Plant Photoreceptors Research Articles

Generation and Characterization of a Specific Polyclonal Antibody against Arabidopsis thaliana Phytochrome-Interacting Factor 3

Phytochrome-interacting factors (PIFs) are a basic helix-loop-helix family of transcriptional regulators that maintain skotomorphogenesis and suppress photomorphogenesis. PIFs are regulated by plant photoreceptors, especially phytochromes. In general, PIFs physically interact with phytochromes, and this interaction induces PIF’s phosphorylation and subsequent degradation, contributing to the initiation of photomorphogenic development. Among the eight members of PIF (PIF1 to PIF8) reported in Arabidopsis thaliana, PIF3 is the first discovered member and plays central roles in de-etiolation and chlorophyll biosynthesis. More recently, PIF3 has been also reported to regulate hormone signaling and cold tolerance in plants. Although PIF3 protein shows dynamic behaviors in plants, its study has been limited due to the lack of an authentic PIF3 antibody. In this study, we produced polyclonal antibodies using inclusion bodies and characterized the PIF3 antibody based on specificity and sensitivity. In addition, we investigated PIF3 phosphorylation and degradation during phytochrome-mediated light signaling in plants. Furthermore, we successfully performed in vitro protein–protein interaction and co-immunoprecipitation assays between phytochrome B (phyB) and PIF3 using the antibody. Therefore, we obtained an authentic PIF3 antibody that could be used as a valuable tool to study the multi-faceted functions of PIF3.

Far-red Fraction: An Improved Metric for Characterizing Phytochrome Effects on Morphology

Phytochrome, a well-studied photoreceptor in plants, primarily absorbs in the red (R) and far-red (FR) regions and is responsible for the perception of shade and subsequent morphological responses. Experiments performed in controlled environments have widely used the R:FR ratio to simulate the natural environment and used phytochrome photoequilibrium (PPE) to simulate the activity of phytochrome. We review why PPE may be an unreliable metric, including differences in weighting factors, multiple phytochromes, nonphotochemical reversions, intermediates, variations in the total pool of phytochrome, and screening by other pigments. We suggest that environmental signals based on R and FR photon fluxes are a better predictor of plant shape than the more complex PPE model. However, the R:FR ratio is nonintuitive and can approach infinity under electric lights, which makes it difficult to extrapolate from studies in controlled environments to the field. Here we describe an improved metric: the FR fraction (FR/R+FR) with a range from 0 to 1. This is a more intuitive metric both under electric lights and in the field compared with other ratios because it is positively correlated with phytochrome-mediated morphological responses. We demonstrate the reliability of this new metric by reanalyzing previously published data.

Optogenetics in plants.

The last two decades have witnessed the emergence of optogenetics; a field that has given researchers the ability to use light to control biological processes at high spatiotemporal and quantitative resolutions, in a reversible manner with minimal side-effects. Optogenetics has revolutionized the neurosciences, increased our understanding of cellular signalling and metabolic networks and resulted in variety of applications in biotechnology and biomedicine. However, implementing optogenetics in plants has been less straightforward, given their dependency on light for their life cycle. Here, we highlight some of the widely used technologies in microorganisms and animal systems derived from plant photoreceptor proteins and discuss strategies recently implemented to overcome the challenges for using optogenetics in plants.

Comparative analysis of two phytochrome mutants of tomato (Micro-Tom cv.) reveals specific physiological, biochemical, and molecular responses under chilling stress

BackgroundPhytochromes are plant photoreceptors that have long been associated with photomorphogenesis in plants; however, more recently, their crucial role in the regulation of variety of abiotic stresses has been explored. Chilling stress is one of the abiotic factors that severely affect growth, development, and productivity of crops. In the present work, we have analyzed and compared physiological, biochemical, and molecular responses in two contrasting phytochrome mutants of tomato, namely aurea (aur) and high pigment1 (hp1), along with wild-type cultivar Micro-Tom (MT) under chilling stress. In tomato, aur is phytochrome-deficient mutant while hp1 is a phytochrome-sensitive mutant. The genotype-specific physiological, biochemical, and molecular responses under chilling stress in tomato mutants strongly validated phytochrome-mediated regulation of abiotic stress. ResultsHere, we demonstrate that phytochrome-sensitive mutant hp1 show improved performance compared to phytochrome-deficient mutant aur and wild-type MT plants under chilling stress. Interestingly, we noticed significant increase in several photosynthetic-related parameters in hp1 under chilling stress that include photosynthetic rate, stomatal conductance, stomatal aperture, transpiration rate, chlorophyll a and carotenoids. Whereas most parameters were negatively affected in aur and MT except a slight increase in carotenoids in MT plants under chilling stress. Further, we found that PSII quantum efficiency (Fv/Fm), PSII operating efficiency (Fq′/Fm′), and non-photochemical quenching (NPQ) were all positively regulated in hp1, which demonstrate enhanced photosynthetic performance of hp1 under stress. On the other hand, Fv/Fm and Fq′/Fm′ were decreased significantly in aur and wild-type plants. In addition, NPQ was not affected in MT but declined in aur mutant after chilling stress. Noticeably, the transcript analysis show that PHY genes which were previously reported to act as molecular switches in response to several abiotic stresses were mainly induced in hp1 and repressed in aur and MT in response to stress. As expected, we also found reduced levels of malondialdehyde (MDA), enhanced activities of antioxidant enzymes, and higher accumulation of protecting osmolytes (soluble sugars, proline, glycine betaine) which further elaborate the underlying tolerance mechanism of hp1 genotype under chilling stress. ConclusionOur findings clearly demonstrate that phytochrome-sensitive and phytochrome-deficient tomato mutants respond differently under chilling stress thereby regulating physiological, biochemical, and molecular responses and thus establish a strong link between phytochromes and their role in stress tolerance.

