Abstract
Signal perception and transmission of the plant hormone ethylene are mediated by a family of receptor histidine kinases located at the Golgi-ER network. Similar to bacterial and other plant receptor kinases, these receptors work as dimers or higher molecular weight oligomers at the membrane. Sequence analysis and functional studies of different isoforms suggest that the ethylene receptor family is classified into two subfamilies. In Arabidopsis, the type-I subfamily has two members (ETR1 and ERS1) and the type-II subfamily has three members (ETR2, ERS2, and EIN4). Whereas subfamily-I of the Arabidopsis receptors and their interactions with downstream elements in the ethylene pathway has been extensively studied in the past; related information on subfamily-II is sparse. In order to dissect the role of type-II receptors in the ethylene pathway and to decode processes associated with this receptor subfamily on a quantitative molecular level, we have applied biochemical and spectroscopic studies on purified recombinant receptors and downstream elements of the ethylene pathway. To this end, we have expressed purified ETR2 as a prototype of the type-II subfamily, ETR1 for the type-I subfamily and downstream ethylene pathway proteins CTR1 and EIN2. Functional folding of the purified receptors was demonstrated by CD spectroscopy and autokinase assays. Quantitative analysis of protein-protein interactions (PPIs) by microscale thermophoresis (MST) revealed that ETR2 has similar affinities for CTR1 and EIN2 as previously reported for the subfamily-I prototype ETR1 suggesting similar roles in PPI-mediated signal transfer for both subfamilies. We also used in planta fluorescence studies on transiently expressed proteins in Nicotiana benthamiana leaf cells to analyze homo- and heteromer formation of receptors. These studies show that type-II receptors as well as the type-I receptors form homo- and heteromeric complexes at these conditions. Notably, type-II receptor homomers and type-II:type-I heteromers are more stable than type-I homomers as indicated by their lower dissociation constants obtained in microscale thermophoresis studies. The enhanced stability of type-II complexes emphasizes the important role of type-II receptors in the ethylene pathway.
Highlights
The gaseous plant hormone ethylene is decisive for many growth and developmental processes in plants, including fruit ripening, senescence, and the control of biotic and abiotic stress responses, such as pathogen defense and wounding (Kieber and Ecker, 1993; O’Donnell et al, 1996; Penninckx et al, 1998; Bleecker and Kende, 2000)
Previous protein-protein interaction studies mainly focused on the prototype type-I receptor A. thaliana receptor ETR1 (AtETR1), whereas related information on subfamily-II receptors is still sparse
A direct interaction of ethylene type-II receptors with either CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) or ETHYLENE INSENSITIVE 2 (EIN2) has been demonstrated by yeast two-hybrid assays and in planta fluorescence resonance energy transfer (FRET) studies (Cancel and Larsen, 2002; Bisson and Groth, 2010), detailed information about these interactions is still missing
Summary
The gaseous plant hormone ethylene is decisive for many growth and developmental processes in plants, including fruit ripening, senescence, and the control of biotic and abiotic stress responses, such as pathogen defense and wounding (Kieber and Ecker, 1993; O’Donnell et al, 1996; Penninckx et al, 1998; Bleecker and Kende, 2000). Most of the current knowledge about ethylene biosynthesis and signal transduction has been obtained by genetic, physiological, and biochemical studies in the model plant Arabidopsis thaliana Based on these studies, a family of mainly endoplasmic reticulum (ER)-membrane bound receptors was identified to catalyze the first step in all ethylene-regulated phenomena. In addition to the GAF and HK domain receptor isoforms, ETR1, ETR2, and EIN4 carry a response regulator domain (RD) at their carboxy (C)-terminus (Chang et al, 1993; Chen et al, 2002; Voet van Vormizeele and Groth, 2008) Despite their similar overall structure, the individual isoforms contain major differences in these modules. In vitro Ser/Thr kinase activity was demonstrated for these isoforms albeit autokinase activity seems to play only a minor role in ethylene signaling (Wang et al, 2003; Moussatche and Klee, 2004)
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