SDS-PAGE and phenotypic analysis of storage proteins in seed endosperm development
The seed setting rate of new double autotetraploid rice is usually very low because of the separation behavior of the homologous chromosome. To study further dynamic changes in protein content and DEPs during different developmental stages, we selected AJNT-4x and AJNT-2x as the research materials. In our research, the seed setting rate of AJNT-4x reached 49.94%. On the other hand, the agronomic traits, such as grain length, grain width, and grain thickness, of AJNT-4x were also better than those of its diploid (Table 1). The grain weight of AJNT-4x increased obviously, and there was a significant difference in grain weight between AJNT-4x and AJNT-2x. In addition, storage proteins in the seed endosperm were divided into albumin, globulin, prolamin, and glutelin fractions according to their solubility. SDS-PAGE analysis showed that our results were the same as those of a previous report. The molecular weights of the albumin and globulin fractions were 14-16 kDa and 26 kDa, respectively. The molecular weights of the prolamin fraction were 10 kDa, 13 kDa, and 16 kDa, but mainly 13 kDa. The molecular weights of the glutelin fraction were 57 kDa, 32 kDa, and 20 kDa; among these molecular weights, 57 kDa was a precursor protein, 32 kDa was an acidic subunit, and 20 kDa was a basic subunit. All the expression levels of the four seed proteins increased gradually with increasing flowering time. The extracted protein fractions were divided into two parts: one was used to measure their contents, and the other was subjected to MS analysis for glutelin protein evaluation.
Dynamic changes in storage protein contents during endosperm development
The contents and proportions of endosperm storage proteins affect the quality of rice (You et al. 2017). In this study, the contents of four storage proteins were measured by the BCA method (Noble and Bailey 2009). The four proteins all began to accumulate, and their contents were almost the same at 5 DAF in AJNT-2x and AJNT-4x.
The accumulation trends of albumin and globulin were almost the same in AJNT-4x and AJNT-2x. From 5 DAF to grain maturity, during the whole endosperm developmental period, the albumin and globulin contents increased only slightly (Figure 1B).
The prolamin content first decreased, followed by a slight increase, and then a rapid increase from 12 DAF. The content reached a high peak at 18 DAF in AJNT-4x, declined again, and finally stabilized. Eighteen days after flowering, slightly later than that in AJNT-2x, the prolamin content reached its maximum of 0.31 mg/mL in AJNT-4x. The prolamin content in AJNT-2x reached a peak of 0.18 mg/mL at 15 DAF. From 12 to 15 DAF, the prolamin content increased much more rapidly in AJNT-4x than in AJNT-2x (Figure 1C). The prolamin content was much higher in AJNT-4x than in AJNT-2x during all the developmental stages, and the prolamin content difference between AJNT-4x and AJNT-2x at maturity was highly significant.
Compared with those of albumin, globulin, and prolamin, the variation range of glutelin was the largest during endosperm development. Glutelin accumulation was almost the same in AJNT-4x and AJNT-2x. There were two rapid accumulation stages: one was from 9 to 11 DAF, and the other was from 18 to 21 DAF in AJNT-4x, presenting two peaks at 11 DAF and 21 DAF (Figure 1D). Then, the glutelin content declined gradually and stabilized. In general, the accumulation rate of glutelin in AJNT-4x was faster than that in AJNT-2x. The glutelin content was highly significantly different between AJNT-4x and AJNT-2x during the rapid accumulation period.
The change in the contents of the four proteins in AJNT-4x was almost the same as that in AJNT-2x, but the total content in AJNT-4x was much higher than that in AJNT-2x because the four proteins accumulated quickly in AJNT-4x (Figure 1E). Glutelin is rich in lysine, which is an essential amino acid for humans, so we further studied changes in the glutelin content during the developmental stages of both AJNT-4x and AJNT-2x. According to the change in glutelin content during endosperm development, samples from several critical developmental stages, namely, samples collected 10, 15, and 20 DAF, were subjected to proteomic analysis by using the iTRAQ technique.
