The Role of Two Tyrosinase-Like Glycoenzymes in Defining the Final Hue of Parrot Plumage

Abstract

Recent advances in avian melanogenesis have pinpointed multiple genetic loci associated with color polymorphisms, predominantly in the plumage of chickens, quails, and pigeons. However, the genetic basis of melaninization in parrot plumage remains elusive. Previously, we showed that mutations in the melanosomal ion-transporter SLC45A2 lead to a complete loss of blue structural color in green parrot feathers, leaving only yellow psittacofulvin. Yet, several color morphs involving partial or complete melanin reduction are common in captive-bred parrots that have not been studied. To bridge this gap, we investigated two new color morphs of parrot plumage: non-sex-linked recessive lutino (NSL), which entirely inhibits blue structural coloration, and the sex-linked recessive cinnamon, which reduces the intensity of blue structural coloration. Our genotypic analysis revealed that tyrosinase (TYR) variants are responsible for the NSL phenotype in Fischer's lovebird and green-cheeked parakeet, while tyrosinase related protein 1 (TYRP1) variants are associated with the cinnamon phenotype in the rose-ringed parakeet. When transfected into HEK293T cells, the candidate substitutions significantly affected tyrosinase enzymatic activity. This study underscores tyrosinase and related enzymes' role in parrot feather coloration, enhancing our understanding of avian melanogenesis as well as the conserved functions of melanogenic components across different species.

Keywords: TYR; TYRP1; eumelanin; feather; parrot.

FIGURE 1

Color morphs analyzed in this study. (a) NSL (b) cinnamon (WT birds are on the left and mutant morphs are at right). Phenotypic changes in the mutant feather are illustrated through overlapping colored circles. Each circle represents psittacofulvin pigmentation or structural color. [Pictures are taken by Shatadru Ghosh Roy and Moty Abdu or available in public domain].

FIGURE 2

Pigment characterization of cinnamon parrot feathers. Confocal Raman microscopy measurements confirmed the melanin types in (a) black feathers of feral pigeons ( C. livia ), known for their high eumelanin content and (b) brown pigeon feathers characterized by their pheomelanin content (Haase et al. 1992). Measurements from feathers of parrot P. krameri (c) wild type and (d) cinnamon mutant revealed spectra similar to those of black pigeon feathers. (e) Multidimensional SVD analysis further demonstrated that the pigment in parrot feathers matches that found in black pigeon feathers. Panels (a)–(d) display feather details, close‐ups of the measured regions, depth‐resolved virtual feather cross‐sections showing the detected molecular signal, and Raman spectra of the pigments. (f) Spectral interpretation of the difference between black pigeon, cinnamon and wild‐type parrot spectra versus brown pigeon spectra. Thus, this differential spectrum illustrates the spectral difference between predominantly eumelanin and predominantly pheomelanin dominant Raman spectra. Panels show (g) magnified view of light microscope images of wild‐type feathers (top‐left) with green barbs (Brb) and yellow to black barbules (Brbl) and cinnmaon feather (top‐right) with pale green barbs (Brb) and yellow to light brown barbules (Brbl) from P. krameri. Vertical sections of a single barb from wild‐type (bottom‐left) feather, showing medullary melanin (Mel) with dark black coloration and from cinnamon (bottom‐right), showing with light brown coloration. [Scale bars for light microscope images are 20 μm.] (h) Biochemical pathway of eumelanin synthesis in feathers. DHI, 5,6‐dihydroxyindole; DHICA, 5,6‐dihydroxyindole‐2‐carboxylic acid; IQ, Indole‐5,6‐quinone; IQCA, Indole‐5,6‐quinone‐2‐carboxylic acid; L‐DOPA, L‐3,4‐dihydroxyphenylalanine.

FIGURE 3

Non‐synonymous variations found in TYR and TYRP1 homologs. (a) Position of affected residues on the domain structure of typical Psittaciform TYR and TYRP1 homologs. A, first metal‐binding site; B, second metal‐binding site; C, cytosolic domain; CR, cysteine‐rich subdomain; SP, signal peptide; TL, tyrosinase‐like subdomain; TM, transmembrane domain. Secondary structure elements were illustrated using the TYRP1 crystal structure (PDB 5M8L) (Lai et al. 2017). (b) Conservation status of substituted amino acid residues found in TYR (top) and TYRP1 (bottom) homologs. Helices comprising affected residues are illustrated respectively.

FIGURE 4

Effect of candidate substitutions on tyrosinase enzymatic activity (a) Graphical representation of transfecting AfTYR gene into HEK293T cells. (b) melanin deposition (marked with red dotted lines) upon transfection of wild‐type AfTYR in HEK293T cells (c) Cell pellets after 48 h., expressing wild‐type or mutated AfTYR under illumination of white light (top) or 488 blue light (bottom). Corresponding AfTYR mutant used for validating PkTYRP1 variant is marked in red (see Figure S2).

FIGURE 5

Current model of key genes known to control melanin pigmentation in parrot feathers. (a) Diagram illustrating a developing feather follicle and its cross‐section. Melanocytes are situated within barb ridges and transfer their melanosomes to adjacent keratinocytes (Lin et al. 2013). (b) schematic representation of putative avian melanogenesis. The enzyme tyrosinase is a critical rate‐limiting factor in converting tyrosine into melanin. Activation of the melanocortin 1 receptor (encoded by the MC1R gene) by α‐melanocyte‐stimulating hormone (α‐MSH) increases the expression of melanogenic enzymes: Tyrosinase (TYR), dopachrome tautomerase (DCT/TYRP1), and tyrosinase‐related protein 1 (TYRP1), leading to the production of dark eumelanin. Key factors also include melanosome transporter proteins encoded by the SLC24A5, SLC45A2, and OCA2 genes, which are involved in transporting small molecules, ions, and regulating pH. The genes SLC24A5, SLC45A2, OCA2, TYR, TYRP1, and TYRP2 exhibit variations in melanogenesis across species. (c) Identified loci of three melanogenic defects in parrot feathers. Loss of function mutation in SLC45A2 (marked in blue as previously described) and TYR causes lutino (complete absence of melanin pigmentation) phenotype of sex‐linked and non‐sex‐linked inheritance, respectively. Loss of function mutation in TYRP1 leads to cinnamon phenotype turning dark black eumelanin into light brown within barb microstructure, that results in pastel shade of green feather.