Fungal phytochrome chromophore biosynthesis at mitochondria.

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and-go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.

Crystal structure of phytochromobilin synthase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phytochrome.

Phytochromobilin (PΦB) is a red/far-red light sensory pigment in plant phytochrome. PΦB synthase is a ferredoxin-dependent bilin reductase (FDBR) that catalyzes the site-specific reduction of bilins, which are sensory and photosynthesis pigments, and produces PΦB from biliverdin, a heme-derived linear tetrapyrrole pigment. Here, we determined the crystal structure of tomato PΦB synthase in complex with biliverdin at 1.95 Å resolution. The overall structure of tomato PΦB synthase was similar to those of other FDBRs, except for the addition of a long C-terminal loop and short helices. The structure further revealed that the C-terminal loop is part of the biliverdin-binding pocket and that two basic residues in the C-terminal loop form salt bridges with the propionate groups of biliverdin. This suggested that the C-terminal loop is involved in the interaction with ferredoxin and biliverdin. The configuration of biliverdin bound to tomato PΦB synthase differed from that of biliverdin bound to other FDBRs, and its orientation in PΦB synthase was inverted relative to its orientation in the other FDBRs. Structural and enzymatic analyses disclosed that two aspartic acid residues, Asp-123 and Asp-263, form hydrogen bonds with water molecules and are essential for the site-specific A-ring reduction of biliverdin. On the basis of these observations and enzymatic assays with a V121A PΦB synthase variant, we propose the following mechanistic product release mechanism: PΦB synthase-catalyzed stereospecific reduction produces 2(R)-PΦB, which when bound to PΦB synthase collides with the side chain of Val-121, releasing 2(R)-PΦB from the synthase.

Marine algae and land plants share conserved phytochrome signaling systems.

Phytochrome photosensors control a vast gene network in streptophyte plants, acting as master regulators of diverse growth and developmental processes throughout the life cycle. In contrast with their absence in known chlorophyte algal genomes and most sequenced prasinophyte algal genomes, a phytochrome is found in Micromonas pusilla, a widely distributed marine picoprasinophyte (<2 µm cell diameter). Together with phytochromes identified from other prasinophyte lineages, we establish that prasinophyte and streptophyte phytochromes share core light-input and signaling-output domain architectures except for the loss of C-terminal response regulator receiver domains in the streptophyte phytochrome lineage. Phylogenetic reconstructions robustly support the presence of phytochrome in the common progenitor of green algae and land plants. These analyses reveal a monophyletic clade containing streptophyte, prasinophyte, cryptophyte, and glaucophyte phytochromes implying an origin in the eukaryotic ancestor of the Archaeplastida. Transcriptomic measurements reveal diurnal regulation of phytochrome and bilin chromophore biosynthetic genes in Micromonas. Expression of these genes precedes both light-mediated phytochrome redistribution from the cytoplasm to the nucleus and increased expression of photosynthesis-associated genes. Prasinophyte phytochromes perceive wavelengths of light transmitted farther through seawater than the red/far-red light sensed by land plant phytochromes. Prasinophyte phytochromes also retain light-regulated histidine kinase activity lost in the streptophyte phytochrome lineage. Our studies demonstrate that light-mediated nuclear translocation of phytochrome predates the emergence of land plants and likely represents a widespread signaling mechanism in unicellular algae.

DEVELOPMENT OF SINGLE-DOMAIN NEAR-INFRARED FLUORESCENT PROTEIN GAF-FP BASED ON BACTERIAL PHYTOCHROME.