Proteome differential analysis at different endosperm developmental stages between AJNT-4x and AJNT-2x
To investigate differential alterations in glutelin expression during seed endosperm development, glutelin fractions from both AJNT-4x and AJNT-2x endosperm were extracted following a TCA-acetone method (Méchin et al. 2007) and subjected to iTRAQ-based global proteomics analysis by LC-MS/MS (Unwin et al. 2010). A total of 841,375 spectra were generated, of which 38,851 were matched to known peptides from the utilized database. Eventually, 4,880 peptides, 4,047 unique peptides and 1,326 protein groups were identified. Comparative analysis between AJNT-4x and AJNT-2x at 10, 15, and 20 DAF was conducted, and 125, 159 and 78 DEPs were found (fold change>1.2 or<0.83, p-value<0.05) (Table 2). There were many DEPs in the different developmental stages of rice endosperm analyzed. A total of 372 DEPs were measured at 10, 15, and 20 DAF AJNT-4x samples, which was much more than the 184 DEPs detected in the AJNT-2x samples.
Principal component analysis (PCA) showed that the autotetraploid rice endosperm had better separation than diploid endosperm, which indicated that more significant alterations in protein expression occurred in autotetraploid endosperm (Figure 2).
Differentially expressed protein GO analysis for AJNT-4x and AJNT-2x
All DEPs were annotated by GO. Comparing DEPs between AJNT-4x and AJNT-2x at 10, 15 and 20 DAF, it was surprising that no GO functions were mutual among the three comparison groups, but there were 15 common DEPs between 15 DAF and 20 DAF, and only one common DEP between 10 DAF and 15 DAF (Figure 3A), which indicated that endosperm glutelin accumulation was regulated mainly by different DEPs during the late stage, and 15 DAF was a critical regulating point for glutelin accumulation.
The GO enrichment results also showed that the critical functions of DEPs exhibited little overlap at 10, 15 and 20 DAF. For instance, at 10 DAF, all DEPs between AJNT-4x and AJNT-2x endosperm samples were related to metabolic processes (Figure 3B), including those of RNA (GO: 0016072, 0034660), peptides (GO: 0006518), proteins (GO: 0019538) and phosphate (GO: 0019220). At 15 DAF, the DEPs AJNT-4x and AJNT-2x represented a much broader range of regulatory functions, such as growth regulation (GO: 0040007). Metabolic processes or functions were still annotated at 15 DAF, but few proteins for RNA, peptide and protein metabolism were enriched (Figure 3C). At 20 DAF, the number of DEPs between AJNT-4x and AJNT-2x endosperm was decreased compared to that at 10 or 15 DAF (Figure 3D). However, the annotated functions of these proteins were still abundant and mainly enriched in stress response (GO: 0006950), carbohydrate metabolism (GO: 1901135), nutrient reservoir (GO: 0045735) and so on. In summary, differences in protein synthesis and metabolism mainly occurred at 10 DAF, but decreased gradually during later developmental stages. DEPs involved in cell growth and development and amino acid biosynthesis were mainly observed at 15 DAF.
KEGG analysis of differentially expressed proteinsbetween AJNT-4x and AJNT-2x
To investigate metabolic processes and feasible signaling pathways, these DEPs were subjected to KEGG analysis. The KEGG pathway results (Figure 4) indicated that the biological pathways for AJNT-4x endosperm proteins were different from those for AJNT-2x endosperm proteins at 10, 15 and 20 DAF. Ribosomal proteins (Figure S1), which were significantly higher in AJNT-4x than in AJNT-2x endosperm, were the major difference at 10 DAF due to alterations in central carbon metabolism and cellular senescence. At 15 DAF, the most enriched pathway was protein processing in the endoplasmic reticulum (ER) (Figure S2), which contained 12 DEPs, and all of them were enriched in AJNT-4x endosperm compared to their expression levels in AJNT-2x. For example, protein disulfide-isomerase and heat shock protein 70 are both critical for protein folding (Gruber et al. 2006; Wisén and Gestwicki 2008). The biosynthesis and metabolism of amino acids was another important series of pathways enriched at 15 DAF, and the enriched proteins were involved in arginine biosynthesis; alanine, aspartate and glutamate metabolism; tyrosine metabolism, cysteine and methionine metabolism; arginine and proline metabolism; phenylalanine metabolism; and phenylalanine, tyrosine and tryptophan biosynthesis. These amino acid biosynthetic and metabolic pathways were much higher in AJNT-4x than in AJNT-2x (Table S1). This result suggested that not only were protein synthesis and processing more active in AJNT-4x endosperm at 15 DAF but there was also a higher level of amino acid accumulation. Interestingly, two upregulated differential lysine proteins (B8AM24 and B8ARJ0) were found in the lysine biosynthesis process (Figure S3), and four upregulated differential proteins (P37866, Q9S768, A0A0N7KTS9 and B8AEL7) were found to regulate alanine, aspartic acid, and glutamic acid metabolism processes (Figure S4).
