Cotton is an important cash crop grown for fiber, fuel and feed in numerous parts of the world， and as the world’s leading textile fiber plant, it forms a vital part of global agriculture and is a mainstay of the economy of many developed and developing coun-tries^[1]. There are various abiotic stress factors affecting cotton growth, development and yield. Extremes of a climatic nature such as high temperature are generally known to have damaging effects on plant growth and general wellbeing, which sometimes results in serious yield losses for most crops^[2]. Heat stress is a prime example of these climate extremes and it is defined as a temperature rise above the highest tolerable level long enough to induce irreversible damage to crop normal development^[3]. However, the resultant disruption of normal growth and metabolism and yield losses varies with sensitivity of each crop species^[4]. The relentless warming driven mostly by heavy industrialization, unrestricted use of automobiles, burning of fossil fuels, and other activities which produce greenhouse gases, is projected to cause the global temperatures to rise between 1.4 ℃ and 5.8 ℃ by the end of this century^[5]. Though it can be generally considered a heat tolerant crop, cotton also suffers heat stress as the best temperature range for optimal metabolic activity is 23 ℃ to 32 ℃ with 28 ℃ being most suitable for photosynthesis, and its growth declines above 35 ℃^[6]. Heat stress usually occurs alongside other environmental stresses like drought and high light intensity that worsens the impact in terms of quantity and quality of cotton fiber^[7]. Besides its well established negative effect on vegetative indices and growth of plants, heat stress is particularly damaging during flowering when it causes anther and pollen malformation leading to male-sterile flowers that produce indehiscent anthers and sterile pollen which are not capable of sexual reproduction^[8]. Other deformities resulting from high temperature stress during flowering are smaller inflorescences, shorter anther filaments and poor synchrony of reproductive organs, and these all negatively affect cotton reproductive performance^[9]. Pollen itself is particularly sensitive to some conditions that disturb protein homeostasis and both short-term high and long-term mildly elevated day and night temperatures being detrimental to pollen development, and the earliest heat-induced develop-mental defects occur at the meiosis stage^[10]. Most cotton growing environments face moderate to very high day and night temperatures, and this is the primary reason for yield losses experienced^[6]. There-fore, heat-tolerant varieties of cotton are important for sustaining cotton production^[11]. A thorough under-standing of plant molecular and physiological mechanisms underlying stress tolerance is an indis-pensable requirement for effective selection in genetic improvement of crops^[12]. Producing satisfactory yield under high temperature stress is hinged on a variety of plant physiological mechanisms contributing to heat tolerance in the field, such as adjustments to critical processes like photosynthesis, and the associated increases in transcripts coding for proteins involved in protection^[2]. The purpose of this experiment is to evaluate whether there are differences in heat tolerance among transgenic cotton germplasms with insect resistance and herbicide tolerance by using physiological and biochemical property changes under heat stress at flowering and seedling stages.

Six transgenic cotton lines (HNZ 1063, HNZ 1068, HNZ 1073, HNZ 1081, HNZ 1088, HNZ 1091) under development at Zhejiang University and the commercial cultivar Zhongmiansuo 49 (ZMS 49) were used in this experiment. Transgenic lines were transformed with two insect-resistant genes (nsCry1Ac and Vip33D) and one herbicide-resistant gene (1174AALdico). Heat tolerance screening tests (membrane thermostability, pollen viability and chlorophyll stability) were applied on field crops, while all other physiological and biochemical parameters were taken on seedlings. Two sets of seedlings were grown in a general growth room with a light phase of 14 h at 25 ℃ and a dark phase of 10 h at 22 ℃. After four weeks, one set was moved to a separate growth chamber with a light phase of 12 h at 42 ℃ and a dark phase of 12 h at 24 ℃ to induce heat stress. It was done for 7 d under a strict watering regime to avoid drought stress.

All photosynthetic parameters which include net photosynthetic rate, transpiration rate, stomatal conduc-tance, and internal carbon dioxide (CO₂) concentration were measured using an infrared gas analyzer-based photosynthesis system (LI-6400, LI-COR, Lincoln, NE, USA). All measurements were taken at photosyn-thetic photon flux density of 1 000 μmol/(m²•s), using a built-in light-emitting diode (LED) light source, flow rate of 400 μmol/s and CO₂ concentration of 400 μmol/mol. Measurements of photosynthetic parameters were done at midday and were performed on the first full upper leaves (the 3rd or 4th leaf).

