Plant growth regulators have been used as a strategy to reduce heat stress in various crops

        Rice production is declining due to climate change and variability in Colombia. Plant growth regulators have been used as a strategy to reduce heat stress in various crops. Therefore, the objective of this study was to evaluate the physiological effects (stomatal conductance, stomatal conductance, total chlorophyll content, Fv/Fm ratio of two commercial rice genotypes subjected to combined heat stress (high day and night temperatures), canopy temperature and relative water content) and biochemical variables (malondialdehyde (MDA) and prolinic acid content). The first and second experiments were carried out using plants of two rice genotypes Federrose 67 (“F67”) and Federrose 2000 (“F2000”), respectively. Both experiments were analyzed together as a series of experiments. The established treatments were as follows: absolute control (AC) (rice plants grown at optimal temperatures (day/night temperature 30/25°C)), heat stress control (SC) [rice plants subjected to combined heat stress only (40/ 25°C). 30°C)], and rice plants were stressed and sprayed with plant growth regulators (stress+AUX, stress+BR, stress+CK or stress+GA) twice (5 days before and 5 days after heat stress). Spraying with SA increased the total chlorophyll content of both varieties (fresh weight of rice plants “F67″ and “F2000″ was 3.25 and 3.65 mg/g, respectively) compared to SC plants (fresh weight of “F67″ plants was 2.36 and 2.56 mg). g-1)” and rice “F2000″, foliar application of CK also generally improved the stomatal conductance of rice “F2000″ plants (499.25 vs. 150.60 mmol m-2 s) compared to the heat stress control. heat stress, the temperature of the plant crown decreases by 2–3 °C, and the MDA content in plants decreases. The relative tolerance index shows that foliar application of CK (97.69%) and BR (60.73%) can help alleviate the problem of combined heat. stress mainly in F2000 rice plants. In conclusion, foliar spraying of BR or CK can be considered as an agronomic strategy to help reduce the negative effects of combined heat stress conditions on the physiological behavior of rice plants.
        Rice (Oryza sativa) belongs to the Poaceae family and is one of the most cultivated cereals in the world along with maize and wheat (Bajaj and Mohanty, 2005). The area under rice cultivation is 617,934 hectares, and the national production in 2020 was 2,937,840 tons with an average yield of 5.02 tons/ha (Federarroz (Federación Nacional de Arroceros), 2021).
        Global warming is affecting rice crops, leading to various types of abiotic stresses such as high temperatures and periods of drought . Climate change is causing global temperatures to rise; Temperatures are projected to rise by 1.0–3.7°C in the 21st century, which could increase the frequency and intensity of heat stress . Increased environmental temperatures have affected rice, causing crop yields to decline by 6–7% . On the other hand, climate change also leads to unfavorable environmental conditions for crops, such as periods of severe drought or high temperatures in tropical and subtropical regions . In addition, variability events such as El Niño can lead to heat stress and exacerbate crop damage in some tropical regions. In Colombia, temperatures in rice-producing areas are projected to increase by 2–2.5°C by 2050, reducing rice production and affecting product flows to markets and supply chains.
        Most rice crops are grown in areas where temperatures are close to the optimum range for crop growth (Shah et al., 2011). It has been reported that the optimal average day and night temperatures for rice growth and development are generally 28°C and 22°C, respectively (Kilasi et al., 2018; Calderón-Páez et al., 2021). Temperatures above these thresholds can cause periods of moderate to severe heat stress during sensitive stages of rice development (tillering, anthesis, flowering, and grain filling), thereby negatively affecting grain yield . This reduction in yield is mainly due to long periods of heat stress, which affect plant physiology . Due to the interaction of various factors, such as stress duration and maximum temperature reached, heat stress can cause a range of irreversible damage to plant metabolism and development .
        Heat stress affects various physiological and biochemical processes in plants. Leaf photosynthesis is one of the processes most susceptible to heat stress in rice plants, as the rate of photosynthesis decreases by 50% when daily temperatures exceed 35°C. Physiological responses of rice plants vary depending on the type of heat stress. For example, photosynthetic rates and stomatal conductance are inhibited when plants are exposed to high daytime temperatures (33–40°C) or high daytime and nighttime temperatures (35–40°C during the day, 28–30°C). C means night) (Lü et al., 2013; Fahad et al., 2016; Chaturvedi et al., 2017). High night temperatures (30°C) cause moderate inhibition of photosynthesis but increase night respiration (Fahad et al., 2016; Alvarado-Sanabria et al., 2017). Regardless of the stress period, heat stress also affects leaf chlorophyll content, the ratio of chlorophyll variable fluorescence to maximum chlorophyll fluorescence (Fv/Fm), and Rubisco activation in rice plants (Cao et al. 2009; Yin et al. 2010). ) Sanchez Reynoso et al., 2014).
        Biochemical changes are another aspect of plant adaptation to heat stress (Wahid et al., 2007). Proline content has been used as a biochemical indicator of plant stress (Ahmed and Hassan 2011). Proline plays an important role in plant metabolism as it acts as a carbon or nitrogen source and as a membrane stabilizer under high temperature conditions (Sánchez-Reinoso et al., 2014). High temperatures also affect membrane stability through lipid peroxidation, leading to the formation of malondialdehyde (MDA) (Wahid et al., 2007). Therefore, MDA content has also been used to understand the structural integrity of cell membranes under heat stress ( Cao et al., 2009 ; Chavez-Arias et al., 2018 ). Finally, combined heat stress [37/30°C (day/night)] increased the percentage of electrolyte leakage and malondialdehyde content in rice (Liu et al., 2013).
