1. INTRODUCTION

Rice (Oryza sativa L.) is one of the most strategic crops worldwide, especially in developing countries, after wheat [1]. Due to its carbohydrate content and nutritional value, it is considered an important source of daily calories for more than 50% of the world’s population [2]. Rice as a semiaquatic plant is one of the most water-consuming crops; one hectare consumes more than 17,000 m³ of water, where continuous submergence is the most effective method of irrigation. It is, therefore, highly affected by drought stress [3]. Drought can be defined as shortage in soil moisture available for plant growth, development, and completion of its life cycle [4]. It is one of the most serious abiotic stresses affecting the biochemical, physiological, and morphological status of the plant [5]. Stomatal closure according to Vanisri et al. [6] is one of the earliest reactions to stress leading to a reduction in CO₂ diffusion from the atmosphere to carboxylation sites inside chloroplast. Electrons that were supposed to be consumed in CO₂ assimilation are accumulated and transferred to O₂ forming reactive oxygen species (ROS) (oxidative stress) in chloroplast and mitochondria [7]. ROS such as superoxide (O₂^-), singlet oxygen (1/2 O₂), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH⁻) are highly active molecules that react and damage all biological molecules in the cell such as lipids, proteins, carbohydrates, nucleic acids, chlorophylls, and enzymes resulting in membrane lipid peroxidation, protein carbonylation, and chlorophyll loss and inactivating the –SH containing enzymes [8]. Thus, photosynthesis, growth, development, and yield are severely affected by drought [9].

Plant response to drought stress depends upon plant stage, stress duration, and plant species [10]. Plant uses any of these different strategies to cope with drought stress: (i) drought escape that will allow the plant to grow, develop faster, and complete its life cycle before severe water shortages, (ii) drought avoidance by decreasing the rate of transpiration (via reduction of stomatal conductance and leaf area) and increasing water uptake, (iii) drought tolerance by maintaining cell turgor by inducing osmolytes accumulation such as proline, glycine betaine, and certain types of sugars that allow plants to retain water, maintain growth, and restore plant function after drought stress [11]. Acclimation of plants to stress is accompanied by an increase in the plant’s antioxidant system, which has the ability to remove oxygen radicals, repair the damage, and keep the concentration of ROS at a relatively low concentration [7]. An antioxidant system includes enzymatic (such as superoxide dismutases and catalases) as well as nonenzymatic compounds (such as phenolic acids and flavonoids and other molecules) which are able to remove ROS and protect the cells from damage like proline. Gao et al. [12] reported that increasing antioxidant activity of plants under stress is considered characteristic of drought-resistant species. The more increasing the efficiency of ROS scavenging during stress, the more increasing the protection of photosynthetic apparatus, which leads to improving photoassimilates production. For instance, sugars (such as glucose, fructose, sucrose, maltose, and galactose) and sugar alcohols (which are also called polyols, such as mannitol, sorbitol, and xylose) are accumulated and act as typical osmoprotectants. Moreover, they play a crucial role in adapting rice plants to drought stress by maintaining osmotic adjustment to prevent intracellular dehydration [11], protecting macromolecules, and stabilizing cellular membranes, proteins, and enzymes as well as scavenging ROS molecules [4], in addition to the role of proline and organic acids in the adjustment of cellular osmotic stress and maintenance of intracellular water.

More efforts have now been made to increase rice crop productivity to more than 40% to meet population requirements [13]. It has become serious in the shade of increasing water scarcity and agricultural land that is expected to dry up all over the world.

The aim of the current work was to (i) study the changes in metabolite profile (including sugars, phenolic acids, flavonoids, organic acids, and phytohormones) in Egyptian rice in response to drought stress and (ii) highlight the differences in strategies that were exhibited by tolerance and sensitive cultivars.

2. MATERIAL AND METHODS

The present experiment was conducted at the botanical garden, Faculty of Science, Ain Shams University, during the summer season of 2018/2019. Two rice cultivars Giza 179 (Gz179) (a drought-tolerant cultivar) and Sakha 101 (Sk101) (a drought-sensitive one) were selected based on our previous study [3] where several physiological parameters were analyzed on six Egyptian rice cultivars to select the most tolerant and the most sensitive cultivars. In plastic pots (12 cm in diameter and 10 cm high), ten grains per pot were sown and watered to 80% of its water holding capacity and exposed to normal daylight and temperature (30°C ± 2°C, 16 hours light/8 hours dark cycle), each pot filled with one kilogram of soil (clay : washed and dried sand) (2:1 wt/wt). Ten-day-old seedlings were irrigated once weekly with a full-strength Hoagland nutrient solution [14]. The water holding capacity was maintained at 80% until 21-day-old seedlings and pots for each rice cultivar were divided into two groups. The first one was watered to 80% water holding capacity (act as control), and the second group was watered to 40% water holding capacity (act as drought-stressed plants). This water regime was maintained until symptoms of leaf rolling were detected as an indicator of drought stress. At the end of the experiment, 28-day-old plants were collected (Fig. 1). Metabolites such as proline, sugar content, phenolic acids, flavonoids, growth regulators, and organic acid contents were determined by high-performance liquid chromatography (HPLC).

