Analysis of grain filling rate
The results showed that the cultivar Xiangzaoxian 42 significantly surpassed the cultivar Xiangzaoxian 24 in grain-filling rate. The grain-filling process was well fitted by the logistic equation for both cultivars, showing a determination coefficient (R2) of 0.991 and 0.987 for Xiangzaoxian 24 and Xiangzaoxian 42, respectively (Fig. 1a). The grain-filling rate, calculated according to the logistic equations, was more than 2.0 times higher in Xiangzaoxian 42 than in Xiangzaoxian 24 during the period of 0–3 days after full heading (Fig. 1b).
Protein profiles revealed differentially expressed proteins between the two rice cultivars during the early grain filling stage
To further uncover the molecular mechanisms underlying the early grain filling period in the two rice cultivars, TMT and LC-MS/MS were employed to characterize the proteomic profiles of grains during the early ripening period (3 days after full heading). Quality control filtering resulted in a total of 8143 and 6808 highly reproducible proteins that could be quantified in the Xiangzaoxian 24 and Xiangzaoxian 42 cultivars, respectively. These highly reproducible proteins were implemented in the analysis of differentially abundant proteins during the early period of grain filling. Protein profiling analysis identified 219 differentially expressed proteins in grains between Xiangzaoxian 24 and Xiangzaoxian 42 at 3 days after full heading. The molecular weight of those proteins ranged between 5.51-123.03 kDa. Number of peptides for each protein ranged between 1–27 peptides. Out of those 219 differentially expressed proteins, 112 were up-regulated in the cultivar Xiangzaoxian 24 and down-regulated in the cultivar Xiangzaoxian 42, while the remaining 107 proteins were down-regulated in the cultivar Xiangzaoxian 24 and up-regulated in the cultivar Xiangzaoxian 42 (Suppl. Table 2). The down- and up-expression ratio of the differentially expressed proteins ranged between 0.11–55.55.
GO and KEGG annotations of deferentially expressed proteins
All deferentially expressed proteins were annotated to the Gene Ontology (GO) database and classified based on their biological process, cellular compartment and molecular function to the GO database. Deferentially expressed proteins covered all three functional categories, i.e., biological process (BP), cellular compartment (CC) and molecular function (MF) (Fig. 2).
The BP category mostly involves metabolic processes, cellular processes, and single-organism process. Proteins with differential abundance levels assigned to CC category are mainly implicated in catalytic activity and binding. The most prominent proteins in the MF category includes cell, membrane and organelle (Fig. 2).
Analysis of the biological metabolic pathways of the 219 differentially expressed proteins using the KEGG databases revealed that those proteins are mainly associated with general function prediction (20), post-translational modifications, protein turnover, chaperons (17), energy production and conversion (12), carbohydrate transport and metabolism (12) and secondary metabolites biosynthesis, transport and catabolism (9) (Fig. 3). Comparing the differential expression of proteins involved in carbohydrate transport and metabolism and secondary metabolites biosynthesis between the two cultivars indicated that they are detected in the Xiangzaoxian 42 cultivar regarding the biological process (Suppl. Table 3).
Functional enrichment of differentially expressed proteins
After the classification of differentially expressed proteins into different categories, the -log10 (P-value) approach was employed to determine their abundance. According to subcellular location, proteins exhibited increased expression during the early ripening period of grain filling stage are mainly enriched in the chloroplasts, cytoplasm, nucleus, mitochondria and plasma membrane, while proteins exhibited downregulated expression are mainly enriched in chloroplast, cytoplasm, extracellular compartments, mitochondria, nucleus and plasma membrane (Suppl. Table 2).
Classification of the identified proteins based on subcellular location
Subcellular localization annotation was implemented to determine the abundance of differentially expressed proteins in each subcellular location (Fig. 4; Suppl. Table 2). Proteins associated with chloroplasts, cytoplasm and nucleus were mainly induced during the early ripening period of grain filling in rice. Noteworthy, differential expression of proteins was detected in the plasma membrane, extracellular compartment and mitochondria as well during the grain frilling period. Subcellular localization indicated that 1, 2, 3, 3, 10, 10, 14, 36, 48 and 92 differentially expressed proteins are localized to the vacuolar membrane, endoplasmic reticulum, cytoskeleton, peroxisome, mitochondria, plasma membrane, extracellular compartment, nucleus, cytoplasm and chloroplast, respectively. Protein domain analysis indicated that Alpha/Beta hydrolase fold, Glycoside hydrolase superfamily and Glycoside hydrolase, catalytic domains were the top three upregulated protein domain groups where they mapped 8,7 and 6 proteins, respectively. Meanwhile, for the downregulated proteins, START-like and aspartic peptidase domains were the most abundant groups (Fig. 5; Suppl. Table 3).