        The use of plant growth regulators (GRs) has been assessed to mitigate the negative effects of heat stress, as these substances are actively involved in plant responses or physiological defense mechanisms against such stress (Peleg and Blumwald, 2011; Yin et al. et al., 2011 ; Ahmed et al., 2015). Exogenous application of genetic resources has had a positive effect on heat stress tolerance in various crops. Studies have shown that phytohormones such as gibberellins (GA), cytokinins (CK), auxins (AUX) or brassinosteroids (BR) lead to an increase in various physiological and biochemical variables (Peleg and Blumwald, 2011; Yin et al. Ren, 2011 ; Mitler et al., 2012; Zhou et al., 2014). In Colombia, exogenous application of genetic resources and its impact on rice crops have not been fully understood and studied. However, a previous study showed that foliar spraying of BR could improve rice tolerance by improving the gas exchange characteristics, chlorophyll or proline content of rice seedling leaves ( Quintero-Calderón et al., 2021 ).
        Cytokinins mediate plant responses to abiotic stresses, including heat stress (Ha et al., 2012). In addition, it has been reported that exogenous application of CK can reduce thermal damage. For example, exogenous application of zeatin increased the photosynthetic rate, chlorophyll a and b content, and electron transport efficiency in creeping bentgrass (Agrotis estolonifera) during heat stress (Xu and Huang, 2009; Jespersen and Huang, 2015). Exogenous application of zeatin can also improve antioxidant activity, enhance the synthesis of various proteins, reduce reactive oxygen species (ROS) damage and malondialdehyde (MDA) production in plant tissues (Chernyadyev, 2009; Yang et al., 2009). , 2016; Kumar et al., 2020).
        The use of gibberellic acid has also shown a positive response to heat stress. Studies have shown that GA biosynthesis mediates various metabolic pathways and increases tolerance under high temperature conditions (Alonso-Ramirez et al. 2009; Khan et al. 2020). Abdel-Nabi et al. (2020) found that foliar spraying of exogenous GA (25 or 50 mg*L) could increase photosynthetic rate and antioxidant activity in heat-stressed orange plants compared to control plants. It has also been observed that exogenous application of HA increases relative moisture content, chlorophyll and carotenoid contents and reduces lipid peroxidation in date palm (Phoenix dactylifera) under heat stress (Khan et al., 2020). Auxin also plays an important role in regulating adaptive growth responses to high temperature conditions (Sun et al., 2012; Wang et al., 2016). This growth regulator acts as a biochemical marker in various processes such as proline synthesis or degradation under abiotic stress (Ali et al. 2007). In addition, AUX also enhances antioxidant activity, which leads to a decrease in MDA in plants due to decreased lipid peroxidation (Bielach et al., 2017). Sergeev et al. (2018) observed that in pea plants (Pisum sativum) under heat stress, the content of proline – dimethylaminoethoxycarbonylmethyl)naphthylchloromethyl ether (TA-14) increases. In the same experiment, they also observed lower levels of MDA in treated plants compared to plants not treated with AUX.
        Brassinosteroids are another class of growth regulators used to mitigate the effects of heat stress. Ogweno et al. (2008) reported that exogenous BR spray increased the net photosynthetic rate, stomatal conductance and maximum rate of Rubisco carboxylation of tomato (Solanum lycopersicum) plants under heat stress for 8 days. Foliar spraying of epibrassinosteroids can increase the net photosynthetic rate of cucumber (Cucumis sativus) plants under heat stress (Yu et al., 2004). In addition, exogenous application of BR delays chlorophyll degradation and increases water use efficiency and maximum quantum yield of PSII photochemistry in plants under heat stress (Holá et al., 2010; Toussagunpanit et al., 2015).
        Due to climate change and variability, rice crops face periods of high daily temperatures (Lesk et al., 2016; Garcés, 2020; Federarroz (Federación Nacional de Arroceros), 2021). In plant phenotyping, the use of phytonutrients or biostimulants has been studied as a strategy to mitigate heat stress in rice-growing areas (Alvarado-Sanabria et al., 2017; Calderón-Páez et al., 2021; Quintero-Calderón et al., 2021). In addition, the use of biochemical and physiological variables (leaf temperature, stomatal conductance, chlorophyll fluorescence parameters, chlorophyll and relative water content, malondialdehyde and proline synthesis) is a reliable tool for screening rice plants under heat stress locally and internationally (Sánchez -Reynoso et al., 2014; Alvarado-Sanabria et al., 2017; However, research on the use of foliar phytohormonal sprays in rice at the local level remains rare. Therefore, the study of the physiological and biochemical reactions of the application of plant growth regulators is of great importance for the proposal of practical agronomic strategies for this. addressing the negative effects of a period of complex heat stress in rice. Therefore, the purpose of this study was to evaluate the physiological (stomatal conductance, chlorophyll fluorescence parameters and relative water content) and biochemical effects of foliar application of four plant growth regulators (AUX, CK, GA and BR). (Photosynthetic pigments, malondialdehyde and proline contents) Variables in two commercial rice genotypes subjected to combined heat stress (high day/night temperatures).
