- Original Article
- Published:
Transcriptomic profiling of maize (Zea mays L.) seedlings in response to Pseudomonas putida stain FBKV2 inoculation under drought stress
Annals of Microbiology volume 68, pages 331–349 (2018)
Abstract
Several mechanisms have been proposed for plant growth-promoting rhizobacteria (PGPR)-mediated drought stress tolerance in plants, but little is known about the molecular pathways involved in the drought tolerance promoted by PGPR. We, therefore, aim to study the differential gene response between Pseudomonas putida strain FBKV2 and maize interaction under drought stress using Illumina sequencing. RNA Seq libraries were generated from leaf tissue of maize seedlings with and without strain FBKV2 subjected to drought stress. The libraries were mapped with maize genome database for the identification of differentially expressed genes (DEGs). The expression studies confirmed the downregulation of ethylene biosynthesis (ET), abscisic acid (ABA) and auxin signaling, superoxide dismutase, catalase, and peroxidase in FBKV2-inoculated seedlings. On the other hand, genes involved in β-alanine and choline biosynthesis, heat shock proteins, and late embryogenesis abundant (LEA) proteins were upregulated, which could act as key elements in the drought tolerance conferred by P. putida strain FBKV2. Another remarkable expression was observed in genes encoding benzoxazinoid (BX) biosynthesis which act as the chemoattractant, which was further confirmed by gfp-labeled P. putida strain FBKV2 root colonization studies. Overall, these results indicate that secretion of BXs attracted P. putida strain FBKV2 resulted in root colonization and mediated drought tolerance by modulating metabolic, signaling, and stress-responsive genes.
Introduction
Plant growth-promoting rhizobacteria (PGPR) are the rhizosphere bacteria that can offer several benefits to host plants such as increasing the availability of nutrients, elimination of deleterious pathogens, and tolerance to abiotic stresses, including drought stress. Several mechanisms have been proposed for PGPR mediated drought stress tolerance in plants. Phytohormone producing Azospirillum sp. enhanced plant growth promotion and induced drought tolerance in wheat (Arzanesh et al. 2011). 2R, 3R-butanediol producing Pseudomonas chlororaphis O6 induced stomatal closure genes and reduced water loss in Arabidopsis thaliana (Cho et al. 2008). Similarly, 1-aminocyclopane-1-carboxylate (ACC)-deaminase producing Achromobacter piechaudii ARV8 lowered ethylene levels and induced drought tolerance in tomato and pepper seedlings exposed to drought stress (Mayak et al. 2004). PGPR P. putida strain GAP-P45 enhanced plant biomass, relative water content, and leaf water potential by the accumulation of proline in maize seedlings exposed to drought stress (Sandhya et al. 2010). Exopolysaccharides producing Rhizobium leguminosarum (LR-0), Mesorhizobium ciceri (CR-30 and CR-39), and Rhizobium phaseoli (MR-2) showed beneficial interaction to wheat (non-legume) under drought stress (Hussain et al. 2014). Furthermore, Bacillus sp. enhanced drought tolerance in maize seedlings by reducing the activity of the antioxidant enzymes such as ascorbate peroxidase (APX) and glutathione peroxidase (GPX) (Vardharajula et al. 2011).
Gene expression profiling has also been used to characterize a variety of genes expressed under drought stress in plants inoculated with PGPR (Kandasamy et al. 2009; Yuwono et al. 2005). Increased mRNA transcripts of a drought-response gene, EARLY RESPONSE TO DEHYDRATION 15 (ERD15) in Paenibacillus polymyxa B2-inoculated A. thaliana enhanced drought tolerance (Timmusk and Wagner 1999). Six differentially expressed stress proteins were identified in pepper plants inoculated with Bacillus licheniformis K11 under drought stress (Lim and Kim 2013). Stress-related genes APX1, SAMS1, and HSP17.8 were induced in wheat inoculated with Bacillus amyloliquefaciens 5113 and Azospirillum brasilense NO40 (Kasim et al. 2013) respectively. Jasmonic acid marker genes, salicylic acid-regulated genes, and the ethylene-response genes were upregulated in A. thaliana colonized with P. chlororaphis O6 (Cho et al. 2013). Recent studies with Illumina sequencing (HiSeq 2000 system) revealed abscisic acid (ABA) signaling genes in sugarcane cv. SP70-1143 inoculated with Gluconacetobacter diazotrophicus conferred drought tolerance (Vargas et al. 2014).
Our previous work has shown that the potential of P. putida strain FBKV2 and GAPP45 colonization-enhanced drought tolerance in maize by the accumulation of cellular metabolites reduced stomatal conductance and antioxidant enzymes (Vurukonda et al. 2016; Vardharajula et al. 2011; Sandhya et al. 2010). Till date, little is known about the molecular pathways involved in the drought tolerance promoted by PGPR that colonize the maize. We, therefore, hypothesized that rhizobacteria inoculation alter the expression of metabolic, signaling, and stress-protective genes in maize leaves. To address this hypothesis and to advance our understanding of maize response to drought stress with and without bacterial inoculation, we adopted RNA Seq transcriptome analysis using Illumina deep sequencing of maize seedlings.
Material and methods
Bacterial strain
In our previous studies, Pseudomonas spp. strain FBKV2, isolated from eggplant (Solanum melongeana L.) rhizosphere, showed multiple PGP traits under both non-stress and drought stress conditions and enhanced drought tolerance in maize. The strain was identified as Pseudomonas putida by 16SrRNA sequence analysis, and the sequence was submitted to GenBank under the accession number KT311002.1 (Vurukonda et al. 2016).
gfp labeling of P. putida strain FBKV2
The derivative of broad-host-range vector pBBR1MCS-5 (Stuurman et al. 2000), which contains the green fluorescent protein (gfp) gene under the control of lac promoter with gentamycin (Gm) resistance, was obtained from Plant Microbe Interaction lab, Centre for Cellular and Molecular Biology (CCMB), Telangana State, India. The wild-type P. putida strain FBKV2 was grown in 5 mL of Luria Bertani (LB) medium at 28 °C under continuous shaking (120 rpm) until the optical density of 0.6 at λ 600 nm. The cells were harvested by centrifugation at 5000 rpm from 5 min, re-suspended in 5 mL of cold sterile water, and centrifuged at 5000 rpm for 5 min. The supernatant was discarded, and the pellet was washed twice (5000 rpm for 5 min) with 10% glycerol. Transformation was performed by electroporation (Gene Pulser, Bio-Rad) in an electroporation cuvette (0.2 cm) containing 100 μL of competent cells (P. putida strain FBKV2), plus 2 μL plasmid DNA (100 ng μL−1). The following pulse conditions were applied: 12.5 kV cm−1, 25 μF, and 200 Ω (Krzyzanowska et al. 2012a, b; Choi et al. 2006). After transformation, 1 mL of LB medium was added; the mixture was incubated for 1 h at 28 °C and plated on to LB agar medium supplemented with gentamycin (40 μg mL−1) and incubated for 48 h at 28 °C. The identification and selection of clones carrying the gfp gene were carried out under UV light.
