Physiological and biochemical responses to short-term cold stimulation of pak choi under heat stress

Heat stress has become a global concern that will seriously affect the growth and production of crops as global warming progresses. At present, we mainly rely on traditional cultivation techniques and the assistance of horticultural facilities to address this problem, but there are many shortcomings, such as the high cost and low income. Therefore, novel approaches are needed for sustainable pak choi production under heat stress. In this work, three water treatments (15, 20, 25 °C) were imposed for one month on pak choi in a fully controlled growth chamber under heat stress. Our results indicated that the biomass of pak choi significantly increased under low-temperature water treatment compared to the control. Low-temperature water treatment inhibited the decrease in chlorophyll, soluble sugar and soluble protein, reduced the degree of membrane lipid peroxidation, and enhanced ROS scavenging enzyme activity in plants. Under low-temperature water treatment, pak choi had a higher gas exchange index, including photosynthetic rate, transpiration rate, intercellular CO2 concentration and stomatal conductance. In addition, low-temperature water treatment alleviated the adverse effects of heat stress on PS activity and electron transport.


Introduction
Temperature is one of the environmental factors that may affect various agronomic characteristics, such as biomass production, phenology and physiology, and yield-contributing traits of horticultural crops (Ahmad et al. 2021a). Each plant has an optimum temperature range for its growth, development and conditions (Nievola et al. 2017;van Leeuwen et al. 2000). Plants exposed to heat stress experience various changes in physiological and biochemical processes. Under high-temperature stress, all the growth stages in plants are adversely affected from germination to growth and development, reproductive phase (Ahmad et al. 2016;Hasanuzzaman et al. 2013), seed yield and seed quality (Ahmad et al. 2021b).
Protected horticulture is the use of appropriate scientific machinery, equipment and advanced technology to create measured with anthrone reagent as previously described (Zheng et al. 2008).
Determination of the growth trait of pakchoi. The sampling for morphological analysis of pakchoi was carried out after giving the cold water treatment 21 days later. Measurements of the growth trait including plant height, plant weight, stem diameter were measured according to a previous method (Zheng et al. 2008). The dry (DW) and fresh weights (FW) were determined using an electronic analytical balance.The total leaf area and number were measured by Image J 1.8.0. The length, surface area and tip number were measured by WinRHIZO.
Measurement of gas exchange parameters. The GFS-3000 measuring systems (Heinz Walz, Effeltrich, Germany) was used to obtain the gas exchange parameters, including net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E), intercellular CO 2 concentration (Ci). The third true leaf of pakchoi seedlings was used for the measurement. The parameter were set according to the manufacturer's protocol. the air velocity was set at 750 µmol s − 1 , the light intensity was set at 600 µmol m − 2 s − 1 , and the leaf area was set as 3 cm 2 .
Measurement of chlorophyll fluorescence, P700 and PQ pool.
The Chlorophyll fluorescence, P700 redox kinetics and PQ pool were measured with a Dual-PAM-100 fluorescence system (WALZ, Effeltrich, Germany). The third true leaf of pakchoi at 37 °C was used for measurement. After 30 min of dark adaptation the fluorescence induction kinetics were measured. The light-adapted curves were recorded after 2 min exposure to light. The light intensity gradient of the RLC was 2, 13, 40, 80, 114, 157, 213, 279, 358, 592, 956, 1207, 1537µmol•m − 2 •s − 1 . The duration of each light intensity was 30 s and the saturation pulse was 20,000 µmol m − 2 s − 1 for 300 ms. A single turnover flash (ST, 50 ms, PQ pools being oxidized) was applied followed by multiple turnover flashes (MT, 50 ms, PQ pools are fully reduced) in the presence of far-red (FR) background light to determine the redox state of P700. The ST and MT areas was used for calculation as the complementary area between the oxidation curve of P700 after ST and MT excitation and the stationary level of P700 + under FR illumination. The functional size of the PQ pool of intersystem electrons on a P700 reaction centre was determined as follows: e − /P700 = MTareas/ST -areas (Joly Jemâa et al。 2010;Zhang et al. 2014;Zivcak et al. 2014;Oukarroum et al. 2015).
Determination of MDA content, relative conductance and antioxidant enzyme activity.
