Individual and Interactive Effects of Nitrogen and Phosphorus on Drought Stress Response and Recovery in Maize Seedlings

Abstract

Plants have an inherent mechanism for perceiving drought stress and respond through a series of physiological, cellular and molecular changes for maintaining physiological water balance. It has been shown that nitrogen (N) and phosphate (P) can help to improve plant tolerance to water limitation by increasing the activities of the photosynthetic machinery and antioxidant enzymes. Maize is highly sensitive to drought stress, especially at the seedling stage. In this study, we used four maize genotypes (HKI-161, HKI-193-1, HQPM-1 and HQPM-7) and studied the effect of N and P application on response to drought stress and recovery at germination and seedling stage. We show that application of N and P had no effect on rate of germination but increased the seedling growth, chlorophyll content, malondialdehyde levels, proline, anthocyanin content, gas exchange parameters and antioxidant enzymes (APX, CAT and GR) during drought stress. The variation in the effect was visible across genotypes, but the observed changes indicate improved drought stress tolerance in the maize seedlings. During drought recovery, seedlings of HKI-161 and HKI-193-1 genotype that did not receive N and/or P treatment or that were pre-supplemented with only P showed rapid transition to flowering stages. Seedlings pretreated with N showed comparatively late transition to flowering. The HQPM-1 seedlings, which received N treatment moved to flowering stage while HQPM-7 seedlings showed only normal vegetative growth under all treatment conditions. Molecular analysis identified 2016 transcripts that are differentially expressed in the drought tolerant and susceptible genotypes. About 947 transcripts showed >3-fold change in expression and were expressed during stress tolerant genotype. Transcripts coding for proteins in P and N metabolism were identified within the drought regulated transcripts. The analysis showed that transcripts related to P metabolism were expressed during stress and recovery phases in the susceptible genotype while transcripts related to N metabolism were down regulated during drought stress and recovery stages in all the genotypes.

1. Introduction

The incessant food demands of an exponentially growing world population and the unprecedented changes in climatic conditions are posing great challenges for food production and sustainable agriculture [1,2,3]. The episodes of water limitations and drought, imposed by climate change, are becoming more frequent and severe. The reduction in soil moisture content reduces nutrient uptake and translocation thereby inflicting multiple harmful effects on plants [4,5]. Water deficit limits the plant growth and development, as it hampers photosynthesis by decreasing CO₂ diffusion to the chloroplast, results in abnormal cell division, alters different physiological characteristics and increases reactive oxygen species (ROS) [5,6]. The plants respond to water deficit by the coordinated regulation of different physiological and biochemical processes to maintain their water relations, gas exchange parameters, photosynthesis and pigmentation [6,7]. Further, there is increase in metabolism of different compounds, adjustments of the membrane system and reprograming of the molecular–genetic networks [7,8]. Moreover, plants accumulate osmolytes and antioxidant enzymes to reduce cytoplasmic osmotic potential and to remove excessive ROS [5,8,9].

Maize is one of the most popular cereal crops, which is grown globally and is particularly affected by drought stress at the seedling stage [10,11]. Water scarcity along with the reduction of nutrient availability; limit maize plant growth and development during drought stress [12,13]. In agricultural fields, nitrogen (N), phosphorus (P) and potassium (K) nutrients are often supplied in the form of chemical fertilizers to increase plant yields [14,15,16]. Several studies have been performed to understand the beneficial role of N and P applications for plant growth, during drought stress [17,18,19,20,21]. N is an important nutrient component of proteins, nucleic acids, chlorophyll and some hormones [20]. P is another major element present in plant tissues and it is important for the normal physiology, morphology and biochemistry of plants [20].

It has been reported that externally supplied N and P favor plant growth and often help the plants tolerate drought stress, by increasing water use efficiency, stomatal conductance, photosynthesis and enzymatic activities [4,15,17,18,22]. The studies have indicated involvement of various interconnected physiological, biochemical and molecular pathways during uptake and translocation of nutrients and response to drought stress [7,23,24]. Applications of N and P as fertilizers alleviated the adverse effects of drought stress in maize and wheat plants by increasing the levels of osmoprotectants and antioxidant enzymes [21,25]. In this study, we present the effect of single and combined applications of N and P on response to and recovery from drought stress using different maize genotypes. Transcript analysis was performed using the publicly available maize data to identify the drought regulated transcripts and identify their role in P and N metabolism.

2. Materials and Methods

2.1. Plant Materials, Growing Conditions and Stress Treatments

Maize genotypes HKI-161, HKI-193-1, HQPM-1 and HQPM-7 were used in this study. The maize seeds were provided by the Indian Institute of Agricultural Research (ICAR), Pusa Campus, New Delhi, India.

Viable seeds were surface sterilized for 2 min with 70% ethanol, 10 min in 50% sodium hypochlorite solution containing 2–3 drops of Tween-20 and washed with Milli-Q water 5–7 times [26] in between both steps and germinated in a growth chamber (Panasonic Healthcare Co., Ltd., Ora-Gun, Oizumi-machi, Gunma, Japan) with controlled environmental conditions: 14 h light/10 h dark, 28 °C temperature, 200 μmol m⁻² s⁻¹ light intensity and relative air humidity of approximately 60%. Fourteen days after germination, maize seedlings were transplanted into plastic pots containing 8 kg soil mix and allowed to grow in the greenhouse at 28 ± 2 °C and approx. 65% relative air humidity. The soil mix contained 241 kg ha⁻¹ (0.095 g/Kg) N, 48 kg ha⁻¹ (0.02 g/Kg) P, 135 kg ha⁻¹ (0.053 g/Kg) K and 0.315% soil organic carbon. The 14-day-old plants were grouped into sets of 3 plants each. For the next 15 days, the pots were irrigated every alternate day with water, N solution, P solution and NP solution, respectively. The N solution contained 44 Kg ha⁻¹ urea, the P solution contained 15 Kg ha⁻¹ orthophospheric acid (pH 7) and NP solution contained both the chemicals at a concentration of 44:15 Kg ha⁻¹. The concentrations of N and P used in the study were as determined in earlier studies on maize [27].

For drought treatments, the irrigation was stopped on the 17th day while the control sets were irrigated regularly. The pot soil moisture content reached 10 ± 1% on the 15th day after irrigation was terminated. This was marked as the starting point of drought stress and for subsequent experiments stress durations considered were 12 h, 24 h and 48 h. For drought recovery, 48 h drought-stressed plants were irrigated for one week and observed for changes. All the experiments in this study were performed in triplicate.

2.2. Germination Assay

To test the effect of drought stress and drought pre-supplemented with individual and combined N and P nutrients on seed germination, a germination assay was performed. Viable seeds were imbibed overnight with water, N solution, P solution and NP solution. The imbibed seeds were kept on germination paper, in a growth chamber (Panasonic Healthcare Co., Ltd., Ora-Gun, Oizumi-machi, Gunma, Japan) at 28 °C. The seed groups for drought stress were treated with 20% Polyethylene glycol (PEG) while the control groups were kept with water or nutrient solutions. On day 4, the seeds were evaluated for germination and data was recorded for plumule and radicle length. The germination percent was determined as described by Awasthi et al. [28].

2.3. Measurement of Plant Growth Parameters

Shoot height and root length of maize seedlings were measured from the collar point. Stem width was measured at mid stem-length region using measuring tape. To study the effect of drought stress on the leaf length and width, the uppermost fully expanded leaf was used for measurements. Leaf area was calculated according to He et al. [29].

2.4. Measurement of Stress Response Parameters

2.4.1. Leaf Relative Water Content (LRWC)

LRWC was measured according to Barrs and Weatherley [30]. Briefly, the uppermost fully expanded leaf was taken, and its fresh weight (FW) was determined. Then, it was dipped in Milli-Q water in falcon tubes and kept at +4 °C for overnight. The excess leaf moisture was blotted out with tissue paper and leaf turgid weight (TW) was measured. The leaf was then dried at 80 °C for 24 h in a hot air oven (Kumar Instruments, New Delhi, India) and measured for dry weight (DW). The weight recordings were performed using an electronic balance (Mettler Toledo, Greifensee, Switzerland). The LRWC was determined using the formula LRWC = 100 × (FW-DW)/(TW-DW).

2.4.2. Chlorophyll Content

Chlorophyll content was determined according to Arnon [31] with slight modifications. Briefly, 100 mg leaf tissue was ground in liquid nitrogen using a mortar and pestle. 10 mL 80% (v/v) acetone was added, and the crude extract was kept in the dark for 48 h. Then, it was centrifuged (Refrigerated Eppendorf Centrifuge 5804R, Hamburg, Germany) for 10 min at 5000 rotations per minute (rpm). The clear supernatant was collected and quantified at 663 nm and 645 nm wavelengths, using Harvard Biochrom Ultrospec™ 2100 pro UV/Visible Spectrophotometer (Fisher scientific, Oy—Ratastie 2, 01,620 Vantaa—Finland). The chlorophyll content (μg/mL) was calculated using the following formulas: chlorophyll a = 12.7 (A663) − 2.69 (A645); chlorophyll b = 22.9 (A645) − 4.68 (A663); total chlorophyll = 20.2 (A645) + 8.02 (A663).

2.4.3. Lipid Peroxidation

Malondialdehyde (MDA) content was measured as reported by Heath and Packer [32] with slight modifications. Briefly, 200 mg leaves were homogenized in 2 mL of 1% trichloroacetic acid (TCA) and 2 mL of 0.5% thiobarbituric acid (TBA) was added to it. The extract was boiled for 10 min and the mixture was centrifuged at 10,800 rpm for 10 min. The supernatant was quantitated at 532 nm and 600 nm, to determine the MDA content using the formula; MDA (mmol g⁻¹) = (A532 − A600) × SN/(155 × W), where SN is the volume of the supernatant, 155 is the absorption of 1 mmol trimethine at 532 nm and W is the 0.2 g leaf sample weight.

