Phytochrome

Phytochromes are a class of photoreceptor proteins found in plants as well as certain fungi and bacteria. Characteristically they use bile pigments to absorb light in the red to far-red region of the spectrum, changing their colour as they do so (see Fig. 1). Phytochromes control many aspects of plant development, inducing seed germination, seedling development (see Fig. 2), shade avoidance and daylength-dependent flowering by regulating the expression of thousands of genes through the action of phytochrome-associated transcription factors. Higher plant genomes encode several phytochromes with significantly different characteristics and functions. Other plant photoreceptors include cryptochromes and phototropins, which respond to blue and near ultraviolet (UV-A) light, and UVR8, which responds to ultraviolet-B light. Phytochromes have been identified in certain fungi and shown to regulate pigmentation and sexual development. Prokaryotic phytochromes regulate very diverse processes including pigment accumulation and fruiting-body assembly. The mechanism by which the phytochrome molecule itself is activated by light and how it then transmits the light signal to the cell are a major research fields in molecular biology. There is also interest in the possibility of exploiting these processes in biotechnology and other areas.

Phytochromes are a class of photoreceptor proteins found in plants as well as certain fungi and bacteria. Characteristically they use bile pigments to absorb light in the red to far-red region of the spectrum, changing their colour as they do so (see Fig. 1). Phytochromes control many aspects of plant development, inducing seed germination, seedling development (see Fig. 2), shade avoidance and daylength-dependent flowering by regulating the expression of thousands of genes through the action of phytochrome-associated transcription factors. Higher plant genomes encode several phytochromes with significantly different characteristics and functions. Other plant photoreceptors include cryptochromes and phototropins, which respond to blue and near ultraviolet (UV-A) light, and UVR8, which responds to ultraviolet-B light. Phytochromes have been identified in certain fungi and shown to regulate pigmentation and sexual development. Prokaryotic phytochromes regulate very diverse processes including pigment accumulation and fruiting-body assembly. The mechanism by which the phytochrome molecule itself is activated by light and how it then transmits the light signal to the cell are a major research fields in molecular biology. There is also interest in the possibility of exploiting these processes in biotechnology and other areas. Up to 1990 it had been generally assumed that phytochromes were found exclusively in plants, despite the fact that Guy Valadon at the University of London had clearly demonstrated the existence of a phytochrome in the fungus Verticillium in 1982. Thus it was a surprise when in 1992 Hansjörg Schneider-Poetsch at Cologne University noticed weak similarities between plant phytochromes and the recently discovered bacterial sensory histidine protein kinase family. The idea of plant phytochromes functioning as histidine kinases was hardly tenable, however, because the necessary histidine residue is not conserved. In 1996 David Kehoe and Arthur Grossman at the Carnegie lab in Stanford identified a gene, RcaE, in the cyanobacterium Fremyella diplosiphon that was involved in light acclimation (Uniprot Q47897). The protein had sequence similarities to both plant phytochromes and sensory kinases but, under investigation in the lab, it failed to bind bilin cofactors and thereby show photochromicity. Thus it remained unclear whether it was a photoreceptor at all. In the previous year, however, the genome sequence of the cyanobacterium Synechocystis 6803 had been published and a gene identified with clear sequence similarities to a moss phytochrome that had been cloned earlier by none other than Schneider-Poetsch. Jon Hughes and Tilman Lamparter in Berlin and Clark Lagarias at UC Davis subsequently showed that the Synechocystis gene, slr0473, indeed encoded a bona fide phytochrome that they named Cph1 (cyanobacterial phytochrome 1) (Uniprot Q55168). Presumably, plant phytochromes are derived from an ancestral cyanobacterial phytochrome, perhaps by gene migration from an endosymbiotic proto-chloroplast to the nucleus. Subsequently, Seth Davis and Rick Vierstra at the University of Wisconsin discovered a more primitive "bacteriophytochrome" in the genome of the non-photosynthetic bacterium Deinococcus radiodurans (Uniprot Q9RZA4). They proposed that the chromophore attachment site was a histidine residue in the GAF domain, but Lamparter subsequently showed that it was instead a cysteine close to the N-terminus. Although DrBphP was assumed to be a light-sensitive histidine kinase, no such enzyme activity was reported. Indeed it was recently shown that it has phosphatase rather than kinase activity . The RcaE paradox has also been explained: later sequence comparisons showed clearly that it is a member of the phytochrome-related cyanobacteriochrome receptor (CBCR) group, but that the start codon is a GTG further upstream than the ATG that had been assumed (Uniprot M1V809, GenBank: BAM83580). The discovery of prokaryotic phytochromes revolutionised the field. Not only did they help to understand phytochrome molecular structure, evolution and function, they were easy to produce using established recombinant genetic techniques in E. coli. Both the Lagarias and Hughes groups were able to engineer biosynthesis of the bilin chromophore in E. coli too, so that Cph1 holoprotein could be produced en masse, then purified and concentrated for biophysical studies. Thus in 2005, Katrina Forest at the University of Wisconsin collaborated with the Vierstra lab to crystallise the nPAS-GAF bidomain of Deinococcus BphP as Pr and solve its 3D structure (PDB 1ztu) by X-ray crystallography, showing, in particular, that the protein chain forms a knot - a highly unusual structure for a protein - around the nPAS domain. The chromophore ring geometry and linkage to the expected cysteine residue near the N-terminus were also revealed. The nPAS-GAF fragment is unable to photoconvert, however, so