Response of Wheat Genotypes Stressed by High Temperature in Terms of Yield and Protein Composition Across Diverse Environments in Australia

Abstract

Global climate change poses a significant threat to wheat (Triticum aestivum L.) production due to rising temperatures. This study aimed to investigate the impact of high temperatures on wheat yield, thousand kernel weight (TKW), colour, and protein composition to inform breeding strategies for heat tolerance. Two field experiments were conducted: one at three locations in Australia (Horsham, (Vic) Narrabri, (NSW) and Merredin, (W.A.)) in 2019, involving two wheat varieties (Berkut (high-heat-tolerant) and Sokoll (medium-heat-tolerant)) sown at normal (TOS1) and late (TOS2) sowing times; and a second experiment at Narrabri in 2019 and 2020, involving three wheat varieties (Cobra (heat-sensitive), Flanker (high-heat-tolerant) and Suntop (medium-heat-tolerant)) sown at normal (TOS1) and late (TOS2) sowing times. There were reductions in yield and TKW under high temperatures (p < 0.05), particularly in late sowing conditions. The glutenin/gliadin ratio decreased, affecting dough strength and elasticity, especially at Merredin. Heat-tolerant varieties like Flanker and Suntop maintained protein quality, with an increase in the glutenin/gliadin ratio, under high temperature. These findings highlight the necessity for breeding heat-tolerant wheat varieties that can sustain both yield and quality. Future research should focus on genetic traits for heat tolerance, advanced molecular techniques, and interdisciplinary approaches to ensure sustainable wheat production in a changing climate.

1. Introduction

As global climate change intensifies, high-temperature stress impacting plant growth and development is becoming a focus of concern. Among these stresses, rising global temperatures present a formidable challenge to agriculture worldwide. Wheat (Triticum aestivum L.), the most extensively cultivated cereal and a major contributor to global grain markets, is particularly vulnerable to these changing conditions. In fact, similarly to the findings of Rivelli et al. [1] in South America, high-temperature stress has been shown to reduce both wheat yield and quality. Wheat now accounts for approximately 20% of global human calorific intake and 20% of daily protein consumption, making its sustainability critical for global food security [2,3]. Wheat’s sensitivity to heat stress is especially pronounced in low-latitude regions, where approximately 100 million ha are cultivated [4,5]. Each 1 °C increase in temperature is associated with an approximate 6% reduction in global wheat production on an annual basis, with regions like Australia facing yield losses of up to 50% under a 2 °C rise [6,7].

Wheat is a crucial source of protein for human consumption, containing approximately 10–18% protein by dry weight. Wheat proteins are categorised into gluten and non-gluten proteins. Gluten proteins, which include glutenins and gliadins, account for approximately 75% of wheat protein content. These proteins are essential for the viscoelastic properties of dough. Glutenins, in particular, play a crucial role in dough elasticity and strength, as they are composed of high-molecular-weight (HMW) and low-molecular-weight (LMW) subunits linked by disulfide bonds [8]. Recent studies show that the protein content of wheat grains is heavily influenced by genetic factors, as well as environmental stresses such as heat and drought, which can affect the proportion of HMW and LMW glutenins [9,10]. Non-gluten proteins, such as globulin and albumin, make up the remaining 25% of wheat proteins. These proteins are involved in various metabolic processes, and are primarily found in the testa.

High temperatures adversely affect wheat protein synthesis, impacting both the quality and quantity of protein within the grain [11]. Heat stress shortens the grain filling period, which is crucial for protein and starch deposition, and thereby reduces the overall protein content and alters its composition [12]. High temperatures may indirectly affect wheat protein synthesis and quality by affecting nitrogen absorption and utilisation, especially by reducing the glutenin/alcohol-soluble protein ratio, thereby reducing dough strength and stability [13,14]. This is in line with studies showing that high temperatures increase total protein content, but the composition of wheat’s primary protein fraction, gluten, is altered, notably with a shift in the ratio of glutenin to gliadin [10,15]. Under heat stress, the synthesis rate of both gliadins and glutenins increases; however, gliadins are synthesised at a faster rate than glutenins. This results in a lower glutenins/gliadins ratio, negatively affecting the dough’s viscoelastic properties and stability [16]. In particular, elevated temperatures during the grain filling stage can increase the proportion of gliadins and reduce the overall quality of gluten, thus impacting the bread-making properties of wheat [17]. Given these challenges, there is a critical need to develop heat-tolerant wheat cultivars through targeted breeding programmes, to ensure stability in wheat production and protein quality in response to the projected future climate. These efforts are essential for sustaining the growing global demand and for the adaptation of wheat production systems to changing environmental conditions.

