The phycocyanobilin chromophore of streptophyte algal phytochromes is synthesized by HY2

Abstract

Summary 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.

Ferredoxin-dependent bilin reductases in eukaryotic algae: Ubiquity and diversity

“…Both of these enzymes share the overall fold of FDBRs, despite limited sequence similarity, and hence provide suitable outgroups for structurally informed phylogenetic analysis (Hagiwara et al, 2006; Sugishima et al, 2009). The RCCR-based outgroup gave more consistent root placement and is similar to that used in our previous FDBR phylogeny (Rockwell et al, 2017). …”

“…We used our previous phylogenetic analysis of HY2 from streptophyte algae (Rockwell et al, 2017) as a starting point for constructing new alignments including both eukaryotic sequences and sequences from more diverse bacteria. Alignments were then used to infer structurally informed ML phylogenies using PhyML-structure (Le and Gascuel, 2010).…”

“…Eukaryotic FDBR sequences were identified using BLAST searches of the Genbank protein, EST (expressed sequence tag), TSA (transcriptome shotgun assembly), and SRA (sequence read archive) databases, of publicly available OneKP (one thousand plants, (Matasci et al, 2014)) transcriptomes, and of transcriptomes generated by the Marine Microeukaryotes Transcriptome Sequencing Project (MMETSP, (Keeling et al, 2014)). In some cases, including EST and SRA data, multiple hits were combined to yield a complete FDBR sequence using the approach we previously applied to PCYA from Cyanophora paradoxa CCMP329 (Rockwell et al, 2017). Accession information for FDBR sequences used in this paper is provided in Supplemental Table 1.…”

“…Cyanobacteria contain as many as three FDBRs: PcyA, PebA, and PebB. Phylogenetic analysis places these three enzymes into distinct lineages (Rockwell et al, 2017). Eukaryotic FDBRs characterized to date also belong to these three lineages.…”

“…For example, HY2 enzymes from streptophytes are part of the PebB lineage, whereas PCYA from chlorophyte algae belongs to the PcyA lineage. PUBS from prasinophyte and streptophyte algae and nonvascular plants belongs to the PebA lineage (Rockwell et al, 2017). Thus, photosynthetic organisms can contain multiple FDBRs producing different reduced bilins, sometimes referred to as phycobilins or phytobilins.…”