Transcriptome Sequencing and iTRAQ of Different Rice Cultivars Provide Insight into Molecular Mechanisms of Cold-Tolerance Response in Japonica Rice

Abstract

Background: Rice (Oryza sativa L.) is one of the most important crops cultivated in both tropical and temperate regions. However, it has a high sensitivity to cold stress and chilling stress limits its nitrogen uptake and metabolism. To identify the genes and pathways involved in cold tolerance, specifically within nitrogen metabolism pathways, we compared gene and protein expression differences between a cold-tolerant cultivar, Dongnong428 (DN), and a cold-sensitive cultivar, Songjing10 (SJ).

Results: Using isobaric tags for relative or absolute quantification (iTRAQ) with high-throughput mRNA sequencing (RNA-seq) techniques, we identified 5549 genes and 450 proteins in DN and 6145 genes and 790 proteins in SJ, which were differentially expressed during low water temperature (T_w) treatments. There were 354 transcription factor (TF) genes (212 downregulated, 142 upregulated) and 366 TF genes (220 downregulated, 146 upregulated), including 47 gene families, differentially expressed in DN under control (CKDN) vs. DN under low-T_w (D15DN) and SJ under control (CKSJ) vs. SJ under low-T_w D15SJ, respectively. Genes associated with rice cold-related biosynthesis pathways, particularly the mitogen-activated protein kinase (MAPK) signaling, zeatin biosynthesis, and plant hormone signal transduction pathways, were significantly differentially expressed in both rice cultivars. Differentially expressed proteins (DEPs) associated with rice cold-related biosynthesis pathways, and particularly glutathione metabolism, were significantly differentially expressed in both rice cultivars. Transcriptome and proteome analysis of the nitrogen metabolism pathways showed that major genes and proteins that participated in γ-aminobutyric acid (GABA) and glutamine synthesis were downregulated under cold stress.

Conclusion: Cold stress conditions during reproductive growth, resulted in genes and proteins related to cold stress biosynthesis pathways being significantly differentially expressed in DN and SJ. The present study confirmed the known cold stress-associated genes and identified new putative cold-responsive genes. We also found that translational regulation under cold stress plays an important role in cold-tolerant DN. Low-T_w treatments affected N uptake and N metabolism in rice, as well as promoted Glu metabolism and the synthesis of ornithine and proline in cold-sensitive SJ.

Keywords: Cold tolerance; Japonica rice; Proteome; Transcriptome.

Fig. 1

Phenotypic and physiological responses of two rice cultivars with low-T_w treatment during reproductive growth. a. The plots of cold water treatment in the field b, Plants of Dongnong428 (DN) and Songjing10 (SJ) cultivars under low-T_w treatment at the full heading stage. c, Nitrogen (N) concentration, and nitrate reductase (NR), glutamate synthase (GOGAT), glutamine synthetase (GS), glutamate dehydrogenase (GDH), aspartate aminotransferase (GOT), and alanine aminotransferase (GPT) activities in the control (CK) plants and after 15 d of the low-T_w treatment. d, Differences in the amino acid contents of the CK and 15 d low-T_w treatments of DN and SJ

Fig. 2

Comparison of the protein and transcript abundances in the roots of the two rice cultivars. a, Congruency between the detected transcripts and the proteins of the rice endosperm. b, Number of differentially expressed proteins in the roots (1.2-fold change with P value < 0.05). c, Number of differentially expressed genes in the roots (absolute value of log2 (FC ≥1) with FDR ≤0.01)

Fig. 3

Enrichment analysis of the cold responsive transcriptome and proteome changes in the two rice cultivars. a, KEGG enrichment analysis of the differentially expressed genes and proteins presented in a bubble chart. b, GO enrichment analysis of the differentially expressed genes and proteins presented as a heat map

Fig. 4

Comparative transcriptome analysis of the two cultivars revealed cold tolerance mechanisms. a, Venn diagram of the downregulated differentially expressed genes in Dongnong428 (DN) under control (CKDN) vs. DN under low-T_w (D15DN) and Songjing10 (SJ) under CKSJ vs. D15SJ. b, Venn diagram of the upregulated differentially expressed genes in CKDN vs. D15DN and CKSJ vs. D15SJ. c, GO enrichment analysis of the specific CKSJ vs. D15SJ differentially expressed genes

Fig. 5

Comparative proteome analysis of the rice cultivars revealed cold tolerance mechanisms. a, Venn diagram of the downregulated differentially expressed proteins in Dongnong428 (DN) under control (CKDN) vs. DN under low-T_w (D15DN) and Songjing10 (SJ) CKSJ vs. D15SJ. b, Venn diagram of the upregulated differentially expressed proteins in CKDN vs. D15DN and CKSJ vs. D15SJ. c, Protein-protein interaction analysis of the specific CKSJ vs. D15SJ differentially expressed genes, performed using Cytoscape

Fig. 6

Identification of the transcription factors (TFs) responding to cold responses

Fig. 7

Expression differences in the amino acid metabolism pathways of the transcriptome and proteome. a, Amino acid metabolism pathway in KEGG pathway database. b, Differentially expressed genes in the amino acid metabolism pathway of the transcriptome. c, Differentially expressed proteins in the amino acid metabolism pathway of the proteome