The different responses of rice genotypes to heat stress associated with morphological, chlorophyll and yield characteristics

Sixteen rice genotypes were planted under normal and high temperature during 2018 and 2019 seasons to understand the mechanisms that make plants tolerant or susceptible to heat stress and methods which may lead to generate new varieties with sustainable yield production. The combined analysis showed significant differences at the level of probability 0.05 and 0.01 among years for all characters except, chlorophyll content and leaf rolling which would indicate wide differences among the weather of both years. Environment’s variance was significant at the level of probability 0.01 over all characteristics. The significant differences at the level of probability 0.01 were recorded among genotypes and G x E interaction variances for all characteristics. The highest values of panicles number plant were obtained from ‘Giza 178’ and ‘Hybrid 2’, however, the highest values of 100-grain weight were recorded with ‘Giza 179’ and ‘Egyptian Yasmine’. The minimum values of sterility% were recorded with ‘Sakha 107’ and ‘IET1444’. Concerning the cultivars performance across two environments, the cultivars ‘Giza 178’, ‘Giza 179’, ‘Sakha 107’ and ‘IET 1444’ gave the best desirable values over natural and heat stress so, those cultivars are considered to play a vital role in breeding program to enhance heat stresses tolerance accompanied with high yield potential.


Introduction
The increasing temperatures linked with global warming may have serious direct and indirect consequences on crop yield, especially in cereals. There are many environmental stress factors affect the crops such as heat (EL Sabagh et al., 2019), salinity , drought , chilling (Elkelish et al., 2020). Heat stress has become important factor affecting crop growth and yield. Moreover, different global circulation models predict that greenhouse gases will gradually increase the average ambient

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Notulae Botanicae Horti Cluj-Napoca Agrobotanici 2 temperature. According to a report of the Intergovernmental Panel on Climate Change (IPCC, 2004), global means temperature will rise 0.3 o C per decade (Jones et al., 1999), reaching approximately 1 and 3 o C above the present value by years 2025 and 2100, respectively. In addition, the climate is expected to be more variable, with frequent episodes of stressful temperatures during the crop-growing season. As a result, crop production may be severely affected by an increase in mean global temperature. The yield decrease is a result of heat stress alert in the physiological, morphological, biochemical and molecular changes (Wang et al., 2003). Morphologically, heat stress decreases the duration of developmental phases leading to fewer and smaller organs, lower light perception due to a reduced life cycle (Saeed et al., 2017). Photosynthesis, respiration and the partitioning of assimilates to different organs within the plant are also negatively affected (Elkelish et al., 2020).
Rice is a staple food for more than half the world's population, so many studies were conducted to improve rice grain yield (Abou Khadrah et al., 2020). Abiotic stress such as extreme temperatures and salinity frequently limits the major crop species' growth and productivity (Elkelish et al., 2020;Hafez et al., 2020). Rice is a very sensitive crop to heat stress, the critical temperature for spikelet fertility in rice is a maximum of 35 •C and moreover, sustained high temperature for more than a week cause severe damage (Melo et al., 2021). As an important cereal crop, rice productivity might decrease globally by about 3.2% per each degree-Celsius increase in global mean temperature (Zhao et al., 2017). As the rice crop is concerned, the optimum temperature is 33 °C (Jagadish et al., 2007). Depending on the specific stage of rice development, heat stress reduces number of tillers/panicles, decreases grain number/plant and lower grain weight, thus negatively impacting yield formation, improving rice crop tolerance to heat stress in terms of sustaining yield stability under high day temperature, high night temperature, or combined high day and night temperature will bolster future food security (Xu et al., 2020). Therefore, high temperatures over 35 °C encourage spikelets sterility and unfilled grain (Ghazy, 2012). Moreover, high temperature affects rice plant growth, for instance, during seed germination, high temperature may slow down or totally inhibit germination, depending on plant species and the intensity of the stress (Yoshida, 1981). Also, high temperature over 35 °C may harmfully affect photosynthesis, water relations, membrane stability and primary and secondary metabolites (Yoshida, 1981;Naveed et al., 2020). Moreover, throughout plant ontogeny, enhanced the expression of heat shock proteins, other stress-related proteins, and reactive oxygen species accumulation (Wahid et al., 2007). Plant tolerance to heat can be divided into escape (successful reproduction before the onset of severe stress), avoidance (maintenance of a high plant water status during stress e.g. caused by stomatal closure, reduced leaf area, senescence of older leaves, etc. or by increased root growth) and tolerance (the maintenance of plant function at limited water availability and/or the recovery of plant water status and plant function after stress) may involve osmotic adjustments, but may also be the result of rigid cell walls or small cells (Kondamudi et al., 2012). The heat stress response depends on plant species and genotypes (Wahid et al., 2007), so very important to identify the tolerant genotypes based on their morphological and physiological characters under heat conditions. Heat stress dominantly reduced fertility when drought and heat stress co-occurred and the pollen count on the stigma reduced in Moroberekan, therefore, not all drought-tolerant cultivars are tolerant to heat stress by default at sensitive stages however, heat stress seems to be the dominant stress at reproductive stage (Costa et al., 2021).
The phenotypic correlation among some heat morphological characteristics and their contribution to grain yield under heat conditions were directly or indirectly expected in rice (Barnabás et al., 2008). The thousand-grain weight and spikelet fertility showed a positive and significant at probability level 0.05 association with yield plant -1 under heat conditions at the genotypic level. Paddy yield had a strong correlation with grain number panicle -1 , days to maturity and 1000-grain weight (Gaballah and Abu El-Ezz, 2019). The effect of heat stress at the seedling and tillering stages of rice led to no panicle development in the cultivars (Azameti et al., 2021). The objective of this study was to evaluate the impact of high temperatures stress on yield and related parameters of rice, to identify rice genotypes tolerance for heat stress, estimate the correlation coefficient based on agro-morphological characteristics and applicability of stress index under high heat stress conditions.

