Integrative microbial and omic-based approaches reveal the molecular mechanisms of composite PGPR-mediated growth promotion in plants

Integrative microbial and omic-based approaches reveal the molecular mechanisms of composite PGPR-mediated growth promotion in plants | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 March 2025 V1 Latest version Share on Integrative microbial and omic-based approaches reveal the molecular mechanisms of composite PGPR-mediated growth promotion in plants Authors : Shuo Han , Yuchun Gou , Chunzhe Yang , Xiangru Wang , Xiangqian Wang , Jianming Zhu , Zhong Hua Cai 0000-0002-9058-1608 , Shuying Feng , and Jin Zhou 0000-0003-0372-2554 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174098418.86496855/v1 267 views 109 downloads Contents Abstract 3.1. Screening and compounding of PGPR strains 3.2. Effects of PGPR strains on plant and soil physicochemical properties 3.3. Effects of PGPR strains on rhizosphere microorganisms 3.4. Molecular mechanisms of PGPR strains for plant growth promotion 3.6. Linking environment and microbe profiles to crop responses 4. Discussion 5. Conclusion Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Plant growth-promoting rhizobacteria (PGPR) are important due to their biostimulant activity. However, most studies focus on epigenetic mechanisms, leaving the microscopic and molecular aspects of multistrain coordination unexplored. In this study, seven PGPR strains were isolated from mangrove forests. These bacteria exhibited various traits such as phosphate solubilization, indole acetic acid production, and siderophore generation. The combination of Sinorhizobium sp. N2-2 and Pseudomonas putida S5 showed the most prominent PGPR activities. Pot experiments and omic-based analyses revealed that PGPR strains significantly promoted the growth of pakchoi and influenced soil physicochemical properties and sucrose enzyme activity. Metagenomic analysis showed that PGPR strains altered the rhizosphere microbial community, enriching genes related to nitrogen cycling, phosphorus transformation, hormone signal transduction, and carbon utilization. Transcriptomic analysis indicated increased energy metabolism and antistress abilities in the host plant. The results suggest that the “composite PGPR” co-promoted plant growth by driving transformations in rhizosphere nutrient cycling, regulating microbial metabolism, and balancing plant physiology. This study provides insights into the coordination between PGPR strains and highlights their potential for sustainable agriculture development. Integrative microbial and omic-based approaches reveal the molecular mechanisms of composite PGPR-mediated growth promotion in plants Shuo Han 1,2,3† , Yuchun Gou 2,3† , Chunzhe Yang 4 , Xiangru Wang 4 , Xiangqian Wang 4 , Jianming Zhu 1,2,3 , Zhonghua Cai 1,2 , Shuying Feng 5** , Jin Zhou 1,2,3* 1 Marine Ecology and Human Factors Assessment Technical Innovation Center of Natural Resources Ministry, Tsinghua Shenzhen International Graduate School, Shenzhen 518055, Guangdong Province, P. R. China. 2 Shenzhen Public Platform for Screening and Application of Marine Microbial Resources, Institute for Ocean Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, Guangdong Province, P. R. China. 3 Shenzhen Key Laboratory of Advanced Technology for Marine Ecology, Institute for Ocean Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, Guangdong Province, P. R. China. 4 Joint Research Center for Microbial Control and Applied Technology, Tsinghua Shenzhen International Graduate School-China Resources Beverage (Holdings) Company Limited. 5 Medical College, Henan University of Chinese Medicine, Zhengzhou 450046, Henan, China † These authors have contributed equally to this work and share first authorship. * Corresponding author: Jin Zhou Address: Room 902, Marine-Building, Shenzhen International Graduate School, Tsinghua University, Xili University Town, Shenzhen City, 518055, Guangdong Province, P. R. China. Tel: +86-755-86953413; Fax: +86-755-86953413 E-mail address: [email protected] ** co-corresponding author: Shuying Feng E-mail address: [email protected] Abstract Plant growth-promoting rhizobacteria (PGPR) are important due to their biostimulant activity. However, most studies focus on epigenetic mechanisms, leaving the microscopic and molecular aspects of multistrain coordination unexplored. In this study, seven PGPR strains were isolated from mangrove forests. These bacteria exhibited various traits such as phosphate solubilization, indole acetic acid production, and siderophore generation. The combination of Sinorhizobium sp. N2-2 and Pseudomonas putida S5 showed the most prominent PGPR activities. Pot experiments and omic-based analyses revealed that PGPR strains significantly promoted the growth of pakchoi and influenced soil physicochemical properties and sucrose enzyme activity. Metagenomic analysis showed that PGPR strains altered the rhizosphere microbial community, enriching genes related to nitrogen cycling, phosphorus transformation, hormone signal transduction, and carbon utilization. Transcriptomic analysis indicated increased energy metabolism and antistress abilities in the host plant. The results suggest that the “composite PGPR” co-promoted plant growth by driving transformations in rhizosphere nutrient cycling, regulating microbial metabolism, and balancing plant physiology. This study provides insights into the coordination between PGPR strains and highlights their potential for sustainable agriculture development. Keywords: composite plant growth-promoting rhizobacteria (PGPR); plant growth promotion; rhizospheric profiles; microbial functions; plant molecular response; sustainable agriculture Introduction In modern agriculture, the need to improve crop quality and yield is escalating. However, traditional chemical fertilizers impose a significant burden on ecosystems. Consequently, microbial agents, particularly plant growth-promoting rhizobacteria (PGPR), have garnered considerable attention because of their ability to enhance plant growth and crop yields (Kaur et al., 2023). PGPRs employ various mechanisms, including phytohormones synthesis (e.g., indole-3-acetic acid, IAA), phosphate solubilization, nitrogen fixation, antibiotic