Family Of Photoreceptors Research Articles

Phototropism: Translating light into directional growth

Phototropism allows plants to align their photosynthetic tissues with incoming light. The direction of incident light is sensed by the phototropin family of blue light photoreceptors (phot1 and phot2 in Arabidopsis), which are light-activated protein kinases. The kinase activity of phototropins and phosphorylation of residues in the activation loop of their kinase domains are essential for the phototropic response. These initial steps trigger the formation of the auxin gradient across the hypocotyl that leads to asymmetric growth. The molecular events between photoreceptor activation and the growth response are only starting to be elucidated. In this review, we discuss the major steps leading from light perception to directional growth concentrating on Arabidopsis. In addition, we highlight links that connect these different steps enabling the phototropic response.

Temperature‐dependent shade avoidance involves the receptor‐like kinase ERECTA

Plants detect the presence of neighbouring vegetation by monitoring changes in the ratio of red (R) to far-red (FR) wavelengths (R:FR) in ambient light. Reductions in R:FR are perceived by the phytochrome family of plant photoreceptors and initiate a suite of developmental responses termed the shade avoidance syndrome. These include increased elongation growth of stems and petioles, enabling plants to overtop competing vegetation. The majority of shade avoidance experiments are performed at standard laboratory growing temperatures (>20°C). In these conditions, elongation responses to low R:FR are often accompanied by reductions in leaf development and accumulation of plant biomass. Here we investigated shade avoidance responses at a cooler temperature (16°C). In these conditions, Arabidopsis thaliana displays considerable low R:FR-mediated increases in leaf area, with reduced low R:FR-mediated petiole elongation and leaf hyponasty responses. In Landsberg erecta, these strikingly different shade avoidance phenotypes are accompanied by increased leaf thickness, increased biomass and an altered metabolite profile. At 16°C, low R:FR treatment results in the accumulation of soluble sugars and metabolites associated with cold acclimation. Analyses of natural genetic variation in shade avoidance responses at 16°C have revealed a regulatory role for the receptor-like kinase ERECTA.

Why flavins are not competitors of chlorophyll in the evolution of biological converters of solar energy.

Excited flavin molecules can photocatalyze reactions, leading to the accumulation of free energy in the products, and the data accumulated through biochemical experiments and by modeling prebiological processes suggest that flavins were available in the earliest stages of evolution. Furthermore, model experiments have shown that abiogenic flavin conjugated with a polyamino acid matrix, a pigment that photocatalyzes the phosphorylation of ADP to form ATP, could have been present in the prebiotic environment. Indeed, excited flavin molecules play key roles in many photoenzymes and regulatory photoreceptors, and the substantial structural differences between photoreceptor families indicate that evolution has repeatedly used flavins as chromophores for photoreceptor proteins. Some of these photoreceptors are equipped with a light-harvesting antenna, which transfers excitation energy to chemically reactive flavins in the reaction center. The sum of the available data suggests that evolution could have led to the formation of a flavin-based biological converter to convert light energy into energy in the form of ATP.