Experiment preparation
Sixteen rice genotypes (Table 1) were used in the study. The experiments were conducted under natural (mild temperatures) conditions at Sakha Agricultural Research Station and high heat stress conditions at Elkharga Agricultural Research station in 2018 and 2019 successive seasons. The average temperature and humidity percentage in 2018 and 2019 years were illustrated at Table 2 according to Sakha and El-Kharga Agro-Meteorological station. The nursery was sown on the 25 th and 26 th of April in the 2018 and 2019 seasons, respectively, and transplanted to the field after 30 days in both locations. Each genotypes seedling was transplanted to 5 rows along 5 m, 20 cm apart between rows, with a distance of 20 cm between the hills within a row, the plot size (1.20 m x 5 m). The design of the experiment was a randomized complete block design with three replicates.  The soil properties in the two locations are presented in Table 3. The soil samples of 0-15 cm depth were collected from selected plots to determine the chemical and physical properties. The soil samples were air-dried, crushed and passed through 2 mm sieve to analyse organic carbon and total N content. Soil organic carbon was analysed by Walkley-Black method (Allison, 1965). One g air-dried sample (passed through 0.5 mm sieve) was taken in a 500 ml Erlenmeyer flask. Ten ml 1N K2Cr2O7 and 20 ml conc. H2SO4 was added to the flask and react for 30 minutes after which 200 ml distilled water and 10 ml H3PO4 was added. One ml orthophenanthroline indicator was added to the flask and was titrated with about 0.5N FeSO4 solution. Blank titration was run to calculate the strength of the FeSO4 solution. Total nitrogen was determined by Microkjeldahl instrument, 0.5 g air-dried soil sample was digested in 3 ml conc. H2SO4 in the presence of 0.5 g of digestion mixture (50: 10: 1 K2SO4: CuSO4.5H2O: metallic selenium). The digested sample was distillate with 40% NaOH and the distillate was collected in 4% boric acid containing three drops of mixed indicator (bromocresol green and methyl red), which was then titrated with 0.05 N H2SO4. C: N ratio was calculated by dividing the results of organic carbon by total nitrogen (Bremner, 1965). All measurements of organic carbon and total nitrogen were done in triplicate. Soil pH in water was measured from a soil: water ratio 1: 2.5 using glass electrode method (Peech, 1965). Twenty gram of air-dried soil sample was taken in 100 ml of plastic bottle and 50 ml of distilled water was added. The suspension was stirred with a glass-rod at regular interval for 30 minutes. A glass electrode pH meter calibrated with buffer pH 7.0 and 4.0 and the pH of soil suspension was measured. The measurement was done in triplicate. Olsen-P was extracted with 0.5M sodium bicarbonate (pH 8.5) (Olsen et al., 1954). P content in the extract was determined using ascorbic acid as reducing agent by a spectrophotometer. Available potassium (NH4OAc-K) was extracted with neutral 1N ammonium acetate (Hanway and Heidel, 1952) and estimated by a flam photometer.

