Nathan C. Rockwell

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.

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.

Eukaryotic algal phytochromes span the visible spectrum

Significance Photosynthetic organisms exploit photosensory proteins to respond to changing light conditions. In land plants, phytochromes use the ratio of red to far-red light to detect shading by neighboring plants, leading to changes in growth and development. Light conditions can be more variable for algae because of the wavelength-dependent attenuation of light by water and because of ocean mixing. We studied phytochromes from taxonomically diverse eukaryotic algae from groups considered important for coastal ecosystems and the global carbon cycle. These proteins detect light throughout the visible spectrum (blue, green, orange, red, and far-red). Extensive spectral tuning has evolved within these algae, presumably reflecting aquatic light environments. These studies should ultimately facilitate engineering of crop plant species for diverse light environments. Plant phytochromes are photoswitchable red/far-red photoreceptors that allow competition with neighboring plants for photosynthetically active red light. In aquatic environments, red and far-red light are rapidly attenuated with depth; therefore, photosynthetic species must use shorter wavelengths of light. Nevertheless, phytochrome-related proteins are found in recently sequenced genomes of many eukaryotic algae from aquatic environments. We examined the photosensory properties of seven phytochromes from diverse algae: four prasinophyte (green algal) species, the heterokont (brown algal) Ectocarpus siliculosus, and two glaucophyte species. We demonstrate that algal phytochromes are not limited to red and far-red responses. Instead, different algal phytochromes can sense orange, green, and even blue light. Characterization of these previously undescribed photosensors using CD spectroscopy supports a structurally heterogeneous chromophore in the far-red–absorbing photostate. Our study thus demonstrates that extensive spectral tuning of phytochromes has evolved in phylogenetically distinct lineages of aquatic photosynthetic eukaryotes.

A second conserved GAF domain cysteine is required for the blue/green photoreversibility of cyanobacteriochrome Tlr0924 from Thermosynechococcus elongatus.

Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C 499D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing P r form of phytochrome. We propose a model photocycle in which Z/ E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.

Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330–680 nm). Two CBCR subfamilies detect near-UV to blue light (330–450 nm) via a “two-Cys photocycle” that couples bilin 15Z/15E photoisomerization with formation or elimination of a second bilin–cysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing 15Z state (15ZPg) and a red-absorbing 15E state (15EPr). We demonstrate that RcaE from Fremyella diplosiphon undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as 15ZPg but protonated as 15EPr. This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a “protochromic photocycle,” in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.

Distinct classes of red/far-red photochemistry within the phytochrome superfamily

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 Pr and far-red-absorbing Pfr 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 Pr and Pfr are conserved in most phytochromes. Using circular dichroism spectroscopy and ab initio calculations, we establish that the Pfr 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.

Phytochrome C plays a major role in the acceleration of wheat flowering under long-day photoperiod.

Significance Plants perceive day length as a critical environmental signal to trigger major changes in development. Multiple light sensors participate in day-length perception, the most important of which are the red/far-red phytochromes. In rice and Arabidopsis, PHYTOCHROME C (PHYC) requires other phytochromes for stability and function. By contrast, wheat PHYC is stable and functionally active even in the absence of other phytochromes. The loss of functional wheat PHYC results in altered expression of circadian clock and photoperiod genes and a dramatic delay in flowering under long days, indicating that PHYC promotes wheat flowering under inductive photoperiods. Our results provide an additional entry point to modify wheat flowering and to accelerate the development of wheat varieties better adapted to new and changing environments. Phytochromes are dimeric proteins that function as red and far-red light sensors influencing nearly every phase of the plant life cycle. Of the three major phytochrome families found in flowering plants, PHYTOCHROME C (PHYC) is the least understood. In Arabidopsis and rice, PHYC is unstable and functionally inactive unless it heterodimerizes with another phytochrome. However, when expressed in an Arabidopsis phy-null mutant, wheat PHYC forms signaling active homodimers that translocate into the nucleus in red light to mediate photomorphogenic responses. Tetraploid wheat plants homozygous for loss-of-function mutations in all PHYC copies (phyCAB) flower on average 108 d later than wild-type plants under long days but only 19 d later under short days, indicating a strong interaction between PHYC and photoperiod. This interaction is further supported by the drastic down-regulation in the phyCAB mutant of the central photoperiod gene PHOTOPERIOD 1 (PPD1) and its downstream target FLOWERING LOCUS T1, which are required for the promotion of flowering under long days. These results implicate light-dependent, PHYC-mediated activation of PPD1 expression in the acceleration of wheat flowering under inductive long days. Plants homozygous for the phyCAB mutations also show altered profiles of circadian clock and clock-output genes, which may also contribute to the observed differences in heading time. Our results highlight important differences in the photoperiod pathways of the temperate grasses with those of well-studied model plant species.

