Family Of Photoreceptors Research Articles

Tomato phyE Is Required for Shade Avoidance in the Absence of phyB1 and phyB2.

The phytochrome (phy) family of red and far-red photoreceptors provides plants with critical information about their surrounding environment and can signal downstream developmental and physiological changes. Neighboring plants compete for limited light resources, and their presence is detected by the phytochrome photoreceptors as a reduced ratio of red: far-red light. One common response to shade is increased elongation of petioles and internodes to compete with their neighbors. While the phytochrome family, phyB in particular, has been well studied in Arabidopsis, information about the other phytochrome family members is limited, especially in sympodial crop plants such as tomato, that have a very different architecture from that of the model plant. To study the tomato phytochrome family we took advantage of several existing mutants and generated an artificial miRNA (amiRNA) line to target SlPHYE, the remaining phytochrome B subfamily member with no currently available mutant line. Here, we characterize internode elongation and shade avoidance phenotypes of the SlPHYE amiRNA line (PHYE amiRNA). In addition, higher order phytochrome subfamily B mutants were generated with the PHYE amiRNA line to investigate the role of SlphyE within the phyB subfamily. We find that the PHYE amiRNA line has no detectable phenotype on its own, however in higher order combinations with SlphyB1 and/or SlphyB2 there are notable defects in shade avoidance. Most notably, we find that the triple mutant combination of SlPHYE amiRNA, SlphyB1, and SlphyB2 has a phenotype that is much stronger than the SlphyB1 SlphyB2 double, showing constitutive shade avoidance and little to no response to shade. This indicates that SlphyE is required for the shade avoidance response in the absence of SlphyB1 and SlphyB2.

Xanthomonas campestris attenuates virulence by sensing light through a bacteriophytochrome photoreceptor.

Phytochromes constitute a major photoreceptor family found in plants, algae, fungi, and prokaryotes, including pathogens. Here, we report that Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease which affects cruciferous crops worldwide, codes for a functional bacteriophytochrome (XccBphP). XccBphP possesses an N-terminal PAS2-GAF-PHY photosensory domain triad and a C-terminal PAS9 domain as its output module. Our results show that illumination of Xcc, prior to plant infection, attenuates its virulence in an XccBphP-dependent manner. Moreover, in response to light, XccBphP downregulates xanthan exopolysaccharide production and biofilm formation, two known Xcc virulence factors. Furthermore, the XccbphP null mutant shows enhanced virulence, similar to that of dark-adapted Xcc cultures. Stomatal aperture regulation and callose deposition, both well-established plant defense mechanisms against bacterial pathogens, are overridden by the XccbphP strain. Additionally, an RNA-Seq analysis reveals that far-red light or XccBphP overexpression produces genomewide transcriptional changes, including the inhibition of several Xcc virulence systems. Our findings indicate that Xcc senses light through XccBphP, eliciting bacterial virulence attenuation via downregulation of bacterial virulence factors. The capacity of XccBphP to respond to light both in vitro and in vivo was abolished by a mutation on the conserved Cys13 residue. These results provide evidence for a novel bacteriophytochrome function affecting an infectious process.

A Protein-Based Genetic Screening Uncovers Mutants Involved in Phytochrome Signaling in Arabidopsis.

Plants perceive red and far-red region of the light spectrum to regulate photomorphogenesis through a family of photoreceptors called phytochromes. Phytochromes transduce the light signals to trigger a cascade of downstream gene regulation in part via a subfamily of bHLH transcription factors called Phytochrome Interacting Factors (PIFs). As the repressors of light signaling pathways, most PIFs are phosphorylated and degraded through the ubiquitin/26S proteasome pathway in response to light. The mechanisms involved in the phosphorylation and degradation of PIFs have not been fully understood yet. Here we used an EMS mutagenesis and luminescent imaging system to identify mutants defective in the degradation of one of the PIFs, called PIF1. We identified five mutants named stable PIF (spf) that showed reduced degradation of PIF1 under light treatment in both luminescent imaging and immunoblot assays. The amounts of PIF1 in spf3, spf4, and spf5 were similar to a PIF1 missense mutant (PIF1–3M) that lacks interactions between PIF1 and phyA/phyB under light. The hypocotyl lengths of spf1 and spf2 were slightly longer under red light compared to the LUC-PIF1 control, while only spf1 displayed weak phenotype under far-red light conditions. Interestingly, the spf3, spf4, and spf5 displayed high abundance of PIF1, yet the hypocotyl lengths were similar to the wild type under these conditions. Cloning and characterization of these mutants will help identify key players in the light signaling pathways including, the light-regulated kinase(s) and the E3 ligase(s) necessary for the light-induced degradation of PIFs.

