Spraying exogenous regulators improves breadmaking quality and modifies the spatial distribution of gluten protein

ABSTRACT Bread is one of the most important processed foods of wheat flour worldwide. Breadmaking quality is determined by grain protein content and its composition and is highly influenced by fertilization management. This study investigated the effects of spraying different combinations of exogenous regulators on breadmaking quality of the flour. Three spraying schemes including Potassium glutamate+Zinc+Urea (G), Methionine+ Zinc+Urea (M), Cysteine+ Zinc+Urea (C) were screened out to improve the yield and total protein contents in the flour. In addition to protein, the amounts of gliadin, glutenin, glutenin subunits, and gluten were elevated in the treatments listed above. Exogenous regulators, on the other hand, increased bread volume by 14% to 17%. In comparison to G and C, M treatment more efficiently increased the protein quality of inner endosperm rather than aleurone, resulting in superior gluten quality and breadmaking quality of the flour. Our results provide an optimizing combination for improving the quality of bread wheat and demonstrate the varied modulations of exogenous regulators on the spatial distribution of protein in wheat grain.


Introduction
Wheat is one of the most important food crops in the world. In 2019, the global output of wheat was about 765 million tons (FAO 2020). Due to the high amount of human consumption of wheat products, the quality of wheat is becoming a key component in the development of the economy and the improvement of the quality of life.
Breadmaking quality, which depends on the viscoelastic properties of dough, is one of the crucial qualities for hard wheat. It is determined by the strength of gluten (from weak to strong gluten) and is affected by the protein content of flour (Ma et al. 2021). Gluten, a colloidal protein unique to wheat flour, is composed of glutenin and gliadin. The quantity and proportion of gliadin and glutenin determine the quality of gluten. Polymeric glutenins form a continuous network that provides strength and elasticity to the dough (Ewart 2010), while the monomeric gliadins are believed to act as plasticizers of the glutenin polymeric system. In terms of breadmaking, an appropriate balance between dough viscosity and elasticity/strength is required. Glutenin is built of high molecular subunit glutenin subunits (HMW-GS) and low molecular subunit glutenin subunits (LMW-GS) that in the grain. Our findings offer an optimizing combination for improving bread wheat's quality as well as in-depth knowledge of how protein gradients and bodies emerge in wheat grains following exogenous regulator spraying.

Experiment design
The experiment was conducted in a wheat field within rice-wheat rotations (2015)(2016)(2016)(2017) in the Yangtze River Basin. The site is at the Suining Experimental Station, Xuzhou, Jiangsu Province, P. R. China (latitude 34°26′ N, longitude 117°20′ E). The climate in this area is typically subtropical humid monsoon. According to the FAO system classification (FAO 2006), the soil was a clay and the properties of the soil at the 0-20 cm depth of each wheat season are listed in Table SA1. The temperature and precipitation during the wheat growing seasons were shown in Table SA2.
Yannong 19, a widely grown bread wheat (Triticum aestivum L.), was used in this study. Wheat seeds were sown on 27th October 2015 and 24th October 2016, respectively. The plot size was 3 m × 5 m with a sowing density of 180 seedlings m −2 and a row space of 0.25 m. For all plots, pure nitrogen, phosphorus and potassium were applied 240 kg ha −1 , 120 kg ha −1 and 120 kg ha −1 , respectively, per season. The field experiment was laid out in a completely randomalized design, with three replicates for each treatment. Eight spraying treatments including (NH 4 ) 2 SO 4 (N), Zinc +Urea (Z), Serine+Urea (Ser), Serine+Zinc+Urea (S), Glutamine+Zinc+Urea (Gln), Potassium gluta-mate+Zinc+Urea (G), Methionine+Zinc+Urea (M), Cysteine+Zinc+Urea (C) were designed and water was sprayed as a control. The amounts of exogenous regulators are shown in Table 1. The amounts of amino acids and urea were calculated in proportion to 6.96 kg of pure nitrogen sprayed per hectare. The amount of trace element zinc was 2.5 kg ha −1 , and the spraying concentration was 0.3%. The amount of water used for each plot was 1.25 kg. A consistent application of exogenous regulators was made at 5 and 7 following days flowering, respectively. To prevent leaf damage from the bright sunlight and high temperatures, spraying was done around 4pm. The field management was carried out in accordance with local practices. The wheat was harvested on 23rd May 2016 and 20th May 2017.