        In this study, two independent experiments were performed. The genotypes Federrose 67 (F67: a genotype developed in high temperatures during the last decade) and Federrose 2000 (F2000: a genotype developed in the last decade of the 20th century showing resistance to white leaf virus) were used for the first time. seeds. and the second experiment, respectively. Both genotypes are widely cultivated by Colombian farmers. Seeds were sown in 10-L trays (length 39.6 cm, width 28.8 cm, height 16.8 cm) containing sandy loam soil with 2% organic matter. Five pre-germinated seeds were planted in each tray. The pallets were placed in the greenhouse of the Faculty of Agricultural Sciences of the National University of Colombia, Bogotá campus (43°50′56″ N, 74°04′051″ W), at an altitude of 2556 m above sea level (a.s.l.). m.) and were carried out from October to December 2019. One experiment (Federroz 67) and a second experiment (Federroz 2000) in the same season of 2020.
        The environmental conditions in the greenhouse during each planting season are as follows: day and night temperature 30/25°C, relative humidity 60~80%, natural photoperiod 12 hours (photosynthetically active radiation 1500 µmol (photons) m-2 s-). 1 at noon). Plants were fertilized according to the content of each element 20 days after seed emergence (DAE), according to Sánchez-Reinoso et al. (2019): 670 mg nitrogen per plant, 110 mg phosphorus per plant, 350 mg potassium per plant, 68 mg calcium per plant, 20 mg magnesium per plant, 20 mg sulfur per plant, 17 mg silicon per plant. The plants contain 10 mg boron per plant, 17 mg copper per plant, and 44 mg zinc per plant. Rice plants were maintained at up to 47 DAE in each experiment when they reached phenological stage V5 during this period. Previous studies have shown that this phenological stage is an appropriate time to conduct heat stress studies in rice (Sánchez-Reinoso et al., 2014; Alvarado-Sanabria et al., 2017).
        In each experiment, two separate applications of the leaf growth regulator were performed. The first set of foliar phytohormone sprays was applied 5 days before the heat stress treatment (42 DAE) to prepare the plants for environmental stress. A second foliar spray was then given 5 days after the plants were exposed to stress conditions (52 DAE). Four phytohormones were used and the properties of each active ingredient sprayed in this study are listed in Supplementary Table 1. The concentrations of leaf growth regulators used were as follows: (i) Auxin (1-naphthylacetic acid: NAA) at a concentration of 5 × 10−5 M (ii) 5 × 10–5 M gibberellin (gibberellic acid: NAA); GA3); (iii) Cytokinin (trans-zeatin) 1 × 10-5 M (iv) Brassinosteroids [Spirostan-6-one, 3,5-dihydroxy-, (3b,5a,25R)] 5 × 10-5; M. These concentrations were chosen because they induce positive responses and increase plant resistance to heat stress (Zahir et al., 2001; Wen et al., 2010; El-Bassiony et al., 2012; Salehifar et al., 2017 ). Rice plants without any plant growth regulator sprays were treated with distilled water only. All rice plants were sprayed with a hand sprayer. Apply 20 ml H2O to the plant to moisten the upper and lower surfaces of the leaves. All foliar sprays used agricultural adjuvant (Agrotin, Bayer CropScience, Colombia) at 0.1% (v/v). The distance between the pot and the sprayer is 30 cm.
        Heat stress treatments were administered 5 days after the first foliar spray (47 DAE) in each experiment. Rice plants were transferred from the greenhouse to a 294 L growth chamber (MLR-351H, Sanyo, IL, USA) to establish heat stress or maintain the same environmental conditions (47 DAE). Combined heat stress treatment was carried out by setting the chamber to the following day/night temperatures: daytime high temperature [40°C for 5 hours (from 11:00 to 16:00)] and night period [30°C for 5 hours] . 8 days in a row (from 19:00 to 24:00). The stress temperature and exposure time were selected based on previous studies (Sánchez-Reynoso et al. 2014; Alvarado-Sanabría et al. 2017). On the other hand, a group of plants transferred to the growth chamber were kept in the greenhouse at the same temperature (30°C during the day/25°C at night) for 8 consecutive days.
        At the end of the experiment, the following treatment groups were obtained: (i) growth temperature condition + application of distilled water [Absolute control (AC)], (ii) heat stress condition + application of distilled water [Heat stress control (SC)], (iii) conditions heat stress condition + auxin application (AUX), (iv) heat stress condition + gibberellin application (GA), (v) heat stress condition + cytokinin application (CK), and (vi) heat stress condition + brassinosteroid (BR) Appendix. These treatment groups were used for two genotypes (F67 and F2000). All treatments were carried out in a completely randomized design with five replicates, each consisting of one plant. Each plant was used to read out the variables determined at the end of the experiment. The experiment lasted 55 DAE.
        Stomatal conductance (gs) was measured using a portable porosometer (SC-1, METER Group Inc., USA) ranging from 0 to 1000 mmol m-2 s-1, with a sample chamber aperture of 6.35 mm. Measurements are taken by attaching a stomameter probe to a mature leaf with the main shoot of the plant fully expanded. For each treatment, gs readings were taken on three leaves of each plant between 11:00 and 16:00 and averaged.
