Bacterial Strains from Saline Environment Modulate the Expression of Saline Stress-Responsive Genes in Pepper (Capsicum annuum)

Salinity stress is one of the most important problems in crop productivity. Plant growth-promoting bacteria (PGPB) can also confer stress tolerance in plants under saline soil conditions. In a previous work, it was reported that bacteria strains isolated from hypersaline sites mitigated salt stress in chili pepper (Capsicum annuum var. Caballero) plants and promoted plant growth in some cases. The aim of this study was to evaluate the modulation of gene expression in C. annuum plants by bacteria strains isolated from saline environments. Two bacteria strains from high salinity ponds in Guerrero Negro, BCS, Mexico (Bacillus sp. strain 32 and Staphylococcus sp. strain 155) and Azospirillum brasilense Cd (DSM 1843) were used. Significant improvement in fresh weight yield (stem (28%), root (128.9%), and leaves (20%)) was observed in plants inoculated with Bacillus sp. strain 32. qPCR analysis showed that both strains modulated the expression of stress-responsive genes (MYB, ETR1, JAR1, WRKY, and LOX2) as well as heat shock factors and protein genes (CahsfA2, CahsfA3, CahsfB3a, CaDNaJ02, and CaDNaJ04). Finally, the expression levels of genes related to early salt stress and ISR showed differences in plants with dual treatment (bacteria-inoculated and salt-stressed) compared to plants with simple salinity stress. This work confirmed the differential modification of the transcriptional levels of genes observed in plants inoculated with bacteria under salinity stress.


Introduction
Salinity stress is one of the major abiotic stresses experienced by plants and one of the most important issues in worldwide crop productivity.The major causes of soil salinity are rising levels of groundwater with high salt content as well as poor-quality drainage and irrigation systems [1].Stress salinity induces physiological changes in plant cells, such as the generation and accumulation of reactive oxygen species (ROS) functioning as signal molecules; in high concentrations, this has a damaging effect on the plant cells [2].ROS mechanisms involve antioxidative defense enzymes (i.e., catalase and superoxide dismutase) participating in scavenging and transforming ROS into non-toxic end products, protecting cells from oxidative damage.This antioxidant mechanism is one of the primary responses to stress in plants [3].On the other hand, abiotic resistance relies on genetic regulation induced by changes in key phytohormone pathways that intersect in a complex manner [4].This resistance response, or adaptation to salt stress, in the host plant requires the integration and coordination of multiple signals including abscisic acid (ABA), jasmonic Plants 2023, 12, 3576 2 of 17 acid (JA), gibberellic acid (GA), ethylene, and salicylic acid [1].Transcriptomic studies have revealed that salt stress causes plant differential expression from hundreds to thousands of genes, some of them categorized as major transcription factor (TF) families, such as MYC2 (master TF of jasmonate signaling), ETR1 (TF that mediates core ethylene signaling), MYB (participate in multiple stress response and ISR), and WRKY (one of the key biological regulators) [1][2][3][4][5][6].
On the other hand, some less characterized proteins such as the DnaJ proteins (heat shock proteins-HSPs) function as molecular chaperones playing critical roles in growth development and multiple stress responses in plants [7].Such proteins have barely been studied in recent years [7,8].It is worth mentioning is that heat-shock transcriptional factors (HSFs) regulate HSPs' expression and can be transcriptionally modulated during salinity stress [9].
Soil salinity is one of the most significant environmental stresses resulting in significant crop reduction.Therefore, it is necessary to improve salinity stress tolerance in crops for global food security [1].An alternative strategy is the use of microorganisms, such as plant growth-promoting bacteria (PGPB), that have been effective at improving vegetal host, abiotic and biotic, stress tolerance [10].PGPB can modulate the expression of endogenous genes.Several studies revealed that TF genes are significantly regulated in plants inoculated with PGPB under abiotic stress conditions.These PGPB-activated TF genes are important to regulate downstream stress-response genes, which can alleviate the inhibitory effects of abiotic stress on gene expression [11,12].Few stress-related genes have been characterized to understand the PGPB-mediated salinity tolerance in the host plant [11].The bacteria with PGPB-mediated salinity tolerance effects are Pseudomonas putida (PS01), which mediates salt tolerance in Arabidopsis thaliana and increases defense genes' expression regulated by the JA pathway via LOX2 genes [12]; Dietzia natronolimnaea that protect Triticum aestivum and Ocimum basilicum plants under salt stress [9]; and Bacillus subtilis (GB03) that enhances salt tolerance in Arabidopsis thaliana [12,13].
Physical or chemical changes related to plant defense caused by PGPB are referred to as induced systemic resistance (ISR) that suppress plant diseases caused by a range of pathogens.Also, few reports have already proposed the term "induced systemic tolerance" (IST) for the enhanced tolerance to abiotic stress induced by PGPB [14].However, PGPB efficiency is determined by different environmental factors like weather conditions, soil characteristics, and interactions with other indigenous bacteria in the soil [14,15].Bacterial strains isolated from saline habitats are more efficient at enhancing plant salt tolerance than PGPB isolated from non-saline habitats.For instance, the inoculation of Waha durum wheat cultivar with A. brasilense NH, isolated from saline soil, improved growth under salt-stress conditions [15].In Capsicum annuum, the halotolerant strains Brevibacterium iodinum RS16, Bacillus licheniformis RS656, and Zhihengliuela alba RS111 mitigated salt stress [14].The search for saline-environment bacteria that can mitigate salt stress in plants is an area of great interest in agriculture, particularly in arid and semi-arid zones.
Peppers (Capsicum spp.) are commercially important crops that are cultivated with the use of fertilizers and pesticides, a practice that can increase soil salinity.In addition to pharmaceutical applications, chili peppers (C.annuum) can be used as a spice, vegetable, and food coloring.In Mexico, its center of origin, its production was estimated at more than two million tons with a market value of more than USD one billion [16].
In a previous work, it was reported that, from 48 strains isolated from hypersaline sites located in Guerrero Negro, Baja California Sur, Mexico, some strains mitigated salt stress in chili pepper (C.annuum var.Caballero) plants, and in some cases, promoted plant growth [17].In this work, from those 48 strains, 2 were selected for the study of gene expression response of C. annuum induced by these strains.Genes involved in enhanced tolerance to abiotic stress (salinity) were targeted.

