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The phytochrome superfamily comprises three groups of photoreceptors sharing a conserved GAF (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) domain that uses a covalently attached linear tetrapyrrole (bilin) chromophore to sense light. Knotted red/far-red phytochromes are widespread in both bacteria and eukaryotes, but cyanobacteria also contain knotless red/far-red phytochromes and cyanobacteriochromes (CBCRs). Unlike typical phytochromes, CBCRs require only the GAF domain for bilin binding, chromophore ligation, and full, reversible photoconversion. CBCRs can sense a wide range of wavelengths (ca. 330-750 nm) and can regulate phototaxis, second messenger metabolism, and optimization of the cyanobacterial light-harvesting apparatus. However, the origins of CBCRs are not well understood: we do not know when or why CBCRs evolved, or what selective advantages led to retention of early CBCRs in cyanobacterial genomes. In the current work, we use the increasing availability of genomes and metagenome-assembled-genomes from early-branching cyanobacteria to explore the origins of CBCRs. We reaffirm the earliest branches in CBCR evolution. We also show that early-branching cyanobacteria contain late-branching CBCRs, implicating early appearance of CBCRs during cyanobacterial evolution. Moreover, we show that early-branching CBCRs behave as integrators of light and pH, providing a potential unique function for early CBCRs that led to their retention and subsequent diversification. Our results thus provide new insight into the origins of these diverse cyanobacterial photoreceptors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cyanobacteria are unique among bacteria in performing oxygenic photosynthesis, often together with nitrogen fixation and, thus, are major primary producers in many ecosystems. The cyanobacterium, Leptolyngbya sp. strain JSC-1, exhibits an extensive photoacclimative response to growth in far-red light that includes the synthesis of chlorophylls d and f. During far-red acclimation, transcript levels increase more than twofold for ~900 genes and decrease by more than half for ~2000 genes. Core subunits of photosystem I, photosystem II, and phycobilisomes are replaced by proteins encoded in a 21-gene cluster that includes a knotless red/far-red phytochrome and two response regulators. This acclimative response enhances light harvesting for wavelengths complementary to the growth light (λ = 700 to 750 nanometers) and enhances oxygen evolution in far-red light., (Copyright © 2014, American Association for the Advancement of Science.)

Ever since the discovery of the role of bacteriophytochrome (BphP) in inducing carotenoid synthesis in Deinococcus radiodurans in response to light the role of BphPs in other non-photosynthetic bacteria is not clear yet. Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours a pair of BphPs out of which AbBphP1 is a homolog of AtBphP1 of Agrobacterium tumefaciens. By overexpression, purification, biochemical and spectral characterization we have shown that AbBphP1 is a photochromic bacteriophytochrome. Phenotypic study of the ΔAbBphP1 mutant showed that it is required for the survival of A. brasilense on minimal medium under red light. The mutant also showed reduced chemotaxis towards dicarboxylates and increased sensitivity to the photooxidative stress. Unlike D. radiodurans, AbBphP1 was not involved in controlling carotenoid synthesis. Proteome analysis of the ΔAbBphP1 indicated that AbBphP1 is involved in inducing a cellular response that enables A. brasilense in regenerating proteins that might be damaged due to photodynamic stress.

Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.

Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens are used as model phytochromes for biochemical and biophysical studies. In biliverdin binding phytochromes the site for covalent attachment of the chromophore lies in the N-terminal region of the protein, different from plant phytochromes. The issue which stereochemistry the chromophore adopts in the so-called Pr and Pfr forms is addressed by using a series of locked chromophores which form spectrally characteristic adducts with Agp1 and Agp2. Studies on light-induced conformational changes of Agp1 give an insight into how the intrinsic histidine kinase is modulated by light. Comparison of the crystal structure of an Agp1 fragment with other phytochrome crystal structures supports the idea that a light induced rearrangement of subunits within the homodimer modulates the activity of the kinase.

The cyanobacterial phytochrome Cph1 can be photoconverted between two thermally stable states, Pr and Pfr. The photochemically induced Pfr --> Pr back-reaction has been followed at low temperature by magic-angle spinning (MAS) NMR spectroscopy, allowing two intermediates, Lumi-F and Meta-F, to be trapped. Employing uniformly (13)C- and (15)N-labeled open-chain tetrapyrrole chromophores, all four states-Pfr, Lumi-F, Meta-F, and Pr-have been structurally characterized. In the first step, the double bond photoisomerization forming Lumi-F occurs. The second step, the transformation to Meta-F, is driven by the release of the mechanical tension. This process leads to the break of the hydrogen bond of the ring D nitrogen to Asp-207 and triggers signaling. The third step is protonically driven allowing the hydrogen-bonding interaction of the ring D nitrogen to be restored. Compared to the forward reaction, the order of events is changed, probably caused by the different properties of the hydrogen bonding partners of N24, leading to the directionality of the photocycle.

