Rice seedlings showed a higher heat tolerance through the foliar application of biostimulants

The use of biostimulants is an agronomic tool to improve plant tolerance to abiotic stress in plants. This study explored the effect of foliar biostimulants sprays such as brassinosteroids (BR), amino acids (AA), nitrophenolates (NP) or a biostimulant based on botanical extracts (BE) on leaf gas exchange parameters [photosynthesis (PN), stomatal conductance (gs) and transpiration (E)], leaf photosynthetic pigments, lipid peroxidation of membranes and proline content of two commercial rice genotypes [‘Fedearroz 67’ and ‘Fedearroz 60’] under heat stress conditions. The established treatments were: i) plants without heat stress and foliar applications of biostimulants (C), ii) plants under heat stress and without foliar applications of biostimulants (HT), and iii) plants with heat stress and three foliar applications with BR (1 mL·L), AA (30 mL·L), NP (15 mL·L) or BE (15 mL·L). The results showed that the application of BR, AA, NP or BE increased the values of PN (~14.5 μmol CO2·m·s), gs (~0.46 mmol·m·s) and E (~43.9 H20 day·plant) compared to plants (both genotypes) not treated with biostimulants under heat stress (9.9 μmol CO2·m·s for PN, 0.31 mmol·m·s for gs, and 27.3 H20 day·plant for E). Foliar biostimulant sprays also caused a lower malondialdehyde and proline production in rice genotypes under heat stress. In conclusion, the biostimulants BR, AA, NP, or BE can be considered an agronomic strategy to help mitigate the adverse effects of heat stress in rice areas where periods of high temperatures are expected during the day in Colombia.


Introduction
Rice (Oryza sativa L.) is one of the most important cereal crops globally, especially in developing countries (Wang et al., 2015). It is considered a staple food because it is an essential source of calories that feeds almost half of the world's population (Hirabayashi et al., 2014;Bahuguna et al., 2017). In Colombia, rice crops occupied 561,073 ha with a production of 2,564,250 t and an average yield of 4.9 t ha -1 in 2019 (Fedearroz, 2020).
Global climate change has generated an increase in the earth's surface temperature in recent decades, and the global mean air temperature is forecast to increase by approximately 2 to 3 °C by 2050 (IPCC, 2014;Wang et al., 2019). Additionally, heat waves or extreme temperature events are expected to be more intense, frequent, and prolonged than what has been observed in recent years (Hatfield and Prueger, 2015). High daytime temperature events would impact crop productivity (Hatfield and Prueger, 2015;Fan et al., 2018). In Colombia, 65% of the rice-growing areas are expected to increase temperatures between 2 and 2.5 °C by 2050 (Ramirez-Villegas et al., 2012).
High daytime temperatures (heat stress) are among the main environmental factors that cause serious adverse effects on rice production (Nigam et al., 2015;Wang et al., 2019). The optimal average temperature for rice growth varies from 25 to 30 °C during the day and from 20 to 22 °C for nighttime highs (Chen et al., 2017a;Kilasi et al., 2018). When the temperature exceeds these ranges, it can cause physiological, biochemical and molecular changes in plants such as an increase in reactive oxygen species (ROS) and lipid peroxidation of membranes (Zafar et al., 2018), alteration in leaf gas exchange parameters (photosynthesis, stomatal conductance, and transpiration) and plant water relations (Sahu, 2013;Vivitha et al., 2018).
Photosynthesis (PN) is one of the most susceptible physiological processes to heat stress in rice plants, showing a decrease in its values with an increase in daytime temperatures above the optimal levels (28 and 22 °C for day and night, respectively) (Radhakrishna et al., 2018). Sonjaroon et al. (2018) observed a reduction of 57% in PN in rice plants subjected to a moderate period (7 days) of high temperature during the day (40 °C). Biochemical markers such as the concentration of leaf photosynthetic pigments (chlorophyll and carotenoids), lipid peroxidation of membranes (malondialdehyde (MDA) production) and accumulation of compatible solutes (proline) have been used to characterize the response of rice plants to stress conditions caused by high daytime temperatures (Sánchez-Reinoso et al., 2014;Thussagunpanit et al., 2015a). According to , an increase in MDA content and a decrease in chlorophyll concentration indicate a negative impact on the integrity of the cell membrane and the photosynthetic efficiency of plants under heat stress conditions. On the other hand, the accumulation of low-molecular-weight compatible solutes such as proline is considered a primary strategy for protecting and acclimatizing plants to heat stress conditions (Harsh et al., 2016;Shin et al., 2016).
