3.1 Observation of the microstructure of pericarp at different developmental stages
According to the structural characteristics of wheat caryopsis, we observed two parts, namely, the abdomen region with a symmetrical structure and the opposite dorsal region (Fig. 1). The pericarp structure indicates that the thicknesses of the abdomen and dorsal pericarps at different grain positions vary. Eight DAA (Figs. 1A–C, a–c), the thickness of the pericarp on the abdomen and dorsal followed the order G3 > G1 > G2. The large thickness cleared the late start of development. The change in pericarp thickness and the large cavities in the mesocarp were mainly due to the programmed cell death during the development of the pricarp cells. Fourteen DAA (Figs. 1D–F, d–f), the thickness of the abdomen pericarp still followed G3 > G1 > G2, while that of the dorsal pericarp changed to G3 > G2 > G1, revealing that the dorsal pericarp of G1 developed faster than those of the other grains. Twenty DAA (Figs. 1G–I, g–i), the dorsal pericarp thickness of the three grain positions was significantly thicker than that of the abdomen, but no difference in the pericarp thickness in the same region was observed. Thirty DAA (Figs. 1J–L, j–l), no significant difference in pericarp thickness between the two regions of the three grain positions was observed. The above results show an obvious sequence of starting points for the pericarp development of different grain positions, and the development duration varied, which is closely related to the filling process of the caryopsis at each grain position.
3.2 Microstructure observation of endosperm cells in different developmental stages
Eight DAA (Figs. 2 A, E, I), the endosperm cells of the three grains all appeared as spindle-shaped starch granules, which were squeezed by vacuoles at the edge of the cells. However, the quantity and size of the starch granules were obviously different. G1 displayed the most and the biggest starch granules and a certain swelling, followed by G2 and G3. Furthermore, no PB deposition was observed in G2 and G3. Fourteen DAA (Figs. 2 B, F, J), more large starch grains and small spherical starch grains have accumulated in the three grain positions. However, the number of small starch grains in G1 was the highest and that in G3 was the lowest. Generally, small starch grains are formed by the splitting of large starch grains [16]. In terms of protein, G1 had the largest number of and enlarged PBs, whereas G2 and G3 mostly had small PBs. Twenty DAA (Figs. 2 C, G, K), the number of large starch granules did not change significantly, while the number of small starch granules increased further, and the number of granules in G1 and G3 was significantly greater than that of G2. Thirty DAA (Figs. 2 D, H, L), G1 was nearly filled with amyloplast and PBs, which were also squeezed to fill the gaps between starches. Meanwhile, regular spherical PBs could still be observed at G2, and many gaps existed between the amyloplast and PBs at G3, indicating that the fullness of G3 was significantly less than that of G1 and G2. The above results demonstrate that the development of endosperm cells follows an obvious sequence of grain positions, which ultimately determines the length of development time.
3.3 Analysis of material content and characteristics in mature stage
3.4 Observation of Grain Morphology and Measurement of Agronomic Characters
In this study, the large spike wheat YN19 was selected to separate the three grains in the spikelet as the fourth and fifth grain positions without grains (Figs. 3A–C), and the grain size was measured and analyzed (Figs. 3D–E). The picture (Figs. 3D–E) shows that the grain length and width of G3 were obviously smaller than those of the other two positions, while the length of G1 was slightly larger than that of G2, and the width of G2 was slightly larger than that of G1. Through precise measurement (Tab. 1), no significant difference was observed in the grain size and weight of G1 and G2, while those of G3 are significantly smaller than those of G1 and G2.
Table1
Size parameters of mature grains at different grain positions
Sample
|
Length (cm)
|
Wide (cm)
|
Thick (cm)
|
1000-grain weight(g)
|
G1
|
7.55±0.15 a
|
3.50±0.04 a
|
3.28±0.09 a
|
55.65±1.75 a
|
G2
|
7.60±0.13 a
|
3.57±0.06 a
|
3.25±0.17 a
|
55.95±0.95 a
|
G3
|
7.06±0.14 b
|
3.20±0.05 b
|
2.78±0.05 b
|
42.68±1.98 b
|
The values in the table are the average of the three replicate values, and the same column data with different letters indicates significant difference between the two (p < 0.05). |
3.5 Determination of Amino Acid Content in Grains
In nutrition, amino acids are classified as either essential or non-essential amino acids [46]. Essential amino acids, also known as indispensable amino acids, are a group of amino acids that humans and other vertebrates cannot synthesize from metabolic intermediates. We found evident differences in amino acids content, which are mainly manifested as the highest Aspartice acid, Serine, Glutamic acid, Cryteine, Valine, Methionine, Isoleucine, Leucine, and Histidine content at G2, which ultimately leads to G2 having the highest content of essential and non-essential amino acids (Fig. 4A). The essential amino acids and total amino acid in G1 were lower than those in G3 (Fig. 4B). The result is the parameter difference under unit mass, which is converted to the protein content within a single grain followed the sequence G2 > G1 > G3 (Fig. 4C).
