first report of this species in Tunisia. Two putative new lineages were identified, since strains 25PS6, 10PS4 and 12PS15 clustered distinctly from known rhizobia species but within the R. leguminosarum complex (Rlc) with the most closely related species being R. indicum with 96.4% sequence identity. Similarly, strains 16PS2, 3PS9 and 3PS18 showed 97.4% and 97.6% similarity with R. sophorae and R. laguerreae, respectively. Based on 16S-23S intergenic spacer (IGS) fingerprinting, there was no clear association between the strains and their geographic locations. According to nodC and nodA phylogenies, strains of Rlc species and, interestingly, strain 8PS18 identified as E. meliloti, harbored the symbiotic genes of symbiovar viciae and clustered in two different clades showing heterogeneity within the symbiovar. All these strains nodulated and fixed nitrogen with pea plants. However, the strains belonging to A. radiobacter and the two remaining strains of E. meliloti were unable to nodulate P. sativum, suggesting that they were non-symbiotic strains. The results of this study further suggest that the Tunisian Rhizobium community is more diverse than previously reported.

Genome-wide analysis and expression profiling of H-type Trx family in Phaseolus vulgaris revealed distinctive isoforms associated with symbiotic N2-fixing performance and abiotic stress response.

Thioredoxins (Trxs) are implicated in plant development and stress tolerance through redox regulation of target proteins. Trxs of Type h (Trxhs) constitute the largest and the most complicated cluster in the Trx family because of their unknown individual functions. Here, we identified and characterized the Phaseolus vulgaris Trxh family during development, mutualistic interactions and in response to abiotic stress. P. vulgaris (common bean) Trxh gene family (PvTrxh) encompasses 12 isoforms (PvTrxh1-h12), subdivided into 3 groups according to their amino acid sequence features. In silico RNA-seq -based expression analysis showed a differential expression of PvTrxh genes during development. RT-qPCR analysis of PvTrxh genes during nodule organogenesis revealed their highest expression in the nodule primordium (NP). Interestingly, in response to symbiosis, specific PvTrxh isoforms (PvTrxh3 and h5) were found to be highly upregulated compared to mock-inoculated plants. In addition, their expression patterns in the NP positively correlated with the symbiotic N2-fixing efficiency of the Rhizobium strain, as revealed by a number of symbiotic efficiency parameters (ARA, leghemoglobin content, biomass, and total soluble proteins), concomitantly with increased amounts of hydrogen peroxide (H2O2). On the other hand, distinctive PvTrxh isoforms were found to be upregulated in plant leaves, where H2O2 amounts were elevated, in response to both salt and drought constraints. When exogenously applied, H2O2 upregulated specific PvTrxh isoforms in plant leaves and roots. These findings point to a specific, rather than redundant, function for Trxh proteins in common bean beside the association of distinctive Trxh isoforms with symbiosis and abiotic stress response.

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia.

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.

Inoculation of Phaseolus vulgaris with the nodule-endophyte Agrobacterium sp. 10C2 affects richness and structure of rhizosphere bacterial communities and enhances nodulation and growth.

Agrobacterium sp. 10C2 is a nonpathogenic and non-symbiotic nodule-endophyte strain isolated from root nodules of Phaseolus vulgaris. The effect of this strain on nodulation, plant growth and rhizosphere bacterial communities of P. vulgaris is investigated under seminatural conditions. Inoculation with strain 10C2 induced an increase in nodule number (+54 %) and plant biomass (+16 %). Grains also showed a significant increase in phosphorus (+53 %), polyphenols (+217 %), flavonoids (+62 %) and total antioxidant capacity (+82 %). The effect of strain 10C2 on bacterial communities was monitored using terminal restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. When the initial soil was inoculated with strain 10C2 and left 15 days, the Agrobacterium strain did not affect TRF richness but changed structure. When common bean was sown in these soils and cultivated during 75 days, both TRF richness and structure were affected by strain 10C2. TRF richness increased in the rhizosphere soil, while it decreased in the bulk soil (root free). The taxonomic assignation of TRFs induced by strain 10C2 in the bean rhizosphere revealed the presence of four phyla (Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria) with a relative preponderance of Firmicutes, represented mainly by Bacillus species. Some of these taxa (i.e., Bacillus licheniformis, Bacillus pumilus, Bacillus senegalensis, Bacillus subtilis, Bacillus firmus and Paenibacillus koreensis) are particularly known for their plant growth-promoting potentialities. These results suggest that the beneficial effects of strain 10C2 observed on plant growth and grain quality are explained at least in part by the indirect effect through the promotion of beneficial microorganisms.

Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a Northern Tunisian field.

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156(T). S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602sp(T) but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.