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.
Discovery Phytochrome discovery was not a single event: rather, its existence was inferred gradually from diverse experiments. Between 1920 and 1940, it was found that flowering in many plants, including important crops, was controlled by daylength and that light also regulated seed germination. Both these responses were stimulated and inhibited by red and far-red (near infra-red) light, respectively, prompting Harry Borthwick to propose in 1952 that one and the same pigment was involved: this had the character of an optical switch that is preferentially activated by red light, but at the same time changes its properties to absorb far-red light and is thereby de-activated. In 1959 Warren Butler was able to demonstrate the predicted red/far-red (R/FR) reversible colour change (see Fig. 1) and also introduced the name "phytochrome". Pure preparations of intact phytochrome from oat were first described in 1983, the corresponding PHYA gene was cloned and sequenced soon afterwards. Prokaryotic phytochromes were first described in 1997. A more detailed description of phytochrome research is given in the final "Historical aspects" section.
Physiology in plants Until about 1990, phytochrome research was focused on plants. Plant phytochromes in dark-grown seedlings are present in their lowest-energy, physiologically inactive, red-light-absorbing state (hence "Pr") with absorbance maximum (λmax) at 665 nm. In light, Pr is converted to the physiologically active state that absorbs predominantly in the far-red region (hence "Pfr") with λmax at 730 nm. Whereas Pr is restricted to the cytoplasm, Pfr is translocated into the nucleus where it binds members of the phytochrome interacting factor (PIF) family. PIFs are dark-acting basic helix-loop-helix transcription factors that bind CACGTG ("G-box") sequences typically found in the promoters of light-regulated genes. In darkness, the products of many of these genes lead to skotomorphogenesis, the etiolated developmental form of seedlings grown in darkness, for example through enhanced production of phytohormones such as auxin that enhance stem extension (see Fig. 2). Pfr binding, however, leads to rapid dissociation from the promoter and proteolysis. This proteolysis is induced by physical binding of the PIF and Mut9-like kinases (MLKs, also known as photoregulatory protein kinases or PPKs) to Pfr, leading to phosphorylation of both PIF and phytochrome at numerous sites, marking them for ubiquitination and subsequent "mutually-assured destruction" in the proteosome. PIF-mediated etiolation is thereby interrupted and photomorphogenesis allowed to proceed (see Fig. 2). Almost a quarter of the gene complement in Arabidopsis is either up- or downregulated at least 3-fold following exposure to light through the action of Pfr.
The description above is oversimplified, however, as higher plants possess several different phytochromes, each with rather different characteristics and modes of action. In particular, whereas phyB is present at very low but fairly constant levels in the cell, phyA accumulates to much higher levels in darkness and thus predominates in de-etiolation (greening) of dark-grown seedlings when exposed to light (see Fig. 2). With the formation of Pfr in the light, the PHYA gene itself is strongly repressed and phyA Pfr efficiently eliminated proteolytically. In the meantime, however, phyA Pfr is able to signal the presence of exceedingly low light levels to the cell, in part because more Pfr molecules are formed from the relatively large amount of phyA Pr initially present. But phytochromes are dimeric (comprising two protomer molecules bound strongly together), thus in light Pr|Pfr heterodimers are first formed: these are responsible for the very low fluence response (VLFR). The role of PIFs and perhaps other partners in phyA signalling is much less well understood than in the case of phyB. Indeed, whereas phyB Pfr binds all six PIFs in Arabidopsis, phyA Pfr binds only PIF1 and PIF3. Moreover, the binding sites on the PIFs are different for phyA and phyB. Additionally, transcriptional regulation by the HY5/SPA1/COP1 system regulates photomorphogenesis via Pfr of both phyA and phyB. The characteristic R/FR photochromicity of phytochromes is itself important in the biological function of phyB. The Pr and Pfr absorption wavelengths coincide with spectral changes in natural daylight associated with leafy shade (see Fig. 3). The R/FR ratio of unfiltered sunlight is quite constant, even during twilight, but is reduced strongly by the absorption of red light by chlorophyll-bearing leaves. This changes the photoequilibrium established between Pr and Pfr, providing the plant with potentially useful information regarding the presence of competitors, probably the selective advantage associated with R/FR photoreversible induction of seed germination: such phyB-mediated effects are collectively termed low fluence responses (LFR).
