Bacterial strain and plant material

In this study, we used seeds of Silihong (Virginia runner type), an early maturing, local peanut variety with a dark red seed coat. The bacterial strain of Tsukamurella tyrosinosolvens was P9, which was isolated in our laboratory and stored at the China Typical Culture Preservation Center (strain preservation number CCTCC AA 2020052) and in our laboratory as well.

Preparation of bacterial solution

An appropriate amount of strain P9 was added to 50 mL LB liquid medium and then incubated at 30°C with shaking at 180 rpm for 16 h to activate. The activated cultures were transferred to LB liquid medium, incubated for 24 h, and then centrifuged at 5000 ×g for 10 min. The supernatant was discarded, and the bacterial precipitate was rinsed twice with 5 mL sterile water. Finally, a bacterial suspension (10⁸ cfu/mL) was prepared in half-strength MS liquid medium.

Construction of the strain-P9–peanut-seedling co-culture system and preparation of transcriptome samples

Peanut seeds were disinfected with 20% H₂O₂ solution for 20 min, washed five times with sterile water, and soaked for 8 h. The seeds were placed in a Petri dish covered with moist sterile filter paper and then stored in a light incubator (16-h/8-h photoperiod) at 28°C for 3 days until germination. Germinated peanut seedlings with consistent growth were selected and transplanted into a sterile culture bottle containing pearl cotton and 195 mL half-strength MS liquid medium. After the leaves had unfolded, one set of peanut seedlings were inoculated with 5 mL of the P9 bacterial suspension (1 × 10⁸ cfu/mL) as the treatment group and cultured in a 28°C light incubator, while the same volume of half-strength MS medium was added to other seedlings as the control group. Treatment and control groups were further divided as follows: CK1 (24-h control group), CK2 (72-h control group), T1-P9 (treatment group co-cultured with P9 for 24 h), and T2-P9 (treatment group co-cultured with P9 for 72 h). After 24 and 72 h of co-culturing, root samples of the different treatment and control groups were frozen in liquid nitrogen for transcriptome sequencing analysis, and seedling growth indicators and chlorophyll contents were measured as well.

Total RNA extraction and transcriptome analysis of peanut roots

Total RNA was extracted from peanut roots using a TRNzol-A total RNA isolation kit (TIANGEN, Beijing, China). The RNA was checked for quality and quantity using a Nano 6000 Assay kit and a 2100 Bioanalyzer system (Agilent Technologies, Palo Alto, CA, USA). After quantification of RNA with a Qubit RNA Assay kit and a Qubit2.0 fluorometer (Life Technologies, CA, USA), a cDNA library was constructed using a NEBNext Ultra RNA Library Prep Kit for Illumina kit (NEB, USA) and then sequenced on the Illumina HiSeqTM2500 platform (Novogene 6000, Beijing, China).

Raw reads were subjected to Trimmomatic software (v0.40) to remove adapters, poly-N-containing reads, and low-quality reads, Q20, Q30, and GC contents of clean data were calculated. The genome of Arachis hypogaea (GenBank accession number GCF_003086295.2_arahy.Tifrunner.gnml.KYV3) was used as a reference sequence [15], and the clean data were mapped against the reference genome using the software Hisat2 (v2.0.1). Sequences with total mapped reads or fragments larger than 70% of the data were selected for subsequent analysis. Gene expression was calculated using the fragments per kilobase of exon model per million mapped fragments (FPKM) method. The threshold FPKM >1 was used as the criterion for identification of DEGs. DEGs were screened using edgeR software (selection criteria: P < 0.05 and |log2(foldchange)| > 1). and their gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed using clusterProfile R package. RNA-seq raw data has been uploaded to the NCBI database (Accession number PRJNA991079, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA991079).

Quantitative real-time PCR verification

Using the results of the KEGG pathway DEG analysis of peanut roots, we selected 24 candidate genes at the two stages (24 and 72 h after inoculation) for quantitative real-time PCR (RT-qPCR) verification. Primers for RT-qPCR were designed using Primer-BLAST (S1 Table) and then synthesized by Shanghai Bioengineering Co. The actin gene was used as an internal control. After RT-qPCR amplification, relative gene expression levels were calculated using the 2^−ΔΔCt method based on the C_t values of each sample [16].

