3.2 Quantitative expression of rice grain proteins under elevated temperature
In order to further explore the regulation mechanism of the effects of increased temperature on rice quality, we conducted the dynamic proteomic analysis of the rice grains in the early stage of grain filling and a total of 23968 unique peptides and 5872 unique proteins (Supplementary Table 1). The expression and annotation proteins identified in each period were listed and Pearson correlation analysis showed the repeatability of these protein samples was above 97%. Furthermore, we enriched all proteins with COG and GO to perform functional analysis, and results showed that the effect of elevated temperature was mainly in regulating the translation, post-translational modification, protein conversion and signal transduction during the grain-filling stage.
Diﬀerentially expressed proteins (DEPs) were defined as proteins with a FOLD CHANGE ≥2 and p VALUE < 0.05. In the present study, DEPs were identified at least twice in three biological replicates and had the same change trend. Based on these criteria, 112 DEPs were found in ET-3d (3d after heading under elevated temperature treatment) and CK-3d (3d after heading under normal temperature treatment) groups, of which 66 were upregulated and 46 were downregulated (Figure 2). In ET-6d and CK-6d treatments, 118 DEPs were identiﬁed, of which 51 were up-regulated and 67 were down-regulated. Comparing to the CK-9d, 65 proteins were up-regulated and 201 proteins were down-regulated in the ET-9d. In ET-12d and CK-12d treatments, 144 DEPs were identified, of which 59 were up-regulated and 85 were down-regulated. In addition, 200 DEPs were found during the 15d after heading, including 30 up-regulated and 170 down-regulated proteins. The volcano plots and protein annotation of DEPs in ET and CK (3d, 6d, 9d, 12d and 15d) treatments are shown in Figure 3 and Supplementary Table 2. According to the GO enrichment analysis, the prominent GO terms for CC enriched by five stages were the cell, cell part, organelle. Identified proteins were predominantly distributed in metabolic process, cellular process and single-organism process. Based on the molecular function, the DEPs were mainly classified into catalytic activity, binding, transporter activity and structural molecule activity (HT vs CK 3d, 6d, 9d 12d). The top GO MF categories that were enriched by HT-6d and CK-6d DEPs, including the catalytic activity, binding, enzyme regulator activity and transporter activity (Figure 4).
Differentially expressed proteins were further classified into five stages with KEGG pathway. Diﬀerentially metabolic process were defined as pathway with p VALUE < 0.05. The proteome of both the treatments revealed changes in major metabolic pathways. The major metabolic pathways in HT-3d and CK-3d were photosynthesis-antenna proteins, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450, photosynthesis, axon guidance, retinol metabolism (Figure 5). In HT-6d and CK-6d, the main pathways were homologous recombination, AMPK signaling pathway, inositol phosphate metabolism, plant hormone signal transduction, NF-kappa B signaling pathway, ether lipid metabolism, MAPK signaling pathway-plant. Among these, the metabolic pathways enriched in HT-9d and CK-9d mainly include mannose type O-glycan biosynthesis, porphyrin and chlorophyll metabolism, tryptophan metabolism, ABC transporters, phenylpropanoid biosynthesis, isoflavonoid biosynthesis, other types of O-glycan biosynthesis, limonene and pinene degradation. Pathway enrichment analysis of HT-12d and CK-12d DEPs identiﬁed signiﬁcantly enriched in ribosome, mitophagy-yeast, homologous recombination, C5-Branched dibasic acid metabolism. Moreover, two major metabolic pathways, fructose and mannose metabolism, and indole alkaloid biosynthesis were identified in HT-15d and CK-15d. Results suggested that temperature had significantly different regulatory effects on different stages of grain development.