J. Clark Lagarias

Phytochrome ancestry: sensors of bilins and light.

Phytochromes were long thought to have evolved in non-motile photosynthetic eukaryotes for adaptation to unfavorable light environments, but recent studies suggest that phytochromes evolved billions of years earlier from a tetrapyrrole sensor protein progenitor. These investigations have identified phytochromes and phytochrome-related proteins in photosynthetic bacteria (cyanobacteria and purple bacteria), nonphotosynthetic eubacteria and fungi - an observation that has opened new avenues for investigating the origins, molecular evolution and biochemical functions of this ecologically important family of plant photoreceptors.

A brief history of phytochromes

Photosensory proteins enable living things to detect the quantity and quality of the light environment and to transduce that physical signal into biochemical outputs which entrain their metabolism with the ambient light environment. Phytochromes, which photoconvert between red-absorbing P(r) and far-red-absorbing P(fr) states, are the most extensively studied of these interesting proteins. Critical regulators of a number of key adaptive processes in higher plants, including photomorphogenesis and shade avoidance, phytochromes are widespread in photosynthetic and nonphotosynthetic bacteria, and even in fungi. Cyanobacterial genomes also possess a plethora of more distant relatives of phytochromes known as cyanobacteriochromes (CBCRs). Biochemical characterization of representative CBCRs has demonstrated that this class of photosensors exhibits a broad range of wavelength sensitivities, spanning the entire visible spectrum. Distinct protein-bilin interactions are responsible for this astonishing array of wavelength sensitivities. Despite this spectral diversity, all members of the extended family of phytochrome photosensors appear to share a common photochemical mechanism for light sensing: photoisomerization of the 15/16 double bond of the bilin chromophore.

Visualization of bilin-linked peptides and proteins in polyacrylamide gels

Biliproteins and bilipeptides subjected to discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of zinc acetate form a complex which fluoresces an orange color when viewed under ultraviolet light. The complex between the bilin chromophore and the zinc ion fluoresces at wavelengths which can be selectively visualized in gels by using a red filter. For the biliproteins phytochrome and C-phycocyanin the minimum detectable quantities are 100 and 50 ng, respectively. This is comparable to the sensitivity of Coomassie blue staining. The technique has been used for selective detection of phytochrome in plant extracts and to distinguish chromophore-bearing peptides from those not containing chromophore in proteolytic digests of phytochrome.Biliproteins and bilipeptides subjected to discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of zinc acetate form a complex which fluoresces an orange color when viewed under ultraviolet light. The complex between the bilin chromophore and the zinc ion fluoresces at wavelengths which can be selectively visualized in gels by using a red filter. For the biliproteins phytochrome and C-phycocyanin the minimum detectable quantities are 100 and 50 ng, respectively. This is comparable to the sensitivity of Coomassie blue staining. The technique has been used for selective detection of phytochrome in plant extracts and to distinguish chromophore-bearing peptides from those not containing chromophore in proteolytic digests of phytochrome.

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

Light perception by the plant photoreceptor phytochrome requires the tetrapyrrole chromophore phytochromobilin (PϕB), which is covalently attached to a large apoprotein. Arabidopsis mutants hy1 and hy2, which are defective in PϕB 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 PϕB synthase activity (i.e., ferredoxin-dependent reduction of biliverdin IXα to PϕB), 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.

Functional Genomic Analysis of the HY2 Family of Ferredoxin-Dependent Bilin Reductases from Oxygenic Photosynthetic Organisms

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 IXα (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.

Genetic engineering of phytochrome biosynthesis in bacteria.

The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme. Two ferredoxin-dependent enzymes contribute to this conversion: a heme oxygenase and a bilin reductase with discrete double-bond specificity. Using a dual plasmid system, one expressing a truncated cyanobacterial apophytochrome 1, Cph1(N514), and the other expressing a two-gene operon consisting of a heme oxygenase and a bilin reductase, these studies establish the feasibility of producing photoactive phytochromes in any heme-containing cell. Heterologous expression systems for phytochromes not only will facilitate genetic analysis of their assembly, spectrophotometric activity, and biological function, but also might afford the means to regulate gene expression by light in nonplant cells.

