The present experiment was carried out during 2021-22 at National phytotron facility, ICAR-Indian Agricultural Research Institute, New Delhi. The materials for present investigation comprised of 10 germplasms of cucumber collected from various parts of India. On the basis of their performance in two rounds of screening and performance under open field conditions, these genotypes are grouped into thermotolerant and thermosensitive. Five thermotolerant ‘TT’ and five thermosensitive ‘TS’ cucumber lines were grown in the pots with standard NPH potting mixture of soil, sand and coco peat in ratio 2:1:1 (v/v) ( Table 1 ; Figure 1 ). Three seeds were sown in each pot and 5 replications were maintained for each genotype in both control and treatment conditions. A completely randomized design (CRD) with three replications per genotype per treatment was used. Data were taken from 3 randomly selected replications from each genotype. Seedlings were irrigated by sprayer cans with water and Hoagland nutrient solution every day morning and kept in such a condition that there was no water deficit. More frequent watering of plants was done under treatment to avoid any moisture stress. To avoid infections with fungal diseases, seedlings were occasionally sprayed with captan 2g/liter.

In control conditions plants were maintained at normal temperature (30°C/25°C day/night) in same glass house compartment throughout the experiments, while for treatment, plants were initially grown under normal conditions for twenty days, later seedlings were transferred to growing chamber for high temperature treatment. Therefore, the first HT treatment was set as 35°C for 12 h in the daytime and 30°C for 12 h in the night for 5 days, later growing chamber temperature raised to 40°C for 12 h in the daytime and 35°C for 12 h in the night for 5 days. Under the growth chamber, the plants were grown in growth media and were maintained without any moisture stress. Application of water and Hogland solution was practiced multiple times in a day to avoid water stress for the plants kept under temperature stress condition. Measurements were done on first or second true leaf of seedlings from three replications of each genotype in both control and treatment conditions. The physio-biochemical measurements were carried out at three different time intervals. First reading was recorded before transferring of the plants to growing chamber for treatment (day 0 control and day 0 stress). Remaining two readings were recorded after transferring of the plants to growing chamber for treatment. Second reading was noted on the last day of moderate stress (35°C/30°C) treatment (day 5 stress) and on same day readings were recorded under control conditions (day 5 control). Third reading was observed on the last day of high temperatures stress (40°C/35°C) treatment (day 10 stress) and on the same day data were recorded in control conditions (day 10 control).

The relative chlorophyll content of the leaves was measured by a SPAD chlorophyll meter (Apogee chlorophyll content meter). The measurements were done on the adaxial surface of the first and second true leaves in a single plant in five points uniformly distributed throughout the leaves and the average values were taken for analysis. The average value of two leaves was used to estimate the chlorophyll content. Chlorophyll was measured in CCI units. The CCI values of the instrument ranges from 1 to 100.

Membrane stability index (MSI) of fresh leaves was determined as per the method suggested by Bailly et al. (1996). For this purpose, two plants of each genotype were randomly chosen per replicate and two leaf samples per plant were taken as follows. One sample from first true leaf and the second sample from second true leaf to represent mature and developing leaves, respectively. The conductivity of solutions was measured using a conductivity bridge meter and MSI calculated using following formulae:

Where C1 conductivity at 40°C; C2 conductivity at 100°C

Leaf samples were used for Relative water content (RWC) assay according to the method described by Barrs and Weatherley, 1962. RWC in leaves of the plants was measured from two randomly chosen fully developed leaves. A 10 cm long segment was excised from the middle portion of the leaf and cut into two equal halves; FW was recorded and the leaf segments were immediately immersed into distilled water in a Petri plate for 4 h at room temperature. The leaf segments were blotted properly and turgid weight (TW) was recorded. Then, the samples were placed in a paper bag and dried in a hot air oven at 70°C for 24 h and the dry weight (DW) was recorded. The fresh weight (W1), turgid weight (W2), and dry weight (W3) of leaves were measured, and the RWC was calculated as follows:

Gas exchange measurements were performed using a portable photosynthesis system (Li-6400; LI-COR, Inc., Lincoln, NE, USA) in the morning between 9:00-10: 30am, on the first fully expanded leaf between 1 and 6 h of the light period on the third day of control (day 3 control), and moderate stress (day 3 stress) and high temperature treated (day 8 stress) plants. Air temperature was between 25°C - 30°C as per the temperature of the growth chamber. The light response curves were measured at ambient CO₂ concentrations (350-400 μmol) during photosynthetic observations. Leaves were illuminated with photon flux densities 1500 μmol photons m^-2s^-1. Net photosynthetic rate (P_N), stomatal conductance (g_s), transpiration rate (E), and intracellular CO₂ concentration (Ci) was measured. Canopy temperature of cucumber leaves was performed using an imaging FLUKE thermal imager.

Morphological parameters like shoot length, fresh weight and dry weight, measurements were taken only once in both control and treatment conditions (10-day control and 10-day stress). Three plants from each genotype were for recording observations. Fresh weight and shoot length were measured immediately after harvesting of genotypes, whereas dry weight was measured by drying the samples in an oven at 85°C for two days.

Determination of malonaldehyde (MDA) content was described by Dhindsa et al. (1981) and modified by Wang et al. (2018). A total of 0.5 g leaves were ground into powder using 0.5% trichloroacetic acid (TCA), then centrifuged at 3000g for 20 min. The supernatant (2 mL) was added the same volume of 0.5% thibabituric acid (TBA). After that the mixture was boiled at 100°C for 30 min to obtain the supernatants. Finally, we recorded the absorption wavelengths of supernatants on 450 532 and 600 nm. MDA activity was expressed as nmolg^-1FW.

Ascorbate peroxidase activity was determined according to Wang et al. (1991) by estimating the decreasing rate of ascorbate oxidation at 290 nm. APOD extraction was performed in 50 mM Tris–HCl (pH 7.2), 2% PVP, 1 mM EDTA, and 2 mM ascorbate. The reaction mixture consisted of 50 mM KH₂2PO₄ buffer (pH 6.6), 2.5 mM ascorbate, 10 mM H₂O₂, and enzyme, containing 100 µg proteins in a final volume of 1 mL. The enzyme activity was calculated from the initial rate of the reaction using the extinction coefficient of ascorbate (E = 2.8 mM cm−1 at 290 nm). APOX activity expressed in terms of µmol min^-1g^-1.

Hydrogen peroxidase contents were determined by the method Ohkawa et al. (1979). For determination of hydrogen peroxide, 0.5 mL of 0.1 M Tris–HCl (pH 7.6) and 1 mL of 1 M KI were added to 0.5 mL of supernatant. After 90 min, the absorbance was measured at 390 nm. A standard curve for hydrogen peroxide was prepared to calculate hydrogen peroxide concentration in each sample. Hydrogen peroxidase is expressed in terms of μmol g^-1FW.

Two contrasting genotypes, WBC-13 and DGPC-59 ( Supplementary Figure 1 ) were used for gene expression analysis under two different stress conditions (35°C/30°C and 40°C/35°C) along with control without any stress (30°C/25°C). Total RNA from cucumber leaves under different stress conditions was extracted using Trizol reagent. RNA was quantified by spectrophotometric analysis and the quality was evaluated through agarose gel electrophoresis. First-strand complementary DNA (cDNA) synthesis was carried out using the user instruction (Promega, USA). Relative expression of 18 important genes associated with heat tolerance were conducted using two contrasting genotypes under two different stress conditions ( Table S1 ). Quantitative real-time PCR was carried out using Light Cycler (Roche) with Light Cycler Fast Start DNA Master SYBR Green kit (Roche). Amplification of stress-related genes was carried out according to the manufacturer’s protocol. Reaction mixture of 20 μl contains 1.5 μl cDNA, 0.3 μl of primer (forward and reverse), 12.5 μl SYBR Premix, and 5.4 μl dH₂O. Expression analysis of all genes were tested in triplicate with appropriate primers along with Actin used as an internal control. The gene expression data were calculated comparative to Actin, and Ct values of the used target genes were normalized using the Ct values of Actin. The levels of mRNA were also normalized with Actin and its value was expressed relative to that of the control, which was given an arbitrary value 1 (Liu et al., 2012). The relative differential gene expression was measured according to the equation 2−ΔΔCt (Livak and Schmittgen, 2001). The final data of RT-PCR were calculated from three experimental replicates.