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Phosphorus and potassium were applied in full dose (150 Kg P2O5 and 100 Kg K2O, respectively) during land preparation in the permanent field and Nitrogen (150 Kg N2O) fertilizer was applied in three splits as a top dressing. Insects and weeds were controlled periodically using pesticides and herbicides, respectively, as required. The analyses of soil physical and chemical properties were done by laboratory of Rice Research and Training Center, Field Crop Research Institute.

Studied characteristics
The randomly selected plants (25 plants) were harvested were harvested manually at harvest, put into a Nylon mesh bag, sun-dried and threshed. Grain yield for each plot was recorded from a 25-hill sample, adjusted to a moisture content of 14% and expressed as g plant -1 . Days to 50% heading (DTH), were determined as the number of days from the date of sowing to the first panicle exertion date. SPAD-chlorophyll content (SPAD; CC) was determined using chlorophyll analytical apparatus (chlorophyll meter SPAD-502 Minolta camera Co. Ltd., Japan). Five flag leaves were measured from the widest part of the leaf of the main culms for each entry in all replications. Leaf rolling (LR) scores and symptoms for leaf rolling and heat resistance at the vegetative stage were done according to De Datta et al. (1988). Plant height (cm; PH) was measured (cm) from the soil surface to the tip of each plant's main panicle. Panicle length (cm; PL) for the main panicle was measured from panicle base up to a piculus of the panicle's uppermost spikelet. The number of panicles plant -1 (NP) was determined by counting the number of panicles plant -1 at harvesting. 100-grain weight (g; HGW) was recorded as the weight of 100 random chosen filled grains plant -1 . Sterility percentage was calculated by account number of unfilled grain panicle -1 divided on number of spikelets panicle -1 . All results were recorded according to the standard evaluation system IRRI (IRRI, 2002).

Statistical analysis
Analysis of variance was conducted for all the characteristics using combined analysis according to Steel et al. (1997). Comparison among genotypes, genotypes and environments, years and environments were determined by Tukey's HSD test (P <0.05). The correlation coefficient was carried out using the formula given by Kown and Torrie (1964). Correlation among values of the different characteristics was performed based on Pearson's Product-Moment. The genetic analysis, phenotypic correlation and genetic variability were performed on combined data across seasons.

Results
The analysis of variance for studied characteristics was presented in Table 4. The environments, genotypes and genotypes × environments variances were found to be statistically significant differences at the level of probability 0.01 for all characteristics. The days to heading, chlorophyll content, leaf rolling, plant height, panicle length, number of tillers and number of panicles plant -1 , 100-grain weight and sterility percentage as well as grain yield were exhibited not significant difference over the years.