Arabidopsis phytochrome mutant에서 빛이 뿌리 생장과 굴중성 반응에 미치는 영향

빛이 굴중성과 뿌리 생장에 미치는 영향을 알아보기 위해 Arabidopsis phytochrome mutant를 이용하여 뿌리 생장과 굴중성 반응을 측정하였다. <TEX>$PhyA$</TEX>의 뿌리 생장은 white light를 제외한 모든 빛 조건에서 WT와 비교하여 촉진되었다. 특히 red light에서 키웠을 때 다른 mutant와는 달리 뿌리 생장이 촉진되었다. 반면에 phyB는 모든 빛 조건에서 키웠을 때 뿌리 생장이 억제되었으며, 특히 double mutant인 <TEX>$phyAB$</TEX>는 white light와 red light에서 키웠을 때 뿌리 생장이 가장 많이 억제되었다. <TEX>$PhyA$</TEX>의 굴중성 반응은 far-red light에서 키웠을 때를 제외하고 모든 빛 조건에서 키웠을 때 WT와 비교하여 촉진되었다. 한편 <TEX>$phyAB$</TEX>는 모든 조건에서 모두 굴중성 반응이 억제되었다. Ethylene 생합성을 조절하는 효소인 ACS transcript 수준은 white light와 red light에서 키웠을 때 <TEX>$phyA$</TEX>가 높게 나타났으며, far-red light에서 키웠을 때는 <TEX>$phyA$</TEX>의 transcript 수준이 억제되었다. 결론적으로 뿌리 생장과 굴중성 반응은 <TEX>$phyB$</TEX>의 <TEX>$P_{fr}$</TEX>에 의해 조절된다. Light, one of the environmental stimuli, is fundamental to the growth and development of plants. Red and far-red light are sensed using the phytochrome family of plant photoreceptors. To investigate the effect of light on root growth and gravitropism, we used the Arabidopsis phytochrome mutants grown in several light conditions. The root growth of <TEX>$phyA$</TEX> reared in all light conditions except white light and was stimulated compared to the WT. The stimulation of root growth was obvious in <TEX>$phyA$</TEX> grown in red light. On the other hand, the root growth of <TEX>$phyB$</TEX> grown in all light conditions decreased, and the lowest rate of decrease was observed in <TEX>$phyAB$</TEX> grown in white and red light. The gravitropic response of <TEX>$phyA$</TEX> was stimulated compared to the WT when it was grown in all light conditions except far-red light. <TEX>$PhyAB$</TEX> grown in all light conditions showed the inhibition of gravitropic response. The transcript level of ACS, one of the enzymes regulating ethylene biosynthesis, increased in <TEX>$phyA$</TEX> grown in white and red light, but not in <TEX>$phyA$</TEX> grown in far-red light. In conclusion, these results suggested that the <TEX>$P_{fr}$</TEX> form of <TEX>$phyB$</TEX> regulates the root growth and gravitropism.

Thiol-Based Photocycle of the Blue and Teal Light-Sensing Cyanobacteriochrome Tlr1999

Cyanobacteriochromes are a spectrally diverse photoreceptor family that binds a bilin chromophore. For some cyanobacteriochromes, in addition to the widely conserved cysteine to anchor the chromophore, its ligation with a second cysteine is responsible for a remarkable blue shift. Herein, we report a newly discovered cyanobacteriochrome Tlr1999 exhibiting reversible photoconversion between a blue-absorbing form at 418 nm (P418) and a teal-absorbing form at 498 nm (P498). Acidic denaturation suggests that P418 harbors C15-Z phycoviolobilin, whereas P498 harbors C15-E phycoviolobilin. When treated with iodoacetamide, which irreversibly modifies thiol groups, P418 is slowly converted to a green-absorbing photoinactive form denoted P552. The absorption spectrum of P498 appears to be unaffected by iodoacetamide, but when iodoacetamide modified, it is photoconverted to P552. These results suggest that a covalent bond exists between the second Cys and the phycoviolobilin in P418 but not in P498. Subsequent treatment with dithiothreitol converts P552 into P418, whereas dithiothreitol reduces P498 to yield P420, a photoinactive form. Site-directed mutagenesis shows that the second Cys is essential for assembly of the photoactive holoprotein and that the photoactivity of this inert mutant is partially rescued by β-mercaptoethanol. These results suggest that the covalent attachment and detachment of a thiol, although not necessarily that of the second Cys, is critical for the reversible spectral blue shift and the complete photocycle. We propose a thiol-based photocycle, in which the thiol-modified P552 and P420 are intermediate-like forms.

