User:Photochrom/sandbox

Early work on phytochrome was a gradual process, thus there was no clear "discovery" of phytochrome. A certainly crucial finding was the discovery that day length regulated flowering of crops and other plants by Garner & Allard at the USA Dept. of Agriculture in 1920^[31]. In the 1930s, Flint & McAlister reported that lettuce seed germination could be induced by red light^[32] or suppressed by far-red light^[33]. Independently, Dieter Meischke in Germany reported similar behaviour in various species, also suggesting that the far-red effect might be associated with the significance of leafy shade in seedling survival^[34]. Flint & McAlister's work lead to the discovery by Sterling Hendricks and Harry Borthwick at the USDA laboratory at Beltsville in Maryland that given a series of red and far-red light pulses, only the final red or far-red pulse determined the outcome, prompting the suggestion that a single, red/far-red photochromic photoreceptor might be responsible^[35]. Light-pulse experiments gave similar results regarding day length regulation of flowering^[36], greatly expanding the significance of the research project. Already in 1950, Time magazine ran a short feature on these discoveries.^[37]. It took several years of frustrating work before an appropriately photochromic pigment was identified, but in 1959 Warren Butler and Harold Siegelman at Beltsville found just that in dark-grown seedlings using a sophisticated absorption difference-spectrophotometer.^[38] Butler was also responsible for the name "phytochrome" at about this time^[30]. Phytochrome even became a pawn during the Cold War when Nikita Khrushchev visited the USA in 1959. On the first day of his guided tour, he was taken to the Beltsville laboratory where the phytochrome work was explained to him by Borthwick. He was also given a demonstration of phytochrome photochromicity using Butler's equipment, presumably as an attempt to prove Western technological superiority in the face of Soviet domination of space exploration.

Various problems arose as phytochrome work progressed. For example, although photoreversiblity with red/far-red pulses was shown in light regulation of anthocyanin accumulation and seedling elongation, much stronger effects were seen under strong, continuous light at around 710 nm, between the Pr and Pfr absorption maxima.^[39] Was there, perhaps, another photoreceptor active in that region? More importantly, as it turned out, questions were raised as to whether the phytochrome from dark-grown seedlings ("bulk phytochrome") that was being studied was the same thing as the one active in light-grown (i.e. green) plants.^[40] In those days, however, the notion of a gene family fulfilling a variety of physiological roles was missing. Following Jacob & Monod's seminal work on gene regulation,^[41] it was easy to imagine that phytochrome could somehow do this too,^[42] but it was puzzling that it could have such profound effects on so many different processes. It took many years to answer these questions.

In the 1970s, on the one hand, protein biochemistry was improving rapidly, allowing phytochrome to be extracted from dark-grown seedlings, purified and concentrated so that its properties could be studied exactly. The chromophore was shown to be a "straight-chain pyrrole" as had already been suggested in 1950^[30] and even the amino acid sequence of the fragment to which it is attached was determined.^[43] It proved difficult to extract phytochrome samples from dark-grown seedlings without damage caused by proteases, but finally in 1983 the laboratories of Peter Quail (then at the University of Wisconsin) and Clark Lagarias (at UC Davis) independently reported the purification of intact phytochrome from oat^[44][45]. On the other hand, in the 70s, interest in the environment and ecological aspects of biology was growing. Thus Harry Smith's group in England took up the idea of phytochrome acting as a photochromic detector of low red/far-red ratios under leafy shade, showing that stem extension in species adapted to open conditions responded strongly, whereas those adapted to life in the shade were indifferent.^[46]

Perhaps the most under-appreciated paper in the phytochrome literature is that of Maarten Koorneef and colleagues in 1980, then at the University of Wageningen in the Netherlands .^[47] He was one of the first to understand the value of mutational genetics for plant biology - in particular in Arabidopsis, a plant with tiny seedlings and a very small genome. Having created a population of potential mutants, he simply looked for individual seedlings that kept elongating in light as though they were in darkness. These hy mutants were "blind". He identified four different hy genes, each of which was later found to encode an important component of the phytochrome system (including phyB itself,^[48] an important transcription regulator,^[49] and two enzymes necessary for chromophore biosynthesis^[50][51]), as well as one that turned out to encode a blue light photoreceptor, cryptochrome.^[52] Discovering all that took a lot of work in many labs worldwide, but back in 1980, no one was interested.

