Among dominant grain cereals produced for human food consumption, common wheat (Triticum aestivum
L.) is one of the most important. Yield and quality of wheat grain are dependent on genotype, soil fertility, growing conditions and crop nutrition. Bread wheat yield potential is largely genetically determined and varies significantly as a result of differences in climate and adequate production systems, which include mineral fertilization [1
]. Utilization of nitrogen fertilization has many benefits. Nitrogen (N) is necessary for wheat growth, and crop yield and quality depend upon a sufficient input of N. A satisfactory supply of N is essential for the grain protein accumulation that is necessary for baking and processing quality [3
]. Grain protein content is an important factor defining wheat end-use quality and usually varies between 6 and 19% and has an impact on the grading of flour quality for traditional bread making and other food products [5
]. In Croatia, in accordance with the “Regulation on quality parameters and quality classes of wheat in the purchase of wheat (2018)”, in the formation of the market price, the final and decisive parameter is protein content (%) and, accordingly, wheat produced exclusively for purchase in the territory of the Republic of Croatia is classified into four qualitative classes: premium (>15%), I class (13.5–14.99%), II class (12–13.49%), III class (10.5–11.99) and IV class (<10.49%). According to their solubility characteristics, wheat grain proteins are classified as albumins and globulins (AGs), gliadins (GLIs) and glutenins (GLUs). GLIs and GLUs are the two main groups of gluten proteins, the endosperm storage proteins, accounting for 60~80% of the total grain proteins. Gluten proteins influence dough rheological properties that determine the processing quality of bread, noodles and cookies [7
]. GLIs belong to the monomeric prolamin fraction, and are classified into three structural groups of polypeptides according to their electrophoretic mobility, α/β-, γ- and ω-GLI. The polymeric glutenin fraction is constituted of high-molecular-weight (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) linked with intermolecular disulphide bonds. The gluten protein accumulation during the grain filling period is largely influenced by environmental growing conditions, particularly N availability. However, the type and distribution of gluten subunits have a strong genetic component [10
]. It is reported that the composition of the protein fractions is more influenced by the N nutrition than by temperature [12
Grain nitrogen content plays an important role affecting the ratio of storage to non-storage proteins, the percentage of the storage protein fractions and, thus, the GLI/GLU ratio and the ratio of HMW-GS/LMW-GS [8
]. To obtain the maximum wheat potential in the field, N is the most limiting nutrient required by plants. The growth of wheat plants begins with protein reserves of the kernel, which serve to maintain the germination and further growth of the seedling until the first leaf appears. After rootlets are developed, additional N is obtained by the rootlet system and at this stage the N uptake is limited by root size. In the Northern Hemisphere, it is considered necessary to administer a first application of N fertilizer before the second leaf emerges. A second important time for N application is the tillering stage, when the N concentration in the tissue impacts the formation of the tillers in each plant. The third N application is the most important and it is critical for biomass formation during the extension stage and later for promoting protein accumulation [4
In Croatia, the annual wheat production was 820,682 t in the last 10 years (2009–2018), with 5.12 t ha−1
]. Since producers in Croatia get a higher price for premium quality wheat (grain protein content above 15%), it is desirable to select high-quality cultivars that offer a competitive advantage by minimizing yield penalty in environments that are not classified as high yielding. A few studies regarding bread-making quality and environmental influence on gluten proteins are being done continuously. However, comprehensive research on nitrogen’s influence in different environments on protein content and composition has not been carried out so extensively. Therefore, the hypothesis and objective of this study is to determine the quantitative changes of quality-related proteins in historical and currently grown wheat cultivars grown in different agro-ecological conditions at two N fertilization levels in order to assess if there is a specific cultivar reaction with nitrogen treatment in terms of their quality potential for breeding and technological purposes.