production, induction of systemic resistance, and interactions with the rhizosphere microbial community (Bukhat et al., 2020). These interactions form a positive feedback loop that promotes nutrient cycling and improves soil structure, thus enhancing plant nutrient uptake and fertilizer use efficiency. In recent years, certain strains, such as Bacillus sp., Pseudomonas sp., and Actinobacteria sp., have been commercialized (Liu et al., 2023). These PGPR products are employed in agricultural production to improve plant growth and alleviate biotic and abiotic stresses, advancing the development of sustainable and environmentally friendly ecosystems (Bhattacharyya et al., 2012). Compared with the instability of single PGPR strains, composite PGPRs have attracted considerable attention because of their ability to exchange metabolites and signals, coordinate activities, and minimize systemic energy consumption through the division of labor, resulting in more efficient nutrient-promoting functions (Oleńska et al., 2020). For instance, Bacillus amyloliquefaciens Bs006 and Pseudomonas fluorescens Ps006 treatments regulated gene expression related to growth regulation, flowering, photosynthesis, glucose catabolism, and plant defense in banana (Gamez et al., 2019). Recently, Zhang et al. (2024) reported that inoculating a composite PGPR strain consisting of Bacillus cereus AR156, Bacillus subtilis SM21, and Serratia marcescens XY21 led to changes in soil microbial communities, suppressing soil-borne diseases and improving soil physicochemical properties. These findings have made developing diverse and efficient PGPR strains a focus of current research efforts. In recent years, mangrove forests, as unique ecosystems, have become important sources for PGPR screening because of their distinctive microbial diversity. Microorganisms in mangrove environments are highly abundant and adapted to extreme conditions such as high salinity, high humidity, and hypoxia, making them potentially valuable for ecological or agricultural applications (Mai et al., 2021). For example, halotolerant bacteria ( Arthrobacter sp., Pseudomonas plecoglossicida , and Kocuria rosea ) from mangroves could be used as potential bioagents in saline soils, with potential antagonistic effect on root rot disease (Pallavi et al., 2023). Similar results have been reported by Alghamdi et al. (2023), which found that Isoptericola sp. AK164, from mangrove sediment, contributes to plant salt tolerance and stress reduction, making it a potential biostimulant for improving agriculture in submerged saline land. Dey et al. (2022) further isolated multiple functional strains ( Priestia megaterium , Bacillus sp., Kocuria palustris , Enterobacter hormaechei , and Pseudomonus fulva ) that exhibited arsenic-resistant behavior with plant-growth-promoting characteristics (IAA, NH 3 , and P-solubilization) that can be used in mangrove reforestation and heavy metal bioremediation. In particular, their effect on soil microecological profiles and the molecular mechanisms underlying promoting crop growth remain poorly explored. In addition, most studies focus on epigenetic mechanisms (Santoyo et al., 2021), and research on the synergistic plant growth promotion mechanisms through which PGPR strains act remains limited, hindering the application and development of composite PGPR strains. This study aims to develop a composite PGPR strain that can significantly promote plant growth by regulating soil microbial profiles to achieve efficient nutrient recycling and utilization. It is hypothesized that specific PGPR strains from mangrove sediment can enhance plant growth by altering soil microbial communities, which improves nutrient recycling and utilization. These positive bacteria could promote plant growth by improving soil enzyme activity, regulating microbial characteristics (diversity, composition, and function), and mediating plant metabolism (including energy utilization, hormone homeostasis, and physiological balance). In order to test this hypothesis, we first detected isolate traits, including N-utilization, P-solubilization, IAA production, and siderophore secrate. Then, their growth-promoting role and agronomic traits were verified through bacterium–pakchoi co-culturing experiments. Finally, rhizosphere microbial metagenomic features and plant transcriptional responses were analyzed. The final goals are to provide insight into the molecular mechanism underlying plants after inoculation with composite PGPRs and to provide guidance for applying microbial agents. Additionally, providing new insights and a theoretical basis for the future development of efficient plant growth promoters from mangrove environments is desirable. 2. Materials and methods 2.1. Screening of PGPR strains from mangrove environments Natural root samples were collected from a mangrove forest in Futian, Shenzhen, China (longitude 114.0549°E, latitude 22.5356°N). Detailed methods are available in Supplementary Information Text S1. 2.2. Pot experiments with PGPR strains The pot experiments were conducted in a controlled indoor environment using soil collected from agricultural fields near Shenzhen, China. The soil, with moderate fertility, slightly acidic pH, and clay loam texture, was sterilized at 121 °C and divided into 1.0 kg portions per pot. Its properties included pH 6.67±0.01, available potassium 64.58±0.98 mg/kg, available phosphorus 80.35±0.91 mg/kg, total nitrogen 16.39±0.42‰, total carbon 63.82±0.51‰, sucrase 4.15±0.43 mg/g-24 h, urease 5.42±0.09 mg/g-24 h, and acid phosphatase 78±3 μg/g-24 h. The soil was exposed to sunlight for 3 days before sieving to remove large particles. Brassica chinensis L. (pakchoi) seeds were sterilized in 75% ethanol for 1 minute, followed by 10% H₂O₂ for 10 minutes, and washed with sterile water. Seedlings were grown in vermiculite for 15 days before transplanting. Healthy, uniform seedlings were selected for the experiment, which included five treatment groups with four replicates each containing three seedlings as shown in Figure 1. Bacterial cultures were grown to the logarithmic phase, centrifuged, washed, and resuspended to 10⁸ CFU/mL. Each pot received 10 mL of bacterial suspension, while the control group received sterile water. The experiment lasted for two months under controlled conditions: 25°C, light intensity of 2800 Lux, and a 12-hour light/dark cycle. 