Fresh leaf samples from pakchoi were harvest for antioxidant enzyme activity assays which were carried out at 4˚C. The H 2 O 2 and O 2 − content was detected as reported previously (Velikova et al. 2000). The APX activity 2021). Biochar production has been widely viewed as an exemplar of the circular economy in the sustainable energy field (Laird 2008). Biochar is mainly produced by either pyrolysis or gasification (Woolf et al. 2010). The waste gas left after the production of biochar can be recycled. We propose that these waste gases can be used to cool water to irrigate agronomic crops.
Pak choi (Brassica rapa ssp. Chinensis) is one of the most important vegetables grown worldwide (Liu et al. 2020). The extremely high temperature in summer has affected the normal growth and development of crops, leading to a reduction in crop yield and quality (Ahuja et al. 2010). To improve the yield of pak choi in summer, we simulated the high-temperature environment in summer in a greenhouse, treated pak choi under heat stress with normal temperature water and cold water, and measured a variety of physiological indicators. In the present study, we attempted to characterize the photosynthetic and antioxidant capacities of pak choi treated with water. We found that low-temperature treatment affected photosynthetic electron transport, gas exchange capacity and antioxidant enzyme activity, which contributed to the growth of pak choi. The results show that cold-water treatment can effectively alleviate the damage to pak choi caused by high temperature.

Materials and methods
Plant materials and growth conditions. An heat resistant variety "xinxiaqing"was selected to conduct this study. The Pak choi seeds were grown in pots containing filled mixture of soil and vermiculite (3:1) and then left in a growth chamber at 24 •C for 16 h day and 8 h night cycles with 300µE m − 2 s − 1 light intensity and 60% relative humidity (RH).
Cold water treatment. When the seedlings reach the four-leaf stage, young soilgrown pak choi were subjected to heat stress in an incubator at 37 •C for 8 h per day. plants was evenly irrigated an equal amount of cold water (15 and 20•C ). The control plants were irrigated with room temperature water per day. The experiment was conducted for 3 weeks.
Determination of chlorophyll and metabolite contents. The chlorophyll content of each plant was measured in accordance with the previous method (Ni et al. 2009). The third fully expanded leaves of each plant of four biological replications of pakchoi was used to measure the chlorophyll index values. A total of 0.2 g of fresh sample was taken and placed into a 5 ml acetone (80%) at 4 ℃ from 1-monthold seedlings. Soluble protein content was measured with coomassie brilliant blue G-250 which was described in a previous study (Bradford 1976). Soluble sugar content was Leaves are responsible for photosynthesis of the crop, and the leaf area directly affects light absorption. We counted the total leaf area of pak choi within the three groups. The results suggested that the irrigation water temperature did not significantly affect the leaf number among the three groups of pak choi. However, the leaf area of the T2 group increased by 13.18% compared with that of the T1 group and increased by 29.74% compared with that of the T3 group (Table 1). Increasing the leaf area helps enhance the accumulation of crop assimilation. The root is an important plant organ that has multiple functions, including the acquisition and fixation of water and nutrients, the perception of environmental changes in soil, and the synthesis of phytohormones . Thus, root branching and root surface are important aspects of the root system architecture that directly affect the absorption of water and nutrients. In our work, the total root surface area, root length, and root tip number of pak choi were measured. The coldwater treatment had no obvious effect on the root surface area, but the root length and root tip number were significantly increased in the T1 and T2 groups. Compared with the control, the root length of T1 and T2 increased by 7.56% and 12.46%, respectively. Concerning root tip number, T1 and T2 increased by 19.01% and 26.84%, respectively ( Table 1). The longer root length and more root tips will help plants absorb more water and nutrients for various life activities of plants.
Chlorophyll is the main pigment for photosynthesis of plants, which functions in harvesting light energy and driving electron transfer . Accumulating evidence has proven that chlorophyll breakdown and the photosynthetic machinery are injured when plants are faced with high-temperature stress . The chlorophyll contents in pak choi under the three water treatments was measured. According to the experimental results, the contents of chlorophyll a, chlorophyll b and carotenoids in T1 and T2 were significantly higher than those in the control. In detail, the levels of Chl a in T1 and T2 seedlings increased by 11.03% and 38.19% compared to the control, activity was measured according to Asada (Asada et al. 1992). The CAT activity was calculated according to a previous report(Aebi 1984). The POD activity was determined according to the protocol as described previously (Trevisan et al. 1997). The SOD activity was measured as previously described. All the experiments were performed for at least four biological replicates (Beauchamp et al. 1971). The method for determing ondialdehyde (MDA) content described by Shah et al. (2001). The relative conductance (REC) was measured according to Aghaie et al. (2018).

