Multiple heme oxygenase family members contribute to the biosynthesis of the phytochrome chromophore in Arabidopsis

The oxidative cleavage of heme by heme oxygenases (HOs) to form biliverdin IXalpha (BV) is the committed step in the biosynthesis of the phytochrome (phy) chromophore and thus essential for proper photomorphogenesis in plants. Arabidopsis (Arabidopsis thaliana) contains four possible HO genes (HY1, HO2-4). Genetic analysis of the HY1 locus showed previously that it is the major source of BV with hy1 mutant plants displaying long hypocotyls and decreased chlorophyll accumulation consistent with a substantial deficiency in photochemically active phys. More recent analysis of HO2 suggested that it also plays a role in phy assembly and photomorphogenesis but the ho2 mutant phenotype is more subtle than that of hy1 mutants. Here, we define the functions of HO3 and HO4 in Arabidopsis. Like HY1, the HO3 and HO4 proteins have the capacity to synthesize BV from heme. Through a phenotypic analysis of T-DNA insertion mutants affecting HO3 and HO4 in combination with mutants affecting HY1 or HO2, we demonstrate that both of the encoded proteins also have roles in photomorphogenesis, especially in the absence of HY1. Disruption of HO3 and HO4 in the hy1 background further desensitizes seedlings to red and far-red light and accelerates flowering time, with the triple mutant strongly resembling seedlings deficient in the synthesis of multiple phy apoproteins. The hy1/ho3/ho4 mutant can be rescued phenotypically and for the accumulation of holo-phy by feeding seedlings BV. Taken together, we conclude that multiple members of the Arabidopsis HO family are important for synthesizing the bilin chromophore used to assemble photochemically active phys.     

The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase

The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.     

Evidence against a regulatory role for heme oxygenase in hepatic heme synthesis

The role of a microsomal enzyme system, heme oxygenase, in the regulation of intracellular heme concentration and of the heme biosynthetic pathway was investigated. It was determined that alterations in heme oxygenase activity were not consistent with the observed alterations of heme biosynthesis produced by the administration of various drugs. It is concluded that heme oxygenase does not play a role in the regulation of heme biosynthesis under these circumstances.     

Root-localized phytochrome chromophore synthesis is required for photoregulation of root elongation and impacts root sensitivity to jasmonic acid in Arabidopsis

Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA.     

Spectral properties and chromophore rotation of phytochrome bound to substituted Sepharose

Brushite purified phytochrome from Avena sativa L. cv. Sol II was bound to phenyl Sepharose, octyl Sepharose, CNBr-activated Sepharose and to anti-phytochrome immunoglobulins immobilized on Sepharose. The spectral properties of phytochrome bound to anti-phytochrome immunoglobulins and to phenyl Sepharose were similar to phytochrome in solution. Phytochrome bound to CNBr-activated Sepharose or to octyl Sepharose showed reduced P_fr formation after red irradiation. The reversal to P_r with far-red light was only partial but a further increase at 667 nm took place slowly in the dark. A peak at 657 nm was seen in the difference spectrum between CNBr-activated Sepharose-bound phytochrome kept in darkness and the identical sample immediately after a far-red irradiation.
The change in linear dichroism at 660 nm and 730 nm, induced by plane polarized red or far-red light, was measured. It was computed that the long-wavelength transition moment of phytochrome had an average rotation angle of 31.5° or 180°–31.5°. The substrate used for immobilization had a limited effect on the rotation angle. Phytochrome immobilized on CNBr-activated Sepharose gave an angle of 27.8° and phytochrome immobilized on phenyl Sepharose gave an angle of 32.6°.     

Complex formation between heme oxygenase and phytochrome during biosynthesis in Pseudomonas syringae pv. tomato

The plant pathogen Pseudomonas syringae pv. tomato carries two genes encoding bacterial phytochromes. Sequence motifs identify both proteins (PstBphP1 and PstBphP2, respectively) as biliverdin IXα (BV)-binding phytochromes. PstbphP1 is arranged in an operon with a heme oxygenase (PstBphO)-encoding gene (PstbphO), whereas PstbphP2 is flanked downstream by a gene encoding a CheY-type response regulator. Expression of the heme oxygenase PstBphO yielded a green protein (λ(max) = 650 nm), indicative for bound BV. Heterologous expression of PstbphP1 and PstbphP2 and in vitro assembly with BV IXα yielded the apoproteins for both phytochromes, but only in the case of PstBphP1 a light-inducible chromoprotein. Attempts to express the endogenous heme oxygenase BphO and either of the two phytochromes from two plasmids yielded only holo-PstBphP1. Relatively small amounts of soluble holo-PstBphP2 were just obtained upon co-expression with BphO from P. aeruginosa. Expression of the operon containing PstbphO:PstbphP1 led to an improved yield and better photoreactivity for PstBphP1, whereas an identical construct, exchanging PstbphP1 for PstbphP2 (PstbphO:PstbphP2), again yielded only minute amounts of chromophore-loaded BphP2-holoprotein. The improved yield for PstBphP1 from the PstbphO:PstbphP1 operon expression is apparently caused by complex formation between both proteins during biosynthesis as affinity chromatography of either protein using two different tags always co-purified the reaction partner. These results support the importance of protein-protein interactions during tetrapyrrole metabolism and phytochrome assembly.     