2.3. Plant and soil sampling Approximately 60 days after seedling emergence, and the root systems were extracted using a spade, taking care to remove the excess soil. The soil was collected on sterilized tinfoil, sieved through a 2 mm sieve, and a portion was stored in 15 mL DNase-free tubes, quickly frozen with liquid nitrogen, and retained at −80 °C for soil metagenomic analysis. Another portion was collected in 50 mL centrifuge tubes for physicochemical analysis. The fresh weight of the plants was recorded, and a portion of the fresh leaves was retained for subsequent chlorophyll and total sugar measurements. The remaining portion was dried in a blast oven at 80 °C to 90 °C for 20 minutes, dried to constant weight at 60 °C to 70 °C, and the dry weight was recorded. The dried samples were ground and sieved through a 40-mesh sieve for measurement of plant indicators such as TN and total phosphorus (TP). 2.4. Determination of plant and soil physicochemical indicators The basic physicochemical parameters, including soluble sugars, total sugar, TC, TN, TP, total potassium, as well as soil sucrase activity were detected. Detailed experimental methods are available in Supplementary Information Text S2. 2.5. Soil DNA extraction and 16s and metagenomic sequencing Detailed methods are in Supplementary Information Text S3. 2.6. Metagenome sequencing data processing Adapter sequences were trimmed and quality-controlled using Fastp (v0.20.0). The following parameters were used for trimming: minimum quality score of Q20, minimum read length of 50 bp, and a sliding window of 4 bases with a quality threshold of Q15. Sequences were assembled with Megahit, and contigs ≥300 bp. Sequences were assembled with Megahit, and contigs ≥300 bp were selected. Open reading frames were predicted with MetaGene, and genes ≥100 bp were translated into amino acids. Non-redundant gene sets were created with CD-HIT (identity ≥95%, coverage ≥90%). To prevent contamination, extraction blanks and mock communities were included in the sequencing process. Extraction blanks were processed in parallel with sample DNA extractions to detect any contamination introduced during DNA extraction. Mock communities, containing known mixtures of bacterial DNA, were also sequenced to verify the accuracy of the sequencing and bioinformatics pipeline. High-quality reads were matched to this gene set using SOAPaligner to calculate gene abundance. Sequences were aligned with the COG database using DIAMOND for COG gene abundance. Functional annotations were done with KOBAS 2.0 using KEGG data. Bacterial community and gene abundance were analyzed with Canoco 5, and Spearman correlation was conducted using SPSS 23.0 (Xie et al., 2011). 2.7. Transcriptomic analysis of plants Transcriptome sequencing was carried out on a NovaSeq 6000 system (Illumina, San Diego, CA, USA). Three root tissue samples from each treatment group were selected randomly for the RNA-seq analysis, and their respective total RNA was extracted using the mirVana™ miRNA isolation kit (Ambion). The integrity of the RNA was assessed using an Agilent 2100 Bioanalyzer system (Agilent Technologies, USA). and RNA Integrity Numbers (RINs) were determined, with samples having a RIN value ≥7 being considered acceptable for sequencing. Next, the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) was used to assemble the library. Specific methodological information can be found in Zhao et al. (2023). After the library was qualified, it was fully sequenced on the Illumina NovaSeq 6000 sequencing platform, an average of 40 reads per sample. To identify differentially expressed genes (DEGs), the DEGseq2 package was used. RNA-seq raw data were deposited in the Genome Sequence Archive database (Accession number: PRJNA1182542). 2.8. Statistical analysis Data were analyzed using one-way ANOVA with SPSS software (version 22.0). Microbial diversity was assessed using QIIME for Chao 1 and Shannon indices. Non-metric multidimensional scaling (NMDS) was performed in R (vegan package). Microbial community structure was compared using Bray-Curtis distances for PCA. Co-occurrence networks were analyzed with the R psych package and Gephi software. Differentially expressed genes (DEGs) were identified by ANOVA with FDR correction. Pathway analysis was conducted using the SPSSAU online tool for PLS-PM. Detailed experimental methods are available in Supplementary Information Text S4. 3. Results 3.1. Screening and compounding of PGPR strains Based on the screening criteria, seven positive PGPR strains were obtained, i.e., N2-2, N4, PM-2, PM-8, M6, S5, and KM-4. After 16S rRNA sequencing and blast, they were identified as Sinorhizobium sp., P. putida , Acinetobacter sp., Enterobacter hormaechei , Enterobacteriaceae sp., Pseudomonas sp., and Comamonadaceae sp., respectively (Table 1). All strains have P-solubilizing and N-fixing abilities, especially the S5 and N2-2 strains. With regard to siderophore production ability, N2-2, S5, PM-2, and PM-8 were found to have relatively stronger capacities, essential for plant growth in iron-limited environments. Meanwhile, except for N4 and PM-2, the other bacteria strains can biosynthesize IAA, with PM-8 and N2-2 displaying the highest values. In addition, N2-2, S5, and M6 also possessed the ability to synthesize ACC(1-aminocyclopropane-1-carboxylate) deaminase, showing its potential as an efficient nitrogen-utilization bacterium suitable for combination with a phosphorus-solubilizing bacterium. In order to obtain the optimum compatibility between PGPR strains, antagonism experiments were conducted. The antagonism tests showed that N2-2 did not antagonize any of the screened bacteria, indicating that N2-2 is a relatively ideal candidate strain. In addition, in the co-culturing experiment, the existence of N2-2 promoted IAA expression. Based on the growth-promoting potential of individual strains and their non-antagonistic interactions, several strain combinations were designed for further testing. After co-culturing with Sinorhizobium sp. N2-2 and P. putida S5, the IAA level could reach 232.04 mg/L, 2.51 times higher than N2-2 alone and 16.81 times higher than S5 alone (Table 2). At the same time, N2-2 and S5 have relatively comprehensive PGPR capabilities and display positive responses in all five measured factors (i.e., P-solubilizing, N-fixing, siderophore production, IAA production, and ACC deaminase synthesis). Based on these results, N2-2 and S5 were selected for compounding because of their excellent performance. 