Kernel growth
Wheat kernel development was studied from anthesis until 46 days after anthesis (DAA) when maturity was reached. Images of developing spikes and kernels as well as their corresponding kernel sizes and kernel dry weights are shown in Fig. 1. While samples taken 7 to 25 DAA showed rather immature ears, spikes and kernels of samples taken 33 to 46 DAA were close to maturity. Caryopses harvested 7 days after full/late flowering (anthesis, BBCH scale 65-67) were in transition from the watery ripe to early milk stage. Caryopses harvested 18 DAA were in transition from late milk to early dough stage. While samples taken 7 to 18 DAA were still decidedly green, samples harvested 25 DAA were right in the soft dough stage, starting to turn yellow. Ears sampled 33 to 46 DAA had lost all their green color. In general, dry kernel weight was increasing from 4.8 mg/kernel to 45.2 mg/kernel with a maximum of 49.0 mg/kernel at 33 DAA. Simultaneous to dry kernel weight, grain size increased steadily during grain filling and maturation from 13.4 mm2 to 20.8 mm2 with a maximum of 24.8 mm2 at 25 DAA. The slight decrease in kernel size during the later growth stages was corresponding to the final dehydration phase before maturation. Based on kernel growth analyses and compositional monitoring across the eight sampling dates, grain development could be divided into three phases: cell division and expansion (anthesis to 14 DAA), grain filling (14-25 DAA), grain maturation and desiccation (25-46 DAA) (Fig. 2).
Accumulation of proteins during grain development
Protein contents of the developing wheat kernels derived by Dumas and Bradford method as well as RP-HPLC are shown in Table 1 and Fig. 2. RP-HPLC chromatograms displaying the separation of non-gluten proteins upon the eight developmental stages can be found in Additional file 1: Fig. S1. In general, crude protein concentration showed considerable variations (12-18%) in the time frame of this study (i.e. 7 to 46 DAA). In the early stages of grain development crude protein content declined from 17.7% to 12.3%, whereas kernel size and dry kernel weight were increasing enormously (Fig. 2). After reaching a minimum of 12.3%, further protein was accumulated resulting in 14.5% at final maturity. While protein content per 100 g of flour was found to be slightly increasing after an initial decrease (Fig. 2a), protein content on a single kernel basis was steadily increasing in the first phase of grain development until a final protein content of 6.6 mg/kernel was reached (Fig. 2b). Concentrations on a single kernel basis for other traits are listed in Additional file 2: Table S1 and Additional file 3: Table S2. On the contrary, the amount of salt-soluble proteins (albumins and globulins) remained relatively constant among the eight maturation stages with values from 1.2% to 1.9% (Table 1).
Table 1 Protein content, composition and characterization of developing kernels of bread wheat cv. ‘Arnold’.
Trait1
|
Days after anthesis
|
7
|
11
|
14
|
18
|
25
|
33
|
39
|
46
|
PROT
|
17.7a
|
14.7b
|
13.0bc
|
12.3c
|
13.3bc
|
13.6bc
|
13.9bc
|
14.5b
|
ALBGLO
|
1.9a
|
1.4bc
|
1.5bc
|
1.7ab
|
1.3bc
|
1.2c
|
1.6abc
|
1.7ab
|
ATI
|
n.d.
|
0.2d
|
0.4c
|
0.5b
|
0.6b
|
0.6b
|
0.7a
|
0.7a
|
TIA
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
<LOQ
|
79.6b
|
89.7a
|
79.1b
|
1 PROT, crude protein content by Dumas method; ALB/GLO, combined albumin and globulin content by Bradford method; ATI, ATI content by RP-HPLC (values of all traits in g/100 g); TIA, trypsin inhibitory activity (mg trypsin inhibited per kg of sample); n.d., not detected; <LOQ, trypsin inhibitory activity below 40%. Means denoted by a different letter indicate significant differences between sampling dates (p < 0.05).
ATI proteins extracted from wheat kernels were quantified by RP-HPLC. No ATIs could be detected in the first and only marginal amounts in the second developmental stage. Hence, initial ATI accumulation occurred after one week after anthesis. Subsequently, the concentration increased gradually until grain maturity and resulted in a maximum ATI concentration of 0.7 g/100 g at 46 DAA (Table 1).
