Subcellular co‐localization of Arabidopsis RTE1 and ETR1 supports a regulatory role for RTE1 in ETR1 ethylene signaling

Abstract

Ethylene is an important plant growth regulator perceived by membrane-bound ethylene receptors. The ETR1 ethylene receptor is positively regulated by a predicted membrane protein, RTE1, based on genetic studies in Arabidopsis. RTE1 homologs exist in plants, animals and protists, but the molecular function of RTE1 is unknown. Here, we examine RTE1 expression and subcellular protein localization in order to gain a better understanding of RTE1 and its function in relation to ETR1. Arabidopsis plants transformed with the RTE1 promoter fused to the b-glucuronidase (GUS) reporter gene revealed that RTE1 expression partly correlates with previously described sites of ETR1 expression or sites of ethylene response, such as the seedling root, root hairs and apical hook. RTE1 transcript levels are also enhanced by ethylene treatment, and reduced by the inhibition of ethylene signaling. For subcellular localization of RTE1, a functional RTE1 fusion to red fluorescent protein (RFP) was expressed under the control of the native RTE1 promoter. Using fluorescence microscopy, RTE1 was observed primarily at the Golgi apparatus and partially at the endoplasmic reticulum (ER) in stably transformed Arabidopsis protoplasts, roots and root hairs. Next, a functional ETR1 fusion to a 5xMyc epitope tag was expressed under the control of the native ETR1 promoter. Immunohistochemistry of root hairs not only showed ETR1 residing at the ER as previously reported, but revealed substantial localization of ETR1 at the Golgi apparatus. Lastly, we demonstrated the subcellular co-localization of RTE1 and ETR1. These findings support and enhance the genetic model that RTE1 plays a role in regulating ETR1.

Differential Control of Ethylene Responses byGREEN-RIPEandGREEN-RIPE LIKE1Provides Evidence for Distinct Ethylene Signaling Modules in Tomato

“…Genetic analysis indicates that AtRTE1 functions at or upstream of the ethylene receptors but acts independently of the RAN1 copper transporter (Resnick et al, 2006. In support of this hypothesis, AtRTE1 colocalizes with ETR1 in the ER and Golgi membranes and interacts with the N-terminal region of the ETR1 and ERS1 receptors (Zhou et al, 2007;Dong et al, 2008Dong et al, , 2010. Interestingly, mutations at the rte1 locus suppress ethylene insensitivity mediated by a subset of, but not all, etr1 mutant alleles, and reduced ethylene sensitivity mediated through AtRTE1 overexpression is primarily dependent on the presence of ETR1 and not the other ethylene receptors (Resnick et al, 2006Zhou et al, 2007).…”

“…S1). The current model of the ethylene signaling pathway would place the localization of the receptors and their interaction with the GR/ RTE1 proteins within the ER, and previous studies have suggested either exclusive localization of AtRTE1, OsRTH1, and OsRTH2 within the Golgi or dual localization within the Golgi and ER (Zhou et al, 2007;Dong et al, 2008;Zhang et al, 2012). These slight discrepancies may be caused by the use of different experimental systems and high-level overexpression of heterologous transgenes that may influence the subcellular localization of proteins.…”

“…In Arabidopsis, ethylene is perceived by a family of five receptors that share homology to bacterial twocomponent His kinase receptors that are localized to the endoplasmic reticulum (ER) and Golgi membranes and form multimeric complexes thought to be composed of homoreceptor and heteroreceptor dimers (Chang et al, 1993;Hua et al, 1995Sakai et al, 1998;Chen et al, 2002Dong et al, 2008;Gao et al, 2008). Loss-of-function analyses indicate that the receptors act in a semiredundant manner to negatively regulate ethylene responses, although the ETR1 receptor appears to play more of a significant role in mediating ethylene responses than the other Arabidopsis receptors Cancel and Larsen, 2002;Hall and Bleecker, 2003;Wang et al, 2003;Binder et al, 2006;Qu et al, 2007).…”

“…SlGR, SlGRL1, and SlGRL2 Are Predominantly Golgi-Localized Proteins Several components of the ethylene signaling pathway are localized within the endomembrane system, and separate studies have reported either dual localization of AtRTE1 within the Golgi and ER membranes or exclusive localization within the Golgi (Chen et al, 2002;Gao et al, 2003;Zhou et al, 2007;Dong et al, 2008;Bisson et al, 2009). To determine the subcellular localization of SlGR and SlGRL1, N-terminal yellow fluorescent protein (YFP) fusions of each protein were constructed and transformed into the etr1-2/rte1-3 double mutant.…”

Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis

“…Recently, numerous studies have expanded the linear view of ethylene signaling pathway. For instance, a new protein, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), which is colocalized with the ethylene receptor ETR1, was identified as a positive regulator of ETR1 function, but the connection between RTE1 and ETR1 is still under investigation (Solano et al 1998;Resnick et al 2006;Dong et al 2008). Additionally, a number of groups found that post-transcriptional regulation of protein levels is a key mechanism of modulating EIN3 activity by ethylene.…”