Plant material and growth conditions
A pot experiment was conducted under greenhouse condition to compare the efficacy of selected bacterial strain P. putida strain FBKV2 (gfp labeled) for promoting the growth of maize under drought stress conditions. The soil used for pot experiments belongs to the Chalkas series and has been classified as “red earths with loamy subsoil” in the Indian soil classification system, which falls under the order Alfisols (Bhattacharyya et al. 2007). The soil was collected from the homogeneous horizon (0–20 cm) of College Farm (Field soil), PJTSAU Campus, Rajendranagar, Hyderabad, India, a semiarid region under rain-fed production system. The soil was air-dried and sieved (<2 mm) before being analyzed for the physicochemical properties. The soil contained 73.2% sand, 5.6% silt, 21.2% clay with 1.43 Mg m−3 bulk density, 2.52 Mg m−3 particle density, 38.4% total porosity, and 39.2% water holding capacity; it had a pH of 6.8 with an electrical conductivity of 0.13 dms; the soil contain Ca2+, Mg2+, K+, and Na+ in the ratio of 3.2, 2.1, 0.31, and 0.25 cmol (p+)/kg with CEC of 16.2 cmol (p+)/kg. The organic C, total N, and total P content of soil were 0.89, 0.13, and 0.09 g/kg, respectively (Vurukonda et al. 2016).
Maize (Zea mays L. var. DHM 117) seeds were surface sterilized with 0.1% HgCl2 (30 s) followed by 70% ethanol for 1 min and six times of repeated washing with sterile water (1 min each time) (Vurukonda et al. 2016). The effectiveness of surface sterilization was checked by plating the seeds and aliquots of the final rinse onto nutrient agar (NA) plates. Seeds were considered positive for sowing when no colonies were observed on the NA plates after incubation for 48 h at 28 °C. Surface-sterilized seeds were treated with gfp-labeled P. putida strain FBKV2 suspension (109 cfu mL−1) for 30 min, and for control, seeds were incubated in sterile distilled water.
Four seeds were sown in pots (surface sterilized) with diameter 17 cm and height 15 cm containing 1.5 kg of sterile soil. Pots were arranged in the greenhouse using a completely randomized design with three replications of each treatment. After 1 week of germination, the seedlings were thinned to two per pot. Soil water content was 15.9% (− 0.3 MPa), determined according to Sandhya et al. (2009) by drying the initially saturated soil at different matric potentials by pressure plate apparatus (Model-1250, Santra Barbara, CA, USA). The soil water content of the pots was maintained at 100% water holding capacity (WHC) during the experiment by daily sprinkling with sterile distilled water, and the nutrients (Hoagland’s no. 2 basal salt mixture, Sigma-Aldrich) were supplied on a weekly basis. Drought stress was induced by discontinuing water after 14 days of planting. After stopping irrigation, plants were observed for signs of wilting and leaves were harvested for further studies (4 days of drought stress).
Leaf relative water content
For measuring relative water content (RWC), the leaves were cut into small discs of 1.5 cm2 and fresh weight (FW) was recorded immediately, followed by hydrating the sample overnight by immersing in water and turgid weight (TW) was recorded after blotting the leaf sample gently. The samples were dried at 70 °C until constant dry weight (DW) was observed. RWC was calculated according to the formula RWC (%) = [(FW − DW) / (TW − DW)] × 100 (Teulat et al. 2003).
Specimen preparation for microscopy
To validate the establishment and colonization of strain FBKV2 on the roots of maize seedlings, root colonization studies were performed using confocal laser scanning microscopy (CLSM). Maize root specimens were prepared by scratching a piece of the root surface, around 1 cm in length, from different parts of the root with a sterile blade. All specimens were softly rinsed with sterile distilled water prior to merging into phosphate-buffered saline (PBS) for microscopic observation.
Confocal laser scanning microscopy
CLSM was performed at Banaras Hindu University, Varanasi, India, with a Carl Zeiss Microscopy LSM780, Axio Imager 2 system. While GFP fluorescence was recorded by using an excitation laser of 488 nm (argon laser) and collecting the emission of 500–600 nm, an excitation with NeHe laser of 561 nm was used and the emission band of 538–624 nm was collected. Images were acquired and reconstructed by Zen 2012 Software.
RNA Seq
RNA isolation and library preparation
Drought stress was induced by discontinuing water after 14 days of planting. After stopping irrigation, plants were observed for signs of wilting. When shrinkage of leaves and stem was clearly visible, plants were harvested (4 days of drought stress) and the youngest leaf (fourth leaf) was collected in RNAlater (RNA stabilizing reagent, Qiagen) and used for Illumina deep sequencing. Two biological replicates were used for all RNA Seq experiments. The total RNA from the leaf tissues was extracted using Trizol reagent (Invitrogen) and purified using the RNeasy Plant Mini kit (Qiagen). The integrity and quality of the total RNA were checked by a NanoDrop, formaldehyde-agarose gel electrophoresis, and Agilent 2100. The poly(A) RNA was isolated from purified total RNA using Oligo (dT) beads. Following purification, the mRNA was fragmented randomly into small pieces using fragmentation buffer, and the cleaved RNA fragments were copied into the first-strand cDNA using reverse transcriptase and random hexamer primers. Second-strand cDNA synthesis was done using synthesis buffer (Illumina), dNTPs, DNA polymerase I and RNaseH, and the cDNA fragments were processed for end repair, an addition of a single “A” base, and ligation of the adapters. These products were then purified and enriched by PCR to create the final cDNA library (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®) and sequenced on the HiSeq 2500 sequencer according, to the manufacturer’s recommendations (Illumina). Paired end sequencing with 2 × 150 bp read length was performed with multiplexed sampling assay. All the RNA Seq experiments were performed at Nucleome Informatics Private Limited, Hyderabad, Telangana State, India.