2.3. Extraction, Separation, and Identification of Phenolic Compounds using HPLC

2.3.1. Extraction of the phenolic compounds

Fresh leaves of rice (0.5 g) were grounded in liquid nitrogen into a fine powder, and 10 ml methanol HPLC grade was added and slightly stirring. Tubes were then centrifugated at 1,500 g for 10 minutes. The liquid phase was collected for quantification by HPLC [17,18].

Figure 1: 28-days old rice seedlings: Gz179 (tolerant) and Sk101 (sensitive). C: 80% WHC (control), T: 40% WHC (drought-stressed). [Click here to view]

2.3.2. Identification of the phenolic compounds

Phenolic compounds were measured using HPLC consisting of a solvent delivery module LG-980-02, detector UV/Vis UV-970, and pump PU-980. The column was Phenomenex, UK, an RP-C18 Luna column, 4.60 mm i.d. × 250 mm and particle size = 5 μm. Detection was carried out at 280 and 340 nm and the compounds were identified by comparing their retention time values with those of the standards [19].

3. RESULTS

3.2. Soluble Sugars

The highest accumulation of sugar was recorded by stressed Gz179 (6.075 μg/g f.wt), 6-fold increase compared with its control, while drought-stressed Sk101 had doubled sugar accumulation compared to its control. Interestingly, Gz179 accumulated stachyose (20-fold), sucrose (12-fold), galacturonic (11.5-fold), rhamnose (10.5-fold), galactose (10.4-fold), and sorbitol (10-fold) compared with its control, while Sk101 accumulated stachyose (5.9-fold) and galacturonic (5.2-fold) as compared to its control (Table 1).

Figure 2: Changes in proline in response to water deficit in two rice cultivars (Gz179 and Sk101) differing in drought tolerance. [Click here to view]

Table 1: Identification and quantification of sugars in Egyptian rice cultivars Gz179 and Sk101 as determined by HPLC. [Click here to view]

3.6. Organic Acids

Results in Figure 4 showed that the accumulation of oxalic acid in both rice cultivars in normal conditions was not changed due to drought stress. Tartaric acid was increased 10-fold in treated Gz179 and two-fold in treated Sk101 compared to control. Succinic acid has been detected in control plants and has shown a slight increase in both cultivars under stress. Moreover, glutaric acid displayed an increase in Gz179 and decrease in Sk101 in response to water deficit. In contrast, the accumulation of citric acid was decreased significantly during the drought period.

Table 2: Identification and quantification of phenols in Egyptian rice cultivars Gz179 and Sk101 as determined by HPLC. [Click here to view]

Table 3: Identification and quantification of flavonoids in Egyptian rice cultivars Gz179 and Sk101 as determined by HPLC. [Click here to view]

4. DISCUSSION

Rice growth and grain development are severely affected by drought [5]. In order to meet growing global demand, a thorough understanding of the impact of drought stress on rice growth and production is therefore crucial to increasing biomass yields [25]. In our previous work [3], we have shown that the sensitive cultivar Sk101 is more affected by oxidative damage caused by water deficiency than tolerant cultivar Gz179 as indicated by increased level of lipid peroxidation (detected by Malondialdehyde (MDA) content as a decomposition product of polyunsaturated fatty acids), increased permeability (as indicated by electrolyte leakage increase), and loss of cellular integrity (decrease in membrane stability index). Consequently, morphological criteria of plants (especially plant length, leaf area, and leaf number per plant) as well as photosynthetic pigment content were significantly affected in Sk101 compared to Gz179 [3].

Figure 3: Changes in plant growth regulators in response to water deficit in two Egyptian rice cultivars (Gz179 and Sk101) differing in their drought tolerance as determined by HPLC. [Click here to view]

In the current work, the physiological and metabolic responses of these two cultivars were compared: drought tolerant (Gz179) and the drought sensitive (Sk101) for better understanding of drought-tolerant strategies in Egyptian rice.