The development of new management practices to induce tolerance to abiotic stress in plants, including heat stress, is acquiring great importance (Baninasab and Ghobadi, 2011;Nguyen et al., 2018). In this regard, the application of biostimulants under unfavorable environmental conditions can induce tolerance to abiotic stress in different crops (Povero et al., 2016;Bulgari et al., 2019). According to du Jardin (2015), a plant biostimulant is "any substance or microorganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrients content". Biostimulants can be classified as of natural origins such as free amino acids, algae, and botanical extracts, effective microorganisms, humic substances, and chitosan; or synthetic, in which plant growth regulators, phenolic compounds, inorganic salts, and essential elements can be found (Przybysz et al., 2014;du Jardin, 2015). Biostimulants' positive effects on plants include growth promotion, modulation of development and quality traits, nitrate assimilation, hormonal activity, and higher tolerance to environmental stress (du Jardin, 2015;Francesca et al., 2020). Some of the biostimulants studied against different types of abiotic stress have been brassinosteroids (Thussagunpanit 3 et al., 2015b), amino acids (Li et al., 2016), nitrophenolates (Przybysz et al., 2014) and botanical extracts .
Brassinosteroids (BR) are polyhydroxylated steroidal plant hormones; they are involved in plant growth regulation through their participation in metabolic processes such as photosynthesis, antioxidant activity, osmolyte accumulation, nitrogen metabolism and plant water relations under stress conditions (Ahanger et al., 2018;Anwar et al., 2018). Exogenous applications of BR have been reported to help mitigate the adverse effects of high daytime temperatures in rice (Thussagunpanit et al., 2015a and b), maize (Yadava et al., 2016) and tomato (Zhou et al., 2014) by modulating the components of the antioxidant defense system and inducing heat shock proteins (HSP).
Nitrophenolates (NP) are phenolic compounds that participate in various plant metabolic processes. For example, they increase the concentration of endogenous auxins, promote growth and development, stimulate the absorption of nutrients and improve the photosynthetic and antioxidant activity (Djanaguiraman et al., 2010;Valero et al., 2014). NP can generate a faster and more effective activation of plant defense mechanisms (as they increase the antioxidant system enzymes' capacity and activity) under stress conditions (Przybysz et al., 2014;Kazda et al., 2015). Gulluoglu et al. (2006) reported that the foliar application of NP decreased the negative effects of heat stress in soybean (Glycine max L.) plants by favouring growth (dry mass accumulation) and yield parameters (yield, number of pods and grain weight).
Commercial products of amino acids (AA) can be constituted by active ingredients such as arginine, proline, tryptophan, glycine, or cysteine. AA can increase tolerance to different types of abiotic stress by activating physiological processes such as scavenging of ROS, osmoprotection, increased nutrient availability, and chelation of metals in the soil (Lucini et al., 2015;Van Oosten et al., 2017). The foliar AA sprays generated a better photosynthetic efficiency (Fv/Fm) and higher levels of chlorophyll and carotenoids in Lolium perenne L. plants under heat stress (Botta, 2013). Finally, botanical extracts (BE) have also been considered as a useful alternative for the mitigation of abiotic stress in recent years. They can help to improve various physiological processes that stimulate plant growth and development under abiotic stress conditions (Bulgari et al., 2015;Van Oosten et al., 2017). Zhang et al. (2010) also observed the positive effect of foliar applications of seaweed (Ascophyllum nodosum Jol.) BE on the chlorophyll content and activity of the enzyme nitrate reductase (NR) of Agrostis stolonifera L. plants under high-temperature conditions. Increases in the intensity and frequencies of high daytime temperature events in Colombia are estimated for the next few years (Ramirez-Villegas et al., 2012). Agronomic management strategies to mitigate the negative effect of heat stress conditions have focused mainly on the evaluation and development of rice genotypes under stress scenarios. These genotypes have been studied to identify physiological response mechanisms that allow strengthening plant breeding programs in Colombia (Restrepo-Díaz and Garces-Varon, 2013;Sánchez-Reinoso et al., 2014). Additionally, crop sowing times have been identified to avoid unfavorable climatic conditions in susceptible phenological stages of the crop (Garcés-Varon and Restrepo-Díaz, 2015;Garces-Varón and Puente, 2019). On the other hand, it has been widely reported that foliar applications of biostimulants decrease the negative impact of different types of abiotic stress in cereals (Przybysz et al., 2014;Thussagunpanit et al., 2015b;Li et al., 2016;Farooq et al., 2017). However, scientific studies on agronomic strategies to mitigate the adverse effect of specific periods of heat stress such as foliar applications of biostimulants (BR, AA, NP, or BE) from a physiological and biochemical point of view remain scarce in the country. This study hypothesizes that foliar applications of four biostimulants can help mitigate the negative effects caused by high daytime temperatures in two rice genotypes (susceptible ('Fedearroz 60') and moderately tolerant ('Fedearroz 67')). For this reason, the objective of this research was to evaluate the effect of foliar applications of four biostimulants (brassinosteroids (BR), amino acids (AA), nitrophenolates (NP) or botanical extracts (BE)) on leaf gas exchange parameters (photosynthesis, stomatal conductance, and transpiration), photosynthetic pigment content, and biochemical responses (MDA production and proline content) of two commercial rice genotypes subjected to a prolonged period of heat stress.