Evaluation of Bacterial Strains for Growth Promotion in Capsicum annuum
Bacteria strains were tested for plant growth-promoting characteristics.Capsicum annuum Var.Caballero was used for the experiment.Seeds were surface-sterilized with 5% hypochlorite solution (v/v) for 15 min and washed five times with sterile distilled water.The seeds were sown in sterile commercial plant substrate (Sunshine #3, SOGEMIX PM ® ) contained in 50-well trays, cultivated with natural photoperiod and temperature (35 • C day/24 • C night in average) under shade conditions and for 30 days.Plants were irrigated every week with full Hoagland's nutrient solution and water.Plants that reached approximately 10 cm in height (1 month after seed sowing) were transplanted into individual bags.Four-week-old plants were used for first inoculation.
Bacterial inoculants were obtained from overnight cultures in nutrient broth (NB) at 30 • C and 150 rpm.Five microliters of each bacterial culture were re-inoculated in 200 mL of NB and cultivated at 30 • C and 150 rpm for 72 h.Subsequently, the liquid cultures were adjusted to a bacterial density of 10 6 CFU/mL with NB.Ten milliliters of the adjusted inoculum were added per plant per treatment.Bacterial inoculation in plants was repeated 15 days after the first application.Plant samples were obtained at the end of the experiment, 30 days after the first inoculation.Treatments corresponding to bacterial inoculation were as follows: Bacillus sp.strain 32, Staphylococcus sp.strain 155, and A. brasilense Cd.Plants were watered to field capacity before bacteria treatments were applied.After, a drip irrigation system was used for the rest of the experiment.Control plants (no bacteria and no saline stress applied) were irrigated with B&D solution [19].The experiment was performed as a complete randomized block design with six replicates per group.Plants were evaluated at the end of the experiment using the following growth parameters: stem length (SL, cm), stem fresh weight (SFW), stem dry weight (SDW), foliar fresh weight (FFW), foliar dry weight (FDW), root length (RL), root fresh weight (RFW), and root dry weight (RDW).Eight plants per treatment were evaluated at the end of the experiment.Weighing (g) was performed using an analytic balance (Mettler Toledo, AG204), and for dry weights an oven with forced air circulation at 70 • C (Shel-Lab ® , FX-5, series-1000203) was used until reaching constant weight.