Five leaf discs (0.7 cm in diameter) were used for assay of chlorophyll a (Chl a), chlorophyll b (Chl b) and total carotenoids. Leaf pigments were extracted in 80% acetone for 12 h in the dark and the extract was assayed according to the WELLBURN’s method^[13]. Spectrophotometric measurements were taken using an ultraviolet-visible (UV-VIS) spectro-photometer (UV2550, Shimadzu, Japan) at 470, 646 and 663 nm, respectively, and corresponding equations were used for calculation of chlorophyll a, chlorophyll b and total carotenoid contents. SPAD values were taken using a Minolta chlorophyll meter (SPAD-502, Japan). Measurements were taken on the 4th or 5th leaf or both. On each individual leaf, four readings were taken and their average was used.

Two sets of leaf samples were collected during the mid-flowering period from five fully expanded leaves from three different plants for each cultivar, and five leaf discs (0.7 cm in diameter) from each sample were placed in vials containing 5 mL of dimethyl sulphoxide. To permit the complete extraction of pigments, sample vials were incubated at the room temperature in the dark for 24 h. After the incubation, absorbance of the extract was measured at 470, 646, and 663 nm, respectively, using a UV-VIS spectrophotometer (Bio-Rad, CA, USA) to calculate the concentrations of Chl a, Chl b, and carotenoid contents^[12]. Total leaf chlorophyll was estimated by summing Chl a and Chl b values. Another set of leaf discs of the same size was collected from each cultivar and incubated at 56 ℃ in a temperature-controlled water bath for 1 h. The chlorophyll content of the heat-treated samples was measured once the sample tubes reached room temperature. The chlorophyll stability index (CSI) was calculated as the ratio of chlorophyll content in heated leaf (56 ℃) to that in fresh leaf expressed as a percentage.

Leaf tissue of 0.5 g was homogenized with 8 mL ice-cold 50 mmol/L phosphate buffer solution (PBS, pH 7.8) containing Na₂HPO₄•12H₂O (16.385 g/L) and NaH₂PO₄•2H₂O (0.663 g/L). The homogenates were centrifuged at 4 ℃, 1.2×10⁴ r/min for 15 min, and the supernatants were used for the determination of malondialdehyde (MDA) content and enzymatic activity.

Lipid peroxidation was determined by mea-suring the MDA content, using the method described by HODGES et al.^[14]. The reaction mixture was comprised of 5% 2, 4, 6-trichloroanisole (TCA) solution containing 2.5 g of 2, 4, 6-thiobarbituric acid (TBA) and enzyme extract. The resulting mixture was heated in a hot water bath at 95 ℃ for 15 min and then was immediately immersed in an ice water bath to stop the chemical reaction. The samples were then centrifuged at 4 800 r/min for 10 min and the absorbance of the obtained supernatant was recorded at 532 nm.

Superoxide dismutase [SOD, electrical conduc-tivity (EC) 1.15.1.1] activity was determined by the method of BEAUCHAMP et al.^[15], by following the photo-reduction of nitroblue tetrazolium (NTB). The reaction mixture contained 50 mmol/L PBS (pH 7.8), 0.1 mmol/L ethylene diamine tetraacetic acid (EDTA), 13 mmol/L methionine, 75 µmol/L NTB, 2 µmol/L riboflavin and 100 µL of the supernatant. Riboflavin was added as the last component and the reaction was initiated by placing the tubes under two 15 W fluorescent lamps. The reaction was terminated after 10 min by removing the reaction tubes from the light source. Non-illuminated and illuminated reactions without supernatant were served as calibration standards. Reaction products were measured at 560 nm.

Five leaf discs, avoiding leaf veins, with 1.2 cm in diameter were cut out using a cork borer and placed into a petri dish filled with deionized water. Equal amounts (20 mL) of deionized H₂O were added into each vial, which was then covered with plastic wrap. Samples were kept in the dark for 48 h before measuring electrical conductivity using a single probe conductivity meter (TDS, China). The conductivity meter was calibrated according to the manufacturer’s instructions. After the conductivity measurements (R₁), the samples were placed in a hot water (95 ℃) bath for 10 min and the conductivity was measured again (R₂). Percent injury was calculated by R₁/R₂×100.