Soil sampling and analysis
Soil samples at the 0-20 cm depth were taken by a soil auger (diameter: 3.2 cm) before wheat sowing. A total of 25 samples were taken and combined into one composite for analysis. The soil was sun-dried before crushing to pass through a 4-mm mesh sieve. Phosphorus and K in soil were extracted and measured with Mehlich-3 extractant and analysis methods, respectively (Mehlich 2008). Saturated paste extraction of 1:1 soil to deionized water was used for soil pH. Available N (the sum of NH 4 + and NO 3 − ), ammonium-N and NO 3 -N were extracted using 2 M KCl (Norman and Bremner 1965) and analyzed using a Lachat QuikChem AE automated flow injection analysis system (Lachat Instruments, Milwaukee, WI, U.S.A). Organic matter and Total N were determined using Carlo Erba NA-1500 dry combustion analyser (Milan, Italy).

Grain milling and pearling
Mature grains were harvested, dried and kept for 2 months. After hand-cleaning and adjusting the moisture to 14.0% on dry base, one kilogram of grains was milled with a Buhler experimental mill Buhler Equipment Engineering Wuxi Co.,Ltd.,China) and sieved at 100-mesh. In addition, the grains were pearled into seven fractions from the surface to the center using the Foodstuff Machine (Streckel and Schrader, Germany) following previous reports with a minor modification (Paola et al. 2011). Briefly, the pearling fractions were collected as flour enriched in husk (P1,7%), aleurone layer (P2, 6%), subaleurone layer (P3, 7%), outer endosperm (P4, 10%), middle endosperm (P5-P6, 20%), and inner endosperm (P7, 50%). The P7 was milled with a universal high-speed grinding machine. Each pearling fractions were sieved at 80-mesh.

Contents of protein and protein components
Protein content was measured on whole kernels by Near Infrared Transmission (DA7250, Perten, Sweden) using the calibration WH0003 with Kjeldahl N. The contents of protein fractions (albumin, globulin, glutenin and gliadin) were determined by the micro-Kjeldahl method of AACC 46-13.01 (AACC 2004).

Secondary structure of gluten protein
Gluten was extracted and purified according to Kieffer, Schurer, Kohler and Wieser (Kieffer et al. 2006). Secondary structures of gluten proteins were studied by Vertex 70 Fourier transform infrared spectroscopy (FTIR) according to Wang et al. (Wang et al. 2013). The spectra were recorded under the same conditions as the background. The data were processed by OmnicV6.1 and Peak Fit 4.12 software.

Glutenin macropolymer (GMP) content
GMP content was measured by the method of Weegels et al. (Weegels et al. 1996). Briefly, 50 mg flour was suspended in 1 ml of SDS (1.5%) solution and then the mixture was centrifuged at 15,500 g at 20°C for 30 min. The sediment was washed twice with SDS solution (1.5%). Then the sediment was dissolved in 2 ml NaOH (0.2%) for 30 min. Afterwards, 3 ml Biuret reagent was added to the solution to evaluate the N content for further calculation of GMP content.

Quantifications of HMW-GS and LMW-GS
Total HMW-GS and LMW-GS were extracted and separated by SDS-PAGE according to our previous methods (Yue et al. 2007). Glutenin extract (10 μl) was loaded in each lane. Quantifications of HMW-GS and LMW-GS were conducted by analysis software QUANTITY ONE. During the quantification procedure, a standard protein (Cat NO. 1610373, Bio-Rad, U.S.A) with given concentration was separately loaded in 5, 10 and 15 μl volumes in three lanes on the same gel. The standard proteins were used to give a standard curve of known concentration. The content of each HMW-GS and LMW-GS in each lane was then quantified.

