**1. Introduction**

Climate change increases the generation and dispersion of abiotic stresses that pose a serious risk to crop production [1]. Heat is an abiotic stress factor that limits plant development and crop yield. Heat stress is described as a temperature increase that exceeds a particular level over a period of time and irreversibly damages plant growth [2]. A temporary temperature rise of 10–15 ◦C above ambient temperatures is evaluated as heat stress [3]. When plants are exposed to heat stress, it inhibits plant growth and production by causing physiological and biochemical disorders in plants [4]. Heat stress leads to the denaturation and aggregation of proteins [2], disruption of membrane structures [5], inhibition of photosynthesis [6], deterioration of photosynthetic pigments [7], and alterations in antioxidant enzymes [8]. The main reason for these adverse effects is the negative effect of heat stress on photosynthetic activity. Photosystem II (PSII) is the most heat sensitive in the photosynthetic apparatus, and PSII activity is significantly reduced under heat stress [9]. Chlorophyll *a* fluorescence (ChlF) transients (OJIP), which can be used to determine the extent of photosynthetic responses of plants to heat stress, are a reliable, non-invasive and powerful tool for assessing photosynthetic electron transport. The signals recorded by ChlF allow the determination of the physiological state of plants, calculation of specific

biophysical parameters, quantum yields, and probabilities that determine changes in PSII units, electron transport chain, and photochemical reactions by light [10–14]. Analysis of ChlF has been widely used in numerous studies to investigate various plant responses under heat stress, including rice [15], alfalfa [9], exotic weeds [16], tall fescue [7], barley [6], and maize [3]. The imbalance between the absorption and consumption of light energy due to heat stress leads to overexcitation of thylakoid membranes, resulting in photoinhibition. Heat stress leads to excessive energy loading of thylakoid membranes and eventually photoinhibition due to the imbalance between light energy absorption and utilization [17]. Photoinhibition is mainly due to the overproduction and accumulation of reactive oxygen species (ROS) such as hydroxyl radical (OH−), superoxide radical (O2 −), and hydrogen peroxide (H2O2) [18]. Subsequently, the presence of excessive amounts of ROS leads to oxidative stress and oxidative stress damages all cellular structures, especially membranes [9]. To alleviate the ROS-induced oxidative injury, plants generate antioxidant defense systems (enzymatic and non-enzymatic) to scavenge the overproduced ROS [19]. The enzymatic antioxidant defense system includes several antioxidant enzymes: superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase, etc. Non-enzymatic antioxidants include the metabolites: ascorbate, carotenoids, anthocyanins, flavonoids, etc. The antioxidant defense mechanism in plants is part of the adaptation to heat and its strength correlates with the acquisition of thermotolerance. Thermotolerance can be achieved by heat acclimation with exposure to a non-lethal heat treatment [5]. Heat acclimation is increased tolerance to the physical and physiochemical exceedances of heat stress. This complex process, which involves physiological and biochemical alterations in plants, including rearrangements in the lipid composition of membranes, changes in the content of compatible metabolites, synthesis and accumulation of antioxidants and protective proteins, changes in hormone levels, and modifications of gene expression [20,21]. Even when heat acclimation is successful, plant susceptibility to heat stress varies with plant genotype and developmental stage; however, susceptibility is largely affected by genotype and species variability, as well as mostly intra- and inter-species variations [22].

Chickpea is a heat-sensitive cool season legume, as its potential yield decreases at temperatures above 35 ◦C [8]. The main growing areas of chickpea are in the arid and semi-arid zones of the world and due to climate change, it will be inevitable that the potential yield of chickpea will decrease due to the increase in the intensity and duration of exposure to high temperatures. Since the chickpea is an economically and agriculturally valuable crop, it was very important to investigate the responses of this crop to heat stress and heat acclimation, which our research group had previously studied under chilling [23,24], freezing [25,26] and drought conditions [26,27]. Karacan et al. [26] studied 18 chickpea cultivars using a multi-criteria decision making method to rank them according to their cumulative tolerance to cold and drought stress conditions, using physiological and biochemical analysis data from previous studies. According to the research results, when chickpea cultivars were ranked according to these two stress responses, Diyar scored quite differently from the other cultivars and was classified as tolerant, while Küsmen-99 was classified as moderately tolerant with an average score. Therefore, the heat stress responses of these two cultivars, classified as drought and cold tolerant (Diyar) and moderately tolerant (Küsmen-99), were investigated. To this end, two chickpea cultivars (Diyar and Küsmen-99) were subjected to heat stress (35 ◦C for 5 days) with or without heat acclimation (30 ◦C for 2 days) to understand the interaction between heat tolerance and heat acclimation on PSII photochemical activity, pigments, membrane stability, and defense mechanisms. The objective of this study was to (1) elucidate the physiological mechanisms, especially the photochemical activity of PSII and antioxidant defense systems in chickpea under heat stress; (2) explain the mitigating effects of heat acclimation on the mechanisms damaged by heat stress; (3) compare the thermotolerance of the cultivars studied; (4) determine the role of the correlation between oxidative stress and endogenous defense systems in the thermotolerance of the cultivars.