Stress Salinity and Bacteria Strains Induced Expression Genes 2.3.1. Bacterial Inoculation after Salt Stress
Four-week-old plants were used for all treatments.Plants were manually watered to field capacity prior to being exposed to salinity conditions.Salt stress was applied using sterile deionized water containing 50 mM and 100 mM NaCl through a drip irrigation system for two minutes four times a day.Ten mL of the corresponding bacteria treatment were applied after 24 h of salt stress initiation: Bacillus sp.strain 32, Staphylococcus sp.strain 155, and A. brasilense Cd or corresponding mock was root-drench-applied.Plants were organized in a total of 4 blocks.Each block contained the following: (I) plants under salt stress (50 or 100 mM NaCl), (II) a control group without salt stress, (III) plants with the corresponding bacteria strain and no salt stress, and (IV) plants with the corresponding bacteria strain and salt concentrations (32 + 50 or 100 mM NaCl, 155 + 50 or 100 mM NaCl, A. brasilense + 50 or 100 mM NaCl).Leaf tissue from a representative group of plants (3 pools with 3 plants each pool), from all treatments, was collected for RNA extraction and differential expression analysis twenty-four hours after bacterial inoculation (T1).Treatments were repeated 15 days after first bacterial inoculation, collecting samples 24 h before (T2) and 24 h after bacteria inoculation (T3).Finally, more samples were collected at the end of the experiment, 30 days after the first inoculation (T4).

Bacterial Strain Inoculation before Salt Stress
The experiment was carried out by first adding the bacterial inoculant and then the salt stress.Prior to bacterial inoculation, plants were manually irrigated to field capacity.Ten mL of the corresponding bacterial treatment was applied 24 h before salt stress.Leaf tissue from a representative group of plants was collected (3 pools with 3 plants each pool), for RNA extraction and differential expression analysis, before salt stress was applied (T1).Afterward, plants were irrigated using sterile deionized water containing 50 mM NaCl solution through a drip irrigation system for two minutes four times a day.Leaf tissue of a representative group of plants from all treatments was collected, for RNA extraction and differential expression analysis, 24 h after the salt stress treatment initiation (48 h after bacterial inoculation, T2).

RNA Extraction
PureZOL RNA isolation reagent (Bio-Rad, Hercules, CA, USA) was used to extract total RNA from leaf tissue samples according to the manufacturer's specifications.Extracted RNA was quantified with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).For qRT-PCR analysis, RNA samples were treated with DNase I (1 U µg −1 DNA, Thermo Fisher Scientific, Waltham, MA, USA).The absence of DNA was confirmed by performing end-point PCR (40 cycles, according to the real-time PCR program) on the DNase I-treated RNA using Taq-DNA polymerase.Total RNA was stored at −80 • C until qRT-PCR assay.

Relative Gene Expression Levels by qPCR Analysis
Total RNA from leaf tissue samples of C. annuum was used for qRT-PCR according to the standard iScriptTM cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA), and the iTaq™ Universal SYBR ® Green Supermix (Bio-Rad, Hercules, CA, USA).A "no DNA" template control was used in each analysis.The qPCR conditions were those recommended by the manufacturer: one cycle of pre-treatment at 50 • C for 2 min, one cycle at 95 • C for 10 min, and 40 cycles at 95 • C for 15 s and at 60 • C for 1 min.The reported results are from three independent (n = 3) biological replicates.Each biological replicate was tested by triplicate and data were normalized with ubiquitin-conjugating protein (UBI-3) reference gene [20].Primers used in this work are enlisted in Table 1.The 2 −∆∆CT method was used for relative quantification, where the ∆∆CT value = ((TT 1Target − TT 1Reference ) − (CT 1Target − CT 1Reference )).The tested genes were CaCAT, CaSOD(Cu,Zn), CaSOD(Mn), CaNPR1, CaMYB72, CaETR1, CaJAR1, CaWRKYa, CaLOX2, CahsfA2, CahsfA3, CahsfB3, CaDNaJ2, and CaDNaJ04.