Fluorescent proteins (FPs) are widely used as genetically encoded markers for noninvasive and quantitative study of biological processes. Development of biomarkers that fluoresce in the near-infrared spectral range allows the study of animals at a deeper level due to high permeability of tissues to light in this wavelength range, compared to the visible light. For widespread use of FPs, such properties as low molecular weight and the monomer become important. In this paper, we developed a FP called the GAF-FP and based on the chromophore- binding domain of bacterial phytochrome from Rhodopseudomonas palustris (RpBphP1). GAF-FP has a molecular mass of ~ 19 kDa, 2 times lower than that of other FP based on BphPs and 1.4 times less than the commonly used GFP-like proteins. Unlike most other near-infrared FP, GAF-FP is a monomer, has high photostability and its structure can withstand the introduction of small peptide inserts. Moreover, GAF-FP can covalently bind two different tetrapyrrole chromophores: phycocyanobilin (PCB) and biliverdin (BV), which is found in mammalian tissues. GAF-FP with BV as a chromophore (GAF-FP­BV) has a main absorption band with a maximum at 635 nm and fluorescence maximum at 670 nm, whereby GAF-FP has a high signal to background ratio even if localized at a depth of several mm below the tissue surface. Apart from the near-infrared absorption band, GAF-FP­BV also has also an absorption band in the violet spectral range with a maximum at 378 nm. This property has been used by us to create a chimeric protein consisting of a modified luciferase from Renilla reniformis (RLuc8) and GAF-FP. We have shown that the chimeric protein is capable of resonance energy transfer from the substrate, which is oxidized by luciferase, to chromophore of GAF-FP­BV. In the absence of energy acceptor, RLuc8 catalyzes the cleavage of the substrate with light radiation having a peak of 400 nm. At the same time, as a part of GAF-FP­RLuc8 chimeric protein, the energy from the substrate is transferred to the chromophore of FP and then emitted in the near-infrared spectral range corresponding to GAF-FP fluorescence. These results open the way for the creation of new small near-infrared FPs based on various natural BphPs with a prospect of their wider use in cell and molecular biology.

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.

Electrostatic interaction of phytochromobilin synthase and ferredoxin for biosynthesis of phytochrome chromophore.

In plants, phytochromobilin synthase (HY2) synthesize the open chain tetrapyrrole chromophore for light-sensing phytochromes. It catalyzes the double bond reduction of a heme-derived tetrapyrrole intermediate biliverdin IXalpha (BV) at the A-ring diene system. HY2 is a member of ferredoxin-dependent bilin reductases (FDBRs), which require ferredoxins (Fds) as the electron donors for double bond reductions. In this study, we investigated the interaction mechanism of FDBRs and Fds by using HY2 and Fd from Arabidopsis thaliana as model proteins. We found that one of the six Arabidopsis Fds, AtFd2, was the preferred electron donor for HY2. HY2 and AtFd2 formed a heterodimeric complex that was stabilized by chemical cross-linking. Surface-charged residues on HY2 and AtFd2 were important in the protein-protein interaction as well as BV reduction activity of HY2. These surface residues are close to the iron-sulfur center of Fd and the HY2 active site, implying that the interaction promotes direct electron transfer from the Fd to HY2-bound BV. In addition, the C12 propionate group of BV is important for HY2-catalyzed BV reduction. A possible role for this functional group is to mediate the electron transfer by interacting directly with AtFd2. Together, our biochemical data suggest a docking mechanism for HY2:BV and AtFd2.

Complete loss of photoperiodic response in the rice mutant line X61 is caused by deficiency of phytochrome chromophore biosynthesis gene.

In rice (Oryza sativa), a short-day plant, photoperiod is the most favorable external signal for floral induction because of the constant seasonal change throughout the years. Compared with Arabidopsis, however, a large part of the regulation mechanism of the photoperiodic response in rice still remains unclear due mainly to the lack of induced mutant genes. An induced mutant line X61 flowers 35 days earlier than its original variety Gimbozu under a natural photoperiod in Kyoto (35°01'N). We attempted to identify the mutant gene conferring early heading to X61. Experimental results showed that the early heading of X61 was conferred by a complete loss of photoperiodic response due to a novel single recessive mutant gene se13. This locus interacts with two crucial photoperiod sensitivity loci, Se1 and E1. Wild type alleles at these two loci do not function in coexistence with se13 in a homozygous state, suggesting that Se13 is an upstream locus of the Se1 and E1 loci. Linkage analysis showed that Se13 is located in a 110 kb region between the two markers, INDEL3735_1 and INDEL3735_3 on chromosome 1. A database search suggested that the Se13 gene is identical to AK101395 (=OsHY2), which encodes phytochromobilin synthase, a key enzyme in phytochrome chromophore biosynthesis. Subsequent sequence analysis revealed that X61 harbors a 1 bp insertion in exon 1 of OsHY2, which induces a frame-shift mutation producing a premature stop codon. It is therefore considered that the complete loss of photoperiodic response of X61 is caused by a loss of function of the Se13 (OsHY2) gene involved in phytochrome chromophore biosynthesis.

