Experimental design and plant growth conditions
The controlled pot experiment was conducted during the winter cultivating season of 2018–2019 at the glasshouse of the College of Agronomy and Biotechnology, Southwest University, Chongqing, China. The experimental area lies at longitude 106◦ 26' 02'' E, latitude 29◦ 49' 32'' N, and altitude 220 m. During the cultivation season, the average minimum and maximum temperatures were 10°C to 26°C, and the relative humidity was between 75–87%. The experiment was implemented in a completely randomized design (CRD) with two factors: two soil water conditions (well-watered condition at 85 % of field capacity, and water-deficient condition at 50 % of field capacity), and four spraying treatments [0.00 double distilled water (CK), 140 mg l− 1 salicylic acid (SA), 4 g l− 1 zinc (Zn), and 11.5 g l− 1 glycine betaine (GB)]. The effective concentrations of these spraying treatments were based on enhancements in the growth and yield of various crops under water-deficient [10, 25, 30, 35, 37]. The experiment included eight treatments, and each treatment involved twelve pots. A winter wheat cultivar Yumai-13, which was bred by Sichuan Agricultural University and Chongqing Academy of Agricultural Sciences in China, was used as plant material, and the seeds were obtained from Wheat Research Institute, College of Agronomy and Biotechnology, Southwest University, Chongqing, China.
Each plastic pot (25 cm diameter, 30 cm depth) was filled with 8 kg air-dried and sieved (0.5 mm) soil, which was collected from the experimental station at the College of Agronomy and Biotechnology, Southwest University. Experimental soil was clay loam, and had the following physical and chemical properties: pH, organic matter, electrical conductivity (EC), bulk density, soil water content at field capacity (FC), total N, available phosphorous, and available potassium were 6.25, 12.58 g kg− 1, 0.45 ds/m, 1.44 g cm− 3, 24.35%, 0.98 g kg− 1, 15.53 mg kg− 1, and 86.11 mg kg− 1, respectively. At the time of soil filling, 2.7 g controlled-release urea (44.6% N), 3.5 g calcium superphosphate (12% P2O5), 1.5 g potassium chloride (60% K2O) were applied for each pot. Fifteen uniform grains were manually sown in the third week of November in pots at depth of 4–5 cm. Thinning was conducted 10 days after germination, and seven uniform seedlings per pot were selected for the subsequent studies. Each pot was irrigated to 85% FC by tap water till the start of drought stress treatments.
Soil Water Conditions
The plants were subjected to two soil water conditions for 30 days, from booting (Feekes 10 stage) until milk (Feekes 11 stage) stages of wheat: well-watered condition (85% of field capacity; WW) and water-deficient condition (50% of field capacity; WD). During the drought period, the pots were weighed daily to keep the required soil water levels by adding proper water volumes. Soil water contents for 85% and 50% field capacity were 22.5 % and 11.25 %, respectively . Soil water content (SWC) was determined using the following equation: SWC %=[(FW-DW)/DW] ×100, where FW was the fresh weight of soil sample from the inner area of each pot, and DW was the dry weight of soil sample after oven drying at 85° C for 3 days .
After 7 and 15 days of drought treatment, the wheat plants under each water condition were sprayed with 0.00 (double distilled water; CK), 140 mg l− 1 SA (hydroxybenzoic acid-2 C7H6O3, MW = 138.12 g mol− 1), 4 g l− 1 Zn (zinc sulfate heptahydrate ZnSO4. 7H2O, MW = 287.54 g mol− 1) and 11.5 g l− 1 GB (betaine C5H11NO2, MW = 117.14 g mol− 1) . Tween-20 (0.05%) was added with foliar applications as a surfactant at the time of treatment.
Measurements And Analysis
Wheat plants were sampled after 15 days of foliar application treatments to measure growth, photosynthetic pigments, RWC, photosynthesis gas exchange, and biochemical assays. Completely expanded, undamaged, and healthy wheat plant leaves (2nd leaf from the top) from all replicates were sampled. After washing, wheat leaves were frozen with liquid N2 immediately and stored at -80°C for biochemical analyses, and analysis of yield attributes were recorded at harvesting time.
Growth, Yield, And Its Attributes
Four pots were randomly selected and plants of each pot were taken to measure plant height, fresh weight pot− 1, dry weight pot− 1, and leaf area pot− 1. Total leaf area pot− 1 was measured with LI-3100 leaf area meter (Li-COR, CID, Inc., USA). The dry weight of plants pot− 1 was estimated following oven drying at 85°C for 48 hours. At full maturity (plants at 160-days old), five pots were randomly selected, and plants of each pot were harvested to measure the number of tillers pot− 1, number of spikes pot− 1, number of grains spike− 1, grain weight spike− 1 (g), 1000-grain weight (g), biological yield pot− 1 (g), grain yield pot− 1 (g) and harvest index (HI). The HI was computed as the percent ratio of grain yield and biological yield according to Donald .