3.2. Effects of PGPR strains on plant and soil physicochemical properties The plant growth promotion results are shown in Figure S1. It shows that plants in the “N2-2+S5” treatment group had fuller foliage and more vigorous growth, indicating that this composite PGPR strain promoted the growth of plants significantly. Figure 2A shows that compared with the CK group, all treatment groups improved the physiological indexes of plants, with the “N2-2+S5” group showing the most significant effect. For example, the fresh weight increased by 80.25%, plant height by 32.93%, root weight by 50%, total chlorophyll by 28.91%, soluble sugar by 27.82%, and total sugar by 33.62%. Notably, although the IAA treatment group promoted plant growth, the effect was less pronounced compared with the PGPR treatment groups. These results indicated that the “N2-2+S5” PGPR strain effectively improved the growth and agronomic features of B. chinensis L. In addition to plants, soil physicochemical parameters also improved. The results (Figure 2A) showed that PGPR inoculation improved the soil profiles. The NS treatment group had reduced total TN and TC contents by 11.56% and 5.43%, respectively ( p < 0.05). Simultaneously, it increased available K by 36.11% and available P by 19%, outperforming individual strain treatments. Soil pH shifted closer to neutral, increasing by 0.10–0.15 units. Additionally, enzyme activities essential for nutrient cycling were stimulated: sucrose enzyme activity increased by 78.13%, and phosphatase activity rose by 73.33%. Compared with the CK group, the “N2-2+S5” and IAA treatment groups reduced inter-root soil TC and TN contents, increased soil pH and available K/P levels, and altered the activity of various soil enzymes (Table S1). Considerable correlations were found between most physicochemical parameters by Spearman’s analysis. For example, TN and TC were clustered and negatively correlated with soil physicochemical indicators such as urease, phosphodiesterase, and sucrase, reflecting improvements in soil physicochemical profiles via microbial activities. pH and urease were clustered, and AP, AK, and sucrase were clustered, with AP and AK positively correlating with the activities of phosphodiesterase and sucrase, suggesting synergistic effects between these soil indicators (Figure 2B). The correlation analysis showed that all plant indicators were negatively correlated with soil TN and TC contents, while soil sucrase, urease, and pH were positively correlated with plant growth and development indicators. AP, AK, and phosphodiesterase were significantly and positively correlated with plant root weight, potassium content, plant TN, total sugar, and phosphorus content (Figure 2C), indicating that soil physicochemical properties significantly affected plant growth and development. 3.3. Effects of PGPR strains on rhizosphere microorganisms PGPR strains changed soil microecological features significantly, including an increased Chao 1 index, altered β-diversity, enhanced dominated species effective colonization, and changed microbial network relationships. Detailed information is available in Supplementary Information Text S5. 3.4. Molecular mechanisms of PGPR strains for plant growth promotion 3.4.1. Enhanced C using efficiency Polysaccharides, as an important carbon source, play a crucial role in plant nutrient uptake. Among the carbohydrate-active enzyme (CAZyme) genes, it was found that the highest percentage in the CK group was 32% for glycosyltransferases (GTs), 30% for glycoside hydrolases (GHs), and 21% for carbohydrate esterases (CEs) (Figure 3C). PGPR inoculation altered the gene abundance of CAZymes. In the N and S individual treatment groups, the former was enriched in GT, oxidoreductase, and cellulase genes, whereas the latter was enriched in polysaccharide lyase (PL) genes. Combining the N and S treatments enriched the abundance of genes such as GT, GH, and carbohydrate-binding modules (CBMs) compared with the individual N or S treatment groups. Specifically, genes related to GHs (GH10, GH77, and GH105), GTs (GT41, GT9, and GT104), and PLs (PL5-1, PL7-2, and PL15) increased significantly in all treatment groups, with the greatest increase observed in the NS treatment group (Figure 3B). The enrichment of enzymes such as GTs and GHs in the NS treatment group suggests that these enzymes may be closely related to the degradation and utilization of specific polysaccharides, e.g., GH-like enzymes are usually associated with hydrolyzing cellulose and hemicellulose (Figure 3A). These results suggest that PGPR inoculation not only altered the CAZyme composition but also enhanced their ability to utilize various polysaccharides. 3.4.2. Improved N and P metabolic abilities of rhizosphere microorganisms The metagenomic sequencing results showed that the KEGG pathways enriched significantly include enzyme pathways related to catalyzing the conversion of nitrate to ammonia via nitrite in all treatment groups (1.7.7.1, 1.7.7.2, and 1.7.2.2). In contrast, the enrichment degree of the enzyme pathway related to catalyzing the conversion of nitrite to NO decreased (1.7.2.1). These pathways were enriched (P<0.05) more in the NS treatment group (3.6-fold compared with the CK group), which had a stronger ability to promote nitrogen conversion (Figure 4A). Gene annotation analysis showed significant enrichment of key nitrogen genes such as nitrogen fixation-related members nifU/B/D/K/H/X that increased significantly in the N-treated group, with increases of 30.2% to 56.8% compared with the CK group. The nitrification-related genes ahbD , mftC , and ahbC were enriched to varying degrees in the N-treated group, with their expression levels increasing by 87.8% to 134% compared with the CK group, indicating their efficient nitrogen fixation ability. Moreover, the expression of N-assimilation–dissimilation genes, such as the cysteine desulfurase gene