Free sulfhydryl groups and disulfide bonds
The contents of free sulfhydryl groups and disulfide bonds were quantified according to the method of Riener et al. with minor modifications (Riener et al. 2002). One hundred milligram of flour was fully mixed with 4 ml 6 M guanidine hydrochloride in Tris-HCl buffer (pH 8.0). For free sulfhydryl groups, 2 ml of mixture abovementioned was taken out and mixed with 3 ml guanidine hydrochloride and 40 μL of 10 mM Ellman's reagent. Absorbance at 412 nm was read after 15 min. For total sulfhydryl groups, 1 ml of mixture abovementioned was taken out and mixed with 0.1 ml β-mercaptoethanol, followed by incubation at 40°C for 1 h. After precipitating protein with trichloroacetic acid, pellet was washed by cold acetone twice and was dissolved in UA buffer (8 M urea and 150 mM Tris-HCl, pH 8.0). Absorbance at 412 nm was read after mixing with 40 μL of 10 mM Ellman's reagent for 15 min. The disulfide bond content was calculated according to Equation (1).

Content of gluten
Contents of wet and dry gluten, and gluten index were determined following the AACC 38-12.02 procedure (AACC 2004) with the Glutomatic 2200, the Centrifuge 2015 and the Glutork 2020 (Perten instruments AB, Stockholm, Sweden).

Scanning electron microscopy of gluten
Starch granules were removed with the Glutomatic 2200 (Perten instruments AB, Stockholm, Sweden). Purified gluten was freeze-dried, sputter coated with gold-palladium and observed at 10kV by the Quanta 200 SEM (FEI Company, Eindhoven, The Netherlands).

Determination of relative areas of protein bodies (PBs)
The stems flowering on the same day were tagged. The grains attached to the tagged stems were separately collected at 20 days after anthesis (20DAA). Then, 2 mm cubic blocks were cut by crosssectioning from wheat caryopses from the central spikelets of the ears. The specimens were first fixed by 2.5% glutaraldehyde in 0.1 M phosphate buffer and 1% paraformaldehyde in a 0.05 M sodium cacodylate buffer solution (pH 7.2) for 3 h. The blocks were washed, dehydrated through an ethanol series of 30-100% and embedded in Spurr's low-viscosity embedding medium. The sections of 1 µm thickness were cut with a glass knife on the Leica Ultracut R (Germany), and were stained with Coomassie brilliant blue for 4 min. The sections were visualized and photographed with Leica DMLS microscopy (Germany). The relative areas of PBs of five representative micrographs were determined using Image-Pro Plus 6.0 software.

Bread-making procedure and quality test
Bread was prepared according to the China National Standard (GB/T 14,611-2008), 'Inspection of grain and oils -Bread-baking test of wheat flour -Straight dough method'. Instrumental Texture Profile Analysis (TPA) was carried out with a TA.XT.plus texture Analyzer (Stable Micro System, Godalming, UK), equipped with a cylinder probe P-Cy25S. Bread samples were cut into slices of 1.5 cm thick. TPA was carried out using Exponent (Version 6.1.9.0) from Stable Micro Systems Ltd. Software. The selected settings were pre-test speed of 2 mm s −1 , test speed of 1 mm s −1 , posttest speed of 2 mm s −1 , 50% deformation of the sample, trigger force of 10 g (AACC 1996). The texture attributes (hardness, springiness, cohesiveness, chewiness, and resistance) of bread crumbs were measured by TPA.

Statistical analysis
All data were subjected to one-way ANOVA using the SPSS (Statistical Product and Service Solutions) Version 22.0. ANOVA mean comparisons were performed in terms of the least significant difference (LSD), at the significance level of P < 0.05.

Yield, protein content and protein secondary structure
Concerning CK, all the exogenous spraying treatments increased wheat yield, with M and G being the most pronounced, which increased yield by 8.08% and 5.69%, respectively ( Table 2). The yield increment in exogenous spraying treatments was ascribed to an increment in 1000-grain weight. Namely, M and G increased 1000-grain weight by 9.73% and 6.86%, respectively, compared to CK. Since spraying treatments were conducted after anthesis, no significant difference was observed in the spike number and the grain number.
The contents of total protein showed large variations between the treatments (Table 2). Overall, exogenous spraying increased protein content in flour concerning CK, with an increment ranging from 3.1% to 9.5%. The increments of protein content in G, M and C were 8.8%, 9.5% and 8.5%, respectively, which were more pronounced than the other spraying combinations. The protein secondary structure, which is closely related to the processing quality of flour, was further studied ( Table 2). The most abundant secondary structures in proteins from wheat gluten were α-helix, βsheet and β-turn, accounting for approximately 70% of the total protein secondary structures. Exogenous spraying increased the contents of β-sheets while decreasing the contents of α-helix significantly. Of the eight treatments, the rising impacts of G, M, and C treatments on yield were the most significant, as were their regulating effects on protein content. Thus, G, M, and C were chosen for additional analysis after considering the yield indices and the protein content throughout the course of two growing seasons collectively (Table 2 and Table SA3).

