Chilling stress response in tobacco seedlings: insights from transcriptome, proteome, and phosphoproteome analyses

Abstract

Tobacco (Nicotiana tabacum L.) is an important industrial crop, which is sensitive to chilling stress. Tobacco seedlings that have been subjected to chilling stress readily flower early, which seriously affects the yield and quality of their leaves. Currently, there has been progress in elucidating the molecular mechanisms by which tobacco responds to chilling stress. However, little is known about the phosphorylation that is mediated by chilling. In this study, the transcriptome, proteome and phosphoproteome were analyzed to elucidate the mechanisms of the responses of tobacco shoot and root to chilling stress (4 °C for 24 h). A total of 6,113 differentially expressed genes (DEGs), 153 differentially expressed proteins (DEPs) and 345 differential phosphopeptides were identified in the shoot, and the corresponding numbers in the root were 6,394, 212 and 404, respectively. This study showed that the tobacco seedlings to 24 h of chilling stress primarily responded to this phenomenon by altering their levels of phosphopeptide abundance. Kyoto Encyclopedia of Genes and Genomes analyses revealed that starch and sucrose metabolism and endocytosis were the common pathways in the shoot and root at these levels. In addition, the differential phosphopeptide corresponding proteins were also significantly enriched in the pathways of photosynthesis-antenna proteins and carbon fixation in photosynthetic organisms in the shoot and arginine and proline metabolism, peroxisome and RNA transport in the root. These results suggest that phosphoproteins in these pathways play important roles in the response to chilling stress. Moreover, kinases and transcription factors (TFs) that respond to chilling at the levels of phosphorylation are also crucial for resistance to chilling in tobacco seedlings. The phosphorylation or dephosphorylation of kinases, such as CDPKs and RLKs; and TFs, including VIP1-like, ABI5-like protein 2, TCP7-like, WRKY 6-like, MYC2-like and CAMTA7 among others, may play essential roles in the transduction of tobacco chilling signal and the transcriptional regulation of the genes that respond to chilling stress. Taken together, these findings provide new insights into the molecular mechanisms and regulatory networks of the responses of tobacco to chilling stress.

Keywords: chilling stress; molecular mechanism; phosphoproteome; proteome; tobacco; transcriptome.

Figure 1

The phenotype of tobacco seedlings and workflow for the transcriptome, proteome and phosphoproteome profiling under chilling stress. (A) Phenotype of tobacco seedlings under chilling stress. K326 seedlings at the six-leaf stage were treated at 4 °C for 24 h (24 h), and the seedlings before chilling treatment (0 h) were used as the control. Scale bar=10 cm. (B) Workflow for the transcriptome, proteome and phosphoproteome profiling of tobacco seedlings under chilling stress.

Figure 2

Proteome and phosphoproteome analyses of tobacco in response to chilling stress. (A) Proteomic identification data of tobacco under chilling stress. Prot, the proteome. S, shoot data. R, root data. (B) Phosphoproteomic identification data of tobacco under chilling stress. Phos, the phosphoproteome. (C) Proportion of the Ser, Thy and Tyr sites in phosphoproteins. Left pie chart, shoot data. Right pie chart, root data. (D) The number of phosphorylation sites in proteins.

Figure 3

Analyses of the DEPs and differential phosphopeptide data and significantly different phosphorylation sites in response to chilling stress. (A) DEPs and differential phosphopeptides in the proteome and phosphoproteome, respectively. Proteins and phosphopeptides that significantly differed quantitatively between the experimental and control groups were defined as DEPs and differential phosphopeptides (P < 0.05, FC > 1.5 or FC < 0.67. (B) Volcano plots of quantified phosphorylation sites. (C) Heatmaps of significantly different phosphorylation sites. The phosphorylation sites were filtered for an average FC of > 1.5 or < 0.67 with P < 0.05. (D) Venn diagram of the DEPs and corresponding proteins of the differential phosphopeptides in the proteome and phosphoproteome.

Figure 4

GO and KEGG enrichment analyses of DEPs and corresponding proteins of the differential phosphopeptides in response to chilling stress. GO (A) and KEGG (C) enrichment analyses of DEPs in the proteome. (A), UDP-glucose: glycoprotein glucosyltransferase activity; (B), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase activity; (C), 2-C-methyl-D-erythritol -2,4-cyclodiphosphate synthase activity; (D), 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase activity. GO (B) and KEGG (D) enrichment analyses of corresponding proteins of the differential phosphopeptides in the phosphoproteome. GO and KEGG enrichment analyses were performed with P < 0.05 as the threshold.

Figure 5

Transcriptome analysis of tobacco in response to chilling stress. (A) PCA plots of the transcriptome samples under chilling treatment. Volcano plots of differentially expressed genes (DEGs) in the shoot (B) and root (C). (D) Comparative analysis of DEGs in the shoot and root under chilling stress. Transcripts with an adjusted p-value (padj) < 0.05 and |log₂FC| > 1 were identified as DEGs. (E) Venn diagram of the DEGs in the shoot and root under chilling stress.

Figure 6

Correlation analysis of the transcriptome and proteome of tobacco under chilling stress. (A) GO and (B) KEGG enrichment analyses of pairs of DEGs/DEPs in the shoot. (C) GO and (D) KEGG enrichment analyses of pairs of DEGs/DEPs in the root.

Figure 7

Schematic representation of a possible comprehensive chilling response model in tobacco. DEPs and differential phosphoproteins of the KEGG pathways significantly enriched in the proteome and phosphoproteome, as well as KEGG pathways that are significantly enriched in the transcriptome, are shown. The abundance of DEPs and differential phosphoproteins of the KEGG pathways was indicated with heatmaps. They were generated with scaled log₂FC. The abundance of chilling-responsive kinases and TFs is indicated in red (increased) and blue (decreased). When tobacco seedlings were exposed to chilling stress, the chilling signal was transmitted into the cells, which triggers MAPK cascades that amplify and transmit chilling signals, activate the chilling-responsive transcription factors, induce the chilling-responsive genes, and lead to alterations in the biological processes related to chilling stress, including RNA transcription and processing, protein metabolism, photosynthesis adjustment, redox homeostasis, energy metabolism, and the accumulation of osmoregulatory compounds.