2.4.4. Osmolyte Accumulation

The amount of free proline in fresh leaf was determined as reported by Bates et al. [33]. About 500 mg leaf was homogenized in 10 mL of 3% aqueous sulfosalicylic acid using mortar and pestle in an ice-cold bath. The homogenate was centrifuged at 10,000 rpm for 15 min at +4 °C. An aliquot (2 mL) of the supernatant was mixed with an equal volume of acetic acid and acid ninhydrin. Ninhydrin was prepared by warming 1.25 g of 1,2,3-indantrione monohydrate in 30 mL glacial acetic acid and 20 mL of 6 M orthophosphoric acid with vortexing and gentle warming. The mixture was incubated for 1 h at 100 °C (Serological water bath, New Delhi, India) until a colored complex was developed. The reaction was terminated in an ice bath and extracted with 4 mL of toluene. The extract was vortexed for 20 s and the chromophore (red-pink in color) was used to determine the absorbance at 520 nm. L-proline was used to plot the standard curve for calculating the concentration of proline as µmole/gm fresh weight.

2.4.5. Anthocyanin Content

Anthocyanin content was determined following Kim et al. [34]. Briefly, 100 mg of leaf tissue was extracted with 5 mL of solution comprising of 1% HCl in methanol, by incubating for 24 h at 4 °C. A quantity of 200 μL of water and chloroform were added to the extract and centrifuged at 4 °C. The absorbance of the supernatant was recorded at 530 nm and 657 nm. Anthocyanin content was estimated as [A530 nm − (0.25 × A657 nm)] × 5.

2.5. Enzyme Activities

2.5.1. Ascorbate Peroxidase (APX)

APX activity was estimated by the rate of oxidation of ascorbate [35]. About 100 mg leaf tissues were homogenized in 1 mL of 100 mM phosphate buffer (pH 7.0). The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C and 600 μL of the supernatant was added into a 3 mL reaction mixture containing 5 mM ascorbate and 0.5 mM H₂O₂. After mixing, the absorbance was read at 290 nm.

2.5.2. Catalase (CAT)

CAT activity was determined [36] by homogenizing about 100 mg of leaf tissues in 1 mL of 50 mM phosphate buffer (pH 7.8). The homogenate was then centrifuged at 10,000 rpm for 15 min at 4 °C. Then, 100 μL of supernatant was added into a 3 mL of 50 mM phosphate buffer (pH 7.8) containing 0.5 mM H₂O₂. After mixing, the absorbance was measured at 240 nm.

2.5.3. Glutathione Reductase (GR)

GR activity was determined based on the oxidation of NADPH [37]. About 100 mg of leaf tissues were homogenized in 1 mL of 50 mM phosphate buffer (pH 7.6). The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C and 50 μL of the supernatant was added into a 1 mL reaction mixture containing 1 mM oxidized glutathione and 2 mM NADPH. After mixing, the absorbance was measured at 340 nm.

2.6. Gas Exchange Measurements

The fully expanded leaves were used for measuring the gas exchange parameters such as photosynthetic rate (PR), transpiration rate (TR), stomatal conductance (SC) and carbon dioxide concentration (Ci) between 11:00 and 15:00 solar time by using portable open-flow gas exchange system (LI-6400, LI-COR Inc., Lincoln, NE, USA). During the recording time, relative air humidity, CO₂ concentration and photon flux density were maintained at 60–70%, 400 µmol mol⁻¹ and 1000 µmol m⁻² s⁻¹, respectively.

2.7. Microarray Data Analysis

The data for drought tolerant and drought susceptible maize genotypes were downloaded from EXPath 2.0, a comparative expression analysis inferring metabolic pathway analysis tool [38]. The differentially expressed transcripts (DETs) under drought stress, and stress recovery conditions were filtered by setting cutoff parameters of fold change: ≥3 and p-value: ≤0.05. The selected DETs were used to generate the heat map and metabolic profiles and predict gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To know the distribution patterns of the DETs Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/) and DrawVenn (https://bioinformatics.psb.ugent.be/webtools/Venn/) were used.

2.8. Statistical Analysis

All the experiments were performed with three replicates, each containing three biological replicates. The results were expressed as the mean ± standard error of the mean (SEM). The statistical analysis was carried out for statistical significance using analysis of variance (ANOVA) test. The p values < 0.05 were considered as significant.

3. Results

3.1. Drought Stress Affects Seed Germination

Seed germination is significantly dependent on the availability of water. Under well-watered conditions (control), germination started after 48 h and by 96 h sufficient shoots and roots had emerged in most seeds of the four maize genotypes, HKI-161, HKI-193-1, HQPM-1 and HQPM-7 (Figure 1a). The percentage germination varied for seeds of the different genotypes from average 71% in HKI-161 and HKI-193-I to 86% in HQPM-7 and 100% in HQPM-1. The shoot length (Figure 1b) at 96 h after germination was longest in HQPM-1 and shortest in HKI-161 seedlings. The root length (Figure 1c) at 96 h after germination was longest in HQPM-1 but shortest in HQPM-7 seedlings. In absence of water, no germination was recorded in the imbibed seeds of all the four maize genotypes.

The germination percentage, shoot length and root length were differentially affected in the presence of N and/or P when compared with only water controls. In HKI-161 genotype, 57.14% germination was recorded in presence of N alone, 71.4% germination in presence of P alone and 85% germination in presence of NP (Figure 1a). The seedling measurements showed that in presence of P there was no significant change in shoot length, but the size decreased in presence of NP and was lowest in presence of N (Figure 1b). In roots the length decreased by 4 cm in presence of N or P and to less than half in presence of NP (Figure 1c).

The HKI-193-1 genotype showed nearly 100% germination in presence of N or P and 85.71% germination under NP treatment. The seedling measurements showed that in presence of N there was no significant change in shoot length when compared with water controls (Figure 1b). However, the size decreased by 3 cm in presence of P and by 4.5 cm in presence of NP (Figure 1b). In roots the length decreased by an average of 4 cm in presence of N or P and by 7 cm in presence of NP (Figure 1c).

The HQPM-1 genotype showed nearly 100% germination in presence of N or P or NP (Figure 1a). The seedling measurements showed that in presence of N or P there was no significant change in shoot length, but the size decreased by 2 cm in presence of NP (Figure 1b). In roots the length decreased by an average of 4 cm in presence of N or P and by 7 cm in presence of NP (Figure 1c).

The HQPM-7 genotype showed 71.43% germination in presence of both NP and P but 28.57% in presence of N (Figure 1a). The seedling measurements showed that in presence of P or NP there was no significant change in shoot length, but the size decreased slightly in presence of N (Figure 1b). In roots, the length decreased slightly in presence of P and by 4 cm in presence of NP but 5 cm in presence of N (Figure 1c).

3.2. Effect of Nitrogen and Phosphorus Pre-Supplementation on Growth of Drought-Stressed Seedlings

The shoot length, root length, stem width and leaf area of seedlings of the four maize genotypes were recorded under drought conditions with and without the pre-supplement of N and/or P and the changes observed in the 4 maize genotypes were plotted (Figure 2).

3.2.1. Shoot Length

Overall decrease in shoot length was observed in seedlings of all the genotypes under drought stress (Figure 2a). The seedlings of HKI-161, HKI-193-1 and HQPM-1 showed no significant changes in the shoot length at 12 h and 24 h drought stress time points but at 48 h stress timing a reduction (10 cm with respect to water control) was observed (Figure 2a). The seedlings of HQPM-7 showed decrease in the shoot length at 12 h followed by increase at 24 h and 48 h drought stress time points (Figure 2a).

In HKI-161 seedlings, the shoot length increased under drought stress in the presence of NP (Figure 2b) and N (Figure 2c) but decreased on addition of P (Figure 2d). At 12 h, the shoot length was shorter as compared to drought stress controls when pre-supplemented with NP and N (Figure 2b–d). In the presence of NP, the shoot length gradually increased at 24 h and 48 h (Figure 2b). In the presence of N, no change in shoot length was observed till 24 h but a sharp increase was observed at 48 h (Figure 2c). In the presence of P, the shoot length decreased till 12 h but showed a small increase at 48 h stress (Figure 2d).

In HKI-193-1 seedlings, the shoot length decreased under drought stress on addition of NP (Figure 2b), but increased in presence of N or P, respectively (Figure 2c,d). At 12 h, the shoot length was shorter as compared to drought stress controls when pre-supplemented with NP and N (Figure 2b–d). In the presence of NP, the shoot length gradually decreased at 24 h and 48 h (Figure 2b). In the presence of N, the shoot length gradually increased at 24 h and 48 h (Figure 2c). In the presence of P, the shoot length decreased till 12 h but rapidly increased at 48 h stress (Figure 2d).

In HQPM-1 seedlings, the shoot length increased under drought stress in the presence of NP (Figure 2b) and P (Figure 2d) but decreased on addition of N (Figure 2c). At 12 h, the no significant change was observed in shoot length when pre-supplemented with N and/or P (Figure 2b–d). In the presence of NP, the shoot length increased at 24 h and decreased at 48 h (Figure 2b). In the presence of N, the shoot length did not show much change till 24 h but decreased by 48 h (Figure 2c). In the presence of P, the shoot length increased at 24 h and 48 h stress (Figure 2d).

In HQPM-7 seedlings, the shoot length increased under drought stress in the presence of NP (Figure 2b) and N (Figure 2c) but decreased on addition of P (Figure 2d). At 12 h, decrease in shoot length was observed when seedlings were pre-supplemented with NP and N (Figure 2b,c). In the presence of NP, the shoot length decreased at 24 h and increased at 48 h (Figure 2b). In the presence of N, the shoot length gradually increased by 48 h (Figure 2c). In the presence of P, the shoot length decreased till 24 h and then did not show much change till 48 h stress (Figure 2d).