The Structure of Phytochrome: A Picture Is Worth a Thousand Spectra

Sensing and responding to light is a central feature of plant biology that is critical for tuning plant metabolism to the light environment. To accomplish this vital task, plants use a number of different photosensory proteins that perceive different bands of the electromagnetic spectrum ([

Phycoviolobilin formation and spectral tuning in the DXCF cyanobacteriochrome subfamily.

Phytochromes are red/far-red photosensory proteins that regulate adaptive responses to light via photoswitching of cysteine-linked linear tetrapyrrole (bilin) chromophores. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. CBCRs and phytochromes share a conserved Cys residue required for bilin attachment. In one CBCR subfamily, often associated with a blue/green photocycle, a second Cys lies within a conserved Asp-Xaa-Cys-Phe (DXCF) motif and is essential for the blue/green photocycle. Such DXCF CBCRs use isomerization of the phycocyanobilin (PCB) chromophore into the related phycoviolobilin (PVB) to shorten the conjugated system for sensing green light. We here use recombinant expression of individual CBCR domains in Escherichia coli to survey the DXCF subfamily from the cyanobacterium Nostoc punctiforme. We describe ten new photoreceptors with well-resolved photocycles and three additional photoproteins with overlapping dark-adapted and photoproduct states. We show that the ability of this subfamily to form PVB or retain PCB provides a powerful mechanism for tuning the photoproduct absorbance, with blue-absorbing dark states leading to a broad range of photoproducts absorbing teal, green, yellow, or orange light. Moreover, we use a novel green/teal CBCR that lacks the blue-absorbing dark state to demonstrate that PVB formation requires the DXCF Cys residue. Our results demonstrate that this subfamily exhibits much more spectral diversity than had been previously appreciated.

Red/Green Cyanobacteriochromes: Sensors of Color and Power

Phytochromes are red/far-red photoreceptors using cysteine-linked linear tetrapyrrole (bilin) chromophores to regulate biological responses to light. Light absorption triggers photoisomerization of the bilin between the 15Z and 15E photostates. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. Several subfamilies of CBCRs have been described. Representatives of one such subfamily, including AnPixJ and NpR6012g4, exhibit red/green photocycles in which the 15Z photostate is red-absorbing like that of phytochrome but the 15E photoproduct is instead green-absorbing. Using recombinant expression of individual CBCR domains in Escherichia coli, we fully survey the red/green subfamily from the cyanobacterium Nostoc punctiforme. In addition to 14 new photoswitching CBCRs, one apparently photochemically inactive protein exhibiting intense red fluorescence was observed. We describe a novel orange/green photocycle in one of these CBCRs, NpF2164g7. Dark reversion varied in this panel of CBCRs; some examples were stable as the 15E photoproduct for days, while others reverted to the 15Z dark state in minutes or even seconds. In the case of NpF2164g7, dark reversion was so rapid that reverse photoconversion of the green-absorbing photoproduct was not significant in restoring the dark state, resulting in a broadband response to light. Our results demonstrate that red/green CBCRs can thus act as sensors for the color or intensity of the ambient light environment.