3.1. Effect of Irrigation and Nitrogen and the Interaction between the Tw, on Protein Content of Sunflower Seed Kernels
We found a significant effect of irrigation (
p < 0.05) and N fertilizer use (
p < 0.05) on the protein content of sunflower seed kernels and a significant interaction (
p < 0.05) (
Table 3).
In the no irrigation treatments (W
0), we observed a significant difference in protein content between N
0, N
52, and N
104 nitrogen application levels in both years (
p < 0.05). This indicated that protein content of seed kernels increased with increasing nitrogen in the absence of irrigation. At the W
45 level of irrigation, we also found a significant difference in protein content amongst all three nitrogen application levels in 2016 (
p < 0.05), whereas in 2017, there were significant differences between the N
0 and N
104 treatments (
p < 0.05) and between the N
52 and N
104 treatments (
p < 0.05). In general, this showed that the protein content of seed kernels increased with increasing nitrogen application, At the W
90 level of irrigation, significant differences in protein content were identified between N
0 and N
52 treatments and between N
0 and N
104 treatments in both years. Again, protein content increased with increasing nitrogen application. Overall, considering mean values, protein content showed an increasing trend with increasing nitrogen application at all irrigation levels; in 2016 and 2017, protein content was highest in W
0N
104, at 25.3% and 24.3%, respectively. The reason for this is that nitrogen is the main component of protein and nucleic acids; therefore, nitrogen fertilizer can promote the synthesis of amino acids in seed kernel proteins [
27]. However, increasing levels of irrigation had a negative effect on seed kernel protein synthesis: as the level of irrigation increased, seed kernel protein content showed a downward trend. In both years, we found a significant interaction between the effect of irrigation and nitrogen application on seed kernel protein content (
p < 0.05;
Figure 1).
The results showed that the protein content of sunflower seed kernels increased with increasing nitrogen application, which is consistent with the findings of previous studies [
28,
29,
30]. Nitrogen is usually a limiting factor for sunflower growth [
9,
13,
23,
31,
32,
33], and different nitrogen fertilizers are required at different stages of reproduction [
34]. Blamey et al. reported that the protein content of sunflower seeds in each season was significantly increased by applying nitrogen fertilizer [
13]. Shoghi-Kalkhoran et al. found that when applying farmyard manure, chemical fertilizer, or a 50:50 combination of the two that the combination had the highest protein content [
16]. These earlier studies focused on different levels and combinations of fertilizers on protein content, whereas we considered the effects of different fertilizer levels in combination with different levels of irrigation on sunflower seed protein content. Under no irrigation treatments, seed kernel protein content increased with increasing nitrogen application. However, water stress is considered to be a key factor influencing the growth, development, and spatial distribution of crops [
7]. The level of irrigation can significantly impact crop growth and quality. Oraki et al. showed that protein content increased with decreasing levels of irrigation [
35]. Jalilian et al. found that a water deficit increased the protein content of sunflower seeds [
36]. Our results are consistent with these previous studies, and showed that different combinations of water and nitrogen have significant effects on the protein content of sunflower seeds.
3.2. Effects of Irrigation and Nitrogen and the Interaction between the Two on Total Amino Acids, and Key Amino Acids Specifically in Sunflower Seed Kernels
In this study, a total of 17 major amino acid components in the kernels of oil sunflower seeds were tested for and all were present. To clarify the spatial correlation amongst the 17 amino acids, we used a systematic method based on Euclidean distance using cluster analysis on the data from samples, and a systematic clustering tree was obtained (
Figure 2).
When the threshold value was 1, the 17 amino acids clustered into four categories: histidine, lysine, tyrosine, threonine, serine, cystine, and methionine; leucine, valine, alanine, proline, isoleucine, glycine, and phenylalanine; aspartic acid and arginine grouped; and glutamic acid. When the threshold value was >2 and <5, the 17 amino acids clustered into three categories: glutamic acid; aspartic acid and arginine; and the remaining 14 in the third category. When the threshold value was >5, the 17 amino acids cluster into two categories: glutamic acid in one category, and the remaining 16 indicators in the other.