Name
Primer Sequence References

Name Primer Sequence References
Designed in this work

Statistical Analysis
Data were analyzed by univariate and multivariate analysis of variance (ANOVA and MANOVA).The least significant differences in Tukey's HSD test were calculated by two-way ANOVA.Differences among means were considered significant at p < 0.05 for all cases.All analyses were conducted using Statistica software version 10.0 for Windows and GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA).

Effect of Bacteria Isolated from Saline Habitats Inoculation on C. annuum Growth
The bacteria Bacillus sp.strain 32, Staphylococcus sp.strain 155, and A. brasilense were tested for growth promotion effects on the C. annuum.
Bacillus sp.strain 32 had a positive effect on root dry weight production (63.3%), and for fresh weight a positive effect on the stem (28%), root (128.9%), and leaves (20%) was also observed.All comparisons were performed against the control treatment (t-test p < 0.05) (Table 2).It is worth noting that A. brasilense Cd did not present any effect on plant growth promotion.

Expression Analysis of Stress-Related Genes in Plants Inoculated with the Bacterial Strains from Saline Sites
Transcriptional modification by Bacillus sp.strain 32, Staphylococcus sp.strain 155, and A. brasilense Cd in fourteen genes in C. annuum was analyzed for their changes in expression (Figures 1 and 2).The effect of bacterial inoculation on the genes' expression in time 1 showed a null or negative response to all three bacteria in all gene groups analyzed except for the CaLOX2, Ca MYB72, CaDnaJ02, CaDnaJ04, CaWRKY, and CaHsfA2 genes.For the CaHsfA2 gene, this upregulation proved to be significant at time 1 (Figure 2d).A positive effect in the expression of all genes was observed in time 2 by Bacillus sp.strain 32 and Staphylococcus sp.strain 155 (Figures 1 and 2).The increase was significant in the CaJAR1 gene with Bacillus sp.strain 32 and the CaDnaJ04 gene with Staphylococcus sp.strain 155.The exception was for the CaLOX2 gene which showed downregulation when plants were inoculated with Bacillus sp.strain 32 (Figure 1f).For plants inoculated with A. brasilense Cd, all showed null or positive changes in the level of expression, being lower, but not significantly, than those observed with the other inoculants.At time 3, greater effects were observed due bacterial inoculants.Overall, the three bacteria induced positive effects, being significant for CaCAT, CaLOX2, CaNPR1, CaDnaJ02, CaHsfA3, and CaHsfB3a genes with Bacillus sp.strain 32, and CaCAT, CaSOD (CuZn), CaLOX2, and CaNPR1 with A. brasilense (Figures 1 and 2).However, it was observed that the three inoculated bacteria caused a downregulation of the CaMYB72 gene and Bacillus sp.strain 32 also caused a downregulation of the CaWRKY and CaHsfA2 genes (Figure 1g,h and Figure 2d).Finally, during time 4, the three inoculants caused a null expression or downregulation in most of the genes, becoming significant in CaCAT, CaSOD(CuZn), CaJAR1, CaETR1, CaLOX2, CaMYB72, CaWRKY, CaNPR1, CaHsfA2, and CaHsfB3a.analyzed except for the CaLOX2, Ca MYB72, CaDnaJ02, CaDnaJ04, CaWRKY, and CaHsfA2 genes.For the CaHsfA2 gene, this upregulation proved to be significant at time 1 (Figure 2d).A positive effect in the expression of all genes was observed in time 2 by Bacillus sp.strain 32 and Staphylococcus sp.strain 155 (Figures 1 and 2).The increase was significant in the CaJAR1 gene with Bacillus sp.strain 32 and the CaDnaJ04 gene with Staphylococcus sp.strain 155.The exception was for the CaLOX2 gene which showed downregulation when plants were inoculated with Bacillus sp.strain 32 (Figure 1f).For plants inoculated with A. brasilense Cd, all showed null or positive changes in the level of expression, being lower, but not significantly, than those observed with the other inoculants.At time 3, greater effects were observed due bacterial inoculants.Overall, the three bacteria induced positive effects, being significant for CaCAT, CaLOX2, CaNPR1, CaDnaJ02, CaHsfA3, and CaHsfB3a genes with Bacillus sp.strain 32, and CaCAT, CaSOD (CuZn), CaLOX2, and CaNPR1 with A. brasilense (Figures 1 and 2).However, it was observed that the three inoculated bacteria caused a downregulation of the CaMYB72 gene and Bacillus sp.strain 32 also caused a downregulation of the CaWRKY and CaHsfA2 genes (Figures 1g,h and 2d).Finally, during time 4, the three inoculants caused a null expression or downregulation in most of the genes, becoming significant in CaCAT, CaSOD(CuZn), CaJAR1, CaETR1, Ca-LOX2, CaMYB72, CaWRKY, CaNPR1, CaHsfA2, and CaHsfB3a.

