Phytochrome evolution: Phytochrome genes in ferns and mosses

We have isolated phytochrome genes from the moss Physcomitrella , the fern Psilotum and PCR-generated phytochrome sequences from a few other ferns. The phytochrome gene of the moss Physcomitrella turned out not to contain the aberrant C-terminal third of the phytochrome from the moss Ceratodon , but the transmitter module-like sequences found in other phytochromes. A series of different phytochrome genes was detected in Psilotum . Differences between the amino acid sequences derived from them ranged from about 5 to more than 22%. Some of these genes are likely pseudogenes. Analysis by phylogenetic tree constructions revealed that higher and lower plant phytochromes evolved with different velocities. Lower plant phytochromes form a separate family characterized by a high degree of similarity. The amino acid differences between phytochrome types detected in a single species of higher plants are about two-fold higher than the differences between phytochromes of species of lower plants belonging to different divisions ( Physcomitrella and Selaginella ). Future studies on phytochrome sequences may eventually also throw light on the significance of Psilotum in the evolution of vascular plants.     

Epigenetics: a landscape takes shape

Epigenetics has recently evolved from a collection of diverse phenomena to a defined and far-reaching field of study. In this Essay, we examine the epistemology of epigenetics, provide a brief overview of underlying molecular mechanisms, and suggest future challenges for the field.     

FhaC takes a bow to FHA in the two‐partner do‐si‐do

FhaC is an outer membrane transporter from Bordetella pertussis belonging to the t wo‐ p artner s ecretion (TPS) pathway with its primary role being the secretion of the virulence factor f ilamentous h aem a gglutinin (FHA). FhaC serves as a model transporter of the TPS pathway and significant work has been done to characterize the role of FhaC in FHA secretion. Recent studies characterized interactions between FHA and the POTRA domains of FhaC, suggesting that secretion may involve a successive translocation mechanism mediated by β‐augmentation and/or electrostatic interactions. Moreover, it was also shown that reconstituted FhaC is necessary and sufficient to transport FHA into proteoliposomes. While the crystal structure of FhaC clearly suggests a role in transport, the putative transport pore is plugged by an N‐terminal α‐helix (H1 helix) that occludes access by FHA. Therefore, it has been proposed that the H1 helix must be expelled from the pore in order for secretion of FHA to occur. However, this has yet to be shown experimentally. Guérin et al. (2014) report the first direct experimental evidence to show that the FhaC H1 helix is quite dynamic and exchanges between closed and open states upon interaction with FHA.     

Gene amplification: yeast takes a turn

The origins of gene amplifications in mammalian cells have been difficult to analyze because of secondary genome rearrangements. Recent studies in budding yeast, including in this issue of Cell, have provided new insights into the role of palindromic sequences in gene amplification.     

Phytochrome radioimmunoassay

A phytochrome radioimmunoassay with a detection limit of about 2 nanograms has been developed. The radioimmunoassay does not suffer from the potential drawbacks of the commonly used spectral assay and requires less than 1 microliter of crude extract from dark-grown plants for quantitation of phytochrome. Measurement of phytochrome in crude extracts by radioimmunoassay gives values about 25% greater than those obtained by spectral assay. The amount of phytochrome detected in crude extracts of light-grown oats by radioimmunoassay is approximately 1% of that detected in comparable extracts from dark-grown oats. General interference by crude plant extracts with radioimmunoassays was also observed and corrected for.     

Phytochrome photoconversion

The spectral properties of native and modified phytochromes and the molecular events during phytochrome photoconversion, , are reviewed. Steady-state and time-resolved absorption spectra of native phytochrome A, as well as recombinant phytochromes (oat and potato phytochrome A and potato phytochrome B) reconstituted with phycocyanobilin and phytochromobilin as chromophores, are analysed. The vinyl double bond, present at position 18 in phytochromobilin and substituted by an ethyl group in phycocyanobilin, has a considerable influence on the photo-transformation kinetics of phytochromes A and B, evidently due to a strong interaction of this region of the chromophore with the protein surrounding. The kinetics of the phototransformation of potato phytochrome B differs from that of oat phytochrome A (wild-type and recombinant), indicating that the chromophore-protein interaction in phytochrome B is different from that in phytochrome A. It remains to be seen whether this difference is due to the di- versus monocotyledon origin of the phytochromes. Optoacoustic spectroscopy, applied to native oat phytochrome A, afforded thermo-dynamic, structural and kinetic parameters of the Pr→I₇₀₀ and the I₇₀₀→Pr phototransformations. Raman and infrared spectroscopic data for wild-type phytochrome A suggest that the protonated chromophore in Pr undergoes torsions around two single bonds in addition to the Z→E isomerization of the 15 ,16 double bond, and that all transients, possibly with the exception of I_bI, are protonated at the central pyrrole ring.     

