Gene expression study of signaling pathway in response to high salinity stress in rice (Oryza sativa L.) seedlings

Salinity as one of the major abiotic stresses influences plant growth and development. Among the cereal, rice is the most sensitive to salinity stress, with 30 mmol NaCl already strongly reducing the growth and yield of rice plants. Over the past few decades, significant efforts have been made worldwide to understand mechanisms of salinity tolerance and to breed salt-tolerant varieties in rice. Plants respond to salt stress through perceiving and transducing the osmotic and ion signals to cell interiors, followed by modification of cellular characteristics.
So far, no specific sensor or receptor for Na+ has been identified in plants. However, the salt overly sensitive (SOS) signaling pathway and calcineurin B-like (CBL)/CBL-interacting kinase (CIPK) pathway has been well characterized in Arabidopsis. Salt-induced elevation in cytosolic Ca2+ activates the SOS2-SOS3 protein kinase complex, which phosphorylates and stimulates the activity of SOS1, a plasma membrane Na+/H+ antiporter. In rice, the OsSOS1, OsSOS2/OsCIPK24 and OsSOS3/OsCBL4 genes have been isolated and the function and relationship between them investigated. Among them, OsCIPK24 and OsCBL4 act in concert to activate OsSOS1. In this research, we used RNA-seq approach to dissect signaling pathway in response to salinity stress using salt-tolerant and sensitive rice cultivars.

The seeds of two rice (Oryza sativa L. ssp. Indica) genotypes with different salinity tolerance were obtained from International Rice Research Institute (IRRI) in Philippines. The plants were grown hydroponically in the greenhouse of Heinrich-Heine-University (HHU), Düsseldorf, Germany. The two-week old seedlings were exposed to 150 mM (15 dSm-1) NaCl salinity. The root and shoot samples were harvested at 6h and 54h post-treatment in three biological replications. 48 samples were sequenced by Illumina platform and the raw filtered reads were mapped on rice reference genome. Tuxedo instruction was applied to identifying the differentially expressed genes (DEGs). MapMan software was used to identify the genes involved in signaling pathway

In RNA-Seq analysis, 48 samples were sequenced by Illumina platform and 15483 differentially expressed genes (DEGs) were identified. Out of the DEGs (from the comparison between the cultivars), 525 and 1472 genes were salt-specific in 6 and 54h time points in roots respectively. Out of the salt-specific DEGs in shoots, 635 and 606 genes were in 6 and 54h respectively. MapMan pathway analysis detected 91 genes in signaling pathway. Out of the genes, 27 genes showed high expression. Out of the genes, 21 genes showed more expression in tolerant cultivar CSR28 compared to sensitive cultivar IR28. The most difference between the cultivars was observed in roots after 54h of salt treatment suggesting the critical role of roots in salt tolerance induction. Receptor like kinase (RLK) proteins played the most important role among the identified signaling genes. Several important genes involved in major signaling processes such as OsSIK1, OsSAPK4, OsCIPK05, OsCIPK14, OsCBL4 and OsPP2C1 were identified in this research.

Salt-tolerant cultivars use better signaling pathways to sensing of stress and having the stronger osmotic and ionic reactions to cope with salinity stress. In the present research, huge number of differentially expressed genes generated using rice tolerant cultivar CSR28 and sensitive cultivar IR28 at 6 and 54h sampling times by RNA-Seq method. The comparison of the cultivars at specific salt stress showed that 91 genes (including 27 high expressed genes) were involved in signaling pathway. Kinase proteins played the most important role among the signaling pathway genes. The important genes identified in this research can be applied in the selecting and developing of salt-tolerant rice cultivars.