Currently, there is a lack of research on the effects of heat stress on the yield, protein content, and composition of different heat-tolerant wheat cultivars commonly grown in major wheat-growing regions of Australia. The hypothesis is that the harvest year, sowing time, location, and wheat cultivars affect wheat yield and protein content as influenced by temperature.

Therefore, the main objective of this study is to assess the impact of heat stress on the yield, protein content, and composition of two wheat cultivars, Berkut and Sokoll, in three locations (Horsham (VIC), Narrabri (NSW), and Merredin (WA)) during a hot year (2019) (Experiment 1), and the same for three other wheat cultivars, Flanker, Cobra, and Suntop, grown in Narrabri over 2019 (hot year) and 2020 (cool year) (Experiment 2).

2. Materials and Methods

2.1. Field Experimental Design

In the first experiment, two cultivars (Berkut and Sokoll) were sown at two times (normal sowing on 21 May and late sowing on 26 July) in three locations (Horsham, Narrabri, and Merredin) in 2019 (Experiment 1). In the second experiment, three cultivars (Cobra, Flanker, and Suntop) were sown at two times (normal sowing and late sowing) in the Narrabri in 2019 and 2020 (Experiment 2). In all experiments, two sowing times were used: normal sowing and late sowing. The late sowing exposed the wheat to higher temperatures during the grain filling and maturation stages.

2.2. Wheat Genotypes

The present study utilised five wheat cultivars extensively cultivated in Australia, namely Berkut (Pedigree: Irena/Babax/Pastor), Sokoll (Pedigree: Pastor/Altar84/OpataM85), Flanker (Pedigree: EGA Gregory//EGA Gregory/Lang), Cobra (Pedigree: Westonia/W29), and Suntop (Pedigree: SUNCO/2*PASTOR//SUN436E), provided by the Plant Breeding Institute, University of Sydney. These cultivars are categorised based on their heat tolerance, based on previous field studies (

Table 1. Summary of wheat cultivars and heat tolerance ratings (based on previous field studies).

Cultivar	Flanker	Berkut	Suntop	Sokoll	Cobra
Rating	Highly tolerant	High	Medium	Medium	Sensitive

).

The experimental design involved two sowing dates for all wheat varieties: normal sowing (time of sowing, TOS1) in May and late sowing (TOS2) in July. The sowing rate was 42 kg/ha for all experiments. Supplementary irrigation was implemented to eliminate the effects of water stress, thereby ensuring that only the high-temperature treatment was the primary abiotic stress. To preserve soil structure, minimum tillage practises were applied. The experimental site was fallowed during the summer and rotated with leguminous crops in alternate years to reduce disease incidence and maintain soil health. The crop field experiment utilised an alpha lattice design, which was a randomised complete block layout with two replicates.

In 2019, the time required from sowing to maturity for TOS1-sown wheat in Horsham, Victoria, Narrabri, New South Wales, and Merredin, Western Australia, was approximately 202 days (2280 GDD (growing degree days)), 152 days (2260 GDD), and 150 days (2332 GDD), respectively, and the time required from grain filling to maturity was approximately 59 days (878 GDD), 48 days (914 GDD), and 44 days (883 GDD), respectively. For TOS2-sown wheat, the required time was approximately 164 days (2236 GDD), 126 days (2202 GDD), and 119 days (2312 GDD), respectively, and the time required from grain filling to maturity was approximately 45 days (872 GDD), 37 days (913 GDD), and 38 days (878 GDD), respectively. In 2020 in Narrabri, the time required from sowing to maturity for TOS1- and TOS2-sown wheat was approximately 159 days (2275 GDD) and 117 days (2314 GDD), respectively.

Temperature and rain data for 2019 in Narrabri, Horsham, and Merredin are illustrated in Figure 1a–c, with the temperature variations for Narrabri displayed across 2019 and 2020. In the first experiment conducted in 2019, Berkut and Sokoll were sown in Narrabri (northwest NSW), Horsham (Victoria), and Merredin (Western Australia), following both normal (TOS1) and late (TOS2) sowing schedules. The second experiment involved planting Flanker, Suntop, and Cobra in Narrabri in 2019 and 2020, again adhering to both TOS1 and TOS2 sowing schedules.

