Effects of Severe Drought Stress on Some Physiological and Biochemical Parameters of AMF Inoculated C. arietinum

: In this study, physiological and biochemical changes caused by mycorrhizal symbiosis in chickpea plants under drought conditions were investigated in both root and leaf. Drought stress reduced leaf water potential, but mycorrhizal symbiosis caused a significant increase in leaf water potential. However, the application of mycorrhiza under drought stress caused an increase in the amount of elements that are very important for the development of the plant in the root and leaf. In our study, drought increased the proline concentration and MDA content, while mycorrhiza application decreased them in both leaf and root. In addition, while mycorrhizal application increased the activity of catalase, it decreased the activity of superoxide dismutase. In general, enzyme activities were found to be higher in the leaf, but no distinct pattern was obtained between root and leaf in other analyzes. The study shows that the responses of mycorrhizal symbiosis in chickpea plants may change depending on the severity of the drought. Especially antioxidant enzyme activities and proline content patterns reveal that more comprehensive studies should be conducted on these issues. However, continuing studies until determining the effects of AMF symbiosis on grain yield under drought may provide more comprehensive results.


Introduction
Legumes (Fabaceae) are a very valuable plant group both agriculturally and economically.Besides being used for nutritional purposes, free nitrogen digestion in the soil also increases the ecological value of this group (Pandey, 2008).According to FAO (2019), Turkey is the most chickpeaproducing (630.000tonnes) country in the world after India.Chickpea, which is one of the most grown legume products in the world and Turkey, is generally grown in semi-arid and arid areas.Although chickpeas have developed mechanisms that can cope with drought, it is known that this stress causes serious product loss in chickpeas (Canci and Toker, 2009).
Plants are continuously exposed to abiotic and biotic stress factors throughout their life in nature.Drought, one of the most important abiotic stress, affects fields, and cause serious yield losses (Sadak et al., 2021).It has been estimated that drought-induced inefficient soil levels for crop production reach up to 28 % of the world's cultivated land (Aroca et al., 2008).Drought stress is one of the most limiting factors for chickpea growth during vegetative and reproductive development stages (Günes et al., 2006).Chickpea is known to be resistant to drought, but the yield loss due to drought is around 45-50 % for chickpeas (Devasirvatham and Tan, 2018;Shah et al., 2020).Studies conducted on this subject have shown that the morphological, physiological and biochemical mechanisms of chickpea are negatively affected by drought stress, resulting in crop losses (Rani et al., 2020).
There are many strategies developed by plants against drought stress, one of which is symbiotic interactions.Many symbiotic interactions occur between plants and other organisms in nature.One of these interactions is between the plant and mycorrhiza fungi, which was established approximately 400 million years ago (Diagne et al., 2020).Arbuscular mycorrhizal fungi (AMF) colonize within the root cortex, producing large amounts of hyphae (mycelia), increasing the surface area of the infected root.This allows the nutrients and water in the form and amount that the plant cannot take from the soil, away from the root, through the mycorrhiza hyphae and transmit it to the upper parts of the plant.Thus, a symbiotic life is established where the mycorrhizal fungus provides water and minerals to the plant and the plant carbon to the mycorrhizal fungus (Wu et al., 2008).
Increasing the surface area of plant roots infected with AMF provides a great advantage for the plant to cope with stress, especially in drought stress conditions (Ortaş, 2012).This advantage is not limited to taking water and mineral substances from the soil; It also includes many physiological and biochemical events such as the mycorrhizal promoting root regeneration, accelerating plant growth, promoting intracellular soluble substance concentration, activating the antioxidant system (Kaya et al., 2009).The symbiotic relationship between AMF and plants is an important topic that has been studied for a long time.Within the scope of these studies, the role of symbiotic relationships under stressful and/or non-stressful conditions is attempted to be understood.In this study, some physiological and biochemical responses caused by drought stress in chickpea plants were investigated.It has been observed that the plant creates different stress responses with the increase of stress intensity.

Plant material and drought treatment
Cicer arietinum (ILC482) seeds were sterilized by soaking in 2.5 % sodium hypochlorite solution for 10 minutes, then washed thoroughly and soaked in distilled water for 1 day.Then they were transferred to plastic pots (2 L) and filled with mineral-poor soil.The soil was autoclaved at 121 °C for 2 hours before use.Half of these seeds are infected with mycorrhiza (Glomus mosseae), approximately 1000 spores of G. mosseae were used for each seed.4 seeds were planted in each of the pots.According to the plant output, 2 plants were developed in each pot.In addition, there were three pots in each group.All pots were watered to %85 of field capacity before sowing.After sowing, all pots were also watered 75 mL every 4 days.Plants were grown at 24 ± 2 °C, 16/8 h photoperiod, irradiance 480 µmol m −2 s −1 , 65 ± 5 relative humidity under controlled conditions for 21 days in the plant growth room.At the end of this period, half of the seedlings were not watered for 12 days and the other half were irrigated as control plants.Afterwards, the leaves and roots were harvested and taken into liquid nitrogen quickly and stored in a freezer at -80 °C until analysis day.

