Introduction

Water stress is the major problem in agriculture and the ability to withstand such stress is of immense economic importance. Water stress tolerance involves subtle changes in cellular biochemistry. It appears to be the result of accumulation of compatible solutes and of specific proteins that can be rapidly induced by osmotic stress (Rhodes 1987). The numerous physiological responses of plant-to-water deficits generally vary with the severity, as well as the duration, of water stress (Mathews et al. 1984; Weber and Gates 1990; Rose et al. 1993; Li 2000; Correia et al. 2001; Pane and Goldstein 2001; Pita and Pardes 2001; Weigh 2001; Shao and Chu 2005; Shao et al. 2005, 2006b, 2007a, b, 2008a, b, c). One of the most important abiotic stresses for crop yields concerns about plant dehydration (Shao et al. 2006b; Boudsocq and Lauriere 2005; Mittler 2006). Plants suffer from dehydration under the condition of drought, high salinity, and low temperatures, all of which cause hyper-osmotic stress characterized by a decreased turgor pressure and water loss (Shao et al. 2003, 2006a, 2007b; Sun et al. 2005; Munns 2005; Humphreys et al. 2006).

Drought stress is well known as a major environmental stress that restricts the growth and yield of crop plants worldwide and can trigger a series of physiological and biochemical responses in plants (Yue et al. 2012). The changes of environment and global climate, drought stress not only seriously affects plant development and evolution, but also reveals the ecological equilibrium of the ecosystem (Zhao et al. 2011; Shi et al. 2014). Drought is a complex physical–chemical process, in which many biological macromolecules and small molecules are involved, such as nucleic acids (DNA, RNA, microRNA), proteins, carbohydrates, lipids, hormones, ions, free radicals, and mineral elements (Munns 2002; Casson and Lindsey 2003; Casati and Walbot 2004). In addition, drought is also related to salt stress, cold stress, high temperature stress, acid stress, alkaline stress, pathological reactions, senescence, growth, development, cell cycle, UV-B damage, wounding, embryogenesis, flowering, signal transduction, and so on (Zhu 2002; Jiang and Zhang 2004). Therefore, drought is connected with almost all aspects of biology. Currently, drought study has been one of the main directions in global plant biology and biological breeding. Plants sense the loss of water and adapt the drought stress which mainly dependent on the regulated expression of drought-related genes.

The expression of various stress response genes is mediated by a number of transcription factors. In the transcription factors interact with specific cis-acting elements existing in target gene promoters and regulate elaborate networks of drought-responsive genes and associated biochemical and physiological adaptations (yang et al. 2014). Most of these adaptation responses are determined by several secondary messengers including some plant hormones (Qin et al. 2011; Du et al. 2013). Diverse sets of genes expressed in response to abiotic stress have been identified, and elucidation of their physiological or cellular roles in terms of either stress tolerance or sensitivity is a critical issue in current plant biology (Huang et al. 2012; Jirschitzka et al. 2013). It is essential to study the functions of stress-responsive genes to ameliorate stress tolerance in crop plants (Cabello et al. 2014; Ismail et al. 2014).

Maize (Zea mays L.) is one of the most important crops in the world (Kresovic et al. 2014). It was first domesticated in South-West Mexico about 6000 years ago. With more than 880 million tons produced in 2011, it is now the crop with the largest production and constitutes the third most important staple crop for human after rice and wheat. Within the European Union, it ranks second in terms of volume of production after wheat—but third in harvested area only to wheat and barley (FAOSTAT 2013). The Maize production in these regions plays a significant role in ensuring food security, but their climates vary dramatically, with the annual accumulated temperature above 10 °C, total sunshine hours, and total precipitation of 2000–4700 °C day, 2100–2900 h, and 3400–800 mm, respectively (Li and Wang 2010). These climatic differences have a significant influence on maize growth and development (Liu et al. 2013).

Triazole compounds have both fungitoxic and plant growth-regulating properties. In addition, they can also protect plants against various environmental stresses. Triazoles affect the isoprenoid pathway and alter the levels of certain plant hormones by inhibiting gibberellin synthesis, reducing ethylene evolution, and increasing cytokinin levels (Jaleel et al. 2008). Plant growth regulators play vital roles in coordination of many growth and behavioral processes in rice, which regulates the amount, type, and direction of plant growth (Anjum et al. 2011). Drought stress amelioration by triazole compounds is of major research interest, because, these compounds have innate potentiality for increasing antioxidant enzymes and molecules in oxidative stressed plants (Fletcher et al. 2000).

