5.1. Plant material and growth conditions
Three genotypes (L1, L2 and L3) of contrasting drought tolerance under field conditions, developed at the International Maize and Wheat Improvement Centre (CIMMYT), Mexico and the Ayub Agricultural Research Institute (AARI) Pakistan, respectively, were selected. Genotype L1 and L3 were drought tolerant and drought sensitive, respectively, while L2 was of intermediate drought response (Table 1). Four seeds were sown in 4 liters pots (filled with peat material, Sphagnum, 32% organic matter, pH = 5.6–6.4 and EC = 0.45 mS cm− 1) and only two seedlings were remained after one week of emergence by thinning. Twenty-four replications for each genotype were grown under well-watered conditions.
4.1. Growth conditions
After few days of emergence automatic fertigation (irrigation + mixture of essential nutrients) was applied to the plants. Furthermore, weight of each pot was kept at the same water level by manual weighing of the pots. At flowering time, 4 replications of each genotype were harvested to study different agro-physiological parameters before applying drought stress. Remaining 20 replications of each genotype were divided into two sets: irrigation was withdrawn during anthesis for one set (10 pots) and water status of the other set (10 pots) was kept at 95% pot water holding capacity. Daily evapotranspiration (ET) of each pot was recorded by weighing. Total transpirable soil water was the change of between the pot weight at 95% water holding capacity (about 3.2 kg pot weight) and when evapotranspiration of the drought plants decreased to 10% of the well-watered plants (when pot weight was ca. 1.6 kg).
5.2. Leaf and spikes sampling
Stress was imposed at anthesis until all the plant available water in the pot was consumed. In genotype L1 and L2 the drought treatment lasted 9 days while in genotype L3 the drought treatment lasted 8 days. At the end of the stress period, samples were taken from both well-watered and stressed plants. Two main tillers of each plant were selected for sampling. Flag leaf and attached spike from each of the tillers were taken and snap frozen in liquid nitrogen after tightly wrapping into aluminum foil. These samples were kept at -80 oC until the further use to analyze antioxidant and carbohydrate metabolic enzyme activities and osmotic potential. Then, 5 replications of each treatment were harvested to study their eco-physiology and dry biomass of the plants (Additional file1).
5.3. Gaseous exchange and plant water relations
Leaf photosynthetic rate (An, µmol m− 2 s− 1) and stomatal conductance (Gs, mol m− 2 s− 1) were determined from fully expanded flag leaves between 11:00 and 14:00 h with a portable photosynthetic system (LiCor-6400XT, Li-Cor, NE, USA). Measurements were performed at 20°C chamber temperature and 1500 ∝mol m− 2 s− 1 photosynthetic active radiation (PAR), and 400 ppm CO2 concentration in cuvette. Relative water content (RWC) were determined in flag leaves according to the method by Jensen et al. (2000). The RWC was calculated as follow:
RWC (%) = [(FW-DW)/(TW-DW)] × 100
where FW and DW are leaf fresh and dry weights, respectively, and TW is leaf turgid weight.
To measure osmotic potential (Ψπ) of the plant tissue, frozen material wrapped in aluminum foil was thawed, squeezed, and a piece of filter paper was dipped into the obtained sap. Ψπ was determined using psychrometers (C-52 sample chambers, Wescor Inc., Logan, UT, USA) connected to a datalogger (Wescor's Dew Point Microvoltmeter, model HR-33T). Likewise, osmotic adjustment (OA) was recorded using following formula;
OA = RWC (well-watered) x Ψπ (well-watered) - RWC (drought) x Ψπ (drought)
5.4. Extraction of samples for enzymes analysis
Samples extraction was done following the protocol by Jammer et al. [34]. Briefly, leaf and 10 spikelets from the middle of the spike excluding rachis homogenized in liquid nitrogen was used. 250 mg leaf and 500 mg spike material, respectively, was extracted with 1 ml of extraction buffer consisting of 40 mM TRIS-HCl pH 7.6, 3 mM MgCl2, 1 mM EDTA, 0.1 mM PMSF, 1 mM benzamidine, 14.34 mM β-mercaptoethanol, 24 µM NADP and milliQ H2O was added into plant material to get dialyzed extract. A piece of dialysis tube for each sample (~ 3 -4cm for 1 mL sample) was cut and sealed with a clip having number on it. This setup of dialysis tube was placed in cold water (4 °C) for 15 minutes. Extracted supernatant was Pipette into the dialysis tubes according to the arrangements. Air bubbles from the dialysis tube were removed before sealing the other end of the tube with another clip. Likewise, 1 ml of high salt buffer comprised of 1 M Tris HCl pH 7.6, 500 mM MgCl2, 250 mM EDTA, 4 M NaCl, ddH2O was added to obtain cell wall extract.
