The phycocyanobilin chromophore of streptophyte algal phytochromes is synthesized by HY2.

Land plant phytochromes perceive red and far-red light to control growth and development, using the linear tetrapyrrole (bilin) chromophore phytochromobilin (PΦB). Phytochromes from streptophyte algae, sister species to land plants, instead use phycocyanobilin (PCB). PCB and PΦB are synthesized by different ferredoxin-dependent bilin reductases (FDBRs): PΦB is synthesized by HY2, whereas PCB is synthesized by PcyA. The pathway for PCB biosynthesis in streptophyte algae is unknown. We used phylogenetic analysis and heterologous reconstitution of bilin biosynthesis to investigate bilin biosynthesis in streptophyte algae. Phylogenetic results suggest that PcyA is present in chlorophytes and prasinophytes but absent in streptophytes. A system reconstituting bilin biosynthesis in Escherichia coli was modified to utilize HY2 from the streptophyte alga Klebsormidium flaccidum (KflaHY2). The resulting bilin was incorporated into model cyanobacterial photoreceptors and into phytochrome from the early-diverging streptophyte alga Mesostigma viride (MvirPHY1). All photoreceptors tested incorporate PCB rather than PΦB, indicating that KflaHY2 is sufficient for PCB synthesis without any other algal protein. MvirPHY1 exhibits a red-far-red photocycle similar to those seen in other streptophyte algal phytochromes. These results demonstrate that streptophyte algae use HY2 to synthesize PCB, consistent with the hypothesis that PΦB synthesis arose late in HY2 evolution.

There and Back Again: Loss and Reacquisition of Two-Cys Photocycles in Cyanobacteriochromes.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Both families use linear tetrapyrrole (bilin) chromophores that are covalently attached to a conserved Cys residue. CBCRs are more spectrally diverse than phytochromes, with known examples detecting light from the near ultraviolet to the edge of the infrared (370-750 nm). Detection of ultraviolet to blue light by CBCRs is mediated by a second Cys residue, which forms a covalent linkage to the bilin C10 atom. Second linkage formation is best understood in a subfamily possessing a conserved Asp-Xaa-Cys-Phe (DXCF) motif. Some DXCF CBCRs can isomerize their phycocyanobilin (PCB) chromophores into phycoviolobilin (PVB), a property not reported for other lineages. Both the DXCF Cys and PVB formation have been lost during evolution of other CBCR subfamilies. Using phylogenetic analysis and characterization of recombinantly expressed CBCRs, we show that the DXCF Cys residue has also been reacquired during CBCR evolution. Guided by this knowledge, we successfully reintroduced a second cysteine into a red/green CBCR, restoring blue-light sensing and PVB formation with two additional substitutions. Our results validate the roles of these residues in CBCR spectral tuning and thus provide new insight into the molecular basis of their spectral diversity.