        RWC was determined according to the method described by Ghoulam et al. (2002). The fully expanded sheet used to determine g was also used to measure RWC. Fresh weight (FW) was determined immediately after harvest using a digital scale. The leaves were then placed in a plastic container filled with water and left in the dark at room temperature (22°C) for 48 hours. Then weigh on a digital scale and record the expanded weight (TW). The swollen leaves were oven dried at 75°C for 48 hours and their dry weight (DW) was recorded.
        Relative chlorophyll content was determined using a chlorophyll meter (atLeafmeter, FT Green LLC, USA) and expressed in atLeaf units (Dey et al., 2016). PSII maximum quantum efficiency readings (Fv/Fm ratio) were recorded using a continuous excitation chlorophyll fluorimeter (Handy PEA, Hansatech Instruments, UK). Leaves were dark-adapted using leaf clamps for 20 min before Fv/Fm measurements (Restrepo-Diaz and Garces-Varon, 2013). After the leaves were dark acclimated, baseline (F0) and maximum fluorescence (Fm) were measured. From these data, variable fluorescence (Fv = Fm – F0), the ratio of variable fluorescence to maximum fluorescence (Fv/Fm), the maximum quantum yield of PSII photochemistry (Fv/F0) and the ratio Fm/F0 were calculated (Baker, 2008; Lee et al. ., 2017). Relative chlorophyll and chlorophyll fluorescence readings were taken on the same leaves used for gs measurements.
        Approximately 800 mg of leaf fresh weight was collected as biochemical variables. Leaf samples were then homogenized in liquid nitrogen and stored for further analysis. The spectrometric method used to estimate tissue chlorophyll a, b and carotenoid content is based on the method and equations described by Wellburn (1994). Leaf tissue samples (30 mg) were collected and homogenized in 3 ml of 80% acetone. The samples were then centrifuged (model 420101, Becton Dickinson Primary Care Diagnostics, USA) at 5000 rpm for 10 min to remove particles. The supernatant was diluted to a final volume of 6 ml by adding 80% acetone (Sims and Gamon, 2002). The content of chlorophyll was determined at 663 (chlorophyll a) and 646 (chlorophyll b) nm, and carotenoids at 470 nm using a spectrophotometer (Spectronic BioMate 3 UV-vis, Thermo, USA).
        The thiobarbituric acid (TBA) method described by Hodges et al. (1999) was used to assess membrane lipid peroxidation (MDA). Approximately 0.3 g of leaf tissue was also homogenized in liquid nitrogen. The samples were centrifuged at 5000 rpm and absorbance was measured on a spectrophotometer at 440, 532 and 600 nm. Finally, the MDA concentration was calculated using the extinction coefficient (157 M mL−1).
        Proline content of all treatments was determined using the method described by Bates et al. (1973). Add 10 ml of a 3% aqueous solution of sulfosalicylic acid to the stored sample and filter through Whatman filter paper (No. 2). Then 2 ml of this filtrate was reacted with 2 ml of ninhydric acid and 2 ml of glacial acetic acid. The mixture was placed in a water bath at 90°C for 1 hour. Stop the reaction by incubating on ice. Shake the tube vigorously using a vortex shaker and dissolve the resulting solution in 4 ml of toluene. Absorbance readings were determined at 520 nm using the same spectrophotometer used for the quantification of photosynthetic pigments (Spectronic BioMate 3 UV-Vis, Thermo, Madison, WI, USA).
        The method described by Gerhards et al. (2016) to calculate canopy temperature and CSI. Thermal photographs were taken with a FLIR 2 camera (FLIR Systems Inc., Boston, MA, USA) with an accuracy of ±2°C at the end of the stress period. Place a white surface behind the plant for photography. Again, two factories were considered as reference models. The plants were placed on a white surface; one was coated with an agricultural adjuvant (Agrotin, Bayer CropScience, Bogotá, Colombia) to simulate the opening of all stomata [wet mode (Twet)], and the other was a leaf without any application [Dry mode (Tdry)] (Castro -Duque et al., 2020). The distance between the camera and the pot during filming was 1 m.
        The relative tolerance index was calculated indirectly using stomatal conductance (gs) of treated plants compared to control plants (plants without stress treatments and with growth regulators applied) to determine the tolerance of the treated genotypes evaluated in this study. RTI was obtained using an equation adapted from Chávez-Arias et al. (2020).
        In each experiment, all physiological variables mentioned above were determined and recorded at 55 DAE using fully expanded leaves collected from the upper canopy. In addition, measurements were carried out in a growth chamber to avoid changing the environmental conditions in which the plants grow.
        Data from the first and second experiments were analyzed together as a series of experiments. Each experimental group consisted of 5 plants, and each plant constituted an experimental unit. Analysis of variance (ANOVA) was performed (P ≤ 0.05). When significant differences were detected, Tukey’s post hoc comparative test was used at P ≤ 0.05. Use the arcsine function to convert percentage values. Data were analyzed using Statistix v 9.0 software (Analytical Software, Tallahassee, FL, USA) and plotted using SigmaPlot (version 10.0; Systat Software, San Jose, CA, USA). The main component analysis was carried out using InfoStat 2016 software (Analysis Software, National University of Cordoba, Argentina) to identify the best plant growth regulators under study.