Plants were harvested after five weeks of growth under the heat stress and control conditions. They were separated into stem and roots then washed with distilled water. Fresh mass of these plant parts was determined. After this, the samples were oven-dried at 70 ℃ for about 48 h and then weighed for dry mass.

Test was done according to the method of SHIVANNA et al.^[16] with slight modifications. Two freshly opened flowers were collected from three plants in each genotype between 09：00 and 10：00 following successive and very hot days ranging from 36 ℃ to 38 ℃. Pollen viability was tested using 2% 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining in deionized water. A drop of TTC solution was placed on a microscope slide to which a small amount of pollen was suspended and distributed it uniformly. A cover glass applied as hanging drop or sitting drop cultures are not suitable since oxygen inhibits reduction of TTC. The preparation was incubated in the dark at 35 ℃ for 60 min with over 95% relative humidity (RH). The preparation was observed under a microscope and scored for percentage of viable pollen grains, i.e., pollen grains that have turned red due to accumulation of formazan. The pollen grains were scored from only the central area in the preparation as pollen grains lying near the margin of the cover glass would show variable degrees of red coloration due to higher oxygen availability. Mean of two flowers from the same plant was used as a single measure.

Data analysis was carried out using SPSS version 20.0. Significant differences between treatments were tested with analysis of variance (ANOVA). Waller-Duncan’s multiple range tests were used for mean separation at P＜0.05. The t-test for independent samples was used to compare the controls with their respective treatments. Figures were plotted in Excel 2013.

Heat tolerance tests were carried out at the flowering stage following some researchers who found them to be more accurate at this stage^[11]. There were significant differences between the genotypes based on the three parameters (Table 1), i.e., membrane thermostability (P＜0.001), pollen viability (P=0.003) and chlorophyll stability index (P＜0.001). Higher pollen viability values were observed in HNZ 1068, HNZ 1091 and ZMS 49, while the lower values were observed in HNZ 1081, HNZ 1073 and HNZ 1063. In terms of membrane thermostability, higher values were in HNZ 1068, HNZ 1091 and HNZ 1073, while the lower values were in HNZ 1081, HNZ 1063 and HNZ 1088, respectively. Higher chlorophyll stability indexes were observed in HNZ 1068, ZMS 49 and HNZ 1063, while the lower values were in HNZ 1081, HNZ 1088 and HNZ 1091. Results from these three parameters show that HNZ 1091, HNZ 1068 and ZMS 49 can be considered as the more tolerant genotypes of the lot, while HNZ 1073 and HNZ 1081 can be considered as susceptible.

There were significant differences in all parameters measured (P＜0.05). Relative to the initial measurements, all photosynthetic parameters were significantly reduced except for internal CO₂ concen-tration (Fig. 1). Three genotypes with the higher photosynthesis rate were ZMS 49, HNZ 1091 and HNZ 1088, respectively, while the three genotypes with lower values were HNZ 1081, HNZ 1073 and HNZ 1063 in that order. Stomatal conductance trend was slightly different from photosynthesis rate. The higher values were observed in ZMS 49, HNZ 1063 and HNZ 1088, while the lower ones were observed in HNZ 1073, HNZ 1081 and HNZ 1068. As a result, internal CO₂ concentrations were higher in HNZ 1068, HNZ 1088 and HNZ 1073, while they were lower in ZMS 49, HNZ 1063 and HNZ 1081. Transpiration rate followed a similar trend to stomatal conductance which was to be expected. The higher transpiration rates were observed in ZMS 49, HNZ 1091 and HNZ 1063, while the lower ones were in HNZ 1073, HNZ 1081 and HNZ 1068.

Significant differences in final pigment levels were observed between the genotypes (P＜0.05). Relative to the control (Fig. 2), there was a significant decline in the photosynthetic pigment parameters under heat stress. This was especially the case with chlorophyll a. Chlorophyll b was only slightly affected and significant differences between the control and treatment were observed in HNZ 1073 and HNZ 1088. The higher chlorophyll a levels were observed on ZMS 49, HNZ 1091 and HNZ 1068, while the lower values were recorded in HNZ 1081, HNZ 1073 and HNZ 1088. SPAD measurements also confirmed the decline in total chlorophyll levels.