Table 2. Physical and chemical characteristics of soil at different locations in 2019.

Soil Characteristics	Horsham	Narrabri	Merredin
Soil Type	Grey Vertosol	Grey Vertosol	Orthic Tenosol
Texture	Clay	Clay	Sandy Loam
PAWC (Plant Available Water Capacity) (mm/m) [18]	160.6	158.7	132.9
Organic Carbon (%)	1.13	0.63	0.96
Nitrate Nitrogen (mg/kg) (soil extract)	19.0	11.5	20.0
Phosphorus—Colwell (mg/kg) (soil extract)	70.5	66.5	42.0
Organic Matter (OM) %	1.95	1.05	1.65
Total Ca (%)	0.74	0.46	0.18
Total Mg (%)	0.70	0.63	0.20
Total Na (%)	0.075	0.039	0.025
Total K (%)	0.76	0.36	0.30
pH (1:5 Water)	8.65	8.65	6.00
pH (1:5 CaCl2)	7.9	7.6	5.5

provides an overview of the soil properties at the three locations.

2.3. Chemical Analyses

All wheat grains intended for analysis were finely ground into flour, utilising a mill (3100 Lab Mill, Perten, Australia) equipped with 500 μm sieve. The colour of wheat flour was evaluated using the Konica Minolta Cr-400 Chromameter (Minolta Co., Ltd., Osaka, Japan). The data were reported in terms of the Commission Internationale de l’Eclairage L*, a*, and b* (CIELAB) values, which describe the transition of flour colour from black to white (L*), green to red (a*), and blue to yellow (b*), respectively [19]. Each colour testing was conducted five times to ensure the repeatability of the measurements.

The protein content in the wheat flour samples was ascertained using the Vario MACrO cube organic elemental analyser (Elementar, Langenselbold, Germany). For this analysis, 40 mg of wheat flour underwent rapid oxidation in an ultra-high-purity oxygen environment at a temperature of 950 °C, leading to the formation of nitrogen dioxide, nitrogen monoxide, and water. The oxidised compounds were subsequently passed through a secondary furnace to eliminate any residual particulates. The resulting nitrogen content was quantified using a thermal conductivity detector, and was utilised to determine the total protein content in the samples. Each sample was analysed twice to improve the precision and reliability of the findings [20]. A conversion factor of 6.05 was used for protein estimation.

The bicinchoninic acid (BCA) protein assay method was employed in this study to determine the quantities of albumin, globulin, gliadins, and glutenins in the samples. This method leverages the principle of protein-induced reduction of Cu²⁺ to Cu⁺ in an alkaline medium, coupled with the sensitive, specific, and colorimetric detection of the copper (I) cation (Cu⁺) by bicinchoninic acid [21].

For albumin extraction, 1 g of flour was mixed with 10 mL of water, stirred at 200 rpm for 30 min, and then centrifuged at 3500 rpm for 20 min. One millilitre of the supernatant was collected and stored at −5 °C, while the remainder was discarded. This extraction process was repeated four times to obtain four separate 1 mL supernatant samples. The residual flour was then extracted four times with 10 mL of 0.4 M NaCl solution to isolate globulin, followed by four subsequent extractions using 10 mL of 70% ethanol for gliadin, and, finally, four extractions with 10 mL of 0.1 M NaOH for glutenin. The collected supernatants were combined with an indicator and incubated at 37 °C for 30 min, and absorbance was measured at 562 nm using a UV-1900 spectrophotometer (Shimadzu, Kyoto, Japan).

2.4. Statistical Analyses

The experimental data were subjected to two-way and three-way analysis of variance (ANOVA) using Statistix 8.1 and Genstat 22nd Edition. Where there were significant differences, post hoc Tukey’s Honestly Significance Difference (HSD) tests were used. Correlations between each component were also determined.

3. Results

3.1. Experiment 1

Table 3. The main interactions of location (Horsham, Narrabri, and Merredin), time of sowing (TOS1 and TOS2), and cultivar (Berkut and Sokoll) and their effects on wheat yield, protein, protein fractions, kernel colour, and maturity days in 2019 were analysed, with corresponding p-values shown.