3. Results and Discussion
The results of ANOVA analysis are shown in Table S2 (Supplementary Materials)
. For all analysed traits, statistically significant differences were found for the main factors (cultivars, N level, year and location) and their interactions, which is in accordance with the results of other authors [17
]. Among the main sources of variation, cultivar was the main contributing factor for the majority of quality-related proteins, therefore, in the data presentation, we further emphasized the differences between cultivars grown under different N treatments. Grain and flour quality of wheat cultivars under low and high N rates are presented in Table 2
. The mean values of the P and WG content for 16 wheat cultivars under low N were relatively low, with means of 11.5% and 22.5%, respectively. Under same low N application, the examined cultivars expressed relatively high gluten strength, with an average GI value of 95. The cultivars OS Olimpija and U-1 showed the highest grain P (13.4% and 13.0%, respectively), while cultivars Graindor, Solehio and Sofru had the lowest (9.9%, 10.2% and 10.3%, respectively). WG ranged from 17.2% (Isengrain) to 26.6% (OS Olimpija), while GI ranged from 89 (U-1) and 99 (Graindor and BC Ljepotica). The P and WG contents were significantly higher under high N levels and were cultivar dependent (Table 2
and Table S2
). Generally, the P (11.5% vs. 12.8%) and WG (22.2% vs. 25.8%) increased by 11.3% and 16.2%, while GI (95 vs. 92) decreased by 3.2%, which is in agreement with Zheng et al. [20
], who also found that processing quality parameters, except gluten index, were remarkably improved after increasing N application. These authors noted that protein content of the four cultivars and four N levels (N0
) increased during two consecutive growing seasons from 10.2% to 13.9% and 9.5% to 12.2%, respectively. Yu et al. [21
], studying six Australian bread wheat cultivars grown under different N treatments over two years at several locations, noted that the genotype was dominant in grain P content in response to N availability, indicating the diversity of cultivars used in the experiment.
The modern cultivars (bred after 1960 in a phase of the Green Revolution) (Table 1
), OS Olimpija, Viktorija and Kraljica, with the highest P, WG and GI, have shown the greatest bread-making quality potential and their increase in the P content under a high N level was 8.8%, 12.5% and 11.0% (Table 2
) and, in accordance with Croatian regulations, belong to the quality class I. It is important to note that all cultivars, except Isengrain and BC Ljepotica, achieved a higher quality class due to the increase in N intake. It is interesting to note that old cultivars (cross-bred until the late 1960s), Libellula, San Pastore and U-1, with P content of 11.6–13.0%, showed a significantly (p
< 0.05) lower GI (89–91) than the modern ones, with a GI from 93 to 99. These cultivars also showed a remarkable increase in P content with increasing N level by 14%, 13.2% and 17.1%, respectively. The old cultivar Bezostaja-1 with 13.7% P under higher N showed the largest decrease in GI (94 vs. 86) as well as cultivars MV Nemere (95 vs. 87) and Libellula (90 vs. 82), meaning that there was a significant decrease in gluten strength under higher N level, which could negatively affect the baking properties of flour. Guerrini et al. [22
] noted that flour from the old wheat cultivars is typically characterized by high yield and poor technological properties and is not suitable for industrial baking but, recently [23
], the old wheat cultivars have been reintroduced in wheat breeding programs as a contribution to biodiversity and nutritional and nutraceutical properties.
As we mentioned before, the protein accumulation during the grain filling period is largely influenced by N fertilization level, however, the composition of protein fractions has strong genetic background [19
]. AG represents approximately 15–20% of the grain proteins and often has structural and metabolic functions with limited impact on the baking properties [27
]. The AG proportion under low N ranged from 13.8% (OS Olimpija) to 22.0% (Isengrain). In relation to N increase (Figure 1
), the AG mean generally decreased by 1.1%, but the N effect on cultivars was not consistent, so, in Bologna, Isengrain and San Pastore, the AG proportion decreased by 15.3%, 11.4% and 10.9%, while in Bezostaje-1, Ingenio and Solehio, it increased by 7.2%, 8.4% and 7.5%, respectively.
