*3.4. Effect of Heat Stress on Chickpea H2O2 Content and Antioxidant Enzyme Activities*

The H2O2 content of the cultivars increased during heat stress treatments, indicating oxidative stress (Figure 4A). While the increase during heat acclimation (A) treatment was not significant in the Diyar cultivar, A treatment caused a 32% increase in Küsmen-99. Moreover, heat stress treatments caused a gradual increase in the H2O2 content of Diyar and Küsmen-99, especially in S treatment, by 31% and 84%, respectively. Superoxide dismutase (SOD) activity increased markedly in all heat-treated (A, A + S, and S) chickpea cultivars, although it was more pronounced in Diyar (3.9-, 6.7-, and 7.1-fold, respectively, of the corresponding control) (Figure 4B). Similar to SOD, all heat treatments resulted in a significant increase in peroxidase activity (POD) of both cultivars compared to the corresponding controls (Figure 4C). However, the increase in activity between A + S and S treatments in Diyar and A and A + S treatments in Küsmen-99 proved to be insignificant compared to each other. In contrast to SOD and POD, the catalase (CAT) activity of cultivars declined sharply in heat stress treatments, regardless of whether they were acclimated or not, while heat acclimation (A) treatment did not cause any significant change in CAT activity (Figure 4D). However, when acclimated and non-acclimated heat treatments (A + S and S) were compared, no significant differences in the CAT activity were determined for either cultivar.

**Figure 4.** Heat stress with or without heat acclimation induced changes in H2O2 content (**A**) and antioxidant enzyme activities (SOD, (**B**); POD, (**C**) and CAT, (**D**)) of chickpea cultivars. The values are presented as the mean ± standard error (SE), *n* = 3. The bars indicate significant differences between treatments and cultivars at *p* < 0.05 according to the LSD test.

#### **4. Discussion**

The objective of the study was to elucidate the physiological and biochemical mechanisms involved in the tolerance of chickpea cultivars to heat stress, either acclimated or non-acclimated. Plants develop different tolerance mechanisms to overcome the deleterious effects of high-temperature stress, especially when acclimated to heat [5]. The effects of heat acclimation on the biochemical and physiological mechanisms of two *Cicer arietinum* L. cultivars subsequently exposed to higher temperatures were studied. Photosynthetic responses to rising temperatures play a critical role in regulating plant heat tolerance. One of the most important responses in regulating plant heat tolerance is the photosynthetic response that the plant develops as temperatures rise. The most heat-sensitive components of the electron transfer chain are the units responsible for photosynthesis and, in particular, the oxygen-evolving complex (OEC) of PSII [6]. Heat stress inhibits photosynthesis by altering the redox balance of electron transport reactions [6,47]. In this study, chickpea cultivars exhibited higher FO values, especially when heat acclimated at 35 ◦C. Elevated FO values represent increased damage to chloroplasts due to heat stress, resulting in the inhibition of energy transfer to PSII and reduced quantum efficiency of PSII [22]. Fluorescence densities FJ, FI, and FP also decreased in phases J, I, and P of cultivars exposed to heat stress. These changes reflect the inhibition of electron transport from the OEC to the PQ pool. The curves show that the I and P phases decreased similarly in both cultivars during heat acclimation (A). In addition, significant differences were determined between the cultivars in both heat stress treatments when the ChlF curves were examined. Non-acclimated heat treatments (S) in both cultivars and acclimated stress treatment of Küsmen-99 made the OJIP curves to disappear on the logarithmic scale. Remarkably, the effects of heat stress on the OJIP curve were much more pronounced in the S treatments. The results show that OEC and reduction/reoxidation of QA and QB are more susceptible to high temperatures in Küsmen-99 than in Diyar. Similar effects of high temperature on photoinhibition of PSII have been reported previously [15,48]. The differential effects of heat on PSII could be due to different cyclic electron flow capacities around PSI [15], which could result from the genetic variation of cultivars. Heat stress treatment resulted in an increase in VOK and VOJ, indicating the alteration of L- and K- bands, respectively. The presence of the L-band provides information about the utilization of excitation energy, while the K-band refers to the stable electron transfer from OEC to P680<sup>+</sup> and subsequently to QA<sup>−</sup> [10,12]. Higher K-band is a heat indicator to predict plant response to heat [16]. Küsmen-99 showed