Effect	Loc (Location)	TOS	Var (Variety)	Loc × TOS	Loc × Var	TOS × Var	Loc × TOS × Var
Yield	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
Thousand kernel weight (TKW)	<0.001	<0.001	<0.001	<0.001	NS	<0.001	0.011
Crude protein	<0.001	<0.001	<0.001	<0.001	<0.001	0.027	0.012
Glutenins/gliadins	NS	<0.001	<0.001	0.01	<0.001	<0.001	0.002
Gliadins	<0.001	<0.001	<0.001	0.041	<0.001	NS	<0.001
Glutenins	<0.001	NS	<0.001	NS	<0.001	0.002	NS
Albumin	0.016	<0.001	NS	NS	NS	0.001	NS
Globulin	NS	NS	<0.001	NS	NS	NS	NS
L* Value	<0.001	<0.001	NS	<0.001	<0.001	NS	<0.001
a* Value	<0.001	<0.001	<0.001	<0.001	<0.001	NS	<0.001
b* Value	<0.001	0.003	<0.001	<0.001	<0.001	<0.001	<0.001
Grain filling to maturity	<0.001	<0.001	NS	<0.001	NS	NS	NS
Sowing to maturity	<0.001	<0.001	NS	<0.001	0.019	NS	NS

Total of 12 treatments = 3 locations × 2 varieties × 2 sowings; number of replications = 4; NS = p > 0.05.

illustrates the interactions that occurred between the location, time of sowing, and cultivar and their effects on yield, thousand kernel weight (TKW), crude protein content, glutenins/gliadins ratio, gliadins, and L*, a* and b* values (p < 0.05). Sokoll had lower yield at all three locations for TOS2 compared with TOS1 (Figure 2a). There was a reduction in yield for Berkut at Merredin for TOS2 compared with TOS1 (p < 0.001). The TKW of Berkut was consistently higher than that of Sokoll across all three locations and both sowing dates, particularly at Merredin (Figure 2b). The crude protein content was higher for TOS2 compared with TOS1 for both varieties (p = 0.012), except for Berkut at Horsham (Figure 2c). At all three locations, the glutenins/gliadins ratio of Berkut for TOS1 was higher than for TOS2, whereas Sokoll grown in Narrabri showed higher glutenins/gliadins for TOS1 (p = 0.002) (Figure 2d). In comparison to TOS1, the gliadins content for TOS2 was consistently higher across the two varieties and three regions. Specifically, there were no differences between TOS2 and TOS1 for Berkut in the Horsham and Narrabri regions (Figure 2e). Compared with TOS1, the L* values for Berkut and Sokoll increased for TOS2, except for Berkut in Narrabri and Sokoll in Merredin (Figure 2f). The highest a* value for Berkut for TOS1 was in Horsham, whereas the lowest for Sokoll for TOS2 occurred in Merredin (Figure 2g). Regarding the b* values, Sokoll consistently exhibited higher values than Berkut (Figure 2h). The grain filling to maturity stage of wheat varied by locations and TOS, and the sowing to maturity stage also differed notably by locations and TOS. In general, wheat required the shortest time to grow in Merredin (warmest location) and the longest time in Horsham (coolest location). The growth time required for TOS2 was shorter than that for TOS1.

The analysis indicates that wheat yield did not exhibit a statistically significant correlation with traits such as TKW, protein content, or fraction. TKW was negatively correlated with crude protein (r = −0.665, p < 0.001), gliadins (r = −0.418, p = 0.042), glutenins (r = −0.433, p = 0.035), and b* value (r = −0.724, p < 0.001). Additionally, crude protein showed a negative correlation with L* value (r = −0.507, p = 0.011) and a positive correlation with b* value (r = 0.574, p = 0.003). Glutenins/gliadins ratio was positively correlated with globulin (r = 0.639, p < 0.001). Gliadins and glutenins were both negatively correlated with globulin (r = −0.709, p < 0.001 and r = −0.683, p = 0.001). Albumin was positively correlated with L* value (r = 0.463, p = 0.023) and negatively with a* value (r = −0.436, p = 0.033). L* value was negatively correlated with a* value (r = −0.77, p < 0.001). The number of days from grain filling to maturity and from sowing to maturity were both negatively correlated with the L* value (r = −0.473, p = 0.019; r = −0.534, p = 0.117, respectively) and the b* value (r = −0.601, p = 0.002; r = −0.432, p = 0.035, respectively), and positively correlated with the a* value (r = 0.745, p < 0.001; r = 0.719, p < 0.001, respectively) (

Table 4. Pairwise correlations between wheat yield, protein, protein fractions, kernel colour, and maturity days for Berkut and Sokoll in three locations in 2019.

