Do nitrogen and zinc application alleviate the adverse effect of heat stress on wheat (Triticum aestivum L.)?

Late-season heat stress (LSH) is a limiting factor for wheat production. Besides, low zinc and poor protein diet usually is a problem in low-income countries. The primary calorie source in such countries is prepared from bread. This study aims to mitigate heat stress by zinc and nitrogen application and improve zinc and protein content in wheat grain. We did the field experiments as a split-split-plot based on a randomized complete block design with four replications to assess zinc and nitrogen’s possible mitigation effect on LSH and protein and zinc enrichment of wheat grain during two years. Factors included LSH by delay in planting date (optimum, late, and very late) as the main plot, nitrogen (0, 75, 150, and 225 kg ha) in subplots, and zinc (0, 10, and 20 kg ha) as sub-subplots. We measured yield, yield components, physiological traits, zinc, and protein contents in the grain. Results showed that the highest relative water content and cell-membrane thermal stability were attained at the optimum planting date, 150 kg N ha and 20 kg Zn ha. The maximum chlorophyll a and carotenoids contents in wheat cells were recorded in the optimum planting date, 225 kg N ha, and 20 kg Zn ha. Heat stress reduced the grain yield. In the second year of the experiment, the grain number per unit area was more than that of the first year; however, the highest grain yield was achieved in the first year owing to the higher mean grain weight. Nitrogen application decreased the adverse effects of heat stress on grain yield by increasing the grain number. Zinc application diminished the adverse effects of heat stress by increasing the mean grain weight. The adverse impact of the LSH on grain yield was more than that of biological yield. Heat stress reduced the hectolitre weight and zinc content of the grain. Meanwhile, it increased grain protein. In general, under LSH, the application of 225 kg N ha and 20 kg Zn ha can reduce the adverse effects of heat on the grain quality and quantity.


Introduction
As the primary source of humans' daily calorie intake, wheat plays a significant role in food security worldwide. This crop is also a critical source of protein, mineral nutrients, and vitamins, particularly B-group vitamins (Halim et al., 2018). Considering a close relationship among soils, crops, and human health nutrition, plant growth in zinc-deficient soils results in low zinc seeds, causing zinc deficiency in humans and animals fed such seeds, which will result in many health-related side effects (Niyigaba et al., 2019).
In the southwest of Iran, wheat is cultivated during late autumn. Under such conditions, the vegetative wheat growth coincides with cool-season precipitations, and no environmental limitations related to weather affect the vegetative growth. However, in the late-season of wheat growth, there exist limitations related to increased air temperature and water deficit, negatively impacting crop growth and production. Moshatati and Mousavi (2017) observed that late-season heat stress reduced wheat grain yields up to 48%. They reported that the highest and lowest grain yield belonged to the early November and early January planting dates. Delay in planting can cause a 65% reduction in grain yield because of late-season heat stress (Asakereh-Nezhad and Lak, 2017). It is concluded that heat stress reduces the biological yield by imposing premature aging on photosynthetic organs and lowering the current assimilates (Pireivatlou et al., 2010). Cell membrane damage, electrical leakage from thylakoid, and reduced chlorophyll content are the adverse effects of heat stress, ultimately decreasing the photosynthesis performance (Rehman et al., 2016). It is essential to use nitrogen and zinc efficiently to mitigate the undesirable influences of heat on crop yield and growth (Hafez and Badawy, 2018).
Nutrient deficiency is an important environmental limitation experienced by wheat during its growth and development. Zinc deficiency in the soil reduces wheat yield and grain Zn content (Niazkhani et al., 2018).
Almost 60% of the cultivated soils globally, especially soils in arid and semi-arid regions, are zinc deficient, leading to a more than 50% reduction in crop yield (Yang et al., 2014). Zinc deficiency leads to the inefficiency of metabolic and enzymatic defence systems associated with stress resistance. Zinc improves the growth of the root and stem of cereals, hence produce vigour plants that will be more tolerant against stress and have more grain yield (Bechoff and Dhuique-Mayer, 2016). Besides, zinc deficiency in soils and plants is a global micronutrient deficiency problem reported in many countries, and it is registered as a risk factor for human health universally. Confirming acceptable levels of zinc in diets and its intake should be a key constituent in reducing child illness, improving physical growth, and decreasing death in developing countries (Chiranjib and Kumar, 2010). Zinc increases the grain protein content by activating protein synthesis enzymes (Saleem et al., 2015). High temperatures reduce photosynthesis because of damage to chlorophyll pigments, a decline in leaf nitrogen, obstruction of PSII reaction centre and electron flow, decreased quantum efficiency, and downregulation of PSII photochemistry (Fahad et al., 2017). In addition to the role of nitrogen in plant growth, it has been shown that high nitrogen regimes induce more heat shock proteins (mitochondrial Hsp60 and chloroplastic Hsp24) that confer heat tolerance to plant (Heckathorn et al., 1996). In irrigated wheat fields, where no nitrogen deficiencies occur, the most influential factor on the grain protein content is the air temperature during the grain-filling period. It has been reported that high temperature enhanced the wheat protein content (Velu et al., 2016).
As the essential food source, wheat experiences high temperatures and low precipitation during the grain filling period in Iran. Also, large areas of wheat cultivated lands have low available zinc for plants. Hence, the current research was designed to assess the probable heat stress mitigation effect of zinc and nitrogen fertilizers on wheat yield and zinc bio-fortification for improving wheat grain quality (protein and zinc content) under control and the heat stress conditions.