The interaction between genotypes and Environments
Regarding the interaction in Table 5, the results indicated that, the shortest days to heading genotypes were observed in 'Sakha 102' under heat conditions while the longest days to heading genotypes were recorded with 'Egyptian Yasmine' and 'Sakha 101' under natural conditions. The highest values of chlorophyll content were found in 'Sakha 103' under natural conditions and the minimum values were found in 'Giza177' under heat conditions. Concerning leaf rolling, the minimum mean values were achieved from 'Giza 178', 'Giza 179' and 'Sakha 107' under heat stress conditions, nevertheless, the highest values were obtained with 'Giza 177', 'Giza 182', 'Sakha 101', 'Sakha 102' and 'Sakha 103' under heat stress conditions. The highest panicles length was recorded with 'Egyptian Yasmine' under natural conditions and the minimum values were achieved with 'Sakha 105' under heat stress conditions.
The interaction between rice genotypes and environments for grain yield and its related characters were shown in Table 6. The maximum values of panicles number plant -1 were achieved with 'Hybrid 2' under natural conditions; however, the minimum values were obtained with 'Sakha 103' under heat conditions. The heaviest 100-grain weight was recorded with 'Egyptian Yasmine' and 'Sakha 104' under natural conditions and the minimum grains weight was obtained with 'Giza 178', 'Sakha 103', 'Sakha 104' and 'Sakha 106' under heat stress. The minimum values of sterility percentage were recorded with 'Sakha 101', 'Sakha 103' and 'Sakha107' under natural conditions, therefore the highest percentage was obtained from 'Sakha 106' under heat stress. Additionally, the highest mean values of grain yield plant -1 were obtained from 'Hybrid 2' under natural conditions, however, the minimum values were obtained with 'Sakha 102' and 'Giza 177' under heat conditions. 7 Regarding days to heading as affected by heat stress (Figure 1), the 'E. Yasmine' was the most affected by high temperature conditions (33.28%) followed by 'Giza 178' (25.62%). Nonetheless, the minimum genotype which affected by high temperature was 'IEET1444' (6.88%). 'Giza 177', 'Sakha 103' and 'Sakha 105' had the most decrease percentage by heat stress in chlorophyll content and most standing cultivars were 'IET1444' and 'E. Yasmine'.

The interaction among genotypes, years and environments
The interaction among genotypes for days to heading and grain yield plant -1 characteristics was presented in Table 7. For days to heading the genotypes 'Sakha 102' gave the shortest days to heading under heat conditions in 2019 growing season. Otherwise, the latest genotype was 'Egyptian Yasmine' at 2018 season under natural conditions. Regarding grain yield plant -1 , the highest values were recorded with 'Hybrid 2' under natural conditions at 2018 growing season, while the minimum values were recorded with 'Sakha 102' under stress at 2019 growing season.