Functional enrichment-based clustering
The KEGG pathways enrichment analysis of differentially expressed proteins identified 22 significantly enriched pathways in the highly expressed proteins including starch and sucrose metabolism, galactose metabolism, fatty acid biosynthesis, fatty acid elongation, MAPK signaling pathway plant, fructose and mannose metabolism, amino acids metabolism, Cyanoamino acid metabolism, oxidative phosphorylation. Likewise, 22 significantly enriched pathways in the low expressed proteins including starch and sucrose metabolism, glycolysis, biosynthesis of secondary metabolites, glycerophospholipid metabolism, pantothenate and CoA biosynthesis, pyruvate metabolism, amino sugar and nucleotide sugar metabolism and amino acids (purine, pyrimidine, glycine, serine, threonine, arginine, proline, tryptophan and glutathione) metabolism were identified (Fig. 6; Suppl. Table 3).
To further identify specific protein families, protein functional domain clustering of the previously identified differentially expressed proteins was analyzed. Proteins with decreased expression levels are enriched in RmlC-like cupin, Zinc finger UBP-type, AMP-binding enzyme C-terminal, START-like, Lysozyme-like and Catalase-like domains. Meanwhile, proteins with elevated expression levels are enriched in NAD(P)-binding, Glycoside hydrolase superfamily, Glycoside hydrolase-catalytic, FAE1/Type III polyketide synthase-like, AMP-binding enzyme C-terminal, NmrA-like, Pyruvate/Phosphoenolpyruvate kinase-like, Glutathione S-transferase, Lysozyme-like, Isopenicillin N synthase-like, Non-haem dioxygenase N-terminal and Alpha/Beta hydrolase fold domains (Fig. 7; Suppl. Table 3).
According to transformed Z score, quantile-based analysis classified proteins into four groups (Q1, Q2, Q3, and Q4) when ranked by -log10 (P-value). Each quantified protein was then assigned to the respective quantile. Accordingly, quantified proteins were allocated to four quantiles Q1 (> 1.3), Q2 (1.2–1.3), Q3 (1/1.2), and Q4 (< 1/1.3) (Fig. 8a&b). Q1 proteins group are enriched in the hydrolase activity acting on glycosyl bonds of the MF protein category. The Q2 proteins are enriched in the lipid transport of BP protein category, and metal ion binding and cation binding of the MF protein category. Proteins belong to the Q3 group are enriched in formaldehyde catabolic process and hexose metabolic process of the BP protein category, and transferase activity, transferring glycosyl group, S-formylglutathione hydrolase activity and ATP-phosphoribosyl transferase activity of the MF protein category. However, the Q4 group of proteins are enriched in regulation of proteolysis and negative regulation of hydrolase activity of the BP protein category and peptidase inhibitor activity, peptidase regulator activity, ADP binding, endopeptidase regulator activity, endopeptidase inhibitor activity, enzyme inhibitor activity, enzyme regulator activity and molecular function regulator of the MF protein category (Fig. 8a&b).
KEGG pathway clustering for four protein groups (Q1, Q2, Q3, and Q4) was also carried out (Fig. 8d). Q1 proteins group are enriched in the Ubiquinone and other terpenoid-quinone biosynthesis KEGG pathway. The Q2 proteins group are enriched in the Pentose and glucuronate interconversions and biosynthesis of secondary metabolites pathways. The Q3 proteins group are enriched in the biosynthesis of secondary metabolites pathway. However, no KEGG pathways assigned to the Q4 proteins group (Fig. 8c).
Protein domain-based clustering revealed that the Q1 group of proteins are enriched in Bifunctional inhibitor/plant lipid transfer, START-like, Glycoside hydrolase superfamily and Glycoside hydrolase catalytic domains. The Q2 proteins group are enriched in Mitochondrial carrier, Carboxyl transferase and Bifunctional inhibitor/plant lipid transfer domains. The Q3 proteins are enriched only in the Alpha/Beta hydrolase fold domain. However, proteins of the Q4 group are enriched in Alpha/Beta hydrolase fold domain, DUF1618 and Ankyrin repeat-containing domains (Fig. 8d).