Statistical analysis
Data processing was conducted on randomly selected samples from four independent biological and technical replicates. All values were shown as mean ± SE (Standard Error); Data were subjected to one-way analysis of variance (ANOVA) using SPSS software (SPSS, Chicago, USA) for mean comparison and significant differences were calculated (Tukey-Kramer test). All graphs were made by graphpad 8.0.

Physiological and biochemical traits in pak choi.
Environmental stresses have become an important constraint affecting grain yields in crops. The perception mechanism of plants involved in heat stress is very complicated and is not yet fully understood. Heat stress signals stimulate plasma membrane sensors and result in an elevated level of Ca 2+ , which can act as a second messenger to induce the expression of heat stress response genes (Ahmad et al. 2021). We supposed that cooling can instantly reduce the plant surface temperature, which can reduce the damage from high temperature to the plant plasma membrane. To investigate this possibility, we imposed water at three temperatures (15, 20 and 25 ℃) on pak choi, which were divided into T1, T2 and T3 groups, respectively. The T3 group was the control. The morphological traits of the three groups of pak choi under heat stress were measured ( Table 1). The phenotypes of the three groups of seedlings are shown in Fig. 1. The T2 seedlings showed the greatest growth vigor among them. The order was T2 > T1 > T3. The results suggested that cold water alleviates the damage caused by heat stress to the growth and metabolism of pak choi. ROS are generated in redox reactions, such as respiration and photosynthesis, in plants. The participate in all aspects of growth and development, such as cell proliferation and differentiation, gravitropism, programmed cell death, seed germination, root hair growth, pollen tube development, and senescence (Singh et al. 2016). In many cases, heat stress contributes to various metabolic changes, known as elevated ROS levels, which are mainly generated in PS II and PSI (Asada et al. 2006). To enhance heat tolerance and detoxify ROS in a high-temperature environment, plants recruit multiple antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). To confirm whether alternative detoxification pathways were activated under cold-water treatment, we measured the antioxidant enzyme activity. Figure 3 shows that the activities of SOD, APX, and CAT in T2 were higher than those in T1 and T3, which is consistent with previous findings. Subsequently, we determined the H 2 O 2 and O 2− contents from the three groups of pak choi. Figure 4 shows that H 2 O 2 production decreased by 32.32% in T2 compared to that in the control. This demonstrated that pak choi under respectively. The Chl a/Chl b ratio in T2 was significantly higher than that in the control. As shown in Fig. 2, a decrease in the carotenoid level was observed in T2 compared to that in the control.
To confirm whether cold-water treatment influences nonenzymatic antioxidants in plant cells, we measured the contents of soluble sugar, soluble protein and ascorbic acid (vitamin C). As shown by the experimental results presented in Fig. 2, T2 plant leaves accumulated an approximately 1.5-fold increase in soluble sugar and an approximately 1.22-fold increase in soluble protein compared with the control. The results showed that more soluble sugar and soluble protein were synthesized in pak choi treated with cold water than in the control. The level of vitamin C was also enhanced under cold-water treatment. Overall, the altered levels of soluble sugars, soluble proteins, and ascorbic acid are advantageous for regulating osmotic pressure and maintaining cell membrane stability. These data suggested that the nonenzymatic antioxidant content of pak choi was improved under cold-water treatment.
Antioxidant enzyme activity and membrane lipid peroxidation. has profound effects on the photosynthetic capacity of plants (Wahid et al. 2007;Allakhverdiev et al. 2008;Berry et al. 1980). It affects the chlorophyll content, photosynthetic enzyme activity, stomatal opening and hormone secretion of plants. High temperatures seriously impede photosynthetic efficiency, diminish productivity and shorten the plant life cycle (Xalxo et al. 2020). The gas exchange parameters, including the net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E) and intercellular CO 2 concentration (Ci), were measured in this work [ Fig. 5]. The Pn of pak choi under cold-water treatment was significantly higher than that of the control. The Pn values of pak choi were recorded to be 12.61 µmol m − 2 •s − 1 (T1), 13.75 m − 2 •s − 1 (T2), 11.33 m − 2 •s − 1 (T3) 6 h after treatment. The E and gs of pak choi under cold-water treatment (T1 and T2) were significantly higher than those of the control, while the Ci values of T1 and T2 were significantly lower than that of the control. The above findings suggested that cold-water treatments can enhance the gas exchange capacity of pak choi under heat stress.