nfs1 and peptide cyclase gene pqqE , were further increased in the NS treatment group (P<0.05). The increased degree is in the range of 2–3 times. These results suggest that strain complexation can further promote the nitrogen fixation capacity of soil microbial communities (Figure 4C). Similarly, notable changes in phosphorus cycling functional genes were also found. According to the Circos plot (Figure 4B), in the CK group, the top three genes in terms of abundance were ugpQ (K01126), ppx (K01524), and pstS (K02040), whereas, in the NS-treated group, the top three genes in terms of abundance were ppx (K01524), gcd (K00117), and pstS (K02040). The dumbbell plot analysis (Figure 4D) showed that the NS treatment group had a higher abundance (P<0.05) of most phosphorus cycle-related genes than the other treatment groups, including phosphorus transformation-related gene gcd (encoding the glucose dehydrogenase) and pstS (substrate-binding protein in phosphate transport system) with 147.9% and 260.2% increases compared with the CK group. The gene pstC/pstA (involved in phosphate transport system permease synthesis) involved in phosphorus uptake increased by 150.6% and 138.8% compared with the CK group. In addition, the phosphorus transport genes phoR and phoD/phoB (the phosphate regulator response modifiers, encoding the phospho-regulator sensor histidine kinase) also showed 28.3%–185.2% increases, suggesting that the composite PGPR strain altered the abundance of functional genes for phosphorus solubilization and nitrogen fixation in root-associated microorganisms, exhibiting more efficient phosphorus utilization and nitrogen fixation activities. Additionally, to further understand what contributes to the above-mentioned function, a species traceability analysis of element cycling was performed. For nitrogen cycling, Escherichia coli , Sphingomonas , Pseudomonas , and Vibrio fibrillaris were the dominant contributors in the NS treatment group (Figure 4E). In terms of phosphorus cycling, Pseudomonas and Sphingomonas were the main members of the NS treatment group (Figure S4). These results suggest that adding PGPR strains resulted in structural plasticity (species abundance and composition) and functional variability (elemental metabolic abilities) for root-associated microorganisms. 3.4.3. Promoted IAA and siderophore secretion The phytohormone-related functions after incubating with the composite PGPR strain were analyzed. The results (Figure 5A) showed that the average abundance of genes related to IAA synthesis from tryptophan via the pyruvate pathway increased significantly (P<0.05) in the experimental groups compared with the CK group. The indole-3-pyruvate synthesis IAA pathway (1.14.13.168) and tryptophan synthesis pathway (4.1.1.28), with the indole-3-pyruvate synthesis IAA pathway enriched significantly in the NS treatment group. This suggests that exposure to the composite PGPR strain promotes IAA biosynthesis in the root environment. Additionally, the indole-3-acetaldehyde pathway (4.1.1.74) was remarkably enriched in all treatment groups, and there was a significant increase in gene abundance in the tryptophan-synthesizing indole pathway (4.1.99.1) involving tryptophanase. This pyridoxal phosphate-dependent enzyme catalyzes the conversion of tryptophan to indole, commonly used as intraspecific or interspecific signaling molecules. Moreover, the S and N treatment groups showed an increased abundance of genes in the tryptophan-synthesizing indole-3-acetamide pathway, potentially impacting hormone synthesis in the plant root system (Figure 5A). These results suggest that PGPR inoculation regulates phytohormone homeostasis via the IAA synthesis pathway. IAA, or indole-3-acetic acid, is a key auxin that regulates plant growth and development, including cell elongation, root initiation, and vascular tissue differentiation. It also helps plants respond to abiotic and biotic stresses, promoting root development under drought or salinity stress and enhancing pathogen resistance. After the species traceability analysis, the NS treatment group exhibited the highest relative abundance of Vibrio , γ-Proteobacteria, and Acidobacteria. Moreover, Pseudomonas significantly increased in abundance in the NS treatment group. In contrast, the N and S treatment groups showed differences in the abundance of Sphingomonas and Xanthomonadaceae (Figure 5B). These results suggest that the above-mentioned species were potential contributors to IAA production and can change the efficiency of phytohormone biosynthesis in the root environment. 3.5. Effects of PGPR strains on the root transcriptome To identify DEGs at the molecular level, RNA-seq was performed. Specifically, in the treatment groups receiving N, S, NS, and IAA, 282, 1210, 612, and 646 significantly upregulated genes, and 467, 530, 864, and 1030 significantly downregulated genes were detected compared with the CK group (p < 0.05, Figure 6A and B). Further, the KEGG enrichment analysis indicated that, in the NS vs. CK comparison, DEGs were enriched significantly in several key pathways, including ”starch and sucrose metabolism,” ”phosphatidylinositol signaling system,” and ”MAPK signaling pathway (plant).” These pathways reached significant P-values. Additionally, in the NS treatment group, significant enrichment was observed in lipid metabolism pathways, such as ”linoleic acid metabolism,” suggesting that the DEGs have potential functions in membrane composition or energy metabolism regulation (Figure 6C). The gene ontology (GO) enrichment analysis further supported these findings, as DEGs in the NS vs. CK comparison were enriched significantly in functions such as ”plant-type cell wall biogenesis,” ”cell structure organization,” and ”cellular component biosynthesis” (Figure 6D). These GO terms highlight the critical roles of DEGs in the formation and maintenance of plant cell wall structures, likely contributing to cellular structural stability and enhanced stress resistance. 