Arabidopsis thalianaTERMINAL FLOWER2 is involved in light‐controlled signalling during seedling photomorphogenesis

Plants respond to changes in the environment by altering their growth pattern. Light is one of the most important environmental cues and affects plants throughout the life cycle. It is perceived by photoreceptors such as phytochromes that absorb light of red and far-red wavelengths and control, for example, seedling de-etiolation, chlorophyll biosynthesis and shade avoidance response. We report that the terminal flower2 (tfl2) mutant, carrying a mutation in the Arabidopsis thaliana HETEROCHROMATIN PROTEIN1 homolog, functions in negative regulation of phytochrome dependent light signalling. tfl2 shows defects in both hypocotyl elongation and shade avoidance response. Double mutant analysis indicates that mutants of the red/far-red light absorbing phytochrome family of plant photoreceptors, phyA and phyB, are epistatic to tfl2 in far-red and red light, respectively. An overlap between genes regulated by light and by auxin has earlier been reported and, in tfl2 plants light-dependent auxin-regulated genes are misexpressed. Further, we show that TFL2 binds to IAA5 and IAA19 suggesting that TFL2 might be involved in regulation of phytochrome-mediated light responses through auxin action.

Computational Analysis of Electron Tunneling Pathways of Blue-Light Photoreceptors

DNA phtolyases and cryptochrome DASH (cry-DASH) enzymes in the blue-light photoreceptor family repair UV-damaged DNA by photo-induced electron transfer reaction. Recently (Miyazawa et al., 2008) we analyzed the electron tunneling pathways in a class I CPD photolyase derived from A. nidulans and identified a key residue, Met-353, which is perfectly conserved in the class I CPD photolyases. Interestingly, this site is switched to histidine in 6-4 photolyases, and to glutamine in cry-DASH enzymes. For instance, Met-353 is replaced to Gln-395 in cry-DASH. It is likely that the amino acid residue at the site controls the enzymatic functions of the blue-light photoreceptor family. Until now, the electron transfer pathways in cry-DASH enzymes remain unclear, while those of CPD photolyases are studied well.To characterize the roles of the amino acid residue at the site, we performed molecular dynamics simulation and electronic state calculations of the CPD photolyase of A. nidulans and the cry-DASH. We analyzed the electron tunneling pathways in the forward and backward electron transfer reactions between FADH- and CPD of these two enzymes. As a result, we observed busy trafficking of electron-tunneling current at both Met-353 of the CPD photolyase and Gln-395 of the cry-DASH.

Tomato plants overexpressing cryptochrome 2 reveal altered expression of energy and stress‐related gene products in response to diurnal cues

In order to sense and respond to the fluctuating light conditions, higher plants possess several families of photoreceptors, such as phytochromes (PHYs), cryptochromes (CRYs) and phototropins. CRYs are responsible for photomorphogenesis and play a role in circadian, developmental and adaptive growth regulation of plants. In tomato (Solanum lycopersicum), CRY2 controls vegetative development, flowering time, fruit antioxidant content as well as the diurnal transcription of several other photoreceptor genes. We applied large-scale molecular approaches to identify altered transcripts and proteins in tomato wild-type (WT) versus a CRY2 overexpressing transgenic genotype, under a diurnal rhythm. Our results showed that tomato CRY2 profoundly affects both gene and protein expression in response to daily light cycle. Particularly altered molecular pathways are related to biotic/abiotic stress, photosynthesis, including components of the light and dark reactions and of starch and sucrose biosynthesis, as well as to secondary metabolism, such as phenylpropanoid, phenolic and flavonoid/anthocyanin biosynthesis pathways. One of the most interesting results is the coordinated up-regulation, in the transgenic genotype, of a consistent number of transcripts and proteins involved in photorespiration and photosynthesis. It is conceivable that light modulates the energetic metabolism of tomato through a fine CRY2-mediated transcriptional control.

Structure of full-length Drosophila cryptochrome.

The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions.

Phosphorylation by CK2 Enhances the Rapid Light-induced Degradation of Phytochrome Interacting Factor 1 in Arabidopsis

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.