Materials and Methods
This research was carried out during two successive growing seasons of 2018 and 2019 in the research field (31º36'N, 48º53'E, and 34m above sea level) of Khuzestan University of Agriculture and Natural Resources, Molassani, Iran. The experimental site was placed in a semi-arid area with an average annual temperature and precipitation of 26.6 ºC and 213 mm, respectively. Some chemical and physical features of the soil, meteorological data of the experimental site, as well as the trend of Tmax during the experiments in relation to wheat growth stages during the study period are given in Tables 1 and 2, and Figure 1 respectively. The research was conducted using a split-split plot based on a randomized complete block design with four replications. Experimental factors were three planting dates, including November 22 (as an appropriate planting date), December 11 (late planting date), and December 31 (very late planting date) as main plots, four nitrogen levels, including 0, 75, 150, and 225 kg ha -1 as subplots, and three zinc levels, namely 0, 10.0, and 20.0

Temperature (ºC)
Tmax 2018-19 T max 2019-20 4 kg ha -1 as sub-sub plots. Urea (containing 46% N) and zinc sulfate (containing 14.7% zinc) were used as fertilizer sources. The field experiment had 144 plots. Each plot area was 4 m 2 (2 m× 2 m with 10 planting rows of 2 m lengths), with 60 cm, 90 cm, and 300 cm wide ridge between two neighbouring sub-sub plots, subplots, and main plots, respectively. The entire experimental plots were 144 each year.

Crop management
Wheat (Chamran2 cultivar) was cultivated on three planting dates. The planting density was 400 plants m -2 . Chamran2 is a cultivar resistant to rust and lodging. Field preparation (plowing and land levelling), weed and pest control, and irrigation were carried out according to typical region practices. All plots were given identical applications of P at 100 kg ha -1 as superphosphate triple and K at 100 kg ha -1 as potassium sulfate, as recommended by soil analysis. Nitrogen treatments were applied three times: one-third before planting at land preparation time, one-third at tillering stage, and one-third at the onset of the stem extension period. Zinc treatment was applied at the mid-stage of tillering. Zinc sulfate was dissolved in water, and the obtained solution was added to the soil close to the plant crown.

Measuring indices and methods
Growth stage recording For all treatments, time to reach a specific growth stage was recorded (Table 3).

Yield
At maturity (when the moisture content of grains reached 14%), all plants in each plot were harvested; above-ground biomass, the number of seed per unit area, weight of 1000-grain, and final grain yields were further determined. Each plot's harvest index was measured by dividing the grain yield by the above-ground biomass (Dai et al., 2016).