3.2.2. Root Length

Overall increase in root length was observed in seedlings of the HKI-161 and HQPM-7 genotypes under drought stress and decrease in root length was observed in seedlings of the HKI-193-1 and HQPM-1 genotypes under drought stress (Figure 2e). The seedlings of HKI-161, HQPM-1 and HQPM-7 showed increase in the root length at 12 h and 24 h drought stress time points but the seedlings of HKI-193-1 did not show much change in length (Figure 2e) when compared with watered controls. The seedlings of HKI-161 and HQPM-7 showed increase in the root length at 48 h drought stress time points while a decrease in length was observed in HQPM-1 seedlings (Figure 2e).

In HKI-161 seedlings, the root length was longer as compared to drought stress controls in the presence of N and/or P at 12 h (Figure 2f–h). In the presence of NP, the root length decreased at 24 h but increased at 48 h (Figure 2f). A Similar pattern was observed in the presence of N (Figure 2g) and P (Figure 2h).

In HKI-193-1 seedlings, the root length was longer as compared to drought stress controls at 12 h in the presence of NP (Figure 2f) and N (Figure 2g). At 24 h, the root length decreased but increased slowly at 48 h (Figure 2f,g). In the presence of P, no significant change in root length was observed (Figure 2h).

In HQPM-1 seedlings, the root length was longer as compared to drought stress controls at 12 h in the presence of N and/or P (Figure 2f–h). In the presence of NP and P (Figure 2g), the root length decreased at 24 h but increased by 48 h (Figure 2f,h). In the presence of N, no significant change in root length was observed (Figure 2g).

In HQPM-7 seedlings, the root length was longer as compared to drought stress controls at 12 h in the presence of N and/or P (Figure 2f–h). In the presence of NP, the root length decreased at 24 h but increased by 48 h (Figure 2f). In the presence of N or P, the root length increased at 24 h but increased by 48 h (Figure 2g,h).

3.2.3. Stem Width

Overall, an enhancement in stem width was also observed in the drought-stressed seedlings of all four genotypes when compared with water controls (Figure 2i). Stem width of HKI-161 and HKI-193-1 seedlings did not show any change at 12 h of drought stress but it slowly decreased till 48 h drought stress. In HQPM-1 and HQPM-7 seedlings, a decrease was observed at 12 h drought stress but it increased by 24 h stress and thereafter not much change was observed (Figure 2i).

In the drought-stressed HKI-161 and HQPM-7 seedlings, the stem width decreased in presence of NP (Figure 2j) and P (Figure 2l) at 24 h but increased in presence of only N (Figure 2k). At 48 h, the stem width increased in presence of NP (Figure 2j) but decreased further in presence of only P (Figure 2l) and only N (Figure 2k).

In the drought-stressed HKI-193-1 seedlings, increase in stem width was observed in presence of NP (Figure 2j) and N (Figure 2k) from 12 h to 48 h time points. In the presence of only P (Figure 2l) the stem width decreased at 24 h but increased at 48 h time points.

In the drought-stressed HQPM-1 seedlings, increase in stem width was observed in presence of NP (Figure 2j) and P (Figure 2l) from 12 h to 24 h time points, while in the presence of only N (Figure 2k) the stem width decreased at 24 h. No further change was observed in the stem width of these seedlings in the presence of N and/or P (Figure 2j–l).

3.2.4. Leaf Area

Overall, no change in leaf area was observed in the drought-stressed seedlings of all four genotypes when compared with water controls (Figure 2m). Leaf area of HKI-161 seedlings increased under drought stress at 24 h but decreased to that observed in watered controls at 48 h of stress. In HKI-193-1 and HQPM-7 seedlings, the leaf area decreased under drought stress at 12 h but increased at 24 h. At 48 h, it again decreased in HKI-193-1 seedlings but did not change for HQPM-7 seedlings. In HQPM-1 seedlings, no change in leaf area was observed under drought stress (Figure 2m).

In the drought-stressed HKI-161 seedlings, the leaf area decreased significantly in presence of N and/or P (Figure 2n–p) at 24 h. Thereafter, it increased at 48 h of drought stress in presence of NP and N (Figure 2n,o) but decreased further in presence of P (Figure 2p).

In the drought-stressed HKI-193-1 seedlings, a decrease in the leaf area was observed in presence of NP (Figure 2n) and P (Figure 2p) up to 24 h time points and it increased thereafter at 48 h. A reverse pattern was observed in the presence of only N (Figure 2o).

In the drought-stressed HQPM-1 and HQPM-7 seedlings, the leaf area did not change significantly in presence of NP (Figure 2n) and P (Figure 2p) but in presence of only N (Figure 2o) it decreased after 24 h of drought stress.

3.3. Effect of Nitrogen and Phosphorus Pre-Supplementation on the Relative Water Content of Drought-Stressed Seedlings

The time kinetics of drought stress showed a decrease in the leaf relative water content (LRWC) in all four maize genotypes (Figure 3a). The response was more rapid in seedlings of HQPM-1 genotype as compared to the others. Among the other three genotypes relatively higher values of LRWC were observed in HQPM-7 followed by HKI-193-1 and HKI-161, in decreasing order.

Upon pre-treatment with N and/or P, the drought-induced reduction in LRWC was less in seedlings of all the genotypes (Figure 3). The HKI-161 seedlings showed lowest values of LRWC among all the genotypes. When drought stress was applied to the control set of seedlings and those pre-treated with NP, the values declined sharply. In seedlings pre-treated with only N the LRWC values decreased by 15% while in seedlings pre-treated with only P the LRWC values decreased by ~5%. The HQPM-1 genotype showed a significant increase in the LRWC upon pretreatment with N or P, though maximum increase was seen upon pretreatment with NP. Moreover, the HKI-193-1 and HQPM-7 seedlings did not show much change in the LRWC on pretreatment with NP. When drought stress was applied to HKI-193-1 seedlings pretreated with N or P, the values declined sharply by 18–20%. When drought stress was given to HQPM-7 seedlings pretreated with N or P, the values declined by ~5%.

3.4. Effect of Nitrogen and Phosphorus Pre-Supplementation on the Physiological and Biochemical Properties of Drought-Stressed Seedlings

3.4.1. Chlorophyll Content

Increasing duration of drought stress reduced the total chlorophyll content in all the genotypes (Figure 4). Under water-irrigated control conditions, HQPM-1 seedlings showed the highest total chlorophyll content (21.14 μg/mL) among the four genotypes. The drought stress related reduction in the total chlorophyll content was observed after 24 h and it reached to the lowest value at the 48 h time point (7.34 μg/mL). The loss in total chlorophyll content under stress was similar in HKI-161, HKI-193-1, and HQPM-7 but the kinetics of response was varied (Figure 4a,e,i,m). During drought stress, significant loss in total chlorophyll was observed after 48 h in HKI-161, while it was seen at 12 h in HKI-193-1 and at 24 h in HQPM-7.

Pre-supplementation of N and/or P resulted in increase in the total chlorophyll content (control samples). In HKI-161 the increase was observed at 12 h of pre-supplementation of N (Figure 4c) and 48 h of pre-supplementation of NP or P (Figure 4b,d). In HKI-193-1 and HQPM-7, the increase was observed after 12 h of pretreatment of N, P or NP (Figure 4f–h,n–p), but maximum increase was observed at 24 h of treatment with N in HKI-193-1 and 12 h of treatment with NP (34.73 μg/mL) in HQPM-7. In HQPM-1, the increase was observed after 12 h of pretreatment of NP (Figure 4j) and at all other time points the values were similar to that of water controls (Figure 4j–l).

The application of drought stress to seedlings pre-supplemented with N and/or P had a differential negative impact on the total chlorophyll content. In drought stressed HKI-161 seedlings, decrease in total chlorophyll content was observed at 12 h of stress in sets pre-supplemented with P (Figure 4d), 24 h of stress in sets pre-supplemented with N (Figure 4c) and 48 h of stress in seed pre-supplemented with NP (Figure 4b).

In drought stressed HKI-193-1 seedlings pre-supplemented with P, decrease in total chlorophyll content was observed at 12 h of stress but the negative impact was reduced at 48 h of stress (Figure 4h). The seedlings pre-supplemented with N showed loss in total chlorophyll content at the 12 h time point (Figure 4g) while pre-supplementation with NP delayed the loss till 48 h of stress (Figure 4f).

In HQPM-1 seedlings, pre-supplementation with NP (Figure 4j) and N (Figure 4k) reduced the drought stress induced loss in total chlorophyll content. The pre-supplementation of seedlings with P delayed the loss in total chlorophyll content to 24 h and 48 h time points (Figure 4l).

In HQPM-7 seedlings, pre-supplementation with N (Figure 4o) and P (Figure 4p) the total chlorophyll levels were high (>25 μg/mL) and some loss of chlorophyll was seen at 12 h of drought stress but this did not increase with the duration of stress. The pre-supplementation of seedlings with NP could significantly prevent the loss in total chlorophyll content (Figure 4n).

3.4.2. Lipid Peroxidation

The membrane lipid peroxidation leads to higher MDA levels, so it was used as an indicator of oxidative damage. Assays for the MDA content revealed that its levels increased in all genotypes during drought stress (without nutrient pre-supplementation) as compared to the control counterparts (Figure 5(a1)). The MDA content was highest in the HQPM-1 genotype, and the drought-stressed seedlings showed a 1.34% increase in MDA content at 24 h. The HQPM-7 genotype came second in terms of the MDA content and under drought stress the seedlings showed 1.74% increase at 24 h and 2% increase at 48 h. The drought stressed seedlings of HKI-193-1 and HKI-161 genotype exhibited 1.51% and 1.39% increase in MDA content at 48 h, respectively.

When drought-stressed seedlings were pre-supplemented with NP, the HQPM-1 genotype showed significantly high MDA content followed by HQPM-7, HKI-193-1 and HKI-161 (Figure 5(a2)). In HQPM-1 seedlings, the change in MDA content (with respect to control) decreased from 2.1% at 12 h to 0.9% at 48 h. In HQPM-7, seedlings the MDA content increased from 0.5% at 12 h to 1.0% at 48 h. In HKI-193-1 and HKI-161 seedlings, there was no significant change in the MDA content at 12 h of stress but at it increased to 1.1% and 1.4%, respectively, at 48 h.

