*2.6. Effects on Physiological Attributes*

Plant growth and development is based on physiological processes such as photosynthesis, membrane permeability, and stomatal conductance [54]. Heat stress adversely affects physiological attributes of plants and limits productivity [55]. The rate of photosynthesis is significantly reduced or even inhibited at high temperatures. The deactivation of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) and increase in ionic conductance of thylakoid membranes are the primary causes of photosynthesis reduction or inhibition in cotton during high temperature conditions [56,57]. Chlorophyll content also decreases when cotton plants are exposed to high temperature that results in a decrease in the rate of photosynthesis [58]. Heat stress changes the permeability of membranes and alters cell differentiation and elongation by causing injuries to cellular membranes and deforming the organization of microtubules and cytoskeleton [59]. Higher cell membrane thermostability is positively associated with heat tolerance in cotton. Therefore, a number of experiments have been conducted to screen for heat tolerant accessions on the basis of cell membrane thermostability [60–62]. Stomatal conductance is directly related to water relations and photosynthesis in plants. High temperature causes opening of stomata and an increase in stomatal conductance that results in a decrease in the water potential of leaf. High stomatal conductance also increases the rate of transpiration and intercellular CO2. Stomatal conductance increases up to 40% in most of plant species when temperature rises from 30 to 40 ◦C [63]. The advantage of higher stomatal conductance is associated with cooling of leaves, which provides tolerance to heat stress [64]. Experimental results have shown that upland cotton has more stomatal conductance and higher rate of photosynthesis under high temperature conditions than compared to pima cotton [65]. Studies revealed that differences in cotton accessions and species for stomatal conductance are under genetic control. Thus, this trait can be improved through breeding and selection [66–68].

#### **3. Mechanisms of Heat Tolerance**

#### *3.1. Antioxidant Activity in Response to Oxidative Stress*

Crop plants face environmental stresses which alter the various metabolic activities in order to ensure balance between production and consumption of energy through oxidation and reduction reactions [69]. This change in metabolism alters the concentration of various molecules. Likewise, metabolic imbalances during high temperature stress promote the extra-accumulation of reactive oxygen species (ROS) into the cellular compartment of plants [70]. ROSs are highly reactive chemicals formed from O2. Excessive production and over-accumulation of ROS in plant cells cause irreversible damage to its organelles through oxidative stress. However, a balanced amount of ROS is required for normal activities such as detoxification of poisonous substances, antimicrobial phagocytosis, and apoptosis. ROS also benefits plants by acting as signaling molecules for activation of numerous genes related to stress tolerance, cell proliferation, seed germination, growth of root hairs, and cell senescence [71,72]. The over-accumulation of ROS during stress conditions results in the oxidation damage of vital molecules such as DNA, proteins, and lipids. This condition is termed as oxidative stress in plants [73]. ROS includes free radicals such as the hydroxyl radical (OH) and superoxide anion (O2 −), as well as non-radicals such as singlet oxygen (1O2) and hydrogen peroxide (H2O2). These species are produced by excitation and reduction in intra-cellular oxygen (O2). The schematic diagram of ROS production is illustrated in Figure 3.

**Figure 3.** Schematic diagram of ROS production in plants.

The concentration of ROS and scavenging capacity of antioxidants in cotton is considered as a selection criterion for heat tolerant accessions [74]. An experiment was conducted using two cotton cultivars, and heat stress was applied gradually from 30 to 45 ◦C on 30 days old seedlings. The results revealed a 206 to 248% increase in hydrogen peroxide content and a 40 to 170% increase in lipid peroxidation under heat stress conditions. The concentration of non-enzymatic antioxidants also increases with increases in temperature, while the activity of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) increase 56–70%, 37–69%, 43–91%, and 22–27%, respectively. It was concluded that genotypic differences exist in cultivars for ROS production and antioxidants response. Higher levels of antioxidants and lower levels of ROS during high temperature are an indication of heat stress tolerance [75]. In another study, the cotton plants were grown at two temperature regimes, i.e., 38 and 45 ◦C. The results indicated non-significant differences in the concentration of hydrogen peroxide at both temperatures, while the concentration of proline decreased rapidly and significantly as the temperature increased from 30 to 45 ◦C. The activity of

SOD declined at 45 ◦C while the activity of CAT, POX, and APX increased with the increase in temperature [76]. It is found that the exogenous application of hydrogen peroxide on cotton plants triggers the activity of SOD and CAT. It was further concluded that foliar applications of H2O2 on field grown cotton can enhance the heat tolerant ability without compromising yield [77]. The effect of high night temperature on biochemistry of leaf and pistil was studied in upland cotton cultivar. The results indicated that glutathione reductase activity in leaves is increased with an increase in night temperature, while no change in the concentration of glutathione reductase was observed in pistils. This shows that the antioxidant mechanism of pistil or floral parts is less sensitive to changes in mean night temperature than compared to leaves or vegetative parts of the cotton plant [78].