Physiological traits
At the flowering stage, cell membrane thermal stability (CMTS), carotenoid content, chlorophyll a, chlorophyll b, relative water content, and wheat canopy temperature were measured as the physiological responses of wheat to the experimental treatments. CMTS was determined based on a method presented by Agarie et al. (1995). For each plot, the flag leaves of 10 plants were selected to measure carotenoids content, chlorophyll a, and chlorophyll b with a spectrophotometer (SPEKOL2000) using the Arnon method (1967). Relative water content was measured using a 5 cm 2 surface of the flag. The samples' fresh weight was measured; the samples were floated on distilled water for seven hours in darkness. The turgid weight of samples was determined. Afterward, to specify the dry weight of samples, they were dried at 75 ºC for 48 h. Relative water was measured using the following equation: RWC = [(FW-DW) / (TW-DW)] × 100 Where DW is dry weight, FW is fresh weight, TW and is the turgid weight.
Grain quality Protein (%) and zinc (mg kg -1 ) contents were determined as grain quality indices. Protein content was measured by the NIR grain analyzer (7250, Perten Company, Sweden). Atomic absorption (240FS AA, Agilent Company, USA) was used to measure grains' zinc content. Hectolitre was determined, according to Hill's (1975) method.

Data analysis
A combined analysis of the data about yield and yield components, physiological traits, and grain quality was carried out using SAS ver.9.4 (SAS Institute Inc., Cary, NC, USA). Because of page limitations, ANOVA tables are not included in the manuscript. However, means comparison is shown. The mean comparisons of various treatments were carried out utilizing Duncan's Multiple Range Test.

Relative water content (RWC) of wheat
The compound analysis showed that year and interaction of planting date × nitrogen × zinc on RWC were significant (P≤0.01, Table 10). Delay in planting caused a significant reduction in leaf RWC, were late and very late planting dates, respectively, had 3.28% and 5.55% lower RWC than the optimal planting date ( Table 4). The highest RWC (83.40%) was obtained at optimal planting date using 150 kg ha -1 N and 20 kg ha -1 Zn. The lowest RWC (61.64%) belonged to the very late planting date with no usage of N and zinc fertilizers (control), which was 26% lower than that of the highest value (Table 4). RWC of the second year of the experiment was higher than that of the first year (Table 5). When wheat was sowed late (late planting date), the highest RWC was attained using 225 kg ha -1 N and 10 kg ha -1 Zinc. Under the conditions of very late planting, the highest RWC was achieved with the application of 150 kg ha -1 N and 20 kg ha -1 zinc (Table 4).

Cell membrane thermal stability (CMTS)
According to the results, year and interaction of planting date × nitrogen × zinc significantly affected CMTS (P≤0.01, Table 10). Delay in planting reduced CMTS, where late and very late planting dates caused 6.45% and 13.1% reductions in CMTS, respectively. The mean comparison of the data showed that the highest CMTS (52.25µmhos.cm -1 ) belonged to the treatment with optimal planting date, 150 kg ha -1 N and 20 kg ha -1 zinc. However, the lowest CMTS (21.63 µmhos. cm -1 ) was observed in a very late planting date with no N and Zinc application (control treatment). There was a 65.79% reduction in CMTS in the second year compared to the first year of the experiment (Table 5). Regarding the interaction of planting date× fertilizer, 225 kg ha -1 N; 20 kg ha -1 Zinc, 75 kg ha -1 N; 10 kg ha -1 Zinc and 225 kg ha -1 N and 20 kg ha -1 Zinc were required to achieve the highest CMTS under the conditions of optimal, late, and a very late planting dates, respectively (Table 4).