Effect of Different NaCl Concentrations on Gene Expression of C. annuum
To determine the effects of salt stress in C. annuum, plants were exposed to 50 and 100 mM of NaCl treatment.Plant tissue was collected after 48 h for analysis (Figures 3 and  4 to 50 mM, and S3 and S4 to 100 mM of NaCl).A greater response in terms of relative expression was observed under 50 mM of saline stress than at 100 mM.In general, an upregulation was observed in the oxidative stress genes group, being higher at times 3 and 4 (Figure 3a-c).For the second group of genes involved in response to both abiotic and biotic stress, the CaJAR1 and ETR1 genes showed downregulation in time 1, to subsequently increase their expression in later times, especially in time 3.For the CaLOX2 gene, an upregulation was observed in times 1 and 3, and downregulation in times 2 and 4 (Figure 3d-f).In the next group, which includes transcriptional regulators of ISR, an apparent up and downregulation of these genes was observed at different times.An upregulation of the CaMYB72 gene was observed at time 1 and for CaWRKY at time 2 and 3. On the other hand, a downregulation at time 3 was observed for CaMYB72, and for CaWRKY at times 2 and 4 (Figure 3g,h).Finally, for the CaNPR1 gene and the groups of HSP and HSF genes (Figure 4), an upregulation was observed, being significantly higher at time 3 for the group of HSP and HSF (Figure 4d).These results indicate that treatment with 50 mM NaCl induces a response in plants detectable up to 48 h after treatment (Figures 3 and 4).

Effect of Different NaCl Concentrations on Gene Expression of C. annuum
To determine the effects of salt stress in C. annuum, plants were exposed to 50 and 100 mM of NaCl treatment.Plant tissue was collected after 48 h for analysis (Figures 3 and 4 to 50 mM, and S3 and S4 to 100 mM of NaCl).A greater response in terms of relative expression was observed under 50 mM of saline stress than at 100 mM.In general, an upregulation was observed in the oxidative stress genes group, being higher at times 3 and 4 (Figure 3a-c).For the second group of genes involved in response to both abiotic and biotic stress, the CaJAR1 and ETR1 genes showed downregulation in time 1, to subsequently increase their expression in later times, especially in time 3.For the CaLOX2 gene, an upregulation was observed in times 1 and 3, and downregulation in times 2 and 4 (Figure 3d-f).In the next group, which includes transcriptional regulators of ISR, an apparent up and downregulation of these genes was observed at different times.An upregulation of the CaMYB72 gene was observed at time 1 and for CaWRKY at time 2 and 3. On the other hand, a downregulation at time 3 was observed for CaMYB72, and for CaWRKY at times 2 and 4 (Figure 3g,h).Finally, for the CaNPR1 gene and the groups of HSP and HSF genes (Figure 4), an upregulation was observed, being significantly higher at time 3 for the group of HSP and HSF (Figure 4d).These results indicate that treatment with 50 mM NaCl induces a response in plants detectable up to 48 h after treatment (Figures 3 and 4).