Leaf water potential
The leaf water potential was measured by using a pressure chamber (PMS Instrument Co., Model 1000).

Determination of root and leaf element contents
Leaf samples (0.5 g) were extracted in a 3:1:1 ratio nitric acid/perchloric acid/hydrochloric acid solution in an oven at 200 °C.These samples were then diluted with 50 mL of ultrapure water and analyzed by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS, Agilent 7500).

Antioxidant enzyme assays
The same extraction method was used for SOD and CAT enzymes.Leaf and root tissues (0.5 g) were homogenized with phosphate buffer (5 mL, pH 6.8) and centrifuged at +4 °C, 5 min, 16.000 g and supernatant was used for measurements.Total SOD activity was determined according to Beyer and Fridovich (1987).One unit of SOD activity was defined as the amount of enzyme that was required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm.CAT activity was determined by measuring the rate of decomposition of H2O2 at 240 nm, as described by Aebi (1983).

Determination of Lipid peroxidation
Lipid peroxidation was determined by measuring the malondialdehyde (MDA) level, according to Ohkawa et al. (1979).Firstly, leaf and root tissues (0.2 g) were homogenized in trichloroacetic acid (5 %) (TCA) solution and centrifuged at 12.000 rpm.The supernatant, thiobarbituric acid (TBA) and 20 % TCA solutions were transferred to the tubes in equal volumes and incubated at 96 °C for 25 min.After that, the tubes were centrifuged at 12.000g for 5 min and the supernatant was measured at 532 and 600 nm.The MDA content was calculated using the extinction coefficient.

Determination of free proline content
Free proline content was determined according to the method of Bates et al. (1973).Leaf and root samples were homogenized in sulfosalicylic acid (3 %) and centrifuged at 3.000 rpm, then the supernatant, acetic acid and ninhydrin were mixed well and boiled for 1h.Then, cold toluene was added to this mixture and the toluene phase was measured at 520 nm.The proline concentration was calculated by using a calibration curve and expressed as μmol proline g -1 FW.

Statistical analysis
Stress and mycorhiza treatments were carried out completely randomized experimental design with two factors.Treatments had three replications with three plants each.Data were subjected to ANOVA and the means were separated using the LSD multiple range test at P<0.05.All the statistical analyses were performed using the JMP8 Software package).

Results
Drought stress significantly reduced leaf water potential.However, AMF inoculation enhanced leaf water potential under drought stress (Figure 1).In the present study, AMF inoculation significantly increased the element contents in the root especially under drought conditions (Table 1).Proline (Figure 2) and MDA (Figure 3) contents of C.arietinum increased with drought stress compared to the control group in both leaf and root.AMF inoculation of C.arietinum resulted in a significant decrease in proline and MDA content in both leaf and root under drought.While the proline content was higher in the roots under drought conditions compared to the leaves, this situation reversed with AMF inoculation under drought.MDA content was found higher in leaves at all conditions.Antioxidant enzymes (SOD and CAT) activities increased under drought stress in both leaf and root.However, AMF inoculation decreased the SOD activity (Figure 4) while increasing the CAT activity (Figure 5) compared to drought stressed group.Also interestingly, AMF increased enzyme activities compared to control.