Materials and Methods
General growth conditions and biological material An experiment was carried out from December 2015 to March 2016 under glass greenhouse and laboratory conditions at the Faculty of Agricultural Sciences of the Universidad Nacional de Colombia, Bogotá campus (4°35'56" N, 74°04'51" W). Initially, seeds of cultivars 'Fedearroz 60' ('F60') (genotype susceptible to high daytime temperatures (Sánchez-Reinoso et al., 2014)) and 'Fedearroz 67' ('F67') (genotype widely cultivated by farmers in Colombia for the last five years) were sown in 1 L plastic pots containing loam soil in the glass greenhouse. The environmental conditions of the greenhouse during the experiment were: 28/20 °C day/night temperature, 60-80% relative humidity and a natural photoperiod of 12 h (1500 μmol·m -2 ·s -1 of photosynthetically active radiation (PAR) at noon). After seed germination (7 days after sowing (DAS)), the seedlings of both cultivars were watered daily with 50 mL of a nutrient solution prepared from a complete liquid fertilizer (Nutriponic ® , Walco SA, Colombia) at a dose of 2.5 mL·L -1 H2O until the end of the experiment (80 DAS). The concentration of the nutrient solution was as follows: 2.08 mM Ca (NO3)2·4H2O, 1.99 mM MgSO4·7 H2O, 2.00 mM NH4H2PO4, 10.09 mM KNO3, 46.26 nM H3BO3, 0.45 nM Na2MoO4·2H2O, 0.32 nM CuSO4·5H2O,9.19 nM MnCl2·4H2O,0.76 nM ZnSO4·7H2O,and 19.75 nM FeSO4·H2O. In general, the plants were always kept at field capacity to avoid water deficit conditions during the experiment.

Heat stress treatments and foliar applications of biostimulants
At 57 DAS, when rice plants showed collar formation on the fifth leaf of the main stem (stage V5) (Counce and Keisling, 2000), they were transferred to growth chambers (MLR-351H, Sanyo, Bensenville, Illinois, USA) to determine the different treatments of daytime temperatures. Rice plants were initially divided into two groups to establish heat stress treatments caused by high daytime temperatures. The first group of plants was established with the initial environmental conditions (28/20 °C day/night) (control without heat stress (C)). The second group was exposed to a temperature of 40 °C between 11:00 and 16:00 for 15 days. The exposure period and the high daytime temperature were selected based on preliminary studies (Restrepo-Diaz and Garces-Varon, 2013;Sánchez-Reinoso et al., 2014). Finally, a 12 h photoperiod and relative humidity of 60-70% were established in the chambers during the period of heat stress.
The group of plants subjected to conditions of heat stress (40 °C) was also subdivided into other five groups to establish foliar treatments with biostimulants, which were as follows: brassinosteroids (BR) at a dose of 1 mL·L -1 , amino acids (AA) at a dose of 30 mL·L -1 (Globafol, Valagro SpA, Atessa, Italy), nitrophenolates (NP) at a dose of 15 mL·L -1 (Atonik ® , Arysta Lifescience SA, Bogotá, Colombia), a commercial product based on botanical extracts, gibberellins, auxins and cytokinins (BE) at a dose of 15 mL·L -1 (Biozyme ® TF, Arysta Lifescience SA, Bogotá, Colombia) and plants without application of biostimulants (HT). Rice plants of treatments C and HT were only sprayed with distilled water. At the end of the experiment (80 DAS), a total of eight treatment groups were obtained depending on the heat stress condition and foliar application of biostimulants, which are summarized as follows: i) plants without stress due to high daytime temperatures (40 °C) and without foliar applications of biostimulants (control without heat stress and foliar application (C)); ii) plants subjected to heat stress and without foliar applications of biostimulants (heat stress without foliar application (HT)); iii) plants with heat stress and with three foliar applications of BR, AA, NP or BE. The concentrations of all biostimulants (BR, AA, NP, or BE) were selected from the doses of the commercial products recommended in rice-producing areas of Colombia. Finally, Table 1 summarizes the characteristics of the biostimulants used in this study.