#### **2. Materials and Methods**

Seeds of chickpea (*Cicer arietinum* L.) cultivars (Diyar and Küsmen-99) were obtained from the Central Research Institute of Field Crops in Ankara, Turkey. To prevent fungal infections, to which chickpea is frequently exposed, seeds were treated with pesticides [Benomyl and Thriam (0.3 g per 100 g of seed)] and were sown in pots (3 seeds each) containing 325 g of air-dried soil. The soil had the following characteristics: Texture, clay [28]; water holding capacity, 20.1% [29]; pH, 7.54 [30]; EC, 258 μS cm−<sup>1</sup> [31]; N, 1.48 g kg−<sup>1</sup> [32]; P, 16.25 mg kg−<sup>1</sup> [33]; and K, 464 mg kg−<sup>1</sup> [33]. 100 μg g−<sup>1</sup> NH4NO3 and 100 μg g−<sup>1</sup> KH2PO4 were added to the soil, because the N, P, and K levels were found to be insufficient for chickpea. Plants were grown for 15 days in a growth chamber under good irrigation, at 25 ± 1 ◦C/20 ± 1 ◦C (day/night), a 16/8 h (day/night) photoperiod, a relative humidity of 60 ± 5%, and a light intensity of 250 <sup>μ</sup>mol m−<sup>2</sup> <sup>s</sup>−<sup>1</sup> and then randomly divided into the following groups to conduct the experiments:

C0 and C, 17- and 22-day-old control seedlings grown under control conditions (25 ± 1 ◦C/ 20 ± 1 ◦C);

A, 17-day old heat-acclimated seedlings (grown under control conditions for 15 days, then exposed to 30 ± 1 ◦C/25 ± 1 ◦C for 2 days);

A + S, 22-day-old heat-treated acclimated seedlings (heat-acclimated and then exposed to 35 ± 1 ◦C/30 ± 1 ◦C for 5 days);

S, 22-day-old heat-treated non-acclimated seedlings (grown for 17 days under control conditions, then exposed to 35 ± 1 ◦C/30 ± 1 ◦C for 5 days).

The central leaves of the seedlings were used for the experimental analyses.

Since no statistically significant difference was found between the 17- and 22-day-old control groups (C0 and C) in all physiological and biochemical analyses examined, the results of the study were evaluated using the 22-day-old control group (C).

#### *2.1. Polyphasic Chlorophyll a Fluorescence (ChlF) Measurement*

ChlF transients were determined in dark-adapted leaves (6 replicates) using a Handy PEA fluorimeter (Plant Efficiency Analyser, Hansatech Instruments Ltd., Norfolk, UK). After a 30-min dark adaptation, leaves were irradiated with light (3000 μmol m−<sup>2</sup> s−1) for one second and the intensity of fluorescence at 20 μs (F0), 300 μs (FK), 2 ms (FJ), 30 ms (FI), and maximum fluorescence (FP) were determined [10]. The JIP test parameters were calculated from obtained fluorescence intensities. The effects of heat stress on cultivars were assessed based on relative fluorescence between the steps O and K [20 and 300 μs, respectively = VOK = (Ft − F0)/ (FK − F0)], O and J [20 μs and 2 ms, respectively = VOJ = (Ft − F0)/(FJ − F0)] and I and P [30 ms and at the peak P of OJIP, respectively = VIP = (Ft − FI)/(FP − FI)] were normalized and given as the kinetic difference VOK = VOK(treatment) − VOK(control), VOJ = VOJ(treatment) − VOJ(control) and VIP = VIP(treatment) − VIP(control), respectively [10,11]. The efficiencies and quantum yields of fluorescence were also calculated: ϕP0, (1 − F0/FM or FV/FM), maximum quantum yield of primary photochemistry; ψE0, (1 − VJ), probability that a trapped exciton moves an electron into the electron transport chain beyond QA−; ϕE0, [(1 − F0/FM) × ψE0], quantum yield for electron transport; ϕD0, (DI0/ABS), quantum yield of energy dissipation; ϕR0, (ϕP0 × ψ<sup>0</sup> × δR0), the quantum yield of electron transport from QA<sup>−</sup> to the PSI end electron acceptors; δR0, (1 − VI)/(1 − VJ), the efficiency with which an electron can move from the reduced intersystem electron acceptors to the PSI end final electron acceptors. The performance indexes (PIABS and PITOTAL) were calculated from the components to determine the difference between the cultivars PIABS, [(RC/ABS) − [ϕP0/(1 ϕP0)] [ψ0/(1 − ψ0)], performance index (potential) for energy conservation from photons absorbed by PSII to the reduction of intersystem electron acceptors; PITOTAL, PIABS [(δR0/(1 − δR0)], performance index (potential) for energy conservation from photons absorbed by PSII to the reduction of PSI end acceptors; DF, log(PIABS), driving force on absorption basis [10,11].

#### *2.2. Water Content and Pigment Analysis*

To determine the percent relative water content (RWC) of leaf segments (R = 0.5 cm and 6 replicates), fresh leaves were weighed (FW) and then incubated in 10 mL distilled water for 24 h to determine the saturated weight (SW), and the leaves were dried at 80 for 48 h, their dry weight (DW) was determined, and the RWC was calculated as (%) = [(FW − DW)/(SW − DW)] × 100 [34]. After extraction of the leaves (0.1 g with 6 replicates) in 100% acetone, they were measured spectrophotometrically (at wavelengths 470, 644.8, and 661.6 nm), and the content of chlorophyll (Chl) (*a* + *b*) and carotenoids (*x* + *c*) (mg g−<sup>1</sup> FW) was calculated [35]. To determine anthocyanin content (mg g−<sup>1</sup> FW) and flavonoid content (%), fresh leaf samples (0.1 g with 3 replicates) were ground in acidified methanol [methanol:water:HCl (79:20:1)] and measured at wavelengths of 530 and 657 nm for anthocyanin and 300 nm for flavonoid, respectively. Anthocyanin was calculated according to the method of Mancinelli et al. [36]. Flavonoid was calculated as a percentage of the content of 22-day-old control plants (C) [37].