The development of enzymatic and nonenzymatic antioxidants that scavenge ROS and turn it into harmless molecules is one of the most effective mechanisms for preventing oxidative stress-induced damage in tolerant plants [26]. In this study, we focused on nonenzymatic molecules (such as phenolic acids and flavonoids). Exposing the plant to drought stress improves the secondary metabolites biosynthesis as a part of the nonenzymatic mechanisms. Phenolic compounds are one of the major groups of secondary metabolites. Phenolic compounds show an increase in their accumulation under drought stress [27]. This is clearly evident from our results (Table 2), where tolerant cultivar showed higher accumulation in phenols than sensitive cultivar under the same stress conditions. Tolerant cultivar is characterized by high levels of isoferulic, cinnamic, and pyrogallol. Plant may tolerate the negative effect of drought stress on membranes by increasing ferulic acid that acts as a precursor for oryzanol (one of phytosterol in plant cell), which increases the stabilizing property and strengthens the cell membrane as a result of its polarity and 3D interaction with lipid bilayer membrane [28]. Ferulic acid is also known as the precursor to lignin that protects cell wall from microbial degradation and imparts rigidity to cell wall [29].

Flavonoids are low molecular weight polyphenolic compounds, which play an important role in the growth, development, reproduction, and defense of plants under stress. The current study showed an increase in total flavonoid content following treatment with drought, which is also consistent with the results obtained by Yuan et al. [30] and Liu et al. [31]. As shown from results (Table 3), tolerant cultivar had a high content of flavonoids under normal conditions and has the ability to accumulate more flavonoids under stress compared to sensitive cultivar that has low flavonoid content under control conditions and recorded no marked increase under drought treatment. The most drought-induced flavonoids in tolerant cultivar were kaempferol and quercetrin which indicated that their accumulation was associated with drought tolerance in rice. Similar results were obtained in tomato [32]. The protective role of flavonoids in plant defense against drought stress was achieved through their strong activity in ROS scavenging. Their role achieved by either preventing the ROS generation [33,34] or rapid scavenging of ROS [35] resulted in the justification of oxidative stress under drought condition [36]. The key cause of this accumulation of phenolic compounds is phenylpropanoid biosynthetic pathway modulation. Many important genes that encode master enzymes in the phenylpropanoid pathway, which induce the development of phenolic compounds, are regulated by drought [27].

Figure 4: Changes in organic acids in response to water deficit in two Egyptian rice cultivars (Gz179 and Sk101) differing in their drought tolerance as determined by HPLC. [Click here to view]

The adaptive role of antioxidants in tolerant cultivar conflict over the efficacy of chlorophyll, photosynthetic rate, and photoassimilate production (such as proline, sugars, sugar alcohols, and organic acid), which accumulated as osmoprotectants. Osmoprotectants are low molecular weight molecules that are highly water-soluble, uncharged, and nontoxic at molar concentrations [37]. They help plants to survive under severe osmotic stress, through osmotic adjustment, which ensures continuous water uptake under drought, maintaining its turgidity and growth. This is another important strategy used by rice plants to cope with reduced water availability under drought stress [38]. Proline highly accumulated in the drought-tolerate cultivar Gz179 (3-fold of control) as one of the metabolic responses; the plants use it under stress. It has several functions as a free radical scavenger, a protective agent for enzymes, and stabilizing the cell membrane and protein structure in addition to its role in osmotic adjustment maintenance [39]. Many researchers have noted a significant increase in proline concentration due to drought stress in many plants such as wheat [40], peanut [41], soybean [42], and rice [43].

Sugars play a vital role in normal plant growth and development, and their contents are greatly increased under drought stress (Table 2), acting as osmoprotective agents to adjust osmotic pressure and overcome water shortages, in addition to their role in secondary metabolites biosynthesis (glycosylation process), by which active sugar hexoses such as glucose, galactose, and rhamnose and pentoses such as arabinose and xylose have been transferred to other molecules such as flavonoid in flavonoid modification process to increase the stability and solubility of hydrophobic flavonoids. In addition, its reaction with lipid to form galactolipid, which was considered to be the major lipid in plant cell membranes and the most abundant in chloroplast membrane, increased its content under drought stress to increase the stability of the thylakoid membranes, thus maintaining the efficiency of photosynthesis [44]. Tolerant rice cultivar is characterized by a significant increase in mannitol accumulation in response to drought that was not shown in sensitive cultivar. In addition, the increase in stachyose, sucrose, and galacturonic in tolerant cultivar was much higher than that detected in sensitive one in response to water deficit. Similar results were obtained by Gundaraniya et al. [45] who noted the accumulation of sugar alcohols such as myo-inositol and D-mannitol in the leaf sample of drought-tolerant peanut (Arachis hypogaea L.) cultivar in response to water stress. Sugar alcohols are osmoprotectants as well as potent ROS quenchers and accumulated in different concentrations under water stress. The hydroxyl group of sugar alcohol can substitute the hydroxyl group of water during interaction with membrane lipids and proteins, maintaining their structure and properties under drought conditions.