Data processing and analysis

Data were analyzed for significant differences using the independent sample t-test in IBM SPSS Statistics 25.0. The following criteria were used to indicate significant and very significant differences, respectively: P < 0.05 and P < 0.01. Bar charts were constructed using Microsoft Excel 2010.

Establishment of a P9–peanut-seedling co-culture system

A hydroponics-based co-culture interaction system was established between Tsukamurella tyrosinosolvens P9 and peanut seedlings. As shown in Figs 1 and 2, the root length, lateral root number, and chlorophyll content of peanut seedlings at the initial stage of co-culturing with strain P9 (24 h after inoculation) were increased by 8.34%, 6.67%, and 5.26%, respectively, compared with those in control seedlings. At the late stage of co-culturing (72 h after inoculation), chlorophyll content was increased by 17.65%, plant height was significantly different (P < 0.05), and fresh weight, root length, root weight, and lateral root number were highly significantly different (P < 0.01) compared with those in the control. Seedling roots were more abundant and thicker, and stem and leaf growth was also significantly better than that of the control. These results demonstrate that strain P9 had positive effects on peanut growth 24 h after inoculation and that the effect was more significant 72 h after inoculation. These two co-culturing time points were therefore used for the preparation of root transcriptome samples.

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Fig 1. Growth status of peanut inoculated with Tsukamurella tyrosinosolvens strain P9 after 24 and 72 h.

https://doi.org/10.1371/journal.pone.0298303.g001

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Fig 2. Growth indices and chlorophyll contents of peanut seedlings co-cultured with Tsukamurella tyrosinosolvens P9.

Error bars show standard deviation (SD) of the mean of three replicates (⁎, P < 0.05; ⁎⁎, P < 0.01).

https://doi.org/10.1371/journal.pone.0298303.g002

Peanut root transcriptome sequencing and quality analysis

As shown in S2 Table, transcriptome sequencing of the 24-h peanut root sample (CK1) yielded an average of 46,253,588 and 44,135,290 raw and clean reads, respectively, while an average of 43,823,152.67 raw reads and 41,972,738 clean reads were generated from the 72-h peanut root sample (CK2). In regard to P9-strain-treated peanut groups, the average number of raw and clean reads obtained from roots inoculated with strain P9 for 24 h (T1-P9) was 44,791,624 and 42,654,016, respectively, whereas the average from roots inoculated with P9 for 72 h (T2-P9) was 45,770,172 and 43,806,935.33, respectively. Q30 values of the data from different treatment groups were all higher than 94.00%, and the percentage of sample sequences matching the reference genome was higher than 94.34%. These results confirm the quality of the data and the suitability of the selected reference genome, thus validating their use in the transcriptome analysis.

Differentially expressed gene (DEG) and KEGG enrichment analyses of peanut roots in response to strain P9

We observed differences in the gene expression of peanut roots co-cultured with strain P9 (S1 Fig). Compared with the genes in the control group at the same time point, 2,271 genes were differentially expressed in peanut roots co-cultured with strain P9 for 24 h (total of 1,559 up-regulated genes and 712 down-regulated genes), and 1,479 genes were differentially expressed in roots after 72 h of co-culturing with strain P9 (total of 912 up-regulated genes and 567 down-regulated genes). In addition, at 24 h post-inoculation, there were 2,206 DEGs shared between treatments, with 1,555 were up-regulated and 651 down-regulated. At 72 h post-inoculation, there were 1,397 DEGs, with 607 up-regulated and 701 down-regulated, respectively. Additionally, there were 38 and 56 unique genes identified at 24 and 72 hours post-inoculation, respectively (Fig 3). At 24 h after inoculation, the roots expressed 38 unique genes, mostly related to transcription factors. At 72 h after inoculation, the roots expressed 56 unique genes involved in multiple metabolic pathways, such as starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, pyruvate metabolism, oxidative phosphorylation, glycine, serine and threonine metabolism, pentose and glucuronate interconversions. These results suggest that inoculation with strain P9 affects the gene expression of peanut seedlings and that this regulation is characterized by temporal differences. The heatmap analysis revealed differences in gene expressions of peanut roots after inoculation with the P9 strain at different time points, especially at 24 and 72 hours (Fig 4).