Biostimulants' applications were carried out at three different times during the experiment: i) foliar application 54 DAS, ii) foliar application 65 DAS, and iii) foliar application 75 DAS. In general, each biostimulant application was carried out between 07:00 and 09:00, using a Style 1.5 compression sprayer (Matabi, Spain) with an application volume of 15 mL H2O per plant, wetting the upper and lower surfaces of leaves. All foliar applications carried a surfactant adjuvant (INEX-A, Cosmoagro, Colombia) at a dose of 0.1% 5 (v/v). The treatments were arranged in a completely randomized design with five replicates, and each replicate consisted of two plants. Finally, the experiment lasted 80 days. Regulates cell division in meristems, as well as cell elongation Leaf gas exchange and water use efficiency One mature leaf per plant was selected to carry out gas exchange readings. The leaves were previously adapted to the surrounding environment, and the leaf surface was not touched to avoid stomatal closure before measurements. Photosynthesis (PN), stomatal conductance (gs), and intercellular CO2 concentrations inside the leaf (Ci) were estimated using a portable photosynthesis meter (LI-COR 6200, Lincoln, NE, USA) between 10:00 and 15:00 h. During the readings, the conditions of the LiCor chamber were as follows: photosynthetically active radiation of 700 μmol•m -2 •s -1 , leaf temperature 27 ± 5 °C, and leaf-air vapor pressure difference 1.8 ± 0.5 kPa. With the values of PN, gs, Ci and ambient CO2 concentration (Ca) we calculated, respectively, the stomatal limitation (1-Ci/Ca = LS) (Yin et al., 2006), the intrinsic water use efficiency given by the ratio (PN/gs=WUEi) and the carboxylation efficiency (PN/Ci) (Flexas et al., 2001). Two measurements were carried out per plant. Finally, the total plant transpiration (E) was obtained using the gravimetric technique described by Sánchez-Reinoso et al. (2014), which consisted of covering pots with plastic to avoid water loss due to evaporation and weighing pots daily before and after irrigation between 57 and 80 DAS. The transpiration rate was expressed in mg H2O• g -1 FW•h -1 .

Leaf photosynthetic pigments
The equations described by Wellburn (1994) were used to estimate chlorophyll total (TChl) and carotenoid (Cx+c) content from 50 mg of the tissue sample from the same leaves used to measure the gas exchange variables. The tissue samples were homogenized in 3 mL of 80% acetone and then centrifuged (Model 420101, Becton Dickinson Primary Care Diagnostics, MD, USA) at 5000 rpm for 10 minutes to remove particles. The supernatant was diluted to a final volume of 6 ml by adding 80% acetone (Sims and Gamon, 2002). Chlorophyll content was determined at 663, and 646 nm and carotenoids were determined at 470 nm using a spectrophotometer (Spectronic BioMate 3 UV-vis Thermo, Madison, WI). Leaf photosynthetic pigments were determined at 80 DAS.

Proline and malondialdehyde content
The same leaves collected for the determination of leaf photosynthetic pigments were used for the proline and MDA content in all treatments. The method described by Bates et al. (1973) was used to estimate 6 the proline content in each of the evaluated treatments. Leaves were collected from the upper third of the plant; samples of 300 mg of this plant material were homogenized in liquid nitrogen and stored for further analysis. Subsequently, 10 mL of a 3% aqueous solution of sulfosalicylic acid was added to the samples and Whatman paper (No. 2) was used to filter them. Then, 2 mL of the filtrate was reacted with 2 mL of ninhydrin acid and 2 mL of glacial acetic acid. The mixture was placed in a water bath at 90 °C for 1 h and incubation on ice was used to stop the reaction. The resulting solution was dissolved in 4 mL of toluene and vortexed (V-1, BOECO, Germany) for 30 s. Finally, the absorbance readings were determined at 520 nm with the same spectrophotometer used in the quantification of leaf photosynthetic pigments (Spectronic BioMate 3 UV-Vis, Thermo, Madison, WI, USA). The proline content was calculated with the fresh weight of the sample using a standard calibration curve (equation 1).
