Phytochromes

What are the phytochromes?

The phytochromes (phys) are a superfamily of sensory photoreceptors. They were discovered in plants over half a century ago, but the era of whole-genome sequencing revealed that phy-related sequences are also widely dispersed across the microbial world (mainly cyanobacteria and eubacteria, as well as some filamentous fungi). The canonical phy molecule is a soluble dimer of chromoprotein subunits, each of which consists of a polypeptide with a single, covalently-linked bilin (tetrapyrrole) chromophore, which is responsible for light perception (Figure 1A). Each polypeptide folds into two major domains: an amino-terminal sensory domain which cradles the chromophore, and a carboxy-terminal ‘output’ or ‘regulatory’ domain. Each domain in turn contains subdomains in various combinations, identified by sequence similarity to several known structural and/or functional domains (Figure 1A). The core sensory domain typically consists of a PAS, GAF and PHY subdomain, with an additional variable amino-terminal extension in certain phys. The carboxy-terminal domain typically contains a subdomain related to those found in two-component histidine kinases (HKRD), with two additional upstream PAS domains in the plant phys. Recent crystallographic and solution NMR studies of the sensory domain of bacterial phys have begun to provide beautiful images of the three-dimensional structure of the photoreceptor molecule.

Figure 1. Phytochrome (phy) domain structure and intracellular signaling pathway.

(A) phy domain structure. Higher plant phy polypeptides, represented by phyB (top), contain two major structural domains (amino- and carboxy-terminal) and several subdomains (N (non-conserved amino-terminal extension), PAS (PER, ARNT, SIM), GAF (cGMP-specific phosphodiesterase, adenylate cyclase, FhlA), PHY (phytochrome) and HKRD (His-kinase related domain)) within each. Small partitioned box above GAF represents the chromophore. Microbial phys, represented by bacteriophytochrome (BphP), have a similar overall architecture but lack the twin caboxy-terminal PAS subdomains. (B) Plant phy photoperception and signal transduction in regulating seedling photomorphogenesis. phy molecules switch reversibly between their Pr and Pfr conformers upon sequential absorption of red (R) and (FR) photons. Pfr formation (signal perception) triggers rapid translocation into the nucleus where the activated molecule interacts with bHLH-class, phy-interacting transcription factors (PIFs), such as PIF3, initiating alterations in gene expression, via a transcriptional network, that culminates in the switch from skotomorphogenesis to photomorphogenesis (seedling de-etiolation). About 10% (2,400) of the genes in the Arabidopsis genome display altered expression within 24 h of first exposure to R light, whereas 1% (250 genes) change expression within 1 h of initial exposure (‘early-response’ genes). (C) The dark side: multiple PIF bHLH factors repress photomorphogenesis in darkness. A quadruple pif1 pif3 pif4 pif5 (pifq) mutant of Arabidopsis displays a constitutively photomorphogenic (cop)-like phenotype in completely dark-grown seedlings. (Figure coutesy of B. Al-Sady, P. Leivar, E. Monte and J. Tepperman.)

What are the biological functions of the phys?

In the most general sense, the function of the phys is to monitor information from the environment in the form of light signals and direct responses in the organism appropriate to the prevailing conditions. More specifically, the phys constantly monitor multiple physical parameters of the impinging light signals (including presence/absence, color (wavelength), intensity (fluence rate) and diurnal duration (photoperiodicity)) and transduce this information via intracellular signaling pathways that elicit molecular and cellular responses specific to the organism and developmental state. In plants, the phys regulate multiple facets of growth, development and reproduction throughout the life cycle, including seed germination, the switch from heterotrophic to photoautotrophic seedling development (termed de-etiolation), juvenile vegetative development and architecture (through a process called neighbor-detection/shade-avoidance), flowering and senescence. A wealth of information has been gathered over the years on the montage of underlying cellular, subcellular, biochemical and molecular processes that drive these overt morphogenic responses, including stimulation or inhibition of cell expansion rates, switching of cell fate, induction of organelle biogenesis, alterations in metabolic pathways, modulation of hormone activities, regulation of the circadian clock and global changes in gene expression. Less is known about the functions of the phys in microorganisms but the data indicate adaptive functions, not only in photosynthetic bacteria, but also potentially in heterotrophic bacteria.

What is the mechanism of photosensory perception?

The sensory function of the phy molecule resides in its capacity to switch reversibly (within milliseconds) between two stable conformers, called Pr (for red light (R)-absorbing) and Pfr (for far-red light (FR)-absorbing), upon sequential photoexcitation by R and FR light (Figure 1B). The Pr form is biologically inactive (at least in plants), whereas the Pfr form is biologically active. With a few bacterial exceptions, the molecule is synthesized in the inactive Pr form, so that initial light-induced Pfr formation is the signal that the host organism has been exposed to light for the first time, as occurs for young seedlings emerging from subterranean darkness after germination. In fully light-exposed organisms, under sustained irradiation conditions, a dynamic photoequilibrium is established between the Pr and Pfr forms, with the proportion of molecules in each form at any instant being determined by the relative levels (photon fluxes) of R and FR wavelengths in the incoming light signal. The phys thus provide a primitive form of color vision to the host organism, especially in the region of the spectrum containing photosynthetically active radiation. A variety of spectroscopic data, including recent NMR-based solution-structural analysis, have shown that photon absorption by the chromophore triggers rapid isomerization-driven ring rotation in the tetrapyrrole, and that this induces conformational changes in the surrounding protein that presumably lead to the reversible switching in biological activity of the photoreceptor.

What is the nature of the intracellular signal transduction pathway?