Fv/Fm and Pm of pak choi. cold-water treatment had a stronger ability to scavenge ROS than did pak choi under room-temperature water treatment.
To detect the extent of lipid peroxidation in pak choi, we also measured malondialdehyde (MDA) content and relative electrical conductivity (REC) in the three water treatments. The results suggested that pak choi has a lower MDA content and lower REC under cold-water treatment. MDA is a compound produced by membrane lipids under the action of reactive oxygen species (Djanaguiraman et al. 2010). It reflects the degree of plasma membrane damage. The RECs of the T1 and T2 leaves were significantly lower than that of the control. In accordance with these findings, our study suggested that low-temperature water treatment, on the one hand, reduces the damage of high temperature to leaf cell membrane lipids; overall, it improves the antioxidant system to scavenge free radicals and cell membrane fluidity.
Gas exchange capacity in pak choi. Multiple environmental stresses can alter the activity of the photosynthetic electron transport chain, which can reduce photochemical reaction efficiency, create excess absorption of light energy, and induce aggravated photoinhibition (Xalxo et al. 2020). Elevated ambient temperature To clarify the potential photosynthetic activity of pak choi under water treatment of varying temperatures, we measured the rapid light curves of plants under different light intensities. The photochemical quenching coefficient (qP) reflects the photosynthetic activity of plants. Figure 7 shows that the qP of pak choi leaves showed a rapid elevation under all illumination conditions. The results indicated that the activity of PSII photochemical reactions under coldwater treatment was higher than that under room-temperature water treatment.
PSII and PSI activities of pak choi leaves. It has been well documented that heat stress damages thylakoid membranes and reduces the activities of membraneassociated electron carriers and enzymes (Zhao et al. 2020;Hasanuzzaman et al. 2013). Under heat stress, the PSII electron transfer efficiency in plants decreased, and the formation of NADPH and ATP was prevented, which further resulted in a reduced photosynthetic rate. Since PSII is the most heat-sensitive complex within chloroplast thylakoid membrane protein complexes (Hasanuzzaman et al. 2013; The Fv/Fm value is an important indicator for studying the effects of photoinhibition or various environmental stresses on photosynthesis (Hejnák et al. 2015a, b). Strong evidence supports that Fv/Fm is inversely proportional to the degree of plant stress. We measured the maximum photochemical efficiency of PSII (Fv/Fm) of pak choi leaves with three water treatments at varying temperatures.
We found that the Fv/Fm of pak choi under cold-water treatment (T1 and T2) was higher than that of the control. Of note, the Fv/Fm of T1 was lower than that of T2 (Fig. 6). This may be explained by the fact that extremely low-temperature water will inhibit plant photosynthetic activity. The maximum oxidation state of PSI (Pm) of the pak choi under low-temperature water treatment was approximately 1.45 times that of the control. These results indicated that cold-water treatment counteracts the inhibition of the redox state of PSI and that photoprotection pathways were activated. Thus, we proposed that short-term heat adjustment improved the maximum light energy conversion efficiency of PS II in pak choi leaves. treatment increased the quantum yield of PSI. In addition, we observed that cold -water treatment significantly reduced the quantum yield of nonphotochemical energy dissipation caused by donor-side restriction (ND), while the quantum yield of PSI nonphotochemical energy by receptor-side restriction (NA) showed no significant difference between treatments (Fig. 7). All of these observations suggest that the cold-water treatment reduces the negative effects of the electron transfer efficiency of PSII and PSI.