Materials and methods

The hybrid seeds of maize variety NK-6240 were obtained from Syngenta India limited and employed for this investigation; Plastic pots of 40 cm diameter and 45 cm height size were used for culture study. The pots were filled with 10 kg of soil mixture containing red soil; sand and farm yard manure in 1:1:1 ratio. Two hundred and fifty pots were arranged in Completely Randomized Block Design (CRBD). Single lot of 50 pots was kept as control, three sets of 150 pots were used for drought with triazole treatment at 4 days interval, and the remaining one set was kept in drought treatment in order to impose drought stress at 4 days interval. Triadimefon (15 mg l−1), Tebuconazole (10 mg l−1), and Propiconazole (15 mg l−1) were used to determine their effect on Zea mays. The treatments were given as soil drenching, 30 days after sowing (DAS). The plants were left for 30 DAS with alternative day irrigation. From day 30 to 60, control plants were irrigated on every alternative day, drought treated, and drought with triazole-treated plants were irrigated at every 4 days interval. After drought treatment, all the pots were irrigated on alternative day and it last up to harvest. Plants were uprooted randomly on DAS 40, 50, and 60, washed with water, and separated into root, stem, and leaf for estimating photosynthetic pigments and biochemical constituents.

Estimation of photosynthetic pigments

Estimation of chlorophyll and carotenoid contents

Chlorophyll and carotenoid contents were extracted from the leaves and estimated according to the method suggested by (Arnon 1949).

Extraction

Five hundred milligram of fresh leaf material was ground with 10 ml of 80 % acetone at 4 °C in a pestle and mortar and centrifuged at 2500 g for 10 min at 4 °C. The residue was re-extracted with 80 % acetone until the green color disappeared in the residue and the extracts were pooled and transferred to graduated tube and made up to 20 ml with 80 % acetone and assayed immediately.

Estimation

Three milliliters aliquots of the extract were transferred to cuvette and the absorbance was measured at 645, 663, and 480 nm with a spectrophotometer (U-200 1-Hitachi) against 80 % acetone as blank. Chlorophyll content was calculated using the formula of Arnon 1949 and carotenoid content was calculated using the formula of Kirk and Allen 1965 and were expressed in milligram per gram fresh weight.

Estimation of anthocyanin content

Anthocyanin content was extracted and estimated by the method of Zhang and Quantick 1997.

In a pestle and mortar, 500 mg of tissue was ground with 10 ml of 1 % methanol and repeated three times. The homogenate was centrifuged at 19,000 g for 15 min. The resultant supernatant was diluted with 1 % HCl–methanol to 50 ml. The absorption of diluents was measured at 530 nm. The anthocyanin contents were expressed in mg/gram fresh weight.

Estimation of biochemical constituents

Estimation of soluble protein content

According to the (Bradford 1976) soluble protein was estimated.

Extraction

One gram of plant material was ground in mortar and pestle with 20 ml of 20 % trichloro acetic acid (TCA). The homogenate was centrifuged for 15 min at 800 rpm. The supernatant was discarded, and to the pellet 5 ml of 0.1 N NaOH was added to solubilize the protein and the solution was centrifuged again at 800 rpm for 15 min. The supernatant was saved and made up to 10 ml with 0.1 N NaOH and used for the estimation of protein content.

Estimation

Protein solution containing 10–50 μg protein in a volume of 0.1 ml was pipetted in 12 × 100 mm test tubes, and to this 5 ml of protein reagent was added and the contents were mixed by vortexing. The absorbance at 595 nm was measured after 2 min against a reagent blank. The reagent blank was prepared with distilled water, 0.1 ml of 0.1 N NaOH and 5 ml of Bradford reagent. The weight of protein was plotted against the corresponding absorbance resulting in a standard curve and it was used to determine the protein in unknown samples and the results were expressed in milligram per gram dry weight.

Estimation of glycine betaine content

The samples were extracted and estimated by the method of Grieve and Grattan 1983.