        Table 1 summarizes the ANOVA showing the experiments, the different treatments, and their interactions with leaf photosynthetic pigments (chlorophyll a, b, total, and carotenoids), malondialdehyde (MDA) and proline content, and stomatal conductance. Effect of gs, relative water content. (RWC), chlorophyll content, chlorophyll alpha fluorescence parameters, crown temperature (PCT) (°C), crop stress index (CSI) and relative tolerance index of rice plants at 55 DAE.
       Table 1. Summary of ANOVA data on rice physiological and biochemical variables between experiments (genotypes) and heat stress treatments.
        Differences (P≤0.01) in leaf photosynthetic pigment interactions, relative chlorophyll content (Atleaf readings), and alpha-chlorophyll fluorescence parameters between experiments and treatments are shown in Table 2. High daytime and nighttime temperatures increased total chlorophyll and carotenoid contents. Rice seedlings without any foliar spray of phytohormones (2.36 mg g-1 for “F67″ and 2.56 mg g-1 for “F2000″) compared to plants grown under optimal temperature conditions (2.67 mg g -1)) showed lower total chlorophyll content. In both experiments, “F67” was 2.80 mg g-1 and “F2000” was 2.80 mg g-1. In addition, rice seedlings treated with a combination of AUX and GA sprays under heat stress also showed a decrease in chlorophyll content in both genotypes (AUX = 1.96 mg g-1 and GA = 1.45 mg g-1 for “F67” ; AUX = 1.96 mg g-1 and GA = 1.45 mg g-1 for “F67″; AUX = 2.24 mg) g-1 and GA = 1.43 mg g-1 (for “F2000″ ) under heat stress conditions. Under heat stress conditions, foliar treatment with BR resulted in a slight increase in this variable in both genotypes. Finally, CK foliar spray showed the highest photosynthetic pigment values ​​among all treatments (AUX, GA, BR, SC and AC treatments) in genotypes F67 (3.24 mg g-1) and F2000 (3.65 mg g-1). The relative content of chlorophyll (Atleaf unit) was also reduced by combined heat stress. The highest values ​​were also recorded in plants sprayed with CC in both genotypes (41.66 for “F67” and 49.30 for “F2000”). Fv and Fv/Fm ratios showed significant differences between treatments and cultivars (Table 2). Overall, among these variables, cultivar F67 was less susceptible to heat stress than cultivar F2000. The Fv and Fv/Fm ratios suffered more in the second experiment. Stressed ‘F2000′ seedlings that were not sprayed with any phytohormones had the lowest Fv values ​​(2120.15) and Fv/Fm ratios (0.59), but foliar spraying with CK helped restore these values ​​(Fv: 2591, 89, Fv/Fm ratio: 0.73). , receiving readings similar to those recorded on “F2000” plants grown under optimal temperature conditions (Fv: 2955.35, Fv/Fm ratio: 0.73:0.72). There were no significant differences in initial fluorescence (F0), maximum fluorescence (Fm), maximum photochemical quantum yield of PSII (Fv/F0) and Fm/F0 ratio. Finally, BR showed a similar trend as observed with CK (Fv 2545.06, Fv/Fm ratio 0.73).
       Table 2. Effect of combined heat stress (40°/30°C day/night) on leaf photosynthetic pigments [total chlorophyll (Chl Total), chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids Cx+c] effect ], relative chlorophyll content (Atliff unit), chlorophyll fluorescence parameters (initial fluorescence (F0), maximum fluorescence (Fm), variable fluorescence (Fv), maximum PSII efficiency (Fv/Fm), photochemical maximum quantum yield of PSII (Fv/F0 ) and Fm/F0 in plants of two rice genotypes [Federrose 67 (F67) and Federrose 2000 (F2000)] 55 days after emergence (DAE)).
        Relative water content (RWC) of differently treated rice plants showed differences (P ≤ 0.05) in the interaction between experimental and foliar treatments (Fig. 1A). When treated with SA, the lowest values ​​were recorded for both genotypes (74.01% for F67 and 76.6% for F2000). Under heat stress conditions, the RWC of rice plants of both genotypes treated with different phytohormones increased significantly. Overall, foliar applications of CK, GA, AUX, or BR increased RWC to values ​​similar to those of plants grown under optimal conditions during the experiment. Absolute control and foliar sprayed plants recorded values ​​of around 83% for both genotypes. On the other hand, gs also showed significant differences (P ≤ 0.01) in the experiment-treatment interaction (Fig. 1B). The absolute control (AC) plant also recorded the highest values ​​for each genotype (440.65 mmol m-2s-1 for F67 and 511.02 mmol m-2s-1 for F2000). Rice plants subjected to combined heat stress alone showed the lowest gs values ​​for both genotypes (150.60 mmol m-2s-1 for F67 and 171.32 mmol m-2s-1 for F2000). Foliar treatment with all plant growth regulators also increased g. On F2000 rice plants sprayed with CC, the effect of foliar spraying with phytohormones was more obvious. This group of plants showed no differences compared to absolute control plants (AC 511.02 and CC 499.25 mmol m-2s-1).