Raw data filtering
DrSeq™ Software suite was used to execute the RNA Seq data analysis. The filtering process was done by Trimmomatic and includes (1) removal of reads containing adapters, (2) removal of reads containing N > 10% (N represents the base cannot be determined), and (3) removal of reads containing low quality (Q score ≤ 5) base which is over 50% of the total base. The detailed statistics for the quality of sequencing data of four libraries (presence of P. putida strain FBKV2 (DS + PP) and uninoculated control (DS) in two replicates each) is shown in Supplementary Table S1.
Mapping reads to a reference genome and transcript assembly
The filtered reads were mapped to the maize genome (http://ftp.ensemblgenomes.org/pub/plants/release-28/fasta/zea_mays/dna/Zea_mays.AGPv3.28.dna.toplevel.fa.gz) using Tophat version v2.0.12 (Kim et al. 2013), which allows the identification of novel exons and novel intergenic transcripts. The mismatch parameter is set to two, and other parameters are set to default. The overview of mapping status of four libraries is shown in Supplementary Table S2.
The Map files generated by Tophat version v2.0.12 were provided as an input file to the software Cufflinks version v2.1.1, which assembles the alignments in the Map file into transfrags. The Cufflinks parameters are set to default. The assembled transfrags are then compared to the reference transcripts to determine if they are sufficiently different to be considered novel.
Gene expression quantification
The gene expression levels were estimated by counting the reads that map to genes or exons. In order for the gene expression levels estimated from different genes and experiments to be comparable, the FPKM (Fragments Per Kilobase of transcript sequence per Million) was used. FPKM is the commonest method of estimating gene expression levels, which considers the effects of both sequencing depth and gene length on-counting of fragments (Trapnell et al. 2010). HTSeq version v0.6.1 (Anders et al. 2015) software was used to analyze the gene expression levels, using the union mode. An FPKM value of 1 was set as the threshold for determining whether the gene is expressed or not. The gene expression data generated by HTSeq was provided as an input file to study the differential gene expression (DEG) using DESeq R package (1.18.0) (Anders and Huber 2010) with padj < 0.05.
GO enrichment
Gene ontology (GO) classification was carried out using BLASTx results in GO Seq, Release 2.12 software for functional classification of GO terms (Young et al. 2010). GO terms were obtained from nr hits using GO Seq software with default parameters during mapping.
KEGG pathway enrichment analysis
KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis identifies significantly enriched metabolic pathways or signal transduction pathways associated with differentially expressed genes compared with the whole genome background. KOBAS v2.0 with corrected P value < 0.05 was used to study the KEGG pathway enrichment analysis.
MapMan analysis
The MapMan software (Usadel et al. 2009) was used to identify the metabolic pathways of interest annotated by groups of participatory entities (maize transcripts), where each entity within a given group is represented by a discrete signal visualized using intensity of the color (Thimm et al. 2004).
qRT-PCR analysis
Real-time PCR was carried out using a Bio-Rad CFX96 Touch Real-Time thermocycler. The comparative CT method of quantitation was performed using maize Actin 2 and actin-binding protein (Zmabp3) as reference genes. Total RNA was extracted from two biological replicates of each sample and converted to cDNA using oligo-dT and random primers mix using QuantiTect Reverse Transcriptase (Qiagen). Three technical replicates of each biological replicates were used for qRT-PCR using QuantiFast SYBR Green PCR Kit (Qiagen). Experiments were performed on five to nine log dilutions of each of the target genes together with the reference genes for equal amplification efficiencies. To each well, 2.5 μL of first strand cDNA, 12.5 μL of SYBR Green solution, 2.5 μL of the forward primer (10 μM), and 2.5 μL of reverse primer (10 μM) were added, along with 5.0 μL of sterile, ultrapure water to bring the final volume to 25 μL in each well. Negative control reactions using untranscribed RNA were also run to confirm the absence of genomic DNA. To determine relative fold differences for each sample in each experiment, the CT value for each gene was normalized to the CT value for the reference gene and was calculated relative to a calibrator using the ΔΔCT method as described (Livak and Schmittgen 2001). Primers used for qRT-PCR were designed with Integrated DNA technologies (https://eu.idtdna.com/site/order/qpcr/predesignedassay). The gene sequence used for primer design of each transcript was aligned to the NCBI maize database, and the transcripts with specific regions were selected for designing primers (Table S3).
Data availability
The sequence data (raw data) generated in this study have been deposited at NCBI Sequence Read Archive (SRA) database (www.ncbi.nlm.nih.gov/sra). The Bioproject accession number for maize seedlings transcriptome is PRJNA362689, and SRA experimental accession numbers are SRX2510677 and SRX2510673.
Results
Evaluation of P. putida stain FBKV2 for growth promotion of maize seedlings under drought stress
In order to investigate the beneficial effects of P. putida strain FBKV2 to maize plants, the length and fresh weight of roots and shoots were evaluated 1 day before drought stress. Leaf samples were labeled as drought stress inoculated with P. putida strain FBKV2 (DS + PP) and uninoculated control under drought stress (DS). Bacterial inoculation significantly enhanced the seedling growth compared to non-inoculated plants (root, shoot, and biomass data not shown). As physiological modifications are the first responses of plants to overcome water deficit, phenotypic analysis of maize seedlings was monitored under drought stress. As shown in Fig. 1a, seedlings started showing visible signals of stress, such as rolling and wilting of leaves, after 4 days of drought stress. The visible signals of stress were lower in inoculated seedlings compared to uninoculated treatment. Furthermore, survival rate rapidly decreased in uninoculated treatment and seedlings died completely at the end of sixth day. However, seedlings inoculated with P. putida strain FBKV2 survived up to 10 days after exposure to drought stress and started wilting thereafter. These results suggest that inoculation with P. putida strain FBKV2 enhanced drought tolerance in maize seedlings compared to non-inoculated seedlings. The positive influence of microbial inoculations was also observed on leaf relative water content (RWC) content. Inoculation with P. putida strain FBKV-2 significantly increased the RWC (71.25 ± 1.05) over uninoculated control (66.05 ± 1.28).