Functional protein interaction networks of differentially abundant proteins
The STRING database v10.5 identified 22 functional protein interaction networks, 9 of which are downregulated while the remaining 13 are upregulated (Fig. 9). Seven protein interaction networks are located in the chloroplast, 6 interactions are located in the cytoplasm, 5 interactions are located in the nucleus, 2 interactions are located in the mitochondria and one interaction located in each of cytoskeleton and extracellular compartment (Fig. 8; Table 1). Seven interaction proteins belong to the chloroplast network, including 4 downregulated proteins, i.e., glutathione S-transferase, heterogeneous nuclear ribonucleoprotein A1/A3, small subunit ribosomal protein S17e and Xylanase inhibitor and 3 upregulated proteins, i.e., Glutathione synthase, Transferase CAF17, mitochondrial and Tropinone reductase I. The cytoplasm protein interaction networks comprise one downregulated interaction, i.e., dUTP pyrophosphatase, and 5 upregulated interactions, i.e., peptide chain release factor subunit 1, small subunit ribosomal protein S2e, large subunit ribosomal protein L24e, tyrosine aminotransferase and glycine hydroxymethyl transferase. The nucleus protein interaction networks include two downregulated interactions, i.e., dUTP pyrophosphatase and translation initiation factor 4A, and two upregulated interactions, i.e., translation initiation factor 3 subunit B, large subunit ribosomal protein L19e and large subunit ribosomal protein L27Ae (Fig. 8; Table 1). Additional information regarding functional protein interaction networks are shown in Suppl. Table 3.
Table 1
Differentially abundant proteins involved in the identified functional protein interaction networks.
Protein accession
|
Regulated Type
|
Gene name
|
Subcellular localization
|
KEGG Gene
|
B8AZE9
|
Up
|
OsI_18978
|
chloroplast
|
GT; Glutathione synthase, substrate-binding, eukaryotic
|
A2WQ51
|
Down
|
OsI_01983
|
chloroplast
|
GST; glutathione S-transferase
|
A2WN71
|
Down
|
OsI_01299
|
chloroplast
|
HNRNPA1_3; heterogeneous nuclear ribonucleoprotein A1/A3
|
B8ALF3
|
Down
|
OsI_09682
|
chloroplast
|
RP-S17e; small subunit ribosomal protein S17e
|
B8B294
|
Up
|
OsI_21522
|
chloroplast
|
IBA57; transferase CAF17, mitochondrial
|
A2XEZ0
|
Up
|
OsI_10905
|
chloroplast
|
TR1; tropinone reductase I
|
A2Y4I3
|
Down
|
OsI_19910
|
chloroplast
|
XI, Xylanase inhibitor
|
A2WYW8
|
Up
|
OsI_05132
|
cytoplasm
|
ETF1, ERF1; peptide chain release factor subunit 1
|
B8B8I7
|
Up
|
OsI_25329
|
cytoplasm
|
RP-S2e; small subunit ribosomal protein S2e
|
B8A8Y8
|
Up
|
OsI_02167
|
cytoplasm
|
RP-L24e; large subunit ribosomal protein L24e
|
B8AFY2
|
Up
|
OsI_06871
|
cytoplasm
|
TAT; tyrosine aminotransferase
|
A2ZJS7
|
Up
|
OsI_38072
|
cytoplasm
|
glyA, SHMT; glycine hydroxymethyl transferase
|
A2XKG3
|
Down
|
OsI_12940
|
cytoplasm
|
DUT; dUTP pyrophosphatase
|
A2 × 2I8
|
Down
|
OsI_06407
|
nucleus
|
EIF4A; translation initiation factor 4A
|
B8BIC4
|
Up
|
OsI_34716
|
nucleus
|
EIF3B; translation initiation factor 3 subunit B
|
A2XIU7
|
Up
|
OsI_12360
|
nucleus
|
RP-L19e; large subunit ribosomal protein L19e
|
A2XHV1
|
Up
|
OsI_11989
|
nucleus
|
RP-L27Ae; large subunit ribosomal protein L27Ae
|
A2 × 1H8
|
Down
|
OsI_06059
|
nucleus
|
RP-L27Ae; large subunit ribosomal protein L27Ae
|
B8B7U0
|
Down
|
OsI_26563
|
mitochondria
|
RP-S13e; small subunit ribosomal protein S13e
|
A2YEZ0
|
Up
|
OsI_23687
|
mitochondria
|
GLDC; glycine dehydrogenase
|
B8BPG2
|
Up
|
OsI_38205
|
cytoskeleton
|
DHFR-TS; dihydrofolate reductase / thymidylate synthase
|
A2Y7Y4
|
Down
|
OsI_21150
|
extracellular
|
CPVL; vitellogenic carboxypeptidase-like protein
|