*2.1. Heat Stress Estimation*

Heat stress was measured in plants that were sown late in the month of April and traits were compared to plants sown earlier in May because temperature in the latter was higher than 46–48 ◦C during the research time period and the heat stress-related 23 morphophysiological traits were observed to be affected by temperature in late-sown irrigated conditions. The heat was estimated by a weather forecast taken from the automated metrological station of cotton research station, Multan, as given in the Table 1:



Heat-susceptible and -resistant varieties (MNH-814 and MNH-886 respectively) were selected on the basis of data for relative water content, osmotic potential, cell injury, and proline concentration. Relative water content was measured by the following [32] formula:

$$RWC = \frac{fresh\ weight - Dry\ weight}{Turing\ weight - Dry\ weight} \times 1000$$

Cell Injury (CIY) was measured when the crop was 55–60 days old, and a sufficient number of leaves was taken from the upper portion and stored in a paper bag. By the use of a punching machine, 15–25 discs of 1 cm diameter were cut. With distilled water, leaf discs were washed three times, were put in test tubes, and then the test tubes were filled up to 40 mL with distilled water. Three sets were made each of 14 test tubes containing leaf discs of 14 cultivars. The first set of test tubes was kept at room temperature as control and the electrical conductivity of the water was noted. The second set was heated at 48 ◦C for 45 min in a water bath. When water was cooled after 6 h, its electrical conductivity was recorded, while the third set of test tubes was autoclaved at 15 lbs (pressure) for 15 min and electrical conductivity was noted when water was cooled.

The greater the EC, greater the damage caused to plant cells due to heat stress as the maximum number of electrolytes came out of the cell due to cell injury. Consequently, cell injury was also greater. Cell injury was expressed in percentage. Proline is an organic compound synthesized from glutamine. It is located in cytoplasm under stressed conditions as nontoxic compatible organic solute to compensate for the dehydrating effects of high osmotic pressure in the vacuole and in the external media. The proline concentration at 700 mol m3 was not inhibitory to enzymes and develops in consequences of poor plant growth under toxic effects. Therefore, its exogenous application should promote

tolerance [33]. Different workers stated that upon heat stress, when starch and protein synthesis are inhibited, proline might be used by the plant for growth [34,35]. Proline from different tissues was measured by Spectrophotometry based on the method of ref. [36].