Root colonization of maize seedlings by strain FBKV2
To visualize the root colonization of maize seedlings by gfp-labeled P. putida strain FBKV-2 under drought stress, root specimens were viewed by CLSM. As shown in Fig. 1b, the fluorescent rod-shaped structures of strain FBKV2 indicated the presence of high numbers of bacterial cells compared to uninoculated seedlings (Fig. 1c), validating the successful root colonization by P. putida strain FBKV2.
RNA Seq analysis of maize seedlings
Between 39 and 52 million reads from each sample were generated using Illumina HiSeq 2500 technology. The high-quality clean reads (uniquely mapped reads; Table S1) were used for mapping to the reference genome and the gene expression level was estimated by FPKM. Figure 2a, showing the violin plot of the log FPKM values, suggests that the quartile and the median values among the samples being compared for differential gene expression are almost identical with a slightly higher density in P. putida strain FBKV2-inoculated treatment. Similarly, the magnitude of variability among the biological replicates was also determined using Pearson correlation coefficient method. The closer the correlation coefficient is to 1, the greater the similarity of the samples. In the present RNA Seq analysis, the R2 is 0.918 (Fig. 2b), indicating slight variability among the biological replicates.
In the present study, cluster analysis was also performed to identify the genes with similar expression patterns under various experimental conditions. From the cluster analysis (Fig. 2c), it is evident that the maize seedlings inoculated with P. putida strain FBKV2 showed differential expression pattern than the uninoculated seedlings under drought stress. A total of 3738 DEGs were obtained. Among them, 1163 genes showed increased transcript abundance and 2575 genes showed decreased transcript abundance in P. putida strain FBKV2-inoculated seedlings (DS + PP) (Fig. 3a).
A comparable gene expression was seen between inoculated and uninoculated treatments in gene ontology (GO) terms related to plant growth and development. GO categories corresponding to various aspects of the biological process such as response to abiotic stress, water stress, water deprivation, abscisic acid, response to hormones, and biological activity were enriched in P. putida strain FBKV2-inoculated seedlings (DS + PP) compared to uninoculated (DS) control (Fig. 3b). Similarly, GO categories related to molecular function (catalytic, glutathione transferase, oxidoreductase activity) and cellular components (cell wall, cell periphery, non-membrane bound organelle, and ribonucleoproteins) were significantly enriched with increased transcript abundance in DS + PP treatment compared to DS (Fig. 3b). Furthermore, MapMan software tool was employed to analyze the statistically significant drought-mediated gene expression data for P. putida strain FBKV2-inoculated seedlings (Fig. 4) for known metabolic pathways, biological processes, and functional categories. Few of the GO categories were selected for detailed examination.
Differential expression of carbohydrate metabolism-associated genes
In the present study, carbohydrate metabolic genes showed altered expression pattern in maize seedlings inoculated with P. putida strain FBKV2 under drought stress. Genes involved in starch synthesis namely plastid ADP-glucose pyrophosphorylase large subunit (GRMZM2G391936), the first step in starch biosynthesis, showed decrease gene expression. Similarly, granule-bound starch synthase-1 (GRMZM2G024993) responsible for amylose synthesis also showed decreased transcript abundance, whereas starch branching enzyme-III (GRMZM2G005298) responsible for amylopectin synthesis showed increased transcript abundance. On the other hand, a large number of genes involved in the starch break down such as β-amylase isoforms (GRMZM2G450125, GRMZM2G035749, GRMZM2G082034, GRMZM2G007939, and GRMZM2G025833) showed decreased transcript abundance, whereas α-amylase (GRMZM2G138468) showed increased gene expression (Table 1).
In sucrose metabolism, genes encoding sucrose synthase 7 (GRMZM2G410704) and sucrose phosphate phosphatase (GRMZM2G097641) showed increased transcript abundance (Table 1). These data suggest that starch biosynthesis and degradation have been suppressed and sucrose accumulation has been enhanced, and at the same time, its degradation into UDP-glucose and fructose serves as a starting point for glycolysis and synthesis of cellulose.
Differential expression of membrane transporters
The dynamic GO enrichment results revealed that a large number of genes belonging to the “membrane transporters” family significantly downregulated in response to P. putida strain FBKV2 inoculation under drought stress (Table 2). The most important functional group of this category is the aquaporin genes. Out of nine genes, six genes annotated as plasma membrane intrinsic proteins (PIPs), one as tonoplast intrinsic proteins (TIPs) and two genes correspond to NOD26-like intrinsic proteins (NIPs). Among the nine aquaporins, gene-encoding PIP2–4 (GRMZM2G154628) of PIP family showed increased transcript abundance, whereas the remaining eight aquaporins showed decreased transcript abundance (Table 2). This result suggested that strain FBKV2 inoculation downregulated aquaporins to minimize the water flow through cell membranes and increase the leaf turgor, which might have enhanced the cell membrane stability with higher relative water content (71.25 ± 1.05) in the seedlings compared to uninoculated control (66.05 ± 1.28).
Differential expression of abiotic stress-responsive genes
The maize expression data annotated by GO category “response to abiotic stimulus” were significantly affected by P. putida strain FBKV2 inoculation. Among the reactive oxygen species (ROS) scavenger genes, three genes encoding glutathione S-transferase (GRMZM2G077206, GRMZM2G096269, GRMZM2G363540), and gene-encoding dehydroascorbate reductase—DHAR4 (GRMZM2G005710)—showed increased transcript abundance; three superoxide dismutase CU-ZN family isomers (GRMZM2G169890, GRMZM2G025992, GRMZM2G058522), catalase isoenzyme B (GRMZM2G088212), and putative glutathione peroxidase (GRMZM2G144153) showed decreased transcript abundance (Table 3). Another important functional group of GO category “response to abiotic stimulus” is heat shock proteins. In the present study, genes encoding chaperonin cpn60 (GRMZM2G042253, GRMZM2G074790), HSP40 (GRMZM2G049373), chaperonin cpn10 (GRMZM2G091189), HSP70 (GRMZM2G137696), and 18.8 kDa class V HSP (GRMZM2G429396) showed increased expression (Table 3). Furthermore, genes involved in β-alanine biosynthesis such as polyamine oxidase (GRMZM2G150248), along with genes associated with choline biosynthesis such as putative phospho ethanolamine N-methyltransferase (PEAMT) (GRMZM2G122296), showed increased transcript abundance (Table 3) in P. putida strain FBKV2-inoculated seedlings compared to uninoculated.