		Yield	TKW	Crude Protein	Glutenins/Gliadins	Gliadins	Glutenins	Albumin	Globulin	L*	a*	b*	Grain Filling to Maturity	Sowing to Maturity
Yield	r =	1
	p =
TKW	r =	NS	1
	p =
Crude protein	r =	NS	−0.665	1
	p =		<0.001
Glutenins/Gliadins	r =	NS	NS	NS	1
	p =
Gliadins	r =	NS	−0.418	NS	−0.909	1
	p =		0.042		<0.001
Glutenins	r =	NS	−0.433	NS	−0.6604	0.899	1
	p =		0.035		<0.001	<0.001
Albumin	r =	NS	NS	NS	NS	NS	NS	1
	p =
Globulin	r =	NS	NS	NS	0.639	−0.709	−0.683	NS	1
	p =				<0.001	<0.001	0.001
L*	r =	NS	NS	−0.507	NS	NS	NS	0.463	NS	1
	p =			0.011				0.023
a*	r =	NS	NS	NS	NS	NS	NS	−0.436	NS	−0.77	1
	p =							0.033		<0.001
b*	r =	NS	−0.724	0.574	NS	NS	NS	NS	NS	NS	NS	1
	p =		<0.001	0.003
Grain filling to maturity	r =	NS	NS	NS	NS	NS	NS	NS	NS	−0.473	0.745	−0.601	1
	p =									0.019	<0.001	0.002
Sowing to maturity	r =	NS	NS	NS	NS	NS	NS	NS	NS	−0.534	0.719	−0.432	0.935	1
	p =									0.007	<0.001	0.035	<0.001

Correlation and significance at p = 0.05, NS = p > 0.05.

).

3.2. Experiment 2

Interactions occurred between sowing period and cultivar concerning crude protein content, glutenins/gliadins, gliadins, glutenins, globulin, and a* value for Cobra, Flanker, and Suntop at Narrabri in 2019 (

Table 5. Main interactions of year, time of sowing, and cultivar, and effects on wheat yield, protein, protein fractions, kernel colour, and maturity days, for Cobra, Flanker, and Suntop in Narrabri in 2019 were analysed, with corresponding p-values shown.

Effect	TOS	Variety	TOS × Variety
Yield	0.027	NS	NS
TKW	0.001	NS	NS
Crude protein	<0.001	<0.001	<0.001
Glutenins/gliadins	NS	0.001	0.006
Gliadins	<0.001	<0.001	0.024
Glutenins	<0.001	0.002	0.024
Albumin	0.024	<0.001	NS
Globulin	0.045	<0.001	<0.001
L* Value	<0.001	<0.001	NS
a* Value	<0.001	<0.001	0.01
b* Value	<0.001	<0.001	NS
Grain filling to maturity	<0.001	0.02	NS
Sowing to maturity	<0.001	NS	NS

Total of 6 Treatments = 3 varieties × 2 sowings; number of replications = 4; NS = p > 0.05.

, (p < 0.05)). The wheat yield ((4.46 t/ha) p < 0.001) and TKW (36.72 g, p = 0.011) were higher for TOS1 than TOS2 (at 3.48 t/ha and 27.18 g, respectively) in 2019 (Figure 3).

Compared to TOS1, the crude protein content (p < 0.001) of the three wheat varieties (Cobra, Flanker, and Suntop) for TOS2 increased, with Cobra and Flanker exhibiting the highest crude protein (Figure 4a). Furthermore, the glutenins/gliadins ratio (p = 0.006) of Cobra and Suntop decreased with TOS2 compared to TOS1, while that of Flanker increased with TOS2 (Figure 4b). The gliadins (p = 0.024) of Cobra, Flanker, and Suntop increased with TOS2 (Figure 4c). The glutenins (p = 0.024) of Cobra and Suntop increased with TOS2, while only Flanker decreased with TOS2 (Figure 4d). Cobra had the highest globulin content (p < 0.001) with TOS2, while Suntop had the lowest globulin content with TOS1 (Figure 4e). Compared with TOS1, the a* value (p = 0.01) of the three wheat varieties increased for TOS2 (Figure 4f).