Discussion and Conclusion
In this study, it was determined that inoculation of chickpea plants with G. mosseae improved plant tolerance for drought stress.Although this situation has been shown in the literature in general, some data obtained from this study have the potential to provide new information to the literature and some issues related to mycorrhizal symbiosis should be studied in more detail.
AMF inoculation increased leaf water potential of C.arietinum under drought stress.When the roots of plants infected with AMF, the surface areas of the roots increase, resulting in the potential for the plant to absorb more water from more areas (Bagyaraj et al., 2015).This gives the plant a great advantage, especially under drought stress conditions.
Under drought conditions, amount of some important macroelements such as phosphorus (P), calcium (Ca) and microelements such as iron (Fe), manganese (Mn), nickel (Ni), copper (Cu) and zinc (Zn) (Table1) increased by mycorrhizal symbiosis.Chen et al. (2020) explained these results by extraradical hyphal network formed in the soil upon AMF colonization.P and Ca are the main mineral elements for plant growth.Increasing the amount of these elements under drought stress leads to an increase in root growth, leaf area, photosynthesis rate, higher membrane stability, and water content (Ahanger et al., 2016).Zn, Mn and Fe are also important micronutrients, which have several vital roles for plants.Babaeian et al. (2011) showed that foliar application of these elements enhanced the yield components and alleviate the effects of drought.Peuke and Rennenberg (2011) also emphasized that Fe, Mn and Zn are important ligands for more than 1500 proteins that have catalytic, (co-)activating and/or structural functions.Ahanger et al. (2016) also well discussed in detail the importance of all these mineral elements in drought tolerance mechanisms.By mycorrhizal symbiosis, the increase in the amount of these elements under drought indicates that this symbiosis will provide an advantage for the C.arietinum plants to cope with stress.
Proline concentration increased with drought stress.Proline is known as a good osmolyte, and accumulation of proline under drought stress is well documented in various plant species in the literature.(Chun and Chandrasekaran, 2018;Çevik et al., 2019).Inoculation of C.arietinum by AMF decreased proline concentration under drought stress.There are different results related to AMF inoculation and proline accumulation under environmental stress in the literature.Some researchers reported that AMF inoculation increased proline content (Begum et al., 2019;Garg and Baher, 2013) while others reported no significant difference (Sohrabi et al., 2013) or decrease (Abdelmoneim et al., 2014) in different plants under stress conditions.Accumulation of proline in plants under drought known as a basic response to stress (Abdelmoneim et al., 2014).There is a good correlation between the increase of proline content and the intensity of the drought.As the severity of the drought increases, the proline content also increases (Keyvan, 2010).In this study, the decrease in proline content with AMF inoculation may indicate that the severity of the drought was reduced with AMF treatment.
Malondialdehyde (MDA), one of the end products of lipid peroxidation, is a good indicator of the level of oxidative stress (Gawel et al., 2004).The data of the present study showed that lipid peroxidation in chickpea plants significantly increased under drought stress.However, AMF treatment decreased lipid peroxidation compared to the drought group.Some researchers determined that the amount of MDA also increased due to the increase of radicals, especially H 202 (Ibrahim and Jaafar, 2012;Hasanuzzaman et al., 2020).As seen in Figure 6, AMF treatment increased the catalase activity.Catalase catalyzes the oxidation of hydrogen peroxide to water and oxygen.Increased activity of catalase with AMF treatment may have caused the scavenging of H202.This situation may have caused a decrease in lipid peroxidation.
Studies have shown that antioxidant enzyme activities increase under various stresses with AMF application (Chang et al., 2018;Duc et al., 2018).However, the decrease in SOD activity may be explained by reducing the intensity of stress with AMF inoculation, but this situation cannot explain the increase in catalase activity.Total enzyme activity analyzes may occasionally lead to such contradictions.Therefore, detailed isoenzyme analyzes can contribute to the solution of this problem.However, in this study, increases in enzyme activities also occurred regardless of the stress conditions.This situation may indicate that different signalling mechanisms are stimulated with the treatment of AMF.Although some studies claimed this may be related to the increase in nutrient intake, more studies should be conducted to clarify this issue.
In conclusion, this study showed that with the inoculation of AMF, the leaf water content and the amounts of some important mineral elements increased, and the proline and MDA content decreased under drought conditions.In addition, while there was a general tendency to increase antioxidant enzyme activities with AMF treatment, the increases in enzyme activities in the control groups also suggested that the antioxidant system could be stimulated in a stress-independent way.Especially the relationships between AMF-proline and AMF-antioxidant system, which have conflicting results in the literature, should be investigated with advanced molecular techniques.In particular, proteomic analysis can provide more data on this subject.

Figure 1 .
Figure 1.Changes in leaf water potential under drought and/or AMF inoculation.

Figure 2 .
Figure 2. Proline content in leaves and roots of chickpea plants under drought and arbuscular mycorrhizal fungal inoculation.

Figure 3 .
Figure 3. MDA content in leaves and roots of chickpea plants under drought and arbuscular mycorrhizal fungal inoculation.

Figure 4 .
Figure 4. Superoxide dismutase activity in leaves and roots of chickpea plants under drought and arbuscular mycorrhizal fungal inoculation.

Figure 5 .
Figure 5. Catalase activity in leaves and roots of chickpea plants under drought and arbuscular mycorrhizal fungal inoculation.

Table 1 .
Effects of mycorrhizal symbiosis on some element content of C.arietinum roots under drought stress ***p<0.001.