A Negative Effector of Blue Light-Induced and Gravitropic Bending in Arabidopsis

Although sessile, plants are able to grow toward or away from an environmental stimulus. Important examples are stem or leaf orientation of higher plants in response to the direction of the incident light. The responsible photoreceptors belong to the phototropin photoreceptor family. Although the mode of phototropin action is quite well understood, much less is known of how the light signal is transformed into a bending response. Several lines of evidence indicate that a lateral auxin gradient is responsible for asymmetric cell elongation along the light gradient within the stem. However, some of the molecular key players leading to this asymmetric auxin distribution are, as yet, unidentified. Previously, it was shown that phototropin gets autophosphorylated upon illumination and binds to a scaffold protein termed NPH3 (for nonphototropic hypocotyl 3). Using a yeast three-hybrid approach with phototropin and NPH3 as a bait complex, we isolated a protein, termed EHB1 (for enhanced bending 1), with a so far unknown function, which binds to this binary complex. This novel interacting factor negatively affects hypocotyl bending under blue light conditions in Arabidopsis (Arabidopsis thaliana) and thus seems to be an important component regulating phototropism. Interestingly, it could be shown that the gravitropic response was also affected. Thus, it cannot be ruled out that this protein might also have a more general role in auxin-mediated bending toward an environmental stimulus.

A eukaryotic LOV‐histidine kinase with circadian clock function in the picoalga Ostreococcus

The marine environment has unique properties of light transmission, with an attenuation of long wavelengths within the first meters of the water column. Marine organisms have therefore evolved specific blue-light receptors such as aureochromes to absorb shorter-wavelength light. Here, we identify and characterize a light, oxygen, or voltage sensing (LOV) containing histidine kinase (LOV-HK) that functions as a new class of eukaryotic blue-light receptor in the pico-phytoplanktonic cell Ostreococcus tauri. This LOV-HK is related to the large family of LOV-HKs found in prokaryotes. Phylogenetic analysis indicates that the LOV domains from LOV-HKs, including O.tauri LOV-HK, and phototropins (phot; plant and green algal LOV serine/threonine kinases) have different evolutionary histories. Photochemical analysis shows that the LOV domain of LOV-HK binds a flavin cofactor and absorbs blue light with a fast photocycle compared with its prokaryotic counterparts. Ostreococcus tauri LOV-HK expression is induced by blue light and is under circadian control. Further, both overexpression and downregulation of LOV-HK result in arrhythmia of the circadian reporter CCA1:Luc under constant blue light. In contrast, photochemical inactivation of O.tauri LOV-HK is without effect, demonstrating its importance for function of the circadian clock under blue light. Overexpression/downregulation of O.tauriLOV-HK alters CCA1 rhythmicity under constant red light, irrespective of LOV-HK's photochemical reactivity, suggesting that O.tauri LOV-HK also participates in regulation of the circadian clock independent of its blue-light-sensing property. Molecular characterization of O.tauri LOV-HK demonstrates that this type of photoreceptor family is not limited to prokaryotes.

Genome‐wide mapping of the HY5‐mediated genenetworks in Arabidopsis that involve both transcriptional and post‐transcriptional regulation

LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology, and defective hormonal and stimulus responses. HY5 thus acts as an integrator that links various gene networks to coordinate plant development. Here we report the experimental mapping of HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate that HY5 binds to over 9000 genes, detectably affecting the expression of over 1100 genes, either positively or negatively. Further, HY5 indirectly regulate many other genes through sub-networks mediated by other regulators. In particular, HY5 regulates eight miRNA genes that in turn control the transcript abundance of specific target genes. Over-expressing HY5-targeted miR408 resulted in phenotypes that are opposite to the hy5 mutants. Together, our results reveal both transcriptional and post-transcriptional components of the HY5-mediated gene networks.

Tripping the light fantastic: blue-light photoreceptors as examples of environmentally modulated protein-protein interactions.

