Spatiotemporal dynamics of the tomato fruit transcriptome under prolonged water stress

Abstract

Water availability influences all aspects of plant growth and development; however, most studies of plant responses to drought have focused on vegetative organs, notably roots and leaves. Far less is known about the molecular bases of drought acclimation responses in fruits, which are complex organs with distinct tissue types. To obtain a more comprehensive picture of the molecular mechanisms governing fruit development under drought, we profiled the transcriptomes of a spectrum of fruit tissues from tomato (Solanum lycopersicum), spanning early growth through ripening and collected from plants grown under varying intensities of water stress. In addition, we compared transcriptional changes in fruit with those in leaves to highlight different and conserved transcriptome signatures in vegetative and reproductive organs. We observed extensive and diverse genetic reprogramming in different fruit tissues and leaves, each associated with a unique response to drought acclimation. These included major transcriptional shifts in the placenta of growing fruit and in the seeds of ripe fruit related to cell growth and epigenetic regulation, respectively. Changes in metabolic and hormonal pathways, such as those related to starch, carotenoids, jasmonic acid, and ethylene metabolism, were associated with distinct fruit tissues and developmental stages. Gene coexpression network analysis provided further insights into the tissue-specific regulation of distinct responses to water stress. Our data highlight the spatiotemporal specificity of drought responses in tomato fruit and indicate known and unrevealed molecular regulatory mechanisms involved in drought acclimation, during both vegetative and reproductive stages of development.

Figure 1

Experimental design and phenotype of tomato plants and fruit exposed to prolonged water stress. A, Schematic illustration of the drought treatments and sample collection. Plants were grown under well-watered conditions (Control, C) or under water limiting conditions at three intensity levels (S1, S2, S3). C: 40%–45% VWC; S: 35% VWC; S2: 30% VWC; S3: 25% VWC. B, Circular dripping system and soil moisture sensor used to monitor and control soil water deficit. C, Equatorial fruit section showing the harvested tissues: pericarp, septum, columella, placenta, jelly, and seeds. D, Representative pictures comparing fruit development under control (C) and water limiting conditions (S1, S2, S3). From left to right: 20 dpa and ripe fruit. From top to bottom: whole fruit, longitudinal section and equatorial section. Scale bar = 1 cm. E, Representative pictures comparing the vegetative phenotype of tomato plants grown under control (C) and water limiting conditions (S1, S2, S3). Left: whole plants. Right top: leaves. Right bottom: leaflets. Scale bar = 2 cm.

Figure 2

Global gene expression changes in tomato leaf and fruit tissues in response to water stress. A, PCA and (B), hierarchical clustering analysis using log2-normalized read values of all genes in the different tissue types, stages, and watering conditions. Leaves and fruit at 20 dpa and at the ripe stage were harvested from control (C) or water stressed plants (S1, S2, S3). Lf: leaf; Pe: pericarp; Se: septum; Co: columella; Pl: placenta; Je: jelly; Sd: seeds. C, Number of DEGs in leaves and in fruit at different water deficit conditions. D, Venn diagram showing the percentage of DEGs shared between the three water deficit conditions. DEGs from all fruit tissues were compiled to obtain the total number of DEGs for each fruit developmental stage. S1: control vs. S1; S2: control vs. S2; S3: control vs. S3.

Figure 3

Specificity of transcriptional responses to water stress in tomato leaves and fruit. A, Venn diagrams showing the overlap between DEGs from leaves, 20 dpa and ripe fruit at different water stress intensity levels (S1, S2, S3). DEGs from every fruit tissue were compiled for each fruit developmental stage. B, Representative GO terms enriched in DEGs specific or common to leaves, 20 dpa and ripe fruit in response to prolonged water stress (S1, S2, S3). Point size represents FDR corrected P-value for upregulated (red) and downregulated (blue) DEGs.

Figure 4

Tissue-specific gene expression changes in the tomato fruit in response to water stress. A, Percentage of total DEGs shared between fruit tissues at different water stress intensity levels (S1, S2, S3). B, Percentage of total DEGs in each fruit tissue. DEGs for each water stress condition (S1, S2 or S3) and developmental stage (20 dpa or ripe fruit) were compiled to obtain the total number of DEGs. C and D, GO enrichment analysis of downregulated and upregulated DEGs in each fruit tissue for each water stress condition. The different water stress intensity levels (S1, S2 and S3) are shown from left to right for each tissue. Pe: pericarp; Se: septum; Co: columella; Pl: placenta; Je: jelly; Sd: seeds. Only representative GO-terms, associated with the highest number of genes, are shown. All GO-terms significantly enriched (FDR < 0.05) in each tissue are listed in Supplemental Table S6.