μmol Proline fresh vegetal material = μg Proline mL × mL Toluene 115.5 μg μmol g sample 5 (1) The thiobarbituric acid (TBA) method described by Hodges et al. (1999) was used to estimate membrane lipid peroxidation (malondialdehyde (MDA)). Samples of 300 mg of leaves from the upper third of the plants from the different treatments were macerated and stored in liquid nitrogen. The samples were centrifuged (Model 420101, Becton Dickinson Primary Care Diagnostics, MD, USA) at 5000 rpm for 10 min, and absorbances were estimated at 440, 532 and 600 nm with a spectrophotometer. Finally, the extinction coefficient (157 M mL -1 ) was used to obtain the MDA concentration. Proline and MDA readings were performed at 80 DAS.
Heat tolerance coefficient (HTC) The heat tolerance coefficient (HTC) was calculated to estimate rice plants' photosynthetic responses to the impact of high daytime temperatures and foliar applications of biostimulants. HTC was calculated by

Statistical analysis
Data were analysed using a factorial arrangement where the first factor corresponded to the rice genotypes ('F60' and 'F67') and the second factor was the biostimulants used in foliar applications (BR, AA, NP or BE). Each treatment group consisted of 10 plants. A principal component analysis was carried out using the InfoStat 2016 program (analytical software, Universidad Nacional de Cordoba, Argentina) to select the best biostimulants used under heat stress conditions. In all cases, an analysis of variance (ANOVA) was carried out and when significant differences (P ≤ 0.05) were observed, a post hoc Tukey test was used for the comparison of means. Percentage values were transformed using the arcsine function. Data were analysed using the Statistix v 9.0 program (Analytical Software, Tallahassee, FL, USA). Figures were performed using Sigmaplot software (version 12.0; Systat Software, San Jose, CA, USA).

8
The variable gs was favoured by foliar sprays of BR (0.54 mmol·m -2 ·s -1 for 'F67' and 0.64 mmol·m -2 ·s -1 for 'F60') or AA (mean values 0.45 mmol·m -2 ·s -1 ) in both genotypes subjected to heat stress conditions. On the other hand, foliar applications of NPs (0.43 mmol·m -2 ·s -1 ) only favoured gs in plants of genotype 'F67' under conditions of high diurnal temperatures compared to HT plants of both genotypes (mean values 0.31 mmol·m -2 ·s -1 ) ( Figure 1B). An increase in the E values of 'F60' plants subjected to heat stress was mainly observed in the treatment with BR sprays (48.6 H20 day -1 ·plant -1 ) compared to HT plants of the same genotype (30.1 H20 day -1 ·plant -1 ). The transpiration of 'F67' plants under heat stress was also stimulated with the foliar application of BE, NP, or BR showing mean values of 47.1 H20 day -1 ·plant -1 compared to HT plants without biostimulant sprays (24.4 H20 day -1 ·plant -1 ) ( Figure 1C). On the other hand, the foliar application mainly of BR (mean values 0.166) or AA (mean values 0.201) favoured LS in plants of both rice genotypes under heat stress compared to HT plants (mean values 0.305) ( Figure 1D). Figure 2 shows that PN/Ci and WUEi showed opposite trends between them. PN/Ci was favoured by foliar applications of BR, AA, NP or BE in both genotypes ('F67' and 'F60'), observing that foliar treatments with these biostimulants helped improve the carboxylation of plants under heat stress (Figure 2A). However, foliar applications mainly of BR caused a decrease in WUEi (28.9 µmol·mmol -1 for 'F60' and 31.7 µmol·mmol -1 for 'F67') compared to control plants under heat stress (HT) with values of 35.1 µmol·mmol -1 for 'F67' and 30.1 µmol·mmol -1 for 'F60' ( Figure 2B). Finally, the plants without heat stress and foliar application of biostimulants (C) always showed higher PN, gs, E and PN/Ci, and lower LS and WUEi in both rice genotypes ('F67' and 'F60') (Figures 1 and 2). Leaf photosynthetic pigments Table 2 shows that differences were only obtained for the factors genotype and biostimulant in the treatments evaluated on the content of total chlorophyll (TChl) (P=0.000 and 0=000, respectively) and carotenoids (Cx+c) (P=0.000 and 0=000, respectively) at the end of the experiment (80 DAS). The content of leaf photosynthetic pigments (TChl and Cx+c) was affected when rice