Extraction

Five hundred milligram of finely ground dry plant sample was mechanically shaken with 20 ml of distilled water for 24 h at 25 °C. The time required for this step was determined by extracting the plant samples for 4, 8, 16, 24, and 48 h. The samples were then filtered through Whitman No.1 filter paper and the filtrate was made up to 20 ml with deionised water and used for estimation immediately.

Estimation

One milliliter of the extract was diluted with one milliliter of 2 N H2SO4 and 0.5 ml of this acidified extract was cooled in ice water for an hour. Later 0.2 ml of cold potassium triiodide solution was added and mixed gently with a vortex mixture, and the tubes were stored at 4 °C for 15 min and then centrifuged at 10,000 g for 15 min. The supernatant was aspirated with a fine tipped glass tube. The per iodide crystals were dissolved in 9.0 ml of 1, 2-dichloroethane with vigorous vortexing. After 2.5 h, the absorbance was measured at 365 nm in a spectrophotometer. Reference standard of glycine betaine was prepared in 1 N H2SO4 and used for estimating the glycine betaine content and the results were expressed in microgram per gram dry weight.

Preparation of reagent: potassium triiodide reagent

15.7 g of iodine and 20 g of potassium iodide were dissolved in 100 ml of distilled water and gently stirred in a vortex mixture.

Statistical analysis

The pot culture was carried out in completely randomized design (CRBD). The data are expressed as mean ± SE for seven samples in each group.

Results

Effect of drought and drought with triazole combination on photosynthetic pigments

Chlorophyll and carotenoid contents

The photosynthetic pigments like chlorophyll-a, chlorophyll-b, and total chlorophyll contents decreased in drought stress and drought with triazole treatments when compared to control (Fig. 1). The carotenoid content decreased significantly in all the treatments when compared to control (Fig. 2).

Fig. 1
figure 1

Effect of drought stress and drought with triazole combination on the chlorophyll content of Zea mays (Expressed in mg/g fresh weight)

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Fig. 2
figure 2

Effect of drought stress and drought with triazole combination on the carotenoid content of Zea mays (Expressed in mg/g fresh weight)

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Anthocyanin content

Drought stress and drought with TDM, TBZ, and PCZ treatments decreased the anthocyanin content when compared to control (Fig. 3).

Fig. 3
figure 3

Effect of drought stress and drought with triazole combination on the anthocyanin content of Zea mays (Expressed in mg/g fresh weight)

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Effect of drought and drought with triazole combination on biochemical constituents

Protein content

The protein content of root, stem, and leaf of maize plants was decreased by drought stress and drought with triazole treatments when compared to control (Fig. 4).

Fig. 4
figure 4

Effect of drought stress and drought with triazole combination on the protein content of Zea mays (Expressed in mg/g dry weight)

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Glycine betaine content

One of the most important mechanisms exerted by higher plants under stress condition is the accumulation of compatible solutes such as glycine betaine. In the present study, the amount of glycine betaine content increased in root, stem, and leaf to a larger extent under drought stress and drought with triazole treatments (Figure 5).

Fig. 5
figure 5

Effect of drought stress and drought with triazole combination on the glycine betaine content of Zea mays (Expressed in μg/g dry weight)

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Discussion

Drought stress decreased the chlorophyll content when compared to control. A reduction in chlorophyll content was reported in drought-stressed Helianthus annuus (Manivannan et al. 2007). The chlorophyll content in the wheat leaf decreased due to chemical desiccation treatments (Shao et al. 2009). A reduction in chlorophyll content was reported in drought-stressed soybean plants by Zhang et al. (2006). The chlorophyll content decreased to a significant level at higher water deficits in maize and wheat plants (Nayyar and Gupta 2006). Similar results were observed in cherry (Centritto 2005). Triazole treatment to the drought-stressed maize plants increased the chlorophyll content when compared to control. Paclobutrazol and drought stress treatment increased the pigments in olive (Thakur et al. 1998). Similar increased results were observed in through difenoconazole and tricyclazole treatments in Lycopersicon esculentum (Mohamadi and Rajaei 2013).