Using gene targeting by homologous recombination in Ceratodon purpureus, we were able to knock out four phytochrome photoreceptor genes independently and to analyze their function with respect to red light dependent phototropism, polarotropism, and chlorophyll content. The strongest phenotype was found in knock-out lines of a newly described phytochrome gene termed CpPHY4 lacking photo- and polarotropic responses at moderate fluence rates. Eliminating the atypical phytochrome gene CpPHY1, which is the only known phytochrome-like gene containing a putative C-terminal tyrosine kinase-like domain, affects red light-induced chlorophyll accumulation. This result was surprising, since no light dependent function was ever allocated to this unusual gene.

Phytochromes are a widespread family of photosensory proteins first discovered in plants, which measure the ratio of red to far-red light to control many aspects of growth and development. Phytochromes interconvert between red-absorbing P(r) and far-red-absorbing P(fr) states via photoisomerization of a covalently-bound linear tetrapyrrole (bilin) chromophore located in a conserved photosensory core. From recent crystal structures of this core region, it has been inferred that the chromophore structures of P(r) and P(fr) are conserved in most phytochromes. Using circular dichroism spectroscopy and ab initio calculations, we establish that the P(fr) states of the biliverdin-containing bacteriophytochromes DrBphP and PaBphP are structurally dissimilar from those of the phytobilin-containing cyanobacterial phytochrome Cph1. This conclusion is further supported by chromophore substitution experiments using semisynthetic bilin monoamides, which indicate that the propionate side chains perform different functional roles in the 2 classes of phytochromes. We propose that different directions of bilin D-ring rotation account for these distinct classes of red/far-red photochemistry.

Phytochromes are photoreceptors that provide plants with circadian, seasonal, and positional information critical for the control of germination, seedling development, shade avoidance, reproduction, dormancy, and sleep movements. Phytochromes are unique among photoreceptors in their capacity to interconvert between a red-absorbing form (absorption maximum of approximately 660 nm) and a far-red absorbing form (absorption maximum of approximately 730 nm), which occur in a dynamic equilibrium within plant cells, corresponding to the proportions of red and far-red energy in ambient light. Because pigments in stems and leaves absorb wavelengths below about 700 nm, this provides plants with an elegant system for detecting their position relative to other plants, with which the plants compete for light. Certain aspects of phytochrome-mediated development outside of flowering plants are strikingly similar to those that have been characterized in Arabidopsis thaliana and other angiosperms. However, early diverging land plants have fewer distinct phytochrome gene lineages, suggesting that both diversification and subfunctionalization have been important in the evolution of the phytochrome gene family. There is evidence that subfunctionalization proceeded by the partitioning among paralogues of photosensory specificity, physiological response modes, and light-regulated gene expression and protein stability. Parallel events of duplication and functional divergence may have coincided with the evolution of canopy shade and the increasing complexity of the light environment. Within angiosperms, patterns of functional divergence are clade-specific and the roles of phytochromes in A. thaliana change across environments, attesting to the evolutionary flexibility and contemporaneous plasticity of phytochrome signalling in the control of development.

Background: Phytochromes are photoreceptors, discovered in plants, that control a wide variety of developmental processes. They have also been found in bacteria and fungi, but for many species their biological role remains obscure. This work concentrates on the phytochrome system of Agrobacterium tumefaciens, a non-photosynthetic soil bacterium with two phytochromes. To identify proteins that might share common functions with phytochromes, a co-distribution analysis was performed on the basis of protein sequences from 138 bacteria., Results: A database of protein sequences from 138 bacteria was generated. Each sequence was BLASTed against the entire database. The homolog distribution of each query protein was then compared with the homolog distribution of every other protein (target protein) of the same species, and the target proteins were sorted according to their probability of co-distribution under random conditions. As query proteins, phytochromes from Agrobacterium tumefaciens, Pseudomonas aeruginosa, Deinococcus radiodurans and Synechocystis PCC 6803 were chosen along with several phytochrome-related proteins from A. tumefaciens. The Synechocystis photosynthesis protein D1 was selected as a control. In the D1 analyses, the ratio between photosynthesis-related proteins and those not related to photosynthesis among the top 150 in the co-distribution tables was > 3:1, showing that the method is appropriate for finding partner proteins with common functions. The co-distribution of phytochromes with other histidine kinases was remarkably high, although most co-distributed histidine kinases were not direct BLAST homologs of the query protein. This finding implies that phytochromes and other histidine kinases share common functions as parts of signalling networks. All phytochromes tested, with one exception, also revealed a remarkably high co-distribution with glutamate synthase and methionine synthase. This result implies a general role of bacterial phytochromes in ammonium assimilation and amino acid metabolism., Conclusion: It was possible to identify several proteins that might share common functions with bacterial phytochromes by the co-distribution approach. This computational approach might also be helpful in other cases.

