Structural Characterization of ETR1 Receptor and Ethylene Signaling in Plants

Abstract

Plant hormones are dynamic organic compounds that are produced by plant metabolism, regulate physiological responses during the process of plant growth and facilitate the reactions to challenges that a plant encounters in a particular environment. Ethylene is a simple gaseous hormone, which is involved in multiple developmental and growth responses such as ripening, seed gemination, seedling growth as well as responses to biotic and abiotic stresses. Ethylene perception and signaling through receptors is of prime importance to frame whole process of plant growth and development. Biochemical and genetic approaches were used to establish the current ethylene signaling pathway in plants. Ethylene perception starts with binding of ethylene to the five-member family of ethylene receptors primarily localized to the ER membrane. Ethylene receptors are characterized in two subfamilies based on their structure. Subfamily-I comprises of ETR1 and ERS1, while subfamily-II receptors include ETR2, ERS2 and EIN4. The overall structure of all the receptors is almost similar containing transmembrane domain at N-terminal, C-terminal transmitter domain and a GAF domain connecting the transmembrane domain and the transmitter domain. Though ETR1 was the first receptor identified in plants in 1993 and structural models for the cytosolic domain of ETR1 and ERS1 have been developed, but the experimental structure of the ethylene binding domain is still not available due to difficulty in determination of crystallographic structure of membrane spanning proteins. Very little information on ethylene binding to its receptor and initiation of the signaling cascade is available, which led to an interest in studying the structure of ethylene binding domain. Several biochemical and genetic studies revealed that the receptors function as disulphide linked homodimers having single ethylene binding site per dimer where ethylene binding facilitated by a Cu(I) co-factor. This copper ligand uses a set of highly conserved Cysteine (Cys) and Histidine (His) residues in ETR1 that are very crucial for ethylene binding as mutations in these residues lead to almost no ethylene binding, for example etr1-1 (a missense mutation in which Cys residue is mutated to Histidine) is unable to bind ethylene conferring the dominant ethylene insensitivity. Research Summary ix Structural Characterization of ETR1 Receptor and Ethylene Signaling in Plants Based on our experimental and computational data we tried to characterize and update the structure of ethylene receptor. We characterized the structure of the ethylene binding/ transmembrane domain of ETR1 receptor by exploiting an evolutionary approach based on multiple sequence alignments (MSAs) of eukaryotic and prokaryotic ethylene binding domains to predict likely contacting residue pairs and then testing the predictions by taking advantage of biochemical and genetic analysis. We determined the critical features of the ethylene-binding domain, by mutating the important residues of the ETR1 receptor and transgenically expressing them in etr1; etr2; ein4 triple background to assess the functionality and ethylene sensitivity of these receptors. Our data summarizes that Aspartate at 25th position (D25) in the first transmembrane helix is an important player that seems to interact with Cu directly for ethylene binding as changing it into different residues Aspartate to Asparagine (D25N), to Glutamic Acid (D25E) and Alanine (D25A) eliminated ethylene binding both in planta and in yeast FY834. All the mutated receptors rescued the phenotype in air but only Aspartate to Asparagine i.e. D25N was able to rescue and facilitate the normal ethylene response while others were insensitive to ethylene as expected. Although D25N rescued and facilitated the triple response phenotype of etr1; etr2; ein4, it did not show ethylene binding that supported the direct interaction of D25 with copper. Additionally, this study supported a model where two Cu atoms are involved rather than one for ethylene binding per receptor. We also provided evidence that Lysine at 91 position (K91) in the third transmembrane domain isinvolved in interactions with D25 and also causes a conformational change to modify the downstream signal. Substitution of Lysine to residues like Arginine (K91R) or Alanine (K91A) resulted in ethylene insensitivity, whereas substitution to Methionine (K91M) conferred hyposensitivity instead. The interaction of D25 in the first transmembrane domain and K91 in the third transmembrane domain is strengthened by the Aspartate and Lysine double mutants asthey exhibited more pronounced ethylene insensitivity as compared to the single mutants. This study aided in clearing the role of Asp25 and Lysine 91 in ethylene binding and thus enhanced our understanding of the structure of the ethylene binding domain of ETR1 receptor. Ethylene is also referred as stress hormone as it is mediator of various stress responses in plants along with role in developmental processes. Ethylene perception and signaling Research Summary x Structural Characterization of ETR1 Receptor and Ethylene Signaling in Plants through receptors has taken limelight in plant research for over 100 years. The second part of this thesis describes how ethylene impacts Arabidopsis thaliana growth in presence of different nanoparticles (NPs) in terms of their beneficial or toxic nature for enhancing crop yield. Therefore, Chapter 3 describes the effects of newly synthesized NPs including ZnO (Zinc Oxide), SiO2 (Silicon Dioxide) and ZnO/SiO2 composite NPs. These nanoparticles were characterized by XRD (X-Ray Diffraction) and FTIR (Fourier Transformed Infrared Spectroscopy) for further confirmation of the functional groups and purity of the NPs. We analyzed seedling behavior by preforming dose response assay of above-mentioned nanoparticles to check their effect on Arabidopsis growth. Different growth parameters including chlorophyll content, root growth and more specifically the expression of ethylene biosynthesis and signaling genes were analyzed to infer their beneficial or toxic role. Our data showed that ZnO was beneficial at lower concentration, but increased concentration have toxic effects. SiO2 promoted plant growth at almost every concentration while composite (Combination of ZnO and SiO2) NPs exhibited combined effects of both the nanoparticles providing more wider range to use ZnO nanoparticles in combination with SiO2 nanoparticles. Ethylene biosynthesis and pathway genes were upregulated in response to ZnO and composite NPs while SiO2 showed no induction and thus confirmed the role of ethylene pathway in mediating stress responses due ZnO NPs. The increased expression of cytokinin response genes due to application of SiO2 NPs implied the beneficial role of SiO2 NPs in seedling growth. These findings are helpful in complete understanding of effects of ZnO, SiO2 and their composite NPs and the pathways mediating toxicity or beneficial effects in plants.