Carotenoids, chlorophyll a, and chlorophyll b
The effect of year and interaction of planting date × nitrogen × zinc on carotenoids, chlorophyll a, and chlorophyll b was significant (P≤0.01, Table 10). Late planting date had a negligible effect on chlorophyll a content, where the difference between optimal and late planting date was only 5% regarding chlorophyll a content. However, a very late planting date (with a difference of 26.27%) significantly reduced chlorophyll a ( Table 4). The same trend was observed in chlorophyll b content, where the difference of optimal planting date with late and very late planting dates was 6.5% and 32%, respectively. Delay in planting resulted in increased carotenoid content, with the flag leaf of wheat having 0.83% and 5.8% higher carotenoids at optimal planting date than the late and very late planting dates, respectively (Table 4). The mean comparison of the interaction of planting date× nitrogen× zinc showed that the highest content of chlorophyll a (27.22 mg g -1 ) was obtained 6 in optimal planting date treatment with 225 kg ha -1 N and 20 kg ha -1 Zinc. No fertilizer application led to the lowest (12.73 mg g -1 ) chlorophyll content of wheat leaf at a very late planting date (Table 4). The highest carotenoid content of flag leaf (27.29 mg g -1 ) was observed at the first planting date with the application of 225 kg ha -1 N and 20 kg ha -1 zinc. With no fertilizers applied and a very late wheat planting, the leaf had the lowest (8.70 mg g -1 ) carotenoids.
Regarding the effect of year, there was a considerable difference (P≤0.01) between the two years of experiment in terms of chlorophyll a and carotenoids content; specifically, the chlorophyll and carotenoids contents of flag leaf were higher in the first year (3% and twice more than that of the second year, respectively) ( Table 5). When wheat was planted with delay, 225 kg ha -1 N and 10 kg ha -1 Zinc had to be applied for high chlorophyll content. Furthermore, to maximize wheat resistance to high-temperature stress through maximizing carotenoid content, 225 kg ha -1 N and 20 kg ha -1 Zinc had to be utilized under late planting conditions (Table 4). However, this amount was reduced to 150 kg ha -1 N and 10 kg ha -1 Zinc at the very late planting date (Table 4). The interaction of year × planting date significantly influenced the chlorophyll b content (P≤0.01). The highest content of chlorophyll b in the two years of the experiment belonged to the LPD. However, the very late planting date had the lowest chlorophyll b content ( Figure 2). The compound analysis revealed that the year's effect and interaction of planting date × nitrogen × zinc on the wheat grain yield were significant (P≤0.01, Table 10). In the second year, the wheat grain yield was significantly (P≤0.01) greater than that of the first year (Table 5). The mean comparison showed that the grain's highest yield was acquired when wheat was planted at optimal planting date using 225 kg N ha -1 and 20 kg Zn ha -1 . A very late planting date with no fertilizer application resulted in the lowest grain yield. Delay in planting was associated with reduced grain yield. At late and very late planting dates, the achieved grain yield was 14% and 35% lower than that of the optimal planting date, respectively. However, under late and very late planting conditions, usage of 225 kg N ha -1 and 150 kg ha -1 and 20 kg Zn ha -1 resulted in the highest grain yield (Table 6).
Grain yield components were reduced by delayed planting; specifically, grain weight and number per unit area experienced 7.86% and 13.37% reduction at the late planting and 12.7% and 25.58% reduction at the very late planting dates, respectively ( Table 6). The highest grain weight (47.13 g) belonged to the optimal planting date applying 150 kg N ha -1 and 20 kg Zn ha -1 . The smallest grains were observed (31.29 g) in the very late planting date with no fertilizers applied, showing a reduction of 33% (Table 6). Using 225 kg N ha -1 and 7 20 kg Zn ha -1 at the optimal planting date resulted in the highest grain number per unit area; in other words, a very late planting date without the use of nitrogen and zinc fertilizers yielded the lowest value of this trait (Table  6). To maximize the grain weight at late and very late planting dates, 150 kg N ha -1 and 10 kg Zn ha -1 was needed. Furthermore, to maximize the grain number per unit area at late and very late planting dates, 150 kg N ha -1 , 10 kg Zn ha -1 , and 225 kg N ha -1 , 10 kg Zn ha -1 had to be applied, respectively (Table 6).   g-n *OPD, LPD, and VLPD indicate optimum, late, and very late planting dates, respectively. Means followed with at least one common letter in each column are not significantly different according to Duncan's multiple range tests (P≤0.01).

9
Hectolitre weight According to the compound analysis, the main effect of year and interaction of planting date × nitrogen × zinc on hectolitre weight was significant (P≤0.01, Table 10). The wheat grain's hectolitre weight was lower in the first year compared with the second year (Table 7).  Delayed planting caused a reduction in hectolitre weight. The highest hectolitre weight (83.00 g) belonged to optimal planting date and the application of 225 kg N ha -1 and 10 kg Zn ha -1 . Very late planting date and no fertilizer treatment (control) yielded the lowest (77.40 g) hectolitre weight of grain, showing a 7% reduction (Table 7). At the late planting date, the fertilizer treatment of 150 kg ha -1 N and 20 kg ha -1 zinc, and at the very late planting date, the fertilizer treatment of 150 kg ha -1 N without zinc enhanced the hectolitre weight of wheat grain (Table 8).