plants were stressed by heat and were not treated with any biostimulant (1.07 mg·mg -1 ·FW for TChl and 0.52 mg·mg -1 ·FW for Cx+c) compared to plants with foliar sprays of BR, AA, BE or NP, with mean values of 1.41 mg·mg -1 ·FW and 0.58 mg·mg -1 ·FW for TChl and Cx+c, respectively. Plants without heat stress and biostimulant sprays (C) showed the highest values of TChl (1.78 mg·mg -1 ·FW) and Cx+c (0.72 mg·mg -1 ·FW) (Figures 3A and B). Leaf proline content and malondialdehyde (MDA) production Figure 4 shows the proline content and malondialdehyde (MDA) production. Differences between the rice genotypes ('F67' and 'F60') and biostimulant sprays were observed in the proline content (P=0.0008) and MDA production (P=0.0078) of rice plants under heat stress conditions at 80 DAS ( Table 2). The highest values of these biochemical variables were recorded in plants of both genotypes under heat stress and without foliar application of biostimulants (HT), with mean values of 9.9 µmol·g -1 ·FW for proline and 12.4 µmol·g -1 ·FW for MDA. In contrast, the exogenous use of biostimulants (BR, AA, BE or NP) caused a reduction in the proline content of around 22%, 34%, 33%, and 42%, respectively, in 'F67' and 'F60' plants subjected to heat stress ( Figure 4A). On the other hand, a decrease in lipid peroxidation (MDA) was also observed with the foliar application of BR, AA, BE or NP, with mean values of 8.4 µmol·g -1 ·FW and 10.5 µmol·g -1 ·FW for 'F67' and 'F60' plants, respectively, under high daytime temperatures ( Figure 4B). Finally, the accumulation of proline (mean values 4.3 µmol·g -1 ·FW) and MDA (mean values 5.9 µmol·g -1 ·FW) was always lower in plants without heat stress (C) in both genotypes evaluated (Figures 4A and B).  Figure 5, the vectors of the physiological (PN, gs, E, LS, PN/Ci, WUEi) and biochemical (TChl, MDA and proline) variables show angles close to the origin, which indicates that there is a high correlation between the plants' behaviour and these variables. Likewise, it was observed that plants of both genotypes under heat stress and without the application of biostimulants (HT) form a single group (I). It can be inferred that this group showed the highest effects due to the imposed heat stress condition. In contrast, the group of plants without the heat stress condition and biostimulant applications (C) (Group IV), was located in the sector opposite to group I. On the other hand, the foliar application of biostimulants showed two differential effects in the analysis: i) plants of both genotypes ('F67' and 'F60') with heat stress and foliar treatment with AA, BE or NP (Group II)

Heat tolerance coefficient (HTC)
The heat tolerance coefficient (HTC) based on PN also corroborated the observations of the biplot analysis. Foliar applications of the biostimulants BR, AA, BE, or NP help tolerate the stress caused by high temperatures in rice genotypes 'F67' and 'F60'. The highest HTC values were observed in stressed plants of both genotypes and treated with foliar applications of BR (0.91 for 'F67' and 0.76 for 'F60'), followed by AA (0.84 for 'F67' and 0.74 for 'F60'), NP (0.80 for 'F67' and 0.67 for 'F60') and BE (0.80 for 'F67' and 0.64 for 'F60'). On the other hand, rice plants without application of biostimulants and subjected to high daytime temperatures showed the lowest values for 'F67' (0.59) and 'F60' (0.43) ( Table 3).

Correlation between physiological parameters and heat tolerance coefficient (HTC)
Highly significant correlations were observed in the leaf gas exchange properties (plant transpiration (E) and stomatal conductance (gs)) and total chlorophyll content (TChl) with the heat tolerance coefficient (HTC) in rice plants under heat stress conditions ( Figure 6). A high positive correlation (r 2 = 0.92 and 0.96, respectively) between E and TChl and the HTC was registered in the rice genotype 'F60', with the highest values of E and TChl in relation to the HTC in the treatments with foliar applications of BR or AA ( Figures   6A and C). Regarding plants of genotype 'F67', the correlations also showed that the treatments with foliar applications of BR showed the highest values of gs (r 2 = 0.85) and TChl (r 2 = 0.97) in relation to the HTC (Figures 6B and D).

