Phytobilins are linear tetrapyrrole precursors of the light-harvesting prosthetic groups of the phytochrome photoreceptors of plants and the phycobiliprotein photosynthetic antennae of cyanobacteria, red algae, and cryptomonads. Previous biochemical studies have established that phytobilins are synthesized from heme via the intermediacy of biliverdin IX alpha (BV), which is reduced subsequently by ferredoxin-dependent bilin reductases with different double-bond specificities. By exploiting the sequence of phytochromobilin synthase (HY2) of Arabidopsis, an enzyme that catalyzes the ferredoxin-dependent conversion of BV to the phytochrome chromophore precursor phytochromobilin, genes encoding putative bilin reductases were identified in the genomes of various cyanobacteria, oxyphotobacteria, and plants. Phylogenetic analyses resolved four classes of HY2-related genes, one of which encodes red chlorophyll catabolite reductases, which are bilin reductases involved in chlorophyll catabolism in plants. To test the catalytic activities of these putative enzymes, representative HY2-related genes from each class were amplified by the polymerase chain reaction and expressed in Escherichia coli. Using a coupled apophytochrome assembly assay and HPLC analysis, we examined the ability of the recombinant proteins to catalyze the ferredoxin-dependent reduction of BV to phytobilins. These investigations defined three new classes of bilin reductases with distinct substrate/product specificities that are involved in the biosynthesis of the phycobiliprotein chromophore precursors phycoerythrobilin and phycocyanobilin. Implications of these results are discussed with regard to the pathways of phytobilin biosynthesis and their evolution.

The timing of the transition to flowering in plants is regulated by various environmental factors, including daylength and light quality. Although the red/far-red photoreceptor phytochrome B (phyB) represses flowering by indirectly regulating the expression of a key flowering regulator, FLOWERING LOCUS T (FT), the mechanism of phyB signaling for flowering is largely unknown. Here, we identified two Arabidopsis thaliana genes, VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2, which are highly conserved throughout land plant evolution, as phyB-interacting factors. voz1 voz2 double mutants, but neither single mutant, showed a late-flowering phenotype under long-day conditions, which indicated that VOZ1 and VOZ2 redundantly promote flowering. voz1 voz2 mutations suppressed the early-flowering phenotype of the phyB mutant, and FT expression was repressed in the voz1 voz2 mutant. Green fluorescent protein-VOZ2 signal was observed in the cytoplasm, and interaction of VOZ proteins with phyB was indicated to occur in the cytoplasm under far-red light. However, VOZ2 protein modified to localize constitutively in the nucleus promoted flowering. In addition, the stability of VOZ2 proteins in the nucleus was modulated by light quality in a phytochrome-dependent manner. We propose that partial translocation of VOZ proteins from the cytoplasm to the nucleus mediates the initial step of the phyB signal transduction pathway that regulates flowering.

Light perception by the plant photoreceptor phytochrome requires the tetrapyrrole chromophore phytochromobilin (P Phi B), which is covalently attached to a large apoprotein. Arabidopsis mutants hy1 and hy2, which are defective in P Phi B biosynthesis, display altered responses to light due to a deficiency in photoactive phytochrome. Here, we describe the isolation of the HY2 gene by map-based cloning. hy2 mutant alleles possess alterations within this locus, some of which affect the expression of the HY2 transcript. HY2 encodes a soluble protein precursor of 38 kD with a putative N-terminal plastid transit peptide. The HY2 transit peptide is sufficient to localize the reporter green fluorescent protein to plastids. Purified mature recombinant HY2 protein exhibits P Phi B synthase activity (i.e., ferredoxin-dependent reduction of biliverdin IX alpha to P Phi B), as confirmed by HPLC and by the ability of the bilin reaction products to combine with apophytochrome to yield photoactive holophytochrome. Database searches and hybridization studies suggest that HY2 is a unique gene in the Arabidopsis genome that is related to a family of proteins found in oxygenic photosynthetic bacteria.

By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. Through the selection of different bilin reductases, apophytochromes were assembled with phytochromobilin, phycocyanobilin, and phycoerythrobilin. The blue-shifted difference spectra of truncated phytochromes were observed with a phycocyanobilin chromophore compared to a phytochromobilin chromophore. When the phycoerythrobilin biosynthetic enzymes were co-expressed, E. coli cells accumulated orange-fluorescent phytochrome. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors.