*3.1. Effect of Heat Stress on Chickpea ChlF Rise and ChlF Parameters*

The OJIP transients measured as ChlF rises in dark-adapted control and stressed chickpea leaves were determined by plotting them on a logarithmic time scale (Figure 1).

The OJIP rise reflects three reduction processes in the electron transport chain (O-J, J-I, and I-P phases) [6,10]. The O-J rise contains information about the antenna size and indicates the reduction on the acceptor side of PSII [45]. The J-I phase refers to the kinetic properties required for the reduction and/or oxidation of the plastoquinone pool (PQ) [46]. The I-P phase represents the re-reduction of plastocyanin and the acceptor side of PSI [6,46]. Exposure to 35 ◦C significantly altered the shape of the typical OJIP curves seen in controls. The reduction in fluorescence intensity was more pronounced in both heat-acclimated (A + S) and non-acclimated (S) treatments of Küsmen-99 (Figure 1B). The S treatment caused the disappearance of the J-I and I-P phases, while the P level approached the O-J phase, indicating photochemical inhibition of PSII. A similar effect was determined in the heat-acclimated stress treatment (A + S) of Küsmen-99.

**Figure 1.** Induction curves of polyphasic ChlF in chickpea cultivars ((**A**,**B**), Diyar and Küsmen-99, respectively) exposed to heat stress with or without heat acclimation. The transients are plotted on a logarithmic time scale (10 μs to 1 s). The mean values of the OJIP transients are plotted, *n* = 6.

ChlF parameters, which provide information about photosynthetic fluxes and quantify the PSII and PSI behaviors are derived from ChlF transients. The parameters representing the relative values of controls were shown by spider plot graphics (Figure 2). Exposure to heat acclimation (30 ◦C for 2 days, A) resulted in slight changes in both cultivars compared to corresponding controls. However, significant changes in almost all selected ChlF parameters were determined in both cultivars exposed to heat stress (35 ◦C for 5 days), whether acclimated (A + S) or non-acclimated (S), compared to the controls. Heat stress resulted in a similar extent increase in both VOK and VOJ parameters in Diyar and Küsmen-99 (Figures 2A and 2B, respectively). The VOK and VOJ parameters are expressed as L- and K-bands, respectively, and reflect the inactivation of the oxygen-evolving-complex (OEC). The VIP values decreased when the cultivars were subjected to 35 ◦C, except S treatment of Diyar (Figure 2A). The decrease in VIP values (G-band) indicates limitations in electron transport on the PSI acceptor side. The maximum quantum yield of the photochemistry of PSII (ϕP0 = TR0/ABS = FV/FM) of chickpea cultivars reduced in both acclimated and nonacclimated heat stress treatments (Figure 2). The highest ϕP0 decreases were determined in A + S (38%) and S (72%) treatments of Küsmen-99 (Figure 2B). The parameter ψE0 (ET0/TR0) explains the probability that captured exciton moves the electron further in the electron transport chain than QA−. The highest decreases in ψE0 values were determined

in Küsmen-99 during heat stress, especially heat acclimated (41% of control). The ϕE0 value that defines the quantum yield efficiency that captured exciton moves electron to the electron transport chain (ϕE0 = ET0/ABS), declined markedly in all cultivars due to heat stress treatments, and the highest decline of ϕE0 results was determined in A + S (63%) and S (81%) treatments of Küsmen-99 (Figure 2B). Heat treatments led to marked increases in the quantum yield of dissipation (ϕD0 = DI0/ABS) values of both cultivars (Figure 2). Küsmen-99 exhibited the highest increment of A + S and S treatments 2.6- and 3.1-fold of control, respectively. Heat reduced quantum yield of electron transport from QA− to the PSI end electron acceptors (ϕR0 = RE0/ABS) values in cultivars, mainly in S treatment of Küsmen-99 (76%). The parameter δR0 (RE0/ET0), which reflected the probability that electron was transferred from intersystem electron carried to electron acceptors at PSI acceptor side was significantly increased by heat stress treatments in all cultivars, except 19% decrease in the A + S treatment of Diyar (Figure 2). The cultivars exhibited a gradual decrease in the values of performance indexes (PIABS and PITOTAL) in both heat acclimation and heat stress treatments (Figure 2). In determining PSII behavior, PIABS refers to energy absorption, capture, and conversion in electron transport steps. Heat acclimation led to a significant decrease in PIABS of both Diyar and Küsmen-99 (21% and 25%, respectively). Additionally, the highest reduction was determined in non-acclimated heat stress treatment of the cultivars, Diyar (94%) and Küsmen-99 (98%). The PITOTAL parameter includes additional electron steps to PIABS, and PSI refers to the measure for performance up to the reduction of final electron acceptors. The extent of the reductions of the PITOTAL was remarkable in both A + S and S treatments for Diyar (84% and 87%, respectively) and Küsmen-99 (91% and 96%, respectively). Likewise, the PIABS and PITOTAL, the total driving force for photosynthesis (DF = log PIABS) values of cultivars declined gradually with heat stress treatments (Figure 2). Among the cultivars, Küsmen-99 had the highest reductions, especially for the S treatment (5-fold of the corresponding control).

