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.