In the late period (20 DAF) of endosperm development, the pathways of DEPs were primarily metabolic pathways, including fructose and mannose metabolism, methane metabolism, and starch and sucrose metabolism (Figure S5). However, the most enriched pathway at 20 DAF was the peroxisome pathway (Figure S6), in which 4 DEPs were downregulated in AJNT-4x when compared to their expression levels in AJNT-2x. As a crucial organelle that regulates large numbers of biological processes, for instance, metabolism and development (Hu et al., 2012), the lower level of the peroxisome pathway in AJNT-4x at this stage suggested reduced metabolic and developmental activity compared with that in AJNT-2x endosperm. Interestingly, KEGG pathway analysis also mapped DEPs to protein processing in the ER (Figure S7) at 20 DAF, but two proteins, namely, protein transport protein Sec61 and translocon-associated protein, had lower expression levels in AJNT-4x than in AJNT-2x.
Protein-protein interaction (PPI) network for DEP analysis
The protein-protein interaction (PPI) network is another important tool to interpret proteomics information. DEPs between AJNT-4x and AJNT-2x were annotated, and PPI networks were generated by the String database (Szklarczyk et al. 2015). As shown in the results, most connected and annotated proteins were ribosomal proteins and metabolic proteins (Figure 5). At 10 DAF, half of the DEPs in the PPI networks were ribosomal proteins, and the other half were metabolic proteins. With increasing endosperm development, ribosomal proteins gradually decreased, and metabolic proteins increased. Until 20 DAF, the proportion of ribosomal proteins was reduced to approximately 1/3, and no ribosomal DEPs were found in the PPI network.
WGCNA analysis of co-expression modules
Traditionally, the analysis of proteomics data has focused on DEPs, which were regarded as largely important participants in the comparison group. However, a large amount of “unchanged” proteins (fold change<1.2) and “nonsignificantly changed” proteins (p-value>0.05) might be ignored because of disadvantages in statistics, although they might result in an important impact on the phenotype. Therefore, a WGCNA approach was applied to construct a global co-expression network without any artificial cut-offs (Pei et al. 2017). First, the identified proteins were divided into 5 different modules (Figure 6A), and then GO and KEGG analysis of each module were performed. Briefly, the blue module contained ribosomal and oxidative phosphorylation-related proteins; the brown module contained proteins related to carbohydrate metabolism, including the metabolism of amino/nucleotide sugars, starch, sucrose, fructose and mannose, and glycolysis; the turquoise module contained proteins related to the proteasome and fatty acid biosynthesis; the yellow and gray modules were similar to the blue module but failed to enrich any KEGG pathways (Figure 6B). During the endosperm development process of AJNT-2x, it can be concluded that proteins in the blue and turquoise modules had high expression levels at 10 DAF that then decreased at 15 and 20 DAF. The proteins in the blue module were expressed much higher in AJNT-4x than in AJNT-2x at 10 DAF but had similar levels at 15 and 20 DAF (Figure 6C). These findings suggested that protein expression and oxidative phosphorylation, which provide energy to biological processes, were highly active in the early stage of development but quickly slowed. In contrast, carbohydrate metabolism processes were less active at 10 DAF than at 15 and 20 DAF but then increased in both AJNT-4x and AJNT-2x. Compared with the differences in expression levels between different developmental stages in AJNT-2x, those in AJNT-4x showed more significant differences.
Confirmation of proteomic data by qRT-PCR analysis
To validate the experimental proteomics data, thirteen randomly selected DEPs, including ten upregulated DEPs and three downregulated DEPs (fold change>1.2 or<0.83, p-value<0.05), were analyzed using real-time quantitative PCR, (Table 3). Among them, an ATP binding protein (A0A0N7KMN9) and a cell redox homeostasis protein (B8AJS5) were significantly downregulated, and a glutelin protein (Q40689) and cell growth protein (A3AI97) were upregulated, contrasting with the results of iTRAQ; the results for the other nine proteins were consistent with the iTRAQ data (Figure 7). Thus, the proteomics data were reliable. Among the nine DEPs with qRT-PCR data correlating with those of iTRAQ, eight were upregulated proteins (Table 3). These proteins are very important to plant growth and development. For example, B8AM24 and B8ARJ0 are related to the lysine biosynthetic process, B8AQM6 regulates cell growth by extracellular stimulus, A2ZCE6 is related to endosperm development, and P55857 is a glutelin protein. These proteins will be subjected to functional experiments in subsequent work.