Discussion
This study showed that plants of both rice genotypes ('F67' and 'F60') without the application of biostimulants and under conditions of heat stress (HT) showed physiological effects such as a reduction in the leaf gas exchange properties (PN, gs, and E), low leaf chlorophyll content, and increased MDA and proline contents (Figures 1-4). Abiotic stress due to high daytime temperatures causes adverse effects on physiological and biochemical parameters that affect the growth and development of cultivable plants (Fahad et al., 2017;Wassie et al., 2019). Exposure of plants to high-temperature stress decreases the exchange properties and the concentration of leaf photosynthetic pigments (total chlorophyll (TChl) and carotenoids (Cx+c)) (Chen et al., 2017b;Ahammed et al., 2018). Heat stress also affects the integrity of the membrane (higher lipid peroxidation (MDA)) and causes an increase in the accumulation of compatible solutes such as proline (Harsh et al., 2016;Ahammed et al., 2018). These physiological and biochemical variables have been used for phenotyping or evaluating management strategies to confer tolerance to rice genotypes under periods of heat stress (Restrepo-Diaz and Garces-Varon, 2013;Sánchez-Reinoso et al., 2014;Sharma et al., 2017).
Agronomic management practices such as the foliar application of biostimulants have been used to improve plant tolerance responses, since they can regulate and/or modify physiological and biochemical processes in plants to stimulate growth, mitigate the limitations induced by abiotic stress and increase yield (Bulgari et al., 2019;Drobek et al., 2019). Our results showed that foliar applications of biostimulants (BR, AA, NP, or BE) helped to mitigate the negative effects caused by heat stress conditions by favouring the physiological and biochemical responses in seedlings of both rice genotypes (Table 3; Figures 1-6). The beneficial role of foliar sprays of these compounds on the physiological and biochemical parameters of plants under periods of high diurnal temperatures has been widely documented for BR in rice (Thussagunpanit et al., 2015a;Sonjaroon et al., 2018), AA in maize (Matysiak et al., 2020) and mung bean (Priya et al., 2019), NP in soybean (Gulluoglu et al., 2006), and BE in tomato (Carmody et al., 2020).
In this study, it was observed that BR mitigated the negative effects of heat stress in rice genotypes 'F67' and 'F60', through an improvement in the gas exchange parameters and biochemical markers, and increased the heat tolerance coefficient (HTC). Sonjaroon et al. (2018) also observed that rice plants with foliar BR treatments and under heat stress showed an increase in physiological variables such as PN, gs, E and LS compared to plants without BR application. It has been reported that BR can induce and improve thermotolerance in both heat-tolerant and heat-susceptible genotypes (Ahammed et al., 2020). The positive response on the gas exchange parameters of plants under heat stress conditions may be associated with the fact that BR participate in the accumulation of carbon metabolites such as soluble sugars, starch or sucrose, and the induction of chlorophyll biosynthesis (Sadura and Janeczko, 2018;Tanveer, 2019). On the other hand, BR applications decreased lipid peroxidation (MDA) values and proline content, since this plant hormone regulates the activity of antioxidant enzymes (ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR)) and favours the expression of stress response genes that can encode phytochelatins, osmolytes, organic acids, metallothioneins, and heat shock proteins (HSP) (Rajewska et al., 2016).
Foliar applications of AA (arginine, proline, tryptophan, glycine, and cysteine) also favoured gas exchange parameters (PN, gs, E, LS, PN/Ci, and WUEi) in plants of genotypes 'F67' and 'F60' under heat stress conditions. The positive effect of foliar AA applications in creeping bentgrass (Agrostis stolonifera) plants has been reported, improving PN, gs, E, and relative water content under heat stress conditions (Li et al., 2016). AA help to mitigate the effects of stress caused by high daytime temperatures because they participate in the metabolism of nitrogen and the balance of the C/N ratio (Price et al., 2012). Additionally, these compounds favor the synthesis of glutamate receptors (GLRs), which help mediate processes such as accumulation of soluble sugars, stomatal movements (stomatal opening), and stimulation of photosynthesis (Teixeira et al., 2018). On the other hand, the exogenous application of AA (arginine, proline, tryptophan, glycine, and cysteine) in plants under conditions of heat stress also increased the content of leaf photosynthetic pigments 14 (TChl and Cx+c) and decreased the concentration of MDA and proline. Vijayakumari1 and Puthur (2016) also found that the application of AA γ-aminobutyric acid caused a significant decrease in the proline content in pepper (Piper nigrum L.) plants under conditions of heat stress. Regarding oxidative damage, the mitigating effect of foliar applications of AA in rice plants could be because these compounds participate in the biosynthesis of chlorophyll , regulation of the activity of antioxidant enzymes such as SOD, CAT, APX and GR and the accumulation of non-enzymatic antioxidants (ascorbic acid (AsA) and glutathione (GSH)) (Li et al., 2016).