higher L- and K-bands than Diyar in acclimated and non-acclimated heat treatments. It has been reported that an increased L-band indicates a loss of connectivity between the reaction centers and their antenna complexes, while elevated K-band value represents an inhibition of OEC due to Mn-complex injury [6,10]. In contrast to the results of VOK and VOJ, the levels of VIP decreased in all treatments, except non-acclimated Diyar. The VIP indicates the changes in the G-band related to electron transfer from PSII to PSI [13]. Thus, these bands (K-, L-, and G-) formed as a result of the heat stress showed that the light reactions of photosynthesis, particularly the acceptor side of PSII were negatively affected. Moreover, heat stress markedly changed the efficiency and quantum yield of PSII *(*ϕP0, ψE0, ϕE0, ϕD0, ϕR0, and δR0) of chickpea cultivars. Among the efficiency and quantum yield parameters, ϕP0, ψE0, ϕE0, and ϕR0 were lower, whereas ϕD0 and δR0 were higher in both acclimated and non-acclimated heat treatments. The only exception was the δR0 parameter in the acclimated heat treatment of Diyar. The increased values of ϕD0 indicated that the trapped energy was probably radiated as heat energy and the connection between the photosynthetic systems was broken [11]. The results showed that electron transfer from PSII was inhibited by heat on both the electron donor and acceptor sides. All heat treatments caused photoinhibition in both cultivars and photoinhibition of PSII was alleviated by heat acclimation. Chickpea cultivars exposed to heat treatments exhibited reductions in both PIABS and PITOTAL. Changes in the efficiencies and quantum yields of the photosystem could be the reason for alterations in the performance indexes, which are multiparametric expressions for successive steps in primary photochemical reactions. While PIABS describes the part up to the reduction of intersystem electron transport of photons absorbed by the PSII reaction centers, PITOTAL describes the part up to the reduction of the PSI final electron acceptor [10,12]. Reductions in performance indexes and DF indicate impairment of the photochemical activities of the reaction centers. The main reason for these disruptions in photochemical activities as a result of heat stress is oxidative stress, which results from the increased formation of ROS in the thylakoid membranes [49].

Heat stress causes wilting, curling and yellowing of leaves as well as a reduction in plant biomass, suggesting that heat stress causes plants to reduce growth and trigger stomatal closure to prevent water loss [4,22]. RWC gradually declined under heat-stress temperatures. Previous reports showed that heat-tolerant wheat [50] and alfalfa [22] cultivars had the highest water content. Since Diyar had higher RWC values, the cultivar could be classified as heat tolerant. Treatments with heat acclimation had higher RWC values than treatments without acclimation, suggesting that the heat acclimation period may play an important role in maintaining the homeostasis of heated cells. When plants are exposed to any stress, this stress is accompanied by oxidative stress. It is well known that chloroplasts are the main source of ROS generation under stress conditions due to the limitation of electron transport [51]. Since chlorophyll is a necessary pigment for photosynthesis, the varying amount of total chlorophyll is a decisive indicator of the level of photosynthesis in plants [4]. The Chl (*a* + *b*) content of cultivars was drastically reduced by heat temperature, regardless of whether acclimated or non, especially in Küsmen-99. Similar results occurred in chickpea plants under heat stress, and this damage to pigment was found to be due to the photooxidation of chlorophyll [8]. Carotenoids, non-enzymatic antioxidants, protect chlorophyll from photooxidation [3]. In this study, the carotenoid content of the cultivars declined in all heat treatments. It was found that the content of photosynthetic pigments was higher in heat-tolerant chickpea genotypes than in the other genotypes [52]. Since Diyar always contains more chlorophyll and carotenoids in heat treatments, the results of this study are consistent with the literature. Contrary to chlorophyll and carotenoids, the increased levels of anthocyanins and flavonoids were determined in heat treatments. Anthocyanins and flavonoids act as antioxidants in plants and maintain chloroplast functionality by protecting chlorophyll from photoinhibition under heat-stress conditions. However, the reduced photosynthetic efficiency of chickpea under heat treatment indicated that the light screening role of anthocyanins and the antioxidant roles of flavonoids was not sufficient to prevent the overexcitation of chloroplasts, especially at Küsmen-99.