Grain protein
The compound analysis showed that the grain protein was meaningfully (P≤0.05) influenced by the year and interaction of planting date × nitrogen × zinc (Table 10). The grain protein was lower in the first year compared with the second year (Table 7). Delayed sowing increased the protein content; in other words, late and very late planting dates resulted in 14.67% and 17.98% more accumulation of protein in wheat grain than the optimal planting date. Mean comparison revealed that the highest protein content of grain (13.71%) belonged to the very late planting date with the usage of 150 kg ha -1 N and 10 kg ha -1 zinc. Furthermore, the lowest values (7.58%) were recorded under optimal planting date and no fertilizer use, showing a 44% reduction. However, at optimal planting date, 225 kg ha -1 N and 10 kg ha -1 zinc improved the grain protein (Table 8).

Grain zinc content
The main effect of year and interaction of planting date × nitrogen × zinc had a meaningful influence (P≤0.01) on the grain zinc content (Table 10). Heat stress, induced by delayed planting, did not affect the zinc content. In the first year, the amount of grain zinc was higher ( Table 7). The highest zinc content (1.9 mg kg -1 ) was achieved under optimal planting date and use of 225 kg ha -1 N and 20 kg ha -1 zinc. The lowest value (1.17 mg kg -1 ) was recorded when wheat was planted very late, and no fertilizers (nitrogen and zinc) were applied.
Wheat grains had higher protein content (20%) and hectolitre weight (3%) in the second year than the first year (Table 7). However, wheat's zinc content in the first year was more significant than in the second year (Table 7).

Physiological traits
Damage to pigments such as chlorophyll is one of the effects of heat stress on plants. Reduction in chlorophyll content under heat stress is due to the increased activity of chlorophyllase (Kamanga et al., 2018).
In the present research, delayed planting reduced the chlorophyll content by inducing heat stress. However, the application of nitrogen fertilizer resulted in increased chlorophyll content. Since nitrogen is one of the main elements of chlorophyll structure, its presence in plants increases chlorophyll synthesis. By affecting the chlorophyll concentration, nitrogen directly influences the photosynthesis rate (Evans, 1983) hence plant growth and yield.
Heat stress reduced chlorophyll biosynthesis and increased its degradation. The chlorophyll biosynthesis inhibition under high temperature is attributed to various enzymes' deactivation (Dutta et al., 2009), such as 5-aminolevulinate dehydratase, an essential enzyme in the pyrrole biosynthesis pathway, that its activity decreases in wheat under heat stress (Mohanty et al., 2006).
Cell membrane usually is the first part to be damaged by environmental stresses, so CMTS is a good indicator of heat stress damage to leaf cells. Delayed planting reduced CMTS (Table 4) as the wheat was exposed to more heat stress. This finding is consistent with the results of Damaris et al. (2016). The decrease in RWC was correlated with a reduction in CMTS (Table 10). Delayed planting and the fact that some growth stages of wheat coincided with heat stress resulted in reduced RWC; this may be attributed to the increasing effect of heat on evaporation and transpiration. When RWC was reduced and stomata closed, the wheat's cooling power decreased, leading to an increase in the temperature of the whole plant. With the rise in the canopy temperature, the internal temperature of cells was further increased, negatively affecting the whole cell, especially the membrane system. The changes in carotenoids content (Table 4) confirms these findings. Carotenoids play a role in the chloroplast as a pigment. However, their most important function is an antioxidant activity because they are responsible for preventing lipid peroxidation and alleviating environmental stress's adverse effects (Altuntas et al., 2018). Accordingly, the increased carotenoid content at delayed planting dates indicates that wheat experienced heat stress at late and very late planting dates, generating more carotenoids to reduce its heat stress effects on cell membranes.
Adverse effects of heat stress on RWC, chlorophyll content, and CMTS were reduced with nitrogen and zinc application (Table 4). Nitrogen can overshadow water losses by affecting stomatal behaviour and leaf area duration and postponing plant maturation (Holík et al., 2018). Zinc application has increased RWC in maize (Tohidi Moghadam et al., 2013). Increasing the amount of nitrogen and zinc resulted in the synthesis of more protein molecules in the cell membrane structure, reducing cellular leakage and increasing membrane stability under stress conditions (Gomez-Coronado et al., 2017). Zinc is involved in the activation of enzymes related to carbohydrate metabolism, maintenance of the integrity of cellular membranes, protein synthesis, regulation of auxin synthesis, and pollen formation (Marschner, 1995). Also, regulation and conservation of the gene expression required for environmental stress tolerance are dependent on zinc (Cakmak, 2000).