Although various plant species adapted to life in shaded habitats are scarcely affected by depressed R/FR ratios, seedlings of ruderal species react dramatically by promoting stem extension in order to raise their leaves above those of the perceived competitor: this shade avoidance response is mediated by phyB, primarily through its interaction with PIF4. For example, Chenopodium album grows as a strong, stocky plant in exposed (unshaded) conditions, but in sugar beet fields it develops monopodially to outgrow the encroaching competitors, emerging above them to form a large, leafy crown that finally sheds thousands of seeds.
A further effect of phyA is the so-called high irradiance response (HIR) in dark-grown seedlings. Although the photoreceptor is activated by red light, hypocotyl elongation is suppressed much more strongly in continuous 710 nm light, between the Pr and Pfr absorbance maxima at 665 and 730 nm, respectively. This results principally from a balance struck over time between suppression of elongation by Pfr and the simultaneous loss of Pfr through its proteolytic destruction. However, light conditions in the natural environment are seldom appropriate to support HIR-like effects for more than an hour, thus it is questionable whether the HIR is important outside the laboratory.
Another difference between phyA and phyB concerns their localisation in the cell. All plant phytochromes are considered to be cytosolic as Pr. When phyA Pr is photoconverted to Pfr it binds the FHY1 adapter protein and is thereby translocated into the nucleus by importin-α. FHY1 is required although phyA proteins themselves carry a nuclear-localisation sequence (NLS). Conversely, no canonical NLS is present in phyB yet it does not require FHY1 for nuclear import. How a protein as bulky as phyB can enter the nucleus is thus unclear. Indeed, whether phyB nuclear import requires Pfr formation at all has been called into question (see ).
Physiology in fungi and bacteria Up to the 1990s phytochrome photoreceptors were generally considered to be restricted to the plant kingdom. In fact, however, Guy Valadon and colleagues at the University of London provided convincing evidence around 1980 that phytochrome regulated the biosynthesis of carotenoid pigments in the fungal genus Verticillium. More recently, pigmentation and sexual development in the fungus Aspergillis nidulans has also been shown to be regulated by phytochrome.
In 1995, a gene (slr0473) in the cyanobacterium Synechocystis 6803 was noticed that encoded a protein with sequence similarity to a moss phytochrome. Moreover, when expressed in E. coli, the product spontaneously attached bilin cofactors to form a R/FR photochromic holoprotein, proving it to be a bona fide phytochrome. R/FR-reversible histidine kinase activity was also shown, in harmony with C-terminal sequence similarity to kinases in "two-component" signalling systems widespread in prokaryotes. However, in contrast to plant phytochromes in which Pfr is the signalling state, kinase activity of Cph1 was found to be associated with Pr rather than Pfr. No physiological function is known to be associated with this prototypical cyanobacterial phytochrome, Cph1. It is generally assumed, however, that early endosymbiotic cyanobacteria introduced phytochrome-encoding genes to the organisms that evolved into plants. The discovery of Cph1 was followed by that of DrBphP, a more primitive bacteriophytochrome in the non-photosynthetic bacterium Deinococcus radiodurans. Although DrBphP was assumed to be a histidine kinase, such activity was not demonstrated. More recently it has been shown that it is instead a phosphatase. DrBphP was reported to regulate carotenoid biosynthesis but this has not been confirmed. Bacteriophytochromes in the plant pathogens Pseudomonas syringae and Agrobacterium fabrum are thought to regulate pathogenicity. Fruiting body formation is controlled by bacteriophytochromes in Stigmatella aurantiaca and several other myxococcal bacteria. The photosynthetic system of Rhodopseudomonas palustris is also thought to be regulated by bacteriophytochrome.
Whereas canonical phytochromes carry a histidine kinase-like C-terminal module, in bacterial PadC photoreceptors this module is replaced by a diguanylate cyclase moeity regulated by a bacteriophytochrome-typical photosensory module. Cyclic-dimeric-GMP is a widely-used "second messenger" in bacteria, regulating diverse responses commonly related to stress.
Molecular structure and function Phytochromes form stable dimers, each protomer comprising a ca. 125 and 85 kDa (ca. 1140 and 750 amino acid) polypeptide in plant- and prokaryotic types, respectively. Understanding of phytochrome molecular function improved greatly following the discovery of prokaryotic phytochromes, not only because samples were easy to prepare using recombinant genetic methods in transgenic E. coli but also because the underlying domain structure of the phytochrome family became clear through comparison with other prokaryotic sequences, providing a guide to likely molecular functions.
Domain structure
Phytochrome protomers comprise several well-defined domains (see Fig. 4).