The data collected were analyzed in R software using one- or two-way ANOVA after verifying data for homogeneity and normality. The correlation among the variables was analyzed using Spearman correlation and a correlogram was constructed for each temperature treatment in the controlled heat-stress experiment and total plant responses from two stages for the field experiment. The multiple comparisons of means were made using Tukey’s HSD (Honestly Significant Difference) under α≤ 0.05. For only the significant main effects of stage, mean separation for the two stages were done within each level of varieties using Tukey’s HSD (Honestly Significant Difference) at α≤ 0.05.

It was evident that the tolerant genotypes were maintaining comparatively less canopy temperature by transpirational cooling than the susceptible genotypes in high temperature treatment conditions ( Figure 2 ; Supplementary Figure 2 ). Lowest canopy temperature was recorded in the tolerant genotypes WBC-13, DGC-103 and DC-83 at moderate temperature stress and the same of set of genotypes also maintained a comparatively lowerer canopy temperature under high temperature stress condition ( Table 3 ). The effects of genotypes, temperature, and genotype × temperature interaction effects on canopy temperature were significant ( Table S2 ).

Leaf Pn, Gs, Ci, and Tr were significantly decreased on exposure to heat stress conditions in susceptible genotypes compared to tolerant genotypes. The net photosynthesis of susceptible genotypes was significantly lower than the tolerant genotypes under temperature stress conditions ( Supplementary Figure 3A ). Percentage decrease of Pn was 29.4% and 67.7% in susceptible groups in contrast to 7.9% and 24.1% in tolerant group under heat stress conditions ( Table 4 ). Similarly, stomatal conductance (Gs) of all genotypes decreased to varied extent at high temperature treatments compared to the controls. Tolerant genotypes expressed higher stomatal conductance compared to susceptible genotypes under heat stress conditions ( Supplementary Figure 3B ). Reduction in stomatal conductance was 23.6% and 37.7% in tolerant group and 55.8% and 80.2% among the susceptible genotypes ( Table 4 ). Tolerant genotypes recorded higher internal CO ₂ concentration (Ci) at high temperature treatments compared to the susceptible genotypes ( Supplementary Figure 4A ). Tolerant plants shown less reduction in Ci percentage (29.5% and 46.1%) as compared to drastic reduction in susceptible genotypes (37.7% and 58.9%) under stress conditions ( Table 4 ). Transpiration rate (Tr) was significantly reduced at high temperature treatment in susceptible genotypes compared to tolerant genotypes ( Supplementary Figure 4B ). High temperature adversely affected transpiration rate of susceptible genotypes (55.5% and 66.3% reduction) compared to tolerant varieties (15.36% and 14.87%) as shown in Table 4 .