The phy molecule is continuously synthesized and accumulates in the cytoplasm in the inactive Pr form until it is exposed to light for the first time. In higher plants, initial photoinduced conversion to the Pfr form triggers rapid (within minutes) translocation into the nucleus where the activated photoreceptor initiates a cascade of changes in gene expression that are also detectable within minutes (Figure 1B). In microbes, the pathway is less well-defined but appears likely to involve a two-component signaling mechanism.

What is the nature of the phy-regulated transcriptional network in higher plants?

About 10% or so of the genes in the genome display phy-regulated changes in expression during the seedling de-etiolation transition triggered by initial exposure of post-germinative seedlings to light (Figure 1B). Unsurprisingly, the predicted or established functions of the large majority of these genes correlate strongly with the various morphogenic changes observed during deetiolation; such genes include numerous photosynthetic genes related to the biogenesis of active chloroplasts, various auxin-, gibberellin-, cytokinin- and ethylene hormone pathway-related genes potentially mediating growth responses, and metabolic genes reflecting the transition from heterotrophic to autotrophic growth. In addition, oscillations in the expression of core circadian clock genes are triggered through the phy system by this initial exposure of seedlings to light, and repetitive diurnal exposure of plants to light at the dawn, dark-to-light transition provides a signal through the phy system that maintains the phase of the clock in sync with the ambient day–night cycle. The loci that respond most rapidly to the initial light signal (‘early-response’ genes) are enriched in genes encoding transcription factors, suggesting a role as direct regulators of downstream genes in the phy-directed transcriptional network.

What is the mechanism of transcriptional regulation by the photoactivated phy molecule?

The phys (primarily phyA and phyB) interact, specifically in the Pfr form, with a subset of constitutively nuclear, basic helix-loop-helix (bHLH) transcription factors termed phytochrome-interacting factors (PIFs), immediately upon light-induced phy translocation into the nucleus. This interaction triggers rapid phosphorylation and subsequent degradation of the PIF protein via the ubiquitin proteasome system. The data suggest, therefore, that the phys regulate at least some target genes by directly modulating the abundance of transcription factors that control the transcriptional activity of those genes. This model is supported by the findings that a quadruple mutant (called pifq), null for four PIF family members (PIF1, 3, 4 and 5), largely phenocopies, when grown in darkness, the morphogenic development of wild-type seedlings grown in the light (Figure 1B,C), and that the global gene expression pattern of this dark-grown mutant also strongly phenocopies the pattern normally induced by light in the wild-type plant. These data also indicate that the PIF proteins function to repress photomorphogenic development in darkness and that phy photoactivation reverses this repression through induced PIF proteolysis.

What is the biochemical mechanism of signal transfer from the activated phy photoreceptor molecule to its immediate signaling partners?

The most prominent current proposed mechanism of phy signaling is that the photoreceptor molecule is a light-regulated protein kinase that transphosphorylates physically interacting transduction-chain partners upon photoactivation. There is strong evidence for this mechanism in the case of the few prokaryotic phys that have been examined. In these cases, the histidine kinase activity predicted from the conserved HKRD domain in these molecules (Figure 1A) has been verified biochemically and shown to be light regulated. In canonical plant phys, however, the issue remains controversial. The first confounding factor is that these phys generally lack the phospho-acceptor histidine residue conserved in the two-component prokaryotic kinases, and no biochemically detectable His-kinase activity has been reported. The plant phys thus appear to have lost this catalytic activity during evolution. This conclusion has led to the alternative proposal that the plant phys have acquired serine/threonine kinase activity, more typical of eukaryotes, during evolution. The rapid in vivo phosphorylation of the PIF proteins triggered by light-induced binding of the phy molecule to the bHLH protein in the nucleus, mentioned above, is consistent with this possibility. Similarly, in vitro kinase assays of purified plant phys have detected the presence of serine/threonine kinase activity, potentially attributable to the phy molecule itself. However, several caveats remain. Firstly, the plant phy sequences lack the canonical signature motifs found in the majority of eukaryotic serine/threonine kinases, invoking the need to propose that they are atypical kinases. Secondly, the potential for the presence of contaminating kinase activity in the in vitro assays has not been rigorously eliminated. Thirdly, and most importantly, evidence that mutagenesis of the plant-phy kinase-like sequence motif results in loss of the protein kinase activity detected either in vitro, or by PIF phosphorylation in vivo, has not been forthcoming to date. In fact, there is evidence that deletion of the entire carboxy-terminal domain (containing the HKRD subdomain; Figure 1A) does not reduce phy activity in vivo. It must be concluded, therefore, that there is currently no unequivocal evidence that the plant phys possess intrinsic, autonomous protein kinase activity. Other alternatives, such as phy-mediated recruitment of third-party kinases to target proteins, remain viable.

What is on the horizon?

Rapid strides are being made in defining the structural features of the phy molecule through crystallographic and solution NMR studies on the sensory domain of bacterial phys. Expansion of this work to include the entire molecule and to solve the structure of a plant phy can be expected to provide invaluable insight into the mechanism of phy signaling. The availability of increasingly economical new-generation high-throughput DNA-sequencing technologies can be expected to permit genome-wide definition of the primary phy-regulated transcriptional network through the use of ChIP-seq and RNA-seq procedures. Proteomic approaches, such as mass spectrometric analysis, may provide an avenue for unravelling the current enigma of the capacity of the phy molecule to induce phosphorylation of signaling partners in vivo, through direct interaction, in the absence of apparent evidence of autonomous protein kinase activity intrinsic to the photoreceptor molecule itself.