Phenotypic characterization of the blue phenotypes belongs to diverse parrot species
Psittacofulvin-based hues range from yellow to red and the absence of any of these colors is designated as the blue phenotype among breeders. In the following segment, we have characterized variations in blue phenotypes among diverse parrot species analyzed in this study. In the blue phenotype of predominantly green parrots, such as P. krameri and P. eupatria, the wild-type green becomes light blue due to the absence of yellow pigment (Fig. 1A). Whereas sexually dichromatic E. roratus, previously shown to use yellow and red psittacofulvins to color their feathers 12, displays the blue phenotype differently (Fig. 1A). In E. roratus, males are mostly bright green, and females are predominantly bright red. In this case, blue males display a uniform light blue color across the entire body while females carrying the blue phenotype appear mostly white throughout the body with dark blue on their back and grey on the wings (Fig. 1A). Both sexes of E. roseicapilla have a pale silver to grey back, a pale grey rump, a pink face, and a light pink mobile crest. However, E. roseicapilla individuals carrying the blue phenotype display a white body lacking the pink pigment with unchanged grey wings (Fig. 1B). Key features of overall plumage coloration in targeted species are summarized in Table S1.
The blue phenotype lacks psittacofulvin pigmentation in the barb cortex
In budgerigar feathers, yellow psittacofulvin pigment has been shown to occur in the barbules and the outer cortex of the ramus 13. We started by examining the status of psittacofulvin pigmentation in a typical parrot feather carrying the blue phenotype. As a standard representative, we examined contour feathers of P. krameri. Under a light microscope, a typical green contour feather from wild type green P. krameri shows green barbs and yellow barbules with black tips. The black tips are likely explained by melanin pigmentation (Fig. 2A). In contrast, light microscopic observation of contour feathers from P. krameri exhibiting the blue phenotype displayed blue barbs and white barbules with black tip (Fig. 2C). To characterize the microstructure responsible for the blue feather, we next examined longitudinal sections of contour feathers from the same green and blue P. krameri samples. We found that the outer cortex of green barbs is filled with yellow pigment while the blue barb lacks yellow pigment. The medullary region showed black melanin pigment surrounding central air vacuoles in both samples (Fig. 2).
Coding SNPs in conserved residues of PKS completely segregate with the blue phenotype
The enzyme MuPKS has been shown to be required for the synthesis of psittacofulvins in budgerigars by an iterative mechanism, homologous to that of vertebrate fatty-acid synthase. This raises a question whether also other parrot species exhibiting similar psittacofulvin-based plumage coloration might follow a similar biochemical pathway 8,12. To test whether PKS is functionally conserved across the order Psittaciformes and if mutations in this gene could also be responsible for blue phenotype in other pet parrots, we characterized the function of PKS in several closely related parrots (Fig. 1). For species with genomes available but with no annotation we identified PKS locus using nucleotide BLAST with MuPKS coding sequence as a query 8. For species without an available genome assembly, we sequenced all PKS exons using primers designed based on the closest related species. Examination of the deduced amino acid sequence of all the identified parrot homologs revealed presence of all conserved domains that characterize the PKS protein reported to date (Fig. 3B). Consequently, to detect non-synonymous variants in protein-coding regions of different PKS homologs, we Sanger-sequenced all six exons of the PKS gene, including the splice sites and adjacent intronic parts, from wild-type and blue mutants of each species and compared them by pairwise alignment.
In blue P. krameri, we found a nonsense mutation (c.2005G > T) at exon six, altering the glutamate 668 into a premature stop codon. We also identified another missense mutation (c.334G > A) at exon three changing glycine 112 to arginine. The blue P. eupatria showed one nonsense mutation (c.2647A > T) at exon six changing the lysine 883 into a premature stop codon and one missense mutation (c.728G > A) at exon five, changing the arginine 243 to lysine. We also found one nonsense mutation (c.5869C > T) at exon six of blue E. roratus PKS, changing glutamine 1957 into a premature stop codon. PKS homolog of blue E. roseicapilla showed two missense mutations at exon 6 (c.1284T > G) modifying the asparagine 428 into a lysine and another (c.2374A > G) changing the amino acid threonine 792 to alanine. A detailed list of the detected mutations across different species has been summarized in Table 1, along with the number of specimens genotyped in this study.
A multiple sequence alignment of diverse species, including major vertebrate classes and invertebrates, showed that three out of four affected amino acids (G112, R243, and N428) are universally conserved in other parrot members analyzed in this study along with chicken (Gallus gallus), anole lizard (Anolis carolinensis), zebra fish (Danio rerio), house mouse (Mus musculus), human (Homo sapiens) and fruit fly (Drosophila melanogaster) (Fig. 3A). T792 showed conservation in the order Psittaciformes, chicken, and anole lizard but not in zebra fish, house mouse, human and fruit fly. Type 1 polyketide synthases have been shown to contain multiple domains with independent catalytic activities 14. Structural analysis showed that three of the non-synonymous polymorphisms (G112R, R243K and N428K) found in this study reside on the ketoacyl synthase domain, whereas T792A maps to the MAT domain (Fig. 3B).