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Fig 3.

Venn diagram of differential gene volcanoes in peanut roots inoculated with Tsukamurella tyrosinosolvens P9 for 24 h (left) or 72 h (right). The non-overlapping region was the number of unique DEGs of peanut inoculated with P9 strain, and the overlapping region was the number of common DEGs.

https://doi.org/10.1371/journal.pone.0298303.g003

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Fig 4. Hierarchical clustering and heatmap of differentially expressed genes in peanut roots inoculated with Tsukamurella tyrosinosolvens P9 for 24 h and 72 h.

The figure displays gene expression values for this group of samples.

https://doi.org/10.1371/journal.pone.0298303.g004

KEGG enrichment analysis (Fig 5) revealed that genes differentially expressed at the early stage of co-culturing were associated with 111 KEGG pathways. Among these pathways, 14 metabolic pathways were significantly enriched in DEGs: flavonoid biosynthesis; circadian rhythm-plant; isoflavone biosynthesis; phenylpropanoid biosynthesis; linoleic acid metabolism; zeatin biosynthesis; biosynthesis of various secondary metabolites-Part 2; pentose and glucuronate interconversions; cysteine and methionine metabolism; carbon fixation in photosynthetic organisms; betalain biosynthesis; cutin, suberin, and wax biosynthesis; mitogen-activated protein kinase (MAPK) signaling pathway-plant; and photosynthesis. At the late stage of co-culturing, 92 metabolic pathways were enriched in DEGs. The following 10 pathways were significantly enriched: pentose and glucuronate interconversions; glycine, serine, and threonine metabolism; zeatin biosynthesis; nitrogen metabolism; alpha-linolenic acid metabolism; ascorbate and aldarate metabolism; glycerolipid metabolism; valine, leucine, and isoleucine biosynthesis; ABC transport system; and isoquinoline alkaloid biosynthesis. Only two pathways, namely, pentose and glucuronate interconversion and zeatin biosynthesis, were significantly enriched during both stages. Furthermore, the metabolic pathways significantly enriched in DEGs in peanut roots at the early stage of co-culturing were mainly associated with flavonoid, isoflavone, and phenylpropanoid synthesis, photosynthesis, and defense responses, whereas those significantly enriched at the late stage of co-culturing were mainly related to nutrient metabolism and energy production. These results indicate that the response mechanisms and metabolic pathways of peanut seedlings interacting with strain P9 were different between the two time periods.

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Fig 5.

KEGG pathways enriched in DEGs in peanut inoculated with Tsukamurella tyrosinosolvens P9 at 24 h (left) and 72 h (right).

https://doi.org/10.1371/journal.pone.0298303.g005

Functional analysis of DEGs

We analyzed DEGs and their functions and found that inoculation with strain P9 significantly affected the transcriptome of peanut roots. Genes differentially expressed in response to strain P9 were mainly related to signal transduction, cell wall modification, plant hormone synthesis, substance and energy metabolism, and plant defense stress, but which genes were differentially expressed and which KEGG pathways were enriched in peanut after inoculation with strain P9 varied according to co-culturing stage.

RT-qPCR verification

Taking into account the results of GO and KEGG enrichment analyses, we selected 24 genes of peanut co-cultured with strain P9 for 24 h and 72 h, involving cell wall pectin degradation and synthesis, photosynthesis, carbon fixation, the synthesis of ascorbic acid, phenylpropanoid, plant hormone, flavonoids, isoflavones, nitrogen metabolism and other pathways, and verified their expressions by RT-qPCR (Fig 6). A consistent trend was observed in the expression of these 24 genes determined by RT-qPCR and RNA sequencing, indicating that the results of RNA-seq were reliable.

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Fig 6. The differential gene expression verified by RT-qPCR.