Characterization of ATIs by MALDI-TOF MS
Sample extracts were analysed by MALDI-TOF MS in order to verify the presence of ATIs. Since ATI aggregates are stabilized by rather low non-covalent forces and thus, easily disrupted by low or high pH, denaturating or organic reagents, ATIs were mainly identified and quantified in their monomeric form. Spectra of the samples are shown in Fig. 3. The α-amylase inhibitor (AAI) standard from wheat (Fig. 3a), purchased from Sigma-Aldrich, showed a strong peak accumulation at around 12-14 kDa and a small peak around 15.5 kDa, which can be assigned as ATIs and CM3 protein. The broad peak at 26-27 kDa represents 2M+H ions of the detected ATIs. Furthermore, distinct signals with lower molecular weights (around 6 kDa and 10 kDa, respectively) were spotted in the AAI standard spectrum, that were resulting from the high amount of impurities present in the AAI standard [13, 14]. The PWG gliadin isolate (Fig. 2b) illustrated a typical gliadin pattern with a majority of α-/ß-gliadins from 30-40 kDa and minor abundance of ω-gliadins from 40-55 kDa. Besides gluten type proteins, the PWG spectrum revealed the presence of ATIs and avenin-like proteins, which was recently confirmed by Lexhaller et al. [15]. As ATIs are rather small proteins that range from 13-18 kDa [13, 16, 17], sample peaks in this area were assigned predominately to ATIs and to other proteins from the prolamine superfamily (e.g. avenin-like proteins) in a minor extent [7]. The first sample from 7 DAA revealed a complete absence of ATIs and other storage proteins. Only an undefined and strong increase of the baseline below 10 kDa was observable, which probably resulted from intermediate peptides and free amino acids [18]. The second sample collected four days later showed weak peak intensities around 13, 16 and 32 kDa, which indicated the biosynthesis of the mentioned proteins. Samples taken 14 and 18 DAA presented a clear onset of ATI accumulation simultaneous to the synthesis of gliadins, which were represented by peaks >30 kDa. Comparison of signal intensities arising from ATIs and gliadins revealed no trend for later developmental stages, which might be influenced by manual sample preparation for MALDI-TOF analysis.
Characterisation of ATI functionality
The biological function of ATIs was described in terms of trypsin inhibitory activity (TIA) by an enzymatic assay [13]. Despite the presence of ATIs at an earlier stage, the onset of enzymatic activity occurred later as the samples taken 7 to 18 DAA showed no detectable TIA (Table 1). An initial inhibitory activity was obtained at 25 DAA, however not quantifiable as the minimum inhibition for a proper quantification (i.e. 40%) was not reached. Samples of later developmental stages showed considerable activities from 79.1 mg/kg to 89.7 mg/kg.
Carbohydrate quantification by HPAEC-PAD
Extraction of soluble mono-, di- and oligosaccharides from the maturing wheat kernels was performed in water without heating. In order to gain further insights into the composition of carbohydrates, the insoluble residues were subjected to a strong acid hydrolysis with 2.5 M TFA. As a result, insoluble carbohydrates were depolymerized to monomers and readily quantifiable by HPAEC-PAD. Changes in carbohydrate compositions of the samples are shown in Fig. 4 and Table 2.
Table 2 Compositional changes in the profile of small water-soluble carbohydrates (SWSC) and non-water-soluble carbohydrates (NWSC) throughout grain development.
Trait1
|
Days after anthesis
|
7
|
11
|
14
|
18
|
25
|
33
|
39
|
46
|
SWSC
|
18.7a
|
8.1b
|
6.7c
|
4.0d
|
2.6e
|
1.7f
|
2.0ef
|
1.7f
|
GAL
|
0.05a
|
0.03b
|
0.03c
|
0.02d
|
0.02d
|
0.02e
|
0.01f
|
0.01f
|
GLU
|
4.82a
|
2.36b
|
1.70c
|
1.09d
|
0.61e
|
0.07f
|
0.05f
|
0.05f
|
FRU
|
8.13a
|
2.94b
|
2.05c
|
1.31d
|
0.68de
|
0.08e
|
0.05e
|
0.05e
|
SUC
|
0.09c
|
0.09c
|
0.09c
|
0.05c
|
0.06c
|
0.59b
|
0.72a
|
0.68a
|
RAF
|
0.24c
|
0.19cd
|
0.13de
|
0.08e
|
0.13de
|
0.38ab
|
0.42a
|
0.32b
|
STA
|
0.02c
|
0.03b
|
0.27a
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
VER
|
2.50a
|
1.29b
|
1.09c
|
0.53d
|
0.07e
|
0.02e
|
0.01e
|
0.01e
|
MAL
|
1.49a
|
0.65d
|
0.92b
|
0.58d
|
0.76c
|
0.32f
|
0.44e
|
0.31f
|
FOS
|
1.33a
|
0.55b
|
0.40c
|
0.34c
|
0.28c
|
0.22c
|
0.28c
|
0.28c
|
NWSC
|
26.0c
|
44.4bc
|
69.1ab
|
83.7a
|
89.4a
|
77.6a
|
80.2a
|
95.3a
|
WU-GAL
|
0.50a
|
0.38ab
|
0.25b
|
0.37ab
|
0.43ab
|
0.55a
|
0.49a
|
0.49a
|
WU-AX
|
5.99c
|
8.32abc
|
11.9ab
|
12.5a
|
9.73abc
|
7.90bc
|
7.62bc
|
7.44c
|
ARA/XYL
|
1.04a
|
0.75a
|
0.49a
|
0.54a
|
0.60a
|
0.83a
|
0.79a
|
0.69a
|
STARCH
|
19.6d
|
35.7cd
|
57.0bc
|
70.8ab
|
79.3ab
|
69.1ab
|
72.1ab
|
87.3a
|
1SWSC, small water-soluble carbohydrates; GAL, galactose; GLU, glucose; FRU, fructose; SUC, sucrose; RAF, raffinose; STA, stachyose; VER, verbascose; MAL, maltose; FOS, short-chain fructooligosaccharides (sum of GF2, GF3 and GF4); NWSC, non water-soluble carbohydrates; WU-GAL, water-unextractable galactose; WU-AX, water-unextractable arabinoxylans (sum of arabinose and xylose after acid hydrolysis); STARCH, starch (values of all traits in g/100 g); ARA/XYL, arabinose/xylose ratio. Means denoted by a different letter indicate significant differences between sampling dates (p < 0.05).