A Negative Feedback Loop between PHYTOCHROME INTERACTING FACTORs and HECATE Proteins Fine-Tunes Photomorphogenesis in Arabidopsis.

The phytochrome interacting factors (PIFs), a small group of basic helix-loop-helix transcription factors, repress photomorphogenesis both in the dark and light. Light signals perceived by the phytochrome family of photoreceptors induce rapid degradation of PIFs to promote photomorphogenesis. Here, we show that HECATE (HEC) proteins, another small group of HLH proteins, antagonistically regulate PIFs to promote photomorphogenesis. HEC1 and HEC2 heterodimerize with PIF family members. PIF1, HEC1, and HEC2 genes are spatially and temporally coexpressed, and HEC2 is localized in the nucleus. hec1, hec2, and hec3 single mutants and the hec1 hec2 double mutant showed hyposensitivity to light-induced seed germination and accumulation of chlorophyll and carotenoids, hallmark processes oppositely regulated by PIF1. HEC2 inhibits PIF1 target gene expression by directly heterodimerizing with PIF1 and preventing DNA binding and transcriptional activation activity of PIF1. Conversely, PIFs directly activate the expression of HEC1 and HEC2 in the dark, and light reduces the expression of these HECs possibly by degrading PIFs. HEC2 is partially degraded in the dark through the ubiquitin/26S-proteasome pathway and is stabilized by light. HEC2 overexpression also reduces the light-induced degradation of PIF1. Taken together, these data suggest that PIFs and HECs constitute a negative feedback loop to fine-tune photomorphogenesis in Arabidopsis thaliana.

Evolutionary aspects of plant photoreceptors.

Plant photoreceptors link environmental light cues with physiological responses, determining how individual plants complete their life cycles. Structural and functional evolution of photoreceptors has co-occurred as plants diversified and faced the challenge of new light environments, during the transition of plants to land and as substantial plant canopies evolved. Large-scale comparative sequencing projects allow us for the first time to document photoreceptor evolution in understudied clades, revealing some surprises. Here we review recent progress in evolutionary studies of three photoreceptor families: phytochromes, phototropins and neochromes.

Advanced lighting technology in controlled environment agriculture

There is a recent awareness of the importance of plants in our everyday lives. Light is a requirement for plants and serves two important roles. It provides energy for growth and provides information that elicits plant responses including, among others, plant shape, pigmentation, nutritional content and resistance to stress. Light is paradoxical to plants, it is a requirement however, in excess it is damaging. Plants sense and interpret light through many families of photoreceptors and through the energy state of the photosynthetic apparatus. Light emitting diodes (LEDs) are quickly replacing traditional light sources for human applications, and currently there is effort being put into tailoring these technology platforms for the plant community. Potential plant sensing pathways and the spectral effects on pigmentation and photochemistry in red lettuce are described.

Illuminating Progress in Phytochrome-Mediated Light Signaling Pathways.

Light signals regulate a plethora of plant responses throughout their life cycle, especially the red and far-red regions of the light spectrum perceived by the phytochrome family of photoreceptors. However, the mechanisms by which phytochromes regulate gene expression and downstream responses remain elusive. Several recent studies have unraveled the details on how phytochromes regulate photomorphogenesis. These include the identification of E3 ligases that degrade PHYTOCHROME INTERACTING FACTOR (PIF) proteins, key negative regulators, in response to light, a better view of how phytochromes inhibit another key negative regulator, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), and an understanding of why plants evolved multiple negative regulators to repress photomorphogenesis in darkness. These advances will surely fuel future research on many unanswered questions that have intrigued plant photobiologists for decades.

Phototropins maintain robust circadian oscillation of PSII operating efficiency under blue light.