The left is the DEGs at 24 h after inoculation; the right is the DEGs at 72 h after inoculation. Among them, LOC112703750 encodes polygalacturonase, LOC112777310 encodes trans-cinnamate 4-monooxygenase, LOC112720495 encodes caffeoyl-CoA O-methyltransferase, LOC112795729 encodes PetF, LOC112741560 encodes chalcone synthase-like, LOC112741528 encodes ent-kaurenoic acid monooxygenase, LOC112727194 encodes indole-3-pyruvate monooxygenase, LOC112802019 encodes aminocyclopropanecarboxylate oxidase, LOC112737957 encodes L-tryptophan decarboxylase, novel.668 encodes starch synthase, LOC112783179 encodes ribulose-phosphate 3-epimerase, LOC112718386 encodes ERF1, LOC112747725 encodes L-ascorbate peroxidase, LOC112785663 encodes Nrt, LOC114924223 and LOC112705586 encode cytokinin dehydrogenase, LOC112790626 encodes pectinesterase, LOC112766121 encodes glucuronokinase, LOC112726583 encodes L-gulonolactone oxidase, LOC114925084 encodes 12-oxoophytodienoic acid reductase, LOC112728121 encodes D-glycerate 3-kinase, novel.1265 encodes glycerol-3- phosphate acyltransferase, LOC112726696 encodes CaM4, LOC112747667 encodes DNA methyltransferase 1.

https://doi.org/10.1371/journal.pone.0298303.g006

Strain P9 promotes signal transduction and initiates the defense response of peanut

The MAPK signaling pathway is closely related to plant growth, development, and stress responses [19]. FLS2, which binds to bacterial receptor flg22, stimulates ROS production and transmits invasion signals to WRKY33 to initiate plant defense mechanisms [20]. To have a growth-promoting effect, PGPR must invade plants and activate plant defense functions [21]. ChiB is a target gene of ERF1, which can induce plant stress responses [22]. After 24 h of co-culturing in our study, genes encoding FLS2, WRKY33, ERF1, and ChiB were up-regulated. The initial colonization of strain P9, a foreign microorganism, obviously initiated a rapid plant defense response. Consistent with our findings, Liu et al. found that plant immunity is triggered by the up-regulation of FLS2, WRKY, MYB, and MYC2 in tobacco roots inoculated with Paenibacillus polymyxa YC0136 [23], and the ERF transcription factor of Arabidopsis thaliana inoculated with Bacillus amyloliquefaciens FZB42 is up-regulated [24]. CNGCs can transport Ca²⁺, an important second messenger, into the cell and pass it via Rboh and CaMCML to induce cell wall thickening and stimulate stomatal closure to resist external environmental pressures [25]. In our study, the up-regulation of CNGCs and CaMCML during the early stage of co-culturing promoted Ca²⁺ signal transduction and a rapid plant response. After cell injury, Rboh is up-regulated to regulate ROS production and help maintain the dynamic balance of ROS [26]. When we extended the co-culturing time to 72 h, genes encoding FLS2, Rboh, and CaMCML were more strongly up-regulated, whereas the WRKY33 gene was significantly down-regulated. In addition, the ChiB gene was less strongly up-regulated compared with that at the early stage, and the expression of ERF1 did not differ from that of the control. All of these results indicate that the ethylene-induced plant defense response was weakened and that the regulation of ROS homeostasis induced by strain colonization was stronger.

Strain P9 stimulates pectin degradation of cell walls to promote cell elongation and facilitates its colonization of peanut

The plant cell wall includes primary and secondary cell walls. The primary cell wall is mainly composed of cellulose, hemicellulose, and pectin. The high pectin polysaccharide content and strong plasticity of plant cells plays an important role in their morphogenesis [27]. PE, PL, and PG can promote the loosening of cell walls, a phenomenon related to cell wall extension and stem elongation [28, 29]. The PL gene of Arabidopsis may be involved in cell elongation and lateral root formation [30]. In the present study, the significant up-regulation of the PE gene during both time periods and the up-regulation of the PL gene at the early stage of co-culturing promoted cell wall elongation and lateral root formation, thereby improving the root development of inoculated peanut (Figs 1 and 2). Because PGPR usually colonize the rhizosphere to exert their growth-promoting effects on plants [31], partial disintegration of cell walls is very important for bacterial adhesion and colonization on root surfaces [32]. The PL gene of rapeseed is also up-regulated after the establishment of a symbiotic relationship with the endophytic fungus Sclerotinia sclerotiorum strain DT-8 [29]. The PE-coding gene of banana seedlings inoculated with B. amyloliquefaciens Bs006 is up-regulated, which promotes pectin degradation and the enhancement of cell adhesion ability, thus facilitating the colonization of banana roots [33]. In our previous study, we found that strain P9 can stably colonize peanut roots and stems [14]. In the present study, we observed that the up-regulated expression of PE and PL genes in co-cultured peanut increased pectin decomposition, thus laying a foundation for colonization by strain P9.

