Grain development and SEM observations
In general, grain size and weight in both Jimai 20 and Zhoumai 16 increased gradually from flowering to maturity, but their development rates and grain sizes were different (Figure
A, B). Zhoumai 16 had a larger grain size and higher grain weight than Jimai 20 at all grain developmental stages except the first. SEM observations on both cultivars indicated that starch granules accumulated continuously until grain maturity (Figure
C). As previously observed
], A (diameter >10
μm) and B (diameter <10
μm) starch granules appeared at 6 DPA (147o
Cd) and 11 DPA (252o
Cd), respectively. The size of A granules as well as grain weight increased rapidly from 11 to 15 DPA (252-353o
Cd), but B granules grew only slowly from 11 to 31 DPA. This indicated that the period 11–15 DPA was a key stage for grain starch synthesis and accumulation.
Figure 1 Grain development during five stages (I, II, III, IV and V) in wheat cultivars Jimai 20 and Zhoumai 16. A. Grain morphological development (the red lines represent 2mm). B. Grain weight accumulation. C. SEM images of transverse grain sections (more ...)
Identification, classification and localization of differentially accumulated proteins during grain development in the two cultivars
Grain proteins extracted at the five developmental stages in the two cultivars were separated by 2-DE with broad-range (pH 3-10
L) IPG strips. The proteome profiles were generally similar in both cultivars at all five stages (Figure
). Most of the proteins on the 2-DE gels were distributed in the pH 3–7 range during the earlier (I and II) development stages. The numbers of basic proteins increased considerably from stage III, especially in the last stage. In total, 174 protein spots showed more than two fold differences in abundance, of which 117 representing 84 unique proteins were identified by MALDI-TOF/TOF-MS (Table
). Their peptide sequences are listed in Additional file
: Table S1.
2-DE maps of proteins extracted from the first sample of Jimai 20.
Zhoumai 16 at five stages of grain development.
Differentially expressed proteins identified by MALDI-TOF/TOF-MS at five grain developmental stages in bread wheat cultivars Jimai 20 and Zhoumai 16
According to their functions, the identified proteins were classified into several main groups, including carbohydrate metabolism (31.6%), stress/defense (25.6%), storage proteins (14.5%), protein synthesis/assembly/degradation (7.7%), transcription/translation (7.7%), nitrogen metabolism (5.1%) and signal transduction (3.4%) as shown as in Figure
. More than 80% were identified as enzymes. Proteins related to carbohydrate metabolism contained five subcategories: glycolysis (18%), TCA pathway (5%), lipid and sterol metabolism (3%), starch metabolism (3%) and alcoholic fermentation (2%).
Distribution of the proteins identified during five grain development stages in Jimai 20 and Zhoumai 16. Nine protein groups were categorized based on putative functions.
Among the differentially accumulated proteins, 25 different kinds were represented by two or more spots, and more than 10 isoforms were identified with different molecular masses or isoelectric points, each having two or three protein spots located at different positions on the same gel (Figure
A), such as phosphoglycerate mutase (spots 1 and 2), glucose-1-phosphate adenylyltransferase (spots 41 and 44), triticin (spots 67 and 68), alpha-amylase inhibitor CM16 subunit (spots 98 and 99), monomeric alpha-amylase inhibitor (spots 119 and 120), class II chitinase (spots 112–116) and peroxidase 1 (spots 121, 122 and 123). Some of these isoforms might have resulted from certain PTMs such as phosphorylation.
Figure 5 Protein isoforms and phosphoproteins identified by 2-DE and Pro-Q diamond staining. A total of 16 protein spots were identified as phosphoproteins, in which 5 spots (a-e) were newly identified phosphorylated proteins. Spot numbers correspond to those (more ...)
