A Quantitative Acetylomic Analysis of Early Seed Development in Rice (Oryza sativa L.)

Abstract

PKA (protein lysine acetylation) is a critical post-translational modification that regulates various developmental processes, including seed development. However, the acetylation events and dynamics on a proteomic scale in this process remain largely unknown, especially in rice early seed development. We report the first quantitative acetylproteomic study focused on rice early seed development by employing a mass spectral-based (MS-based), label-free approach. A total of 1817 acetylsites on 1688 acetylpeptides from 972 acetylproteins were identified in pistils and seeds at three and seven days after pollination, including 268 acetyproteins differentially acetylated among the three stages. Motif-X analysis revealed that six significantly enriched motifs, such as (DxkK), (kH) and (kY) around the acetylsites of the identified rice seed acetylproteins. Differentially acetylated proteins among the three stages, including adenosine diphosphate (ADP) -glucose pyrophosphorylases (AGPs), PDIL1-1 (protein disulfide isomerase like 1-1), hexokinases, pyruvate dehydrogenase complex (PDC) and numerous other regulators that are extensively involved in the starch and sucrose metabolism, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle and photosynthesis pathways during early seed development. This study greatly expanded the rice acetylome dataset, and shed novel insight into the regulatory roles of PKA in rice early seed development.

Keywords: Rice (Oryza sativa L.); acetylome; early seed development; post-translational modification.

Figure 1

(A) Seed morphology of the pistil (S0), seed at three DAP (Day after pollination) (S3) and seven DAP (S7) from left to right. Scale bar = 2 mm; and (B) Western blot analysis of the acetylation dynamics in early rice seed development by using anti-acetyl lysine antibodies. Equal amount of proteins (20 µg) were used and the sample order from left to right: S0, S3 and S7. Anti-tubulin was used as an internal control for normalization. Molecular mass markers were shown (kDa).

Figure 2

(A) The counts of acetylsites, acetylpeptides and acetylproteins in S0, S3 and S7, respectively; and (B) Venn diagram showing the overlap between our identified acetylproteins and the published acetylomes data in rice suspension cells and young seedlings [20,23], respectively.

Figure 3

(A–F) Motif-X analysis of over-represented motifs around the acetylsites of the identified rice seed acetylproteins: A, (DxkK); B, (Kh); C, (kY); D, (Yk); E, (Fxk); F, (kT); and (G) Icelogo heat map of the 21 amino acid compositions of the acetylated site showing the frequency of the different amino acids in specific positions flanking the acetylated lysine. The different colors of blocks in (G) represent the preference of each residue in the position of a 21 amino acid-long sequence context.

Figure 4

GO analysis of differentially acetylated proteins in terms of: biological process (A); molecular function (B); cellular component (C); and subcellular location (D), and (E) acetylation quantitation heat map of differentially acetylated (DA) proteins in S0, S7 and S3. The sample order from left to right: S0, S7 and S3. Color bar at the bottom represents the log2 acetylation site quantitation values. Green, black and red indicate the low, medium and high acetylation intensity, respectively. The abbreviations in (D) represent as follows: E.R.: endoplasmic reticulum; extr: extracellular matrix; cyto: cytoplasm; mito: mitochondrial; chlo: chloroplast; vacu: vacuole; cysk: cytoplasmic skeleton; cyto_nucl: cytoplasm nuclear; plas: plasma membrane; nucl: nuclear.

Figure 5

Enrichment analysis of DA proteins based on: protein domain (A); and kyoto encyclopedia of genes and genomes (KEGG) pathway (B).

Figure 6

Protein–protein interaction (PPI) network of DA proteins identified in this study. (A) the whole PPI network of all DA proteins; the red circles represent the sub-network groups of DA proteins; (B) sub-network I of DA proteins associated with histone proteins; (C) sub-network II of DA proteins associated with ribosomal proteins; (D) sub-network III of DA proteins involved in glycometabolism and photosynthesis; and (E) sub-network IV of DA proteins associated with the enzymes catalyzing PKA.