Comparative Transcriptome Analysis of Grafted Tomato with Drought Tolerance

Abstract

Grafting is a method used in agriculture to improve crop production and tolerance to biotic and abiotic stress. This technique is widely used in tomato, Solanum lycopersicum L.; however, the effects of grafting on changes in gene expression associated with stress tolerance in shoot apical meristem cells are still under-discovered. To clarify the effect of grafting, we performed a transcriptomic analysis between non-grafted and grafted tomatoes using the tomato variety Momotaro-scion and rootstock varieties, TD1, GS, and GF. Drought tolerance was significantly improved not only by a combination of compatible resistant rootstock TD1 but also by self-grafted compared to non-grafted lines. Next, we found the differences in gene expression between grafted and non-grafted plants before and during drought stress treatment. These altered genes are involved in the regulation of plant hormones, stress response, and cell proliferation. Furthermore, when comparing compatible (Momo/TD1 and Momo/Momo) and incompatible (Momo/GF) grafted lines, the incompatible line reduced gene expression associated with phytohormones but increased in wounding and starvation stress-response genes. These results conclude that grafting generates drought stress tolerance through several gene expression changes in the apical meristem.

Keywords: drought stress; grafting; phytohormones; tomato; transcriptomics.

Figure 1

Grafting in tomato plants increases survival rate through the shoot apical meristem and apical dominance recovery from the axillary bud. (a) Schematic representation of the drought stress experimental design, marking the points of sample collection for RNA sequencing. D0 indicated before treatment and D3 indicates 3 d of drought stress treatment. GH = grafting healing, DS = drought stress. (b) Survival rates of all tomato lines after drought stress treatment. (c) Phenotype of self-grafted Momotaro (Momo/Momo) tomato recovery after 12 d of drought stress treatment through apical meristem, apical dominance, or no recovery. Control: TTM-079 [TD1 (n = 30)], green guard [GS (n = 30)], green force [GF (n = 30)], and Momo (n = 30). Grafted: Momo/TD1 (n = 19), Momo/GF (n = 8), and Momo/Momo (n = 16). Green = apical meristem, brown = apical dominance, and gray = no recovery. White scale = 1 cm.

Figure 2

Control and grafted tomato lines show different gene expression pattern under normal and stress conditions. (a) Dendrogram of control and grafted tomato lines under D0 and D3. Brackets indicated sample lines that were grouped as control and grafted, and D0 and D3. (b) Heatmap of the gene expression in logarithm counts per million (Log₂ CPM) of the different grafted combinations and non-grafted tomato lines. Red and blue indicate high and low expressions, respectively. Top brackets group the gene expression similarities between tomato lines. D0 and D3 indicates before drought stress and day 3 during drought stress treatment.

Figure 3

Comparison of the differentially expressed genes (DEGs) in grafted and control before drought stress treatment. (a) Volcano plot representation of DEGs up-regulated and down-regulated by grafting. The x axis shows Log₂ fold change (FC) between Grafting vs. Control and y axis shows −Log10 (p-value). (b) Bar plot showing gene ontology (GO) enrichment analysis (Panther, false discovery rate < 0.05) for biological process in DEGs by grafted plants. Red and blue color indicate high and low gene expressions on significant DEGs (Adj. p < 0.05, Log₂FC ≥ |1|). The x axis shows the number of genes categorized in each GO term (y axis).

Figure 4

Differentially expressed genes (DEGs) and enriched pathways in grafted and control shoot apical meristem before drought stress and day 3 during stress. Volcano plot representation of DEGs up-regulated and down-regulated by drought stress treatment in (a) control and (b) grafted samples. The x axis shows logarithm fold change (Log2FC [D3/D0]) and y axis shows –Log10 (p-value). Significant DEGs (Adj. p < 0.05, Log2FC ≥ |1|) are in red and blue for up-regulation and down-regulation. (c) Bar plot showing gene ontology (GO) enrichment analysis (Panther, false discovery rate (FDR) < 0.05) for biological processes on significant DEGs of the control and grafted samples. Empty bars indicate the control and fill bars indicate the grafted samples. The x axis shows the number of genes categorized in each GO term (y axis). (d) Comparison of the DEGs in the grafted and control samples during stress. The x axis shows logarithm fold change (Log2FC [Grafting/Control]) and y axis shows –Log10 (p-value). (e) Bar plot showing GO enrichment analysis (Panther, FDR < 0.05) for biological process in DEGs by the grafted samples. Red and blue color indicate higher and lower gene expression on significant DEGs (Adj. p < 0.05, Log2FC ≥ |1|). The x axis shows the –Log10 (p-value) categorized in each GO term (y axis).

Figure 4

Differentially expressed genes (DEGs) and enriched pathways in grafted and control shoot apical meristem before drought stress and day 3 during stress. Volcano plot representation of DEGs up-regulated and down-regulated by drought stress treatment in (a) control and (b) grafted samples. The x axis shows logarithm fold change (Log2FC [D3/D0]) and y axis shows –Log10 (p-value). Significant DEGs (Adj. p < 0.05, Log2FC ≥ |1|) are in red and blue for up-regulation and down-regulation. (c) Bar plot showing gene ontology (GO) enrichment analysis (Panther, false discovery rate (FDR) < 0.05) for biological processes on significant DEGs of the control and grafted samples. Empty bars indicate the control and fill bars indicate the grafted samples. The x axis shows the number of genes categorized in each GO term (y axis). (d) Comparison of the DEGs in the grafted and control samples during stress. The x axis shows logarithm fold change (Log2FC [Grafting/Control]) and y axis shows –Log10 (p-value). (e) Bar plot showing GO enrichment analysis (Panther, FDR < 0.05) for biological process in DEGs by the grafted samples. Red and blue color indicate higher and lower gene expression on significant DEGs (Adj. p < 0.05, Log2FC ≥ |1|). The x axis shows the –Log10 (p-value) categorized in each GO term (y axis).

Figure 5

Changes in gene expression of 4 genes differentially expressed by grafting or drought stress assessed by quantitative real-time PCR. (a) Heat shock protein (Solyc08g062450); (b) Ascorbate peroxidase (Solyc09g007270); (c) Chlorophyll a–b binding protein (Solyc08g067330); and (d) 9-cis-epoxycaratenoid dioxygenase (Solyc07g056570). Gray bars represent before treatment (D0) and black bars represent 3 days drought stress treatment (D3). Asterisks denote significant differences according to t-test (Wilcoxon test) between D0 and D3 of each line, where * indicate p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. No significance is denoted as ns. Error bars represent the standard error from 3 biological replicates with 3 technical replicates.

Figure 6

Compatible [Momotaro shoot (Momo)/TTM-079 (TD1) and Momo/Momo] and incompatible grafted lines [Momo/green force (GF)] show differentially expressed genes revealed by transcriptomic analysis. Gene ontology (GO) analysis classification on (a) reduced gene expression on DEGs by Momo/GF (fold change ≥ 2, Momo/TD1, Momo/Momo vs. Momo/GF) and (b) increased gene expression on DEGs by Momo/GF (fold change ≤ 0.5, Momo/TD1, Momo/Momo vs. Momo/GF). The x axis shows the number of genes of each GO cluster classification, and the y axis shows the GO ontology term.