Figure 5

WGCNA of the response of fruit and leaves to water stress. A, Heat map showing cluster-tissue/stage associations of the 44 WGCNA generated coexpression modules. Each row corresponds to a module and each column corresponds to a tissue and watering condition. From left to right for each tissue and developmental stage: C (control), S1, S2, S3. Color denotes ME values. Examples of stage and tissue-specific modules are shown in red (upregulated), blue (downregulated), and black (mixed pattern). B, Tissue- and stage-specific expression profile of a gene (LTP2, Solyc10g075110) representative of module M25. Images displaying expression levels (RPKM) were extracted from the TEA database (https://tea.solgenomics.net/). C, Network showing connections between core genes in the columella-associated module M29. Each colored circle (node) represents one gene. Diamond shapes represent TFs. Orange node color indicates greater connectivity within the network. The 500 strongest gene–gene interactions are shown. Only edge correlation values > 0.8 are shown and values > 0.9 are indicated in red. The expression profile of underlined genes is shown in (D). D, Expression profiles of hub genes in the columella-associated module M29. Expression data (RPKM) are means (±SD) of three biological replicates. Pe: pericarp; Se: septum; Co: columella; Pl: placenta; Je: jelly; Sd: seeds.

Figure 6

Epigenetic-related gene expression changes in ripe fruit and transgenerational effect of water stress on seedling performance. A, Heat-map showing gene expression in each fruit tissue under control or water stress conditions. The different watering conditions are shown from left to right for each tissue (Control, S1, S2, S3). Normalized RNA-seq expression was transformed into Z-score. Color key: blue, low level of expression; red, high level of expression. Gene identifiers and expression data (RPKM) are listed in Supplemental Table S12. B, Differences in recovery after water stress between seedlings grown from control and from water stressed (S3) seeds. Water stress was applied to 25-d-old seedlings by soaking pots in water to reach full pot capacity, and subsequently withholding water until all control plants showed a strong wilting phenotype. All plants were then rewatered at full pot capacity and pictures were taken 6 h after rewatering. Eight randomly placed control and S3 seedlings were used for each experiment, and the experiment was repeated 3 times with similar results.

Figure 7

Jasmonate- and ethylene-related gene expression changes and hormone levels in tomato fruit under water stress. Heat-maps show gene expression in each fruit tissue at control or water stress conditions for 20 dpa fruit (A) and ripe fruit (B). From left to right for each tissue: C, S1, S2 and S3. Pe: pericarp; Se: septum; Co: columella; Pl: placenta; Je: jelly; Sd: seeds. Normalized RNA-seq expression was transformed into Z-score. Color key: blue, low level of expression; red, high level of expression. Jasmonate (JA)-related genes are in green and ethylene (ET)-related genes are in orange. Gene identifiers and expression data (RPKM) are listed in Supplemental Table S13. C, Pathway for JA biosynthesis showing in red genes involved in the enzymatic steps and upregulated by water stress in columella and/or jelly tissues of 20 dpa fruit. D, JA was measured in columella and jelly fruit tissues at the 20 dpa stage from control and water stressed (S3) plants. Data are means (±SD) of six biological replicates. Statistically significant differences were determined using Student’s t test: **P<0.01). E, Ethylene was measured in fruit at the ripe stage from control (C) and water stressed (S1, S2 and S3) plants. Data are means (±SD) of eight biological replicates. Statistically significant differences were determined using Student’s t test: *P<0.05; **P<0.01.

Figure 8

Effect of water stress on fruit color and carotenoid biosynthesis in the pericarp of ripe fruit. A, Effect of water stress on fruit color. Box plots representing differences in fruit color between plants exposed to different water stress intensity. Fruit color was determined by measuring the lightness factor (L*), the color intensity factor (Chroma, C*), and the actual color appearance factor (Hue angle, °h). Each dataset represents at least 20 biological replicates. Statistically significant differences between control (C) and each of stress condition (S1, S2, S3) were determined using the Student’s t test. The bold line in the center of the boxplots represents the median, the box edges represent the 25th (lower) and 75th (upper) percentiles, and the whiskers extend to the most extreme data points that are no more than 1.5× the length of the interquartile range. B, Effect of water stress on carotenoid content. Data are the mean (±SD) of four biological replicates. Statistically significant differences between control (C) and each of stress condition (S1, S2, S3) were determined using the Student’s t test: *P<0.05; **P<0.01; ***P<0.0001. C, Effect of water stress on carotenoid-related gene expression. Expression data (RPKM) are the mean (±SD) of three biological replicates. PSY: phytoene synthase; ZDS: ζ-carotene desaturase; b-LCY: lycopene ß-cyclase. Statistically significant differences between control (C) and each of stress condition (S1, S2, S3) were determined using the Student’s t test: *P<0.05; **P<0.01.