According to the test results, glutamate, aspartic acid, arginine, glycine, and valine were relatively high amongst the 17 amino acids from sunflower seed kernels. The correlation analysis of these five main amino acid components is shown in
Table 4.
Differences in the total quantity of amino acids (excluding ammonia) and the quantity of each of the five main amino acids (glutamic acid, aspartic acid, arginine, glycine, and valine) in the sunflower seed kernels sampled from different treatments in 2016 to 2017 were analyzed by ANOVA. The two main factors were amount of irrigation and quantity of nitrogen fertilizer. We also considered the interaction between irrigation and nitrogen (
Table 5). The level of irrigation had a greater effect than nitrogen level on the total amino acid content and on each of the five main amino acids individually (
p < 0.05). From Duncan’s multiple comparisons of means, we found no significant difference between those marked with the same letters; lower case letters represent
p < 0.05 in
Figure 3,
Figure 4,
Figure 5,
Figure 6,
Figure 7,
Figure 8 and
Figure 9.
In the no irrigation treatments (W0), we found significant differences in the total amino acid content of kernels amongst all three nitrogen treatments in 2016 (p < 0.05); in 2017, there were significant differences between the N0 and N52 treatments and between the N0 and N104 treatments (p < 0.05), but no significant difference between the N52 and N104 treatments (p > 0.05). In general, the total amino acid content increased with increasing nitrogen application. At the W45 level of irrigation, results were consistent in both years: there was no significant difference in total amino acid content between the N0 and N52 treatments (p > 0.05), but there were significant differences in the total amino acid content between N0 and N104 treatments and between N52 and N104 treatments (p < 0.05). Overall, we found a trend of increasing total amino acid content with increasing nitrogen application. At the W90 level of irrigation, there was a significant difference in the total content of amino acids amongst all three nitrogen application levels in 2016 (p < 0.05); total amino acid content increased first and then decreased. In 2017, significant differences were identified in the total amino acid content between N0 and N52 and between N0 and N104 nitrogen treatments (p < 0.05), but no significant difference between N52 and N104 treatments (p > 0.05).
Overall, for both years, total amino acid content decreased as the irrigation amount increased from 45 to 90 mm at the N104 nitrogen application level. Therefore, at the N104 level of nitrogen, 45 mm irrigation benefits seed kernel amino acid synthesis, but when irrigation increased to 90 mm, the excess water was detrimental to amino acid synthesis. Natural rainfall during the experimental period was significantly higher in 2016 (101 mm) than in 2017 (50.52 mm). In particular, rainfall from the budding period to the flowering period was 58.40 mm in 2016, and only 15.80 mm in 2017. These rainfall levels were sufficient to meet the normal growth and development needs of the crop in 2016, meaning that the maximum total amino acid content (22.74%) could be achieved in the absence of irrigation at the highest nitrogen application rate (W0N104). However, in 2017, 45 mm irrigation was required at the same nitrogen application rate (W45N104) to achieve a similar total amino acid content (21.59%).
In the no irrigation treatments (W0), results for both years showed that there was a significant difference in glutamic acid content amongst all nitrogen treatments (p < 0.05); glutamic acid content increased with increasing nitrogen application. At the W45 level of irrigation, there was no significant difference in glutamic acid content between N52 and N104 treatments in 2016 (p > 0.05), but there were significant differences in glutamic content between the N104 treatment and the other two treatments (N0, N52) (p < 0.05). In 2017, there were significant differences in glutamic acid content amongst all three nitrogen application levels (p < 0.05). At the W90 irrigation level, we observed significant differences in glutamic acid content amongst all nitrogen treatments in both years (p < 0.05). In 2016, glutamic acid content increased first and then decreased with increasing nitrogen application. In 2017, glutamic content increased with increasing nitrogen application but, compared with the W45 level of irrigation, glutamic acid content decreased by 0.01%. Overall, at all irrigation levels, increasing the amount of nitrogen applied was conducive to the synthesis of glutamic acid. As water hydrolyzes nitrogen in the soil and makes it available for plants, appropriate irrigation is conducive to the synthesis of glutamic acid. However, with higher irrigation (90 mm), glutamic acid content declined because the dissolved is leached from the soil by the excess water, limiting the synthesis of glutamate. For the reasons described previously, differences in natural rainfall in the two years meant that glutamic acid content was the highest in the W0N104 treatment (5.45%) in 2016 and in the W45N104 treatment (5.12%) in 2017.