Under drought stress, the HQPM-7 seedlings pre-supplemented with N showed significantly high MDA content followed by HQPM-1, HKI-161 and HKI-193-1 (Figure 5(a3)). In HQPM-7 seedlings, the MDA content (with respect to control) decreased from 1.4% at 12 h to 0.9% at 48 h. In HQPM-1 seedlings, the MDA content decreased from 1.1% at 12 h to 0.6% at 48 h. In HKI-193-1 and HKI-161 seedlings, the MDA content increased from 0.9% and 0.5%, at 12 h to 2.2% and 0.8%, at 48 h, respectively. Therefore, at 48 h, the maximum MDA content was seen in HKI-161 followed by HQPM7, HQPM-1 and HKI-193-1, respectively.

The drought-stressed HQPM-7 seedlings pre-supplemented with P showed significantly high MDA content followed by HQPM-1, HKI-161 and HKI-193-1 (Figure 5(a4)). In HQPM-7 seedlings, the MDA content (with respect to control) increased from 0.4% at 12 h to 0.6% at 48 h, while in HQPM-1 seedlings, the MDA content decreased from 1.0% at 12 h to 0.2% at 48 h. In HKI-161 and HKI-193-1 seedlings, the MDA content increased to 0.3% and 0.2%, respectively, at 12 h and this level was maintained after 48 h of stress.

3.4.3. Proline Accumulation

Significant changes in the content of the osmolyte proline were observed during drought stress in all the genotypes (Figure 5b). When drought stress was provided to seedlings grown under control (water) conditions, high free proline accumulation (1882.6% at 24 h and 4841.5% at 48 h) was seen in the seedlings of genotype HQPM-1 (Figure 5(b1)). An increment in proline accumulation of 221.1% in HKI-161, 675.8% in HKI-193-1 and 976.0% in HQPM-1 was observed at 48 h, respectively, as compared to the control (Figure 5(b1)).

After N and/or P pre-supplementation, the genotypes HQPM-7 and HKI-161 showed pronounced free proline accumulation when drought stressed for 24 h or more (Figure 5b(2–4)). At 48 h of stress the proline levels were higher in HQPM-7 seedlings pretreated with NP (Figure 5(b2)) and P (Figure 5(b4)) and HKI-161 seedlings pretreated with N (Figure 5(b3)). Interestingly, both the genotypes showed nearly similar values at 24 h of stress. In HQPM-1, high levels of proline accumulation were seen at 48 h of drought stress in seedlings pretreated with N and/or P (Figure 5(b2–4)). In HKI-193-1 high levels of proline accumulation was seen at 48 h of drought stress in seedlings pretreated with N (Figure 5(b3)) and P (Figure 5(b4)).

3.4.4. Anthocyanin Content

Under control (watered) conditions the HQPM-1 seedlings showed the lowest levels of anthocyanin and were followed by HQPM-7, HKI-161 and HKI-193-1, in increasing order (Figure 5(c1)). Drought stress induced significant increase in the anthocyanin content in seedlings and the levels were also enhanced by the application of N and/or P in watered controls (Figure 5(c2–4)).

In the HKI-161 seedlings, the levels of anthocyanin showed a 5% change under increasing duration of drought stress. When HKI-161 seedlings were pre-supplemented with NP, the anthocyanin levels increased by 26% (Figure 5(c2)), but pretreatment with N caused an increase in the levels by 16% (Figure 5(c3)) and pretreatment with P caused an increase in the levels by 42.2% at 24 h and 28.5% by 48 h (Figure 5(c4)).

Under drought stress, the anthocyanin levels in HKI-193-1 increased by 17% at 48 h (Figure 5(c1)). Pretreatment with NP increased the levels by 16.1% at 24 h and 25.9% at 48 h under drought stress (Figure 5(c2)). Upon pretreatment with only N (Figure 5(c3)) or P (Figure 5(c4)), the anthocyanin levels in drought-stressed seedlings increased by 16% at 24 h. At 48 h of stress, further increase in levels was not observed in seedlings pretreated with N (Figure 5(c3)), but in seedlings pretreated with P, the anthocyanin levels increased by 19% (Figure 5(c4)).

The anthocyanin levels in drought-stressed HQPM-1 seedlings increased by 29.6% at 48 h (Figure 5(c1)). In seedlings pretreated with NP (Figure 5(c2)), N (Figure 5(c3)) and P (Figure 5(c4)) the levels increased by 24–26% after 48 h of drought stress.

In drought-stressed HQPM-7 seedlings, the levels increased by 10.6% at 12 h, to 49.4% at 48 h, respectively (Figure 5(c1)). Pretreatment with NP increased the levels by 13% at 24 h and 44.58% at 48 h drought stress, respectively (Figure 5(c2)). Pretreatment with N increased the levels by 51.5% at 12 h, 70.2% at 24 h and 80.1% at 48 h drought stress, respectively (Figure 5(c3)), while pre-treatment with P increased the levels by 12–13% only even after c48 h drought stress (Figure 5(c4)).

3.4.5. Gas Exchange Parameters

Drought-induced effects on the gas exchange parameters of the four maize genotypes were assessed by measuring the photosynthetic rate (PR), stomatal conductance (SC), transpiration rate (TR) and carbon dioxide concentration (CI). During drought stress without N and/or P pre-supplementation, the PR, SC and TR were reduced as compared to the control. The HKI-193-1 seedlings showed highest PR, SC and TR under both well-watered (without N and/or P) and drought-stressed conditions while HKI-161 and HQPM-7 showed the highest CI under these conditions (

Table 1. The effect of drought stress with and without N, P and NP pre-supplementation on the photosynthetic parameters of maize seedlings as measured by the using a portable open-flow gas exchange system (LI-6400, LI-COR).