Bacterial Inoculation after Salt Stress
Plants were inoculated with the corresponding bacteria 24 h after being treated with 50 mM NaCl (Figures 5 and 6).In general, a similar response was observed for dual treatment samples (stress + bacteria) in comparison with those treated only with saline stress.The first analyzed group was that of the genes related to the oxidative stress response (CaCAT, CaSOD(CuZn), and CaSOD(Mn)).Generally speaking, in T1 no changes in expression were observed, except in certain cases wherein there was an increase in expression (Figure 5a-c).For T2, increases in the expression of these three genes were observed, being greater in dual treatments.For T3, the highest increase in the expression levels of the CaCAT, CaSOD(CuZn), and CaSOD(Mn) genes was observed.Finally, in T4 the expression levels of these genes were similar to those observed in T2, except for CaCAT treated with saline stress + strain 155 wherein the greatest increase in expression levels was observed.sion (Figure 5a-c).For T2, increases in the expression of these three genes were observed, being greater in dual treatments.For T3, the highest increase in the expression levels of the CaCAT, CaSOD(CuZn), and CaSOD(Mn) genes was observed.Finally, in T4 the expression levels of these genes were similar to those observed in T2, except for CaCAT treated with saline stress + strain 155 wherein the greatest increase in expression levels was observed.The genes involved in response to both abiotic and biotic stress (CaJAR1, CaLOX2, and CaETR1) showed a similar behavior between the four applied treatments.However, a downregulation of the three genes' expression by bacterial inoculants was observed at T3.The genes related to transcriptional regulators of ISR response (CaMYB72 and CaWRKY) showed some changes between treatments.The CaMYB72 increased its expression with the inoculants and when subjected to saline stress.The greatest increase in expression was with A. brasilense Cd (Figure 5g).In the case of CaWRKY, similar behavior was observed in its expression with all treatments (Figure 5h).The transcriptional coregulators NPR1, HSP, and HSF showed similar expression levels in all four treatments (Figure 6).Finally, CaDnaJ02, CahsfA2, and CahsfB3a showed a pattern of downregulation due The genes involved in response to both abiotic and biotic stress (CaJAR1, CaLOX2, and CaETR1) showed a similar behavior between the four applied treatments.However, a downregulation of the three genes' expression by bacterial inoculants was observed at T3.The genes related to transcriptional regulators of ISR response (CaMYB72 and CaWRKY) showed some changes between treatments.The CaMYB72 increased its expression with the inoculants and when subjected to saline stress.The greatest increase in expression was with A. brasilense Cd (Figure 5g).In the case of CaWRKY, similar behavior was observed in its expression with all treatments (Figure 5h).The transcriptional coregulators NPR1, HSP, and HSF showed similar expression levels in all four treatments (Figure 6).Finally, CaDnaJ02, CahsfA2, and CahsfB3a showed a pattern of downregulation due to the effect of bacterial inoculums on T3 similar to that observed in CaJAR1, CaLOX2, and CaETR1 (Figure 6b,d,f).

Bacterial Strain Inoculation before Salt Stress
The transcriptional expression of genes related to early salt stress responses such as CaMYB (transcription factor), CaJAR1 (JA signaling), and CaLOX2 (JA synthesis) was remarkably upregulated in salt stress compared to the control; similar expression patterns were observed in the genes encoding for HSF CahsfA2 and CahsfA3.The analyses show no differences in CaETR1, CaDNaJ2, and CaDNaJ4 expression in seedlings under NaCl treatment.By contrast, in dual treatment, CaJAR1, CaMYB, CaETR1 and genes encoding for heat shock proteins were downregulated.It is worth noting that the expression levels of CaLOX2 in dual-treatment plants with B. pumilus and A. brasilense Cd were higher than with saline stress only (Figures S1 and S2).

