Identification of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in situ promoter analysis

Abstract

Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.

Ectopic Expression of OsSta2 Enhances Salt Stress Tolerance in Rice

“…These proteins contain a conserved AP2/ERF domain (Riechmann et al, 2000; Sharoni et al, 2011; Licausi et al, 2013). One of the best-studied is a group of CBF/DREBs that activate the expression of many stress-related genes and improve drought, salt, and cold tolerance (Stockinger et al, 1997; Liu et al, 1998; Kasuga et al, 1999; Sakuma et al, 2006; Lata and Prasad, 2011; Schmidt et al, 2013; Zhuang et al, 2013; Rong et al, 2014; Yang et al, 2014, 2016; Duan et al, 2015). …”

Applications and roles of the CRISPR system in genome editing of plants

“…Serial 5 0 deletions and sitespecific mutations in the promoter region of OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for induction of the salt response. The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 system, indicating that the GT-1 element directly controls the salt response of OsRAV2 (Basak and Nithin 2015;Duan et al 2015). Thus, a comprehensive molecular toolbox for multifaceted CRISPR/ Cas9 applications in rice (Oryza sativa) now provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for rice using Golden Gate and Gateway cloning methods.…”

“…Basak and Nithin 2015;Duan et al 2015;Jameson and Song 2015;Jiang et al 2013;Lowder et al 2015;Ma et al 2015; Mikami et al 2015a, b;Schiml et al 2014;Seo et al 2015;Shan et al 2014;Sugano et al 2014;Wang et al 2015;Woo et al 2015;Xie et al 2015;Xie and Yang 2013;Xu et al 2014;Zhang et al 2016b;Zhang et al 2014; Zhou et al Gao et al 2015a;Li et al 2014; Upadhyay et al Ali et al 2015;Jiang et al 2013;Johnson et al 2015;Lowder et al 2015;Nekrasov et al 2013;Upadhyay et al 2013) MaizeZea mays ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4 Liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), acetolactate synthase (ALS) genes (ALS1 and ALS2)(Basak and Nithin 2015; Liang et al 2014; Puchta and Fauser 2013; Schuster et al 2015; Svitashev et al 2015; Xing et al Basak and Nithin 2015; Shan et al 2014; Sugano et al 2014; Upadhyay et al 2013; Wang et al 2014) Soybean Glycine max Green fluorescent protein (GFP) transgene GmPDS11GmPDS18(Du et al 2016;Jacobs et al 2015;Michno et al 2015;Ricroch and Henard-Damave 2015) Potato Solanum tuberosum Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4) Phytophthora infestans resistance genes(Butler et al 2015;Richter et al 2012; Schaart et al glucuronidase (GUS) transgene(Michno et al 2015) …”

Synthetic strategies for plant signalling studies: molecular toolbox and orthogonal platforms

“…Recently, the general applicability of the system was demonstrated by efficient editing in woody plants (Fan et al ., ). Nevertheless, applications of the CRISPR/Cas9 technology to the study of signalling pathways have already started to be reported (Duan et al ., ). The transcriptional regulation mechanism of the gene of OsRAV2 [ Oryza sativa related to abscisic acid insensitive 3 (ABI3)/viviparous 1 (VP1) 2], which in rice is exclusively expressed under salt stress, was investigated in an effort to identify the salt responsive element within its promoter.…”