Missense variants disrupt feather pigment synthesis in yeast
Previously, it has been shown that organic extracts from yeast cells expressing chicken PKS (GgPKS; LOC420486) exhibited a yellow pigment, sharing similar pigment components with yeast expressing MuPKS as detected by LC-MS analysis 8. Notably, all missense variants identified in our study were found to be conserved in GgPKS. Utilizing this fact, we decided to validate our changing residues by using heterologous expression of GgPKS in the BJ5464-NpgA strain of Saccharomyces cerevisiae. This strain contains a promiscuous phosphopantetheinyl transferase, NpgA that converts various polyketide synthase apoenzymes to holoenzymes 15. The expression of GgPKS in yeast was confirmed through western blot analysis (Fig. 4B and S1).We found that extracts from yeast expressing the GgPKS wild-type (WT) allele displayed a yellow pigment, contrasting with clear extracts from yeast expressing an empty control vector (Fig. 4A, 4B). This difference confirmed GgPKS's ability to synthesize the yellow pigment. Additionally, extracts from yeast expressing the wild-type allele exhibited notable fluorescence under UV-A illumination as observed in the yellow feathers of certain parrot species 16 as well as in octadecaoctaenal purified from red parrot feathers 11. Interestingly, extracts from three independent yeast strains respectively expressing GgPKS with substitutions G112R, R243K, or N428K remained clear (Fig. 4B), while extracts from a yeast strain expressing GgPKS carrying the T792A substitution showed a yellow coloration like the WT extract (Fig. 4B). This indicates that G112, R243, and N428 are vital residues for the biosynthetic activity of GgPKS while, despite residing at the important MAT domain, T792A appears non-essential in PKS function, supporting its partial conservation status across the species.
To investigate the effect of G112R, R243K, or N428K mutations on psittacofulvin expression in finer detail, we have subjected the yeast extracts to a sensitive analysis using ultra-high performance liquid chromatography with photodiode array detector coupled to high-resolution accurate-mass mass spectrometry (UHPLC-PDA-HRAM MS). The UHPLC-PDA results confirmed the presence of psittacofulvins in WT and their absence in the empty vector control (Fig. 4C). The psittacofulvins in WT extract were eluting as two major peaks at retention times 8.75 min., and 9.67 min., and were characteristic by absorbance spectra with peak of maximum absorbance at 402nm, 414nm respectively (Fig. 4D). Subsequent analysis by HRAM-MS confirmed the identities of the two major compounds as C16-carboxyl psittacofulvin (243.1385 m/z) and C18-carboxyl psittacofulvin (269.1542 m/z) which correspond to compounds found in extracts from feather and yeast expressing wild-type MuPKS (Cooke et al. 2017). In the extracts from yeast expressing GgPKS with substitutions G112R and R243K we have detected no or negligible level of known psittacofulvins respectively (Fig. 4C and Table S2). Surprisingly, in the extract from yeast expressing GgPKS with substitution N428K we have detected the same compounds as in the WT (Fig. 4C) characteristic by absorbance with peaks of maximum absorbance at 400nm and 414 nm (Fig. 4D), and the same molecular masses as the psittacofulvins in WT. Comparison of the total area under the two peaks at 8.75 and 9.67min. at 414nm corrected for dry weight of the yeast pellet shows that N428K yeast produce approximately 31% of the WT psittacofulvins (Table S2). Thus, the findings suggest, despite the lack of fluorescence after UV-A excitation, at least partial functionality of the PKS with N428 mutation.
Structural analysis of causal SNPs
Taking advantage of sequence conservation among fatty acid synthases, we were able to map three SNPs causing loss of PKS function found in this study onto the high-resolution structure of porcine fatty acid synthase 17 (Fig. 5A, 3B). These three SNPs reside at the conserved ketoacyl synthase (KS) domain (Fig. 5B) of mammalian fatty acid synthase (mFAS). The KS domain within the mFAS protein has been shown to catalyse the condensation reaction crucial for fatty acid biosynthesis. Affected Glycine 112 in the PKS homolog of blue P. krameri is conserved among vertebrate fatty acid synthases and corresponds to G94 in porcine fatty acid synthase (Fig. 3A). This residue resides at a loop at the interface between the KS domain and a linker domain, presumably providing flexibility to create a turn in the polypeptide chain (Fig. 5C). Replacing this conserved glycine residue with a much larger residue, i.e., arginine, may disrupt the local structure of the loop and the packing of the KS domain and the linker domain due to steric hindrance. R243 in blue P. eupatria PKS corresponds to R224 in porcine FAS (Fig. 3A). R224 is positioned near the three residues that create the entrance of the substrate binding tunnel (Fig. 5D), suggesting that this arginine residue participates in substrate recognition or positioning relative to the active site 17. The SNP identified a change of an arginine to a lysine, a difference that would not be expected to have a major effect due to the similarity between these amino acids. Nevertheless, if this arginine is indeed involved in substrate binding, even such a small change in the properties of the residue could lead to decreased substrate binding and consequently to the dramatic effect seen in pigment synthesis. Finally, N428 in blue E. roseicapilla corresponds to N399 in porcine fatty acid synthase (Fig. 3A). This asparagine residue is conserved also among fatty acid synthases of lower organisms (Fig. 3A), is placed in a β sheet near one of the substrate-binding tunnel residues and is buried under an α helix (Fig. 5E). Replacement of this asparagine to a longer residue, i.e., a lysine, may create a steric clash with the nearby helix, presumably leading to local unfolding and possibly to disruption of the substrate binding tunnel.