Overall, these data suggest that genes encoding chaperonins, β-alanine, choline biosynthesis, and few ROS genes were more induced by strain FBKV2 inoculation and might have played important roles in seedling tolerance to drought stress.
Differential expression of hormonal metabolism-associated genes
In the present study, differential expression pattern of genes for biosynthesis, signaling, and response to hormones was analyzed. A large number of genes involved in the biosynthesis and signaling of ABA in maize seedlings colonized by P. putida strain FBKV2 were mainly repressed. This repression was indicated by the downregulation of pyrabactin resistance-like protein 3 (GRMZM2G154987), bzip transcription factor ABI5 (GRMZM2G479760), and protein phosphatase family proteins (GRMZM2G134628, GRMZM2G059453, GRMZM5G818101, GRMZM2G001243, GRMZM2G300125, GRMZM2G159811, GRMZM2G082487, and GRMZM2G177386), whereas three genes encoding serine/threonine protein kinase (GRMZM2G147051, GRMZM2G147857 and GRMZM2G084806) showed upregulation (Table 4).
A remarkable effect was observed in the genes associated with ethylene (ET) biosynthesis and signaling. Two genes encoding 1-aminocyclopropane-1-carboxylate oxidase 1 (GRMZM2G052422) and 1-aminocyclopropane-1-carboxylate synthase 2 (GRMZM2G164405) and two genes encoding ethylene-responsive transcription factors (GRMZM2G171569, GRMZM2G131281) showed decreased transcript abundance in seedling colonized with P. putida strain FBKV2 (Table 4). The data suggest that genes encoding ABA and ET biosynthesis, signaling, and response are mostly downregulated in inoculated seedlings indicating a low level of drought stress.
The GO enrichment results revealed that genes involved in auxin signaling (AUX/IAA7) (GRMZM2G121309) and responses GH3.8 (GRMZM2G053338), GH3 (GRMZM2G378106), and SAUR56 (GRMZM2G113135) showed decreased transcript abundance (Table 4) in P. putida strain FBKV2-inoculated seedlings, whereas five genes encoding late embryogenesis-abundant (LEA) (GRMZM2G014419, GRMZM2G017991, GRMZM2G087094, GRMZM2G331027, and GRMZM2G358540) proteins showed increased transcript abundance. The results suggest that the downregulation of auxins and upregulation of LEA proteins in FBKV2-inoculated maize seedlings could contribute to a higher level of drought tolerance.
Microbial signaling-associated genes
Two genes encoding indolin-2-one monooxygenase (CYP71C2) and indole-2-monooxygenase (CYP71C4) (GRMZM2G167549, GRMZM2G085661) showed increased transcript abundance in maize seedlings inoculated with strain FBKV2. CYP71C2 and CYP71C4 are involved in the biosynthesis of plant heteroaromatic metabolite benzoxazinoids (BXs) (Dutartre et al. 2012) which recruit P. putida (Neal et al. 2012).
Quantitative real-time-PCR validation of differentially expressed genes from RNA Seq
To investigate the accuracy of Illumina RNA Seq, 18 DEGs from each GO category were randomly selected from RNA Seq data and were analyzed by quantitative real time PCR (qRT-PCR). Correlation between RNA Seq and real-time PCR was evaluated using log2-fold change measurements. The results showed that the expression pattern of the 18 DEGs was significantly similar (R2 = 0.985) and confirms the accuracy of the Illumina RNA Seq (Fig. 5).
Discussion
Drought stress affects almost every developmental stage of maize plants. However, phenophases such as germination, seedlings, and flowering are the major traits for crop establishment and are more prone to drought stress (Delachiave and Pinho 2003; Ashagre et al. 2014; Khayatnezhad et al. 2010; Aslam et al. 2015). Although several reports have come on microorganism-mediated drought tolerance at seedling stages (Grover et al. 2013; Sandhya et al. 2009) and our previous studies demonstrated that inoculation of maize seedlings with P. putida strain FBKV2 improved plant growth and resilience to drought tolerance (Vurukonda et al. 2016), little is known at molecular level. The present study was aimed to determine the potential of P. putida strain FBKV2 in the regulation of maize genes under drought stress condition.
Growth promotional studies under drought stress
To understand the molecular responses of maize seedlings to drought stress, two different plant treatments were analyzed: (i) plants in association with P. putida strain FBKV2 and drought stress (DS + PP) and (ii) uninoculated plants and drought stress (DS). Our results clearly show that inoculation with P. putida strain FBKV2 significantly enhanced the root and shoot length, soil moisture, and relative water content of seedlings compared to uninoculated treatment. These results validate the findings of Vurukonda et al. (2016), Grover et al. (2013), and Sandhya et al. (2010), who observed that rhizobacteria inoculation enhanced the plant growth, soil moisture, and relative water content of maize and sorghum seedlings. Furthermore, seedlings inoculated with P. putida strain FBKV2 survived up to 10 days after exposure to drought stress, whereas uninoculated plants died completely at the end of the sixth day. These results indicate that maize seedlings colonized with P. putida strain FBKV2 become more tolerant to drought stress than uninoculated seedlings. Drought tolerance promoted by rhizobacterial inoculation was already reported. Inoculation of maize with PGPR P. putida GAP-P45 (Sandhya et al. 2010) and Azospirillum lipoferum (Bano et al. 2013) improved plant growth through the accumulation of free amino acids and soluble sugars compared to non-treated plants under drought stress. Inoculation with ACC deaminase producing Bacillus licheniformis K11 alleviated drought stress in pepper plants (Hui and Kim 2013). Similarly, soybean plants inoculated with rhizobacterium P. putida H-2–3 improved plant growth performance under drought stress conditions (Sang-Mo et al. 2014).
Root colonization studies
Root colonization is the primary requirement to ensure an intimate association with the plant and thus supports rhizobacterial efficacy against water stress (Marasco et al. 2012). To check the ability of P. putida strain FBKV2 to colonize the maize root system, root colonization studies were performed. The results showed that plants were highly colonized by gfp-labeled P. putida strain FBKV2 after 4 days of drought stress and induced resilience to drought tolerance. Colonization of P. putida and Bacillus amyloliquefaciens in chickpea rhizosphere has been visualized using gfp labeling, and the synergistic effect of both the strains mitigated drought stress in chickpea (Kumar et al. 2016). Sultana et al. (2016) demonstrated successful root colonization of sorghum with gfp-labeled P. putida and Azotobacter chroococcum which enhanced nutrient uptake in sorghum. Furthermore, Fan et al. (2012) demonstrated colonization capability of gfp-labeled B. amyloliquefaciens in Zea mays, Arabidopsis thaliana, and Lemna minor under desert ecosystem.