The wheat yield had positive correlations with TKW (r = 0.753, p = 0.005), glutenins (r = 0.621, p = 0.031), grain filling to maturity (r = 0.699, p = 0.012), and sowing to maturity (r = 0.656, p = 0.021). TKW was positively correlated with glutenins (r = 0.597, p = 0.04), grain filling to maturity (r = 0.798, p = 0.002), and sowing to maturity (r = 0.801, p = 0.002). Glutenins/gliadins ratio was positively correlated with L* value (r = 0.629, p = 0.029), and negatively correlated with gliadins (r = −0.898, p < 0.001), a* value (r = −0.598, p = 0.04), and sowing to maturity (r = −0.787, p = 0.002), respectively. Gliadins were positively correlated with glutenins (r = 0.747, p = 0.005), grain filling to maturity (r = 0.65, p = 0.022), and sowing to maturity (r = 0.82, p = 0.001), and negatively correlated with L* value (r = −0.703, p = 0.011). Glutenins had positive correlations with grain filling to maturity (r = 0.716, p = 0.009) and sowing to maturity (r = 0.657, p = 0.02). The L* value had negative correlations with a* value (r = −0.591, p = 0.043), b* (r = −0.924, p < 0.001), grain filling to maturity (r = −0.736, p = 0.006), and sowing to maturity (r = −0.689, p = 0.013). The a* value was positively correlated with b* value (r = 0.761, p = 0.004). The b* value had positive correlations with grain filling to maturity (r = 0.637, p = 0.026) and sowing to maturity (r = 0.613, p = 0.034). Grain filling to maturity was positively correlated with sowing to maturity (r = 0.91, p < 0.001) (

Table 6. Pairwise correlations between wheat yield, protein, protein fractions, kernel colour, and maturity days for Cobra, Flanker, and Suntop in Narrabri in 2019.

		Yield	TKW	Crude Protein	Glutenins/Gliadins	Gliadins	Glutenins	Albumin	Globulin	L*	a*	b*	Grain Filling to Maturity	Sowing to Maturity
Yield	r =	1
	p =
TKW	r =	0.753	1
	p =	0.005
Crude protein	r =	NS	NS	1
	p =
Glutenins/Gliadins	r =	NS	NS	NS	1
	p =
Gliadins	r =	NS	NS	NS	−0.898	1
	p =				<0.001
Glutenins	r =	0.621	0.597	NS	NS	0.747	1
	p =	0.031	0.04			0.005
Albumin	r =	NS	NS	NS	NS	NS	NS	1
	p =
Globulin	r =	NS	NS	NS	NS	NS	NS	0.58	1
	p =							0.048
L*	r =	NS	NS	NS	0.629	−0.703	NS	NS	NS	1
	p =				0.029	0.011
a*	r =	NS	NS	NS	−0.598	NS	NS	NS	NS	−0.591	1
	p =				0.04					0.043
b*	r =	NS	NS	NS	NS	0.621	NS	NS	NS	−0.924	0.761	1
	p =					0.031				<0.001	0.004
Grain filling to maturity	r=	0.656	0.801	NS	NS	0.65	0.716	NS	NS	−0.736	NS	0.637	1
	p=	0.021	0.002			0.022	0.009			0.006		0.026
Sowing to maturity	r=	0.699	0.798	NS	−0.787	0.82	0.657	NS	NS	−0.689	NS	0.613	0.91	1
	p=	0.012	0.002		0.002	0.001	0.02			0.013		0.034	<0.001

Total of 6 Treatments = 3 varieties × 2 sowings; number of replications = 4; NS = non-significant.

).

The year 2020 was characterised by lower than average temperatures, which resulted in minimal differences in wheat performance between the two sowing times, TOS1 and TOS2.

4. Discussion

4.1. Impact of High Temperatures on Wheat Yield and Thousand Kernel Weight (TKW)

High temperatures reduced wheat yield and TKW, particularly under late sowing conditions (TOS2) compared with normal sowing (TOS1). This reduction was most pronounced in Merredin, the hottest location in the study, aligning with previous research indicating that elevated temperatures accelerate phenological development, reducing the grain filling period essential for kernel development [22,23]. The reduction in yield and TKW observed in 2019 for both Berkut and Sokoll, especially under TOS2, highlights the adverse effects of high temperatures on wheat growth and productivity. The shorter grain filling days (GFDs) under high temperatures reduced TKW, as shorter GFDs provided less time for biomass accumulation and grain filling. This was particularly evident in Merredin, where grain filling times were shortest across all cultivars. Shortened GFDs due to heat stress impair nutrient translocation to the grain, contributing to smaller kernels and lower yields. There was little difference between the early and late sowing times within any location for growing degree days (GDDs) from sowing to maturity and from anthesis to maturity.