The circadian system allows plants to coordinate metabolic and physiological functions with predictable environmental variables such as dusk and dawn. This endogenous oscillator is comprised of biochemical and transcriptional rhythms that are synchronized with a plant's surroundings via environmental signals, including light and temperature. We have used chlorophyll fluorescence techniques to describe circadian rhythms of PSII operating efficiency (Fq'/Fm') in the chloroplasts of Arabidopsis thaliana. These Fq'/Fm' oscillations appear to be influenced by transcriptional feedback loops previously described in the nucleus, and are induced by rhythmic changes in photochemical quenching over circadian time. Our work reveals that a family of blue photoreceptors, phototropins, maintain robust rhythms of Fq'/Fm' under constant blue light. As phototropins do not influence circadian gene expression in the nucleus our imaging methodology highlights differences between the modulation of circadian outputs in distinct subcellular compartments.

The origin and evolution of phototropins

Plant phototropism, the ability to bend toward or away from light, is predominantly controlled by blue-light photoreceptors, the phototropins. Although phototropins have been well-characterized in Arabidopsis thaliana, their evolutionary history is largely unknown. In this study, we complete an in-depth survey of phototropin homologs across land plants and algae using newly available transcriptomic and genomic data. We show that phototropins originated in an ancestor of Viridiplantae (land plants + green algae). Phototropins repeatedly underwent independent duplications in most major land-plant lineages (mosses, lycophytes, ferns, and seed plants), but remained single-copy genes in liverworts and hornworts—an evolutionary pattern shared with another family of photoreceptors, the phytochromes. Following each major duplication event, the phototropins differentiated in parallel, resulting in two specialized, yet partially overlapping, functional forms that primarily mediate either low- or high-light responses. Our detailed phylogeny enables us to not only uncover new phototropin lineages, but also link our understanding of phototropin function in Arabidopsis with what is known in Adiantum and Physcomitrella (the major model organisms outside of flowering plants). We propose that the convergent functional divergences of phototropin paralogs likely contributed to the success of plants through time in adapting to habitats with diverse and heterogeneous light conditions.

CUL4 forms an E3 ligase with COP1 and SPA to promote light-induced degradation of PIF1.

Plants undergo contrasting developmental programs in dark and light. Photomorphogenesis, a light-adapted programme is repressed in the dark by the synergistic actions of CUL4(COP1-SPA) E3 ubiquitin ligase and a subset of basic helix-loop-helix transcription factors called phytochrome interacting factors (PIFs). To promote photomorphogenesis, light activates the phytochrome family of sensory photoreceptors, which inhibits these repressors by poorly understood mechanisms. Here, we show that the CUL4(COP1-SPA) E3 ubiquitin ligase is necessary for the light-induced degradation of PIF1 in Arabidopsis. The light-induced ubiquitylation and subsequent degradation of PIF1 is reduced in the cop1, spaQ and cul4 backgrounds. COP1, SPA1 and CUL4 preferentially form complexes with the phosphorylated forms of PIF1 in response to light. The cop1 and spaQ seeds display strong hyposensitive response to far-red light-mediated seed germination and light-regulated gene expression. These data show a mechanism by which an E3 ligase attenuates its activity by degrading its cofactor in response to light.

Professor Harry Smith (1935-2015).

Professor Harry Smith (1935-2015).

Electron transfer quenching in light adapted and mutant forms of the AppA BLUF domain.

