Combined transcriptomic and metabolomic analysis reveals a role for adenosine triphosphate-binding cassette transporters and cell wall remodeling in response to salt stress in strawberry

Abstract

Strawberry (Fragaria × ananassa Duch) are sensitive to salt stress, and breeding salt-tolerant strawberry cultivars is the primary method to develop resistance to increased soil salinization. However, the underlying molecular mechanisms mediating the response of strawberry to salinity stress remain largely unknown. This study evaluated the salinity tolerance of 24 strawberry varieties, and transcriptomic and metabolomic analysis were performed of 'Sweet Charlie' (salt-tolerant) and 'Benihoppe' (salt-sensitive) to explore salt tolerance mechanisms in strawberry. Compared with the control, we identified 3412 differentially expressed genes (DEGs) and 209 differentially accumulated metabolites (DAMs) in 'Benihoppe,' and 5102 DEGs and 230 DAMs in 'Sweet Charlie.' DEGs Gene Ontology (GO) enrichment analyses indicated that the DEGs in 'Benihoppe' were enriched for ion homeostasis related terms, while in 'Sweet Charlie,' terms related to cell wall remodeling were over-represented. DEGs related to ion homeostasis and cell wall remodeling exhibited differential expression patterns in 'Benihoppe' and 'Sweet Charlie.' In 'Benihoppe,' 21 ion homeostasis-related DEGs and 32 cell wall remodeling-related DEGs were upregulated, while 23 ion homeostasis-related DEGs and 138 cell wall remodeling-related DEGs were downregulated. In 'Sweet Charlie,' 72 ion homeostasis-related DEGs and 275 cell wall remodeling-related DEGs were upregulated, while 11 ion homeostasis-related DEGs and 20 cell wall remodeling-related DEGs were downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed only four KEGG enriched pathways were shared between 'Benihoppe' and 'Sweet Charlie,' including flavonoid biosynthesis, phenylalanine metabolism, phenylpropanoid biosynthesis and ubiquinone, and other terpenoid-quinone biosynthesis. Integrating the results of transcriptomic and metabolomics analyses showed that adenosine triphosphate-binding cassette (ABC) transporters and flavonoid pathway genes might play important roles in the salt stress response in strawberry, and DAMs and DEGs related to ABC transporter and flavonoid pathways were differentially expressed or accumulated. The results of this study reveal that cell wall remodeling and ABC transporters contribute to the response to salt stress in strawberry, and that related genes showed differential expression patterns in varieties with different salt tolerances. These findings provide new insights into the underlying molecular mechanism of strawberry response to salt stress and suggest potential targets for the breeding of salt-tolerant strawberry varieties.

Keywords: ABC transporter; cell wall remodeling; salinity tolerance; salt stress; strawberry.

FIGURE 1

Salt damage index of 24 strawberry varieties under different concentration of NaCl. Strawberry plants with 6–7 leaves were irrigated with five different concentrations of salt solutions: 0, 50, 100, 150, or 200 mM NaCl for 50 days. The salt damage index grades were scaled into six levels from 0 to 5, according to different damage symptoms in the leaves after salt treatment. Based on the salt damage indices, tested varieties were clustered into three groups: salt tolerant, salt sensitive, and salt hypersensitive.

FIGURE 2

Relative total biomass of 24 strawberry varieties under different concentrations of NaCl. Strawberry plants with 6–7 leaves were irrigated with five different concentrations of salt solutions: 0, 50, 100, 150, or 200 mM NaCl for 50 days. Relative biomass accumulation was counted as the dry weight of the whole plant treated with different concentrations of salt solutions (50, 100, 150, or 200 mM) divided by the dry weight of the whole plant treated with fresh water. Data are means (±SD) of three independent experiments, *p < 0.05 or **p < 0.01.

FIGURE 3

Physiological indices analysis of strawberry under salt stress. ‘Sweet Charlie’ and ‘Benihoppe’ plants with 6–7 leaves were treated with 0 mM (control) or 100 mM NaCl solution, respectively. After 10 days of treatment, the young leaves were collected for physiological indices determination. The content of MDA (A), H₂O₂ (B), and O₂^•– (C) under control and salt stress treatment in strawberry leaves. The activities of CAT (D), SOD (E), and POD (F) under control and salt stress treatment in strawberry leaves. Data are means (±SD) of three independent experiments. Different letters indicate significant differences (P < 0.05).