In the no irrigation treatment (W0), there were significant differences in aspartic acid content amongst all three nitrogen treatments in both years (p < 0.05). Overall, aspartic acid content increased with increasing nitrogen application. At the W45 irrigation level, there were significant differences in aspartic acid content amongst all three nitrogen treatments in 2016 (p < 0.05), with the highest value for the year in the W0N104 treatment (2.26%), but there was no significant difference in aspartic acid content between the N52 and the N104 treatments (p > 0.05). In 2017, we found significant differences in aspartic acid content amongst all three nitrogen treatments, with the highest value for the year in the W0N104 treatment (2.06%). At the W90 level, there were significant differences in aspartic acid content amongst all the nitrogen treatments in 2016; aspartic acid content first increased and then decreased. In 2017, significant differences were observed in aspartic acid content between N0 and N52 and between N0 and N104 treatments, but no significant difference between the N52 and N104 treatments; the trend was observed for increasing aspartic acid content with increasing nitrogen application. At all irrigation levels, the general trend was that increasing nitrogen application was beneficial for the synthesis of aspartic acid. At all nitrogen levels, 45 mm irrigation creased arginine content and was beneficial for the synthesis of aspartic acid. However, when irrigation increased to 90 mm, there was a downward trend in arginine content, likely because nitrogen dissolved in the water and was leached from the soil.
In the no irrigation treatments (W0), we observed a significant difference in arginine content amongst all three nitrogen treatments in both years (p < 0.05). Overall, arginine content increased with increased nitrogen application, indicating increased nitrogen application was beneficial to the synthesis of arginine. At the W45 irrigation level, we found no significant difference in arginine content between the N52 and N104 treatments in both years (p > 0.05) but there were significant differences in arginine content between the N0 treatment and the other two nitrogen treatments (p < 0.05). Arginine content increased with increasing nitrogen in both years and was highest in the W45N104 treatment (2.09%) in 2017. At the W90 irrigation level, there were significant differences in arginine content amongst the three nitrogen application levels in 2016 (p < 0.05). Arginine content first increased and then decreased. In 2017, there were significant differences in arginine content between the N0 and N52 treatments and between the N0 and N104 treatments (p < 0.05), but no significant difference between N52 and N104 treatments (p > 0.05). Overall, the trend was for arginine content to increase with increasing nitrogen application level. At all irrigation levels, the general trend was for increasing nitrogen application to benefit the synthesis of arginine. At all nitrogen levels, irrigation at levels of 45 mm increased arginine content and benefitted the synthesis of arginine. However, when irrigation increased to 90 mm, we observed a downward trend in arginine content, likely because nitrogen dissolved in the water and was leached from the soil.
In the no irrigation treatments (W0), we found significant differences in the valine content amongst all three nitrogen treatments in both years (p < 0.05); valine content increased with increasing nitrogen application in the absence of irrigation. At the W45 irrigation level, there were significant differences in the valine content between the N0 and N52 treatment and between the N0 and N104 treatment in 2016 (p < 0.05), but none in the valine content between the N52 and N104 treatments (p > 0.05). In 2017, we noted a significant difference in valine content amongst all three nitrogen treatments (p < 0.05); overall, valine content increased with increasing nitrogen levels. At the W90 irrigation level, valine content was significantly different amongst all three nitrogen treatments in 2016 (p < 0.05). In 2017, there were significant differences in the valine contents between the N0 and N52 treatment and between the N0 and N104 treatment (p < 0.05), but no significant difference in valine content between the N52 and N104 treatments (p > 0.05). Overall, at the W90 irrigation level, valine content showed a trend of first increasing with nitrogen application and then decreasing in 2016; in 2017, it continuously increased with N application rate. From the perspective of mean valine values, valine content increased with increasing nitrogen application as increasing nitrogen application benefitted the synthesis of valine. However, when irrigation levels increased to 90 mm, glycine content declined at the highest nitrogen application rate because the water leached the nitrogen from the soil. For the reasons described previously, differences in natural rainfall in the two years meant that valine content was the highest in the W0N104 treatment (1.45%) in 2016 and in the W45N104 treatment (1.30%) in 2017.