Simultaneous analyses of the rates of photoinduced charge separation and recombination between the excited flavin and tryptophans in some flavoproteins: Molecular dynamics simulation

Ultrafast transient absorption (TA) spectroscopy has been one of the most powerful experimental tools to study the mechanism of photoinduced electron transfer (ET) as in photosynthetic and flavin photoreceptor systems in plants. However, no work has been reported on their quantitative mechanisms. Apparent rates of charge separation (CS) from tryptophans (Trps) to the excited isoalloxazine (Iso*) and charge recombination (CR) from the produced ion pairs to Trps and Iso in the ground states are reported to be 0.25 ps and 3.2 ps in medium-chain acyl-CoA dehydrogenase (MCAD), and 0.15 ps and 6.6 ps in flavin mononucleotide binding protein (FMN-bp), obtained by an ultrafast TA method. The decays of the CS and CR processes were for the first time simultaneously analyzed with an ET theory and structures obtained by molecular dynamics simulation. MCAD and FMN-bp form a tetramer and dimer, respectively. The CS and CR rates of an individual donor and various related physical quantities were numerically obtained. It was found that both CS and CR rates were fastest from Trp166 among four Trps in MCAD and those from Trp106 among two Trps in FMN-bp. Logarithmic CS rates in MCAD were dependent on the donor–acceptor distance (Rc) with parabolic functions, while those of CR rates linearly decreased with Rc. Reasons why CS rates were faster than CR rates in both MCAD and FMN-bp were elucidated in terms of pre-exponential factors in the theory. The present method could be useful to understand the precise mechanisms of initial steps of biological functions of photoreceptors in plants.

Role of Arabidopsis BBX proteins in light signaling

Light regulates numerous aspects of plant growth and development like seed germination, seedling de-etiolation, pigment accumulation, cotyledon opening and flowering. Specific photoreceptors in plants sense different wavelengths of light and initiate signaling cascade leading to light-regulated responses. Between these photoreceptors and final response there are a web of transcription factors involved directly or indirectly in the signal transduction. B-Box (BBX) family of transcription factors is one of the largest family of light-regulated proteins acting as signal transducers in the light signaling pathway. B-box proteins are known for their role in light-mediated regulation of seed germination, hypocotyl elongation, flowering, circadian movements and stress tolerance. This review is an up to date compilation of the roles of BBX proteins in Arabidopsis in regulating physiological processes through red/far-red, blue and UV-B light signaling pathways in Arabidopsis. It also summarizes the roles of BBX proteins in modulating developmental pathways and stress-related signaling events in Arabidopsis and some crop species.

Influence of red light on the expression of genes on stomatal formation in maize seedlings

Red light significantly affects the expression of plant photoreceptor genes and influences stomatal development through crosstalk of the constitutive photomorphogenic 1–cryptochrome–phytochrome signaling pathway. When blue light was replaced with red light, the expression levels of ZmCry1, ZmPhyB1, ZmEPF2, and ZmEPFL9 were enhanced, whereas that of ZmCOP1 was restricted. Moreover, the expression levels of ZmSPCH and ZmMUTE were also enhanced, but they were generally lower than those under white light. Consequently, stomatal formation, which was determined by net photosynthesis, stomatal conductance, intercellular CO2 concentration, and transpiration rate, was inhibited through decreased stomatal index and stomatal density. We conclude that red light positively regulates EPFL9 in the intercellular signaling but reduces the positive regulation of blue light on COP1 and epidermal patterning factor 2 in the intracellular and intercellular signaling; therefore, though red light promotes the gene’s function on stomatal development of seedling maize, blue light maybe dominant to red light in seedling stage.