The exogenous use of NP helped the evaluated physiological and biochemical parameters of rice genotypes exposed to a high daytime temperature condition. Przybysz et al. (2014) observed that foliar applications of NP also caused an increase in the photosynthetic efficiency, E and water uptake, and a decrease in the stomatal resistance of Arabidopsis thaliana L. plants under drought conditions. Likewise, Djanaguiraman et al. (2009) reported that cotton (Gossypium hirsutum L.) plants treated with a commercial product based on NP (Atonik) also had lower lipid peroxidation of membranes (MDA) and a higher TChl during the leaf senescence process (oxidative stress). Additionally, Swaefy and El-Ziat (2017) concluded that foliar applications of NP decreased proline values in calendula (Calendula officinalis) plants under salinity conditions. It has been reported that NP can increase the photosynthetic plant capacity by reducing stomatal resistance, guaranteeing a more effortless and higher flow of CO2 to the chloroplast, increasing the maximum photochemical efficiency of PSII, and promoting chlorophyll biosynthesis or slowing down its degradation (Przybysz et al., 2014;Kazda et al., 2015). These compounds are also involved in the capture of free radicals by phenolic compounds (inhibiting lipid and protein oxidation) and ROS scavenging through increased peroxidase activity (Djanaguiraman et al., 2010;Szparaga et al., 2018).
BE applications on plants of both rice genotypes ('F67' and 'F60') under conditions of stress caused by high daytime temperatures decreased the adverse effects on gas exchange parameters. Rashid et al. (2018) recorded that quinoa plants treated with Moringa oleífera BE increased gas exchange parameters (PN and gs) and water use efficiency under heat stress conditions. This positive response may be associated with the fact that BE are rich in glycine betaine, which delays the loss of photosynthetic activity by inhibiting chlorophyll degradation. Additionally, they participate in chlorophyll biosynthesis and regulate S and N metabolism (Jannin et al., 2013;Yao et al., 2020). BE also caused lower lipid peroxidation and proline synthesis, and a higher concentration of leaf photosynthetic pigments in heat-stressed plants. Zhang and Ervin (2008) and Zhang et al. (2010) observed that applications based on seaweed BE promoted higher TChl values and a lower concentration of MDA in creeping bentgrass (Agrostis stolonifera L.) plants subjected to moderate periods of heat stress (>14 days). BE have been reported to help plants under abiotic stress because they improve enzymatic activity (SOD, APX, and CAT) and non-enzymatic antioxidant metabolism, such as phenolic compounds. They also favor the primary metabolism of plants by increasing the levels of free amino acids, proteins, carbohydrates, and leaf photosynthetic pigments (Mansori et al., 2016;Santaniello et al., 2017;Yakhin et al., 2017). This was observed in the present study with a higher content of TChl and Cx+c and lower lipid peroxidation of membranes (MDA).
Finally, the positive effects of the foliar application of the different biostimulants used (BR, AA, NP or BE) on rice plants ('F67' and 'F60') under conditions of high temperatures during the day are the result of the combination of physiological (PN, gs, E, LS, PN/Ci, and WUEi) and biochemical (TChl, Cx+c, MDA and proline) attributes which are reflected in a higher HTC (Table 3). This indicates that these biostimulants are signaling molecules that can interact with cellular transcription factors to regulate different physiological and biochemical processes involved in tolerance to abiotic stress conditions in plants (Van Oosten et al., 2017;Bulgari et al., 2019;Kerchev et al., 2020).

Conclusions
In the current study, the results obtained in this study showed that biostimulants' foliar application (BR, AA, NP, or BE) helped to ameliorate the negative effects of heat stress in rice genotypes 'F67' and 'F60'. Foliar applications of biostimulants helped the leaf gas exchange parameters (net photosynthesis, stomatal conductance, total transpiration, carboxylation efficiency, and water use efficiency), and concentration of leaf photosynthetic pigments (chlorophylls and carotenoids). The use of these biostimulants also showed a significant reduction in the lipid peroxidation of membranes (MDA) and proline content, suggesting an antioxidant activity in rice plants subjected to heat stress. This research allows us to conclude that BR, AA, NP or BE are compounds that can generate a beneficial effect on rice plants under conditions of heat stress. Foliar applications of these biostimulants can be considered an agronomic strategy to mitigate the adverse effects caused by heat stress conditions in rice-producing areas where episodes of increases in mean daytime temperatures could be expected.