Electron transport rate of pak choi leaves. The electron transfer rates of PSII [ETR (II)] and PSI [ETR (I)] were measured to evaluate the photosynthetic performance. As shown in Fig. 8, the ETRI and ETRII of pak choi leaves rapidly increased with increasing light intensity for all treatments. ETR (I) was significantly higher with CWT compared to those of control plants under high light intensity. We also found that cold-water treatment significantly improved the cyclic electron flow around PSI (CEF) (Fig. 1). Previous studies demonstrated that CEF contributed to a high level of Y (NPQ) and depleted the excess reducing power of NADPH via the NADPH dehydrogenase-dependent pathway. CEFs compel excess electron flow to NADPH and O 2 , avoiding the generation of ROS. The Szymańska et al. 2017), we measured the photosynthetic quantum yields for PSI and PSII (Fig. 8). We observed that the effective quantum yield of PSII gradually decreased with increasing light intensity in the three groups, while the Y(II) was significantly higher for the cold-water treatments than for the control (Fig. 8). Figure 1 shows that cold-water treatment caused a modest elevation in the quantum yield of nonregulated energy dissipation in PSII [Y(NO)]. In comparison with T1 and T2, the higher Y(NO) level of T3 (control) indicated that it lost the ability to inspire self-protection in excess light. Y (NPQ) represents the energy dissipated into heat through the regulatory photoprotection mechanism. Researchers have found that plants can mitigate ROS damage caused by excessive light excitation through antioxidant systems and NPQ mechanisms (Zhang et al. 2014). In this study, NPQ increased at almost all light intensities in the three treatments. The quantum yield of regulated energy dissipation in PSII [Y(NPQ)] was highest in T2 seedlings. This result indicated that cold-water treatment enhanced the capacity to induce photoprotection of PSII. Additionally, the quantum yield of PSI [Y(I)] increases and then decreases rapidly with increasing light intensity. The Y(I) of T2 was higher than that of T1 and T3, especially under high light intensities. This result suggested that the low cold-water limiting factors for plant growth and photosynthesis. Heat stress has become a great menace and ever-looming threat to fruitful crop production around the globe (Hatfield et al. 2018;Tariq et al. 2018). Heat stress speeds up crop growth and does not allow the proper completion of crop growth stages, which leads to immature development and perturbing carbon assimilation (Rehman et al. 2021). Great efforts have been made by breeders and physiologists to develop heat stress tolerance traits in oil crops. It is well known that genetic engineering is one of the best economic approaches to develop heat stress tolerance. However, the process of the development of new varieties is laborious and time-consuming. In addition, the heat sensitivity also varies among different species. To increase heat tolerance by reducing the injurious effects of high temperature, cold-water irrigation combined with agricultural machinery is a promising approach.
Plants exposed to high temperature experience various changes in physiological and biochemical processes. Plants have several self-protection mechanisms to minimize plant cell damage from heat stress. Among these physiological mechanisms, the antioxidant protection system and photosynthetic system were highlighted in our research. Multiple lines of evidence support that the photosynthesis process is above supports our view that the CWT can stimulate CEF to protect PSI from photodamage.
Redox state of PSI and PQ pool size. The response of P700 redox kinetics in pak choi leaves (T1, T2 and T3) was measured with applications of single turnover flashes followed by multiple turnover flashes in an FR light background. The maximum oxidation state of P700 was induced after FR light exposure. As shown in Fig. 6, the maximum oxidation state of P700 was higher for the coldwater treatment than for the the control, while the reduction progress showed no differences among all treatments. The size of the PQ pools was estimated as the ratio of MTarea/ ST-area. In the experimental results, the PQ pool size in the pak choi leaves (T2) exhibited a 1.48-fold larger functional PQ pool in comparison with the control. We supposed that the reduction in the PQ pool in control leaves is responsible for blocking of electron transport.

Discussion
Contemporary agriculture is confronted with unprecedented environmental pressure and stress caused by climate change (Argosubekti et al. 2020). Temperature is one of the main ). In the current study, we surveyed the photosynthetic quantum yields, electron transport rate, redox state of PSI, and PQ pool size under water treatment at different temperatures, which demonstrated that photosynthetic function was significantly suppressed in the control plants. IIn cold water treated plants, photosynthetic efficiency and electron transport rate was enhanced significantly compared to control. The Fv/Fm ratio in cold water treated plants increased 1.3 fold change compare to control plants. It means that the maximum light energy conversion efficiency of PS II is significantly increased.
Heat stress leads to the production of ROS in various organelles, including in peroxisomes, chloroplasts, and mitochondria (Smirnoff et al. 2008). ROS signaling is involved in the activation of antioxidant enzymes, heat the most significant response to heat stress (Blum 2018). Heat stress damages photosystems II and I as well as the oxygen complex, impairs the integrity of the thylakoid membrane, decreases carbohydrate and protein synthesis, reduces carbon metabolism, and alters microtubule organization (Bita et al. 2013). Moreover, electron transport and ATP synthesis were disrupted because of thermal damage to PSII during the photosynthetic process . Under high-temperature environments, PSII was damaged, which contributed to the disruption of electron transport and ATP synthesis during the photosynthetic process . Analogously, cold stress blocks both electronic transport in thylakoids and carbon fixation, resulting in the reduction of photosynthetic rates (Nievola et al. 2017;Ensminger et al. 2006;Hajihashemi, 2018; Adams III et al. cellular water levels and reduce dehydration, which leads to a rich photosynthetic capacity. Ahmad et al. (2021b, c) reported that total soluble protein helped improve heat stress tolerance in oilseed crops by improving plant water relations and gas exchange properties. Our results also support this finding; higher levels of Pn, E and gs were recorded in cold water-treated pakchoi leaves. The photosynthetic rate of plants will be inhibited under both heat stress and cold stress. The higher concentrations and activities of enzymatic and nonenzymatic antioxidants contribute to the balance of redox state, the stabilization of cell membranes and gas exchange in pak choi treated with cold water.