3.6. Linking environment and microbe profiles to crop responses The effects of PGPR inoculation, rhizosphere microorganisms, and soil physicochemical properties on plant growth were analyzed using PLS-PM methods. The goodness-of-fit values for the PLS-PM models were 0.7599, indicating they adequately explained the influential effect pathways (Figure 7A and B). Notably, soil physicochemical properties were the main factors triggering changes in the bacterial community in the NS treatment group, which interfered with the soil microbial function and ultimately affected the biomass of B. chinensis L ( p < 0.05). In contrast, in the individual N or S treatment groups, the responses of the root system were mediated by its bacterial communities ( p < 0.05), indirectly affecting the growth. A cascading process was observed, i.e., the microbial community had a significant direct effect on root growth-promoting genes (path coefficient = 0.849), growth-promoting genes greatly affected plant growth (path coefficient = 0.474), and soil enzyme activity (path coefficient = 0.667). Moreover, the soil enzyme activity remarkably affected plant nutrient elements (path coefficient = 0.653), and plant nutrient elements significantly affected plant growth (path coefficient = 0.773). To sum up, NS strains could affect the composition of rhizosphere microbial communities by altering the soil physicochemical properties and root enzyme activities, thus affecting the gene response and growth of B. chinensis L . 4. Discussion 4.1. PGPR strains changed the soil parameters and agronomic characteristics All PGPRs in this study were selected from mangrove environments, exhibiting multifunctional, such as nitrogen-fixing, phosphorus-solubilizing, producing iron carriers, expressing ACC deaminase, and secreting IAA (Table 1). The relative diversity of PGPRs indicates that mangrove environments are a potential pool of PGPR strains from which to draw biotrophic bacteria. This study demonstrated that PGPR application significantly altered soil physicochemical properties, notably improving pH and available potassium (AK) levels compared to the control (CK) group (Table S1). Previous studies revealed that organic matter decomposition can be accelerated via microbes to alleviate soil acidification (Boguta et al., 2019). Through a path model analysis, He and Chen (2013) determined that soil organic matter and pH values can affect the dynamic balance of potassium, and an increase in organic matter or pH value can promote the release of potassium to improve the availability of potassium in the soil. In this study, the correlation analysis shows that soil AK, organic matter, and pH were characteristic factors affecting B. chinensis L growth. Among these parameters, AK has the highest factor loadings and the largest contribution to plant growth (Figure 2B and C). Hence, these results show that PGPRs increase the availability of potassium and provide a necessary element for plant growth. In addition, this study revealed that adding PGPRs reduced the nitrate concentration in crops significantly (Figure 2A), probably because the soil microbial amendment reduced the nitrate concentration via increasing nitrate reductase activity, thus improving the quality of vegetables (Harindintwali et al., 2021). Jin et al. (2022) indicated that applying B. subtilis , a beneficial bacteria, enriched the organic matter concentration in plant bodies, increasing the quality of lettuce. Similar results were observed in the present study, where soluble sugar levels increased in the NS treatment group (Figure 2A). This may be related to the effective promotion of nutrient metabolism coordination so as to ensure a better quality of crops (Brunetti et al., 2019). 4.2. PGPR strains improved the soil microecological profiles In this study, the NS combination could efficiently colonize inter-root soils and change the microbial structure of the soil. Decreases in the abundance of Micromonosporales and Xanthomonadales were observed (Figure S2D). The former is a denitrifying bacterium that can reduces nitrogen nutrient availability in the soil and is detrimental to plant growth (Cao et al., 2015). The latter is an important pathogenic plant bacterium that can invade different plant organs through water pores, stomata, and wounds, causing various diseases such as wilt and black rot (Timilsina et al., 2020). In contrast, the abundance of Burkholderiales and Pseudomonadales increased significantly (Figure S2D). Colavolpe et al. (2020) found that Burkholderia can defend against phytopathogens and promote plant growth. Pseudomonadales are considered to be soil phosphorus solubilizers and can produce organic acids and utilize soil phosphatases or other nutrients in the soil, which in turn promotes plant growth (Ha-tran et al., 2021). This species was riched significantly, especially in the NS treatment group (Figure S2D). These results indicated that PGPR strains changed the proportion of beneficial and harmful bacteria in soil and created a healthy rhizosphere microbial environment. Most edges in the network relationships in the treatment group showed positive correlations, suggesting that beneficial interactions were stronger after applying PGPRs and that microorganisms could colonize and grow in the soil environment through cooperation (Bhattacharyya et al., 2012). In addition, this high connectivity implies high network complexity, which is important for the ecological buffer capability and microbial homeostasis (Kang et al., 2022). The more complex network model and shorter average network path length were observed in the NS treatment group (Figure S2C and Table S2), suggesting that PGPR bacteria improved the co-occurrence efficiency and response of bacteria to environmental changes (Kang et al., 2021). Nitrogen and phosphorus are essential nutrients for plant growth and exist in various forms in soil. They are converted by microorganisms into organic nitrogen and quick-acting phosphorus to be supplied to plants for growth. PGPR strains effectively promote the conversion of nitrogen and phosphorus in plants (Mortinho et al., 2022). Rangasamy et al. (2022) isolated a strain of Rhizobium RSKVG 02 from tea root soil with exceptionally high nitrogen-fixing enzyme activity and the ability to promote phosphorus solubilization and increase soil phosphate content, which can be used as a biofertilizer to improve soil fertility. This is similar to the results in the present study (Figure 2 and Table S1), indicating that the composite PGPR strain effectively