Blue-light photoreceptors play a pivotal role in detecting the quality and quantity of light in the environment, controlling a wide range of biological responses. Several families of blue-light photoreceptors have been characterized in detail using biophysics and biochemistry, beginning with photon absorption, through intervening signal transduction, to regulation of biological activities. Here we review the light oxygen voltage, cryptochrome, and sensors of blue light using FAD families, three different groups of proteins that offer distinctly different modes of photochemical activation and signal transduction yet play similar roles in a vast array of biological responses. We cover mechanisms of light activation and propagation of conformational responses that modulate protein-protein interactions involved in biological signaling. Discovery and characterization of these processes in natural proteins are now allowing the design of photoregulatable engineered proteins, facilitating the generation of novel reagents for biochemical and cell biological research.

On the midpoint potential of the FAD chromophore in a BLUF-domain containing photoreceptor protein

The redox-midpoint potential of the FAD chromophore in the BLUF domain of anti-transcriptional regulator AppA from Rhodobacter sphaeroides equals ∼−260mV relative to the calomel electrode. Altering the structure of its chromophore-binding pocket through site-directed mutagenesis brings this midpoint potential closer to that of free flavin in aqueous solution. The redox-midpoint potential of this BLUF domain is intermediate between those of LOV domains and Cryptochromes, which may rationalize the primary photochemistry observed in these three flavin-containing photoreceptor families. These results also imply that LOV domains, among the flavin-containing photosensory receptors, are least sensitive to intracellular chemical reduction in the dark.

The Contribution of a Covalently Bound Cofactor to the Folding and Thermodynamic Stability of an Integral Membrane Protein

The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol − 1 to 23.4 ± 1.5 kcal mol − 1 at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol − 1 towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol − 1 to 10.9 ± 1.0 kcal mol − 1 , indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3–6 kcal mol − 1 . Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k u H2O, from ~1 × 10 − 16 s − 1 to ~1 × 10 − 4 s − 1 at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol − 1 in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein–retinal interactions that increase the rigidity and compactness of the polypeptide chain.

Integration of phot1, phot2, and PhyB signalling in light-induced chloroplast movements

In Arabidopsis thaliana, chloroplasts move towards the periclinal cell walls upon exposure to low blue light intensities and to anticlinal walls under high light. The regulation of these chloroplast movements involves members of both the phototropin and phytochrome families of photoreceptors. Examination of fluence-rate response dependencies in phot1 and phot2 mutants revealed that although both photoreceptors are capable of inducing chloroplast accumulation under low-light conditions, the signals from these photoreceptors appear to be antagonistic. Chloroplast movements in wild-type plants were intermediate between those of the single phot mutants, consistent with each operating through separate signalling cascades. Mutants in phot2 showed transient chloroplast avoidance responses upon exposure to intense blue light, and slow but sustained chloroplast avoidance under intense white light, indicating that in the absence of phot2, phot1 is capable of generating both a low and a high-light response signal. Mutations in phytochrome B (phyB) caused an enhanced avoidance response at intermediate and high light intensities. Examination of phyB, phot1phyB, and phot2phyB mutants indicated that this enhancement is caused by PhyB inhibition of the high-light avoidance response in wild-type plants. In addition, our results suggest that the inhibition by PhyB is not exclusive to either of the phot1 or phot2 signalling pathways.

Structural Guidance of the Photocycle of Channelrhodopsin-2 by an Interhelical Hydrogen Bond

Channelrhodopsin-2 (ChR2) is a light-gated cation channel and a member of the family of retinylidene photoreceptors. Since the demonstration of light-induced depolarization of ChR2-expressing animal cell membranes, it was increasingly exploited for light triggering of action potentials. ChR2 conducts cations upon light absorption that embodies retinal isomerization as the primary reaction and a structurally unknown opening mechanism. It is evident from spectroscopic data that protonation reactions at the Schiff base are part of the photocycle, comparable to other microbial-type rhodopsins. However, the connection between the processes at the chromophore site and the channel's pore remained enigmatic. Here, we use slow mutants of ChR2 that we generated by disturbing a postulated hydrogen bond when mutating C128 in the transmembrane (TM) helix 3 and D156 in TM helix 4. The lifetime of the mutants' open state is increased more than 100 times. We investigated the spectral properties of the slow mutants. Whereas the deprotonation of the Schiff base (yielding P390) occurs on the same time scale as that of the wild type, reprotonation to P520 is retarded in the slow mutants and their photocycle is split, leading to the presence of two photointermediates, P390 and P520, in the open state. The photoreactions of P390 and P520 lead to a quenching of the current in electrophysiological measurements. We conclude that the putative hydrogen bond between C128 and D156 is an important structural determinant of the channel's closing reaction. Furthermore, we show that the D156A mutant is even more suitable for light control of excitable cells than C128A.