Drought induced a reduction in chlorophyll ‘b’ content in leaves of maize when compared to control plants. Similar results were observed in rice (Farooq et al. 2009). A reduction in chlorophyll content was reported in drought-stressed Vaccinium myrtillus (Tahkokorpi et al. 2007) and sunflower plants (Kiani et al. 2008). Triazole treatment to the drought-stressed maize plants increased the chlorophyll ‘b’ content when compared to control. Triazole with drought stress treatment increased the pigments in rose (Jenks et al. 2001). Similar results were observed in maize (Ren et al. 2007) and in Lycopersicon esculentum. (Shanmugapriya et al. 2013). Drought stress decreased the total chlorophyll content to a larger extent when compared to control. A reduction in chlorophyll content was reported in drought-stressed soybean plants (Zhang et al. 2006). Triazole treatment to the drought-stressed maize plants increased the total chlorophyll content when compared to drought stress. Triazole-treated leaves were dark green due to high chlorophyll a and b in Chrysanthemum (Wood 1984).

In maize, the carotenoid content decreased significantly in all the treatments when compared to control. Among the drought treatments, the highest reduction was observed in carotenoid content when compared to other treatments. Carotenoids are a large class of isoprenoid molecules that are synthesized de novo by all photosynthetic and many non-photosynthetic organisms. They are divided into the hydrocarbon carotenes, such as lycopene and β-carotene, or into xanthophylls, typified by lutein. Carotenoids are also exploited as coloring agents, furnishing distinctive yellow, orange, and red colors to flowers, fruits, and roots, where they probably act as attractants to pollinators and for seed dispersal. The colors provided by the pigments are of important agronomical value in many horticultural crops (Shao et al. 2009). Reduced carotenoid content under drought was reported in sunflower (Gimenez et al. 1992). Triazoles treatment increased the carotenoid content in maize when compared to control and drought-stressed plants. Paclobutrazol with drought stress treatment increased the pigments in olive (Thakur et al. 1998), Triamidefon and tricyclazole applications raised the pigment content in Lycopersicon esculentum (Mohamadi and Rajaei 2013). The anthocyanin content was decreased by drought stress treatment in maize. In drought-stressed plants, the anthocyanin content was low as compared to control plants. Similar results were observed in tomato (Still and Pill 2004). Triazole treatment increased the anthocyanin content in maize plants, but drought stress has no significant effect upon this. Triadimefon increased the chlorophyll and anthocyanin content in radish cotyledons (Lichtenthaler 1979).

Drought stress caused decrease in protein content of all the parts maize plants to a large extent. Similar results were observed in wheat (Gong et al. 2005). The reduction in protein content in the chilling-stressed plants of maize was correlated with increased proline accumulation (Cheng et al. 1999). This may be due to the hydrolysis of protein or the inhibition of protein synthesis by oxidative stress, leading to the accumulation of proline (Feng et al. 2003). Protein metabolism has been associated with adaptation of plants to environment changes. Only a few reports discussing the leaf proteins of sunflower are available. Rodriguez et al. (2002) reported a decrease in leaf soluble proteins in sunflower due to water stress. However, the nature of plant species (Terri et al. 1986) and the types of tissue modulate the concentration of soluble proteins under water stress (Irigoyen et al. 1992; Shao et al. 2009). Triazole with drought stress treatment resulted in increased protein content in maize when compared to drought stress but it was lower than that of control. Paclobutrazol-treated wheat seedlings had more soluble protein (Kraus et al. 1995).

Drought stress caused an increase in the content of glycine betaine in all parts of the maize plant when compared to control. Betaines are known to be one of the major osmoregulating compounds, forming inner salts with quaternary ammonium groups and a carboxyl group. Among them, glycine betaine is the most familiar and widespread in plants and bacteria. The accumulation of glycine betaine occurs in some, but not in all, higher plants, and most of the glycine betaine is synthesized in chloroplasts by two enzymes, viz. choline monooxygenase (CMO) and betaine aldehyde dehydrogenase, responsible for glycine betaine synthesis chloroplastically. Glycine betaine synthesis can be induced by both drought and salt stress by the overexpression of CMO, and by betaine aldehyde dehydrogenase in barley (Nakamura et al. 2001) and stress tolerance in plants (Jun et al. 2000). The glycine betaine content increased under drought stress in sunflower (Manivannan et al. 2007). Glycine betaine is considered to be one of the most abundant quaternary ammonium compounds produced in higher plants under stressful environments (Shao et al. 2009). Triazole treatment to the drought-stressed plants decreased glycine betaine content, but it was higher than that of control. Similar results were observed in Abelmoschus esculentus (Rabert et al. 2013).