Ethylene signaling pathway

Isoforms of ethylene receptor in Arabidopsis thaliana.

Ethylene binds to it specific transmembrane receptor present on the cell membrane of endoplasmic reticulum.[8][9] There are different ethylene receptor isoforms. Five isoforms are known in Arabidopsis thaliana which are named ethylene response/receptor 1 (ETR1), ethylene response sensor 1 (ERS1), ETR2, ERS2, and ethylene insensitive 4 (EIN4).[10] The ETR1 is similar (conserved sequence) in different plants but with slight amino acid differences.[11][12] A. thaliana receptors are classified into two subfamilies based on genetic relationship and common structural features, namely subfamily 1 that includes ETR1 and ERS1, and subfamily 2 that consists of ETR2, ERS2, and EIN4.[13] In tomato there are seven types of ethylene receptors named SlETR1, SlETR2, SlETR3, SlETR4, SlETR5, SlETR6, and SlETR7 (Sl for Solanum lycopersicum, the scientific of tomato).[14]

All ethylene receptors have similar organisation: a short N-terminal domain, three conserved transmembrane domains towards the N-terminus, followed by a GAF domain of unknown function, and then signal output motifs in the C-terminal region.[10] The N-terminus is exposed on the lumen of the endoplasmic reticulum, and the C-terminus that is exposed to the cytoplasm of the cell. The N-terminus contains the sites for binding of ethylene, dimerization and membrane localization.[15][16] Two similar receptors combine to form a homodimer through a disulfide bridge forming a cysteine-cysteine interaction.[17] However, the main membrane localization is done by the transmembrane domain, which can also bind ethylene with the help of copper as a cofactor.[7] Copper ion is supplied by a transmembrane protein responsive-to-antagonist 1 (RAN1) from antioxidant protein 1 (ATX1) via tiplin,[18] or directly by copper transport protein.[19]

Although the receptors are functionally active as dimers, only one copper ion binds to such dimer, indicating that one receptor dimer binds only one ethylene molecule.[7] Mutations in the binding sites stop ethylene binding and also make plants insensitive to ethylene.[20] Cys-65 in the protein helix 2 is particularly important as the binding site of copper ion as mutation in it stops copper and ethylene binding.[1] The C-terminus is basically a bacterial two-component system with kinase activity and response regulator.[15] ETR1 has histidine kinase activity, whereas ETR2, ERS2, and EIN4 have serine/threonine kinase activity, and ERS1 has both.[1] The histidine kinase in ETR1 is not required for ethylene signaling.[21]

Ethylene signaling pathway in Arabidopsis thaliana.

Two proteins are crucial for interacting ethylene with the receptors, namely constitutive triple response 1 (CTR1) and ethylene insensitive 2 (EIN2). CTR1 is a serine/threonine protein kinase that functions as a negative regulator of ethylene signalling. It is a member of the signaling protein mitogen-activated protein kinase (MAPK) kinase kinase.[10] EIN2 is required for ethylene signalling and is part of the NRAMP (natural resistance-associated macrophage protein) family of metal transporters; it comprises a large, N-terminal portion containing multiple transmembrane domains (EIN2-N) in the ER membrane and a cytosolic C-terminal portion (EIN2-C).[1] Other proteins such as reversion to ethylene sensitivity 1 (RTE1), cytochrome b5 and tetratricopeptide repeat protein 1 (TRP1) also play important roles in ethylene signaling. RTE1 is a highly conserved proteins in plants and protists but absent in fungi and prokaryotes.[27] TRP1 is genetically related to transmembrane and coiled-coil protein 1 (TCC1) in animals that is involved F actin function and competes with Raf-1 for Ras binding.[28]

Unlike in most signal transductions where the ligands activate their receptors to relay their signals, ethylene acts as the suppressor of its receptor, and the receptor being the negative regulator in ethylene responses. Ethylene receptor is active in the absence of ethylene. Without ethylene, the receptor binds to CTR1 at its C-terminal kinase domain. The kinase activity of CTR1 becomes activated and phosphorylates the neighbouring EIN2.[1] As long as EIN2 remains highly phosphorylated, it remains inactive and there never is an ethylene signal relay. In ETR1, the receptor histidine kinase is required for binding with EIN2.[29] RTE1 can bind to and activate ETR1 independent of CTR1.[30] There is evidence that cytochrome b5 aids or acts similar to RTE1.[31]

Ethylene binding to the receptor disrupts the EIN2 phosphorylation. It does not cause any particular change in the structural feature of the receptor-CTR1-EIN2 complex or stop the phosphorylation. In fact, at low level of ethylene there is increased receptor-CTR1-EIN2 complexes, which is then reduced as ethylene level rises.[32] The turnover process is not yet fully understood. The only consequence of ethylene binding is reduced phosphorylation of EIN2. Under such condition EIN2 is activated and is cleaved to release EIN2-C from the membrane-bound EIN2-N portion. The enzyme that causes the cleavage is yet unknown.[1] The role of EIN2-N is also unknown in A. thaliana. But in rice, its homologue OsEIN2-N (Os for Oryza sativa, the scientific name for rice) interacts with another protein, mao huzi 3 (MHZ3), a mutation of which gives rise to insensitivity to ethylene.[33]

EIN2-C is the main component that mediates ethylene signal in the cell. It acts in two ways. In one, it binds the mRNAs that encode for EIN3-binding F-box proteins, EBF1 and EBF2 to cause their degradation.[34] In another, it enters the nucleus to bind with EIN2 nuclear associated protein 1 (ENAP1) to regulate transcriptional and translational activities of EIN3 and the related EIL1 transcription factor to cause most of the ethylene responses.[35]