Soluble carbohydrate profile
In general, the total amount of soluble carbohydrates was decreasing throughout grain development from 18.7% to 1.7%. Concentrations of all mono-, di- and oligosaccharides present were decreasing, except of sucrose, which was found only in traces at the beginning of grain development, but showed a final amount of 0.7%. The range of raffinose concentrations remained relatively stable over 46 days, whereas other galacto-oligosaccharides such as stachyose and verbascose could not be detected in later developmental stages or only in very small amounts. The course of changes in glucose and fructose concentrations were almost identical and were the highest during the phase of cell division and expansion (7 DAA) with maximum values of 4.8% for glucose and 8.1% for fructose. During grain development both concentrations decreased significantly resulting in poor amounts of 0.05% for glucose and fructose in the mature kernels. Sucrose concentrations were similar to glucose and fructose decreasing slightly during grain filling, but significantly increasing during grain maturation and desiccation. Due to the opposing behavior of glucose, fructose and sucrose, a shift in the monosaccharide (sum of glucose and fructose) to sucrose ratio could be observed. Besides free glucose and fructose concentrations, changes in the fructan content were found to be the most striking alteration during grain development. The amount of low molecular weight fructans (GF2-GF4) was rapidly decreasing during the milky stage (7 to 11 DAA) from an initial concentration of 1.3 to 0.6% (Table 2). After the milky stage only a minimal reduction to a final amount of 0.3% occurred.
Insoluble carbohydrate composition
As described by Pritchard et al. [19], the sum of arabinose and xylose monosaccharides represents the amount of arabinoxylans. In this case, water-unextractable arabinoxylan (WU-AX) content was determined, which is usually twice that of water-extractable arabinoxylans (WE-AX) in wheat flour [20]. In general, WU-AX concentrations were increasing during grain filling and maturation from 6.0% to 7.4% with a maximum of 12.5% at 18 DAA. Arabinoxylans can be classified according to their molecular structure, which is typically expressed by the arabinose to xylose ratio (ARA/XYL). ARA/XYL of the investigated samples were found between 0.5 and 1.0. Besides WU-AX, low amounts (0.3% to 0.6%) of water-unextractable galactose were detected in the grain samples.
Insoluble wheat starch in the grain samples was represented by the amount of glucose after acid hydrolysis as the first extraction with water should have removed free glucose and β-glucan present in the grain sample. Thus, glucose in the water-insoluble residue derived predominantly from starch. In contrast to the classical method for starch quantification, the approach used in this study might also cover other insoluble glucose-containing polysaccharides, such as cellulose, to a minor extent. Throughout grain filling and maturation starch concentrations were increasing from 19.6% to 87.3% (Table 2). While the amount was enormously increasing during the first two phases of grain development (from 19.6% to 79.3%), samples of the grain maturation and desiccation phase showed rather constant values between 69.1% and 87.3% (Fig. 2a). A similar course of accumulation was monitored on a single kernel basis (Fig. 2b). In general, the course of starch accumulation in the grain was highly correlated to the corresponding dry kernel weight. The crude protein content was also found to be negatively correlated with starch concentrations (Table 3).
Table 3 Pearson correlation coefficients between major kernel components and protein fractions. Probabilities of significance are indicated as superscripted values.
Major compositional characteristics
|
|
Kernel dry weight
|
Crude protein content
|
Starch content
|
Kernel size
|
0.6690.005
|
-0.7550.001
|
0.7090.002
|
Kernel dry weight
|
|
-0.5570.025
|
0.8740.000
|
Crude protein content
|
|
|
-0.7140.002
|
Protein characteristics
|
|
ALBGLO
|
ATI
|
TIA
|
Crude protein content
|
0.4360.092
|
-0.1800.537
|
0.6210.101
|
ALBGLO1
|
|
0.2390.411
|
0.2330.579
|
ATI
|
|
|
0.6120.107
|
1 ALBGLO, sum of albumins and globulins; ATI, amylase-trypsin inhibitor; TIA, trypsin inhibition activity