Strain P9 affects plant hormone synthesis and transport to promotes peanut growth and development

Strain P9 affects the synthesis of various hormones in peanut plants. In the early stage of co-culturing, we observed that strain P9 induced the up-regulation of ALDH and indole-3-pyruvate monooxygenase to increase IAA synthesis and that the up-regulation of the gene encoding CKX accelerated the decomposition of cytokinin. Furthermore, the up-regulation of ACO, ACS, and S-adenosylmethionine synthetase increased ethylene production, and the induced expression of LOX, OPR3, acetyl-CoA acyltransferase 1, and hydroperoxide dehydratase promoted the synthesis of jasmonic acid. Finally, the slight down-regulation of ent-kaurenoic acid monooxygenase resulted in a slight decrease in GA synthesis. The development of plant roots is the result of the crosstalk of multiple hormones. IAA plays a leading role in plant root development, with other plant hormones mostly regulated by their coordination or antagonism with IAA [34]. The antagonistic relationship between cytokinin and auxin is well known [35], the balance between these two hormones is a major determinant of cell fate during hormone-induced root organogensis [36], and the overexpression of this CKX in rice roots promotes adventitious root formation [37]. In addition, the distribution of auxin will be disturbed by ethylene signal, the effects of ethylene and auxin are synergistic in root elongation and antagonistic in lateral root formation [38]. Ethylene promotes auxin transport to the elongation zone by regulating auxin polar transport proteins. It inhibits the growth of root meristems, promotes the formation of lateral roots, and induces the occurrence of adventitious roots and root hairs [39, 40]. The up-regulation of the ACS and ERF affects soybean root cortex thickening [41]. GA can regulate stem elongation, and exogenous application of gibberellin has inhibitory effects on root development [42]. Endogenous jasmonic acid can promote the lateral root formation of wild-type Arabidopsis thaliana [43]. In our study, increased IAA, ethylene, and jasmonic acid synthesis and decreased cytokinin and GA synthesis obviously promoted peanut root development and the formation of lateral roots and root hairs during the interaction with strain P9. Root lengths and lateral root numbers of seedlings during the early stage of co-culturing were therefore increased compared with those in the control (Figs 1 and 2). Inoculation with P. fluorescens significantly increases the concentration of IAA in roots of Sedum alfredii and significantly reduces gibberellic acid, trans-zeatin, ethylene, and jasmonic acid concentrations, thereby inducing lateral root formation [44]. In addition, inoculation with Burkholderia phytofirmans PsJN induces the up-regulation of IAA, GA, and SA synthesis-related genes in Arabidopsis roots, in turn promoting root hair formation and increased root weights and chlorophyll contents [45]. Moreover, Paenibacillus polymyxa YC0136 induces the up-regulation of genes related to IAA, zeatin, and GA synthesis in tobacco roots [23]. Some studies showed that signaling substances secreted by PGPR can affect root development by regulating endogenous hormone pathways of plants [46, 47].