Photosynthetic pigments and RWC
Chlorophyll (Chl. a, Chl. b and total Chl.) contents were determined in the 2nd leaf from the top according to Peng and Liu . The extraction of a 200 mg leaf blade sample was done with 10 ml ethanol-acetone (1:2, v/v), and the extract was moved to a 15 ml centrifuge tube. The tubes were put in the dark to avoid light for 24 hours until the sample changed into a white color. The chlorophyll content was calculated by the following equation: Chlorophyll a content (mg/g tissue) = (12.7D663‒2.69D645) × V/ (1000 × W), Chlorophyll b content (mg/g tissue) = (22.7 D645‒4.68D663) × V/ (1000 × W), and total chlorophyll (mg/g tissue) = D652 × V/ (34.5×W) / Chl a + Chl b, where, D663, D645 and D652, respectively are the corresponding wavelengths of the light density value, V is the volume of extracting liquid and W is the weight of fresh leaf. The RWC of wheat leaves was measured according to Barrs and Weatherley . Fresh leaves were cut into small segments (1.5 cm length), weighed fresh weight (FW), then floated in distilled water for 4 h under low light to register saturated weight (SW), and then dried in an oven until constant weight at 80 °C for 24 hours to record dry weight (DW). RWC was computed as: RWC = (FW - DW)/ (SW - DW) ×100 %.
Net photosynthesis rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO2 concentration (Ci) were registered using a portable infrared gas analyzer-based photosynthesis system (LI-6400; LiCor, Inc., Lincoln, NE, USA) at 09:30-11:30 am from the fully expanded leaf (2nd leaf from top). Air relative humidity and ambient CO2 concentrations were about 78 % and 370 µmol CO2 mol-1, respectively during the collection of data.
Assay of enzymatic antioxidants and lipid peroxidation
Antioxidant enzyme activity was reported using commercial kits for glutathione reductase (GR, A111), superoxide dismutase (SOD, A500), catalase (CAT, A501), ascorbate peroxidase (APX, A304), and peroxidase (POD, A502), by following the manufacturer’s instructions (Sino Best Biological Co., Ltd., China). The absorbance readings of GR, SOD, CAT, APX, and POD were detected at 340 nm, 560 nm, 240 nm, 290 nm, and 470 nm, respectively using an ultraviolet (UV)-visible spectrophotometer, and their activities were expressed as units per fresh weight (U g-1 FW). One unit of GR activity was expressed as the amount of enzyme depleting 1 µmol NADPH in 1 min, one unit of SOD activity was defined as the amount of enzyme needed to reduce the reference rate to 50% of maximum inhibition, one unit of CAT activity was measured as the amount of enzyme that decomposes 1nmol H2O2 at 240 nm min-1 in 1 g fresh weight, one unit of APX was estimated as the amount of enzyme required for catalyzing 1μmol ASA at 290 nm 2 min−1 of 1 g fresh weight in 1 ml of a reaction mixture, and one unit of POD activity was demonstrated as the absorbance change of 0.01 at 470 nm min−1 for 1 g fresh weight in 1 ml of a reaction mixture . Lipid peroxidation in wheat leaves was assayed as MDA content, and was measured by thiobarbituric (TBA) method using MDA Detection Kit (A401). Lipid hydroperoxide degradation products could condense with thiobarbituric acid (TBA) to yield red compounds . The absorbance for MDA content was recorded at 532 and 600 nm and expressed as nmol g-1 fresh weight.
Determination of reactive oxygen species accumulation
Hydrogen peroxide (H2O2) and superoxide anion radical (O2•−) contents in the wheat leaves were noted using the commercial ‘H2O2 Detection Kit (A400)’ and ‘O2•− Detection kit (A407)’, respectively, according to the manufacturer’s instructions. H2O2 content was estimated at 415 nm and represented as μmol g-1 fresh weight. Super oxygen anion serotonin reacted with hydrochloride to produce NO2-. The NO2- interacted with amino benzene and alpha-pyridoxine to produce red compounds at 530 nm which had a characteristic absorption peak . The content of O2•− was measured at 530 nm and expressed as μmol g-1 fresh weight.
Estimation of osmolytes accumulation
Proline and soluble sugar contents in wheat leaves were determined using commercial kits for proline (PRO, A605) and soluble sugar contents (SSC, B602), according to the manufacturer’s instructions (Sino Best Biological Co., Ltd., China). The absorbance reading of the toluene layer was estimated at 520 nm, on a spectrophotometer, and proline (Sigma, St Louis, MO, USA) was used for the standard curve . Proline content was expressed as µg g-1 fresh weight. The absorbance reading of SSC was detected at 620 nm using an ultraviolet (UV)-visible spectrophotometer . Soluble sugar content was articulated as mg g-1 fresh weight.
The collected data were analyzed following the analysis of variance (ANOVA) according to the Two-way Factorial Design using Statistical Software Package MSTAT-C . The significant differences among mean values were estimated according to Least significant difference test (L.S.D) at a 95% confidence level . Sigma Plot 10.0 (Systat Software Inc., San Jose, CA, USA) was used for the graphical presentation of the data.