Protein components and gluten morphology
The protein in wheat flour consists of four main components: albumin, globulin, gliadin and glutenin. No significant difference was observed in albumin and globulin contents between treatments (Table 3). However, in the three exogenous spraying treatments, gliadin content separately increased Note: N, Z, Ser, S, Gln, G, M, C and CK indicate the treatment of (NH 4 ) 2 SO 4 , Zinc+Urea, Serine+Urea, Serine+Zinc+Urea, Glutamine +Zinc+Urea, Potassium glutamate+Zinc+Urea, Methionine+Zinc+Urea, Cysteine+Zinc+Urea and water respectively (see details in Table 1). All data were subjected to one-way analysis of variance (ANOVA) to determine the significant differences between treatments. Small letters in the same row indicate significant differences at P < 0.05.  Table 1). All data were subjected to one-way analysis of variance (ANOVA) to determine the significant differences between treatments. Small letters in the same row indicate significant differences at P < 0.05. HMW-GS, high molecular weight glutenin subunits. LMW-GS, low molecular weight glutenin subunits. GMP, glutenin macropolymers. -S-S-, disulfide bonds. -SH, free sulfhydryl groups.
by 13.8%, 7.6% and 13.8%, and glutenin content increased by 12.8%, 15.7% and 10.2% concerning CK, indicating that the contents of gliadin and glutenin in the treatments with exogenous regulators contributed to the increase of total flour protein. In addition, the total amino acid contents were increased from 2.4% to 8.4% (Table 3). The increments observed in gliadin, glutenin and total amino acid contents were inconsistent with those of protein contents. Glutenin macropolymer (GMP) is the largest glutenin polymer, which mainly determines the strength of dough and the volume of bread (Don et al. 2003). Overall, the contents of GMP, HMW-GS and LMW-GS showed fairly high increments in comparison with CK. Namely, three exogenous spraying treatments increased GMP from 21.9% to 40.8%. Similarly, HMW-GS and LMW-GS contents were both elevated by more than 10% in exogenous spraying treatments. That is, compared to CK, the contents of HMW-GS and LMW-GS were increased by 15.1% (C) to 22.4% (M) and 12.7% (C) to 20.7% (M) compared to CK. It is worth noting that HMW-1Dx5, which is reported to be crucial for gluten strength and baking quality (Horvat et al. 2002), increased by 26.6% (G) to 47.6% (M) than in CK. Disulfide bond, which connects HMW-GS and LMW-GS, also increased in three spraying treatments, but only reached a significant level with an increment of 17.6% in M. In addition, exogenous regulators increased the content of free sulfhydryl groups in wheat flour by 7.40-10.87%.
Exogenous spraying significantly increased wet and dry gluten content and gluten index (Table 3). Compared with CK, wet gluten content increased by 19.0% and dry gluten content increased by 16.7% in M treatment. Exogenous spraying increased the gluten index by 2.6% (C) to 5.2% (G). Utilizing scanning electron microscopy, we examined the morphology of wheat freeze-dried gluten in order to visually evaluate the impacts of exogenous spraying on the gluten network. As shown in Figure 1a, the pores of gluten varied in size and showed non-uniform distribution in CK. In contrast, the pore size was smaller and the distribution of pores was relatively uniform, resulting in a denser gluten network in exogenous regulator treatments.  Table 1). Data are means ± SD.