Characterization of the haem oxygenase protein family in Arabidopsis thaliana reveals a diversity of functions.

HOs (haem oxygenases) catalyse the oxidative cleavage of haem to BV (biliverdin), iron and carbon monoxide. In plants, the product of the reaction is BV IXalpha, the precursor of the PHY (phytochrome) chromophore and is thus essential for proper photomorphogenesis. Arabidopsis thaliana contains one major biochemically characterized HO (HY1) and three additional putative HOs (HO2, HO3 and HO4). All four proteins are encoded in the nucleus but contain chloroplast translocation sequences at their N-termini. The transit peptides of all four proteins are sufficient for chloroplast translocalization as shown by GFP (green fluorescent protein) reporter gene fusions. Overall, all four proteins can be divided into two subfamilies: HO1 and HO2. Here we show that all members of the HO1 subfamily (HY1, HO3 and HO4) are active monomeric HOs and can convert haem to BV IXalpha using spinach Fd (ferredoxin) as an electron donor. Addition of a second electron donor, such as ascorbate, led to a 10-fold increase in the haem conversion rate. Furthermore, haem turnover is also promoted by light when spinach thylakoids are present. All HO1 family members displayed similar kinetic parameters indicating they all have a possible involvement in PHY chromophore biosynthesis. HO2 did not yield sufficient amounts of soluble protein and therefore required the construction of a synthetic gene adapted to the codon usage of Escherichia coli. HO2 is unable to bind or degrade haem and therefore it is not a haem oxygenase. However, HO2 shows strong binding of proto IX (protoporphyrin IX), a precursor for both haem and chlorophyll biosynthesis. A possible function of HO2 in the regulation of tetrapyrrole metabolism is discussed.

Dynamic intracomplex heterogeneity of phytochrome.

Low temperature single-molecule fluorescence emission spectroscopy on individual phytochromes from Agrobacterium tumefaciens corroborates findings from ensemble spectroscopy concerning intercomplex heterogeneity. Furthermore, time-dependent intracomplex heterogeneity has been observed.

The phytofluors: a new class of fluorescent protein probes.

BACKGROUND: Biologically compatible fluorescent protein probes, particularly the self-assembling green fluorescent protein (GFP) from the jellyfish Aequorea victoria, have revolutionized research in cell, molecular and developmental biology because they allow visualization of biochemical events in living cells. Additional fluorescent proteins that could be reconstituted in vivo while extending the useful wavelength range towards the orange and red regions of the light spectrum would increase the range of applications currently available with fluorescent protein probes. RESULTS: Intensely orange fluorescent adducts, which we designate phytofluors, are spontaneously formed upon incubation of recombinant plant phytochrome apoproteins with phycoerythrobilin, the linear tetrapyrrole precursor of the phycoerythrin chromophore. Phytofluors have large molar absorption coefficients, fluorescence quantum yields greater than 0.7, excellent photostability, stability over a wide range of pH, and can be reconstituted in living plant cells. CONCLUSIONS: The phytofluors constitute a new class of fluorophore that can potentially be produced upon bilin uptake by any living cell expressing an apophytochrome cDNA. Mutagenesis of the phytochrome apoprotein and/or alteration of the linear tetrapyrrole precursor by chemical synthesis are expected to afford new phytofluors with fluorescence excitation and emission spectra spanning the visible to near-infrared light spectrum.

Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli.

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.

Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes.

Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs.

Mosses do express conventional, distantly B-type-related phytochromes. Phytochrome of Physcomitrella patens (Hedw.).

We have screened a cDNA library of the moss Physcomitrella patens (Hedw.) for phytochrome sequences. The isolated sequences turned out to encode a phytochrome dissimilar to the phytochrome type postulated for the moss Ceratodon [(1992) Plant Mol. Biol. 20, 1003-1017] Physcomitrella phytochrome was completely alignable to fern phytochrome (Selaginella) and phytochromes of higher plants. The frequency of clones encoding this phytochrome indicated that a Ceratodon-like type should only be expressed, if at all, with lower frequencies than the sequenced phytochrome cDNA. Sequence differences between lower plant phytochromes are small as compared to phytochrome types of higher plants.

Recombinant holophytochrome in Escherichia coli.