The heme-oxygenase family required for phytochrome chromophore biosynthesis is necessary for proper photomorphogenesis in higher plants

The committed step in the biosynthesis of the phytochrome chromophore phytochromobilin involves the oxidative cleavage of heme by a heme oxygenase (HO) to form biliverdin IXalpha. Through positional cloning of the photomorphogenic mutant hy1, the Arabidopsis HO (designated AtHO1) responsible for much of phytochromobilin synthesis recently was identified. Using the AtHO1 sequence, we identified families of HO genes in a number of plants that cluster into two subfamilies (HO1- and HO2-like). The tomato (Lycopersicon esculentum) yg-2 and Nicotiana plumbaginifolia pew1 photomorphogenic mutants are defective in specific HO genes. Phenotypic analysis of a T-DNA insertion mutant of Arabidopsis HO2 revealed that the second HO subfamily also contributes to phytochromobilin synthesis. Homozygous ho2-1 plants show decreased chlorophyll accumulation, reduced growth rate, accelerated flowering time, and reduced de-etiolation. A mixture of apo- and holo-phyA was detected in etiolated ho2-1 seedlings, suggesting that phytochromobilin is limiting in this mutant, even in the presence of functional AtHO1. The patterns of Arabidopsis HO1 and HO2 expression suggest that the products of both genes overlap temporally and spatially. Taken together, the family of HOs is important for phytochrome-mediated development in a number of plants and that each family member may uniquely contribute to the phytochromobilin pool needed to assemble holo-phytochromes.     

Spectroscopic properties and chromophore conformations of the photomorphogenic receptor: phytochrome.

Fluorescence lifetimes of 'large (mol. wt. 120,000) and 'small' (mol. wt. 60,000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of 'large' phytochrome was significantly shorter than that of 'small' phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of 'small' phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very hight (0.4) at the main absorption band and is negative (--0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of 'large' rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of 'small' phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in 'large' phytochrome but not in 'small' Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr leads to Pfr phototransformation have been proposed on the basis of the above results.     

Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme.

An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 heme oxygenase gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The protein binds equimolar free hemin to catabolize the bound hemin to ferric-biliverdin IX alpha in the presence of oxygen and reducing equivalents, showing the heme oxygenase activity. During the reaction, verdoheme intermediate is formed with the evolution of carbon monoxide. Though both ascorbate and NADPH-cytochrome P450 reductase serve as an electron donor, the heme catabolism assisted by ascorbate is considerably slow and the reaction with NADPH-cytochrome P450 reductase is greatly retarded after the oxy-heme complex formation. The optical absorption spectra of the heme-enzyme complexes are similar to those of the known heme oxygenase complexes but have some distinct features, exhibiting the Soret band slightly blue-shifted and relatively strong CT bands of the high-spin component in the ferric form spectrum. The heme-enzyme complex shows the acid-base transition, where two alkaline species are generated. EPR of the nitrosyl heme complex has established the nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase-1 complex. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform-1 is different from that of rat heme oxygenase isoform-1, rather resembling that of bacterial heme oxygenase, H mu O.     

Expression of a Brassica napus heme oxygenase confers plant tolerance to mercury toxicity

Plant heme oxygenases (HOs) regulate biosynthesis of phytochrome which accounts for photo‐acceptance and ‐morphogenesis. Recent studies have demonstrated that plant HOs also regulate many other physiological processes including response to environmental stimuli. To elucidate the mechanism by which HOs regulate plant adaptation to heavy metal exposure, three novel HOs genes were isolated from rapeseed (Brassica napus) and their expression patterns were analysed. Alignment of deduced protein sequences revealed that the three BnHOs share high identity with their corresponding orthologos (AtHO1‐3) from Arabidopsis. To investigate whether the BnHO regulates plant tolerance to Hg toxicity, we constructed B. napus transgenic plants overexpressing BnHO‐1. Under Hg stress, the transgenic plants had 1.41–1.59 folds higher biomass than the untransformants. However, overexpression of BnHO‐1 resulted in less accumulation of Hg in some lines of transformants than in untransformants. The transgenic plants show lower abundance of reactive oxygen species and attenuated oxidative injury compared with the untransgenic plants. We cloned the promoter sequences of BnHO‐1 from B. napus. Analysis revealed that the 1119 bp fragment contains a conserved Cd responsive element (CdRE) and others responding to multiple environmental stimuli. Transient expression in tobacco leaves showed differential responses to heavy metals (Zn, Cu, Pb, Hg and Cd).     