After 72 h of co-culturing, down-regulation of the gene encoding ACO had reduced ethylene synthesis, and peanut plants had adapted to colonization by strain P9. Significant up-regulation of CKX and CYP735A reduced cytokinin levels and promoted root growth [48], and significant up-regulation of OPR3, DOX, and ACX genes increased jasmonic acid synthesis. The down-regulation of gibberellin 2 beta-dioxygenase (GA2ox), a key enzyme in the degradation of active gibberellin [49], led to the accumulation of GA, and the combined action of the proteins encoded by these genes promoted plant root and stem growth. In particular, the significant down-regulation of the indole-3-pyruvate monooxygenase gene significantly decreased IAA synthesis. Because plants are sensitive to IAA, IAA can act as a signal molecule to interfere with plant hormone synthesis when IAA-producing bacteria interact with plants [50]. IAA secreted by some PGPR strains can also be applied to plants to promote plant growth [51]. In our previous studies, we found that strain P9 has strong IAA-secretion ability [13] and that root exudates of peanut significantly up-regulate the tryptophan metabolic pathway of strain P9 and significantly increase IAA synthesis in this strain [52]. In the present study, we also observed that the genes encoding ABC transporter family proteins ABCB1 and ABCB10 were significantly up-regulated in the late stage of co-culturing. The ABCB protein family is mainly located in plant vascular tissue and is closely related to the polar transport of IAA [53]. Auxin signaling and transport play a key role in the root structural changes of Arabidopsis stimulated by PGPR strains [54]. We speculate that the IAA secreted by strain P9 and the enhanced IAA polar transport of peanut seedlings are sufficient to meet and promote plant growth; peanut plants therefore maintain IAA homeostasis by regulating their own IAA synthesis reduction. IAA secreted by Azospirillum brasilense has been reported to promote the development of lateral roots and root hairs by regulating IAA homeostasis in plants [55]. In a metabolomic analysis of the interaction co-culture system in our study, we indeed found that the IAA content of the co-culture system increased to varying degrees but was more significant during the early stage of co-culturing. PGPR can regulates the synthesis and homeostasis of plant hormones such as ethylene, auxin, and cytokinin, abscisic acid and gibberellin, which can influence plant growth [56]. So, the combined effects of these hormones significantly promoted seedling root development, and the heights and root characteristics of inoculated plants were significantly improved compared with the findings in the uninoculated controls (Figs 1 and 2).

Strain P9 enhances peanut metabolism and energy production and promotes plant growth

In the early stage of co-culturing with strain P9, 60 genes involved in circadian rhythm (including GI, CHS, and CK2β), 11 genes participating in the carbon fixation pathway, and 7 genes encoding photosynthetic proteins were significantly up-regulated. Expression of the light-harvesting chlorophyll protein complex LHC subunit was also induced. GI is a plant-specific nuclear protein that plays a role in various physiological processes, such as photosynthesis and circadian rhythm [57]. Antenna proteins can capture solar energy and drive photosynthesis [58], while up-regulated Lhcb1 and Lhcb3 are directly related to increased chlorophyll content [59]. The CHS gene is rhythmically expressed in Arabidopsis seedlings, which makes the regulation of hormones time-dependent [60]. CK2β can act on a variety of substrates and affects light signaling, circadian rhythm, and cell cycle control [61]. During the early stage of co-culturing with strain P9, light capture, photosynthesis, carbon fixation, CO₂ intake, and circadian rhythm regulation were significantly enhanced in peanut. Moreover, the up-regulation of starch synthase led to an increase in starch synthesis, energy storage, and carbon assimilation products. In addition to light energy, plants can directly produce bio-energy through sugar metabolism. Multiple genes related to starch and sucrose metabolism, glycolysis/gluconeogenesis, amino sugar and ribose metabolism, and pyruvate metabolism were all induced during both co-culturing stages. Genes in pathways related to fatty acid metabolism, acetyl-CoA acyltransferase, acetyl-CoA carboxylase1 FabH, long-chain acyl-CoA synthetase, and acyl-CoA thioseterase 1/2/4 were up-regulated, and the synthesis of long-chain fatty acids was increased. Fatty acids are an important source of energy storage in plants and an important component of membrane lipids. Fatty acids can be used as a carbon source by symbiotic mycorrhizal fungi and are considered to be essential for the establishment of symbiotic relationships [62]. Unsaturated fatty acids also maintain the relative fluidity of the cell membrane [63]. Up-regulation of phosphatidate phosphatase LPIN and phospholipid:diacylglycerol acyltransferase increased the production of triglycerides. During the late stage of co-culturing, GLYK was significantly up-regulated, and glyceric acid catalyzed the synthesis of large amounts of 3-phosphoglycerate, which was channeled into the glycolysis pathway. Multiple genes involved in sugar metabolism were significantly more highly up-regulated compared with those at the early stage of co-culturing. The production of CO₂ and ATP increased significantly, and multiple genes participating in oxidative phosphorylation were also up-regulated, thus indicating that the large amount of ATP obtained by biological oxidation can provide energy for peanut growth and development and various life activities. Moreover, GPAT is the first acylesterase used in the synthesis of phosphatidylglycerol, which significantly up-regulates the synthesis of cell membrane component, namely, 1,2-diacyl-sn-glycerol-3-P. GPAT can also control the formation and storage of membrane lipids in Arabidopsis thaliana [64].