Relative to the control, SOD activity decreased under the heat stress condition (Fig. 3). Significant differences were observed across genotypes (P＜0.001). However, in the two genotypes HNZ 1063 and HNZ 1088 there were no significant differences in the SOD activity between the treatment and the control. HNZ 1063, HNZ 1068 and HNZ 1088 had the higher activity of SOD under heat stress. This implies better efficiency of the enzyme in removing ROS. The lower activities were observed in HNZ 1081, HNZ 1073 and HNZ 1091, respectively. The genotypes which maintained higher SOD activity can be considered to have a more robust antioxidant system. Lipid peroxidation as shown by MDA accumu-lation increased under heat stress with significant differences observed (P=0.001). The higher MDA accumulation was observed in HNZ 1063, ZMS 49 and HNZ 1081, while the lower accumulation was observed in HNZ 1091, HNZ 1068 and HNZ 1088. Relative to the control, there was a significant increase in MDA accumulation in all genotypes except for HNZ 1091 only. Total carotenoid levels were signifi-cantly different between the genotypes (P=0.004). Relative to the control, there was a significant decline in all genotypes with the exception of HNZ 1073. However, the ranked list obtained from heat screening tests did not fully conform to other physiological observations like photosynthesis rates. ZMS 49 ranked lower based on membrane thermostability test but it maintained the highest photosynthesis rate. The same also applied for HNZ 1088. Inversely HNZ 1073 scored high on the thermostability test but it had very low photosynthesis rate. The results in other genotypes tended to conform.

Significant differences were observed between all growth parameters both on a fresh and dry mass basis (P＜0.05). There were significant differences between the control and treatment for all genotypes in all parameters both on the fresh and dry mass basis (Fig. 4). The higher shoot masses were observed in HNZ 1068, ZMS 49 and HNZ 1091, respectively, while the lower values were observed in HNZ 1073, HNZ 1063 and HNZ 1081. Root fresh masses also followed a similar trend. Slight differences were in the root dry masses where ZMS 49 had the highest mass followed by HNZ 1068.

The three main parameters for heat tolerance (membrane thermostability, pollen viability and chlorophyll stability index) gave conclusive results in some genotypes (ZMS 49, HNZ 1068, HNZ 1091, HNZ 1081), while it was not entirely conclusive in others (HNZ 1088, HNZ 1063, HNZ 1073). There are a lot of factors which determine final plant tolerance to heat stress since it is a complex trait controlled by additive action of many genes^[11]. This explains the mixed results in some genotypes and shows that though these quick physiological tests can provide useful pointers, there is still need to broaden the range of selection tools for improved accuracy. The decrease in net photosynthesis rates in all genotypes under heat stress can be explained by various factors. Chief among them is the damage to photosystem Ⅱ （PSⅡ) and the reduced activity of photosynthetic enzymes^[17]. Inhibited photorespiration was also observed to cause photosynthetic damage to plants under heat stress^[18]. It is supported by the observed higher internal CO₂ concentration after the treatment relative to the control suggesting conditions suppressing photorespiration. However, the same observation also rules out CO₂ shortage due to the decline in stomatal conductance leading to lesser gas diffusion in and out of the plant as a limiting factor to photosynthesis under heat stress. This corroborates the theory of direct damage to the photosynthetic system since the plants were also well watered during the treatment period meaning all three primary raw materials (water, carbon dioxide, light) for photosynthesis were not limiting. In particular, the inhibited recycling of Rubisco enzyme due to disruption of the electron transport chain was pinpointed as the main reason for reduced photosynthesis under heat stress^[19]. It has been widely reported that heat damage arises from inactivation of the highly sensitive water-splitting reaction, disconnection of PSⅡ centers from the bulk pigments, thermal uncoupling of photophosphory-lation, and bio-membrane lesions^[20]. The downward trend in transpiration rates also fits in with the observed decrease in stomatal conductance. Closed stomata means there is no way for gasses and water vapour to diffuse freely in and out of the plant. Plants are known to increase their transpiration rates under short-lived high temperature stress conditions but it is reduced under long-term stress. Since this was a full week of heat treatment, the observed decline in transpiration rate confirms this theory. Four genotypes (ZMS 49, HNZ 1068, HNZ 1088 and HNZ 1091) maintained significantly higher photosynthesis rate with ZMS 49 maintaining the highest mean photosynthesis rate. This suggests better protection of their photosynthetic apparatus against heat-induced damage.