Locked Chromophore Analogs Reveal That Photoactive Yellow Protein Regulates Biofilm Formation in the Deep Sea Bacterium Idiomarina loihiensis

Idiomarina loihiensis is a heterotrophic deep sea bacterium with no known photobiology. We show that light suppresses biofilm formation in this organism. The genome of I. loihiensis encodes a single photoreceptor protein: a homologue of photoactive yellow protein (PYP), a blue light receptor with photochemistry based on trans to cis isomerization of its p-coumaric acid (pCA) chromophore. The addition of trans-locked pCA to I. loihiensis increases biofilm formation, whereas cis-locked pCA decreases it. This demonstrates that the PYP homologue regulates biofilm formation in I. loihiensis, revealing an unexpected functional versatility in the PYP family of photoreceptors. These results imply that I. loihiensis thrives not only in the deep sea but also near the water surface and provide an example of genome-based discovery of photophysiological responses. The use of locked pCA analogs is a novel and generally applicable pharmacochemical tool to study the in vivo role of PYPs irrespective of genetic accessibility. Heterologously produced PYP from I. loihiensis (Il PYP) absorbs maximally at 446 nm and has a pCA pK(a) of 3.4. Photoexcitation triggers the formation of a pB signaling state that decays with a time constant of 0.3 s. FTIR difference signals at 1726 and 1497 cm(-1) reveal that active-site proton transfer during the photocycle is conserved in Il PYP. It has been proposed that a correlation exists between the lifetime of a photoreceptor signaling state and the time scale of the biological response that it regulates. The data presented here provide an example of a protein with a rapid photocycle that regulates a slow biological response.

Molecular Cloning and Linkage Mapping of Cryptochrome Multigene Family in Soybean

The cryptochromes are a family of blue light photoreceptors that play important roles in the controls of plant development. Seven full‐length cryptochrome cDNAs (GmCRY1a, GmCRY1b, GmCRY1c, GmCRY1d, GmCRY2a, GmCRY2b, and GmCRY2c) were isolated by cDNA library screening and reverse transcriptase–polymerase chain reaction from ‘Williams’ soybean [Glycine max (L.) Merr.], indicating that soybean cryptochrome genes comprise a multigene family. They had homologies ranging from 60 to 89% with CRY1 and CRY2 genes of Arabidopsis and pea (Pisum sativum L.). Two types of transcripts were isolated in GmCRY1b, GmCRY1c, GmCRY1d, and GmCRY2a. One type was derived from four exons, whereas the other type was derived from five exons. Occurrence of the former transcript could be explained by retention of the fourth intron, suggesting existence of alternative splicing. Gene sequences were compared between a soybean line, Tokei 780, and an accession of soybean wild relative, Glycine soja, Hidaka 4. Based on a 10‐bp indel, an amplicon length polymorphism (ALP) marker was designed for mapping of GmCRY2b. For mapping of the other cryptochrome genes, derived cleaved amplified polymorphic sequence (dCAPS) markers were constructed. The cryptochrome genes were individually assigned to different molecular linkage groups (MLG) (GmCRY1a: MLG C1; GmCRY1b: C2; GmCRY1c: B2; GmCRY1d: F; GmCRT2a: O; GmCRY2b: D1b; GmCRY2c: I). The distribution of cryptochrome genes that was deduced from the soybean genome database was consistent with mapping results.