In the no irrigation treatment (W0), there were significant differences in the glycine contents amongst all three nitrogen treatments in both years (p < 0.05); glycine content increased with increasing nitrogen application. At the W45 irrigation level, there were significant differences in the glycine contents between all three nitrogen treatments in 2016 (p < 0.05); in 2017, significant differences were observed in glycine content between N0 and N52 and between N0 and N104 (p < 0.05), but no significant difference between N52 and N104 treatments (p > 0.05). Overall, we found a trend for glycine content to increase with increasing nitrogen application rate. At the W90 irrigation level, we identified significant differences in glycine content amongst all three nitrogen levels (p < 0.05); in 2017, it was similar, except there was no significant difference between N52 and N104 treatments (p > 0.05). From the perspective of mean glycine values, glycine content increased with increasing nitrogen application. However, when irrigation levels increased to 90 mm, glycine content declined at the highest nitrogen application rate because the water leached the nitrogen from the soil. For the reasons described previously, differences in the natural rainfall in the two years meant that glycine content was the highest in the W0N104 treatment (1.28%) in 2016, and in the W45N104 treatment (1.19%) in 2017.
The results were similar for arginine and aspartic acid; both had the highest values in the W
0N
104 treatment in 2016 and the W
45N
104 treatment in 2017 due to a positive correlation between arginine and aspartic acid (
Table 4). In the cluster diagram, these two amino acids were significantly grouped together. We found significant differences in valine and glycine contents in relation to their interactions with irrigation and nitrogen application. The effect of nitrogen application on valine content was significantly stronger than the effect of irrigation level. Both valine and glycine had their highest levels in the W
0N
104treatmentin 2016 and the W
45N
104 treatment in 2017 due to a positive correlation between them (
Table 4). In the cluster diagram, valine and glycine were classified into one category. Our findings showed that under different levels of irrigation, an increase in topdressings of nitrogen was associated with an increase in total amino acid content, and the contents of glutamic acid and arginine, which is consistent with the study of Steer et al. [
22].
In total, 17 amino acids contributed to the total amino acid content; this did not include ammonia, which was present but analyzed separately. ANOVA showed that irrigation and nitrogen had a significant effect on the ammonia content of sunflower seed kernels, as did the interaction between them (
Table 6). We also performed a multiple comparisons of means (
Figure 9).
In the no irrigation treatments (W0), we found significant differences in ammonia content between the N0 and N52 treatments and between the N0 and N104 treatments (p < 0.05), but not between the N52 and N104 treatments in both years (p > 0.05). Ammonia content increased with increasing nitrogen application. At the W45 irrigation level, there was a significant difference in ammonia content amongst all three nitrogen treatments in both years (p < 0.05); ammonia content increased with increasing nitrogen application. At the W90 irrigation level, ammonia content showed a trend of increasing first and then decreasing in 2016, achieving its highest level in the W90N45 treatment (0.74%). In 2017, significant differences were recorded in ammonia content amongst all three nitrogen treatment (p < 0.05); ammonia content increased with increasing nitrogen application, achieving its highest level in the W90N104 treatment (0.61%). From the perspective of mean values, ammonia content increased with increasing nitrogen application; therefore, nitrogen application favored ammonia synthesis in sunflower seed kernels.
To more clearly identify the impact of the different treatments of irrigation and fertilizer on the quality of sunflower seeds, principal component analysis was performed on all experimental data from 2016 to 2017 (
Figure 10).
The interaction responses of the contents of protein, total amino acids, five main amino acids, and ammonia in sunflower seed kernel to water and nitrogen were the same in different years. For the above eight quality indicators, the difference was significant for different water and nitrogen treatments. The protein content of sunflower seed kernels increased with increasing nitrogen application. Increasing nitrogen application was directly related to increased total amino acid content and the synthesis of the five key amino acids in sunflower seeds.