Complex biochemical and physiological strategies have been established by rice plants to allow them to respond to sudden changes in climate [46]. Most of these adaptations are regulated by plant hormones that respond to changing environmental conditions by altering signal transduction to control and organize both growth and/or stress tolerance in order to promote survival or escape from environmental stress [47]. Du et al. [48] found that the equilibrium between auxin and ABA biosynthesis played a key part in the plant’s response to stress. Auxin biosynthesis is known to be monitored through the downregulation of YUCCAs (IAA biosynthesis genes), as well as the upregulation of IAA-conjugated genes (GH3s) [49]. The current results proved that auxins (IAA and IBA) content was significantly decreased, whereas ABA contents were markedly elevated in response to water deficit (Fig. 2); the changes were much more pronounced in sensitive cultivar. The observed changes in phenolic concentration such as kaempferol and quercetrin (increases with 5.3- and 2.7-fold, respectively, in tolerant cultivar) as shown in Table 2 acting as auxin transport inhibitor that influences the cycling of auxin efflux carriers and alters the apoplastic pH affecting the intake and distribution of auxin [50]. Meanwhile, the associated increment in ABA level under drought stress was attributed to upregulation of nine-cis-epoxycarotenoid dioxygenase 3 (NCED3), which acts as a key enzyme in ABA biosynthesis in Arabidopsis [51] and in rice [52] and to stimulate ABA-glucose deconjugation [53]. This increase in ABA concentration led to an increase in potassium ion efflux, a loss of turgor in guard cells, and, consequently, stomatal closure [54]. ABA signaling pathway can be considered important to the drought resistance of rice since higher levels of ABA were observed in drought-tolerant cultivar under both normal and stress conditions in the present study. Similar results were obtained by You et al. [55].

SA plays multiple roles in cell metabolism and also plays a key role in modulating the response of plants to abiotic stress by enhancing antioxidant enzymes and reducing ROS molecules [56]. SA, like ABA, is involved in stomatal regulation through Ca2+-dependent protein kinases located downstream of the peroxidase-mediated ROS signaling pathway in Arabidopsis guard cells [57]. In the current study, a further increase (as a percentage of control) in SA was detected in sensitive cultivar Sk101 that could be a coping strategy to overcome the impact of oxidative damage caused by drought.

Organic acids present in normal cells as intermediates to primary and secondary metabolite pathways and increase under drought stress to act as osmoprotectants. According to the results obtained (Fig. 4), tartaric acid showed a significant increase in the tolerant cultivar compared to that was detected in the sensitive one. Furthermore, the tolerant rice cultivar Gz179 was characterized by an increase in glutaric acid that was not detected in Sk101. Guo et al. [58] worked on wheat and stated that, in order to avoid drought stress, tartaric acid accumulates to sustain intracellular ion equilibrium and nutrient uptake.

5. CONCLUSION

Developing genetically modified rice plants with improved resistance and yield under adverse conditions remains a challenge. One major task is to define the metabolomic and transcriptomic changes that are responsible for the tolerance of abiotic stress in rice. The current study investigated the metabolomic differences between tolerant and sensitive Egyptian rice cultivars in response to drought stress. We found that the tolerant cultivar is characterized by a vast array of metabolites that have not been detected or less accumulated in the sensitive cultivar and appears to have a key role in ROS scavenging, osmotic adjustment, strengthening cell cytoskeleton, and stabilizing cell membrane properties, and hence drought tolerance. These metabolomes include both primary and secondary metabolites, namely, proline, sugars (mannitol, stachyose, sucrose, and galacturonic), organic acids (tartaric and glutaric), phenols (isoferulic, cinnamic, and pyrogallol), and flavonoids (kaempferol and quercetrin). Induction of these metabolites is responsible, at least in part, for drought stress tolerance in Egyptian rice cultivar Gz179 and can be used as efficient biomarkers.

7. FUNDING

The work was supported by Ain Shams University.

8. AUTHOR CONTRIBUTIONS

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.

9. CONFLICTS OF INTEREST

The authors report no financial or any other conflicts of interest in this work.

10. ETHICAL APPROVALS

Not applicable.

11. PUBLISHER’S NOTE

This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

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