	HKI-161				HKI-193-1				HQPM-1				HQPM-7
	PR	SC	CI	TR	PR	SC	CI	TR	PR	SC	CI	TR	PR	SC	CI	TR
D-C	5.43 ± 0.31	0.07 ± 0.001	248.40 ± 8.58	2.47 ± 0.02	12.03 ± 0.36	0.10 ± 0.009	197.54 ± 12.31	1.86 ± 0.14	6.30 ± 0.16	0.05 ± 0.0	172.84 ± 5.12	1.74 ± 0.002	6.36 ± 0.26	0.05 ± 0.003	156.40 ± 5.32	1.70 ± 0.08
12 h D	5.08 ± 0.09	0.03 ± 0.002	131.89 ± 14.77	1.15 ± 0.08	6.66 ± 0.46	0.04 ± 0.002	135.44 ± 9.77	0.80 ± 0.02	3.32 ± 0.14	0.02 ± 0.0	168.78 ± 9.18	0.90 ± 0.003	3.5 ± 0.13	0.03 ± 0.0	163.66 ± 6.04	0.97 ± 0.0
24 h D	2.93 ± 0.09	0.02 ± 0.001	164.65 ± 5.82	0.81 ± 0.03	5.36 ± 0.35	0.05 ± 0.002	211.66 ± 9.3	0.94 ± 0.03	2.37 ± 0.37	0.02 ± 0.0	223.62 ± 25.11	0.89 ± 0.016	1.8 ± 0.15	0.01 ± 0.0	195.04 ± 11.98	0.57 ± 0.01
48 h D	4.21 ± 0.22	0.01 ± 0.004	361.79 ± 172.53	0.54 ± 0.09	5.29 ± 0.09	0.06 ± 0.005	230.71 ± 14.84	1.14 ± 0.09	1.08 ± 0.11	0.02 ± 0.0	299.06 ± 8.91	0.78 ± 0.008	1.23 ± 0.26	0.03 ± 0.002	441.53 ± 23.13	1.04 ± 0.05
12 h NP-C	9.15 ± 0.5	0.05 ± 0.004	106.45 ± 16.96	1.9 ± 0.12	8.98 ± 0.52	0.08 ± 0.004	207.02 ± 2.62	1.91 ± 0.09	6.85 ± 0.05	0.07 ± 0.0	225.68 ± 1.32	2.55 ± 0.002	6.4 ± 0.06	0.04 ± 0.0	143.75 ± 3.57	1.59 ± 0.00.0.
12 h NP + D	8.72 ± 0.67	0.05 ± 0.004	109.44 ± 13.01	1.88 ± 0.16	9.10 ± 0.34	0.08 ± 0.005	201.78 ± 6.44	2.02 ± 0.11	7.25 ± 0.25	0.06 ± 0.0	180.34 ± 7.48	2.08 ± 0.005	0.65 ± 0.06	0.01 ± 0.0	296.68 ± 8.12	0.47 ± 0.01
24 h NP-Cont.	5.98 ± 1.77	0.05 ± 0.004	197.48 ± 43.81	1.70 ± 0.13	6.68 ± 0.26	0.06 ± 0.001	213.89 ± 5.51	1.66 ± 0.02	6.93 ± 0.06	0.14 ± 0.001	300.37 ± 0.88	4.97 ± 0.028	5.18 ± 0.20	0.03 ± 0.001	92.09 ± 6.85	1.07 ± 0.04
24 h NP + D	1.22 ± 0.27	0.007 ± 0.001	427.71 ± 119.6	0.27 ± 0.03	2.68 ± 0.24	0.03 ± 0.001	246.05 ± 9.58	0.87 ± 0.02	3.75 ± 0.09	0.03 ± 0.0	203.53 ± 05.75	1.22 ± 0.013	3.46 ± 0.67	0.02 ± 0.002	125.04 ± 18.13	0.77 ± 0.09
48 h NP-C	8.87 ± 0.96	0.03 ± 0.004	435.62 ± 205.07	0.96 ± 0.14	8.02 ± 0.45	0.06 ± 0.003	159.39 ± 15.25	1.67 ± 0.09	7.29 ± 0.08	0.14 ± 0.001	299.23 ± 1.16	4.37 ± 0.016	5.37 ± 0.17	0.04 ± 0.002	168.88 ± 5.31	1.56 ± 0.07
48 h NP + D	1.36 ± 0.27	0.02 ± 0.008	374.72 ± 44.93	0.91 ± 0.3	3.18 ± 0.5	0.02 ± 0.001	102.89 ± 29.46	0.49 ± 0.03	1.89 ± 0.28	0.03 ± 0.0	278.03 ± 14.01	1.06 ± 0.009	1.98 ± 0.04	0.02 ± 0.0	187.06 ± 3.75	0.63 ± 0.0
12 h N-C	7.09 ± 1.29	0.08 ± 0.014	225.48 ± 4.39	2.62 ± 0.42	8.46 ± 0.55	0.05 ± 0.005	126.94 ± 5.14	1.52 ± 0.12	6.63 ± 0.07	0.07 ± 0.0	240.23 ± 1.43	2.74 ± 0.006	6.46 ± 0.63	0.04 ± 0.006	142.36 ± 11.83	1.73 ± 0.22
12 h N + D	3.07 ± 0.2	0.02 ± 0.001	138.50 ± 12.79	0.79 ± 0.03	3.51 ± 0.42	0.02 ± 0.002	78.69 ± 14.67	0.50 ± 0.04	4.19 ± 0.22	0.04 ± 0.0	212.7 ± 8.18	1.44 ± 0.008	1.46 ± 0.09	0.02 ± 0.001	259.39 ± 3.96	0.76 ± 0.03
24 h N-C	9.97 ± 1.57	0.10 ± 0.019	194.35 ± 13.33	3.94 ± 0.72	8.53 ± 0.42	0.06 ± 0.006	143.87 ± 20.27	1.642 ± 0.15	6.71 ± 0.11	0.1 ± 0.0	271.92 ± 1.55	3.27 ± 0.011	6.19 ± 1.14	0.04 ± 0.004	148.99 ± 19.85	1.51 ± 0.14
24 h N + D	5.13 ± 0.48	0.05 ± 0.007	207.27 ± 14.6	2.17 ± 0.29	4.53 ± 0.34	0.02 ± 0.001	60.79 ± 10.29	0.60 ± 0.02	4.24 ± 0.34	0.04 ± 0.0	206.33 ± 13.19	1.41 ± 0.009	0.22 ± 0.04	0.02 ± 0.0	365.50 ± 6.82	0.71 ± 0.0
48 h N-C	4.01 ± 0.53	0.03 ± 0.003	197.77 ± 10.79	1.45 ± 0.13	6.90 ± 0.27	0.03 ± 0.001	66.47 ± 8.5	0.89 ± 0.03	5.62 ± 0.16	0.11 ± 0.0	295.29 ± 2.44	3.92 ± 0.006	1.37 ± 0.05	0.02 ± 0.0	273.74 ± 4.24	0.80 ± 0.0
48 h N + D	0.56 ± 0.09	0.008 ± 0.001	276.98 ± 14.44	0.26 ± 0.02	3.98 ± 0.27	0.02 ± 0.003	189.09 ± 50.65	0.45 ± 0.09	4.37 ± 0.08	0.03 ± 0.0	179.9 ± 4.61	1.27 ± 0.009	1.23 ± 0.11	0.02 ± 0.0	251.26 ± 12.06	0.60 ± 0.01
12 h P-C	5.74 ± 0.27	0.06 ± 0.002	222.36 ± 5.35	1.80 ± 0.06	7.06 ± 0.21	0.05 ± 0.001	177.30 ± 5.28	1.58 ± 0.02	7.33 ± 0.11	0.06 ± 0.0	186.89 ± 3.32	2.20 ± 0.005	6.93 ± 0.05	0.05 ± 0.0	141.79 ± 1.86	1.77 ± 0.0
12 h P + D	1.91 ± 0.4	0.03 ± 0.003	455.83 ± 59.29	1.01 ± 0.1	4.45 ± 0.57	0.03 ± 0.002	120.27 ± 22.9	0.82 ± 0.07	5.81 ± 0.1	0.09 ± 0.0	274.71 ± 1.91	3.22 ± 0.002	0.82 ± 0.10	0.01 ± 0.00	283.92 ± 11.22	0.53 ± 0.0
24 h P-C	5.60 ± 0.08	0.06 ± 0.001	225.62 ± 2.43	2.16 ± 0.04	5.94 ± 0.17	0.04 ± 0.001	152.97 ± 2.71	1.34 ± 0.03	4.29 ± 0.06	0.03 ± 0.0	180.08 ± 2.98	1.24 ± 0.002	3.7 ± 0.12	0.04 ± 0.0	230.49 ± 5.62	1.50 ± 0.0
24 h P + D	6.61 ± 0.2	0	42.29 ± 10.48	1.19 ± 0.07	2.25 ± 0.56	0.06 ± 0.003	348.87 ± 30.85	1.97 ± 0.09	4.69 ± 0.66	0.06 ± 0.006	252.46 ± 7.54	2.03 ± 0.194	3.77 ± 0.16	0.02 ± 0.0	122.83 ± 8.15	0.87 ± 0.01
48 h P-C	6.40 ± 0.39	0.05 ± 0.005	177.87 ± 5.88	1.97 ± 0.18	5.87 ± 0.19	0.04 ± 0.002	140.60 ± 5.33	1.45 ± 0.07	4.86 ± 0.23	0.06 ± 0.002	246.006 ± 2.21	2.06 ± 0.084	3.48 ± 0.05	0.03 ± 0.0	173.56 ± 4.38	1.0. ± 0.0
48 h P + D	1.47 ± 0.07	0.02 ± 0.002	378.49 ± 44.68	0.84 ± 0.08	3.04 ± 0.81	0.02 ± 0.004	223.71 ± 47.84	0.90 ± 0.13	2.78 ± 0.44	0.03 ± 0.003	252.18 ± 13.62	1.18 ± 0.120	3.18 ± 0.17	0.04 ± 0.0	255.87 ± 6.59	1.51 ± 0.0

The abbreviations used are drought (D); control (C); nitrogen (N) and phosphorous (P). The units of different measurements detailed above are photosynthetic rate (PR): µmol CO₂ m² s⁻²; stomatal conductance (SC): mmol H₂O m² s⁻¹; carbon dioxide concentration (CI): µmol CO₂ mole⁻¹; transpiration rate (TR): mmol H₂O m² s⁻¹.

).

In drought-stressed HKI-193-1, HQPM-1 and HQPM-7 genotypes, a significantly linear reduction in PR was observed under drought stress. In HKI-161 seedlings the SC also showed a linear reduction but in HKI-193-1 seedlings an increment in the SC was observed. The HQPM-1 seedlings showed similar values for SC at all the three stress time points, while HQPM-7 seedlings maintained the SC during increasing drought stress. The TR showed a linear reduction in HKI-161 and HQPM-1 seedlings, but in HKI-193-1 and HQPM-7 seedlings, the TR was reduced at the 12 h and 24 h time points and increased at 48 h stress. All the genotypes showed an exponential increase in the CI with increase in time period of drought stress.

Pre-supplementation with N and/or P under irrigated (control) conditions resulted in relative changes in the gas exchange parameters of the four maize genotypes as compared to water controls. In most cases the values obtained in NP were higher than those obtained for N or P alone. In HKI-161, when NP or N or P was supplied under control conditions, an increase in the PR, but decrease in the CI and TR was observed. There was increase in the SC values and these were similar in NP and N but higher in P. In HKI-193-1 seedlings, pre-supplementation of NP or N under control conditions caused an increase in the PR and decrease in the SC, CI and TR. In presence of P, a decrease in the PR and SC but increase in the CI and TR were observed. In HQPM-1 seedlings, the PR, TR and CI values showed an increase upon pretreatment of NP or N under control conditions. In presence of P, a decrease in the PR, CI and TR but no changes in SC were observed. In HQPM-7 seedlings, the PR, TR and CI values showed a decrease upon pretreatment of NP or N under control conditions. In these genotypes the values of SC did not show much change, but CI was less. In presence of P, an increase in the PR, CI and TR but no changes in SC were observed (Table 1).

When seedlings pre-supplemented with NP were exposed to drought stress, the HKI-161, HKI-193-1 and HQPM-1 seedlings showed a pronounced increase in PR at 12 h stress time points but it reduced with increasing stress exposure. In HQPM-7 seedlings, the PR showed a reduction at 12 h but increased slowly at 24 h and 48 h stress time points. The PR values were higher under drought stress in HQPM-1 and HKI-193-1 seedlings pretreated with N or P while negligible effect was seen in HQPM-7 seedlings pretreated with N. The HQPM-7 seedlings pretreated with P showed a pronounced reduction in PR at 12 h of drought stress but it increased with the increase in duration of stress. In HKI-161 seedlings pretreated with N, the PR was extensively reduced with the increase in drought stress, while the seedlings pretreated with P showed negligible changes in PR.

The HKI-161, HQPM-1 and HQPM-7 seedlings showed a significant increase in the SC during drought stress when pre-supplemented with N and/or P but the effect was more pronounced in the presence of P. In HKI-161, HKI-193-1 and HQPM-1, the CI values increased till 24 h of drought stress point but showed decrease at 48 h of drought stress, in the presence of NP. The HQPM-7 seedlings pretreated with NP showed reduction in the CI with increasing drought stress. When the seedlings were pretreated with P, the HKI-161 and HQPM-7 seedlings showed an increase in CI values till the 24 h of drought stress point but showed a decrease at 48 h of drought stress. The HKI-193-1 and HQPM-1 seedlings pretreated with P showed a reduction in CI with increasing drought stress.

The HQPM-1 seedlings pre-supplemented with N and/or P showed a higher score for TR under drought stress, but the effect was more pronounced in the presence of P. The seedlings of HQPM-7 showed exponential increase and decrease in the TR during drought stress when pretreated with P and N, respectively. The presence of NP caused an increase in TR until 24 h of drought stress but the rates declined at 48 h of stress. The seedlings of HKI-193-1 and HKI-161 showed an increase in the TR during drought stress when pretreated with P or N, but a decrease in TR when pretreated with NP (Table 1).