it was significant that, in 2008, two groups around Lars-Oliver Essen and Jon Hughes in Germany and Xiaojing Yang and Keith Moffat in Chicago published the 3D crystal structures of the complete, photochemically competent nPAS-GAF-PHY photosensory module. One structure was of Cph1 from Synechocystis as Pr (PDB 2vea), the other of PaBphP from Pseudomonas aeruginosa (Uniprot Q9HWR3) as Pfr (PDB 3c2w). They showed that the PHY domain is a GAF relative, held at a distance from the GAF domain itself by a long helix, but that also an unusual tongue-like hairpin extension of the PHY domain stretches back to make contact with the surface of the GAF domain near the chromopore. Later, the Essen lab proposed that Pr→Pfr photoconversion is associated with refolding and shortening of the tongue that would shift the position of the PHY domain, thereby possibly affecting the activity of the histidine kinase module dimer. Although supporting evidence was presented soon after by Heikki Takala and colleagues at the Universities of Jyväskylä and Gothenburg in Scandinavia, later cryo-EM structures of complete bacteriophytochromes as Pr and Pfr indicate that the shifts are much more subtle. Work on plant phytochromes had continued in the meantime, in particular regarding their mode of action. In the early 1990s, the Chua lab at the Rockefeller University in New York City published results implying that plant phytochromes signal via G-protein, calcium/calmodulin and cyclic nucleotide systems perhaps similar to those known in animals. Although the findings were considered a great breakthrough at the time, other workers have not been able to repeat the results. However, the notion of cyclic nucleotides being involved in phytochrome signalling correlates with structural similarity of the phytochrome family to "tandem-GAF" proteins. These include two GAF-like domains separated by a long helix, very similar to the GAF-PHY arrangement seen in phytochromes: a general characteristic of tandem-GAF proteins is their involvement in cyclic nucleotide metabolism, either as cyclases or phosphodiesterases. Although it now seems unlikely that phytochrome action involves cyclic nucleotides, the tandem-GAF configuration might have evolutionary significance. An important question was the location of plant phytochromes in the cell, as that would provide clues as to their mode of action. In the 1980s, Lee Pratt's lab at the University of Georgia had used antibodies directed principally against phyA to label it within the cell, finding that whereas Pr was distributed throughout the cytoplasm, photoconversion to Pfr lead to rapid sequestration of the protein to small areas, followed by its disappearance. What these sequestered areas represent has not been established. The picture changed dramatically in 1999, however, when workers in Japan and Germany using transgenic phytochromes tagged with fluorescent moieties showed that both phyA and phyB as Pfr are imported into the nucleus where they form "speckles", currently thought to be associated with signalling and/or proteolysis. Nuclear localisation of Pfr fitted well with the reasonable idea that phytochrome acts by regulating gene expression. Indeed, at about the same time, Weimin Ni in the Quail lab in Berkeley identified the PIF family of p hytochrome- i nteracting transcription f actors that are thought to be the primary route of phytochrome signalling (see ). As PIF3 was initially identified through its binding to a C-terminal phyB fragment comprising the PAS repeat and histidine kinase-like modules, it was a great surprise to find that the primary interaction is instead with the Pfr state of photosensory module. Indeed, Tomonao Matsushita in the Nagatani lab in Kyoto showed that the phyB photosensory module alone, when dimerised in the nucleus, functions at least as effectively as the complete phyB molecule. Despite some initial confusion, it was established that the PIFs, in particular PIF3, act as regulators of genes responsible for the etiolated developmental form (skotomorphogenesis) of seedlings grown in darkness, partly through production of phytohormones such as auxin that enhance stem extension. This was made clear by the creation of pifq, a multiple PIF gene knockout mutant: pifq seedlings grown in total darkness developed similarly to wild-type seedlings grown in the light. Generally speaking, PIFs promote skotomorphogenesis and repress photomorphogenesis. Solving the 3D crystal structures of the prokaryotic phytochrome photosensory module (see above) encouraged similar studies of plant phytochromes. The first success was in 2014 for the photosensory module of Arabidopsis phyB Pr by Sethe Burgie in the Vierstra lab (PDB 4our). Equivalent Pr structures of other plant phytochromes at higher resolution were published subsequently. Although the sequence identity relative to the photosensor of Cph1 is only about 30%, the 3D structures are remarkably similar. As in the case of prokaryotic phytochromes, it has not been possible to crystallise a complete plant phytochrome, but the emergence of cryo-electron microscopy (cryo-EM) allowed near-complete 3D structures of phyA (PDB 8f5z and others) and phyB (PDB 7rzw) in the Pr state to be solved by single-molecule imaging. How light and thus Pfr formation regulates photomorphogenesis is a centrally important question in plant biology. Weimin Ni in the Quail lab established that phyB Pfr binds PIF3 along with Mut9-like kinases in the nucleus; the PIF is thereby rapidly phosphorylated, marking it for ubiquitination and destruction in the proteasome. The structural basis of this Pfr-dependent binding is still unclear, but recent cryo-EM studies of phyB Pfr in complex with a fragment of PIF6 (PDB 8yb4 and 9jlb) show a domain architecture radically different from that of Pr. How the remodelling is brought about is unknown. MLKs are probably not the only kinases involved in the plant phytochrome system, indeed there are suggestions that plant phytochromes themselves posses kinase activity. Furthermore, transcription factors other than PIFs are involved: much less well understood is the converse function of HY5, a Pfr-dependent transcriptional co-activator of photomorphogenesis.

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