Some authors [12
] noted that AG remained nearly unaffected under the higher N level, while Rekowski et al. [29
] reported that late N supply significantly increased AG by 8–9%.
Storage GLI and GLU fractions constitute 80–85% of total grain proteins and contribute to rheological, pasting and textural properties of dough [27
]. Polymeric glutenins predominantly define the dough elasticity while monomeric gliadins are responsible for dough extensibility [30
]. Significant variability among cultivars under a low N level was observed in the proportion of GLI fractions, with the cultivar Isengrain having the lowest proportion of total GLI (40.0%) and the cultivar U-1 having the highest (55%). The proportion of minor ω-GLI ranged from 4.0% (Sofru) to 9.4% (Kraljica), while the proportion of major α-GLI ranged from 20.4% (Isengrain) to 29.5% (U-1) (Table 3
). All GLI fractions were affected by the higher N supply and, generally, the total GLI (46.1% vs. 47.0%), α-GLI (24.2% vs. 25.1%) and γ-GLI (16.0 vs. 16.1) increased by 1.9%, 3.7% and 1.3%, respectively, while ω-GLI (5.9 vs. 5.8) decreased by 1.7%, meaning that the dominant α-GLI reacted the most to the N level increase (Table 3
). Hurkman et al. [33
] and Rossmann et al. [26
] found that α-GLI and ω-GLI proportions increased under higher N levels, while the γ-GLI proportion decreased. Variations in composition of GLI fractions were strongly cultivar dependent (Table 3
and Table S3 in Supplementary Materials
), with the cultivars San Pastore and Sofru achieving the largest increase in total GLI (6.3% and 5.5%, respectively), while in cultivar U-1, the total GLI proportion decreased by 1% under a higher N level. A similar inconsistency was observed for other GLI factions.
The old cultivars Libellula, San Pastore and U-1 under both N levels achieved a significantly higher total GLI and α-GLI compared to the modern ones (Table 3
). De Santis et al. [34
], who studied only gluten proteins, without AG, reported that the old and modern Italian cultivars showed similar α/γ-GLI expression (61% vs. 58%), while a marked decrease in ω-GLI expression was observed in the modern ones (11% vs. 4%), which is not so clear among our cultivars (Table 3
). The composition and quantity of the GLU are the major determinants of wheat bread-making quality because a higher content of GLU is related to an increase in gluten strength that allows the dough to maintain the desired final shape and maintains the desired appearance of the final baking products [31
]. The total GLU proportion under a low level ranged from 27.7% (MV Nemere) to 42.0% (Sofru). The proportion of LMW-GS as a major GLU fraction ranged from 18.4% (San Pastore) to 30.8% (Sofru) and for minor HMW-GS from 7.8% (U-1) to 13.2% (Bologna) (Table 4
Analysis of the data revealed significant effects of N levels on the GLU components and they were greatly cultivar dependent (Table 4
and Table S4 in Supplementary Materials
). In relation to the N increase (Table 4
), the HMW-GS mean increased by 0.9% (10.9% vs. 11.0%), while total GLU (36.2% vs. 35.5%) and LMW-GS (25.3% vs. 24.5%) decreased by 1.8% and 3.0%, respectively, meaning that major LMW-GS reacted most sensitively to increased nitrogen intake. These results are in agreement with others [26
], who showed that proportions of low-S proteins (such as ω-GLI and HMW-GS) increase, while proportions of S-rich proteins (such as LMW-GS) decrease with increasing N fertilization rates. The largest decrease in the total GLU proportion under higher N was observed for the cultivars BC Ljepotica and Srpanjka (5.0% and 5.4%, respectively), while Bologna and U-1 showed an increase in GLU by 3.0%. It has been reported that HMW-GS have a crucial impact on wheat baking quality even though they constitute only 10% of the total grain P. Among cultivars, significant variations between low and high N levels were observed for HMW-GS, ranging from −6.6% (BC Ljepotica) to + 7.5% (U-1) (Table 4
), which is consistent with others who found that the expression of storage proteins as well as HMW-GS is strongly associated with genotype [19
]. Hurkman et al. [33
] analyzed the effect of different temperature regimes with and without fertilizer on individual gluten proteins (quantitative 2-DE) of one cultivar and concluded that under moderate temperature, the majority of individual protein spots of HMW-GS, ω-GLI and some α-GLI increased in the range of 25.2–49.2%, 56.0–107.6% and 21.5–50.2%, respectively, while two LMW-GS spots and a minor γ-GLI spot decreased by 23.1%, 31.4% and −28%, respectively. Xue et al. [37
] reported that globulins, LMW-GS and α- and γ-GLI were more sensitive to split N application, while Zhong et al. [38
] noted that α-GLI and HMW-GS were commonly dependent on nitrogen top dressing timing.
