Transcriptome and network analysis pinpoint ABA and plastid ribosomal proteins as main contributors to salinity tolerance in the rice variety, CSR28

Abstract

Salinity stress is a major challenge for rice production, especially at seedling stage. To gain comprehensive insight into the molecular mechanisms and potential candidate genes involved in rice salinity stress response, we integrated physiological, transcriptome and network analysis to investigate salinity tolerance in two contrasting rice genotypes. The root and shoot samples were collected at two timepoints (6 hours and 54 hours) of high salt treatment. Element assay showed that the tolerant genotype CSR28 had lower Na+/K+ ratio in both organs than in those of the sensitive genotype IR28 under salinity stress. A total of 15,483 differentially expressed genes (DEGs) were identified from the RNA-Seq analysis. The salt-specific genes were mainly involved in metabolic processes, response to stimulus, and transporter activity, and were enriched in key metabolic pathways such as, biosynthesis of secondary metabolites, plant hormone signal transduction, and carotenoid biosynthesis. Furthermore, the results showed that the differential genes involved in abscisic acid (ABA) biosynthesis were specifically up-regulated in the tolerant genotype. Network analysis revealed 50 hub genes for the salt-specific genes in the roots of CSR28 which mainly encodes ribosomal proteins (RPs). Functional validation of the nine hub genes revealed three plastid RPs (PRPs), including OsPRPL17, OsPRPS9 and OsPRPL11, which contributes to protein synthesis, chloroplast development and stress signaling. Our findings suggested that ABA and PRPs play key roles to enhance of salinity tolerance in CSR28. Our study provides valuable information for further investigations of the candidate genes associated with salt tolerance and the development of salt-tolerant rice varieties.

Fig 1. Graphical workflow of experimental design and integration of RNA-Seq and PPI network analysis for identifying key genes and pathways associated with salt tolerance in rice seedling, as well as functional validation of hub genes.

Fig 2. Clustering and heatmap of samples and genes.

(a) clustering of samples according to Euclidean distances and Average-linkage method. Data are standardized RPKM values for all genes. (b) Heatmap of 45 highly expressed genes (log10 FC ≥ 3 or log10 FC ≤ -3) in response to salinity stress (comparison of control and salinity samples). Red and green colors indicate increased and decreased expression in response to stress, respectively. R: root, S: shoot, CT: control, SS: salt stress, 6 h: 6-hour timepoint, 54 h: 54-hour timepoint, CSR28: salt-tolerant genotype, IR28: salt-sensitive genotype.

Fig 3. Numbers and functional classification of DEGs.

(a) and (b) Venn diagram analysis showing specific and common genes for DEGs of 6-h and 54-h timepoints, respectively. R: root, S: shoot, CT: control, SS: salt stress, 6 h: 6-hour timepoint, 54 h: 54-hour timepoint, CSR28: salt-tolerant genotype, IR28: salt-sensitive genotype. (c) and (d) Significant GO terms for salt-specific genes in roots and shoots, respectively. (e) and (f) Significant KEGG pathways for salt-specific genes in roots and shoots, respectively.

Fig 4. Protein-protein interaction (PPI) network analysis for up-regulated salt-specific genes in the roots of salt-tolerant CSR28 at 54-hour timepoint.

Top 50 hub genes are indicated by color intensity. Red nodes display highly dens interactions with other proteins.

Fig 5. Functional validation of three hub genes encoding plastid RPs in response to salinity stress.

A significant relationship was shown between dry weight and expression value of three hub genes, including OsPRPL17 (R²= 0.79, P-value= 0.001), OsPRPS9 (R²= 0.91, P-value= 0.000) and OsPRPL11 (R²= 0.77, P-value= 0.001).

Fig 6. Molecular mechanism of rice salt tolerance according to the critical genes identified in the present study.

Salt-induced elevation of signaling molecules activated transcriptional factors followed by several key genes involved in ABA biosynthesis, secondary metabolite biosynthesis, ribosomal proteins, osmolytes and ion transporters, and consequently resulted to Na⁺ detoxification, ion homeostasis and osmotic adjustment in the cells.