High temperatures not only reduce the grain filling period, but also impair pollen tube formation and promote pollen sterility, further contributing to yield losses [24]. The negative correlations found between TKW and crude protein, gliadins, and the b* value, along with the positive correlation with the glutenins/gliadins ratio, suggest a complex interplay between yield components and protein composition under heat stress. These correlations indicate that high temperatures not only reduce yield and TKW, but also alter the protein balance within the grain, affecting both quantity and quality. These findings are critical, as they underscore the need for breeding heat-tolerant wheat varieties that can maintain yield and grain quality.

4.2. Impact of High Temperatures on Wheat Colour

High temperatures influence the colour of wheat flour, as evidenced. Flour colour is an important quality attribute, with high L* values indicating lighter flour and high b* values indicating more yellow flour, both of which are desirable traits in many wheat products [22]. TOS2 generally resulted in higher L* values compared with TOS1, except in specific instances, such as Berkut in Narrabri and Sokoll in Merredin. This increase in L* values under late sowing may be due to the accelerated maturation process induced by high temperatures, which affects the pigment composition and distribution within the grain [25]. The correlation between GFDs and colour traits further supports these findings, as shorter grain filling times resulted in lighter (higher L*) and less yellow (lower b*) flour. This is likely due to accelerated grain maturation under heat stress, which alters pigment biosynthesis.

The correlations between anthesis to maturity duration and flour colour suggest that the shortened grain filling period under heat stress not only affects yield, but also impacts the visual quality of the flour. The negative correlations between L* and b* values with the duration from anthesis to maturity imply that shorter grain filling periods result in lighter and less yellow flour. This finding aligns with previous studies that have shown that heat stress can accelerate grain maturation, leading to lighter and less yellow flour due to changes in pigment concentration and composition [26].

4.3. Impact of High Temperatures on Wheat Protein Content and Composition

The results demonstrate interactions between location, year, sowing time, and variety, influencing the crude protein content and the glutenins/gliadins ratio, which are essential determinants of wheat quality. Late sowing times (TOS2) generally resulted in higher crude protein content compared to normal sowing times (TOS1) across all varieties, locations, and years. For instance, in experiment 1, Berkut and Sokoll exhibited higher crude protein content for TOS2 in Narrabri and Merredin in 2019, except for Berkut at Horsham; in experiment 2, Cobra, Flanker and Suntop had significantly higher crude protein content in Narrabri for TOS2 in 2019. This increase in protein content under late sowing conditions aligns with previous studies indicating that heat stress can lead to an increase in protein concentration due to the reduced grain filling period [10]. The grain filling period is critical for protein accumulation. Under high temperatures, the shortened GFDs contributed to smaller grains with a higher concentration of protein, as starch deposition is more sensitive to heat than protein synthesis. This is evident from the negative correlation between TKW and crude protein content observed (r = −0.6652, p < 0.001). The shorter GFDs also affected the protein composition, leading to higher proportions of gliadins relative to glutenins, which weaken dough quality.

Gliadins and glutenins are the two major types of proteins in wheat that contribute to its unique baking qualities. Heat stress alters the composition of wheat proteins, particularly the ratio of gliadins to glutenins. Gliadins confer extensibility, while glutenins provide dough strength and elasticity [17,18]. This study also showed that the glutenins/gliadins ratio varied with sowing time, location, and variety. For experiment 1, the glutenins/gliadins ratio of Berkut and Sokoll was higher for TOS1 compared to TOS2 across all three locations, while Berkut showed a higher ratio in Merredin. Under heat stress, there was an increase in the proportion of gliadins relative to glutenins, particularly with TOS2, which can adversely affect the viscoelastic properties of the dough [11,16]. This decrease in the gliadins/glutenins ratio leads to weaker dough, which is less suitable for bread-making. The increase in crude protein content under heat stress was accompanied by changes in the protein fractions. The wheat cultivars used in this experiment are more characteristic of high-protein wheats; therefore, despite the reduction in the glutenins/gliadins ratio under high temperatures for TOS2, most varieties still exhibited a relatively high glutenins/gliadins ratio. Only the dough strength and elasticity of the flour will decrease, and the baking quality will be affected to a certain extent. Dough strength and elasticity are key characteristics of bread and other fermented products [27].

