Determination of biochemical responses to drought stress in cultivated and exotic wheat genotypes

Ten wheat genotypes of locally cultivated and exotic origins were grown under drought condition to determine the biochemical responses. Plants were grown in hydroponics in modified Hoagland solution in box container in the Growth Room with temperature, light and humidity control at the Plant Stress Breeding Laboratory (PSBL), at the dept. of Genetics and Plant Breeding, BAU, Mymensingh. Nutrient solution was refreshed after every ten days. Wheat plants were subjected to drought stress, induced by 3% and 6% polyethylene glycol 6000 (PEG 6000) 6000 in hydroponic solution after 47 days of transferring seedling in full strength nutrient solution. Plants were kept in drought treatment for 42 days and on harvesting, leaves were collected for biochemical assays. Six biochemical traits were determined from leaf tissues following standard protocol. Concentration of leaf biochemical viz. Catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), hydrogen peroxide (H2O2), malondialdehyde (MDH) and proline were found in both increasing and decreasing trend of fluctuation. Not a single genotype was found with the entire biochemical traits with favorable actions, but several genotypes demonstrated relatively better performance compared to control. The relationship among the biochemical traits were also determined and found a positive significant correlation between CAT and POD. A ranking table was also constructed considering mean performance of the genotypes under different PEG 6000 treatment. Considering mean performance, % change of bio-chemicals under drought condition and ranking table, genotypes Sonalika, BARI Gom 21, BARI Gom 29, BARI Gom 30, Summa and Burr were found with better biochemical mechanisms under both 3% and 6% PEG 6000 induced drought and recommend to consider for future wheat improvement program for drought tolerance. Article history: Received: 23 April 2020 Accepted: 29 May 2020 Published: 30 June 2020


Introduction
Wheat (Triticum aestivum L.) is the 2 nd of the three major cereals (wheat, rice, and maize) with a global annual production of 756.40 million metric tons from 222.68 million hectares of land, in -2017(USDA, 2019. Wheat is grown under a wide range of climatic conditions covering temperate, Mediterranean to tropical areas. Wheat in tropical areas mostly grown in winter season which is predominantly dry with lower precipitation. This led wheat plants to suffer with drought stress. In developing countries, about 45% of the 120 million hectares are prone to drought (Macharia and Ngina, 2017). The situation will go further adverse for wheat production as drought affected area will be increased twofold by mid 21 st C, threefold by end of 21 st C for many regions (Sheffield and Wood, 2008). Seasonally about 250 -350 mm water is necessary for wheat production with 1.5 -4.0 mm as daily evapo-transpiration (Sattar, 2004). Drought stress reduces the efficiency of photochemicals, reduces the Rubisco efficiency, gathers of stress metabolites, antioxidative enzymes, such as: peroxidase, catalase, ascorbate peroxidase, glutathione reductase, malondoaldyhide, and reactive oxygen species (ROS) accumulation etc. (Nezhadahmadi et al., 2013). These ingredients may initiate disturbing lipid peroxidation, chlorophyll, protein oxidation, and nucleic acids. Plants demonstrate various responses from drought stress, of which some response confers tolerance to drought in different extent (Raffi and Asaduzzaman, 2015). Drought tolerance is a complex mechanism which has a cumulative relation with morphological, physiological and biochemical activities. Among the biochemical mechanisms, antioxidant systems scavenge or detoxify of excess ROS which eventually reduces the damages of cellular activities due to oxidation (Noctor and Foyer, 1998). Antioxidative enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione-S-transferase (GST), glutathione peroxidase (GP), mono dehydro ascorbate reductase (MDHAR) are produced which eventually contribute to the scavenging of accumulated ROS in plants under water stress (Sairam et al., 1998;Nezhadahmadi et al., 2013). Biochemical traits show high variability and heritability as well as association with grain yield that can be exploited as selection indicators to enhance selection efficiency of breeding programs (Saed-Moucheshi et al., 2019). This experiment was aimed at understanding of biochemical mechanisms for drought tolerance in local and exotic wheat genotypes in response of induced drought stress.

