2.1 Plant material, stress treatment and library construction
The tolerant desi chickpea cultivar BG-362 was selected for the experiment. The seeds were initially washed with RO water and further with 0.1% (w/v) mercuric chloride solution for surface sterilization and after washing three to four times with sterile Milli-Q water, the seeds were washed with 70% ethanol and repeatedly washed with sterile Milli-Q and soaked overnight. The overnight water imbibed seeds were placed in Petridishes on wet autoclaved Whatman No. 1 filter paper and incubated in growth chamber for 3-4 days. The germinated seeds were transferred to Hewitt media containing hydroponic trays under standard growth conditions for two weeks. After two weeks the experiment was designed for library construction involving 1% RA suspension inoculation in two trays for 24h and the rest two trays were kept uninoculated. The bacterial culture of RA available in our institute was grown with conditions at 250 rpm and 28°C. After 24h, the uninoculated and inoculated seedlings were subjected to drought stress using 20% (w/v) polyethylene glycol (PEG) 6000. The untreated non-stressed condition was maintained for control. The treated and untreated plants were harvested at 0, 72 and 168h time intervals. The time intervals for physiological and biochemical studies involve 0, 24, 72 and 168h. Hence, four set of plants included control, drought, drought+RA and RA. All the tissues were harvested in three independent biological replicates and were immediately in liquid nitrogen and preserved carefully after labelling in −80°C for further experimental analysis.
2.2 Morphological analysis
All four set of plants were evaluated for various morphological parameters in terms of drought stress response evaluation. The number of biological replicates of plants used for experimental analysis at each time interval was five. The samples were wiped carefully after removing from hydroponics medium and different parameters were recorded thereafter. After the fresh weight analysis, the samples were kept in blotting sheets and were dried in hot air oven at 60°C for 4 days and then dry weight was recorded. The morphological parameters were studied and noted as described elsewhere (Tiwari et al. 2016).
2.3 Electrolyte Leakage (EL)
The electrolyte leakage (EL) of treated and untreated roots of chickpea plants was measured according to method explained by Lata et al (2011) and Tiwari et al (2016) with minor modifications. About 100 mg of fresh root samples were taken from all sets and further incubated in 20 ml sterile deionized Milli-Q water in 50 ml sterile Falcon tubes for 40 mins at 120rpm at room temperature. After 1h, the initial electrolyte conductivity (E1) was measured using Orion 5star conductivity meter (Thermo Scientific, USA). The tubes were further kept in boiling water with temperature 90°C in a water bath for 40 min and after cooling for some time, the final electrolyte conductivity (E2) was recorded. EL was calculated as by the formula mentioned in previous reports (Tiwari et al. 2016).
2.4 Relative water content (RWC)
For the assessment of relative water content in both control and treated samples, the leaf samples were taken in triplicate (Lata et al. 2011; Tiwari et al. 2016). The leaf samples of similar size were collected and their fresh weight (FW) was recorded immediately. Afterwards, these samples were soaked in 30 ml sterile Milli-Q water for 4h in Petridish after which their turgid weight (TW) was recorded. These samples were then kept for drying in hot air oven at 60°C for 48h after which their dry weight (DW) was recorded. The readings were then subjected to calculation of RWC using formula: RWC% = (FW-DW)/(TW-DW)*100 (Barrs and Weatherley 1962).
2.5 Lipid peroxidation assay
The modified protocol of Heath and Packer (1968) was used for estimation of lipid peroxidation. The aldehyde product, malondialdehyde (MDA) was measured using 2-thiobarbituric acid (TBA) reaction. 0.1% (w/v) TCA solution was prepared from which 500 µl was used for homogenization of ~100 mg leaf tissue samples from all sample sets in triplicates. The mixture was centrifuged at 13,000 g at 4°C for 10 min. The 500 µl supernatant was mixed with 1.5 ml of 0.5% TBA and incubated for 25 min at 95°C. The reaction was inhibited after 5 min incubation on ice and the absorbance was measured at 532 nm and 600 nm in a microplate reader.
2.6 Proline estimation assay
The amino acid proline estimation was measured using the protocol of Carillo and Gibbon (2011). 1ml of 70% ethanol was used to homogenize ~100 mg of leaves. 50 µl ethanolic extract was mixed with reaction mixture prepared by mixing 1% w/v ninhydrin in 60% (v/ v) acetic acid and 20% (v/v) ethanol. The reaction was incubated at 95°C for 20 min followed by 5 min on ice and then absorbance was recorded in microplate recorder at 520 nm.
2.7 Total RNA isolation for control and treated samples
Total RNA was isolated using mirPremier® microRNA Isolation Kit (Sigma-Aldrich, USA) according to the manufacturer's instructions. The root samples were taken for quantitative real-time PCR analysis of all four sample sets for miR166h and its target ATHB15expression analysis. For tissue-specific expression analysis, total RNA was isolated from different tissues as well as root, shoot and leaves with 15 days old seedlings harvested at time intervals 0 h, 1 h, 24 h and 72 h with different abiotic stress treatments like 20% polyethylene glycol (PEG) (6000), 100mM NaCl and 10µM ABA. Purity and quantity of isolated RNA samples was checked using Nano-Drop (Nanodrop 1000, Thermo Scientific, USA) and 1.2% formaldehyde-agarose gel electrophoresis. Turbo DNA-free™ kit (Ambion, Life Technology) was then used for DNase treatment of 5 µg RNA.