The tolerant genotype, DC-83 (19.2 μmol CO ₂ m ^-2 s ^-1, 16.1 μmol CO ₂ m ^-2 s ^-1) and WBC-13(18.3 μmol CO ₂ m ^-2 s ^-1, 16 μmol CO ₂ m ^-2 s ^-1) had stable net photosynthesis in moderate and high temperature stress conditions, respectively. Drastic reduction in photosynthesis was recorded in Baropatta (12.7 μmol CO ₂ m ^-2 s ^-1), WBC-22 (13.2 μmol CO ₂ m ^-2 s ^-1) at moderate heat stress conditions and DGPC-59 (4.5 μmol CO ₂ m ^-2 s ^-1) and EC-753493 (5.1 μmol CO ₂ m ^-2 s ^-1) in peak stress conditions. Maximum stomatal conductance was observed in DC-83 (0.57 mol H ₂ Om ^-2 s ^-1) followed by WBC-13(0.57 mol H ₂ Om ^-2 s ^-1) and minimum was seen in DGPC-59 (0.24 mol H ₂ Om ^-2 s ^-1), followed by DC-206 (0.25 mol H ₂ Om ^-2 s ^-1) in moderate stress. Similarly, WBC-13 (0.53 mol H ₂ Om ^-2 s ^-1) and DC-83 (0.49 mol H ₂ Om ^-2 s ^-1) had high stomatal conductance where as drastic drop in stomatal conductance was recorded in DGPC-59 (0.08 mol H ₂ Om ^-2 s ^-1) and Baropatta (0.11 mol H ₂ Om ^-2 s ^-1) at peak stress conditions ( Table 5 ).

Maximum internal CO ₂ concentration was observed in DC-83 (558.9 µmolCO ₂ mol ^-1) followed by DARL 106 (548.9 µmolCO ₂ mol ^-1) and minimum was recorded in DGPC-59 (407.9 µmolCO ₂ mol ^-1), followed by WBC-22 (457.4 µmolCO ₂ mol ^-1) in moderate stress. WBC-13 (490.9 µmolCO ₂ mol ^-1) and DGC-103 (413.0 µmolCO ₂ mol ^-1) had high internal CO ₂ concentration where as drastic drop in stomatal conductance was recorded in DGPC-59 (272.3 µmolCO ₂ mol ^-1) and WBC-22 (260.4 µmolCO ₂ mol ^-1) at peak stress conditions. Similarly, highest transpiration rate was recorded in DC-83 (4.8 m mol H ₂Om ^-2 s ^-1) followed by WBC-13 (4.5 m mol H ₂Om ^-2 s ^-1) and minimum was recorded in WBC-22 (2.3 m mol H ₂Om ^-2 s ^-1) followed by DGPC-59 (2.4 m mol H ₂Om ^-2 s ^-1) in moderate stress and WBC-13 (4.7 m mol H ₂Om ^-2 s ^-1), DC 83 (4.7 m mol H ₂Om ^-2 s ^-1) shown highest transpiration rate whereas WBC-22 (1.8 m mol H ₂Om ^-2 s ^-1) followed by DGPC-59 (1.9 m mol H ₂Om ^-2 s ^-1) had lowest transpiration rate at peak stress conditions ( Table 5 ).

Genotype, temperature and genotype × temperature interaction had an impact on photosynthetic pigment and leaf gas exchange−related parameters and significant effects were observed ( Table S3 ).

Shoot length, fresh weight and dry weight of all genotypes as well as percentage change are presented in Table 6 . Fresh weight, shoot length and dry weight were significantly reduced in seedlings grown under high temperature stress condition in all the genotypes. However, tolerant group showed less reduction (32.1%) in shoot length whereas higher reduction was recorded in susceptible group (71.7%) under high temperature stress condition. Significant reduction was also observed in fresh weight in both tolerant and susceptible genotypes. But tolerant genotypes were able to maintain the fresh weight in treatment conditions in contrast to susceptible genotypes. Reduction in fresh weight was higher in susceptible group of genotypes (87.0%) whereas in tolerant group shown much lower (62.7%) reduction in treatment conditions. Significant reduction in dry weight was recorded in all genotypes, however, percent of reduction was high in susceptible genotypes (67.4%) compared tolerant group (36.3%) under heat stress conditions.

It was found that highest shoot length was observed in WBC-13 (20 cm), DARL-106 (15.3 cm) and lowest in DGPC-59 (6.6 cm), DC-206 (8.7cm) under stress conditions. Fresh weight was higher in WBC-13 (21.1 g), DGC-103 (15.5 g) and much lower low in DGPC-59 (3.1 g), DC-206 (3.2 g) under high temperature conditions. Highest dry weight was recorded in WBC-13 (2.6g) and WBC 39-1 (1.8 g) and lowest in WBC-22 (0.2 g) and EC-753493 (0.4g) under stress conditions. Significant effects were seen (p< 0.05) for the effects of genotype, temperature, and genotype × temperature interaction on shoot length, fresh weight, and dry weight ( Table 7 ; Table S4 ).