Grain yield
The results showed that grain yield was negatively affected by a delay in planting. The induced heat stress by delayed sowing reduced the plant's photosynthetic potential, leading to reduction of chlorophyll content, RWC (Table 4), and grain filling-period (Mondal et al., 2015;Moshatati and Mosavi, 2017). These processes ultimately reduced grain number per unit area and the mean grain weight as two main wheat grain yield components. In the second year, the grain number per unit area was more than that of the first year. However, grain yield was lower in the first year, which can be attributed to the lower grain weight in the first year. It seems that climatic parameters are useful in determining wheat grain yield. In the second year, precipitation and relative humidity were significantly higher than the first year (Table 2), resulting in yellow rust disease in the post-pollination stage (data not shown). This adverse condition and reduced sunny hours reduced assimilate allocation to grains, hence grain weight reduction (Table 6).
Grain weight depends on the rate and duration of the grain-filling period and is strongly affected by the environment. Therefore, any factor that reduces the duration or rate of the grain filling period will reduce grain weight (Joshi et al., 2016). Another reason for the loss of grain weight under heat conditions is the plant's inability to transport and convert photosynthetic materials to starch under heat stress conditions (Ahmed et al., 2017).
A decrease in the grain-filling period reduces mean grain weight under heat stress conditions. Although the grain filling rate increases under such an adverse condition, this increase cannot reimburse reducing grain weight due to the reduced grain filling period (Gupta et al., 2015). By inducing kernel abortion, heat stress leads to grain number and grain yield reduction (Zaki and Radwan, 2011). Similarly, in the present study, delayed planting caused heat stress on wheat and reduced the number of grains per unit area.
Nitrogen application alleviated the adverse effects of heat stress on grain number per unit area; specifically, at late and very late planting dates, with the usage of 225 kg ha -1 N, the number of grains per unit area enhanced by 55% and 21%, respectively. Given the positive correlation between grain number per unit area and grain yield (Table 9), nitrogen increased grain yield by 60% and 53% under late and very late planting dates, respectively (Table 6). There is a reciprocal relationship between the number of grains per unit area and mean grain weight (Kutlu and Olgun, 2015); therefore, nitrogen application led to grain weight reduction in the present study. Another reason for the decrease in grain weight under increased nitrogen application is the 12 prolonged vegetative growth period. This exposes the grain filling period to higher temperatures and exacerbated heat stress effects (Lotfi, 2013).  Zinc has positive effects on the enzymatic systems of the plant, having catalytic or structural parts in protein synthesis and increased production capacity of photo-assimilates; therefore, zinc plays a significant role in spike formation and increasing yield of the grain through enhancing the yield components (Ahmadi et al., 2017;Singh, 2014). Application of zinc under heat stress conditions increases the grain yield (Kamaei et al., 2018). Zinc can also mitigate the effects of environmental stresses on plants by improving cellular stability (Torrion and Stougaard, 2017) and enhancing antioxidant activity (Yadavi et al., 2014). In the recent study, zinc alleviated the adverse effects of heat stress on grain yield components; in other words, at late and very late planting dates, the weight of the grain and grain number per unit area was improved by 7%, 11% and 26% 33%, respectively. This ultimately led to a 26% (late planting date) and 33% (very late planting date) increase in grain yield. Because zinc had been applied when the plant had not yet experienced the heat stress; so maybe additionally stored zinc in the plant could play a beneficial role in increasing the grain weight (probably through increasing assimilates transfer to grain (Ma et al., 2017) and participate in the biosynthesis of growth regulators (Zand et al., 2009).