The lower pigment levels in the treatment relative to the control can be a result of inhibited pigment biosynthesis, breakdown of available pigments or both. Chlorophyll catabolism is part of the programmed cell death (PCD) phenomenon for remobilization of resources and streamlining metabolic activities^[21]. Stress-induced chlorophyll catabolism also feeds the tocopherol biosynthesis pathway which then acts as a lipid antioxidant for enhanced self-defense^[22]. This decline in pigment concentration can also explain the decline in photosynthesis as chlorophyll is a critical component of the process. High chlorophyll a level was observed to be correlated with general stress tolerance. Drought experiments showed strong correlations between chlorophyll a and dry matter production under both water-limited and well-watered treatments of cotton^[23]. Some wheat experiments showed that high chlorophyll genotypes were generally better performers all around. The stay-green trait was associated with heat tolerance in wheat and similarly high chlorophyll content was associated with heat tolerance of sister lines in some wheat crosses^[24]. This is in some agreement with findings of this research where the genotypes which showed higher tolerance also had higher chlorophyll a level. Carotenoids which are an important part of the antioxidant defense network also declined under heat stress. The observation is also consistent with other researchers who noted that short-term stress increases carotenoid concentration but prolonged stress results in a net decline.

Decline in activity suggests an overpowering of the SOD antioxidant system by the various oxidants generated during the stress period. Manageable stress results in a net increase in SOD activity as the apparatus is rolled out to scavenge oxidative agents^[25]. This is the case when stress is short term or mild. Since the treatment temperature was 40 ℃ which is known to be detrimental to most enzymes, it can also be postulated that the dismutase enzyme was directly affected rather than being overwhelmed by the amount of oxidative agents. This can be explained by the observed failure in protective systems like SOD leading to membrane lipid damage. Low damage in cotton leaf cells as indicated by MDA accumulation was observed where defense systems were not compromised^[25]. It is also worth noting that even though ZMS 49 could be considered as heat tolerant, it had higher relative MDA accumulation even in comparison with the supposedly susceptible ones. This proves the importance of using a variety of tests to come up with conclusive results. According to this research, antioxidant activity and lipid peroxidation cannot give conclusive evidence to classify genotypes. ZMS 49 maintained a relatively high SOD activity but also had high lipid peroxidation rate. HNZ 1063 despite being considered of moderate tolerance also had high SOD activity and high lipid peroxidation rate. Generally, high SOD activity did not necessarily guarantee reduced lipid peroxidation.

The decline in dry matter accumulation is linked to a combination of the aforementioned factors. Reduction in photosynthesis implies lesser availability of assimilates for dry matter production. This in turn leads to growth retardation. It is also an established fact that when stressed, plants prioritize survival to growth. Most of the energy produced by plants is channeled towards active processes for defense rather than powering photosynthesis. As all the processes occurring in plants are mostly enzyme controlled, the reduced dry matter accumulation can also be linked to reduced enzyme activities. Root development was also severely affected by heat stress. Stress tolerance in plants is associated with extensive root systems and vigorous shoot growth. This is also supported in this research where the order of shoot and root masses was HNZ 1068＞ZMS 49＞HNZ 1091. The differences between transgenic lines suggest that plant transformation can influence other untargeted plant properties in both positive and negative ways. Thorough selection and vetting becomes critical for advancement of lines with positive added features. Gene insertion (three in this case) is a random phenomenon and it has been reported to affect other plant traits. Introduction of a sugar beet chitinase Ⅳ gene into silver birch will yield transgenic lines that carry the transgene at random locations in birch genome and show considerable variation both in the target (fungal disease resistance) and non-target traits like growth, leaf phenology, general condition^[26]. In a similar research with maize, LASERNA et al.^[27] suggested that differences in traits among the transgenic versions of the same hybrid, may suggest a possible novel role of the introduced genes on the physiology and the phenotype of the plants. In cotton, off-target mutations were reported resulting in phenotypic variations, even after using modern specific gene editing tools^[28].

The differences observed in physiological, biochemical and reproductive parameters under heat stress were useful for grouping genotypes. Specifically, ZMS 49, HNZ 1068 and HNZ 1091 did considerably better on most of the tests conducted. HNZ 1073 and HNZ 1081 consistently performed badly, while HNZ 1063 and HNZ 1088 exhibited moderate to low tolerance when compared with the other genotypes. Genetic transformation therefore affected heat tolerance mechanisms within the plants.