FIGURE 4

Gene Ontology enrichment analysis and KEGG pathway analysis of differentially expressed genes in strawberry leaves under salt stress. ‘Sweet Charlie’ and ‘Benihoppe’ plants with 6–7 leaves were treated with 0 mM (control) or 100 mM NaCl solution, respectively. After 12 h of treatment, the young leaves were collected for RNA-seq analysis. GO enrichment analysis and KEGG pathway analysis were performed to explore the functional significance of DEGs. Top 20 GO enrichment terms of differentially expressed genes in ‘Benihoppe’ (A) and ‘Sweet Charlie’ (B) under salt stress. Top 20 KEGG pathways of differentially expressed genes in ‘Benihoppe’ (C) and ‘Sweet Charlie’ (D) under salt stress.

FIGURE 5

Verification of differentially expressed genes by qRT-PCR. ‘Sweet Charlie’ and ‘Benihoppe’ plants with 6–7 leaves were treated with 0 mM (control) or 100 mM NaCl solution, respectively. After 12 h of treatment, the young leaves were collected for RNA extraction. Twelve genes were randomly selected to validate the RNA-seq results via qRT-PCR. Data are means (±SD) of three independent experiments.

FIGURE 6

The statistics of differentially accumulated metabolites and KEGG enrichment under salt stress in strawberry. ‘Sweet Charlie’ and ‘Benihoppe’ plants with 6–7 leaves were treated with 0 mM (control) or 100 mM NaCl solution, respectively. After 12 h of treatment, the young leaves were collected for metabolite extraction. Non-targeted metabolomic analysis was performed to expose the metabolomic changes of ‘Benihoppe’ and ‘Sweet Charlie’ in response to salt stress. The volcano map of differentially accumulated metabolites in ‘Benihoppe’ (A) and ‘Sweet Charlie’ (B) leaves under salt stress. KEGG pathways of differentially accumulated metabolites in ‘Benihoppe’ (C) and ‘Sweet Charlie’ (D) under salt stress.

FIGURE 7

Differentially expressed genes related to ion channel and ion transporter, aquaporin, NRT1/PTR FAMILY, and ABC transporters, and differentially accumulated metabolites related to ABC transporters in ‘Benihoppe’ (left) and ‘Sweet Charlie’ (right) under salt stress. The relative expression levels as described by log₂FC are represented by a color gradient from low (blue) to high (red). Under salt stress, the DEGs with log₂FC > 0 represent upregulated expression, log₂FC < 0 represents downregulated expression, and log₂FC = 0 represents unchanged expression. Accumulation of metabolites as described by log₂FC is represented by a color gradient from low (green) to high (yellow). Under salt stress, the DAMs with log₂FC > 0 represent upregulated accumulation, log₂FC < 0 represents downregulated accumulation, and log₂FC = 0 represents unchanged accumulation. Gray squares indicate no differentially expressed genes in this term.

FIGURE 8

Differentially expressed genes related to cell wall remodeling in ‘Benihoppe’ (left) and ‘Sweet Charlie’ (right) under salt stress. The relative expression levels as described by log₂FC are represented by a color gradient from low (blue) to high (red). Under salt stress, DEGs with log₂FC > 0 represent upregulated expression, log₂FC < 0 represents downregulated expression, and log₂FC = 0 represents unchanged expression. Gray squares indicate no differentially expressed gene or differentially accumulated metabolite in this term.

FIGURE 9

Differentially expressed genes and differentially accumulated metabolites related to flavonoid pathways in ‘Benihoppe’ (left) and ‘Sweet Charlie’ (right) under salt stress. The relative expression levels as described by log₂FC are represented by a color gradient from low (blue) to high (red). Under salt stress, the DEGs with log₂FC > 0 represent upregulated expression, log₂FC < 0 represents downregulated expression, and log₂FC = 0 represents unchanged expression. The accumulated of metabolites as described by log₂FC are represented by a color gradient from low (green) to high (yellow). Under salt stress, the DAMs with log₂FC > 0 represent upregulated accumulation, log₂FC < 0 represents downregulated accumulation, and log₂FC = 0 represents unchanged accumulation. Gray squares indicate no differentially expressed gene or differentially accumulated metabolite in this term.