RNA Seq
In the present study, we employed Illumina HiSeq 2500 sequencing technology to profile the transcriptome changes in the leaf tissues of inoculated and uninoculated maize seedlings under drought stress. A total number of 3738 DEG were identified in the maize seedlings inoculated with P. putida strain FBKV2. Among 3738 DEG, 1163 genes showed increased transcript abundance and 2575 genes showed decreased transcript abundance.
Response to carbohydrate metabolism
Carbohydrate metabolism is the major metabolism, which provides the essential saccharides and energy that the plant needs. The changes in the expression of genes involved in carbohydrate metabolism may induce the regulation that plants undergo during drought stress (Min et al. 2016). In our study, genes encoding starch biosynthesis and degradation showed decreased transcript abundance in P. putida strain FBKV2-inoculated seedlings; however, α-amylase and starch branching enzyme-III showed increased transcript abundance. The breakdown of starch in plants is catalyzed by amylase and with the interactive action of debranching enzyme, the stored starch breakdown into small oligosaccharides which provides energy to the plants under drought stress (Kaplan and Guy 2004).
The increase in the expression levels of the two Suc synthases encoding enzymes in FBKV2-inoculated seedlings suggests the possibility of sucrose synthesis which may help in osmotic adjustment and maintain homeostasis, allowing the plant to perform its normal cellular functions under drought stress (Krasensky and Jonak 2012), and sucrose hydrolysis results in the accumulation of glucose and fructose which serves as a starting point for glycolysis and synthesis of other saccharides. Our results validate the findings of Gagné-Bourque et al. (2016), who demonstrated that inoculation of timothy grass with Bacillus subtilis strain B26 increased sucrose concentrations in shoots under both non-stress and drought-stress conditions. Furthermore, inoculation with symbiotic Neotyphodium coenophialum in tall fescue plants alleviated drought stress, due to rapid accumulation of compatible solutes such as sucrose, glucose, and fructose (Nagabhyru et al. 2013).
Response to membrane transporters
Aquaporins are membrane channel proteins which facilitate the transport of water across membranes in living organisms (Maurel et al. 2008). Few aquaporins can also facilitate the transport of other essential molecules such as CO2, silicon, boron, urea, or ammonia (Li et al. 2014). Plant aquaporins belong to the gene family of major intrinsic proteins (MIPs), which is subdivided into five subfamilies based on amino acid sequence similarity: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs), and uncharacterized intrinsic proteins (XIPs), each group being also divided into several groups (Chaumont et al. 2001; Maurel et al. 2008; Gupta and Sankararamakrishnan 2009; Marulanda et al. 2010). Among the MIPs, PIPs are the major determinant in regulating water uptake by plants (Siefritz et al. 2002; Javot et al. 2003; Postaire et al. 2010). In the last few years, much effort has been concentrated on investigating the function and regulation of aquaporins upon inoculation with bacteria and fungi. Porcel et al. (2006) found a reduction in the expression of a plasma membrane aquaporin gene in the roots of soybean plants inoculated with the nitrogen-fixing bacteria Bradyrhizobium japonicum under both well water and water-deprived conditions. In our study, P. putida strain FBKV2 inoculation downregulated the expression of gene-encoding PIP1; 3 and the results are in agreement with recent findings by Quiroga et al. (2017) who demonstrated that inoculation of drought sensitive and tolerant cultivars of maize with Arbuscular mycorrhizal fungi (AMF) downregulated the expression of ZmPIP1; 3 gene. Similarly, Bárzana et al. (2014) reported that AMF inoculation downregulated ZmPIP1; 3 and upregulated the expression of ZmPIP2; 4 gene in maize leaf and the same gene was confirmed by Armada et al. (2015) in root tissue under drought stress. Furthermore, two genes encoding ZmPIP2; 2 and ZmPIP2; 4 were upregulated in AMF inoculated drought tolerant cultivars (Quiroga et al. 2017). In our study, gene-encoding PIP2; 4 showed increased transcript abundance, whereas PIP1; 5, PIP2; 1, PIP2; 2, and PIP2; 6 showed decreased transcript abundance. Arbuscular mycorrhizal symbiosis with maize plants has been shown to regulate aquaporin expression differentially in drought-tolerant and drought-sensitive maize cultivars especially ZmTIP1; 1, ZmTIP2; 3, and NIP2; 1 that showed downregulation in both drought tolerant and sensitive cultivars, whereas TIP4; 1 showed upregulation in both the cultivars under drought stress (Quiroga et al. 2017). In our study, NIP, NIP1; 1, and TIP4; 2 showed decrease transcript abundance.
These results suggest that inoculation with P. putida strain FBKV2 downregulated aquaporins which might be minimized water flow through cell membranes and uphold leaf turgor thereby helping the seedlings from being affected by drought stress compared to the uninoculated seedlings.
Response to abiotic stress
Exposure of plants to drought stress leads to the generation of reactive oxygen species (ROS), including superoxide anion radicals (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (OH), singlet oxygen (O12), and alkoxy radicals (RO). ROS react with proteins, lipids, and deoxyribonucleic acid causing oxidative damage and impede the normal functions of the plant (Vurukonda et al. 2016). In order to overcome the oxidative stress, plants develop antioxidant defense systems comprising both enzymatic and non-enzymatic antioxidants which prevents ROS accumulation and alleviate the oxidative damage (Miller et al. 2010; Vurukonda et al. 2016). Enzymatic antioxidants are ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), and peroxiredoxin (PrxR). Non-enzymatic components contain ascorbate and glutathione.
In the present study, the activity of enzymatic antioxidant SOD, CAT, and GPX in P. putida strain FBKV2-inoculated seedlings was significantly reduced as compared to uninoculated seedlings growing under drought stress. Our results are consistent with those reported by Naseem and Bano (2014), Vardharajula et al. (2011), and Sandhya et al. (2010) for maize where exopolysaccharide producing Proteus penneri (Pp1), Pseudomonas aeruginosa (Pa2), and Alcaligenes faecalis (AF3), as well as Bacillus and Pseudomonas species inoculation reduced the CAT and GPX activity under drought stress. Recently, Bulegon et al. (2016) and Saeed et al. (2016) reported that inoculation of Urochloa ruziziensis and Brassica napus with Azospirillum species decreased SOD activity under drought stress.