COMPARATIVE PHOTOCHEMICAL ANALYSIS OF HIGHLY PURIFIED 124 KILODALTON OAT and RYE PHYTOCHROMES in vitro

Abstract A direct comparison of the photochemical interconversions between red (Pr‐) and far‐red (Pfr‐) absorbing forms of highly‐purified 124 kDa oat and rye phytochromes under identical experimental conditions was performed. In two different buffer systems at 5°C, the quantum yields for the Pr to Ptr and Pfr to Pr phototransformations under constant red and far‐red illumination, φr and φfr respectively, were determined to be 0.152‐0.154 and 0.060‐0.065 for oat preparations and 0.172‐0.174 and 0.074‐0.078 for rye preparations. These values as well as the wavelength dependence of the photoequilibrium produced under continuous illumination throughout the visible and near‐ultraviolet spectrum were based on the absorption spectra of the two phytochrome preparations and revised molar absorption coefficients. The molar absorption coefficients were estimated by quantitative amino acid analysis and shown to be identical for the two monocot phytochromes (i.e. 132 mM−1 cm−1 at the red absorption maximum for the Pr form). Because these measurements were performed under identical experimental conditions, including buffer, temperature, light fluence rate, and instrumentation, the differences observed must reflect structural features inherent to the two different monocotyledonous phytochromes.

Ultrafast excited-state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy

Photochemical interconversion between the red-absorbing (Pr) and the far-red-absorbing (Pfr) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The Pr-to-Pfr transition commences with a rapid Z-to-E photoisomerization at the C15C16 methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C15–H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted CC stretch and NH in-plane rocking modes within 500 fs, reveal that initial distortion of the C15C16 bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to Pr in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C15–H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C15C16 Z-to-E isomerization occurs on the excited-state surface.

Diverse two-cysteine photocycles in phytochromes and cyanobacteriochromes

Phytochromes are well-known as photoactive red- and near IR-absorbing chromoproteins with cysteine-linked linear tetrapyrrole (bilin) prosthetic groups. Phytochrome photoswitching regulates adaptive responses to light in both photosynthetic and nonphotosynthetic organisms. Exclusively found in cyanobacteria, the related cyanobacteriochrome (CBCR) sensors extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. Blue/green light sensing by a well-studied subfamily of CBCRs proceeds via a photolabile thioether linkage to a second cysteine fully conserved in this subfamily. In the present study, we show that dual-cysteine photosensors have repeatedly evolved in cyanobacteria via insertion of a second cysteine at different positions within the bilin-binding GAF domain (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) shared by CBCRs and phytochromes. Such sensors exhibit a diverse range of photocycles, yet all share ground-state absorbance of near-UV to blue light and a common mechanism of light perception: reversible photoisomerization of the bilin 15,16 double bond. Using site-directed mutagenesis, chemical modification and spectroscopy to characterize novel dual-cysteine photosensors from the cyanobacterium Nostoc punctiforme ATCC 29133, we establish that this spectral diversity can be tuned by varying the light-dependent stability of the second thioether linkage. We also show that such behavior can be engineered into the conventional phytochrome Cph1 from Synechocystis sp. PCC6803. Dual-cysteine photosensors thus allow the phytochrome superfamily in cyanobacteria to sense the full solar spectrum at the earth surface from near infrared to near ultraviolet.

RcaE is a complementary chromatic adaptation photoreceptor required for green and red light responsiveness.

The recent discovery of large numbers of phytochrome photoreceptor genes in both photosynthetic and non‐photosynthetic prokaryotes has led to efforts to understand their physiological roles in environmental acclimation. One receptor in this class, RcaE, is involved in controlling complementary chromatic adaptation, a process that regulates the transcription of operons encoding light‐harvesting proteins in cyanobacteria. Although all previously identified phytochrome responses are maximally sensitive to red and far red light, complementary chromatic adaptation is unique in that it is responsive to green and red light. Here, we present biochemical and genetic evidence demonstrating that RcaE is a photoreceptor and that it requires the cysteine at position 198 to ligate an open chain tetrapyrrole covalently in a manner analogous to chromophore attachment in plant phytochromes. Furthermore, although the wild‐type rcaE gene can rescue red and green light photoresponses of an rcaE null mutant, a gene in which the codon for cysteine 198 is converted to an alanine codon rescues the red light but not the green light response. Thus, RcaE is a photoreceptor that is required for both green and red light responsiveness during complementary chromatic adaptation and is the first identified phytochrome class sensor that is involved in sensing and responding to green and red light rather than red and far red light.