Discussion
The use of bacteria with the capacity to promote plant growth (PGPB) is a potential biological tool to alleviate abiotic stress, including salinity.Under highly saline conditions, bacterial diversity is reduced in comparison with other environments [25][26][27].Nevertheless, bacteria isolated from hypersaline sites are candidates for plant growth promotion and more efficient at enhancing plant tolerance to salt stress than those isolated from non-saline habitats [28].The effects of PGPB with the rapid activation of transcriptional expression of genes related to stress response led to a faster and stronger primed stress response at the transcriptional level.
In this study, two bacteria strains from ponds with high salinity from Guerrero Negro, Baja California Sur, Mexico, were used (Bacillus sp.strain 32 and Staphylococcus sp.strain 155).Bacillus is one of the most abundant and diverse bacteria in agroecosystems including extreme hypersaline environments.Moreover, Staphylococcus was isolated from marine environments in the East coast of India, salinity soils of Sharkia (Egypt), halophyte plants, or fermented fish [29][30][31][32][33][34][35].
This present study demonstrated that Bacillus sp.strain 32 can stimulate the growth of C. annuum under shade conditions, which is not the case for Staphylococcus sp.strain 155 or PGPB A. brasilense Cd (Table 2).Bacillus sp. has been reported to promote growth in some plant species, and C. annuum is among them [36].Additionally, B. pumilus has been reported to promote plant growth in Solanum lycopersicum, Rose hibrida, Vicia faba, Oryza sativa, and Atriplex lentiformis [25,[37][38][39][40].The above indicates that Bacillus has a positive effect on the growth of a wide range of plants despite being isolated from extreme environments, as seen in this work.On the other hand, very little is known about the Staphylococcus effect on plant growth.Few reports registered a positive effect on the root lengths of A. thaliana (Brassicaceae family) [35].In another study, it was observed that S. equorum did not significantly affect growth promotion in S. lycopersicum (Solanaceae family) [41], which is similar to results obtained in the present work with Staphylococcus strain 155 in C. annuum.As other PGPB, the growth promotion effect by the Staphylococcus genus could be specific to plant species.
Multiple genes have been reported to be involved in plant responses to salinity stress where the expression level depends on NaCl concentration and stress time exposure [42,43].In this study, salinity response and ISR genes (CaSOD(Cu,Zn), CaSOD(Mn), CaCAT, CaMYB, CaWRKY, CaJAR1, and CaLOX2) were stimulated by NaCl 50 mM 48 h after saline stress was initiated; these results are consistent with previous reports [5,11,44,45].
It is hypothesized that stress tolerance driven by PGPB is accompanied by bacteriaprimed transcriptional activation of multiple stress-responsive factors before plant exposure to salt stresses, in a similar response to ISR against pathogens [46].In this work was observed a differential modulation in the genes' expression related to salt-stress responses.The PGPB A. brasilense Cd upregulated the expression of CaMYB, CaJAR1, CaWRKY, and CaLOX2 genes, while Bacillus sp.strain 32 and Staphylococcus sp.strain 155 downregulated them or did not affect them 24 h after inoculation.A. brasilense Cd did not act as a PGPB, at least under current experimental conditions, but activated genes related to the ISRtype response (CaMYB72).Nevertheless, molecular mechanisms and the main signaling pathways involved in the defense response triggered by beneficial microorganisms have not been well characterized and vary from one beneficial bacterial species to another [47].An example is the transcription factor MYB72 that is suggested as a node of convergence in the ISR signaling pathway triggered by different beneficial microbes as Pseudomonas spp., Trichoderma spp., and halotolerant Dietzia natronolimnaea [11,46,47].In this study, plants inoculated with bacteria and treated with saline stress did not modify the expression of its genes, except in time 3, where a downregulation was observed.The results obtained in the present study are similar to those obtained by other authors in response to similar treatments (bacteria + stress), where bacteria conferred tolerance to stress [12,[48][49][50].
HSFs regulate the expression of heat shock proteins (HSPs) and other chaperone genes.In Arabidopsis, the hsfA4A transcriptional factor binds to promoters of target genes encoding the small heat shock protein HSP17.6A, as well as the WRKY30 and ZAT12 genes, indicating that hsfA4A's role is as a stress-response downstream transcription regulator [10,51].The ROS controlled by the HSFA4A gene are induced by salinity, elevated temperature, and a combination of these conditions [51].Little is known about the Hsf family in Capsicum.Some Hsf were upregulated under salt stress [21].CaHsfA3 presented a similar pattern of expression in this work with NaCl 50 mM after 24 h.It is worth noting that, in this study, the CahsfA2 gene expression level was significantly higher in plants under salinity stress, and this gene has been previously reported to be downregulated under salinity conditions (NaCl) [21].
Evidence of the role of DnaJ proteins (heat shock proteins) on stress response in plants has increased in recent years.Cis-elements in the promoter regions of CaDnaJ genes have been identified.The major groups of cis-elements include stress response and hormone response (SA, IAA, GA, MeJA, ABA, and ethylene) [7].In the present work, only A. brasilense induced the expression of CaDnaJ02 and CaDnaJ04 genes under saline-stress conditions.CaDnaJ02 and CaDnaJ04 genes have cis-response elements to the MeJa hormone in their promoter region [7].The expression of the genes related to this phytohormone was observed when inoculating them with the PGPB A. brasilense.JA synthesis pathways are important during the interaction with beneficial bacteria and salinity stress [33].

