Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)-Based Comparative Proteome Analysis of the Response of Ramie under Drought Stress

Abstract

In this study, we conducted the first isobaric tags for relative and absolute quantitation (isobaric tags for relative and absolute quantitation (iTRAQ))-based comparative proteomic analysis of ramie plantlets after 0 (minor drought stress), 24 (moderate drought stress), and 72 h (severe drought stress) of treatment with 15% (w/v) poly (ethylene glycol)6000 (PEG6000) to simulate drought stress. In our study, the association analysis of proteins and transcript expression revealed 1244 and 968 associated proteins identified in leaves and roots, respectively. L1, L2, and L3 are leaf samples which were harvested at 0, 24, and 72 h after being treated with 15% PEG6000, respectively. Among those treatment groups, a total of 118, 216, and 433 unique proteins were identified as differentially expressed during L1 vs. L2, L2 vs. L3, and L1 vs. L3, respectively. R1, R2, and R3 are root samples which were harvested at 0, 24, and 72 h after being treated with 15% PEG6000, respectively. Among those treatment groups，a total of 124, 27, and 240 unique proteins were identified as differentially expressed during R1 vs. R2, R2 vs. R3, and R1 vs. R3, respectively. Bioinformatics analysis indicated that glycolysis/gluconeogenesis was significantly upregulated in roots in response to drought stress. This enhancement may result in more glycolytically generated adenosine triphosphate (ATP) in roots to adapt to adverse environmental conditions. To obtain complementary information related to iTRAQ data, the mRNA levels of 12 proteins related to glycolysis/gluconeogenesis in leaves and 7 in roots were further analyzed by qPCR. Most of their expression levels were higher in R3 than R1 and R2, suggesting that these compounds may promote drought tolerance by modulating the production of available energy.

Keywords: comparative proteome analysis; drought stress; isobaric tags for relative and absolute quantitation (iTRAQ); ramie.

Figure 1

(a) After two-week propagation from stem cuttings, the plantlets of “Huazhu No. 5” were transplanted into half-strength Hoagland's solution for 20 days. Leaf and root samples of the same sizes were harvested at 0 h (b), 24 h(c), and 72 h (d) after materials had been treated with 15% (w/v) PEG6000 to induce drought stress. They were cultured under cool white fluorescent light in 16/8 h (light/dark) with a relative humidity of 50%–70% and temperatures about 25 ± 2 °C in the daytime and 20 ± 2 °C at night. Scale bar = 2 cm.

Figure 2

The correlation coefficients were calculated between two biological replicates. The ratios of quantified proteins were log-transformed and plotted. (a) Leaves; (b) roots. Tags 113 and 115 in Figure 2a represent repeat (1) and repeat (2) of 0 h of leaf treatment with 15% (w/v) PEG6000, respectively. Tags 116 and 117 in Figure 2a represent repeat (1) and repeat (2) of 24 h of leaf treatment with 15% (w/v) PEG6000, respectively. Tags 118 and 119 in Figure 2a represent repeat (1) and repeat (2) of 72 h of leaf treatment with 15% (w/v) PEG6000, respectively. Tags 113 and 115 in Figure 2b represent repeat (1) and repeat (2) of 0 h of root treatment with 15% (w/v) PEG6000, respectively. Tags 116 and 117 in Figure 2b represent repeat (1) and repeat (2) of 24 h of root treatment with 15% (w/v) PEG6000, respectively. Tags 118 and 119 in Figure 2b represent repeat (1) and repeat (2) of 72 h of root treatment with 15% (w/v) PEG6000, respectively. The ratios (L2L3 vs. L1 and R2R3 vs. R1) were then log-transformed and plotted against each other.

Figure 3

The categorization of proteins is based on gene ontology (GO) annotation. The category number is displayed with biological process, cellular components, and molecular functions. y-axis (left) represents percentages of proteins identified, y-axis (right) represents the protein number.

Figure 4

COG function classification of all leaf (a) and root (b) proteins.

Figure 4

COG function classification of all leaf (a) and root (b) proteins.

Figure 5

Hierarchical display of data from differentially expressed protein of leaves under drought stress. Upregulated proteins are in red; downregulated proteins are in green (for interpretation of the color references in the figure legend).

Figure 6

Hierarchical display of data from differentially expressed protein of roots under drought stresses. Upregulated proteins are in red; downregulated proteins are in green (for interpretation of the color references in the figure legend).

Figure 7

Association clustering analysis of differentially expressed proteins and its corresponding transcripts. Protein (left) represents the expression levels of differentially expressed proteins, Transcript (right) represents the expression profile of the corresponding genes encoding differentially expressed proteins. (a) leaf of ramie; (b) root of ramie. Upregulated proteins are in red; downregulated proteins are in green (for interpretation of the color references in the figure legend).

Figure 8

Regulatory changes in the pathway of glycolysis/gluconeogenesis. Colored circle nodes correspond with the ramie genes detected in the isobaric tags for relative and absolute quantitation (iTRAQ) data. (a) upregulated or downregulated genes in leaves; (b) upregulated or downregulated genes in roots. The above network model is generated with a cytoscape web application, based on information gained from up to four levels of functional analysis: fold change of gene/protein, protein-protein interaction, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and biological process enrichment. Circle nodes: genes/proteins; rectangle nodes: KEGG pathway or biological process. Pathways are colored in a gradient color from yellow to blue; yellow indicates a lower p-value, and blue indicates a higher p-value. Biological processes are colored in red. In the case of fold change analysis, genes/proteins are colored in red (upregulation) and green (downregulation). A default confidence cutoff of 400 was used: interactions with a higher confident score are shown as solid lines between genes/proteins; dashed lines indicate otherwise.

Figure 9

qPCR data for the mRNA expression levels of genes for drought-responsive proteins mapped in glycolytic/gluconeogenetic pathway in ramie leaves. The values represent relative mRNA levels against control groups (0 h samples), values of which were all set to 1 unit. Statistically significant differences in gene expression are indicated with asterisks: * p < 0.05.

Figure 9

qPCR data for the mRNA expression levels of genes for drought-responsive proteins mapped in glycolytic/gluconeogenetic pathway in ramie leaves. The values represent relative mRNA levels against control groups (0 h samples), values of which were all set to 1 unit. Statistically significant differences in gene expression are indicated with asterisks: * p < 0.05.

Figure 10

qPCR data for the mRNA expression levels of genes for drought-responsive proteins mapped in glycolytic/gluconeogenetic pathway in roots of ramie. The values represent relative mRNA levels against control groups (0 h samples), values of which were all set to 1 unit. Statistically significant differences in gene expression are indicated with asterisks: * p < 0.05.