Materials and Methods
Ten wheat genotypes (seven local: BARI Gom 20, BARI Gom 21, BARI Gom 28, BARI Gom 29, BARI Gom 30, BARI Gom 31, Sonalika and three exotic: Macya, Burr and Summa) with three replications in Randomized Completely Block Design (RCBD) design were used to study their biochemical mechanisms against drought condition. Seven local genotypes were primarily introduced or developed from CIMMYT lines by Wheat Research Centre (Now, Bangladesh Wheat and Maize Research Institute) for their better agronomic features. These genotypes are well adapted to Bangladesh and can be used to improve with new characteristics from exotic materials. The exotic materials are varieties brought from Saudi Arabia considering dry agro-climatic condition. Catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), hydrogen peroxide (H202), malondialdehyde (MDA) and proline are the biochemical traits which were considered in this study. For seed germination and seedling establishment, standard procedures were followed as described by Malik et al. (2009). Plants were grown in 20 litre plastic containers popularly known as Milk Crate by RFL Plastic Industries Bangladesh, filled with hydroponic solution (Hoagland and Arnon, 1950) in controlled environment facilities at the Plant Stress Breeding Laboratory (PSBL) at the Department of Genetics and Plant Breeding, BAU. Optimum growth condition was maintained throughout the growing period of wheat genotypes (14h/10h light/dark period, 22 o C/18 o C day/night temperature, 60%/65% day/night RH and 4500-5500 lux light intensity) for their normal growth and development. The pH of the nutrient solution was maintained at 6.5. Nutrient solution was refreshed after every 10 days. Sometimes, humidifier was used to maintain the relative humidity of the growth room. Air bubbler was used to ensure availability of oxygen to the root zone inside the container. Drought was induced by applying PEG 6000 (Mark, India) at 0%, 3% and 6% in the nutrient solution on 47 th day of transferring seedlings (Zadoks Scale 22;Zadoks et al., 1974) to full strength solution and kept for 6 weeks (Zadok's Scale 41). A schematic presentation of the activities done for the experiment was presented in Fig 1.

Biochemical analysis
Flag leaf was selected for biochemical data collection. This leaf sample was collected after 42 days of PEG 6000 treatment. Fresh leaf sample were used for biochemical analysis.

Sample extraction for CAT, POD and APX
Fifty milligrams of fresh plant sample was collected and homogenized with 3 ml of 50mM potassium phosphate buffer (p H 8.0) in a mortar and pestle. The homogenate was centrifuged at 12,000 rpm for 10 min. In all stage 4˚C temperature was maintained. The clear supernatant was used for assaying CAT, POD and APX activity.

Determination of CAT activity
CAT activity was determined by following the method of Aebi (1984). 0.7 ml of 50 mM potassium phosphate buffer (p H 8.0), 0.1 ml of EDTA and 0.1 ml H2O2, were added in an eppendorf and mixed well. In all stage 4˚C temperature maintained. Reaction was started by adding 0.1 ml of enzyme extract and changes in absorbance were recorded immediately at 240 nm at 30 seconds interval for two minutes. The activity of catalase was calculated from the decrease in absorbance in per minute according to following formula:

Determination of POD activity
POD activity was determined by following the method of Nakano and Asada (1981). In an eppendorf tube, 0.6 ml of 50 mM potassium phosphate buffer (p H 8.0), 0.1 ml of EDTA, 0.1 ml of H2O2 and 0.1 ml of Guaiacol were added and mixed well. Reaction was started by adding 0.1 ml of enzyme extract and changes in absorbance were recorded immediately at 470 nm at 30 seconds interval for two minutes.  Determination of APX activity: APX activity was determined by following the method of Nakano and Asada (1981). In an eppendorf tube, 0.6 ml of 50 mM potassium phosphate buffer (p H 8.0), 0.1 ml of EDTA, 0.1 ml of H2O2 and 0.1 ml of Guaiacol were added and mixed well. Reaction was started by adding 0.1 ml of enzyme extract and changes in absorbance were recorded immediately at 290 nm at 30 seconds interval for two minutes. The activity of ascorbate peroxidase was calculated from the decrease in absorbance in per minute according to following formula. Determination of H2O2 activity: H2O2 content was determined using the method given by Velikova et al., (2000). About 0.1 g of fresh leaf sample was homogenized in a pre-chilled mortar with pestle using 1ml of 0.1% tri-chloro-acetic acid (TCA) and homogenize it at 4˚C. The homogenate was centrifuged at 12,000 rpm for 15mins. 0.5m supernatant of sample was then mixed with 0.5mL of 10mM PO4 3buffer (pH 7) and 1.0mL of 1M potassium iodide. The absorbance was recorded at 390nm and calculation was done according to following formula.