The Blue Light Using Flavin (BLUF) domain proteins are an important family of photoreceptors controlling a range of responses in a wide variety of organisms. The details of the primary photochemical mechanism, by which light absorption in the isoalloxazine ring of the flavin is converted into a structure change to form the signalling state of the protein, is unresolved. In this work we apply ultrafast time resolved infra-red (TRIR) spectroscopy to investigate the primary photophysics of the BLUF domain of the protein AppA (AppABLUF) a light activated antirepressor. Here a number of mutations at Y21 and W104 in AppABLUF are investigated. The Y21 mutants are known to be photoinactive, while W104 mutants show the characteristic spectral red-shift associated with BLUF domain activity. Using TRIR we observed separately the decay of the excited state and the recovery of the ground state. In both cases the kinetics are found to be non-single exponential for all the proteins studied, suggesting a range of ground state structures. In the Y21 mutants an intermediate state was also observed, assigned to formation of the radical of the isoalloxazine (flavin) ring. The electron donor is the W104 residue. In contrast, no radical intermediates were detected in the studies of the photoactive dark adapted proteins, dAppABLUF and the dW104 mutants, suggesting a structure change in the Y21 mutants which favours W104 to isoalloxazine electron transfer. In contrast, in the light adapted form of the proteins (lAppABLUF, lW104) a radical intermediate was detected and the kinetics were greatly accelerated. In this case the electron donor was Y21 and major structural changes are associated with the enhanced quenching. In AppABLUF and the seven mutants studied radical intermediates are readily observed by TRIR spectroscopy, but there is no correlation with photoactivity. This suggests that if a charge separated state has a role in the BLUF photocycle it is only as a very short lived intermediate.

A new type of dual-Cys cyanobacteriochrome GAF domain found in cyanobacterium Acaryochloris marina, which has an unusual red/blue reversible photoconversion cycle.

Cyanobacteriochromes (CBCRs) form a large, spectrally diverse family of photoreceptors (linear tetrapyrrole covalently bound via a conserved cysteine) that perceive ultraviolet to red light. The underlying mechanisms are reasonably well understood with, in certain cases, reversible formation of an adduct between a second cysteine and the chromophore accounting, in part, for their spectral diversity. These CBCRs are denoted as dual-Cys CBCRs, and most such CBCRs had been shown to reversibly absorb blue and green light. Herein, we report the structural and mechanistic characterization of a new type of dual-Cys CBCR, AM1_1186, which exhibits reversible photoconversion between a red-absorbing dark state (λmax = 641 nm) and a blue-absorbing photoproduct (λmax = 416 nm). The wavelength separation of AM1_1186 photoconversion is the largest found to date for a CBCR. In addition to one well-conserved cysteine responsible for covalent incorporation of the chromophore into the apoprotein, AM1_1186 contains a second cysteine in a unique position of its photosensory domain, which would be more properly classified as a red/green CBCR according to its sequence. Carboxyamidomethylation and mutagenesis of the cysteines revealed that the second cysteine forms an adduct with the tetrapyrrole, the phycocyanobilin, that can be reversed under blue light. The proline immediately upstream of this cysteine appears to determine the rate at which the cysteinylation following photoexcitation of the dark state chromophore can occur. We propose a possible reaction scheme and color-tuning mechanism for AM1_1186 in terms of its structure and its place in a phylogenetic tree.

Crystal structure of the photosensing module from a red/far-red light-absorbing plant phytochrome.

Many aspects of plant photomorphogenesis are controlled by the phytochrome (Phy) family of bilin-containing photoreceptors that detect red and far-red light by photointerconversion between a dark-adapted Pr state and a photoactivated Pfr state. Whereas 3D models of prokaryotic Phys are available, models of their plant counterparts have remained elusive. Here, we present the crystal structure of the photosensing module (PSM) from a seed plant Phy in the Pr state using the PhyB isoform from Arabidopsis thaliana. The PhyB PSM crystallized as a head-to-head dimer with strong structural homology to its bacterial relatives, including a 5(Z)syn, 10(Z)syn, 15(Z)anti configuration of the phytochromobilin chromophore buried within the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain, and a well-ordered hairpin protruding from the Phy-specific domain toward the bilin pocket. However, its Per/Arnt/Sim (PAS) domain, knot region, and helical spine show distinct structural differences potentially important to signaling. Included is an elongated helical spine, an extended β-sheet connecting the GAF domain and hairpin stem, and unique interactions between the region upstream of the PAS domain knot and the bilin A and B pyrrole rings. Comparisons of this structure with those from bacterial Phys combined with mutagenic studies support a toggle model for photoconversion that engages multiple features within the PSM to stabilize the Pr and Pfr end states after rotation of the D pyrrole ring. Taken together, this Arabidopsis PhyB structure should enable molecular insights into plant Phy signaling and provide an essential scaffold to redesign their activities for agricultural benefit and as optogenetic reagents.

Competing neighbors: light perception and root function.