        Figure 1. Effect of combined heat stress (40°/30°C day/night) on relative water content (RWC) (A), stomatal conductance (gs) (B), malondialdehyde (MDA) production (C), and proline content . (D) in plants of two rice genotypes (F67 and F2000) at 55 days after emergence (DAE). Treatments assessed for each genotype included: absolute control (AC), heat stress control (SC), heat stress + auxin (AUX), heat stress + gibberellin (GA), heat stress + cell mitogen (CK), and heat stress + brassinosteroid. (BR). Each column represents the mean ± standard error of five data points (n = 5). Columns followed by different letters indicate statistically significant differences according to Tukey’s test (P ≤ 0.05). Letters with an equal sign indicate that the mean is not statistically significant (≤ 0.05).
        MDA (P ≤ 0.01) and proline (P ≤ 0.01) contents also showed significant differences in the interaction between experiment and phytohormone treatments (Fig. 1C, D). Increased lipid peroxidation was observed with SC treatment in both genotypes (Figure 1C), however plants treated with leaf growth regulator spray showed decreased lipid peroxidation in both genotypes; In general, the use of phytohormones (CA, AUC, BR or GA) leads to a decrease in lipid peroxidation (MDA content). No differences were found between AC plants of two genotypes and plants under heat stress and sprayed with phytohormones (observed FW values ​​in “F67” plants ranged from 4.38–6.77 µmol g-1, and in FW “F2000” plants “observed values ​​ranged from 2.84 to 9.18 µmol g-1 (plants). On the other hand, proline synthesis in “F67″ plants was lower than in “F2000″ plants under combined stress, which led to an increase in proline production. in heat-stressed rice plants, in both experiments, it was observed that the administration of these hormones significantly increased the amino acid content of F2000 plants (AUX and BR were 30.44 and 18.34 µmol g-1) respectively (Fig. 1G).
        The effects of foliar plant growth regulator spray and combined heat stress on plant canopy temperature and relative tolerance index (RTI) are shown in Figures 2A and B. For both genotypes, the canopy temperature of AC plants was close to 27°C, and that of SC plants was around 28°C. WITH. It was also observed that foliar treatments with CK and BR resulted in a 2–3°C decrease in canopy temperature compared to SC plants (Figure 2A). RTI exhibited similar behavior to other physiological variables, showing significant differences (P ≤ 0.01) in the interaction between experiment and treatment (Figure 2B). SC plants showed lower plant tolerance in both genotypes (34.18% and 33.52% for “F67” and “F2000” rice plants, respectively). Foliar feeding of phytohormones improves RTI in plants exposed to high temperature stress. This effect was more pronounced in “F2000” plants sprayed with CC, in which the RTI was 97.69. On the other hand, significant differences were observed only in the yield stress index (CSI) of rice plants under foliar factor spray stress conditions (P ≤ 0.01) (Fig. 2B). Only rice plants subjected to complex heat stress showed the highest stress index value (0.816). When rice plants were sprayed with various phytohormones, the stress index was lower (values ​​from 0.6 to 0.67). Finally, the rice plant grown under optimal conditions had a value of 0.138.
        Figure 2. Effects of combined heat stress (40°/30°C day/night) on canopy temperature (A), relative tolerance index (RTI) (B), and crop stress index (CSI) (C) of two plant species. Commercial rice genotypes (F67 and F2000) were subjected to different heat treatments. Treatments assessed for each genotype included: absolute control (AC), heat stress control (SC), heat stress + auxin (AUX), heat stress + gibberellin (GA), heat stress + cell mitogen (CK), and heat stress + brassinosteroid. (BR). Combined heat stress involves exposing rice plants to high day/night temperatures (40°/30°C day/night). Each column represents the mean ± standard error of five data points (n = 5). Columns followed by different letters indicate statistically significant differences according to Tukey’s test (P ≤ 0.05). Letters with an equal sign indicate that the mean is not statistically significant (≤ 0.05).
        Principal component analysis (PCA) revealed that the variables assessed at 55 DAE explained 66.1% of the physiological and biochemical responses of heat-stressed rice plants treated with growth regulator spray (Fig. 3). Vectors represent variables and dots represent plant growth regulators (GRs). The vectors of gs, chlorophyll content, maximum quantum efficiency of PSII (Fv/Fm) and biochemical parameters (TChl, MDA and proline) are at close angles to the origin, indicating a high correlation between the physiological behavior of plants and them. variable. One group (V) included rice seedlings grown at optimal temperature (AT) and F2000 plants treated with CK and BA. At the same time, the majority of plants treated with GR formed a separate group (IV), and treatment with GA in F2000 formed a separate group (II). In contrast, heat-stressed rice seedlings (groups I and III) without any foliar spray of phytohormones (both genotypes were SC) were located in a zone opposite to group V, demonstrating the effect of heat stress on plant physiology. .
        Figure 3. Bigraphical analysis of the effects of combined heat stress (40°/30°C day/night) on plants of two rice genotypes (F67 and F2000) at 55 days after emergence (DAE). Abbreviations: AC F67, absolute control F67; SC F67, heat stress control F67; AUX F67, heat stress + auxin F67; GA F67, heat stress + gibberellin F67; CK F67, heat stress + cell division BR F67, heat stress + brassinosteroid. F67; AC F2000, absolute control F2000; SC F2000, Heat Stress Control F2000; AUX F2000, heat stress + auxin F2000; GA F2000, heat stress + gibberellin F2000; CK F2000, heat stress + cytokinin, BR F2000, heat stress + brass steroid; F2000.