Based on the performance and analysis of physio-biochemical characters in 10 different genotypes, two genotypes namely, WBC-13 (heat tolerant) and DGPC-59 (heat susceptible) were selected for gene expression studies of selected heat responsive genes. Expression profiling of 18 selected heat responsive genes was conducted in two contrasting genotypes under three different temperature conditions (Control: 30°C/25°C, Moderate stress: 35°C/30°C and High stress: 40°C/35°C). Genes used in study are mentioned in Table S5 . Rubisco S gene showed highest expression of 6-7 folds at moderate heat stress in DGPC-59 whereas WBC-13 showed highest expression of 4-5 folds at high stress condition. Analysis of result showed that Rubisco L gene was 5-6 folds upregulated in DGPC-59 at high heat stress whereas WBC-13 showed increase in 5-6 folds expression at moderate heat stress ( Figure 3A ).

A remarkable up-regulation in relative accumulation of some HSPs under high stress conditions were recorded in tolerant genotypes. At the same time, few HSPs were shown down regulation under high heat stress conditions in the tolerant genotype, WBC-13 ( Figure 3B ). In control conditions, no significant differences were observed between the genotypes except for the HSP70 which showed higher expression in susceptible genotypes. It was observed that at moderate heat stress condition, only HSP 90.6 shown higher expression in WBC-13 as compared with other stress condition. At high heat stress condition, HSP 90.1, HSP 70 and HSP 17.6A shown higher expression in tolerant genotype, WBC-13. Some HSPs like HSP90.3, HSP90.6, HSP 23.A, HSP 90.5 shown upregulation in DGPC-59 whereas HSP90.4 shown no significant difference between the genotypes at high heat stress condition. Additionally, the genes corresponding to signal transduction were also studied under control and heat stress conditions. In control and treatment conditions, significant differences were seen among the genotypes ( Figure 3C ). Under high heat stress conditions tolerant genotype, WBC-13 expressed downregulation for CsTIP1;3, CsTIP3;2, CsTIP1a whereas WBC-13 shown upregulation for CsTIP1b gene ( Figure 5 ). In case of Calmodulin gene expression, both contrasting genotypes had same level of expression with no significant difference. G-protein-α and oxygen evolving enhancer genes were upregulated in DGPC-59 at high heat stress condition and downregulated in WBC-13.

