| Popis: |
C-TERMINALLY ENCODED PEPTIDEs (CEPs) control diverse plant responses including root development, root system shape, nitrogen demand signalling, and nutrient allocation influencing yield. There is evidence that different CEP ligands impart different phenotypic responses. Thus, a need exists for a simple method that determines if different CEP family peptides bind the same receptor and validates bona fide CEP hormone-receptor pairings in vivo. The method developed used formaldehyde or photo-activation to cross-link fluorescently tagged group 1 or group 2 CEPs to receptors in semi-purified Medicago truncatula or Arabidopsis thaliana leaf vascular tissues. We used this method to verify that COMPACT ROOT ARCHITECTURE 2 (CRA2) is the major Medicago CEP receptor and to investigate if sequence diversity of ligands within the CEP family influences receptor binding. Formaldehyde cross-linked the fluorescein isothiocyanate (FITC)-tagged Medicago group 1 CEP (MtCEP1) to wild-type Medicago or Arabidopsis vascular tissue cells, but not to the corresponding CEP receptor mutants, cra2 or cepr1. Binding competition showed that unlabelled MtCEP1 displaces FITC-MtCEP1 from CRA2. By contrast, the group 2 CEP, FITC-AtCEP14, bound to vascular tissue independently of CEPR1 or CRA2, and AtCEP14 did not complete with FITC-MtCEP1 to bind CEP receptors. The binding of a photo-activatable FITC-MtCEP1 to the periphery of Medicago vascular cells suggested that CRA2 localises to the plasma membrane. We extracted and visualised a fluorescent 105 kDa protein corresponding to photo-cross-linked FITC-MtCEP1-CRA2 complex using SDS-PAGE. Mass spectrometry identified CRA2-specific peptides in this protein band. These results indicate that FITC-MtCEP1 binds to CRA2, MtCRA2 and AtCEPR1 are functionally equivalent, and that the binding specificity of group1 and group 2 CEP ligands are distinct. Using formaldehyde or photo-activated crosslinking of biologically-active, fluorescently-tagged ligands may find wider utility by identifying CEP-CEP receptor pairings in diverse plants. |