Determination of proline activity
Proline was determined according to the method described by Bates et al. (1973). Approximately 0.5g of fresh leaf was homogenized in 10 ml of 3% aqueous sulfosalicyclic acid and filtered through Whatman's No. 2 filter paper. Two ml of filtrate was mixed with 2 ml acid-ninhydrin and 2 ml of glacial acetic acid in a test tube. The mixture was placed in a water bath for 1 hr at 100°C. The reaction mixture was extracted with 4 ml toluene and the chromophore containing toluene was aspirated, cooled to room temperature, and the absorbance was measured at 520 nm. A standard curve was prepared with analytical grade proline and proline contents in sample were calculated by using the standard curve. The percentage of proline present in the leaves was expressed as mg/100g fresh leaves. Data were managed with MS Excel spreadsheet and analysed using STAR 2.0.1 (IRRI) software.

Results and Discussion
The results of the experiment conducted on ten wheat genotypes at PEG 6000 induced drought condition were analysed, presented, and discussed in the following section under respective sub-headings. Here, mean performances of ten genotypes under control and drought stress for six biochemical traits were presented in Table 1 and per cent changes of biochemical traits due to drought stress in Fig. 2.1 to 2.6.

Catalase content
Catalase detoxify plant cell by converting H2O2 to H2O or O2 (Abid et al., 2018). In a study, Islam et al. (2015) found that leaf CAT concentration was produced more in drought tolerant variety than drought susceptible variety in drought condition. Here, synthesis of catalase in leaf was found with both increasing and decreasing trends under PEG treatments ( Furthermore, highest per cent increase of catalase in 6% PEG over 3% PEG was observed in BARI Gom 21 (260%) and lowest in Summa (-96.16%). In summary, genotypes with higher CAT activity under drought stress were Sonalika, BARI Gom 21, Summa and Burr and therefore, recommended for future drought tolerance improvement program.

Ascorbate peroxidase (APX) content
According to Mittler (2002), APX content found in increased amount in drought tolerant plant against drought condition compared to control. APX uses ascorbate as the electron donor for the reduction of H2O2 to scavenge, especially when CAT is absent (De Gara et al., 2003, Huseynova, 2012

Peroxidase (POD) content
Drought stress in the plant tissue is alleviated by a concerted action of both enzymatic and non-enzymatic antioxidant systems which includes POD enzymes (Prochazkova et al., 2001). Islam et al. (2015) found that POD content was produced more in drought tolerant genotype than drought susceptible in drought condition. Synthesis of POD in leaf was found with both increasing and decreasing trends under PEG treatments (

Hydrogen peroxide (H2O2) content
Drought stress reduces the carbon assimilation results in an imbalance between electron excitation and utilization through photosynthesis, which results in the production H2O2 (Abid et al., 2018). At higher concentration, H2O2 is deleterious to cell, but at lower concentration, it acts as a signaling molecule to indicate drought stress (Caverzan et al., 2016). Accumulation of H2O2 in leaf was found with both increasing and decreasing trends under PEG treatments ( Considering % change of H2O2 production upon PEG treatments ( Fig. 2.4), all the genotypes except BARI Gom 20 and BARI Gom 21 demonstrated increase in production of H2O2 at 3% PEG over control, where Burr demonstrated lowest production (-88.61%) followed by Summa (-63.61%). At 6% PEG over control, production of H2O2 increased in BARI Gom 20, BARI Gom 21 and BARI Gom 28, and decreased in other genotypes. Here, genotype BARI Gom 30 (-66.78%) demonstrated decrease in production of H2O2 followed by BARI Gom 29 (-60.93%). By contrast, only four genotypes demonstrated decrease in concentration of H2O2 at 6% PEG compared to 3%, viz. BARI Gom 29 (-31.87%), BARI Gom 30 -40.25%), BARI Gom 31 (-8.29%) and Macya (-7.55%) compared to other genotypes.
The decreasing rate of H2O2 accumulation in genotypes might be due to scavenging of H2O2 by the anti-oxidants molecules produced from PEG treatments. In summary form Table 1 and Fig. 2.4, genotypes BARI Gom 29, BARI Gom 30, Burr and Summa could be recommended for exploitation to improve H2O2 related drought response mechanism in wheat.