Baking quality
To assess if exogenous regulator spraying increases breadmaking quality, we baked bread ( Figure 1b) with white flour obtained from different treatment and evaluated texture parameters by TPA (Figure 1c). Bread volume increased in a range from 1.14 (G) to 1.17 (M) folds in exogenous regulator spraying treatments than in CK. Hardness and chewiness of bread slices were reduced by all exogenous regulator spraying treatments in comparison with CK, while no significant difference was observed in springiness, resilience and cohesiveness between treatments (Figure 1c).

Contents of protein, GMP, glutenin subunits in different pearling fraction
To explore how the exogenous regulators modified the protein distribution in grain, we pearled the grains into seven layers from outer to inner (P1 to P7) and quantified the contents of protein and its components in each layer. The variation trend of total protein content in different parts of wheat grains was first increased and then decreased. The protein content in P3 layer (mainly subaleurone) was the highest, while that in P7 layer was the lowest, and the protein content in P5-P7 layer decreased significantly (Figure 2). Application of exogenous regulators significantly increased the protein contents in flour from each layer of grains with an exception of P7 in treatment C. As can be seen from the increments of protein in each layer, the treatment of M had the most significant effects among different treatments, especially on total protein in the inner layer of the grain (P6-P7) (Figure 2). The content of GMP peaked at the P2 (aleurone), then dropped down gradually from  Table 1). Data are means ± SD.
P2 to P7, which was consistent with protein distribution. Different exogenous regulators had varied effects on GMP content in different layers of the grain. For example, among three treatments, G treatment had the greatest increase in GMP content in P3 layer, while M treatment could significantly increase GMP content of inner endosperm (P7 layer) ( Figure 2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS−PAGE) was used to separate and determine the contents of HMW-GS and LMW-GS. A representative SDS-PAGE diagram of both HMW-GS and LMW-GS in each pearling fraction is shown in Figure SA1. Overall, contents of HMW-GS and LMW-GS showed a unimodal curve from the outermost to the innermost layer, peaking at P3 layer (Figure 2), which were in line with the curve of protein content. Spraying exogenous regulators improved the contents of HMW-GS and LMW-GS in different parts of wheat grain. M treatment had a significant advantage in increasing the contents of HMW-GS and LMW-GS in P5 to P7 layers compared with the other two exogenous regulators. G treatment showed better regulation effects on HMW-GS and LMW-GS in the outer layer (P1) compared with M and the C treatments. The increments of both HMW-GS and LMW-GS in different layers were relatively balanced from P1 to P5 under C treatment.

Protein body distribution and morphology at 20 DAA
To observe the distribution of protein bodies in the endosperm, the section of developing caryopsis at 20DAA was stained with coomassie brilliant blue (Figure 3b). The protein bodies in five areas including dorsal outer endosperm (DO), dorsal inner endosperm (DI), abdominal outer endosperm (AO), abdominal middle endosperm (AM), and abdominal inner endosperm (AI) were quantified ( Figure 3a) and expressed as a percentage of the corresponding area (Figure 3c).  Table 1).
Overall, the size of protein bodies of DO was significantly larger than that of DI ( Figures 3B and C). In the abdomen of caryopsis, the area of protein bodies gradually decreased from the endosperm cells in the outer endosperm cells to the central endosperm cells. After spraying the exogenous regulator, the area of protein body increased significantly compared with that of the control group. Different spraying treatments showed spatial preferences in regulating the size of protein body. All three treatments consistently increased protein body area of AO, while the protein bodies of AM were not modified by spraying treatments. More protein bodies accumulated in DO under G treatment, while the more protein body accumulation in AI could be perceived visually under M treatment.