Ferritin is not a required intermediate for iron utilization in heme synthesis

Pulse-chase analysis of newt (Triturus cristatus) erythroblasts has shown that ferritin is not a primary source of iron for heme synthesis. During chase incubation with and without non-radioactive plasma iron in the medium, no transfer of 59Fe from ferritin to hemoglobin was detected although the integrity of heme synthesis was maintained. In puromycin-inhibited cells where iron uptake was drastically curtailed, heme synthesis continued to occur, though at reduced levels; incorporation of 59Fe from the plasma appeared initially in heme and hemoglobin without any prior labelling of ferritin. These results indicate that ferritin is neither an obligatory iron intermediate in heme synthesis nor a cytosolic transport molecule involved in mobilization of iron from the transferrin-receptor complex. The most likely role for erythroid ferritin is storage of excess iron.     

Purification and properties of heme oxygenase from rat liver microsomes.

Heme oxygenase was purified to apparent homogeneity from liver microsomes of rats which had been treated with either cobaltous chloride or hemin to induce heme oxygenase in the liver and the purified preparations from either rats showed an apparent molecular weight of about 200,000 when estimated by gel filtration on a column of Sephadex G-200, and gave a minimum molecular weight of about 32,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hepatic heme oxygenase could bind heme to form a heme . heme oxygenase complex showing an absorption peak at 405 nm, and the extinction coefficient at 405 nm of the heme . heme oxygenase complex was 140 mM-1 cm-1. The heme bound to the hepatic heme oxygenase protein was easily converted to biliverdin when the complex was incubated with the NADPH-cytochrome c reductase system in air. The hepatic heme oxygenase appears to have characteristics essentially similar to those of the splenic heme oxygenase (Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224 and 4230). The heme oxygenase preparation which was purified from the cobalt-treated rats contained a small amount of cobaltic protoporphyrin, indicating that cobalt protoporphyrin was synthesized in these rats.     

Expression of heme oxygenase and its RNA in mouse liver after injection of heme and splenectomy.

Heme is known to activate the HO (heme oxygenase) gene in cultured cells, but little is known about the effect of heme on the HO gene in intact organisms. The expressions of HO and its RNA in mouse liver were measured using mouse HO cDNA and HO antibody after injection of heme or splenectomy. The antibody was prepared against a beta-galactosidase-HO hybrid protein made in Escherichia coli. The HO mRNA level increased to a maximum 15 h after heme injection. In contrast, expression of HO was maximal about 45 h after heme injection. Essentially the same results were obtained in mice after splenectomy. These results suggest that the HO gene in mouse liver was activated by the injection of heme and splenectomy.     

Crystal structure of the dioxygen-bound heme oxygenase from Corynebacterium diphtheriae: implications for heme oxygenase function

HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 microm(-1) s(-1) and 0.22 s(-1), respectively, yielding an O(2) affinity of 21 microm(-1), which is approximately 20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3-4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor O(2) binding. Resonance Raman spectra show that the Fe-O(2) vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe-O-O geometry that is characteristic of the oxy forms of heme oxygenases. In the crystal structure of the oxy form the Fe-O-O angle is 110 degrees, the O-O bond is pointed toward the heme alpha-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O(2) and the amide nitrogen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation. In addition, the O-O bond is orthogonal to the plane of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin alpha-meso-carbon by preventing premature O-O bond cleavage.     

Intracellular site of synthesis of microsomal heme oxygenase in pig spleen

In the pig spleen the specific activity of heme oxygenase was two to three times higher in smooth microsomes than in rough microsomes, whereas the total heme oxygenase activities recovered in the two microsomal fractions were similar. Free and bound polysomes were isolated from pig spleen and nascent peptides on these polysomes were analyzed by employing [3H]puromycin and a heme oxygenase-specific rabbit antibody (IgG). It was shown that free polysomes are the major site of heme oxygenase synthesis. In addition, cell-free synthesis of heme oxygenase was performed in a reticulocyte lysate system with free and bound polysomes isolated from pig spleen, and the results obtained again indicated that heme oxygenase is synthesized predominantly on free polysomes. The heme oxygenase newly synthesized on free polysomes may be incorporated first into the rough portion of endoplasmic reticulum either before or after its release from polysomes, although the specific activity of this enzyme at the steady state is considerably higher in the smooth region.