The Arabidopsis HY2 Gene Encodes Phytochromobilin Synthase, a Ferredoxin-Dependent Biliverdin Reductase

Abstract

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.

Phycocyanobilin:Ferredoxin Oxidoreductase ofAnabaena sp. PCC 7120

“…PCC 7120 pcyA was cloned into the Escherichia coli expression vector pGEX-6-P1 (Amersham Biosciences) to produce pGEXpcyA (4). E. coli strain DH5␣ containing pGEXpcyA was induced to express glutathione S-transferase-PcyA, which was purified according to instructions supplied by the manufacturer and protocols described earlier (10). Proteolytic cleavage with the PreScission™ protease yielded the native protein with the N-terminal amino acid extension GPLGSPEF and with the initiator methionine residue changed to isoleucine.…”

“…2 Flavodoxin was quantified by absorption at 467 nm and an absorption coefficient of ⑀ 467 nm 9,500 M Ϫ1 cm Ϫ1 (13), whereas putidaredoxin and putidaredoxin reductase were quantified by absorption at 454/415 nm, respectively, using the absorption coefficients of ⑀ 454 nm 10,000 M Ϫ1 cm Ϫ1 and ⑀ 415 nm 11,100 M Ϫ1 cm Ϫ1 . 2 Standard Bilin Reductase Activity Assay-Assays for bilin reductase activity were performed as described previously (10,14). Standard assays contained 1.5 M PcyA, 4.8 M ferredoxin, and 5 M BV IX␣ in 25 mM TES-KOH, pH 7.5 (assay buffer), and were incubated for 30 min at 28°C under green safe light unless otherwise specified.…”

“…To verify that pigment I was a bona fide intermediate in the formation of PCB, it was collected and tested for its ability to bind to apo-Cph1 using a coupled phytochrome assembly assay (10,14) and for its ability to be further metabolized by PcyA to PCB using HPLC. As shown in Fig.…”

TheElm1(ZmHy2) Gene of Maize Encodes a Phytochromobilin Synthase

“…There are a number of known mutants in which linear tetrapyrrole synthesis is disrupted. These include the hy1 and hy2 mutants of Arabidopsis (Koornneef et al, 1980;Davis et al, 1999;Muramoto et al, 1999;Kohchi et al, 2001), the partially-etiolated-in-white-light1 (pew1) and pew2 mutants of tobacco (Nicotiana plumbaginifolia; Kraepiel et al, 1994), the phytochrome chromophore-deficient1 (pcd1) and pcd2 mutants of pea (Pisum sativum; Weller et al, 1996Weller et al, , 1997, the aurea (au) and yellow-green2 (yg-2) mutants of tomato (Lycopersicon esculentum; Koornneef et al, 1985;Terry and Kendrick, 1996), the photoperiodic sensitive5 (se5) mutant of rice (Oryza sativa; Yokoo and Okuno, 1993;Izawa et al, 2000), and the elongated mesocotyl1 (elm1) mutant of maize (Sawers et al, 2002). PFB is synthesized from 5-aminolevulinic acid, a precursor to both heme and chlorophyll (Elich and Lagarias, 1987).…”

“…It is not known whether the isomerization of 3Z-PFB to 3E-PFB is enzymemediated or whether it occurs spontaneously. Of these three activities, genes encoding the first two have now been cloned (Davis et al, 1999;Muramoto et al, 1999;Kohchi et al, 2001). The HO1 (HY1) gene encodes heme oxygenase, which is targeted to the plastid (Muramoto et al, 1999).…”