Heat stress leads to lipid peroxidation of cell membranes and membrane injury in numerous plants [5,19,22,53]. The heat stress treatments led to a marked increase in RLR and MDA results of the cultivars, but this increase remained lower increase due to heat acclimation. The increase in the RLR ratio indicates electrolyte leakage and loss of membrane stability. The excessive accumulation of MDA is due to the induction of lipid peroxidation in cell membranes by ROS, which is formed and accumulated by heat stress. Oxidative stress, which occurs as a result of increased ROS production and accumulation due to metabolic disorders, is known to be an important indicator of stress in plants. H2O2 is one of the ROS and is highly toxic to plant tissues [5]. Chickpea cultivars were found to have significantly increased H2O2 levels under heat stress. However, the increase in H2O2 levels was lower in acclimated cultivars than in non-acclimated cultivars. Although H2O2 is a stimulant to increase antioxidant capacity under stress conditions, its presence in cellular components above a threshold level is an indicator of oxidative stress [19]. Plants have antioxidant defense mechanisms to prevent excessive ROS production and improve tolerance to oxidative stress. It was found that plants with high levels of antioxidant enzymes also have a high tolerance to ROS-induced oxidative damage [4]. The results showed that both cultivars exhibited higher activity of antioxidants in all heat stress treatments, except CAT. Disturbances in the photochemical activity of PSII due to heat stress may lead to increased production of superoxide radicals (O2 −), the secondary product of the electron transfer chain, and thus to an increase in the activity of SOD [54]. SOD plays a role as the first defense against ROS, by converting O2 − to O2 and H2O2 [55]. Subsequently, POD, one of the antioxidants that detoxify H2O2, removes H2O2 by oxidizing components such as phenolic compounds and/or antioxidants. In the present study, although both SOD and POD activities increased with heat stress, the elevated RLR, MDA, and H2O2 levels indicated insufficient to remove oxidative stress in cultivars. In addition, ascorbate peroxidase (APX) and glutathione reductase (GR) are enzymes of the Halliwell–Asada pathway, one of the metabolic systems responsible for the detoxification of H2O2. It was reported that the activities of APX and GR increased in maize exposed to high temperatures [56]. Moreover, heat tolerance at lethal temperatures was found to be associated with increased activities of SOD and APX [57]. In contrast to SOD and POD, the activity of CAT exhibited a pronounced decline under heat treatments. CAT degrades H2O2 to H2O and O2 and is primarily located in peroxisomes. The decrease in the activity of CAT under heat stress is due to the photoinactivation of catalase and decreased catalase synthesis. In addition, high H2O2 may lead to a decrease in the activity of CAT due to substrate inactivation. The reduced activity of CAT may contribute to H2O2 accumulation, which leads to lipid peroxidation under heat-stress conditions [53].

According to the research results, it was found that heat acclimation in chickpea cultivars increased heat tolerance at higher temperatures to which the cultivars were later exposed. The increased tolerance was found to be associated with the enhancement of protective mechanisms such as anthocyanins, flavonoids, and antioxidant enzymes. Acquisition of thermotolerance by prior heat acclimation reduced cellular leakage and membrane injury. Therefore, oxidative damage and heat injury was reduced in treatments subjected to heat acclimation. Tolerance differences among cultivars became more evident in seedlings exposed to heat stress, especially acclimation. Diyar, which is known to be cold and drought tolerant, responded similarly to heat stress as the acclimated Küsmen-99, although it was not acclimated in all parameters. However, heat tolerance of both cultivars increased significantly with acclimation. According to the polyphasic chlorophyll *a* fluorescence data, Diyar showed photosynthetic activity under heat stress that approached the control by acclimation, whereas Küsmen-99 did not improve photosynthetic activity. This could be due to the fact that Diyar is more successful in maintaining water, chlorophyll, and carotenoid content and increases its anthocyanin content under heat-stress conditions. In addition, the increase in antioxidant enzyme efficiencies while maintaining membrane damage and the lower H2O2 content are physiological changes that make Diyar more tolerant to heat stress. The cultivar Diyar was more successful than Küsmen-99 in coping with the negative effects of heat stress.

**Funding:** This research was supported by the Scientific Research Unit (Project FEN-BAP-A-220413-39) of Giresun University.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are contained within the article.

**Acknowledgments:** The author would like to thank Yasemin Ekmekçi for her guiding contributions to the research.

**Conflicts of Interest:** The author declares no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