Glutathione S-transferases (GST) are important phase II, GSH-dependent ROS-scavenging enzymes found in the plants. GST catalyzes the conjugation of GSH to electrophilic sites on a wide range of phytotoxic substrates (Labudda and Safiul Azam 2014). Inoculation of strawberries with P. polymyxa RC05, P. fluorescens RC77, P. fluorescens RC86, P. putida RC06, P. putida 29/2, and Rhodococcus erythropolis RC9 enhanced the activity of GST compared to uninoculated control under drought stress. Similarly, under non-stress condition, Arabidopsis thaliana inoculated with Piriformospora indica upregulated two proteins with homology to GST (Pesškan Berghӧfer et al. 2004). In our study, gene-encoding glutathione S-transferases—12, 26, and 27—showed increased transcript abundance in P. putida strain FBKV2-inoculated seedlings compared to uninoculated treatment.
Dehydroascorbate reductase (DHAR) catalyzes the reduction of dehydroascorbate (DHA) to ascorbic acid (Asc) and provides protection against oxidative damage in plants (Chen and Gallie, 2006). Recently, Kim et al. (2014) reported that overexpression of a rice cytosolic DHAR gene conferred enhanced salt stress tolerance to rice plants by maintaining the Asc pool (Kim et al. 2014). Similarly, transgenic tobacco overexpressing Arabidopsis cytosolic DHAR showed enhanced tolerance to ozone and drought stresses (Eltayeb et al. 2006). Our results are in agreement with the findings of Hasna et al. (2016) who reported that PGPR inoculated okra plants showed higher expression of DHAR than uninoculated control plants and confer salinity tolerance in okra. In our study, gene-encoding DHAR4 showed increased transcript abundance in maize seedlings inoculated with strain FBKV2.
Plants possess a number of adaptive mechanisms to deal with drought stress to maintain tissue turgor pressure (Ashraf 2010; Gou et al. 2015). One of the most efficient mechanisms is osmotic regulation through the accumulation of water-soluble compounds known as “compatible solutes” that enable plants to cope with drought stress (Ashraf and Foolad 2007; Ashraf 2010; Gou et al. 2015). Among the compatible solutes, glycine betaine (GlyBet) and choline (Cho) are the important osmoprotectants that confer tolerance to abiotic stresses in plants (Rhodes and Hanson 1993; Gorham, 1995; Sakamoto and Murata 2000; Zhang et al. 2010). Choline plays a critical role in plant stress resistance, mainly for enhancing GlyBet synthesis and accumulation (Zeisel 2006; Zhang et al. 2010). Plant Cho is synthesized in the cytosol via stepwise methylation of ethanolamine derivatives (Zeisel and Blusztajn 1994). The cytosolic enzyme phosphoethanolamine N-methyltransferase (PEAMT) catalyzes the methylation steps in the Cho biosynthetic pathway (Nuccio et al. 1998, 2000; Zhang et al. 2010). Evident reports on the induced role of Bacillus subtilis GB03 in Arabidopsis showed obvious enhancements in biosynthesis and accumulation of choline as a precursor in GB metabolism (Zhang et al. 2010). The relative expression of PEAMT gene induced in Arabidopsis by Bacillus subtilis GB03 was almost 3-fold as compared with uninoculated plants under osmotic stress, resulting in an elevated metabolic level of choline together with GB in osmotically stressed plants (Zhang et al. 2010). Furthermore, inoculation of maize with PGPR strains Klebsiella variicola F2, P. fluorescens YX2, and Raoultella planticola YL2 induced higher accumulation of choline and GB than those plants without inoculation under different degrees of drought stress (Gou et al. 2015). In our RNA Seq experiment, gene-encoding putative phosphoethanolamine N-methyltransferase (PEAMT) was upregulated in P. putida strain FBKV2-inoculated seedlings compared to uninoculated treatment. These observations, in the light of available literature, suggest the induced role of P. putida strain FBKV2 in conferring drought tolerance by expressing PEAMT in maize seedlings.
Drought stress induce the accumulation of osmolytes such as polyamines (Zhou et al. 2017; Sperdouli and Moustakas 2012; Groppa and Benavides 2008). Polyamines (PAs), widely present in living organisms, are now regarded as a new class of growth substances which includes spermidine (Spd, a triamine), spermine (Spm, a tetramine), and their precursor putrescine (Put, a diamine) which play an important role in the regulation of plant developmental and physiological processes (Kusano et al. 2007; Gill and Tuteja 2010). Furthermore, accumulation of cellular PAs significantly enhances the tolerance of plants to various abiotic stresses due to the upregulation of stress-related genes (Zhou et al. 2017, 2015; Wen et al. 2008). In our study, gene-encoding polyamine oxidase responsible for the conversion of spermine to spermidine showed increased transcript abundance in P. putida strain FBKV2-inoculated seedlings. Zhou et al. (2017) investigated the effects of spermidine producing Bacillus megaterium BOFC15 on Arabidopsis plants under drought stress. Inoculation with BOFC15 enhanced primary and lateral roots than the control plants. Furthermore, the cellular Spd and Spm contents were higher in the BOFC15-inoculated plants and displayed stronger ability to tolerate drought stress than control plants. Our data suggest that P. putida strain FBKV2 inoculation upregulated the expression of polyamine oxidase that resulted in the accumulation of spermidine, conferring drought tolerance in maize seedlings.
Protein dysfunction is a common consequence of abiotic stresses. Plants respond to abiotic stresses, through a number of mechanisms to maintain their proteins in a functional form. Several proteins such as heat shock proteins (HSPs) are synthesized by the plants, and these proteins are responsible for protein folding, localization, accumulation, and degradation and thus are believed to play an important role in cellular processes and impart tolerance to abiotic stresses (Feder and Hofmann 1999; Wang et al. 2004; Wahid et al. 2007). In the present study, six HSPs (Cpn60, Cpn10, Hsp40, Hsp70, and Hsp18.8 class V) showed increased transcript abundance in FBKV2-inoculated seedlings compared to uninoculated control. These results validate the findings of Lim and Kim (2013), who observed a higher level of expression of sHSP in pepper plants inoculated with Bacillus licheniformis K11 under drought stress. Similarly, priming of wheat seedlings with A. brasilense NO40 significantly alleviated the deleterious effect of drought stress by upregulating the gene-encoding Hsp17.8 (Kasim et al. 2013).