Conclusions
In the present study, we evaluated the gene expression in C. annuum plants induced by Bacillus sp.strain 32, Staphylococcus sp.strain 155 (both isolated from saline environment), and PGPB A. brasilense, through RT-qPCR.This study determined that the transcription of CaJAR1, CaMYB72, WRKY, and CaLOX2 genes was induced by strains; meanwhile, only the CaLOX2 gene was involved in the response to dual treatments with all three strains.Such results indicate that the JA signaling pathway is involved in the response to treatments.However, further studies are needed to confirm that the JA pathway is responsible for the effect observed in the strains.These results contribute to improve our understanding of the molecular mechanisms involved when plants are inoculated with bacteria.

Figure 4 .Figure 4 .
Figure 4. Effects of salt concentration on C. annuum plants in NPR1, heat shock protein, and factor genes that regulate genes that encode heat shock proteins.Quantitative RT-PCR determinations of relative expression levels of the genes: CaNPR1, CaDNaJ2, CaDNaJ04, CahsfA2, CahsfA3, and CahsfB3.The data represent means of biological triplicates and experimental replicates; error bars represent SEM.The asterisk indicates statistically significant differences between treatments (range test p < 0.05*, and 0.01***).

Figure 6 .
Figure 6.Gene expression analysis of NPR1, heat shock protein genes, and factor genes that regulate genes that encode heat shock proteins of C. annuum plants inoculated with bacteria after 24 h of salt stress (50 mM NaCl).Quantitative RT-PCR determinations of relative expression levels of the genes: CaNPR1, CaDNaJ2, CaDNaJ04, CahsfA2, CahsfA3, and CahsfB3.The data represent means of biological triplicates and experimental replicates; error bars represent SEM.The asterisk indicates statistically significant differences between treatments (range test p < 0.05*, 0.025**, 0.01***, and 0.001****).

Figure 6 .
Figure 6.Gene expression analysis of NPR1, heat shock protein genes, and factor genes that regulate genes that encode heat shock proteins of C. annuum plants inoculated with bacteria after 24 h of salt stress (50 mM NaCl).Quantitative RT-PCR determinations of relative expression levels of the genes: CaNPR1, CaDNaJ2, CaDNaJ04, CahsfA2, CahsfA3, and CahsfB3.The data represent means of biological triplicates and experimental replicates; error bars represent SEM.The asterisk indicates statistically significant differences between treatments (range test p < 0.05 *, 0.025 **, 0.01 ***, and 0.001 ****).

Table 1 .
Primers used in this work.

Table 2 .
Stem, root, and leaf length as well as fresh and dry weight of C. annuum plants inoculated with potentially growth-promoting isolates (strain 32, 155) and PGPB A. brasilense.
Effects of bacterial inoculation on C. annuum plants in NPR1, heat shock protein, and factor genes that regulate genes that encode heat shock proteins.Quantitative RT-PCR determinations of genes relative expression levels: CaNPR1, CaDNaJ2, CaDNaJ04, CahsfA2, CahsfA3, and CahsfB3.Data represent means of biological triplicates and experimental replicates; error bars represent SEM.The asterisk indicates statistically significant differences between treatments (range test p < 0.05*, 0.025**, and 0.001****).