Eleven carbohydrate metabolic enzymes were selected to check their activity within leaf and spike tissue. Dialyzed extract was used for the estimation of vacInv, cytInv, AGPase, UGPase, HXK, FK, PGM, PGI, PFK, Aldolase, and cell wall extract was used to determine the activity of cwInv.
5.5. Carbohydrate metabolic enzyme assays
Method described by Jammer et al. [34] was used to determine the activity of invertases. Concisely, 5 µL of the extract were added in flat bottom 96-well plates to determine the activity of all invertases. While, 5 µl of 100 mM sucrose and 5 µl of reaction buffer pH 4.5 (454 mM Na2HPO4/273 mM citric acid) was added into dialyzed and cell-wall extract to determine the activity of vacuolar invertase (vacInv) and cell wall invertase (cwInv) respectively while reaction buffer with pH 6.8 (772 mM Na2HPO4/114 mM citric acid) was added into dialyzed extract to determine the activity of cytoplasmic invertase (cytInv). sucrose was not added into control. Likewise, calibration curve was added by glucose standard (0–50 nmol). These plates were incubated at 37 °C for 30 minutes after adding the distilled water to raise the total reaction volume of 50 µl. Plates were put at room temperature for 20 minutes after removing from incubator. 200 µl of GOD-POD reagent (10 U ml− 1 GOD, 0.8 U ml− 1 POD, and 0.8 mg ml− 1 ABTS in 0.1 M potassium phosphate buffer, pH 7.0 was added in each well. The absorbance was measured at 405 nm of plate reader. Principle of Sung et al. [79] was used to determine the activity of all the invertase enzymes.
All remaining carbohydrate enzyme activities were determined using higher throughput method described by Jammer et al. [34]. For the activity of HXK and FK was determined following the principle of Petreikov et al. [80]. Moreover, 100 mM fructose, 50 mM NAD, 100 mM ATP, 3500 U ml− 1 PGI, 1000 U ml− 1 G6PDH (from Leuconostoc mesenteroides) and common buffer (composed of 1 M Tris HCl with pH 8.0, 0.25 M EDTA, 0.5 M MgCl2) was used to determine the activity of FK. TPI was not used and 100 mM fructose was replaced with 100 mM glucose to assay the activity of HXK. For the activity of UGPase and AGPase, principle of Pelleschi et al. [7] and Appeldoorn et al. [81] was used. Again, glucose and fructose were omitted from the control. For the activity of AGPase and UGPase, common buffer, 10% BSA, 100 mM Na-PPi, 10 mM NADP, 50 mM 3-PG, 1.28 U ml− 1 G6PDH from Saccharomyces cerevisiae, 1000 U ml− 1 PGM, 50 mM ADP-Glucose (for AGPase) and 100 mM UGP-glucose (for UGPase), was used to determine the activity of AGPase and UGPase respectively. However, for the control samples ADP-glucose and UDP-glucose were omitted. Similarly, principle of Manjunath et al., [82] was used to determine the activity of PGM. In continuation, to assay the activity of PGM 1 M Tris-HCl pH 8.0, 0.5 M MgCl2, 500 mM DTT, 10 mM Glc-1,6-bisP, 100 mM Glc-1-P*, 10 mM NADP, 6000U ml− 1 G6PDH (from S. cerevisiae) was used. Activity of PGI was determined following the principle of Zhou and Cheng (2008). However, to determine the action PGI 10 mM glc-1,6-bisP and 100 mM glc-1-P*, were replaced with fruct-6-P* and; glc-1-P* and fruct-6-P*. Mastermix was prepared using common buffer, 25 mM fruct-1,6-bisP*, 25 mM NADH, GPDH 2100 U ml− 1, TPI 6000 U ml− 1. Additionally, activity of PFK was determined following the principle of Klotz et al. [84]. Similarly, apart from common buffer, 100 mM fruct-6-P*, 25 mM NADH, 100 mM ATP, 372 U ml− 1 aldolase, GPDH 2100 U ml− 1, TPI 6000 U ml− 1 was used for the activity of PFK. Fruct-6-P* was omitted as substrate in the control samples. and activity of aldolase was determined following the principle of Schwab et al. [85]. The absorbance was studied at 340 nm for 30 minutes and deviation of readings/peaks was monitored during this period and calculation of specific enzyme activity in nkat g FW− 1. Gen5 v3.04.17 software (Biotek Instruments. Inc) was used to measure the absorbance of different enzymes.