        Variables such as chlorophyll content, stomatal conductance, Fv/Fm ratio, CSI, MDA, RTI and proline content can help understand the adaptation of rice genotypes and evaluate the impact of agronomic strategies under heat stress (Sarsu et al., 2018; Quintero-Calderon et al., 2021). The purpose of this experiment was to evaluate the effect of application of four growth regulators on the physiological and biochemical parameters of rice seedlings under complex heat stress conditions. Seedling testing is a simple and rapid method for simultaneous assessment of rice plants depending on the size or condition of available infrastructure (Sarsu et al. 2018). The results of this study showed that combined heat stress induces different physiological and biochemical responses in the two rice genotypes, indicating an adaptation process. These results also indicate that foliar growth regulator sprays (mainly cytokinins and brassinosteroids) help rice adapt to complex heat stress as favor mainly affects gs, RWC, Fv/Fm ratio, photosynthetic pigments and proline content.
        Application of growth regulators helps improve the water status of rice plants under heat stress, which may be associated with higher stress and lower plant canopy temperatures. This study showed that among “F2000” (susceptible genotype) plants, rice plants treated primarily with CK or BR had higher gs values ​​and lower PCT values ​​than plants treated with SC. Previous studies have also shown that gs and PCT are accurate physiological indicators that can determine the adaptive response of rice plants and the effects of agronomic strategies on heat stress (Restrepo-Diaz and Garces-Varon, 2013; Sarsu et al., 2018; Quintero). -Carr DeLong et al., 2021). Leaf CK or BR enhance g under stress because these plant hormones can promote stomatal opening through synthetic interactions with other signaling molecules such as ABA (promoter of stomatal closure under abiotic stress) (Macková et al., 2013; Zhou et al., 2013). 2013). ). , 2014). Stomatal opening promotes leaf cooling and helps reduce canopy temperatures (Sonjaroon et al., 2018; Quintero-Calderón et al., 2021). For these reasons, the canopy temperature of rice plants sprayed with CK or BR may be lower under combined heat stress.
        High temperature stress can reduce the photosynthetic pigment content of leaves (Chen et al., 2017; Ahammed et al., 2018). In this study, when rice plants were under heat stress and not sprayed with any plant growth regulators, photosynthetic pigments tended to decrease in both genotypes (Table 2). Feng et al. (2013) also reported a significant decrease in chlorophyll content in leaves of two wheat genotypes exposed to heat stress. Exposure to high temperatures often results in decreased chlorophyll content, which may be due to decreased chlorophyll biosynthesis, degradation of pigments, or their combined effects under heat stress (Fahad et al., 2017). However, rice plants treated mainly with CK and BA increased the concentration of leaf photosynthetic pigments under heat stress. Similar results were also reported by Jespersen and Huang (2015) and Suchsagunpanit et al. (2015), who observed an increase in leaf chlorophyll content following application of zeatin and epibrassinosteroid hormones in heat-stressed bentgrass and rice, respectively. A reasonable explanation for why CK and BR promote increased leaf chlorophyll content under combined heat stress is that CK may enhance the initiation of sustained induction of expression promoters (such as the senescence-activating promoter (SAG12) or the HSP18 promoter) and reduce chlorophyll loss in leaves. , delay leaf senescence and increase plant resistance to heat (Liu et al., 2020). BR can protect leaf chlorophyll and increase leaf chlorophyll content by activating or inducing the synthesis of enzymes involved in chlorophyll biosynthesis under stress conditions (Sharma et al., 2017; Siddiqui et al., 2018). Finally, two phytohormones (CK and BR) also promote the expression of heat shock proteins and improve various metabolic adaptation processes, such as increased chlorophyll biosynthesis (Sharma et al., 2017; Liu et al., 2020).
        Chlorophyll a fluorescence parameters provide a rapid and non-destructive method that can assess plant tolerance or adaptation to abiotic stress conditions (Chaerle et al. 2007; Kalaji et al. 2017). Parameters such as the Fv/Fm ratio have been used as indicators of plant adaptation to stress conditions (Alvarado-Sanabria et al. 2017; Chavez-Arias et al. 2020). In this study, SC plants showed the lowest values ​​of this variable, predominantly “F2000” rice plants. Yin et al. (2010) also found that the Fv/Fm ratio of the highest tillering rice leaves decreased significantly at temperatures above 35°C. According to Feng et al. (2013), the lower Fv/Fm ratio under heat stress indicates that the rate of excitation energy capture and conversion by the PSII reaction center is reduced, indicating that the PSII reaction center disintegrates under heat stress. This observation allows us to conclude that disturbances in the photosynthetic apparatus are more pronounced in sensitive varieties (Fedearroz 2000) than in resistant varieties (Fedearroz 67).
        The use of CK or BR generally enhanced the performance of PSII under complex heat stress conditions. Similar results were obtained by Suchsagunpanit et al. (2015), who observed that BR application increased the efficiency of PSII under heat stress in rice. Kumar et al. (2020) also found that chickpea plants treated with CK (6-benzyladenine) and subjected to heat stress increased the Fv/Fm ratio, concluding that foliar application of CK by activating the zeaxanthin pigment cycle promoted PSII activity. In addition, BR leaf spray favored PSII photosynthesis under combined stress conditions, indicating that application of this phytohormone resulted in decreased dissipation of excitation energy of PSII antennae and promoted the accumulation of small heat shock proteins in chloroplasts (Ogweno et al. 2008; Kothari and Lachowitz ). , 2021).