promotes nitrogen and phosphorus recycling for use by plants. With regard to the molecular mechanism, the metagenome showed that nitrogen fixation members such as cysteine desulfurase gene ( nfs1 ), peptide cyclase gene ( pqqE ), and nitrogen-fixing enzyme genes (nifU/B/D/K/H/X ) were highly enriched in the NS treatment group (Figure 4C). Additionally, the nitrogen fixation efficiency of microorganisms could be improved significantly by providing iron–sulfur clusters, enhancing the antioxidant capacity, and ensuring the synthesis and function of nitrogen-fixing enzymes (Dos Santos et al., 2012). Specifically, the nfs1 gene is involved in the biosynthesis of iron–sulfur clusters, an essential component in the active center of nitrogen-fixing enzymes, ensuring their correct assembly and function (Johnson et al., 2005). The pqqE gene is involved in the biosynthesis of pyrroloquinoline quinone. This antioxidant enhances the antioxidant capacity of microorganisms and protects nitrogen-fixing enzymes from oxidative damage (Barr et al., 2016). The nsf1 and pqqE abundances in the NS treatment group were more than twice that of the CK group (, indicating that it plays a crucial role in ensuring the synthesis of nitrogenase. Additionally, this increase enhancing the availability of nitrogen for plant uptake, which serves as a potential mechanism for promoting plant growth. Meanwhile, phosphorus-solubilizing genes such as glucose dehydrogenase gene ( gcd ), phosphate regulator sensor histidine kinase gene ( phoR ), and phosphate regulator response modifiers ( phoD and phoB ), increased significantly in the NS treatment group (Figure 4D). Specifically, the gcd gene encodes a glucose dehydrogenase enzyme and showed a 147.9% increase compared with the CK group, indicating an enhanced ability to produce gluconic acid, which reduces soil pH, dissolves phosphate minerals, and increases the availability of quick-acting phosphorus (Rodriguez et al., 2003). The regulatory members phoR/D/B were also enriched, with increases ranging from 28.3% to 185.2% in the NS treatment group, indicating improved phosphate sensing and regulation in response to environmental conditions. This occurs because PhoR activates PhoB under low-phosphate conditions to trigger phosphate uptake responses, while PhoD hydrolyzes organic phosphorus compounds, releasing inorganic phosphate for uptake (Hsieh et al., 2010). Additionally, the phosphate transport system genes ( pstS, pstC, and pstA ) were also upregulated significantly in the NS treatment group. PstS , the substrate-binding protein, increased by 260.2%, while pstC and pstA , responsible for forming transmembrane channels that transport phosphate into the cell, increased by 150.6% and 138.8%, respectively (Figure 4D). This enrichment suggests that the NS treatment improved phosphate capture and transport, enhancing phosphorus delivery to plants and microorganisms (Zhao et al., 2022). In addition to N and P, adding PGPR strains changed C metabolism by regulating CAZyme genes. The enrichment of GT, GH, CBM, and PL genes appeared in the NS treatment group, indicating that the NS treatment exhibited a broader spectrum of CAZymes and greater functional diversity in polysaccharide utilization than the individual N or S treatments. In addition, the NS treatment group had the highest percentages of GTs (32%), GHs (30%), and CEs (21%) (Figure 3C). Meanwhile, genes associated with GH-like enzymes (e.g., GH10, GH77, and GH105) and GT-like enzymes (e.g., GT41, GT9, and GT104) were also upregulated significantly (Figure 3C), correlating with improved hydrolysis of cellulose and hemicellulose (Lairson et al., 2008). This suggests that the NS treatment improved carbon availability, contributing to the observed increases in soil fertility and plant health. In addition to improving the metabolism of dominant elements, PGPR strains also regulate the metabolism of trace elements, such as phytohormones and iron carriers. IAA is an important phytohormone that regulates plant growth and development, including the processes of cell division, elongation, and differentiation (Rehan et al., 2023). In this study, the N2-2 and S5 strains produced 92.4 mg/L and 13.8 mg/L of IAA, respectively, and their combination increased IAA production to 232 mg/L (Table 2), indicating a synergistic effect on IAA synthesis. In addition, the N2-2 and S5 bacterial strains increased the efficiency of IAA synthesis by soil microorganisms significantly. In particular, the gene abundance of IAA synthesis pathways, such as the indole-3-acetaldehyde pathway (4.1.1.74) and tryptophan-synthesizing indole pathway (4.1.99.1), was enriched significantly in the NS treatment group. The ipdC gene in the indole pyruvate-synthesizing indole-3-acetaldehyde pathway encodes an indole-3-pyruvate decarboxylase capable of converting indole-3-pyruvate to indole-3-acetaldehyde, a key intermediate in the synthesis of IAA. This process is considered a major pathway for IAA biosynthesis (Spaepen et al., 2007). The tryptophan enzyme encoded by the tnaA gene in the tryptophan-synthesizing indole pathway can convert tryptophan to indole, which can subsequently be further converted to IAA through a variety of pathways (Kanda et al., 2020). The enrichment of these pathways suggests that rhizospheric microorganisms significantly improve the efficiency of IAA synthesis by increasing the expression of these key enzymes, thus promoting plant growth by hormone drive. Moreover, IAA synthesis is closely related to the conversion processes of nitrogen and phosphorus, and many rhizospheric microorganisms can use tryptophan as a precursor to synthesize IAA and provide nitrogen to crops via nitrogen-fixing (Bhattacharyya et al., 2012). For example, Pseudomonas and Bacillus dissolve insoluble phosphate in soil by secreting organic acids and enzymes to increase the effective phosphorus content in soil. In addition, these microorganisms synthesize and secrete IAA to promote plant growth (Alori et al., 2017), suggesting that root-associated microorganisms often function synergistically to produce a variety of growth-promoting factors that promote plant growth in concert. Based on the PLS-PM results (Figure 7), PGPR strains have a close relationship with microbial features; the optimized microbial community not only influenced the expression of growth-promoting genes but also increased soil bioactivity and nutrient cycling efficiency. This aligns with previous studies stating that soil microbial diversity enhances soil biological functions and promotes plant nutrient uptake (Van der Heijden et al., 2007). Previously, Abbasi et al. (2022) verified that a Streptomyces composite PGPR strain is capable of modifying the rhizospheric microbial community, inducing plant defense initiation, and improving the quality of sweet pepper fruits. The PLS-PM results in this study also found similar synergistic effects and further support the multiple mechanisms by which PGPR act on plant growth promotion. 