In our present experiment, analysis of variance for different physiological and biochemical parameters was significant indicating the existence of significant genetic variability among the genotypes and differential response of genotypes to heat stress conditions. In chloroplast, the chlorophyll is harbored by thylakoid which are considered as the most heat labile cell structures (Vacha et al., 2007). Any damage to thylakoid caused by heat is expected to result in chlorophyll loss. This showed that chlorophyll is linked with dry matter accumulation and can be utilized in screening the genotypes for high-temperature tolerance at seedling stage of cucumber. We found variations in the percent degradation of chlorophyll in the present investigation suggesting that some of the cucumber genotypes were able to maintain more chlorophyll under stress conditions. Chlorophyll content in leaves decreased as HT days increased, and the decrease was faster in a heat-susceptible cultivar compared to tolerant cultivar as was observed in other crops like hot pepper (Arnaoudova et al., 2020) and tomato genotypes (Bhattarai et al., 2021). The higher chlorophyll content in heat-tolerant cultivar gives better photosynthetic stability than heat-susceptible tomato cultivars (Zhou et al., 2017). By elevating unsaturated fatty acids and making the plasma membrane more fluid, HS causes the plasma membrane to become disorganized (Hofmann, 2009). It also affects cellular processes by starting a signal cascade (Firmansyah and Argosubekti, 2020; Hassan et al., 2021). The ability to adapt to high temperatures appears to be governed by a stable cell membrane system that keeps working under heat stress. High temperature stress can directly affect membrane integrity through photochemical modifications during photosynthesis or ROS (Bita and Gerats, 2013). It is also suggested that the membrane disruption may alter water, ion and organic solutes movement across the plant membrane which interferes with photosynthesis and transpiration. In this study we evaluated the membrane stability index and it was found that tolerant genotypes maintained high stability index. Recently, it was demonstrated that tolerant genotype of cucumber shown less relative conductivity and less damage of membrane lipid (Wang et al., 2020). The thermosensitive cucumber genotypes had high relative conductivity after high-temperature stress compared to tolerant genotypes (Yu et al., 2022). Similarly, cucumber genotypes with less electrolyte leakage ability tend to be more heat tolerant than genotypes with more electrolyte leakage at a high temperature of 40°C (Ali et al., 2019). Heat-tolerant cultivars of cauliflower expressed more cell membrane thermostability than susceptible ones (Aleem et al., 2021). Under different stress conditions, plants maintain their physiological balance through higher RWC values particularly under higher rates of transpiration. In the present study, plants subjected to heat stress conditions displayed decreased RWC values suggesting the sensitivity of cucumber plants towards heat stress. The difference in RWC under stress conditions did not differ significantly among the tolerant and susceptible genotypes suggesting its limited role in heat tolerance in cucumber. Raja et al. (2020) reported decreased RWC in tomato plants under high-temperature stress.

Chlorophyll fluorescence is a rapid, reliable, and inexpensive procedure for predicting photosynthetic performance under HS. Reduced Fv/Fm values indicate damage to the light-harvesting complex (Moradpour et al., 2021). Chlorophyll fluorescence has been used as screening tool in common bean (Stefanov et al., 2011) and okra (Hayamanesh, 2018). Several recent studies also supported our finding that tolerant genotypes shown higher Fv/Fm ratio with stable photosynthetic system under heat stress conditions. High temperature seriously impaired the photosynthetic system of susceptible plants as compared to the tolerant genotypes (Yu et al., 2022).

In our study, HT significantly decreased the photosynthetic rate in thermos-sensitive genotypes but stable rate of photosynthesis was maintained in the tolerant cultivar. The stable photosynthetic rate of heat-tolerant cultivar in HT might be due to the increased stomatal conductivity and transpiration rate. Under the condition of 40°C/35°C heat treatment, rate of photosynthesis and stomatal conductance were drastically reduced in the susceptible genotypes in contrast to the tolerant genotypes suggesting the ability of the tolerant genotypes to sustain the photosynthetic activities even under high-temperature stress conditions. Yu et al. (2022) demonstrated thermo-tolerant plants showed stable photosynthesis under high-temperature stress whereas sensitive plants had extremely unstable photosynthesis in cucumber. Photosynthetic rate was significantly reduced in susceptible cultivar but not in tolerant seedlings even with the exposure to 42°C in hot pepper. Similarly, stomatal conductivity and transpiration rate was significantly higher in tolerant genotypes compared to susceptible genotypes (Rajametov et al., 2021). In tomato, high stomatal conductivity and transpiration rate under heat stress facilitate reduced canopy temperature in heat-tolerant genotypes, providing better protection for chlorophyll and maintaining a relatively high photosynthetic rate (Zhou et al., 2017). Therefore, it was concluded that the ability of the tolerant genotypes to maintain a stable photosynthetic rate was one of the important factors for thermotolerance.