The GLI/GLU ratio is related to the balance of dough strength and extensibility [32
]. The negative influence of a higher GLI/GLU ratio on dough rheological properties is well known [31
]. The GLI/GLU ratio under a low N level varied from 0.98 (Sofru) to 1.98 (U-1) and, under higher N supply, the GLI/GLU ratio was generally increased by 4.6% (1.32 vs. 1.38) (Table 4
). The highest increase in the GLI/GLU ratio was noticed in the cultivars San Pastore, BC Ljepotica and Sofru (10.8%, 9.6% and 9.3%, respectively). In the old cultivars, the total P increase resulted in an increase in the GLI/GLU ratio (San Pastore: 1.72 vs. 1.91 and Libellula: 1.58 vs. 1.68), while the cultivar U-1 retained the highest GLI/GLU value despite its reduction by 3% under higher N (1.98 vs. 1.92). In the modern cultivars under a high N level, the GLI/GLU ratio ranged between 1.08 (Sofru) and 1.50 (Kraljica), except for cultivar MV Nemere which had a high GLI/GLU ratio at both N levels (1.81 vs. 1.87). De Santis et al. [34
] reported that the better technological performance of modern Italian cultivars compared to old ones was found to be due to the presence of superior HMW-GS and LMW-GS alleles and to the differential expression of specific storage proteins and because the higher GI (55.3 vs. 9.6 for old cultivars in the first crop season and 46 vs. 7.5 in the second crop season) observed in modern genotypes was correlated with a decreased GLI/GLU ratio (1.6 vs. 2.6 for old cultivars in the first crop season and 1.8 vs. 2.9 in the second crop season). From a breeder’s perspective, in order to hold their seed market share, there are presently two major concerns: to extend the target regions and to increase the end-use quality [40
]. To fulfill those requirements, there is a practical consequence of the higher cost of breeding programs, due to an increased number of field trials and an increased number of laboratory tests to assess. This means that breeders are faced with the dilemma that increasing the number of trials with additional N levels would increase their costs, but, on the other hand, there is a penalty in terms of missing information about the agronomic and quality performance and responsiveness of new genotypes. Sylvester-Bradley et al. [41
] suggested that we should select for wheat cultivars showing a low N optimum and that are highly responsive to N at lower doses while, simultaneously, high N responsiveness is kept under high N conditions. In our study, N treatment had an effect on both wheat protein content and composition, but not equally on all protein fractions. The effect on GLI was more pronounced than on GLU, while AG were not as increased. Furthermore, there was a cultivar-specific reaction to N treatment. This could offer breeders a blueprint for selecting genotypes more responsive to N treatment by selecting for those protein fractions that contribute to the overall wheat quality under increased N levels, while, on the other side, selecting other protein fractions that could be more important under reduced N regimes. Ideally, such an approach could contribute to the selection of new genotypes with improved stability of quality under different soil fertility and N fertilization regimes.