During plant growth, roots mainly obtain nitrogen sources by absorbing nitrate and ammonium salts from the soil via nitrate and ammonium transporters. Nitrate and nitrite are transported into cells by nitrate/nitrite transporter (NRT), whose activity in roots can affect nitrogen acquisition, plant growth, and seed development [65]. NR, the rate-limiting enzyme in nitrate assimilation, is the main compound used by many plants and organisms to obtain nitrogen sources [66]. Overexpression of the NR gene can improve the absorption and utilization of nitrogen nutrition in plants [67]. NRT-encoding genes are significantly up-regulated in wheat roots inoculated with Azospirillum brasilense inoculated with Phyllobacterium strain STM196 [68]. In the present study, the NR gene was up-regulated at both co-culturing stages. This gene was significantly up-regulated at the later stage of co-culturing, which indicates that peanut nitrogen uptake and nitrate assimilation were further enhanced and that plants could better obtain sufficient nitrogen sources. AtNRT1.1 is involved in lateral root development and root hair formation in Arabidopsis [69], and OsNRT2.1 contributes to adventitious root elongation in rice [70]. When the duration of co-culturing with strain P9 was increased, carbon, nitrogen, and lipid metabolism in peanut was therefore accelerated, ATP production was increased, and nitrogen uptake and assimilation were accelerated. Consequently, the promotive effect of P9 on plant growth was more obvious, and root development was better than that of the control group.

Our previous study confirmed that the strain P9 has a good ability to dissolve phosphorus and produce siderophore. Phosphorus is an essential element for the growth and development of plants. In order for plants to use insoluble phosphate in the environment, it must first be dissolved through the action of organic acids [71]. During the early stage of co-culture with strain P9, the PDC and ALDH genes, which catalyse the production of pyruvate and acetic acid, respectively, were up-regulated. Additionally, the LPIN, PDAT, phospholipase C and lysophospholipase II genes, which are involved in glycerophospholipid and glycerolipid metabolism, were also up-regulated. In the later stage, the genes encoding PDC and ADH was up-regulated to a greater extent (7.92-fold and 8.35-fold, respectively), and the genes encoding D-lactate dehydrogenase, malate dehydrogenase and pyruvate decarboxyase, which catalyse the production of lactic acid and malic acid, and the genes encoding choline-phosphate cytase and GPAT, which are involved in the synthesis of phospholipids and phosphoinositides, were significantly up-regulated. Our additional study also confirmed that organic acids are one of the main components of root extracts from peanut seedlings inoculated with the P9 strain [52]. So, it was speculated that the P9 strain not only promoted the formation of organic acids in peanut roots, but also provided more soluble phosphorus for peanuts through its phosphorus solubilsing ability, which obviously promoted plant root development. In addition, iron is closely related to chlorophyll synthesis and photosynthesis, and the regulation of iron homeostasis greatly affects cell signal transduction, which in turn affects root growth [72, 73], but the efficiency of iron utilization by plants is very low. Siderophore secreted by PGPR are an effective strategy for plants to absorb and utilize iron [74]. After inoculation with the P9 strain, the chlorophyll content of the leaves increased, root development was more vigorous, and the expression of photosynthesis-related proteins PetC and PetF was up-regulated. It was speculated that the siderophore secreted by P9 provided sufficient iron for the growth of peanut seedlings, promoting the development of peanut roots and an increase in chlorophyll content. This was consistent with the good plant growth-promoting ability of the P9 strain.

