Plant materials and experimental conditions
Two tolerant (Co 98014, Co 0118), and two sensitive (CoJ 85, Co 89003) sugarcane genotypes of semi-arid region of India that have contradictory response to water stress as per their physio-biochemical and antioxidant activities  were chosen under this study. Experimental trial was conducted under rain out shelter, field laboratory, Department of Biotechnology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, U.P., India and qRT-PCR for AQPs genes was performed at DUVASU, Mathura, India. Each genotype was planted in plastic pots (39 × 33 cm2) containing 25.0 kg of soil and 5.0 kg farm yard manure (FYM) in completely randomized design. Healthy and infection free sugarcane setts (2 budded) were prepared and treated prior to planting with 0.2 percent mixture of mancozeb + carbendazim for 10 min followed by chlorpyrifos 20 EC for 5 min. The soil pH, electrical conductivity and organic carbon were 7.2, 1.3 dSm-1 and 4.3 g/kg. Particle size distribution (%) was 58 (sand), 25 (silt) and 22 (clay). Three setts (2 budded) were planted in each pot with three replications for each genotype. After 60 days of planting, thinning was done by leaving only three plants in each pot. Moisture of soil contents was determined as per gravimetric method. Soil moisture content (0 – 20 cm soil depth) obtained was 23.9% at the time of sowing and it was 18.1%, 5.9% on 135th and 150th day, respectively. The similar management inputs like fertilizer and insecticides for proper growth and disease control were provided. On 120th day, samples were collected and marked as control, after that plants were imposed to water withholding. Next samplings were done on 135th and 150th day after imposing water deficit. The third expanded leaf from top was collected as sample in aseptic manner in each case by minimizing activity of ribonucleases at 09:00 h, frozen in LN2 and immediately stored at -80 °C. Samples were collected in two replications from each pot, one for analysis of antioxidant activity and another one for AQPs expression.
Malondialdehyde (MDA) assay was done by the measuring thio-barbituric acid reactive substance and expressed as μM MDA/g FW using extinction coefficient of 155 mM−1cm−1 . H2O2 content was measured by the peroxidase coupled assay . Superoxide dismutase (SOD) measurement was performed by nitroblue tetrazolium (NBT) method and expressed as units/mg protein .
RNA extraction and cDNA synthesis
Total RNA was isolated using TRIZOL reagent (Invitrogen, USA) with some modifications. The 200 mg of leaf sample was crushed in LN2 and 1.5 ml of TRIZOL was added and transferred to RNase free tube. Then, 30 µl β-mercaptoethanol (BME) and 40 µl dithiothreitol (DTT) was added and mixed vigorously by pipetting by passing several times through tip of pipette. Samples were incubated for 5 min at room temp by adding chloroform (300 µl) and centrifuged (12000g, 15 min at 4 °C). Upper aqueous phase was separated carefully without disturbing interphase and precipitated by adding chilled isopropanol (250 µl), 1.5 M NaCl (125 µl), 0.8 M potassium acetate (125 µl) and incubated for 10 min. The samples were centrifuged (12000g, 10 min, 4 °C) and supernatant was decanted. RNA pellet was washed twice with 1 ml of 75% ethanol and pellet was air dried and dissolved in 75 µl nuclease free water (NFW). Residual genomic DNA contamination was removed using DNase I (GeNei, Bangalore) as per the manufacturer instructions. The concentration of RNA was determined using Biophotometer (Eppendorf, Germany). The integrity of RNA was analyzed using 0.08% agarose gel electrophoresis. A total of 36 samples of RNA were isolated and reverse transcribed to cDNA in a final volume of 20 µl using m-mulv RT-PCR kit (GeNei, Bangalore) by taking 1µl oligo dT, 1µl random Hexamer primers and 2 µg of total RNA. The volume was finalized to 12 µl by NFW and incubated at 65 °C for 10min. The tubes were ice chilled and master mix was prepared by taking 10 µl of 5X reaction buffer, 1 µl m-mulv RT, 0.5µl each dNTPs and 1 µl RNase inhibitor. To minimize pipetting error, the master mix was prepared in one PCR tube (0.2 ml) by taking multiple values of required reagents as per the number of samples and mixed. The 8 μl of master mix was mixed to each tube and incubated with 1 µl oligo dT and 1 µl of random hexamer primers. Reaction was carried out in thermal cycler at 37 °C for 1 h followed by 95 °C for 5 minutes. The cDNA was stored at -20 °C.
Primer for sugarcane AQPs used under study were taken from already published primer sequences . These sequences were further aligned using PRIMER-BLAST at NCBI for specificity. β-tubulin  was amplified as internal reference gene (Table 1).
Quantitative real-time PCR (qRT-PCR)
The qRT-PCR was performed with Hi-SYBr Master Mix (MolBio-HIMEDIA) using CFX-96 real-time thermal cycler C-1000 (BIO-RAD) for gene expression in a final volume of 20 µl containing 10 µl of Hi-SYBr Mix (MolBio-HIMEDIA), 1 µl each forward and reverse primer and cDNA template (≤100ng). A total of three biological samples with three technical replicates were used for each gene. The thermal profiles were set with an initial temp 95 °C for 3 min followed by 36 cycles of PCR (95 °C for 30 sec, 56.5 °C for ShPIP2;1, 51 °C for ShPIP2;5, 57 °C for ShPIP2;6 and 58.5 °C for b-tubulin for 30 sec with plate read) and 72 °C for 30 sec. The melt curve was obtained between 65 °C to 95 °C with an increment of 0.5 °C per 5 sec and plate read. No template control (NTC) also included. For normalization, b-tubulin was taken as housekeeping gene. The differential expression was calculated by obtaining cycle threshold (Ct) value using the equation 2(-ΔΔCt) .
Data analysis was estimated using software SPSS (SPSS 20.0 for windows, Chicago, USA). Analysis of Variance (ANOVA) was performed following Tukey’s-b multiple range test and significant difference was determined at probability level of 5%.