Correlation coefficient
The correlation coefficient had different associated between pairs traits under natural and heat stress conditions. The days to heading had positive and significant at probability 0.01 correlation with leaf rolling, panicle length, tillers number plant -1 and panicles number plant -1 . Nonetheless, the days to heading under stress conditions had positive and significant (≥ 0.05) correlation with chlorophyll content while negative correlation was found with tillers number plant -1 (Table 8).  0.07 0.25 -0.66 ** -0.14 0.35 * 0.36 * 0.43 ** -0.11 -0.50 ** *,** is statistically significant differences at the level of probability 0.05 and 0.01, respectively Chlorophyll content was correlated negative and significant ≥ 0.01 with sterility percentage but positive with plant height and grain yield plant under natural conditions whereas, it was correlated had a positive and significant correlation (≥ 0.05) with panicle length, number of panicles plant -1 and negative with sterility percentage under heat conditions. Under natural conditions the leaf rolling had positive correlation and significant (≥ 0.01) with panicle length, tillers number, panicles number plant -1 and grain yield plant -1 while under heat conditions had negative correlated with panicle length, panicles number plant -1 and grain yield plant -1 as well as positive correlation with sterility percentage. Plant height was correlated positive and significant (≥ 0.01) with 100-grain weight and negative with sterility percentage under natural conditions. Panicle length showed positive and highly significant correlation with number of tillers plant -1 and number of panicles plant -1 under natural conditions thus positive correlation and significant (≥ 0.05) with grain yield plant -1 under stress. The highly correlation were found between the number of tillers plant -1 and number of panicles plant -1 , 100-grain weight and grain yield plant -1 under natural conditions. However, under stress conditions, the tillers number plant -1 has correlated positively and significantly (≥ 0.05) with grain yield. The 100-grain weight was negative correlated and significant (≥ 0.05) with sterility percentage under both nature and heat stress conditions. The negative correlation and significant (≥ 0.05) was found between sterility percentage and grain yield plant -1 under both nature and heat stress condition. Under natural conditions the grain yield plant -1 had highly correlation and positive with chlorophyll content, leaf rolling, tillers number plant -1 and panicles number plant -1 , while the negative and significant (≥ 0.01) correlation was confirmed with sterility percentage under natural conditions. Therefore, under stress conditions the positive correlations were found among grain yield and panicle length, tillers number and panicles number plant -1 however, the correlation was significant (≥ 0.01) and negative with leaf rolling and sterility percentage.