In order to confirm the presence of phosphorylated proteins with isoforms, grain proteins at stage V were separated by 2-DE and then subjected to Pro-Q Diamond staining (Invitrogen) to detect putative phosphorylated proteins. Many spots stained in different intensities on the gels, indicating they were phosphorylated (Figure
B). Sixteen spots with intense signals in both cultivars were identified by MALDI-TOF/TOF-MS (Table
); their peptide sequences are listed in Additional file
: Table S2. These phosphoproteins were mainly involved in stress and defense and the isoforms were particularly well stained, such as class II chitinase (gi/62465514) at spots 112, 113 and 114, and peroxidase 1 (gi/22001285) at spots 122 and 123 (Figure
Phosphorylated proteins stained by Pro-Q Diamond and identified by MALDI-TOF/TOF-MS during grain filling
Phosphorylated modification sites on serine, threonine and tyrosine were predicted by NetPhos 2.0 Serve
]. Generally, the predicted results were in accordance with those from Pro-Q Diamond dye staining (Table
). For example, class II chitinase (spots 112, 113 and 114, gi/62465514) was predicted to possess 9 serine, 3 threonine and 1 tyrosine phosphorylated modification sites, whereas peroxidase 1 (spots 122 and 123, gi/22001285) had 11, 5 and 2 phosphorylated modification sites of serine, threonine and tyrosine, respectively. Phosphorylated protein staining showed that both proteins had strong staining signals (Figure
Through sub-cellular localization of the identified proteins during grain development, a large number of proteins appeared to locate in the cytosol (Table
). Most of the identified enzymes with higher abundance, and involved in glycolysis-the TCA pathway-nitrogen metabolism were located in the cytosol especially during the first two stages. Until stage III many storage proteins appeared on the endoplasmic reticulum (ER). Stress-related proteins were mainly located in the plastids and were extracellular at all development stages. Additionally, some enzymes involved in starch synthesis also appeared in plastids during the early development stages.
Protein expression profiles during grain development
The expression profiles of the 117 protein spots were investigated by hierarchical cluster analysis (Figure
). Five main expression patterns (A-E) were present and clearly reflected three distinct grain development phases: differentiation (I-II), grain filling (II-IV) and desiccation/maturation (IV-V) as shown in Figure
Hirerarchical clustering analysis of differentially accumulated protein spots in Jimai 20 (left) and Zhoumai 16 (right). Red color: the higher abundance of protein spots; blue color: the lower abundance of protein spots.
Protein expression patterns in Jimai 20 and Zhoumai 16 during five grain developmental stages (A-E).
Expression pattern A included 19 spots in Jimai 20 and 24 in Zhoumai 16 exhibiting down-regulated modes during grain development. For example, triticin precursor (spot 69) was highly accumulated in the first two stages but was down-regulated at grain filling and maturity. Most of the proteins involved in the TCA pathway and glycolysis showed this expression pattern, such as isocitrate dehydrogenase NADP-dependent and its precursor (spots 32 and 33), and phosphoglycerate mutase (spots 1 and 2). Preprotein of glucose-1-phosphate adenylyltransferase (spot 35) also displayed this pattern in both cultivars.
Expression pattern B included the largest proportion of identified proteins and showed up-regulated expression, especially during the late grain development stages. Totals of 48 spots in Jimai 20 and 44 in Zhoumai 16 were in this expression group, which contained most of the storage proteins, many stress/defense-related proteins and two enzymes involved in alcoholic fermentation. In general, storage proteins including globulins (spots 70, 71, 80 and 82), gliadins (spots 74, 75, 76 and 77) and glutenins (spot 78), triticins (spots 67 and 68) and avenin-like protein (spot 73) accumulated significantly at the later developmental stages, but only had trace expression levels during the earlier developmental stages in both cultivars. The same responses also occurred for formate dehydrogenase (spot 38 and 39) involved in alcoholic fermentation, and class II chitinase (spots 112–116) involved in stress/defense pathways. Three spots (121, 122 and 123) were identified as peroxidase 1, of which 122 displayed B expression pattern in both cultivars.