Indeed, the revolution of molecular genetics had hardly begun even in 1984 when the first phytochrome gene was cloned and sequenced by Howard Hershey in the Quail lab.^[53][54] This was a great technical achievement by any standards, and at that time it was easily the longest plant gene sequence known. It was precocious, however: no sequences with significant similarity were known at that time, so it was almost impossible to derive any conclusions from it. By 1987 work with monoclonal antibodies had shown clearly that more than one type of phytochrome existed in the plant cell, namely type I ("bulk") predominant in dark-grown seedlings, and type II predominant in green plants. Once again, however, it was molecular genetics in the Quail lab that provided the accurate picture as Bob Sharrock cloned and sequenced the five phytochrome genes (PHYA - E) in Arabidopsis^[55]: phyA is type I phytochrome, the rest are type II, within which phyB predominates.

By that time it had become possible to create transgenic plants, so several groups introduced modified phytochrome genes from one plant into another, just to see what happened. The various transgenics that overproduced one or other of the phytochromes were much shorter and more strongly pigmented than the wild-type plants, reflecting enhanced responses to the high levels of Pfr.

Up to 1990 it had been generally assumed that phytochromes were found exclusively in plants, despite the fact that Guy Valadon^[56][57] at the University of London had clearly demonstrated the existence of a phytochrome in the fungus Verticillium n 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.^[58] The idea of plant phytochromes functioning as histidine kinases was hardly tenable, however, because the necessary His 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.^[59] The protein had sequence similarities to both plant phytochromes and sensory kinases but, under investigation in the lab, it failed to bind chromophore cofactors and thereby show photochromicity. Thus it remained unclear whether it was a photoreceptor at all. In the previous year, however, the complete genome sequence of the cyanobacterium Synechocystis 6803 had been published^[60] 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 indeed encoded a bona fide phytochrome that they named Cph1 (cyanobacterial phytochrome 1).^[61][62] Presumably plant phytochromes are derived from an ancestral cyanobacterial phytochrome, perhaps by gene migration from the chloroplast to the nucleus. Subsequently, a more primitive "bacteriophytochrome" (BphP) group was discovered in the genome of the non-photosynthetic bacterium Deinococcus radiodurans^[63]. The chromophore attachment site was proposed to be a His residue in the GAF domain, although Lamparter subsequently showed that the attachment site in bacteriophytochromes is a Cys close to the N-terminus^[64]. 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.^[65] The RcaE paradox has also been explained: later sequence comparisons show clearly that it is a member of the phytochrome-related CBCR photoreceptor group, but that the start codon is a GTG further upstream than the ATG that had been assumed.^[66]

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^[67][68], 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 and solve its 3D structure (PDB 1ztu) by X-ray crystallography^[17], 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 Cys 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)^[18]; the other of BphP from Pseudomonas aeruginosa as Pfr (PDB 3c2w)^[69]. They showed that the PHY domain is a GAF relative, connected to the GAF domain itself by a long helix, but that also a remarkable tongue-like hairpin extension of the PHY domain stretches back to make contact with the GAF domain. 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 function of the histidine kinase module^[70] Although supporting evidence was presented soon after by Heikki Takala et al.^[71] at the Universities of Jyväskylä and Gothenburg in Scandinavia, later cryo-EM structures of complete bacteriophytochromes as Pr and Pfr imply that the shifts can be quite subtle.^[72][73]

Work on plant phytochromes had of course continued in the meantime, especially regarding their cell biology and signalling mechanism/s. An important question was the location of phytochromes in the cell, as that was expected to provide clues as to their likely function. Lee Pratt's lab at the University of Georgia used antibodies directed 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^[74]. What these sequestered areas represent has not been established. Later, however, in 1999, workers in Japan and Germany using transgenic methods showed that both phyA and phyB as Pfr are imported into the nucleus where they form "speckles" perhaps associated with signalling and/or proteolysis.^[75][76] Nuclear localisation of Pfr fitted well with the reasonable idea that phytochrome acts by regulating gene expression. Indeed, at about the same time, the Quail lab identified the PIF family of phytochrome-interacting transcription factors that are thought to be the primary route of phytochrome signalling.^[77] Interestingly, however, the Chua lab at the Rockefeller University in New York City published results in the early 90's implying that plant phytochromes signal via G-protein, calcium/calmodulin and cyclic nucleotide systems perhaps similar to those known in animals.^[78][79] Although the findings were considered a great breakthrough at the time, other workers have not been able to repeat the results. The notion of cyclic nucleotide involvement in plant phytochrome signalling is nevertheless intriguing, however, because phytochromes show structural similarity 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. Remarkably, tandem-GAF proteins are generally involved in cyclic nucleotide metabolism, either as cyclases or phosphodiesterases.^[18] Although it now seems unlikely that phytochrome action involves cyclic nucleotides, the tandem-GAF configuration might have evolutionary significance.

nuclear loc: FHY1? | maybe not in phyB!

thermosensor too?