Heat stress often damages membrane stability and causes a decrease in leaf chlorophyll content (Bita et al. 2013;Hejnák et al. 2015a, b). We found that the Chl a and Chl b contents were significantly higher in pak choi irrigated with cold water (15 and 20 °C) than in the control. A reduction in photosynthetic pigments was also observed in cold-stressed plants. Almeselmani et al. (2006) proposed that the tolerance levels of crops were linked to the level of chlorophyll synthesis and breakdown under heat stress. The decreased chlorophyll level suppresses the process of carbon fixation shock proteins (HSPs), and dysfunction of the plant cell membrane (Bohnert et al. 2006). Thus, the induction of thermotolerance for plant protection under heat stress is directly linked to the ability to detoxify and scavenge radical ROS. In this study, the antioxidant enzyme activity of pak choi treated with cold water and room-temperature water was measured. We found that the activities of SOD, APX, and CAT in pak choi treated with cold water were higher than in pak choi treated with room-temperature water. Meanwhile, we detected that the release of oxygen radicals (H 2 O 2 and O 2− ) decreased under cold-water treatment. The increasing amount of H 2 O 2 under heat stress can create oxidative damage in the plant. Based on these results, we inferred that cold-water treatment helped enhance the activities of antioxidants, which helped decrease the H 2 O 2 level. Some nonenzymatic antioxidants also play a role in resisting heat stress, such as soluble sugar, protein and ascorbic acid. A large body of literature has revealed that an elevation in sugar plays an important role in the acquisition of cold tolerance (Hajihashemi et al. 2020). It accumulated more in pak choi under cold-water treatment compared to in the control. We supposed that these nonenzymatic antioxidants regulate heat tolerance acquisition (Ahmad et al. 2021). Huang et al. (2019) reported that GmHsp90A2 positively regulated heat stress in soybean. It interacted with GmHsp90A1 and acquired increased tolerance to heat stress through higher chlorophyll and lower MDA contents in plants. Ca 2+ channels are putative heat sensors that interact with HSFs (heat stress transcription factors) via CBK and CaM 3 (Ahmad et al. 2021). Ca 2+ also regulates HSPs and HSFs and triggers enzyme activity to increase heat stress tolerance in plants. We supposed that short-term thermal adjustment affects Ca 2+ channels and HSP expression, further modulating intracellular ROS signaling, osmotic adjustment and the photosynthetic apparatus. These hypotheses require further validation at the molecular and protein levels. Nevertheless, there are some common stress resistance pathways between cold and heat stress, we hoped that our research in the future can provide some new ideas for plants to resist temperature stress in winter and summer. We can also use some waste gas and other resources to achieve energy recycling, which is conducive to the development of a low-carbon economy. in photosynthesis. Malondialdehyde (MDA) is an indicator of lipid peroxidation. ROS generation leads to a high amount of MDA production, causing oxidative damage and damaging membrane integrity (Ahmad et al. 2021). A decreased MDA concentration and electrolyte leakage were detected in cold water-treated plants, which revealed that cold-water treatment can effectively alleviate plant lipid peroxidation.
An earlier study suggested that Canola growth was impeded under heat stress by reducing plant height, root length, and biomass accumulation due to an impaired photosynthetic rate and stomatal conductance (Waraich et al. 2022). According to the experimental data, greater biomass accumulation was observed in pak choi under cold-water treatment than in control plants. The results of this investigation indicated that cold water helps reduce adverse effects on plant growth resulting from heat stress. The reduced photosynthetic rate is responsible for the reduced biomass in the control. In addition to direct physiological and biochemical responses, plants also respond to heat stress at the molecular level. High temperature can significantly activate the expression of some key genes involved in the heat stress response. Heat shock factors (HSFs) and heat shock proteins (HSPs) are involved in heat stress signaling for