Plant responses to competition have often been described as passive consequences of reduced resource availability. However, plants have mechanisms to forage for favorable conditions and anticipate competition scenarios. Despite the progresses made in understanding the role of light signaling in modulating plant-plant interactions, little is known about how plants use and integrate information gathered by their photoreceptors aboveground to regulate performance belowground. Given that the phytochrome family of photoreceptors plays a key role in the acquisition of information about the proximity of neighbors and canopy cover, it is tempting to speculate that changes in the red:far-red (R:FR) ratio perceived by aboveground plant parts have important implications shaping plant behavior belowground. Exploring data from published experiments, we assess the neglected role of light signaling in the control of root function. The available evidence indicates that plant exposure to low R:FR ratios affects root growth and morphology, root exudate profiles, and interactions with beneficial soil microorganisms. Although dependent on species identity, signals perceived aboveground are likely to affect root-to-root interactions. Root systems could also be guided to deploy new growth predominantly in open areas by light signals perceived by the shoots. Studying interactions between above- and belowground plant-plant signaling is expected to improve our understanding of the mechanisms of plant competition.

PHYTOCHROME INTERACTING FACTOR1 Enhances the E3 Ligase Activity of CONSTITUTIVE PHOTOMORPHOGENIC1 to Synergistically Repress Photomorphogenesis in Arabidopsis.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a RING/WD40 repeat-containing ubiquitin E3 ligase that is conserved from plants to humans. COP1 forms complexes with SUPPRESSOR OF PHYTOCHROME A (SPA) proteins, and these complexes degrade positively acting transcription factors in the dark to repress photomorphogenesis. Phytochrome-interacting basic helix-loop-helix transcription factors (PIFs) also repress photomorphogenesis in the dark. In response to light, the phytochrome family of sensory photoreceptors simultaneously inactivates COP1-SPA complexes and induces the rapid degradation of PIFs to promote photomorphogenesis. However, the functional relationship between PIFs and COP1-SPA complexes is still unknown. Here, we present genetic evidence that the pif and cop1/spa Arabidopsis thaliana mutants synergistically promote photomorphogenesis in the dark. LONG HYPOCOTYL5 (HY5) is stabilized in the cop1 pif1, spa123 pif1, and pif double, triple, and quadruple mutants in the dark. Moreover, the hy5 mutant suppresses the constitutive photomorphogenic phenotypes of the pifq mutant in the dark. PIF1 forms complexes with COP1, HY5, and SPA1 and enhances the substrate recruitment and autoubiquitylation and transubiquitylation activities of COP1. These data uncover a novel function of PIFs as the potential cofactors of COP1 and provide a genetic and biochemical model of how PIFs and COP1-SPA complexes synergistically repress photomorphogenesis in the dark.

Oxidative control of oligomeric structure formation in Vibrio cholerae cryptochrome 1 (549.2)

Cryptochrome is a member of the blue‐light photoreceptor family, and it appears to play a role in the regulation of the circadian rhythm in organisms. The oligomeric structure of the protein appears to play a key role in the signaling mechanism. Vibrio cholerae Cryptochrome 1 (VcCry1) will be used as model protein for this study; it may be involved in both DNA repair and blue‐light signaling in vivo. From our preliminary data, the oligomeric structure in VcCry1 appears to be related to the oxidation state of the active site FAD cofactor. Chemical cross linking will be used to examine the link between FAD oxidation state and oligomeric structure.

Conservation of Dark Recovery Kinetic Parameters and Structural Features in the Pseudomonadaceae “Short” Light, Oxygen, Voltage (LOV) Protein Family: Implications for the Design of LOV-Based Optogenetic Tools