        MDA and proline contents often increase when plants are under abiotic stress compared to plants grown under optimal conditions (Alvarado-Sanabria et al. 2017). Previous studies have also shown that MDA and proline levels are biochemical indicators that can be used to understand the adaptation process or the impact of agronomic practices in rice under daytime or nighttime high temperatures (Alvarado-Sanabria et al., 2017; Quintero-Calderón et al. . , 2021). These studies also showed that MDA and proline contents tended to be higher in rice plants exposed to high temperatures at night or during the day, respectively. However, foliar spraying of CK and BR contributed to a decrease in MDA and an increase in proline levels, mainly in the tolerant genotype (Federroz 67). CK spray can promote the overexpression of cytokinin oxidase/dehydrogenase, thereby increasing the content of protective compounds such as betaine and proline (Liu et al., 2020). BR promotes the induction of osmoprotectants such as betaine, sugars, and amino acids (including free proline), maintaining cellular osmotic balance under many adverse environmental conditions (Kothari and Lachowiec, 2021).
        Crop stress index (CSI) and relative tolerance index (RTI) are used to determine whether the treatments being evaluated help mitigate various stresses (abiotic and biotic) and have a positive effect on plant physiology (Castro-Duque et al., 2020; Chavez-Arias et al., 2020). CSI values ​​can range from 0 to 1, representing non-stress and stress conditions, respectively (Lee et al., 2010). The CSI values ​​of heat-stressed (SC) plants ranged from 0.8 to 0.9 (Figure 2B), indicating that rice plants were negatively affected by combined stress. However, foliar spraying of BC (0.6) or CK (0.6) mainly led to a decrease in this indicator under abiotic stress conditions compared to SC rice plants. In F2000 plants, RTI showed a higher increase when using CA (97.69%) and BC (60.73%) compared to SA (33.52%), indicating that these plant growth regulators also contribute improving the response of rice to the tolerance of the composition. Overheat. These indices have been proposed to manage stress conditions in different species. A study conducted by Lee et al. (2010) showed that the CSI of two cotton varieties under moderate water stress was about 0.85, whereas the CSI values ​​of well-irrigated varieties ranged from 0.4 to 0.6, concluding that this index is an indicator of the water adaptation of the varieties. stressful conditions. Moreover, Chavez-Arias et al. (2020) assessed the effectiveness of synthetic elicitors as a comprehensive stress management strategy in C. elegans plants and found that plants sprayed with these compounds exhibited higher RTI (65%). Based on the above, CK and BR can be considered as agronomic strategies aimed at increasing the tolerance of rice to complex heat stress, as these plant growth regulators induce positive biochemical and physiological responses.
        In the last few years, rice research in Colombia has focused on evaluating genotypes tolerant to high daytime or nighttime temperatures using physiological or biochemical traits (Sánchez-Reinoso et al., 2014; Alvarado-Sanabria et al., 2021). However, in the last few years, the analysis of practical, economical and profitable technologies has become increasingly important to propose integrated crop management to improve the effects of complex periods of heat stress in the country (Calderón-Páez et al., 2021; Quintero-Calderon et al., 2021) . Thus, the physiological and biochemical responses of rice plants to complex heat stress (40°C day/30°C night) observed in this study suggest that foliar spraying with CK or BR may be a suitable crop management method to mitigate adverse effects. Effect of periods of moderate heat stress. These treatments improved the tolerance of both rice genotypes (low CSI and high RTI), demonstrating a general trend in plant physiological and biochemical responses under combined heat stress. The main response of rice plants was a decrease in the content of GC, total chlorophyll, chlorophylls α and β and carotenoids. In addition, plants suffer from PSII damage (decreased chlorophyll fluorescence parameters such as Fv/Fm ratio) and increased lipid peroxidation. On the other hand, when rice was treated with CK and BR, these negative effects were mitigated and the proline content increased (Fig. 4).
        Figure 4. Conceptual model of the effects of combined heat stress and foliar plant growth regulator spray on rice plants. Red and blue arrows indicate the negative or positive effects of the interaction between heat stress and foliar application of BR (brassinosteroid) and CK (cytokinin) on physiological and biochemical responses, respectively. gs: stomatal conductance; Total Chl: total chlorophyll content; Chl α: chlorophyll β content; Cx+c: carotenoid content;
        In summary, the physiological and biochemical responses in this study indicate that Fedearroz 2000 rice plants are more susceptible to a period of complex heat stress than Fedearroz 67 rice plants. All growth regulators assessed in this study (auxins, gibberellins, cytokinins, or brassinosteroids) demonstrated some degree of combined heat stress reduction. However, cytokinin and brassinosteroids induced better plant adaptation as both plant growth regulators increased chlorophyll content, alpha-chlorophyll fluorescence parameters, gs and RWC compared to rice plants without any application, and also decreased MDA content and canopy temperature. In summary, we conclude that the use of plant growth regulators (cytokinins and brassinosteroids) is a useful tool in managing stress conditions in rice crops caused by severe heat stress during periods of high temperatures.
       The original materials presented in the study are included with the article, and further inquiries can be directed to the corresponding author.