3.6 Determination of starch particle size distribution in different grain positions
The granule size distribution in wheat starch is an important factor affecting the end-use quality. According to the granule size, the endosperm starch was divided into B-type granules (diameter < 9.9 μm) and A-type granules (diameter > 9.9 μm) [47]. As shown in Figure 5A, the volume-type diameter parameters of the different grain positions of wheat show a weak difference between G1 and G2, but G3 exhibited a significant difference between the two ranges, namely, 2.8–9.9 μm and 22.8–42.8 μm. The results indicate that the number of small starch granules in G3 was less than those in G1 and G2, but the number of large starch granules was greater than those of the two. Similar results can be seen from the results of the scanning electron microscopy of starch (Figs. 5B, C, D). Interestingly, the data indicates that A-type, Area average granule size and the Volume average granule size were significantly different among the three positions and followed the sequence G3 > G1 > G2, whereas the B-type starch granule size followed the order G2 > G1 > G3 (Tab. 2).
Table 2
Particle size analysis of starch granules
Sample
|
B-type (< 9.9 μm)
|
A-type
(> 9.9 μm)
|
Area average granule size D[3,2]
|
Volume average granule size D[4,3]
|
G1
|
36.02b
|
63.98b
|
7.043b
|
17.799b
|
G2
|
38.22a
|
61.78c
|
6.995c
|
17.263c
|
G3
|
28.60c
|
71.40a
|
7.572a
|
18.997a
|
The values in the table are the average of three replicate values, and the same column data with different letters indicates a significant difference between the two (p < 0.05). |
3.7 Analysis of the structure of starch in different grain positions
According to previous studies, the structural characteristics of starch are mainly reflected in three aspects: the content of components, the order of surface structure, and the degree of crystallinity. The components are mainly amylose and amylopectin. In this study, the apparent amylose content of G1 was the lowest, followed by G2, and that of G3 was the highest (Tab. 3). 13C CP/MAS NMR spectroscopy is widely used for studying the structure of starch samples, kinetics, and correlation. The single- and double-helix contents formed a crystalline structure, and the amorphous region formed an amorphous structure. Figure 6A indicates that the 13C CP/MAS NMR spectrum of starch has four main resonance peaks (i.e., 103, 82, 73, and 62 ppm) in the range of 50–120 ppm. Software analysis shows that the amorphous starch proportion of G2 was the highest, followed by G1 and G3; the single-helical starch ratio of G3 was the highest, followed by G2 and G1, and the double helix ratio followed the order G3 > G1 > G2 (Tab. 3). The results of Fourier transform infrared spectroscopy show that G1 > G2 = G3 in the ratio of 1045/1022 and G1 < G2 = G3 of the 1022/995 ratio (Fig. 6C, Tab. 3). The crystallinity of starch was analyzed through the X-ray diffraction spectrum. Obvious characteristic peaks were observed at 15°, 17°, 18°, and 23° of the spectrum, which are typically A-type crystal peaks (Fig. 6D). Data processing results show that G3 exhibited the highest relative crystallinity, followed by G1, and that of G2 was the lowest (Tab. 3). The above results indicate significant differences in the order degree of the surface and crystal structures of starch at different grain positions.
Table 3
Relative proportions of starch single helix, double helix and amorphous structure
Sample
|
Amorphous ratio
|
Single-helical ratio
|
Double-helix ratio
|
1045/1022 (cm-1)
|
1022/995 (cm-1)
|
RC (%)
|
AAC (%)
|
G1
|
48.58±0.74b
|
5.18±0.08a
|
46.24±0.71b
|
0.702±0.042a
|
1.132±0.038b
|
14.98±0.10b
|
20.75±1.16 c
|
G2
|
50.21±0.66a
|
5.03±0.22a
|
44.76±0.65c
|
0.612±0.031b
|
1.201±0.033a
|
12.68±0.21c
|
23.53±0.48 b
|
G3
|
45.77±0.81c
|
4.15±0.21b
|
50.08±0.68a
|
0.558±0.025b
|
1.252±0.045a
|
15.45±0.13a
|
25.24±1.02 a
|
The values in the table are the average of three replicate values, and the same column data with different letters indicates significant difference between the two (p<0.05).
|
3.8 Hydrolysis of starch
The hydrolysis process of starch is divided into two stages: the early rapid and late slow hydrolysis stages. The results of the study indicate that the three hydrolysis modes exhibited two stages, namely, the rapid and slow hydrolysis stages (Fig. 7). In the rapid hydrolysis stage, the hydrolysis times of the three granular starches were 0–4, 0–6, and 0–6 days (Fig. 7A), the hydrolysis times using porcine pancreatic alpha-amylase (PPA) were 0–12, 0–24, and 0–8 h (Fig. 7B), and the hydrolysis times through Aspergillus niger amyloglucosidase (AAG) were 0–8, 0–6, and 0–8 h (Fig. 7C). In the same hydrolysis mode, the order of final hydrolysis of starch was G2 > G1 > G3 (Fig. 7).