Expression pattern C was the second largest group of identified proteins, represented by 41 spots in Jimai 20 and 40 in Zhoumai 16, many of which included glycolysis and stress/defence-related proteins and showed up-regulated expression at the early development stages, and were then down-regulated with advancement of grain filling and maturity. Representative proteins involved in this expression group were phosphoglucomutase (spot 18) and glucose-1-phosphate adenylyltransferase (spot 41) related to starch synthesis.
Expression pattern D, unlike pattern C, displayed a down to up-regulated expression trend. Only spots 36, 54 and 55 representing two proteins (peptidylprolyl isomerase Cyp2 and cyclophiliwere) showed this pattern. The remaining two spots (26 and 100) in Jimai 20 and three (26, 88 and 100) in Zhoumai 16 accumulated a complicated pattern E that fluctuated during gain development (Figure
Comparative proteome characterization in Jimai 20 and Zhoumai 16 during grain development
Comparative proteomic analysis demonstrated a considerable proteome expression difference between Jimai 20 and Zhoumai 16 during grain development. A total of 20 protein spots co-accumulated in both wheat cultivars, but with different expression patterns (Figure
). For example, starch-rating enzyme glucose-1-phosphate adenyl-transferase (AGPase, spots 44 and 45) displayed A expression patterns in Jimai 20, but C patterns in Zhoumai 16. Spots 121 and 123 were identified as peroxidase I that showed expression pattern C in Jimai 20, and patterns A and B in Zhoumai 16. In addition, 27
K protein (spot 86) and superoxide dismutase (spot 127) gradually accumulated in Zhoumai 16, but showed little change in Jimai 20 during all five stages.
Protein spots with two fold changes, or greater, in abundance at particular times between the two cultivars were considered to be cultivar-different proteins. Twenty seven spots showed cultivar-different during the five development stages and they were mainly involved in carbohydrate metabolism, stress/defense, protein storage and transcription/translation. Among them, spots 8, 56 and 79 (glyceraldehyde-3-phosphate dehydrogenase, cyclophilin and S-type low molecular weight glutenin L4-55) only accumulated in Jimai 20, but were not detected in Zhoumai 16 when subjected to ImageMaster™ 2D Platinum Software analysis. Another two spots, 49 and 120 (aspartate aminotransferase and monomeric alpha-amylase inhibitor), only accumulated in Zhoumai 16. In the remaining protein spots, 14 accumulated in higher abundance in Jimai 20 than in Zhoumai 16, whereas 8 were more abundantly accumulated in Zhoumai 16. For example, important proteins displaying higher levels of expression in Jimai 20 than in Zhoumai 16 included isocitrate dehydrogenase NADP-dependent (spot 33), triticin precursor (spot 69), LMW-s glutenin subunit (spot 78), and replication factor C like protein (spot 91) as shown in Figure
. Some proteins displayed higher expression abundances in Zhoumai 16 than in Jimai 20, for example, phosphoglucomutase (spots 18 and 19).
Differential expression of six protein spots in Jimai 20 and Zhoumai 16 during five grain development stages. Spot 18 (II/III); spot 33 (II); spot 69 (II); spot 78 (V); spot 91 (I); spot 127 (IV).
Transcriptional expression analysis by qRT-PCR
Since wheat grains in stage V were approaching maturity and the reference gene accumulated unstably, the transcriptional expression profiles of ten representative genes from stages I to IV were investigated by qRT-PCR with specific primers ( Additional file
: Table S3). As shown in Figure
, the transcriptional expression patterns of only four protein genes (phosphoglucomutase, thaumatin-like protein, superoxide dismutase and monomeric alpha-amylase inhibitor) in both cultivars and two genes (glucose-1-phosphate adenylyltransferase and alpha amylase inhibitor protein) in Zhoumai 16 were consistent with their protein expression models. The remaining protein genes showed poor consistency between their transcriptional and translational levels. Interestingly, alpha-amylase inhibitor was shown to be phosphorylated when stained by Pro-Q Diamond dye (Table
), a feature that might be responsible for lower consistency between transcriptional and translational expression patterns.
Transcriptional expression profiles of 10 representative protein genes from four grain development stages (I-IV) determined by qRT-PCR.