Strain P9 enhances the defense ability of peanut and maintains ROS homeostasis

The phenylpropanoid pathway is a rich source of plant metabolites; it can not only synthesize lignin, but is also the starting point for the production of flavonoids [75]. This pathway plays an important role in plant resistance to biotic and abiotic stress [76]. The accumulation of lignin, a major component of plant secondary cell walls, helps improve the mechanical strength of cells and is crucial to plant structure and defense [77]. CYP73A catalyzes the first oxidation step of the phenylpropanoid pathway in higher plants, whereas F5H is the only link in S-type lignin synthesis, and CCoAOMT is an important regulatory protein for G-type lignin synthesis [78]. CAD catalyzes the last step of lignin biosynthesis before oxidative polymerization of cell walls [79]. 4CL, a key enzyme involved in the synthesis of lignin and flavonoids, regulates the direction of carbon flow in the downstream branch metabolic pathway of the phenylpropane [80]. In the present study, the up-regulation of CYP73A, F5H, CAD, and CAGT at the early stage and that of 4CL, CCoAOMT, and other genes at the late stage all increased lignin synthesis in peanut roots. This increase promoted the formation of secondary structure and vascular bundles, laid a foundation for the development of lateral roots, and enhanced plant mechanical strength. In another study, the up-regulation of 4CL and CCoAOMT genes in tobacco roots after co-culturing with Paenibacillus polymyxa YC0136 for 20 h was found to promote cell wall differentiation and thickening and improved stress resistance of tobacco [23].

Flavonoid and isoflavones are secondary metabolites with multiple biological functions. In addition to playing a key role in stress protection and plant defense, these compounds act as signal transduction molecules for the interaction of transcription factors and kinases [81, 82]. During the early stage of co-culturing in our study, 86 differentially expressed genes in the flavonoid and isoflavone biosynthesis pathway were up-regulated compared with those in the control, especially, the first key enzyme in flavonoid biosynthesis, CHS, showed significant induction of up-regulated expression. The up-regulated expression of CYP93C and CYP81E (I2′H), members of the cytochrome P450 (CYP) superfamily, can protect plants from stress injury [83]. In the early stage of co-culturing in our study, the up-regulation of multiple genes related to flavonoid and betaine synthesis pathways would therefore have significantly improved the defense ability of peanut. In the later stage of co-culturing, the biosynthesis of isoquinoline alkaloids in seedlings was increased. In addition, chitinase plays an important role in plant disease resistance [84]. The Chic-coding gene of peanut co-cultured with strain P9 was up-regulated at both stages, thus indicating that inoculation with this strain improved the disease resistance of peanut.

A complex environment will affect plant growth and development and thus increase ROS content in cells. APX and AO are important oxidases in the ascorbate metabolic pathway [85]. Most ascorbate present in the cytoplasm binds to APX to form monodehydroascorbate and decomposes H₂O₂ to remove ROS [86], whereas AO mainly oxidizes ascorbate in apoplasts and catalyzes its transformation into L-dehydroascorbate, which is transported to the cytoplasm to maintain the oxidation/reduction state of apoplasts [87]. After co-culturing with strain P9 for 24 h, AO was significantly up-regulated, but APX was down-regulated, and APX and GULO genes were up-regulated at 72 h. According to this result, the ability of peanut seedlings to scavenge ROS was significntly enhanced by prolongation of the strain P9 inoculation time. Additionally, in In the later stage of co-culturing, the significant down-regulation of GST-encoding genes not only maintained the intracellular glutathione (GSH) level, but also improved the defense abilities and detoxification of plant cells [88]. In addition, multiple coding genes of peroxidase were significantly up-regulated during the two co-culturing stages. Kandaswamy et al. have reported that Pseudomonas putida RRF3 enhances plant defense by inducing the up-regulation of peroxidase genes in rice roots [89]. Co-culturing with strain P9 obviously enhanced a variety of defense substances and antioxidant enzymes, such as peroxidase, GST, APX, and AO in peanut seedlings, thereby facilitating the rapid removal of excess ROS. The expression of stress-related genes in plants, mediated by PGPR, can improve plant resistance to biotic and abiotic stresses.