We have successfully co-expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism to produce holophytochrome in Escherichia coli. Heme oxygenase was used to convert host heme to biliverdin IXalpha which was then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase, presumably with the aid of host ferredoxin. In this host environment Cph1 apophytochrome was able to autoassemble with the phycocyanobilin in vivo to form fully photoreversible holophytochrome. The system can be used as a tool for further genetic studies of phytochrome function and signal transduction as well as providing an excellent source of holophytochrome for physicochemical studies.

Recombinant phytochrome of the moss Ceratodon purpureus: heterologous expression and kinetic analysis of Pr-->Pfr conversion.

The phytochrome-encoding gene Cerpu;PHY;2 (CP2) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P phi B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [Pr]/far-red-absorbing phytochrome [Pfr]) for the PCB adduct, and 659/724 nm for the P phi B-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that P phi B is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophytochrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the Pr-->Pfr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I700), decaying with a time constant of approximately 110 microseconds. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the Pfr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.

Biosynthesis of phycobilins. Formation of the chromophore of phytochrome, phycocyanin and phycoerythrin.

Phycobiliproteins play important roles in photomorphogenesis and photosynthesis. The light-absorbing chromophores of the phycobiliproteins are linear tetrapyrroles (bilins) very similar in structure to the mammalian bile pigments. 5-Aminolaevulinate (5-ALA) is the first committed intermediate in phycobilin synthesis. The biosynthesis of 5-ALA, destined for phycobilins, occurs via the five-carbon pathway, now well established for tetrapyrrole synthesis in plants and distinct from the mammalian pathway. The phycobilins are formed by reduction of biliverdin which results from the synthesis and degradation of haem. This haem is an essential intermediate in the biosynthesis of phycobilins. Phycocyanobilin, the blue-green pigment found in certain algae and cyanobacteria, is formed from biliverdin via phytochromobilin, the chromophore of phytochrome. This leads to the likelihood that phytochromobilin is formed as an end product, or intermediate, in the synthesis of all phycobilins.

Expression of recombinant full-length plant phytochromes assembled with phytochromobilin in Pichia pastoris.

We have successfully developed a system to produce full-length plant phytochrome assembled with phytochromobilin in Pichia pastoris by co-expressing apophytochromes and chromophore biosynthetic genes, heme oxygenase (HY1) and phytochromobilin synthase (HY2) from Arabidopsis. Affinity-purified phytochrome proteins from Pichia cells displayed zinc fluorescence indicating chromophore attachment. Spectroscopic analyses showed absorbance maximum peaks identical to in vitro reconstituted phytochromobilin-assembled phytochromes, suggesting that the co-expression system is effective to generate holo-phytochromes. Moreover, mitochondria localization of the phytochromobilin biosynthetic genes increased the efficiency of holophytochrome biosynthesis. Therefore, this system provides an excellent source of holophytochromes, including oat phytochrome A and Arabidopsis phytochrome B.

Chlorophyll deficiency in the maize elongated mesocotyl2 mutant is caused by a defective heme oxygenase and delaying grana stacking.

BACKGROUND: Etiolated seedlings initiate grana stacking and chlorophyll biosynthesis in parallel with the first exposure to light, during which phytochromes play an important role. Functional phytochromes are biosynthesized separately for two components. One phytochrome is biosynthesized for apoprotein and the other is biosynthesized for the chromophore that includes heme oxygenase (HO). METHODOLOGY/PRINCIPAL FINDING: We isolated a ho1 homolog by map-based cloning of a maize elongated mesocotyl2 (elm2) mutant. cDNA sequencing of the ho1 homolog in elm2 revealed a 31 bp deletion. De-etiolation responses to red and far-red light were disrupted in elm2 seedlings, with a pronounced elongation of the mesocotyl. The endogenous HO activity in the elm2 mutant decreased remarkably. Transgenic complementation further confirmed the dysfunction in the maize ho1 gene. Moreover, non-appressed thylakoids were specifically stacked at the seedling stage in the elm2 mutant. CONCLUSION: The 31 bp deletion in the ho1 gene resulted in a decrease in endogenous HO activity and disrupted the de-etiolation responses to red and far-red light. The specific stacking of non-appressed thylakoids suggested that the chlorophyll biosynthesis regulated by HO1 is achieved by coordinating the heme level with the regulation of grana stacking.

Investigating tissue- and organ-specific phytochrome responses using FACS-assisted cell-type specific expression profiling in Arabidopsis thaliana.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL4. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

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.