Screening and Genetic Analysis of Ethylene-Response Mutants in Etiolated Rice Seedlings

Background

The key molecular elements of ethylene-signaling pathway have been identified by molecular genetics and genomic approaches in Arabidopsis (Guo and Ecker, 2004). Ethylene plays important roles in rice growth, development, and environmental adaptation, including coleoptile and shoot elongation, aerenchyma development, and submergence response (Xu et al., 2006; Fukao and Bailey-Serres, 2008; Ma et al., 2010). Rice may have a distinct mechanism of ethylene-signaling pathway due to the different ethylene-regulated biological processes in rice and Arabidopsis (Yang et al., 2015a). The forward genetic analysis is essential to reveal fully the mechanism of ethylene signaling pathway in rice. So, it is prerequisite to develop an efficient method to screen ethylene-response mutants in rice. Genetic screens that are based on the triple-response have been extensively conducted on Arabidopsis (Johnson and Ecker, 1998; Stepanova and Ecker, 2000). However, the genetic screens in rice have been hamper owing to the lack of ethylene-response phenotypes.

Ethylene promotes rice coleoptile elongation of dark-grown seedlings (Ku et al., 1970). In our experimental conditions, root elongation can be inhibited by ethylene in etiolated rice seedlings. Over the past decade, based on the double-response of etiolated rice seedling, including ethylene-induced coleoptile growth promotion and root growth inhibition, several unique rice mutants have been identified. We named these ethylene-response mutants maohuzi (mhz) (Ma et al., 2013). Loss-of-function of MHZ7/OsEIN2 rice plants display insensitive to ethylene, including ethylene-insensitive coleoptile elongation and root growth. Conversely, MHZ7/OsEIN2-overexpression transgenic lines display ethylene hypersensitivity (Ma et al., 2013). Characterization of MHZ6/OsEIL1 revealed that MHZ6/OsEIL1 and its homolog gene OsEIL2 have functional diversification in rice ethylene response (Yang et al., 2015b). Identifications of MHZ4/ABA4 (Ma et al., 2014) and MHZ5/CRTISO (Yin et al., 2015) revealed that ethylene inhibits root growth requiring ABA pathway in rice, which is different with that in Arabidopsis. The study of MHZ2/SOR1 provides a candidate mechanism that auxin acts downstream to modulate ethylene inhibition of root growth in etiolated rice seedlings (Chen et al., 2018). The research of MHZ3 found that ethylene-induced MHZ3 stabilizes MHZ7/OsEIN2 and impeding protein ubiquitination to facilitate ethylene signaling pathway (Ma et al., 2018). Here, we describe the method in detail for genetic analysis of ethylene response mutants in etiolated rice seedlings. This protocol can also be used in other monocotyledonous plants to detect ethylene response (Yang et al., 2015a). Besides, the longer mesocotyl and coleoptile mutant gaoyao1(gy1) was also discovered by this method (Xiong et al., 2017).