4.3. Plant response mechanisms for PGPR strain addition The transcriptomic analysis of different treatment groups compared with the CK group reveals significant insights into the molecular responses elicited by each treatment. Significant enrichment of genes involved in plant resilience was observed (Figure 6). Akbar et al. (2022) found that applying PGPR strains improved cotton plant salt and drought tolerances significantly, accompanied by substantial changes in the gene expression profiles in roots, particularly of genes related to metabolism, hormone signaling, and antioxidant systems. Similarly, in this study, PGPR treatment may enhance carbohydrate utilization or storage, providing additional energy and metabolic intermediates for growth and defense. Moreover, the enrichment of phosphatidylinositol signaling and the MAPK signaling pathway further supports the influence of PGPR strains on plant cellular signaling systems. The MAPK signaling pathway regulates plant responses to biotic and abiotic stresses, suggesting an enhanced capacity of plants treated with PGPR strains to respond to environmental fluctuations. Chang et al. (2019) noted that microbial stimuli significantly activated the MAPK signaling pathway in plants, thus enhancing pathogen defense, aligning with the PGPR strain-induced effects observed in this study (Figure 6C). The GO enrichment analysis shows that DEGs in the NS treatment group are mainly involved in cell wall biogenesis and cell structure organization, consistent with the role of PGPR strains in enhancing plant physical barrier capabilities (Figure 6D). Previous research has shown that PGPR treatment may increase cell wall thickness and structural stability by affecting key genes involved in cell wall biosynthesis, thus enhancing plant resistance to mechanical stress and pathogens (Meena et al., 2020). Additionally, the enrichment of the linoleic acid metabolism pathway observed in the NS treatment group emphasizes the importance of lipid signaling in cell stress tolerance (Figure 6D). This finding aligns with the hypothesis proposed by Abe et al. (2018) that plants under adverse conditions increase linoleic acid accumulation to enhance membrane stability and fluidity. 5. Conclusion This study demonstrates that PGPR strains from mangrove environments, particularly the combination of Sinorhizobium sp. N2-2 and P. putida S5, can effectively enhance soil microbial functionality and plant resilience. By modulating microbial diversity and enriching beneficial taxa, these strains improved nutrient cycling, soil quality, and plant stress tolerance (Figure 7). The findings suggest that the observed improvements in crop growth result from the interplay of enhanced microbial activities and plant molecular responses. These results provide valuable insights into the potential of mangrove-derived PGPR strains as targeted bioinoculants for sustainable agriculture. Acknowledgements This work was supported by the NSFC [grant number 41976126]; Projects of Shenzhen Science and Technology Innovation Committee [grant numbers JCYJ20230807111759016, ZDSYS20230626091459009, and KCXFZ20230731093402005]; and the Innovation Team Project for Guangdong’s Universities [grant number 2023KCXTD052]. CRediT authorship contribution statement SH and YG: Conceptualization, experiments, bioinformatics analysis, writing-original draft. CY, XW, and JZ data curation and methodology. JZ and ZC: experimental design and methodology. SF: funding. SF, XW, and JZ: resource, review, editing, and funding. Data availability statement Data will be made available on request. Conflicts of Interest The authors have nothing to declare. References Abbasi, S., Kafi, S. A., Alipour, S., 2022. Streptomyces consortium improved quality attributes of bell pepper fruits, induced plant defense priming, and changed microbial communities of rhizosphere under commercial greenhouse conditions. 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Potential PGPR strains from mangrove root microorganisms. Note: Different numbers of plus signs (+) indicate strength and ND indicate that the data were not determined. Table 2. IAA production of each strain and composite strains. IAA mg/L ND 92.4 99.2 149.4 13.8 ND 26.6 ND 106.4 158.1 232 93.7 Note: ND indicate that the data were not determined. Supplementary Material File (figs1.pdf) Download 28.53 MB File (ga(1).pdf) Download 12.35 MB Information & Authors Information Version history V1 Version 1 03 March 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords composite plant growth-promoting rhizobacteria (pgpr) growth microbial functions plant growth promotion proteome rhizospheric profiles Authors Affiliations Shuo Han Tsinghua University View all articles by this author Yuchun Gou Tsinghua University View all articles by this author Chunzhe Yang Tsinghua University View all articles by this author Xiangru Wang Tsinghua University View all articles by this author Xiangqian Wang Tsinghua University View all articles by this author Jianming Zhu Tsinghua University View all articles by this author Zhong Hua Cai 0000-0002-9058-1608 Tsinghua University View all articles by this author Shuying Feng Henan University of Chinese Medicine View all articles by this author Jin Zhou 0000-0003-0372-2554 [email protected] Tsinghua University View all articles by this author Metrics & Citations Metrics Article Usage 267 views 109 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shuo Han, Yuchun Gou, Chunzhe Yang, et al. 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