3.4.6. Antioxidant Enzyme Activities

During drought stress, the activities of enzyme ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) were measured. The activities of APX (Figure 6(a1)), CAT (Figure 6(b1)) and GR (Figure 6(c1)) were increased in all the genotypes in response to drought stress. The HKI-193-1 and HQPM-1 genotypes recorded highest (6.62%) and lowest (1.51%) APX activities, respectively, at 12 h of drought stress (Figure 6(a1)). After 48 h of stress, APX activity was maximum in HQPM-1 (52.42%) while minimum in HQPM-7 (4.54%). Pretreatment with N and/or P caused a further increase in APX activity in the drought-stressed seedlings. In presence of NP, HKI-193-1 showed minimum (9.58%) and HKI-161 showed maximum (31.8%) activities at 48 h of drought stress (Figure 6(a2)). In presence of N, HQPM-1 showed minimum (6.25%) and HKI-193-1 showed maximum (53.75%) activities at 48 h of drought stress (Figure 6(a3)). In presence of P, HKI-193-1 and HQPM-7 showed minimum (~28.0%) and HKI-161showed maximum (67.95%) activities at 48 h of drought stress (Figure 6(a4)).

The HKI-161 and HQPM-7 genotypes recorded highest (47.04%) and lowest (9.8%) catalase activities, respectively, at 12 h of drought stress (Figure 6(b1)). After 48 h of stress, catalase activity was maximum in HKI-161 (94.08%) while minimum in HQPM-1 (42.14%). Pretreatment with N and/or P caused a further increase in catalase activity in the drought-stressed seedlings. In presence of NP, HKI-161 showed minimum (21.56%) and HQPM-7 showed maximum (153.85%) activities at 48 h of drought stress (Figure 6(b2)). In presence of N, HQPM-1 showed minimum (39.2%) and HQPM-7 showed maximum (98.98%) activities at 48 h of drought stress (Figure 6(b3)). In presence of P, HKI-193-1 showed minimum (53.9%) and HKI-161showed maximum (108.78%) activities at 48 h of drought stress (Figure (6b4)).

The HKI-161 and HQPM-1 genotypes recorded highest (5.53%) and lowest (1.08%) GR activities, respectively, at 12 h of drought stress (Figure (6c1)). After 48 h of stress, GR activity was maximum in HKI-161 (12.69%) while minimum in HQPM-1 and HQPM-7 (~7%). Pretreatment with N and/or P caused a further increase in GR activity in the drought stressed seedlings. In presence of NP, HQPM-1 showed minimum (5.1%) and HKI-193-1 showed maximum (18.65%) GR activities at 48 h of drought stress (Figure (6c2)). In presence of N, HQPM-7 showed minimum (3.47%) and HKI-161 showed maximum (11.06%) GR activities at 48 h of drought stress (Figure (6c3)). In presence of P, HQPM-1 showed minimum (1.52%) and HKI-161showed maximum (34.27%) GR activities at 48 h of drought stress (Figure 6(c4)).

3.5. Drought Recovery

The seedlings exposed to drought stress for 17 days were watered for one week and monitored for recovery. After one week of rewatering, all the genotypes showed drought recovery (Figure 7) but seedlings of HKI-161 and HKI-193-1 genotypes showed rapid transition to flowering stage. During drought recovery, the HKI-161 seedlings that were not pre-supplemented with nutrients and/or pre-supplemented with only P showed rapid transition to tasseling and silking stages (Figure 7a). Seedlings pretreated with NP and N showed comparatively late transition to flowering during recovery (Figure 7b). Tassels emerge when the plants reach maturity and begin to shed pollen. The tasseling stage is followed by the silking stage, when silk hairs emerge from the ear to receive pollen to begin the process of fertilization. The HQPM-1 seedlings that received N treatment moved to flowering stage (Figure 7c) while HQPM-7 seedlings showed only normal vegetative growth under all treatment conditions (Figure 7d).

3.6. Differential Expression of Drought Responsive Genes in Maize

A total of 2654 and 3032 transcripts were identified on comparing data of tolerant and susceptible maize genotypes, under drought stress and drought recovery (rewatering), respectively. About 2016 transcripts were expressed in both the genotypes and these were differentially expressed under drought stressed and recovery conditions (Figure 8a). It was seen that drought stress induced the expression of 800 transcripts specifically in the susceptible genotype and 452 transcripts specifically in the drought stress-tolerant genotype (Figure 8a). During drought recovery, 152 transcripts were expressed in the drought-tolerant genotype and 109 in the susceptible genotype (Figure 8a).

Transcripts that were differentially regulated during drought recovery after rewatering were identified and compared across the tolerant and susceptible genotypes (Figure 8b). This analysis showed that 155 transcripts were upregulated and 162 transcripts were downregulated in the drought-tolerant genotype, while 273 transcripts were upregulated and 203 transcripts were downregulated in drought-susceptible genotype. It was observed that 165 transcripts were downregulated while 155 transcripts were upregulated in both genotypes during drought recovery. Interestingly, four transcripts were downregulated in the drought-tolerant genotype but upregulated in the drought-susceptible genotype during the recovery phase.

During drought recovery on rewatering, about 34 transcripts (15 were upregulated and 19 downregulated) were expressed in the tolerant genotype while about 62 transcripts (37 upregulated and 25 downregulated) were expressed in the drought-susceptible genotype (Figure 9a and Supplementary File S1). Heatmap analysis of the DEGs also revealed that most of the transcripts in both the tolerant and sensitive genotypes were expressed during severe drought stress Transcripts (GRMZM2G008108, GRMZM2G016312, GRMZM2G018375, GRMZM2G028665, GRMZM2G067702, GRMZM2G074097, GRMZM2G103945, GRMZM2G107101, GRMZM2G148453, GRMZM2G150248, GRMZM5G815358, GRMZM6G182520, GRMZM6G423719, GRMZM2G047368, GRMZM2G068557, GRMZM2G081843, GRMZM2G093096, GRMZM2G145041, GRMZM2G302245, GRMZM2G326270 and GRMZM5G863645) showed similar change in expression pattern (up or down regulation) with subtle difference in the fold change in both the genotypes (Supplementary File S1). Based on the expression profiles the DETs could be grouped into 10 clusters (Figure 9b).

3.7. GO and KEGG Pathway Analysis of Drought Responsive Transcripts

To define their biological function, the DETs in response to drought stress, were sorted into GO term categories. It was found that the DETs were clustered into 810 GO terms in the cellular component, 179 in molecular function and 866 GO terms in the biological process categories (Figure 10). Within the biological process category, GO terms such as nucleosome assembly, macromolecular complex assembly, chromosome organization, cellular component assembly, response to hydrogen peroxide, response to stress, DNA metabolic process and others were highly significant. Within the cellular component category, nucleosome, chromosome, extracellular region and others and in the molecular function, protein heterodimerization activity and others with their respective order were most significant terms (Supplementary File S2).

The drought-responsive DETs were associated with a total of 172 KEGG pathway terms (with 73 in the tolerant and 98 in the susceptible genotypes) while the DETs identified under drought recovery associated with 21 KEGG terms (5 in the tolerant and 16 in the susceptible). The statistical analysis indicated that most of the transcripts were related to metabolism, followed by environmental information processing. The drought-downregulated transcripts mapped to the pathways involving fatty acid elongation (2.69 × 10⁻⁴), biosynthesis of secondary metabolites (9.10 × 10⁻³), pentose and glucuronate interconversions (6.34 × 10⁻³), limonene and pinene degradation (5.51 × 10⁻⁴), phagosome (6.18 × 10⁻³), nucleotide excision repair (4.14 × 10⁻³) and benzoxazinoid biosynthesis (1.04 × 10⁻³). The drought-upregulated transcripts mapped to the pathways involving alanine, aspartate and glutamate metabolism (7.91 × 10⁻³), protein processing in endoplasmic reticulum (3.93 × 10⁻³), DNA replication (7.57 × 10⁻³), homologous recombination (6.13 × 10⁻³) and mismatch repair (4.83 × 10⁻³) (Supplementary Figure S1 and Supplementary File S3).

The KEGG IDs of DETs during drought stress and recovery were used to identify the transcripts involved in nitrogen and phosphate metabolic pathways, respectively. The nitrogen responsive GRMZM2G376957 (Histone 3.2) was downregulated in both drought-tolerant and -susceptible genotypes during drought stress. In plants showing recovery after drought stress, GRMZM2G074097 (thiamine thiazole synthase 2, chloroplastic) and GRMZM2G150248 (lysine-specific histone demethylase 1) were downregulated in the susceptible genotype and the transcripts GRMZM2G424857 (uncharacterized protein), GRMZM2G028665 (uncharacterized protein), GRMZM2G018375 (thiamine thiazole synthase 1, chloroplastic), and GRMZM2G150248 (lysine-specific histone demethylase 1) were downregulated in the tolerant genotype. The DETs such as GRMZM2G326270, GRMZM2G043191 and GRMZM2G071630 were predicted to be involved in phosphate signaling. Phosphate-responsive transcripts such as GRMZM2G155253 (Histone H3.2) GRMZM2G071630 (Glyceraldehyde-3-phosphate dehydrogenase) were downregulated in the sensitive genotype during drought stress while GRMZM2G155242 (myo-inositol-1-phosphate synthase) was upregulated in the sensitive genotype during recovery after rewatering (Supplementary Files S1–S3). The KEGG analysis results showed that these DETs in the nitrogen metabolic pathways grouped under the GO categories of biological process (cellular nitrogen compound metabolism) and molecular function (glyceraldehyde-3-phosphate dehydrogenase-NAD+ phosphorylating activity), while the DETs in the phosphate metabolism were involved in the oxidative and reductive phosphate pentose pathway and inositol phosphate metabolism (Figure 11).

