ABSTRACT
Plant glutamate receptors (GLRs) are integral membrane proteins that function as non-selective cation channels, involved in the regulation of developmental events crucial in plants. Knowledge of these proteins is restricted to a few species and their true agonists are still unknown in plants. Using tomato SlGLRs, a search was performed in the pepper database to identify GLR sequences in habanero pepper (Capsicum chinense Jacq.). Structural, phylogenetic, and orthology analysis of the CcGLRs, as well as molecular docking and protein interaction networks, were conducted. Seventeen CcGLRs were identified, which contained the characteristic domains of GLR. The variation of conserved residues in the M2 transmembrane domain between members suggests a difference in ion selectivity and/or conduction. Also, new conserved motifs in the ligand-binding regions are reported. Duplication events seem to drive the expansion of the species, and these were located in the evolution by using orthologs. Molecular docking analysis allowed us to identify differences in the agonist binding pocket between CcGLRs, which suggest the existence of different affinities for amino acids. The possible interaction of some CcGLRs with proteins leads to suggesting specific functions for them within the plant. These results offer important functional clues for CcGLR, probably extrapolated to other Solanaceae.
ABSTRACT
Capsaicinoids are responsible for the pungency in Capsicum species. These are synthesized by the Capsaicin synthase (CS) encoded by the AT3 gene, which catalyzes the transference of an acyl moiety from a branched-chain fatty acid-CoA ester to the vanillylamine to produce capsaicinoids. Some AT3 gene copies have been identified on the Capsicum genome. The absence of capsaicinoid in some nonpungent accessions is related to mutant AT3 alleles. The differences between CS protein copies can affect the tridimensional structure of the protein and the affinity for its substrates, and this could affect fruit pungency. This study characterized 32 AT3 sequences covering Capsicum pungent and non-pungent accessions. These were clustered in AT3-D1 and AT3-D2 groups and representative sequences were analyzed. Genomic upstream analysis shows different regulatory elements, mainly responsive to light and abiotic stress. AT3-D1 and AT3-D2 gene expression was confirmed in fruit tissues of C. annuum. Amino acid substitutions close to the predictable HXXXD and DFGWG motifs were also identified. AT3 sequences were modeled showing a BAHD acyltransferase structure with two connected domains. A pocket with different shape, size and composition between AT3 models was found inside the protein, with the conserved motif HXXXD exposed to it, and a channel for their accessibility. CS substrates exhibit high interaction energies with the His and Asp conserved residues. AT3 models have different interaction affinities with the (E)-8-methylnon-6-enoyl-CoA, 8-methylnonanoyl-CoA and vanillylamine substrates. These results suggested that AT3-D1 and AT3-D2 sequences encode CS enzymes with different regulatory factors and substratum affinities.Communicated by Ramaswamy H. Sarma.