Malondialdehyde (MDA) content
The inter-cellular accumulation of MDA, a product of fatty acid peroxidation, in plants due to cellular membrane lipid peroxidation is a measure of oxidative stress induced membrane damage during water stress (Farooq et al. 2010;Abid et al., 2018). Therefore, increased MDA content in drought stressed cell indicates more oxidative damage, which eventually refrains plants to tolerate drought stress. Accumulation of MDA in leaf was found with both increasing and decreasing trends under PEG treatments ( Considering Table 1 and Fig. 2.5, BARIGom20, BARI Gom 29, BARI Gom 30 could be recommended for improving MDA related traits in wheat.

Proline content
Plant brings osmotic adjustment by producing more proline which a compatible solute and helps plant to maintain turgor pressure and cell volume at lower water potential for facilitating metabolic functions (Hammad and Ali, 2014;Abid et al., 2018). Therefore, increase of proline in drought suffered cells offers plants an ameliorating option to keep cellular activities by increasing the osmotic potential lowered due to drought (Errabii et al., 2006). Synthesis of proline in leaf was found with both increasing and decreasing trends under PEG treatments (Table 1 and Fig. 2.6). Genotypes showed differences in proline synthesis even in control condition indicating differences in inherent capability for proline synthesis. It is evident from the above results that not all the mechanisms conferring drought tolerance are present in a single genotype, which eventually depicts the gradient for drought tolerance in the genotypes studied. Drought tolerance is a complex mechanism and can be achieved with the interplay of different traits with different degree of contributions (Chaves et al., 2003). Different genotypes showed tolerances due to different mechanisms (Bansal et al., 2016). For instances, some genotypes might have better osmoprotectant abilities, whereas some might have better ROS scavenging abilities (Abid et al., 2018), or some might have both (Laxa et al., 2019). Therefore, it is really important to find out interrelationships between the biosynthesis of different biochemicals towards bringing drought tolerance. In the present study, positive and significant linear relationship was observed between POD and CAT in 3% drought condition but not in control condition, indicating that, these traits have synergistic relationship only when drought is induced. However, they did not show significant linear relationship in higher drought level, which indicated their possible independent role to drought tolerance in severe drought condition (Gong et al., 2005).   (Table  3) and presented. Genotypes with better performance were scored with higher grade based on the number of letters found while grading. Trait based scores of a genotype were then summed to obtain total score, and based on that, genotypes were ranked. Considering the above approach, cumulatively better biochemical mechanisms under drought stress were found in Sonalika, followed by Summa and BARI Gom 21, at 3% PEG.
At 6% PEG, BARI Gom 29, followed by BARI Gom 30 and BARI Gom 21 were found to have better biochemical responses under drought condition. In general, BARI Gom 21, exotic genotypes Summa and Burr demonstrated consistently better biochemical responses under drought compared to other genotypes. The above-mentioned genotypes, therefore, should be evaluate in more detail with yield data at different level of drought in different stages of growth before considering for future hybridization program to improve drought tolerance in wheat considering biochemical mechanism.

Conclusion
Biochemical parameter has effective responses against drought stress in wheat plants and demonstrated higher variability among the genotypes at different level of drought stress. So, these are very important indicators to recognize the drought stress tolerant variety. This performance profile of genotypes for different biochemical traits could be helpful to select genotypes for hybridization program for drought tolerance in future drought stress breeding program for wheat.