Structure of the Cyanobacterial Phytochrome 2 Photosensor Implies a Tryptophan Switch for Phytochrome Signaling

Phytochromes are highly versatile photoreceptors, which occur ubiquitously in plants as well as in many light-responsive microorganisms. Here, photosynthetic cyanobacteria utilize up to three different phytochrome architectures, where only the plant-like and the single-domain cyanobacteriochromes are structurally characterized so far. Cph2 represents a third group in Synechocystis species and affects their capability of phototaxis by controlling c-di-GMP synthesis and degradation. The 2.6-Å crystal structure of its red/far-red responsive photosensory module in the P_r state reveals a tandem-GAF bidomain that lacks the figure-of-eight knot of the plant/cph1 subfamily. Its covalently attached phycocyanobilin chromophore adopts a highly tilted ZZZssa conformation with a novel set of interactions between its propionates and the GAF1 domain. The tongue-like protrusion from the GAF2 domain interacts with the GAF1-bound chromophore via its conserved PRXSF, WXE, and W(G/A)G motifs. Mutagenesis showed that the integrity of the tongue is indispensable for P_r → P_fr photoconversion and involves a swap of the motifs'' tryptophans within the tongue-GAF1 interface. This “Trp switch” is supposed to be a crucial element for the photochromicity of all multidomain phytochromes.     

Phytochrome Modification and Light-enhanced, In Vivo-induced Phytochrome Pelletability

Phytochrome that was induced by red irradiation in vivo to pellet with subcellular material and that was released from the pellet by removal of divalent cations exhibited altered characteristics. Compared to phytochrome extracted in a soluble red-absorbing form from etiolated tissue, pelleted and released phytochrome, which was also assayed in the red-absorbing form even though pelleted in the far-red-absorbing form, showed 50% greater micro complement fixation activity, eluted closer to the void volume of a Sephadex G-200 column, and electrophoresed more slowly on sodium dodecyl sulfate-polyacrylamide gels. Data presented here document that phytochrome pelleted in the far-red-absorbing form differs from soluble phytochrome extracted from nonirradiated tissue. These data, however, do not permit the conclusion that there is a causal relationship between pelletability and phytochrome modification.     

Mitotic motors: kinesin-5 takes a brake

A kinesin-5-dependent 'sliding filament' mechanism is commonly used to actively push apart the poles during mitotic spindle assembly and elongation, but a recent study now shows that, in C. elegans, kinesin-5 is deployed as a brake to slow down spindle-pole separation.     

Phytochrome behaves as a dimer in vivo

Abstract It is well established that phytochrome exists as a dimer in vitro. A comparison of the relative photoequilibrium concentrations of P_rP_r, P_rP_fr and P_frP_fr, with the relative sizes of the P_fr-pools which undergo dark reversion in the intact plant, leads to the hypothesis that phytochrome also exists as a dimer in vivo, This hypothesis is in accordance with kinetic properties of the phytochrome system under continuous irradiation. Additional support for this view is provided by the observation that P_fr-destruction after a red light flash, which should favour the formation of P_rP_fr dimers, is paralleled by a decay of P_r, even if the presence of P_r cycled through P_fr can be excluded. Preliminary observations could indicate an interaction of the subunits of a phytochrome dimer during the process of phototransformation.     

Phytochrome Characterization by Rabbit Antiserum against High Molecular Weight Phytochrome

Both small and large sizes of phytochrome purified from Garry oat (Avena sativa L. ev. Garry) as well as large phytochrome purified from Newton oat (A. sativa L. cv. Newton), rye (Secale cereale L. cv. Balbo), barley (Hordeum vulgare L. cv. Harrison), and pea (Pisum sativum L. cv. Alaska) seedlings are characterized by a specific antiserum against large Garry oat phytochrome. A spur is observed by double diffusion assay against large and small Garry oat phytochrome indicating only partial identity. In micro-complement fixation assays, large Garry oat phytochrome yields greater activity than small Garry oat phytochrome. In addition, the peak of activity is shifted to a higher antigen concentration with small phytochrome. Phytochrome, red-absorbing form, and phytochrome, far redabsorbing form, are indistinguishable by both double diffusion and micro-complement fixation assay. The different grass phytochromes are antigenically identical by double diffusion assay. Immunoelectrophoretic analyses of oat and rye large phytochrome, after proteolysis, suggest that there are one or a few regions of the molecule especially susceptible to hydrolysis by a wide variety of endopeptidases.