Discussion
Bread has been one of the major constituents of the human diet for several thousand years, and wheat is by far the most important cereal in breadmaking. Therefore, it is of great importance to produce flour with the best desired qualities (i.e. high protein quality and proper gluten strength). In this study, a variety of spraying schemes were adopted, and finally, the mixed treatment of potassium glutamate, methionine and cysteine with urea and zinc, respectively, showed optimal regulating effects on protein contents of bread wheat (Table 2 and Table SA3). The primary building blocks for protein synthesis are amino acids. Exogenous foliar spraying can ensure a sufficient supply of amino acids, enhance protein synthesis, and increase the protein content in grains since there is a high need for amino acids in wheat during grain filling (Popko et al. 2018). It has been proved that the plant's uptake of zinc is significantly positively correlated with the absorption of nitrogen (Kutman et al. 2011). A significant quantity of zinc elements entered wheat grains as zincnicotinamide (Zn-NA) after zinc was sprayed on the leaves. Zn-NA catalyzed the conversion of NA into amino acids and proteins in the grains, increasing the protein content in wheat grains (Yilmaz et al. 1997). In addition, urea, as an important nitrogen fertilizer, can be directly absorbed by wheat leaves as nitrogen nutrition, and it also acts as a softener to the cuticle, thus improving the absorption and utilization of other nutrient elements by plants (Bremner 1995). Therefore, it is not surprising that the combination of amino acid, zinc and urea increased the protein content and breadmaking quality significantly in our study.
The baking quality of bread depends on several factors. First, the volume of bread made by wheat flour is linearly related with flour protein content and the secondary structure of gluten protein (Johansson et al. 2001). Spraying treatments in this study had a positive effect on the total amount of protein (Table 2). Consistently, the bread volumes were increased significantly with exogenous spraying (Figure 1c). The dough that has a higher proportion of intermolecular β-sheets plus antiparallel β-sheets and lower proportion of α-helix had greater gluten strength. Cultivars containing a higher amount of β-sheets resulted in dough with higher the dough stability, which is beneficial for breadmaking quality (Gao et al. 2016). Here, though the contents of intermolecular βsheets decreased with exogenous sprayings, either the sum of antiparallel β-sheets and intermolecular β-sheets or the contents of all β-sheets increased while the contents of α-helix decreased significantly under spraying treatments, which were consistent with enhancing dough index (Table 2) and denser gluten matrix (Figure 1a).
Second, because this protein fraction grows substantially more than the non-gluten protein fraction with increasing grain protein content, wheat flour breadmaking performance is linearly related to the gluten protein concentration. In the current study, spraying treatments greatly increased the amounts of gliadin and glutenin in the flour, which in turn increased the amount of gluten (Table 2). Third, gluten quality, e.g. the ratio of glutenin/gliadin and HMW-/LMW-GS, is crucial for breadmaking quality since an adequate balance of viscosity and elasticity/strength is required. Here, we did not observe significant changes in glutenin/gliadin and HMW-/LMW-GS in the G and C treatment, wheres both ratios showed a small increase in the M treatment (Table 3). Therefore, the improvement of the breadmaking quality was mainly ascribed to the increments of protein and gluten protein contents under exogenous regulator treatments.
Interestingly, though both G and M showed similar regulating effects from the view of grain (Table 2 and Table SA3), M showed better regulating effects on the quality indices of flour (Table 3). The mechanism of exogenous regulators on flour quality was explored from a spatial view. G showed more significant modifications on flour from outer layers, and this regulating effect was similar to previous reports on protein gradients affected by nitrogen. He et al. compared the spatial patterns of proteins and found this gradient was much steeper in the grain grown at higher amount of nitrogen (He et al. 2013). Zhong et al. also reported that protein contents in outer layers (aleurone and outer endosperm) were prior to being modulated by nitrogen topdressing timing (Zhong et al. 2018). However, during the milling process, outer layers (aleurone, pericarp, testa and part of outer endosperm) containing high protein content are always separated from starchy endosperm, and the purest flour fractions milled from the starchy endosperm occupied around 75% of the grain weight. Hence, enhancing the protein content of the inner layer of the grain is an economic solution for alleviating the 'loss' of protein during commercial milling. In contrast with G and previous reports, spraying Met, zinc plus urea has significantly positive effects on total protein, GMP and LMW-GS in the inner layer of wheat grains (Figures 2 and 3). In comparison with C and G, the optimal breadmaking quality of flour obtained from M could be explained by a more positive response of inner layers to spraying M treatment.
In conclusion, the foliar application of exogenous regulators had significant positive effects on wheat protein quality and gluten structure, and the spraying combination of methionine + urea + zinc showed the most desirable breadmaking quality of white flour. The responses of protein content and glutenin subunit content in different layers of the grain varied under different exogenous regulators treatments, which provides new insights into regulating the quality of white flour through modifying the protein spatial distribution of the grain.