2.8 cDNA synthesis of miR166h and ATHB15
The purified RNA samples were used for cDNA preparation using Taqman® MicroRNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer's protocol. Stem-loop reverse transcription primer of miR166h and mature miR166h forward primer were designed according to the previous study (Jatanet al. 2019). Universal reverse primer was designed as described (Kramer et al. 2011). The target cDNA was prepared using Verso cDNA synthesis kit (Thermo Scientific, USA) according to the manufacturer’s protocol. The information regarding target gene prediction and its functions was gathered as per Jatan et al (2019).
2.9 Stem-loop quantitative real-time PCR (SL-qRT) for car-miR166 expression analysis
The SL-qRT-PCR analysis was performed to validate the expression levels of selected conserved car-miR166h in control and treated samples. Real-time PCR was done using Applied BiosystemsTM SYBRTM Green PCR Master mix on a 7500 fast real-time PCR machine (Applied Biosystems, USA). Real time primers were designed as described by Jatan et al (2019). U6 snRNA was used as an internal control gene for miR166h expression data normalization (Jain et al. 2014). The relative expression of the miR166h in different treated samples vs. control was calculated using 2−ΔΔCt method (Livak and Schmittgen 2001).
2.10 qRT-PCR expression analysis of predicted target ATHB15
To analyse the relative expression of respective target gene ATHB15in control and treated samples, chickpea glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as a reference gene for transcript normalization (Garg et al. 2010; Jatan et al. 2019). qRT-PCR analysis was performed on 7500 fast real-time PCR machine (Applied Biosystems, USA). The relative expression of the target genes was determined using 2−ΔΔCt method (Livak and Schmittgen 2001).
2.11 Phylogenetic analysis of target
The target was subjected to evolutionary phylogenetic analysis in different leguminous crops in comparison to chickpea using Molecular Evolutionary Genetics Analysis (MEGA X) (Kumar et al. 2018). The sequences were retrieved from NCBI using BLAST and were aligned further using CLUSTAL W and sequentially the data was analysed by neighbour-joining tree method using bootstrap test.
2.12 Phylogenetic analysis of pre-miR166h
Sequences of miR166 precursors (pre-miR166h) of different crops plants were collected from NCBI using BLAST application and then aligned using Clustal W. Subsequently, phylogenetic tree was constructed with the bootstrap value calculated with 1000 replicates using maximum likelihood method on MEGA-X.
2.13 Promoter analysis of mir166
For prediction of transcription start site (TSS) in miR166 gene, TSSP software from Softberry(http://linux1.softberry.com/berry.phtml?topic=tssp&group=programs&subgroup=promoter)was used.A1500 bps upstream of precursor sequence was selected for promoter analysis and the cis regulatory motifs were identified using Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) as described by Li et al (2017).
2.14 Detection of miR166 cleaved target ATHB15 mRNA by 5´RLM RACE
For the validation of predicted target of miR166h, modified 5´ RACE was performed. For this validation, the FirstChoice RLM-RACE Kit (Ambion, USA) was used with slight modifications. 1 µg of total RNA from RA inoculated drought treated root samples were isolated, checked for purity and quantity and further subjected to adapter ligation. This was followed by cDNA synthesis in which ligated product was used as a template. Specific gene reverse primers were designed for 5′ RLM-RACE and checked by OligoEvaluator™ (Sigma) (Supplementary Table 1). PCR reactions for cDNA amplifications using primer combinations with PCR cycling conditions set according to manufacturer’s instructions were performed. The annealing temperature was optimized and the single PCR fragments were cloned into the pGEM-T Easy Vector (Promega, USA) and sequenced to identify the 5´end of the amplified target gene.
2.15 Relative expression analysis of miR166 in different chickpea tissues
cDNA for miR166h, U6 and target were synthesised from different tissue samples of chickpea and further studied for relative expression analysis. Stem-loop primers for miR166h and U6 were used for reverse transcription. For transcript normalization, GAPDH was used (Garg et al. 2010). Expression analysis of miR166h and its target was performed in triplicates. The relative expression of the miR166h and target gene in different samples was calculated using 2−ΔΔCt method (Livak and Schmittgen 2001).
2.16 Tissue-specific expression analysis in abiotic stresses
The leaves, shoot and root samples were harvested from control and different abiotic stress treatments including drought, salinity and ABA treatments according to the manufacturer’s instructions. The relative expression of the miR166h and target gene in different treated samples compared to the control was calculated using 2−ΔΔCt method (Livak and Schmittgen 2001).
2.17 Statistical analysis
Relative expression data for miR166 and its target gene, ATHB15 from three independent biological replicates were calculated as the mean with standard error (mean ± SEM). One-way analysis of variance (ANOVA) using Duncan's multiple range tests (DMRT) was used for significant differences in variance between average values of control and treated plants with the analysis of significant difference between the means (p < 0.05). Standard deviation (SD) values were calculated using the mean of the replicates.