In the present study, plants were subjected to high temperature stress conditions exhibited reduction in shoot length, fresh weight and dry weight values suggesting the effect of heat stress on biomass of the plants. However, the tolerant genotypes were able to maintain the higher biomass compared to susceptible genotypes. Reduction in plant growth under high temperatures varied among the genotypes and tolerant genotypes exhibited significantly better morphological traits when compared with the sensitive genotypes in tomato (Shaheen et al., 2016). Lower height reduction under heat-stress in tolerant genotypes signified that their ability to maintain their growth properly when exposed to heat stress conditions. The changes in plant diameter under heat stress may be related to changes in stem tissue hydration. Reduction in growth because of reduced water content in cell and cell size is common when plants are exposed to high temperature stress conditions (Ashraf and Hafeez, 2004; Rodríguez et al., 2005). Besides, retarded relative growth in the susceptible genotypes because of reduction in net assimilation rate (NAR) was reported in maize, millet (Wahid et al., 2007) and sugarcane (Srivastava et al., 2012).

Variability in increasing the activities of these antioxidants across cucumber genotypes indicates their differential ability to acquire thermo-tolerance. Even the heat tolerance was found directly linked with the percent increase in catalase, superoxide dismutase, guaiacol peroxidase accumulation in most genotypes. Thus, our results show that tolerance mechanism for heat stress exists in cucumber genotypes for a variable extent. Proline serves as a membrane protectant, and due to its zwitter ion character, accumulates in high-concentration in cell cytoplasm under stress conditions without interfering with cellular structure or metabolism. Proline in plants functions as an osmoprotectant and allows them to tolerate stress (Akram et al., 2018; Alzahrani et al., 2018). Higher levels of proline accumulation in plants occur under stress conditions. In this study, we evaluated the contents of proline and found that tolerant genotypes accumulated significantly higher proline content under heat stress in comparison to susceptible genotypes. Tomato genotypes accumulated high amount of proline under heat stress conditions (Raja et al., 2020). Proline content was also significantly higher in tolerant genotypes compared to susceptible genotypes after heat treatment in hot pepper (Rajametov et al., 2021). Several recent studies reported that proline accumulation occurs in plants with exposure to stress conditions (Kaur and Asthir, 2015; Moreno-Galván et al., 2020) because of its property to stabilize subcellular structures, scavenging free radicals and buffer cellular redox potential (Hazman et al., 2015; Dar et al., 2016; Nurdiani et al., 2018). Heat stress is known to accompany with the formation of reactive oxygen species such as H₂O₂ and OH^-, which damage membranes and macromolecules. SOD is usually considered as the first line of defense against oxidative stress. In plants, we found significant difference between genotypes with respect to SOD accumulation and SOD activity was increased under heat stress conditions in tolerant genotype. No significant difference in superoxide dismutase enzyme (SOD) activity was detected between contrasting genotypes under normal conditions, whereas tolerant genotypes exhibited significant increase in SOD activity during heat stress compared with susceptible genotype (Wang et al., 2020). Tolerant genotypes of brinjal were reported to have higher amount of superoxide dismutase, peroxidase and catalase (Faiz et al., 2020). Superoxide dismutase (SOD) and peroxidase (POD) are two necessary antioxidant enzymes that protect plants from heat-induced oxidative stress. Catalase and peroxidises are the most important enzymes involved in regulation of intracellular level of H₂O₂. They convert H₂O₂ into OH^- along with the regeneration of NADP, thus helping the plants under stress conditions (Sairam et al., 1997; Xu et al., 2008). We found higher accumulation of catalase and peroxidises in tolerant genotypes under heat stress conditions compared to susceptible genotypes. The heat tolerance was found directly linked with the percent increase in catalase/superoxide dismutase/guaiacol peroxidase accumulation in wheat genotypes. During present study, MDA content was increased in susceptible genotypes as compared to tolerant genotypes under heat stress conditions. Potential resistance mechanisms of plants exposed to heat stress may involve higher osmotic regulation capacity related to an increase in leaf protein content (Ding et al., 2016). We also observed increased protein levels in tolerant genotypes under heat stress conditions was instrumental in conferring heat tolerance in cucumber genotypes.