Phys (phytochromes) are a superfamily of photochromic photoreceptors that employ a bilin-type chromophore to sense red and far-red light. Although originally thought to be restricted to plants, accumulating genetic and genomic analyses now indicate that they are also prevalent among micro-organisms. By a combination of phylogenetic and biochemical studies, we have expanded the Phy superfamily and organized its members into distinct functional clades which include the phys (plant Phys), BphPs (bacteriophytochromes), Cphs (cyanobacterial Phys), Fphs (fungal Phys) and a collection of Phy-like sequences. All contain a signature GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, which houses the bilin lyase activity. A PHY domain (uppercase letters are used to denote the PHY domain specifically), which helps stabilize the Pfr form (far-red-light-absorbing form of Phy), is downstream of the GAF region in all but the Phy-like sequences. The phy, Cph, BphP and Fph families also include a PLD [N-terminal PAS (Per/Arnt/Sim)-like domain] upstream of the GAF domain. Site-directed mutagenesis of conserved residues within the GAF and PLD motifs supports their importance in chromophore binding and/or spectral activity. In agreement with Lamparter, Carrascal, Michael, Martinez, Rottwinkel and Abian [(2004) Biochemistry 43, 3659-3669], a conserved cysteine within the PLD of several BphPs was found to be necessary for binding the chromophore via the C-3 vinyl side chain on the bilin A ring. Phy-type sequences were also discovered in the actinobacterium Kineococcus radiotolerans and collections of microorganisms obtained from marine and extremely acidic environments, thus expanding further the range of these photoreceptors. Based on their organization and distribution, the evolution of the Phy superfamily into distinct photoreceptor types is proposed.

Phytochromes are chromoproteins found in plants and bacteria that switch between two photointerconvertible forms via the photoisomerization of their chromophore. These two forms, Pr and Pfr, absorb red and far-red light, respectively. We have characterized the biophysical and biochemical properties of two bacteriophytochromes, RpBphP2 and RpBphP3, from the photosynthetic bacterium Rhodopseudomonas palustris. Their genes are contiguous and localized near the pucBAd genes encoding the polypeptides of the light harvesting complexes LH4, whose synthesis depends on the light intensity. At variance with all (bacterio)phytochromes studied so far, the light-induced isomerization of the chromophore of RpBphP3 converts the Pr form to a form absorbing at shorter wavelength around 645 nm, designated as Pnr for near red. The quantum yield for the transformation of Pr into Pnr is about 6-fold smaller than for the reverse reaction. Both RpBphP2 and RpBphP3 autophosphorylate in their dark-adapted Pr forms and transfer their phosphate to a common response regulator Rpa3017. Under semiaerobic conditions, LH4 complexes replace specifically the LH2 complexes in wild-type cells illuminated by wavelengths comprised between 680 and 730 nm. In contrast, mutants deleted in each of these two bacteriophytochromes display no variation in the composition of their light harvesting complexes whatever the light intensity. From both the peculiar properties of these bacteriophytochromes and the phenotypes of their deletion mutants, we propose that they operate in tandem to control the synthesis of LH4 complexes by measuring the relative intensities of 645 and 710 nm lights.

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate-zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.

The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.

We have isolated and sequenced overlapping genomic and cDNA clones encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 77% identity to the Arabidopsis phyB and 50% identity to the potato phyA open reading frame, we suggest that these clones encode phyB phytochrome. However, the size of the deduced open reading frame of 1133 amino acids is smaller than the size of the other two phyB open reading frames characterized so far in higher plants, which contain 1171 or 1187 amino acids. The intron/exon structure within the coding region is conserved in phyA and phyB genes of various species. Southern blot analysis indicates that potato phyB is a single-copy gene. PhyB mRNA levels do not differ among different organs or different light regimes. Transcription initiation starts from two different start points which are 63 bp apart.