5.6. Activity of antioxidants enzymes
Methodology described by Garcia-Lemos et al. [86] was used to determine the activities of different antioxidant and 96-well plates format was utilized while, the activities were determined photometrically. Briefly, activities for ascorbate peroxidase (APX) was determined based upon the principle of Yoshimura et al. [87]. For the reactions, dialyzed extract was used. Master mix comprised of 50 mM KPO4 buffer pH 7.6, 0.25 mM ascorbate and 0.5 mM H2O2 was used and absorbance was recorded at 290 nm. Likewise for control H2O2 was omitted [86]. For the activities for catalase (CAT) principle of Aebi [88] was followed. Master mixed containing 50 mM KPO4 buffer pH7, 0.001% antifoam agent 204 and 100 mM H2O2 was mixed with dialyzed extract and absorbance was recorded at 240 nm. Likewise, for control reactions H2O2 was omitted as mentioned by Garcia-Lemos et al. [86]. To determine the activity of peroxidase (POX) or cell wall peroxidase (cwPOX) principle of Polle et al. [89] was used. For determination of POX activity method described by Garcia-Lemos et al. [86] was used. Again, dialyzed extract was mixed with master-mix containing 100 mM KPO4 buffer pH 7, 2 mM guaiacol and 0.15 mM H2O2 was used. Absorbance was measured at 450 nm and H2O2 was omitted for control reactions. However, cell wall extract was used for the activity of cwPOX. The activity of superoxide dismutase (SOD) were determined following the principle of McCord and Fridovich [90]. Similarly, dialyzed extract was used to mix with master mix containing 50 mM KPO4 buffer pH 7.8, 0.1 mM EDTA, 0.05 mM cytochrome c, 10 mM xanthine and 0.0002 U mg− 1 xanthine oxidase. Absorbance was recorded at 550 nm as described by Garcia-Lemos et al. [86]. However, xanthine was omitted in control reactions. Activities of glutathione reductase (GR) principle of Edwards et al. [91] was used. Dialyzed extract was mixed with master mix containing 100 mM buffer of Tris HCl with pH 7.8, 25 mM NADPH and 30 mM glutathione oxidized (GSSG). Absorbance was detected at 340 nm for 40 minutes and GSSG was omitted for control reactions. For the activity of dehydroascorbate reductase (DHAR) principle of Dalton et al. [92] was followed. Again, dialyzed extract was mixed with master mix comprised of 100 mM KPO4 with pH 6.5, 50 mM glutathione reduced (GSH) and 50 mM dehydroascorbic acid (DHA). The activity was determined at 290 nm for 40 minutes and DHA was not used in control reactions. To determine the activity of monodehydroascorbate reductase (MDHAR) principle described by Arrigoni et al. [93] was followed. Dialyzed extract was mixed with reaction mixture comprised of 50 mM KPO4 buffer with pH 7.2, 25 mM NADH, 5U µl− 1 ascorbic acid oxidase (OAA) and 50 mM ascorbate. Activity was measured at 340 nm for 40 minutes and ascorbate was omitted in control reactions. Additionally, the activities of Glutathione S-transferase (GST) were determined following the principle of Li et al. [94]. Again dialyzed extract was mixed with reaction mixture (100 mM KPO4 buffer with pH 7.4, 50 mM GSH and 2,4-dinitrochlorobenzene (CDNB)). Absorbance was measured at 334 nm for 30 minutes and CDNB was not used for control reactions.
5.7. Abscisic acid assay
ABA concentration in leaf and spike samples was determined through an enzyme linked immunosorbent assay (ELISA) using a monoclonal antibody for ABA (AFRC MAC252) (Asch, 2000).
5.8. Agronomic traits measurement
At the end of drought treatment pots from each treatment were re-watered until the maturity of the plants. Plant maturity stage was determined as described by Zadoks et al. [95]. Harvesting was done at maturity and following traits were recorded:
- number of grains spike-1 (NGS): Five spikes from each replication were taken and their averages were recorded;
- thousand grain weight (TKW): Thousand grains were counted from each replication and their weight was recorded in grams (g);
- Kernel abortion (KA): KA was recorded using following formula: (number of grains spike-1 /number of florets spike-1) x 100
- Plant biomass pot-1 (BM): Both plants from each pot were harvested from soil level and weight of whole plant was expressed in grams (g);
- Grain yield pot-1 (GY): Spikes from the pot were threshed into grains and their weight was expressed in grams (g);
- harvest index (HI) were recorded using following formula: Grain yield (g)/Biomass (g) x 100
5.9. Statistical analysis
Analysis of variance (two-way ANOVA) was done using RStudio 1.0.153.exe to reveal the significance of the effect of genotype, water and their interaction on the measured variables at P = 0.05 level. Similarly, regression analysis was done in Microsoft excel 2016. PCA and Biplot analysis was done in MetaR-v6.0_BASE_setup.exe software and “L” and “S” are indicating leaf and spike antioxidants or carbohydrate metabolic enzymes or phytohormones in biplot figure like, “L-aldolase” was used for leaf aldolase enzymes and “S-aldolase” was used for spike aldolase enzyme.