Grain quality
In the second year of the experiment, the grain's protein content was greater than that in the first year, which can be attributed to the smaller grain (Table 6) in the second year. About 70% of the grain weight of wheat comprises starch; thus, decreasing the accumulation of starch in the grain results in smaller and lighter grains (Hu et al., 2018). The enzymes involved in starch production are susceptible to high temperatures during the period of grain-filling. Reducing these enzymes' activity at temperatures above 22 °C results in the reduction of starch accumulation (Farooq et al., 2011). There is an inverse relationship between the amount of starch and grain protein. Also, protein production has a lower sensitivity to higher temperatures than starch (Kutlu and Olgun, 2015); therefore, grains had lower starch and higher protein in the second year of the test (Table 7).
Nitrogen fertilizers increase protein storage compared to carbohydrates in the grain (Joshi et al., 2016). El-Habbal et al. (2010) concluded that the increase in the grain's protein content using nitrogen fertilizer was related to the increase in essential amino acids, leading to higher nutritional values. Zinc can effectively determine the protein content of grain. In other words, it can increase the grain protein content associated with increased enzymatic activity (Aziz et al., 2019). Zandipour et al. (2018) showed the positive effect of zinc on the increased protein content of wheat grain. The present study's findings follow this because the correlation between protein and zinc content was positive and significant (Table 9). Giunta et al. (2019) concluded that zinc sulfate could increase the grain protein content, which agrees with the present research work results. However, the influence of zinc on the protein content of grain was affected by heat stress. At optimal and late planting dates, zinc application resulted in a 13% and 6% increase in the grain protein content, respectively; however, zinc had no positive influence on the wheat grain protein content at a very late 14 planting date. In other words, the highest effect of zinc on the wheat grain protein content occurred under optimal planting dates and no heat stress imposed on the plant. Hectolitre weight of grains is an indicator of grain yield and depends on uniformity in shape, density, and moisture content. Heat stress may reduce hectolitre weight, and grains damaged by stress also have a lower weight (Motaghi et al., 2012). Similar results were observed in the present study, where delayed planting (which induced heat stress on wheat) caused a reduction in hectolitre weight. This can be due to the negative effect of heat on starch accumulation and the increased bran to endosperm ratio (Ghodyeh Zarinabadi and Ehsanzadeh, 2014). On the other hand, nitrogen had no meaningful influence on hectolitre weight. In other words, nitrogen could not alleviate the negative influences of heat stress on hectolitre weight. These findings are in line with the results of Kazemzadeh et al. (2013), who stated that nitrogen had no effects on the hectolitre weight of wheat ('Alvand' cultivar). The present research work results showed a positive relationship between hectolitre weight and grain protein content (Table 9), which is consistent with Kazemzadeh et al. (2013).

Conclusions
Quantitative (grain kg/ha) and qualitative yields (protein and zinc content) of wheat were improved by integrating nitrogen and zinc fertilizers. The extent to which these fertilizers are used to maximize quantity and quality depends on the planting date. To achieve the highest grain yield, optimal planting date (November 22) and 225 kg ha -1 N and 20 kg ha -1 zinc is the best treatment. The highest protein content was obtained at a very late planting date with 150 kg ha -1 N and 10 kg ha -1 zinc. When zinc is considered a qualitative trait of wheat grain, the most optimal treatment is the first planting date (22 November), 225 kg ha -1 N and 20 kg ha -1 zinc. The treatment recommended for the maximum hectolitre weight is optimal planting date, 225 kg ha -1 N, and 10 kg ha -1 zinc. The role of nitrogen in protein synthesis (HSPs to increase heat tolerance and gluten to increase seed quality) and development of area and photosynthesis efficiency is essential here. Zinc, by its various roles in cell metabolism, especially enzymatic reactions and carbohydrate metabolism, improved the quantity and quality of yield under normal and heat stress conditions.
According to the results of this two-year experiment, wheat's grain yield is affected by climatic conditions. Thus, the wheat grain yield was higher in the first year because, in the second year, the climatic parameters did not fully correspond to the plant's needs. In the second year of the experiment, climatic conditions such as higher rainfall, less sunshine, and higher relative humidity during the grain filling period resulted in reduced plant photosynthetic potential and increased pest damage (yellow rust, especially flag leafdata not shown), reducing the grain yield.

Authors' Contributions
Conceptualization: HRE and SNM; Investigation: NA and AM; Methodology: HRE, AM, and NA; Formal analysis: AI; Writing-original draft: SNM; and Writing-review and editing: HRE and SNM.
All authors read and approved the final manuscript.