Response to hormonal signaling
Abscisic acid (ABA) is the best-known plant hormone in conferring tolerance to abiotic stresses including drought and salinity (Wasilewska et al. 2008; Llanes et al. 2016). Three protein classes such as pyrabactin resistance/pyrabactin resistance-like/regulatory component of ABA receptor (PYR/PYL/RCARs) proposed to be the ABA receptors, protein phosphatase 2Cs (PP2Cs) which act as negative regulators, and SNF1-related protein kinase 2 s (SnRKs) which are positive regulators (Mustilli et al. 2002; Park et al. 2009; Schweighofer et al. 2004; Umezawa et al. 2009; Yoshida et al. 2006) are involved in ABA signal transduction pathways. In the present study, strain FBKV2 inoculation decreased transcript abundance of major key regulators such as PYL3 and PP2C isoforms such as PP2C, 2C 50, 2C ABI1, 37, and 68. Our results are inconsistent with that of Vargas et al. (2014) who reported the downregulation of PYL8, PP2C, and SnRK2 in sugarcane inoculated with Gluconacetobacter diazotrophicus PAL5, whereas in our study, FBKV2 inoculation enhanced SnRK2 family proteins such as plant-specific Ser/Thr kinases. In the presence of ABA, the PYR/PYL/RCAR forms a complex with PP2C, allowing the activation of SnRKs which target ion channels, membrane proteins, and transcription factors and facilitate transcription of ABA-responsive genes, thereby conferring drought tolerance.
In the biosynthetic pathway of ethylene, S-adenosylmethionine (S-AdoMet) is converted by 1-aminocyclopropane-1-carboxylate synthase (ACS) to 1-aminocyclopropane-1-carboxylate (ACC), the immediate precursor of ethylene (Grover et al. 2011). Under both biotic and abiotic stress conditions, the plant hormone ethylene endogenously regulates homeostasis of plants resulting in reduced growth. Plant ACC is sequestered and degraded by ACC deaminase producing bacteria to supply nitrogen and energy (Vurukonda et al. 2016). Furthermore, by removing ACC, the bacteria reduce the deleterious effect of ethylene, ameliorating plant stress and promoting plant growth (Glick 2005). Several reports showed the role of ACC deaminase producing bacteria in mitigating drought stress in crops (Mayak et al. 2004; Dodd et al. 2005; Zahir et al. 2008; Shakir et al. 2012; Sharma et al. 2013; Hui and Kim 2013). In the present study, ACC deaminase producing P. putida strain FBKV2 inoculation lowers the expression of ethylene biosynthesis genes ACO35 and ACS47. Our results are in agreement with Vargas et al. (2014) who reported that inoculation of sugarcane with G. diazotrophicus lowers the expression of ACO and ACS6 genes.
Auxin plays an important role in all the aspects of plant growth and development. Among the most commonly studied auxin signaling gene families are SAURs, GH3s, and Aux/IAAs. Auxin has already been reported as a negative regulator of drought tolerance. In wheat, drought stress tolerance was accompanied by a decrease in IAA content (Xie et al. 2003; Vargas et al. 2014). In the present study, FBKV2 inoculation decreased transcript abundance of genes encoding GH3, GH3.8, SAUR56, and AUX/IAA in maize seedlings under drought stress. Similar results were reported by Vargas et al. (2014) in sugarcane roots inoculated with G. diazotrophicus PAL5 under drought stress.
Downregulation of IAA was found to facilitate the accumulation of late embryogenesis-abundant (LEA) mRNA, leading to drought stress adaptation in plants (Zhang et al. 2009; Vargas et al. 2014). In our studies, FBKV2 inoculation increased the transcript abundance of genes encoding LEA proteins. The results suggest that the downregulation of auxin signaling and response pathway and accumulation of LEA proteins could contribute to drought tolerance in maize seedlings inoculated P. putida strain FBKV2.
Plant-microbe signaling-associated genes
Benzoxazinoids (BXs), such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are heteroaromatic metabolites with benzoic acid moieties which are produced during relatively early, vulnerable plant growth stages (Neal et al. 2012). Exposure of P. putida KT2440 to DIMBOA induced bacterial genes with putative functions in chemotactic responses; furthermore, in vitro chemotaxis assays also proved that P. putida KT2440 displayed taxis towards DIMBOA (Neal et al. 2012). In our study, two genes encoding CYP71C2 and CYP71C4 showed increased transcript abundance in maize seedlings inoculated with strain FBKV2. The upregulation of CYP71C2 and CYP71C4 may enhance the biosynthesis of BXs and attracted P. putida strain FBKV2 and resulted in root colonization there by imparting drought tolerance in maize seedlings.
Conclusions
Summarizing our data, the major outcome of this study is that the P. putida strain FBKV2 inoculation triggered drought tolerance in early stages of maize seedlings. By studying the leaf transcriptome using RNA Seq technology, we could provide data on transcripts for the major genes involved in carbohydrate, membrane transporters, detoxification, and hormonal signaling and discussed their potential role in maize drought tolerance. The data from our study can be utilized to better understand the gene expression networks involved in the other plant-microbe interaction under drought stress and could provide tools to maximize the benefits for crop production.
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The authors wish to acknowledge funding from Science and Engineering Research Board (SERB), Govt. of India for providing the financial assistance in the form of “Start-Up Research Grant (Young-Scientist)—SB/YS/LS—12/2014.”
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SkZ and S.V designed the experiments. SkZ and S.S.K.P.V optimized and performed the experiments. SkZ performed the data analysis. SkZ and S.V wrote the manuscript.
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SkZ, A., Vardharajula, S. & Vurukonda, S.S.K.P. Transcriptomic profiling of maize (Zea mays L.) seedlings in response to Pseudomonas putida stain FBKV2 inoculation under drought stress. Ann Microbiol 68, 331–349 (2018). https://doi.org/10.1007/s13213-018-1341-3
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DOI: https://doi.org/10.1007/s13213-018-1341-3