Furthermore, in Experiment 1, high temperatures could have altered the molecular structure of gluten proteins, thereby affecting their functionality. At Merredin, under TOS2, the glutenins/gliadins ratio showed a greater decrease in Berkut and a smaller decrease in Sokoll, indicating that dough quality varied between cultivars under higher heat stress. This finding is corroborated by previous research, which showed that high temperatures disrupt the synthesis of glutenins more than that of gliadins, resulting in a disproportionate increase in gliadins [16]. However, in Experiment 2, the glutenins/gliadins ratio of Flanker under TOS2 at Narrabri in 2019 was higher than that under TOS1. This suggests that, for some heat-tolerant wheat cultivars, the glutenins/gliadins ratio may be resilient and not decrease even if the crude protein content increases under high temperature. It appears that the glutenins/gliadins ratio in wheat is influenced by genotype. Heat stress increased the extractable gliadins and decreased the unextractable glutenins, which affects the formation of strong gluten networks. This alteration is critical for the baking industry, as it directly impacts the quality of baked goods. High temperatures during grain filling increase the proportion of low-molecular-weight (LMW) glutenins and reduce high-molecular-weight (HMW) glutenins, further weakening the gluten network [26]. These changes in protein composition under heat stress underscore the importance of selecting and breeding wheat varieties that can maintain a balanced glutenin/gliadin ratio and high protein quality under high temperatures. The development of heat-tolerant wheat varieties should focus not only on yield and thousand kernel weight (TKW), but also on maintaining or improving protein quality to ensure the production of high-quality wheat flour suitable for baking.

This research is novel as it was conducted across three distinct locations in Australia—Narrabri, Merredin, and Horsham—representing a diverse range of growing conditions on a continent larger than Western Europe. These locations encompass a broad spectrum of environments that are likely to be affected by global warming, making the findings relevant to many regions of Australia, China, India, USA, and around the world. Specifically, it highlights the critical effects on glutenins/gliadins ratio under heat stress. These findings offer important implications for breeding strategies aimed at improving heat tolerance in wheat cultivars.

4.4. Future Prospects for Wheat Research

This study provides several important directions for future wheat research. The impact of high temperatures on yield, grain quality, and protein composition emphasises the urgent need for breeding programmes to develop heat-tolerant wheat cultivars. Future research should focus on identifying and incorporating genetic traits that confer heat tolerance, such as enhanced pollen viability, efficient stem reserve mobilisation, and robust carbohydrate metabolism under stress [24]. Moreover, advances in molecular biology and genomics offer new opportunities to understand the genetic basis of heat tolerance in wheat. Techniques such as marker-assisted selection and genomic selection can accelerate the breeding process by allowing for the precise incorporation of heat-tolerance traits into high-yielding cultivars. Additionally, exploring the role of epigenetics in heat stress responses could provide insights into how wheat plants can be better prepared to cope with rising temperatures.

Another important area for future research is the detailed study of protein composition changes under heat stress. Using advanced techniques like size-exclusion high-performance liquid chromatography and mass spectrometry, researchers can gain a deeper understanding of how heat stress affects the structure and functionality of gluten proteins. This knowledge will be crucial for developing wheat cultivars with superior baking quality, even under adverse climatic conditions.

5. Conclusions

This study underscores the impact of high temperatures on wheat yield, thousand kernel weight (TKW), colour, and protein composition. Elevated temperatures reduce yield and TKW, particularly under late sowing conditions, and modify wheat colour and protein balance, thereby affecting flour quality.

The protein quality of highly heat-tolerant varieties, such as Flanker, sown under TOS2 did not decline, and even increased, in the TOS2 hot conditions of 2019. These findings highlight the necessity of breeding heat-tolerant wheat varieties that can maintain both yield and quality under increasing temperatures. Future research should focus on identifying genetic traits associated with heat tolerance, employing advanced molecular techniques, and adopting interdisciplinary approaches to ensure sustainable wheat production in the face of a changing climate.