The acquisition of plant heat tolerance is closely associated with the synthesis of chaperone proteins and the levels of non-enzymatic antioxidants in response to HT (Kotak et al., 2007; Wahid et al., 2007). Heat shock proteins play an essential role in the regulation of HSFs and subsequently, the expression of heat responsive genes associated with heat tolerance. The plants generally activate and accumulate a large amount of the HSPs to response to heat shock exposure to maintain the stability of cells under stress conditions (Richter et al., 2010; Li et al., 2018). In our study, HSP 90.1 and HSP 70 shown seven-fold and six-fold increased expression, respectively in tolerant genotype in response to heat stress conditions. Earlier, Yu et al. (2018) also reported the role of heat shock proteins in thermotolerance in cucumber. Besides, lower expression of HSP70 has been recorded in susceptible genotypes of chilies in response to heat stress while the tolerant ones showed overexpression of HSP70 which enhanced the thermostability of cell membranes (Usman et al., 2015). Similarly, significant increase in BoHSP70 in cabbage, HSP60/CPN60, HSP70, HSP90, HSP100/ClpB, and HSP90 activator and HSP70/HSP90 organizing protein in spinach and ClHSP11.1A, ClHSP50.3, and ClHSP17.4 in watermelon are reported to play key role in response to high temperature stress (Park et al., 2013; He et al., 2018; Zhao et al., 2018). Recent studies revealed that chilli specific many HSPs including CaHSP70, CaHSP60, CaHSP20, and CaHSP16.4 are upregulated in pepper under HS and significant difference between the genotypes ( Guo et al., 2015; Usman et al., 2015).

The AQPs channel proteins to facilitate the transport of water primarily through the plasma and tonoplast membranes in the plant cells (Chaumont and Tyerman, 2014). They are often designated as plasma membrane intrinsic proteins (PIPs) or tonoplast intrinsic proteins (TIPs) (Danielson and Johanson, 2008). Participation of AQPs in different abiotic stress responses is reported earlier (Matsumoto et al., 2009; Sreedharan et al., 2013; Wang et al., 2017). Besides, the differential expression of aquaporin isoforms is reported under different stress conditions (Alexandersson et al., 2010). Regulation of TIP controlled water transport across vacuolar membranes by different environmental signals is determined both at the transcriptional and the post-transcriptional levels (Li et al., 2014). Salinity, drought, gibberellic acid, and abscisic acid level are associated with regulation of TIPs (Hachez et al., 2006, Maurel et al., 2008; Moshelion et al., 2009; Hachez et al., 2012; Zarrouk et al., 2016). Transgenic approaches demonstrate that the overexpression of aquaporins in plants confers enhanced tolerance against abiotic stress, including drought (An et al., 2018; Lu et al., 2018; Wang et al., 2018; Patankar et al., 2019; Rosales et al., 2019; Wang et al., 2019). In the present study, varied expression of cucumber specific CsAQPs was recorded in the genotypes with contrasting response to heat stress. In control conditions, significant difference was found for genes CsTIP3.2, CsTIP1a in contrast to high temperature stress conditions. Therefore, CsTIP3.2, CsTIP1a were instrumental in providing heat stress response in the cucumber genotypes. No significant difference in calmodulin genes in the contrasting genotypes under the heat stress conditions indicated its limited role in conferring heat tolerance in cucumber. Upregulation of the G-protein-α and oxygen evolving enhancer genes in the susceptible genotype, DGPC-59 at high heat stress condition indicated their role as negative feedback in heat tolerance in cucumber. In pea and Chinese pear, induced level of the in response to the heat stress has been reported (Misra et al., 2007; Bhardwaj et al., 2020). Negative role of the GPA1 was also reported in Arabidopsis mutants in response to the heat stress (Chakraborty et al., 2015). Inhibition of electron transport from the oxygen evolving complex (OEC) of PSII is because of its dissociation by heat stress (Havaux and Tardy, 1996; Allakhverdiev et al., 1997; Wahid et al., 2007; Allakhverdiev et al., 2008). Therefore, negative feedback of the G-protein and OEC is established in cucumber in relation to the heat stress tolerance.