Discussion
Climate change has been an increasingly significant factor behind fluctuations in the yield and quality of rice (Oryza sativa L.). The development and use of heat tolerant varieties is an effective way to reduce each type of grain damage based on the existence of each varietal difference (Kown and Torrie, 1964). Mean squares due to environments were found to be significant at probability level 0.01 for all studied characteristics indicating that; all environments showed significant (≥ 0.05) effects on all characteristics. The significant differences among rice genotypes in Table 4 showed the presence of genetic variability in the used material and provided a good opportunity for yield improvement. This specified that, the studied genotypes were different from environment to the other and ranked differently from natural to heat stresses conditions. This could show that further improvement through selection for all characters studied may be effective. Gaballah and Abu El-Ezz (2019) mentioned that the analysis of variance exposed significant differences due to environments, genotypes and their interaction for all studied characteristics in both seasons.
Concerning the genotypes mean performance over the two years and two environments the 'Hybrid 2', 'Sakha 107' and 'Giza 178' recorded the superior values for grain yield and related characteristics, refer to those three cultivars have good tolerance in both natural and heat stress conditions and could use in breeding programs to enhance the tolerance of cultivars against heat stress. High night-temperature stress during the reproductive stage decreases fertility of spikelet and yield by inhibiting the physiological processes. Abdallah et al. (2011) reported that high temperature prompts sterility, and reduced average grain yield by 22% as well as grain yield was adversely correlated with spikelet sterility under heat stress therefore, potential donors identified based on at least one trait included 'ARC 11094', 'Pinulupot 1', 'Tupa 729', 'Shinriki', 'Kameji' and 'Giza 178'. Heat and drought stress may cause harmful effects on growth characters and plant development, the main phase being injured is the reproductive phase, particularly, anthesis or grain filling phase (Faseela et al., 2020). All genotypes showed spikelet sterility above 90% and indicated that increased temperature may limit grain yield by affecting fertility of spikelet and grain filling as well as net reduction in grain yield reach to 30.4% (Kumar et al., 2015). Salt stress resulted significant decrease of growth and physiological characteristics in all rice genotypes; therefore, the lowest reduction was observed in some salt-tolerant genotypes namely Ghunsi, Nonabokra, Hogla, Holdegotal, Vusieri, and Kanchon (Rasel et al., 2020). Maruyama et al. (2015) stated the increasing temperatures both accelerated and delayed the timing of heading. Extreme temperatures (>4 °C daily maximum) increased the days to heading.
The most standing genotypes under stress conditions, which gave the minimum reduction were 'IET1444' and 'Sakha 107' while 'Sakha 102', 'Giza 177', 'Sakha 108' and 'Sakha 104' for most studied characteristics. 'NH219' is more tolerant to heat stress than wild type 'N22' as its percentage yield reduction is less than 'N22' (Poli et al., 2013). Brestic et al. (2018) found that the selection for high yields was accompanied by the increase of photosynthetic productivity through unintentional improvement of leaf anatomical and biochemical characteristics including tolerance to non-optimal temperature conditions. Leaf area determination at earlier date was highly correlated with the total shoot biomass at the higher temperature (Pasuquin et al., 2013). Hammer et al. (2020) revealed that the high temperature tolerance genotypes are more important and it is approach to genotype × management for effective use of water through the crop cycle to maintain the productivity. At maturity, post-anthesis drought and heat stress significantly decreased the grain yield by 21.3%-65.2%, which was due to the changes of grain number per spike and thousand grain weight (Ahmad et al., 2019). Improvement the efficacy of transpiration offered the best potential for adaptation relevant to climate changes. Manigbas et al. (2014) reported that the genetic difference in sterility % among the selected backcross populations grown in high temperature showed that great numbers of plants may be identified and selected with lower sterility%. Wang et al. (2020) cleared that OsNAC006 mutant expression profiles showed that growth was inhibited following drought and high temperature stress. Also, Paul et al.
(2020) reported the two indica and one of the tropical japonica accessions exhibited severe heat sensitivity with 13 increased seed abortion; three tropical japonicas showed moderate heat tolerance, while temperate japonicas exhibited strong heat tolerance.
From correlation coefficient in Table 9, the results indicated that the leaf rolling under high temperature is remarkable to identify the genotypes as good tolerance were highly correlated and negative with grain yield, followed by sterility percentage and panicles number plant -1 . Gaballah and Abu El-Ezz (2019) recorded highly significant and positive phenotypic correlations between yield index and leaf rolling, sterility percentage, geometric mean productivity and stress susceptibility index, otherwise tillers number, panicles number plant -1 and panicle weight, as yield attributes had a significant (≥ 0.01) negative correlation with the yield index. From our results we can conclude that the genotypes can be scored as heat tolerant, based on days to heading, leaf rolling, tillering productivity and spikelets sterility percentage and might be used as selection criteria for identifying heat tolerant genotypes. We could summarize the impact of heat stress on rice genotypes compared to natural conditions; heat stress led to induced earliness by 20.16%, reduction percentage by 21. 77, 18.94, 19.03, 32.25, 35.56, 16.92 and 47.98 for chlorophyll content, plant height, panicle length, tillers number, panicles number plant -1, 100-grain weight and grain yield plant -1 , respectively otherwise increasing in leaf rolling 316.70% and sterility percentage 492.45%. The significant correlation of grain yield with days to heading, days to anthesis and days to maturity was recorded with Sharma et al. (2013).

Conclusions
Breeding heat tolerant rice cultivars is one of the most important approaches used to alleviate the effect of heat stress, particularly in high temperature regions. The screening and selection approaches for heat conditions could differentiate the genotypes according to heat tolerance. The desirable characteristics mean values for panicles number plant -1 , 100-grain weight, sterility percentage and grain yield plant -1 were recorded with 'Giza 178', 'Hybrid 2', 'Giza 179' and 'Sakha 107'. However, the grain yield plant -1 had highly positive correlation with chlorophyll content, leaf rolling, tillers number and panicles number plant -1 , however, negative and significant (≥ 0.01) was confirmed with sterility percentage under natural conditions. Moreover, under heat stress conditions the positive correlations were found among grain yield, panicle length, tillers number and panicles number plant -1 and had significant (≥ 0.05) and negative with leaf rolling and sterility percentage. Heat stress led to induce earliness by 20.16%, decrease ratio for panicles number plant -1 (35.56%), 100-grain weight (16.92%) and grain yield plant -1 (47.98%) in rice genotype.