In bacteria and fungi, various light, oxygen, voltage (LOV) sensory systems that lack a fused effector domain but instead contain only short N- and C-terminal extensions flanking the LOV core exist. In the prokaryotic kingdom, this so-called "short" LOV protein family represents the third largest LOV photoreceptor family. This observation prompted us to study their distribution and phylogeny as well as their photochemical and structural properties in more detail. We recently described the slow and fast reverting "short" LOV proteins PpSB1-LOV and PpSB2-LOV from Pseudomonas putida KT2440 whose adduct state lifetimes varied by 3 orders of magnitude [Jentzsch, K., Wirtz, A., Circolone, F., Drepper, T., Losi, A., Gärtner, W., Jaeger, K. E., and Krauss, U. (2009) Biochemistry 48, 10321-10333]. We now present evidence of the conservation of similar fast and slow-reverting "short" LOV proteins in different Pseudomonas species. Truncation studies conducted with PpSB1-LOV and PpSB2-LOV suggested that the short N- and C-terminal extensions outside of the LOV core domain are essential for the structural integrity and folding of the two proteins. While circular dichroism and solution nuclear magnetic resonance experiments verify that the two short C-terminal extensions of PpSB1-LOV and PpSB2-LOV form independently folding helical structures in solution, bioinformatic analyses imply the formation of coiled coils of the respective structural elements in the context of the dimeric full-length proteins. Given their prototypic architecture, conserved in most more complex LOV photoreceptor systems, "short" LOV proteins could represent ideally suited building blocks for the design of genetically encoded photoswitches (i.e., LOV-based optogenetic tools).

Shedding New Light on Retinal Protein Photochemistry

The ultrafast spectroscopic investigation of novel retinal proteins challenges existing notions concerning the course of primary events in these natural photoreceptors. We review two illustrations here. The first demonstrates that changes in the initial retinal configuration can alter the duration of photochemistry by nearly an order of magnitude in Anabaena sensory rhodopsin, making it as rapid as the ballistic photoisomerization in visual pigments. This prompted a reinvestigation of the much studied bacteriorhodopsin, leading to a similar trend as well, contrary to earlier reports. The second involves the study of xanthorhodopsin, an archaeal proton pump that includes an attached light-harvesting carotenoid. Pump-probe experiments demonstrate the efficient transfer of energy from carotenoid to retinal, providing a first glimpse at a cooperative multichromophore function, which is probably characteristic of many other proteins as well. Finally, we discuss measures required to advance our knowledge from kinetics to mode-specific dynamics concerning this expanding family of biological photoreceptors.

Cyanobacteriochromes in full color and three dimensions

Sensory photoreceptors occur in all kingdoms of life, eliciting diverse organismal adaptations in response to incident light. The recently identified cyanobacteriochromes (CBCRs) mediate photochromatic and phototactic responses in cyanobacteria (1⇓–3). Great strides toward a molecular understanding of photoreception and signal transduction in this spectrally diverse and exciting photoreceptor family have now been taken by Narikawa et al. (4), who report high-resolution structures of two CBCR photosensor modules in PNAS. CBCRs are related to plant and bacterial phytochromes (Phys), with which they share the intrinsic ability to form thioether linkages to linear tetrapyrrole (bilin) chromophores via conserved cysteine residues. Moreover, these photoreceptor families use a unifying photochemical mechanism (Fig. 1 A ): photoisomerization of the chromophore between 15Z and 15E configurations with concomitant rotation of the terminal bilin D-ring (5⇓–7). The 15Z and 15E states differ in their absorption properties (photochromism) and modulate the behavior of output domains and downstream signal transduction pathways. Despite similar chromophores, photochemistry, and self-assembly, Phys and CBCRs differ in several striking ways. Most phytochromes require a three-domain PAS-GAF-PHY architecture [GAF domain, cGMP-phosphodiesterase/adenylate cyclase/FhlA (8); PAS domain, Per/ARNT/Sim; PHY domain, phytochrome-specific] for reversible photoconversion (3, 5, 7, 9). CBCRs instead achieve fully reversible photochemistry with a lone chromophore-binding GAF domain. Multiple CBCRs often occur in tandem within a single protein, allowing integration of multiple light signals at a single C-terminal output domain (10). Whereas Phys predominantly respond to the red/far-red spectral region, CBCRs display a rich variety of photocycles spanning the entire visible and near-UV spectrum (2, 11⇓–13). At least four subfamilies of CBCRs can be distinguished on the basis of their underlying photochemistry and primary structure. Fig. 1. Cyanobacteriochrome structure and function. ( A ) Bilin chromophores and … [↵][1]1To whom correspondence should be addressed. E-mail: andreas.moeglich{at}hu-berlin.de. [1]: #xref-corresp-1-1