**Figure 2.** The radar-plot presentation of selected OJIP parameters in chickpea cultivars ((**A**,**B**), Diyar and Küsmen-99, respectively) exposed to heat stress with or without heat acclimation. The mean values of the parameters were plotted in relation to the corresponding controls, *n* = 6.

#### *3.2. Effect of Heat Stress on Chickpea Water and Pigment Contents*

The relative water content (RWC) of the leaves of the cultivars declined sharply in all heat treatments, including heat acclimation (Diyar and Küsmen-99, 9% and 20%, respectively) (Table 1). Exposure to 35 ◦C with heat acclimation (A + S) resulted in significant reductions (Diyar and Küsmen-99, 35% and 46%, respectively), while the non-acclimated heat stress treatment (S) resulted in the highest reductions (Diyar and Küsmen-99, 41% and 57%, respectively). All heat treatments significantly reduced the Chl (*a* + *b*) content of the cultivars (Table 1). The extent of Chl (*a* + *b*) reduction caused by heat acclimation was not as great as that by heat stress treatments. Heat acclimation led 17% and 12% reduction in control levels for Diyar and Küsmen-99, respectively. In addition, the magnitude of the reduction in Chl (*a* + *b*) content for the A + S and S treatments was 23% and 33%, respectively, for Diyar and 48% and 56%, respectively, for Küsmen-99. Similarly, all treatments resulted in a gradual decrease in the carotenoid content of the cultivars. The A + S treatment resulted in a 47% and 57% reduction in carotenoid content of Diyar and Küsmen-99, respectively, with the highest reduction determined in the S treatment of Diyar (60%) and Küsmen-99 (66%). In contrast to the results for Chl (*a* + *b*) and carotenoids, anthocyanin and flavonoid contents of cultivars subjected to heat treatments significantly increased (Table 1). The increase in anthocyanin content was more pronounced in all treatments (A, A + S, and S) of Diyar (3.3-, 5.7- and 4.9-fold of the corresponding control, respectively), while the highest flavonoid content was determined in the heat treatments (A + S and S) of Küsmen-99 (87% and 85%, respectively).

**Table 1.** Relative water content (RWC) (%), chlorophyll (Chl) (*a* + *b*) (mg g−<sup>1</sup> FW), carotenoid (mg g−<sup>1</sup> FW), anthocyanin (mg g−<sup>1</sup> FW), and flavonoid (%) content of chickpea cultivars subjected to heat treatments.


<sup>1</sup> Each value is presented as the mean ± SEs, *<sup>n</sup>* = 6 (for RWC, Chl and carotenoid) or 3 (for anthocyanin and flavonoid). Different letters indicate significant differences between treatments and cultivars at *p* < 0.05 according to LSD 5%.

#### *3.3. Effect of Heat Stress on Chickpea Membrane Integrity and Lipid Peroxidation*

The heat acclimation period did not cause any membrane damage in the leaves of cultivars according to the relative leakage ratio (RLR) and malondialdehyde (MDA) results (Figure 3). Heat treatments, both heat acclimated and non-acclimated, led to a dramatic increase in the RLR, indicating loss of membrane integrity in the leaf cells of chickpea cultivars (Figure 3A). RLR increased 4.9- to 5.6-fold in Diyar and 10- to 10.9-fold in Küsmen-99 under A + S and S treatments, respectively. Similar results were obtained for MDA contents that reflect the lipid peroxidation of cellular membranes. The MDA levels increased 2.1- and 2.8-fold in heat treatments (A + S and S, respectively) in Diyar and 5.1- and 6.2-fold in Küsmen-99 compared with control (Figure 3B).

**Figure 3.** Heat stress with or without heat acclimation resulted in changes in RLR (**A**) and MDA contents (**B**) in chickpea cultivars. The values are presented as the mean ± standard error (SE), *n* = 3. The bars and different letters indicate significant differences between treatments and cultivars at *p* < 0.05 according to the LSD test.