4. Discussion

Climate change is causing a detrimental effect on the agriculture sector [1,3]. The increase in global temperatures is aggravating the effect of water shortage and hampering crop production and food security at large [1,39]. Among the different abiotic stresses, drought stress has become a major cause of concern for cultivated crops including maize [6,40]. Drought stress severely affects seed germination potential and inhibits all the aspects of a plant’s growth and development at the seedling stage [10]. Plants have evolved various response mechanisms to tackle ever-changing environmental conditions and stresses [41]. Each species has specific and well-monitored sets of response pathways to survive in harsh conditions [42]. Several reports have shown the role of externally applied N or P in alleviating plant stress [21,25]. N has been shown to positively influence photosynthesis and related pathways in a genotype-dependent manner. It was reported that N pre-supplementation stimulated leaf growth via the synthesis of proteins involved in cell division and growth [43,44,45]. P is also one of the vital components of plant cell and the genetic material. It is involved in photosynthesis, energy transfer and nutrient translocation [46,47,48]. Therefore, in this report, we presented the impact of drought-stress on maize seedlings of HKI-161, HKI-193-1, HQPM-1 and HQPM-7 genotypes, grown in presence and absence of N and/or P.

We observed that seeds of all four genotypes showed no germination during drought, but in control conditions, germination started after 48 h. The germination percentage varied from an average of 71% in HKI-161 and HKI-193-I to 100% in HQPM-1. The four maize genotypes showed variations in growth potential under individual and combined application of N and/or P in water controls. The HQPM-1 seedlings had the longest shoot and root lengths while HKI-161 had shortest shoots and HQPM-7 seedlings had shortest roots. In all the genotypes (except HQPM-7), percentage of germination was higher but root and shoot length was lower in presence of N and/or P, as compared with the water-irrigated seedlings. The difference in the seed germination potential and the shoot and root growth morphologies might be associated with variations in response of genotypes and their nutrient utilization efficiency [45,46,49]. Seedlings of all the genotypes pretreated with P and the HKI-161, HKI 93-1 and HQPM-1 genotypes pretreated with N showed better root growth than NP pretreatments. This is in accordance with earlier observations that P and N can significantly increase root development [50,51,52]. The role of N and/or P on the shoot increment of the maize genotypes was also explored. The application of N and NP resulted in increased seedling height in all genotypes. This was in accordance with earlier reports that N alone and in combination with P can enhance growth in a variety of plants [11,50,52,53,54]. This also indicates that plants of different genotypes might have different nutrient preferences and up-taking mechanisms [55,56,57].

During drought stress, seedlings of all the genotypes exhibited decrease in shoot length but extension of roots as compared to their control counters. Earlier research has shown that the increase in the root area, biomass and length during drought stress is to facilitate search for water and nutrients [58,59,60]. Drought stress also caused reduction in leaf area and this response was mainly to tackle the loss of water [7,18]. Pre-supplementation with N and/or P caused variable changes in the shoot and root lengths of the drought-stressed seedlings. The application of N and/or P also increased the stem width and leaf area [61,62,63]. The maximum changes were observed in the HQPM-1 and HK-161 seedlings. N and NP pre-supplementation caused greater increase while variable results were observed upon providing only P. This might mainly be due to the role of N in stem growth [61]. The difference in genotypic physiology may result in variations in response in different plant species. For instance, N alleviates drought stress in Leymus chinensis [64], Chinese fir [65], maize [25] and other grasses [66] whereas P application improves drought stress tolerance in soybean [67]. N and P together were found to limit growth of maize and other plants grown under drought stress [13,22].

Drought stress affected the biochemical properties of the maize seedlings. During drought stress, the LRWC was reduced, possibly due to decline of water absorption, water and nutrient assimilation and reduction in photo-assimilation potential. The negative impact of drought on plant water relations has been observed in different plant species [6,68]. The application of N and/or P was reported to increase the LRWC under drought stress [12,69]. In our study, HQPM-1 showed a positive response to N and/or P by displaying improved LRWC under drought stress. In the other three genotypes, the drought-stressed seedlings of the control (without N and/or P) showed better LRWC than the nutrient pre-supplemented ones. This might be due to the difference in the water retention potential and the physiology of the maize genotypes [70]. To understand the difference in responses a number of physiological and biochemical investigations were performed.

It has been reported that the total chlorophyll content is reduced under drought stress and the effect gets more pronounced with increase in the degree of stress [71,72]. Under water control conditions, the genotype HQPM-1 showed the highest total chlorophyll content (21.14 μg/mL), but with increase in duration of drought stress, its chlorophyll content fell to the lowest minimum. In the other three genotypes, nearly the same levels of total chlorophyll content were observed under control (~15 μg/mL) and drought-stressed conditions (~9 μg/mL at 48 h of drought stress). N and/or P pre-supplementation caused an increase in chlorophyll content in all maize genotypes under study. The maximum chlorophyll content was recorded in drought-stressed seedlings pre-supplemented with NP and the genotypes HQPM-7 and HQPM-1 exhibited the highest values. The enhanced chlorophyll levels were also visible in seedlings pretreated with N and P, respectively, though the levels in N > P. The improvement in chlorophyll content by nutrient application has been reported earlier [55,73]. Higher chlorophyll content is indicative of higher photosynthetic capacity, thereby indicating tolerance to drought [73,74]. The increase in chlorophyll pigments during stress has an advantage to plants, as they assist the ROS scavenging pathways for maintaining the photosynthetic balance [74,75].

The lipid peroxidation content of all the genotypes was increased under drought stress. ROS accumulation can result in the increase of membrane lipid peroxidation leading to higher MDA levels, which is used as an indicator of oxidative damage [75]. The genotypes HQPM-1 and HKI-193-1 showed higher MDA content during drought stress when pretreated with NP while genotypes HQPM-7 and HKI-161 showed higher MDA content during drought stress when pretreated with only N. The application of only P has a relatively less effect on the MDA peroxidation score in all the genotypes. Plant species showed variations in response to nutrient application under drought stress. Earlier studies showed that the application of N and NP increased the lipid peroxidation potential during drought stress, but P alone has lesser effect in increasing MDA content [65,71,75,76].

Drought also causes the accumulation of proline in plants [77] as a mechanism to increase osmotic stress tolerance [77,78,79,80]. Proline increases stress tolerance by protecting photosynthetic activities, quenching the ROS and acting as an osmoprotectant [79,81,82]. The roles of N and P in alleviating drought stress by increasing proline accumulation have been reported in different plants [20,83,84]. A statistically significant increase in the proline accumulation was also observed in drought-stressed maize seedlings of all four genotypes and maximum accumulation seen in HQPM-1. However, when the seedlings were pretreated with N and/or P, a reduction in proline content was observed. HKI-161 and HQPM-7 showed a manyfold increase in proline when supplemented with nutrients before drought stress. These variations in maize genotypes in response to drought stress in presence or absence of N and/or P might be associated with the genotypic physiologies [42,83].

Studies have shown that during drought and other abiotic stress the anthocyanin accumulation increases as a measure to check the oxidative damage and improve the rate of photosynthesis [8,85,86]. The role of nutrient application in increasing the anthocyanin accumulation was also reported [87]. An increase in anthocyanin content was also recorded in all the genotypes under all treatments. The magnitude of increment was more pronounced with the increasing duration of stress. The genotypes HQPM-7 and HKI-161 showed higher levels of anthocyanin pigment under N and P, respectively. The main role of anthocyanins in mediating stress responses are associated with their antioxidant properties for ROS detoxification [88]. The stress-induced anthocyanin accumulation has been detected in many plants [89]. The generation of ROS signals the activation of specific transcription factors, which activate the expression of anthocyanin biosynthesis genes [90].

The variations in the morphological, physiological, biochemical and molecular characters of plants are reflected as differences in their photosynthetic potential [91,92,93]. The four maize genotypes also showed differences in the photosynthetic gas exchange parameters during controlled and drought-stressed conditions with and without N and/or P pretreatment. The photosynthetic rate of HKI-193-1 and HKI-161 genotypes was higher while HPQM 1 and HQPM-7 was lower during drought stress without N and/or P pre-supplementation. However, an opposite response was observed with seedlings pre-supplemented with individual and combined N or P. In drought-stressed seedlings, an increase in SC was seen in HKI-161 and HQPM-7 seedlings pre-supplemented with P, and HQPM-1 seedlings pretreated with N and/or P. However, with N and/or P pre-supplementation, HQPM-1 also showed a high TR and the effect was more pronounced in the presence of only P.

Drought stress pointedly affected the gas exchange parameters in plants [94,95]. The stomata play a vital role in monitoring the evapotranspiration by which they close or limit the boundary and save water during stress. Several earlier reports have shown that the optimum nutrient pre-supplementation increased photosynthetic efficiency under normal and stress conditions [96,97,98]. The augmentation in PR may be due to an increase in the rate of formation of chlorophyll pigment and supply of minerals for proper leaf functioning. This is influenced by the leaf SC, the rate at which CO₂ is entering, and TR, the rate of water exiting out of the leaf via regulating the opening or closing of the stomata [99,100,101,102].

During drought stress, plant species reduce their SC to avoid water loss [103,104], leading to an increase in CI and decrease in TR [95,103]. SC and TR determine the water holding capacity and photosynthetic potential of leaves [103,105]. During stress, the leaf SC increases until certain threshold, which might increase the PR [103,105]. An increase in SC during extreme heat can lead to drought induced death of susceptible plant species [106]. The level of increase is dependent on the genotypes and the maturity level of the leaf [70]. The increase in TR might be associated with the susceptibility of the genotypes for drought stress while those showing decrease in TR could have better tolerance capacity [107]. In mulberry seedlings, the increase in CO₂ concentration was reported to enhance water use efficiency and PSII function [108]. Similar report was presented in maize, indicating the improvement of PSII function with increasing CO₂ during drought stress [109,110].

In all four maize genotypes, a reduction in SC and TR and an increase in CI were observed during drought stress without N and/or P pre-supplementation. Under drought conditions, HKI-193-1 and HQPM-1 under N and NP pre-supplementation showed a decrease in TR with increasing drought stress, whereas all the drought stress genotypes pre-supplemented with P showed more TR. The difference in the gas exchange parameters of the maize genotypes under different set of nutrient treatment might be associated with the flexibility in the drought stress tolerance scale, concentration of nutrients applied, as well as genotypic differences. Drought stress reduces the availability of nutrients, and the pre-supplementation of nutrients aids in plant growth under stressful conditions by reducing the rate of transpiration [111,112]. In our study, similar results were obtained, and the differences observed between genotypes might be due to genotypic variability to nutrient application [70,113,114].

Drought stress increased the activity of antioxidant enzymes [115,116]. An increase in the APX, CAT and GR enzymes was observed in all the treatment groups of all the genotypes. All the genotypes showed nearly similar APX content, but HQPM-1 without N and/or P pre-supplementation and HKI-161 under P pre-supplementation showed more APX accumulation. The high accumulation of CAT was experienced by the HKI-193-1 genotype under drought stress without N and/or P pre-supplementation and drought stress seedlings pre-supplemented with N, HQPM-7 under drought stress pre-supplemented with NP and HKI-161 and HKI-193-1 pre-supplemented with P. The magnitude of increment significantly correlated with increase in drought stress time points. Moreover, the drought-stressed HKI-161 genotype under all treatments showed the highest GR activity. During drought stress, the higher production of H₂O₂, ROS, superoxides and radicals causes obstructive damage to the cellular structures and retards plant growth and development. The increase in these antioxidant enzymes helps plants to detoxify H₂O₂ and scavenge the ROS in order to facilitate stress tolerance [75,117,118,119,120,121,122]. It is evident that CAT [123,124], APX [122,124] and GR [125,126,127] play a vital role in abiotic stress tolerance by maintaining their intracellular pool especially functions as an antioxidant.

In this study, the drought-stressed plants were rewatered for one-week and the recovering plants exhibited a clear drought escape mechanism like early flowering. The HKI-161 genotype, the control sets of drought stressed seedlings and those pre-supplemented with P showed early flowering, tasseling, and silking. In the HKI-193-1 genotype, the control sets of drought-stressed seedlings and those pre-supplemented with NP and P also showed early flowering. In the HQPM-1 genotype, the N pre-supplemented drought-stressed seedlings showed relatively late flowering. It has been shown that plants might use early flowering and stage transition as a mechanism to avoid drought stress [128,129].

Drought stress is a quantitatively controlled trait, so the adaptive and escape responses shown by plants associate with various morphophysiological and molecular pathways. The cellular mechanisms involve regulation of a cascade of gene expression at different stages of growth and development [41]. The microarray data analysis showed that several transcripts were differentially regulated in response to drought stress and during recovery. Drought stress specifically induced the expression of 800 transcripts in the susceptible genotype and 452 transcripts specifically in the drought stress-tolerant genotype, while during drought recovery, 152 transcripts were expressed in the drought-tolerant genotype and 109 in the susceptible genotype. During drought stress, the transcript GRMZM2G345700, coding for 36.4 kDa proline-rich protein was downregulated, and seven transcripts, namely GRMZM2G148272 (putative uncharacterized protein (GRMZM2G148272_T01)), GRMZM2G154747 (plasma membrane associated protein (GRMZM2G154747_T01)), GRMZM2G305446 (aquaporin TIP3-1 (GRMZM2G305446_T02); aquaporin TIP3-1 (GRMZM2G305446_T01), uncharacterized protein (GRMZM2G061450_T01), late embryogenesis-abundant protein, group 3 (GRMZM2G096475_T01), uncharacterized protein (GRMZM2G041039_T01), ferredoxin-6 (GRMZM2G063126_T01) were upregulated with a more than sixfold change in expression in the drought-susceptible genotypes. The transcripts coding for a plasma membrane-associated protein (GRMZM2G154747_T01), ferredoxin-6 (GRMZM2G063126_T01), late embryogenesis abundant protein group 3 (GRMZM2G096475_T01) and an uncharacterized protein (GRMZM2G061450_T01) were significantly upregulated during severe drought stress in the tolerant genotype.

The transcripts showing upregulation implied their significant role in the drought stress response. For instance, aquaporins, which may exist in different isoforms, play a role in stress response by controlling transport of water and other molecules in the membrane region [130,131,132]. Late embryogenesis-abundant (LEA) proteins and membrane-associated proteins are reported to protect plants from desiccation due to drought stress [133,134,135,136]. Plant-based ferredoxins are also important in mitigating drought stress by acting over the photosynthesis process. They transfer electrons from Photosystem I to ferredoxin NADP (+) reductase in which NADPH is produced and is then utilized for CO₂ assimilation. It also interacts with several enzymes in the system to drive the metabolic process [137,138]. Most of the stress-regulated transcripts are categorized under the GO term biological process. In the category of molecular function, the enzymatic and biosynthesis pathways were mostly expressed.

According to KEGG enrichment analysis, the genes coding for the biosynthesis of secondary metabolites, protein processing in the endoplasmic reticulum, carbon metabolism, nitrogen metabolism and others showed high expression in the stress-tolerant genotype. When exposed to stress, plants accumulate certain metabolites to withstand and survive the stressful condition [77,85]. The accumulation of metabolites helps plants to grow and survive under stress [40,84]. The KEGG terms of the pentose phosphate pathway and inositol phosphate metabolism were more expressed in the susceptible genotype during stress and rewatering, respectively. It was reported that the activities of G-6-PDH and 6-PGDH in the pentose phosphate pathway could be involved in the survival mechanisms used by plants during drought [139]. Inositol trisphosphate can act as a secondary messenger in plants under stress affecting drought tolerance, carbohydrate metabolism and phosphate-sensitive biomass increase [140,141].

The KEGG IDs of DETs during drought stress and recovery identified the transcripts involved in nitrogen and phosphate metabolic pathways, respectively. The DETs were associated with various aspects of cellular nitrogen and phosphate metabolism, phosphate pentose pathway and inositol phosphate metabolism. It was observed that nitrogen-responsive lysine-specific histone demethylase 1 (GRMZM2G15024) was downregulated in the tolerant genotype. It has been reported that the dimethylation of histone H3 at lysine 9 (H3K9me2) is associated with heterochromatinization and transcriptional gene silencing in plants. The role of H3K9 methylation in drought-stress responses remains unclear [142]; still, reduced H3K9me2 levels indicate activation of gene expression in response to drought stress. In rice plant, the OsJMJ703 (lysine-specific histone demethylase 1) mutant showed enhanced tolerance to drought stress [143]. The phosphate-responsive glyceraldehyde-3-phosphate dehydrogenase transcripts (GRMZM2G071630) were downregulated in the sensitive genotype during drought stress. The glyceraldehyde-3-phosphate dehydrogenases are ubiquitous proteins that play important roles in plant metabolism and stress response. These enzymes were shown to mitigate drought stress in wheat by triggering ROS detoxification and stomatal closure [144].

In plants showing recovery after drought stress, the thiamine thiazole synthase (GRMZM2G074097) was upregulated. The role of thiazole synthase has been reported in guard cells in response to ABA and drought response in Arabidopsis [145]. Arabidopsis plants overexpressing the thiamine thiazole synthase gene showed an activation of stomatal closure and reduced rate of water loss for enhanced drought tolerance [145]. In maize, this gene was activated in response to cold stress and phosphorus deficiency [146]. The phosphate-responsive myo-inositol-1-phosphate synthase (GRMZM2G155242) was upregulated during drought recovery. The enzyme catalyzes a key rate limiting enzyme in myo–inositol biosynthesis. The myo-inositol-1-phosphate synthase gene has been shown to improve tolerance to abiotic stresses in several plant species. In sweet potato, the gene IbMIPS1 had a positive role in salt and drought stress tolerance and stem nematode resistance [147]. It was also reported that myo-inositol-1-phosphate synthase is crucial for mediating ethylene response and regulating the growth and heat stress response in wheat [148].

5. Conclusions

Drought stress severely affects maize seedlings, but the response is varied across genotypes (HKI-161, HKI-193-1, HQPM-1 and HQPM-7). Drought-stressed seedlings exhibited a reduction in shoot length and increase in root length. Pre-supplementation with N and NP increased the height, root length, stem width and leaf area of maize seedlings under irrigated conditions. During drought stress the seedlings of HKI-161, HQPM-1 and HQPM-7 genotypes pre-supplemented with NP, showed no significant change in shoot length while HKI-193-1 seedlings showed an exponential decrease. However, in all these seedlings, a significant increase in root length, stem width and leaf area were seen.

Under drought stress, the chlorophyll content was reduced in all the genotypes. Individual and combined application of N and P greatly increased the total chlorophyll content during drought stress. The MDA content of all the genotypes was higher during drought stress as compared to the control (regularly watered) counterparts. The drought-stressed seedlings of HQPM-1 and HQPM-7 genotypes showed very high MDA content as compared to the nutrient pre-supplemented counterparts. In contrast, the HKI-161 seedlings pre-supplemented with N and NP showed increased MDA peroxidation at 48 h of drought stress. The proline levels were higher in seedlings of HQPM-1 genotype under drought stress but after individual N and P pre-supplementation, drought-induced proline accumulation was seen in HKI-161 and HQPM-7. Positive effects of NP pretreatment on anthocyanin content, gas exchange parameters and antioxidant enzymes (APX, CAT and GR) was observed in the drought stressed seedlings of all the genotypes. The N and/or P pretreated seedlings also exhibited faster transition in developmental stages during drought recovery and the effect was more pronounced in HKI-161 and HKI-193-1.

Transcript analysis was performed to screen for differential gene regulation in drought-tolerant and -susceptible genotypes. The DETs were searched for transcripts coding for proteins in P and N metabolism and this analysis showed that transcripts related to P metabolism were expressed during stress and recovery phases in the susceptible genotype while transcripts related to N metabolism were significantly expressed upon drought recovery in the tolerant genotype. The findings provided a glimpse into the role of N and P addition on drought response of maize seedlings and identified key modules for further experimentation to understand and improve the genetic response of plants to drought stress.