MYB30 Is a Key Negative Regulator of Arabidopsis Photomorphogenic Development That Promotes PIF4 and PIF5 Protein Accumulation in the Light.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants, and PHYTOCHROME-INTERACTING FACTORS (PIFs) are a group of basic helix-loop-helix family transcription factors that play central roles in repressing photomorphogenesis. Here, we report that MYB30, an R2R3-MYB family transcription factor, acts as a negative regulator of photomorphogenesis in Arabidopsis (Arabidopsis thaliana). We show that MYB30 preferentially interacts with the Pfr (active) forms of the phytochrome A (phyA) and phytochrome B (phyB) holoproteins and that MYB30 levels are induced by phyA and phyB in the light. It was previously shown that phytochromes induce rapid phosphorylation and degradation of PIFs upon R light exposure. Our current data indicate that MYB30 promotes PIF4 and PIF5 protein reaccumulation under prolonged R light irradiation by directly binding to their promoters to induce their expression and by inhibiting the interaction of PIF4 and PIF5 with the Pfr form of phyB. In addition, our data indicate that MYB30 interacts with PIFs and that they act additively to repress photomorphogenesis. In summary, our study demonstrates that MYB30 negatively regulates Arabidopsis photomorphogenic development by acting to promote PIF4 and PIF5 protein accumulation under prolonged R light irradiation, thus providing new insights into the complicated but delicate control of PIFs in the responses of plants to their dynamic light environment.

Shedding light on the chromatin changes that modulate shade responses.

Perception of vegetation proximity or plant shade informs of potential competition for resources by the neighboring vegetation. As vegetation proximity impacts on both light quantity and quality, perception of this cue by plant photoreceptors reprograms development to result in responses that allow plants to compete with the neighboring vegetation. Developmental reprogramming involves massive and rapid changes in gene expression, with the concerted action of photoreceptors and downstream transcription factors. Changes in gene expression can be modulated by epigenetic processes that alter chromatin compaction, influencing the accessibility and binding of transcription factors to regulatory elements in the DNA. However, little is known about the epigenetic regulation of plant responses to the proximity of other plants. In this manuscript, we review what is known about plant shade effects on chromatin changes at the cytological level, that is, changes in nuclear morphology and high order chromatin density. We address which are the specific histone post-transcriptional modifications that have been associated with changes in shade-regulated gene expression, such as histone acetylation and histone methylation. Furthermore, we explore the possible mechanisms that integrate shade signaling components and chromatin remodelers to settle epigenetic marks at specific loci. This review aims to be a starting point to understand how a specific environmental cue, plant shade, integrates with chromatin dynamics to implement the proper acclimation responses.

Plant photoreceptor absorption as a quantitative measure for plant photomorphogenic characteristics of incident spectra

Plant photoreceptor absorption as a quantitative measure for plant photomorphogenic characteristics of incident spectra

OPTOGENETICA: CONTROLLARE I NEURONI CON LA LUCE

Optogenetics is a booming technique in neuroscience that allows manipualtion of neuronal activity by light. Neuronal activation can be achieved by the activation of light-gated cation channels while neuronal inhibition relies on the activation of chloride pumps and channels. We have engineered a light-gated potassium (K+) channel (BLINK) by linking the plant photoreceptor module LOV2 to the viral channel Kcv. Upon illumination with blue light (450 nm), BLINK channel hyperpolarizes neurons to EK and this effect persists for several minutes even in the dark. Pilot experiments in a mouse pain model show that this protracted light-off activity can be successfully employed for long term pain relief after activation of the channel by a short pulse of light.

Photooligomerization Determines Photosensitivity and Photoreactivity of Plant Cryptochromes

Plant and non-plant species possess cryptochrome (CRY) photoreceptors to mediate blue light regulation of development or the circadian clock. The blue light-dependent homooligomerization of Arabidopsis CRY2 is a known early photoreaction necessary for its functions, but the photobiochemistry and function of light-dependent homooligomerization and heterooligomerization of cryptochromes, collectively referred to as CRY photooligomerization, have not been well established. Here, we show that photooligomerization is an evolutionarily conserved photoreaction characteristic of CRY photoreceptors in plants and some non-plant species. Our analyses of the kinetics of the forward and reverse reactions of photooligomerization of Arabidopsis CRY1 and CRY2 provide a previously unrecognized mechanism underlying the different photosensitivity and photoreactivity of these two closely related photoreceptors. We found that photooligomerization is necessary but not sufficient for the functions of CRY2, implying that CRY photooligomerization is presumably accompanied by additional function-empowering conformational changes. We further demonstrated that the CRY2–CRY1 heterooligomerization plays roles in regulating functions of Arabidopsis CRYs in vivo. Taken together, these results suggest that photooligomerization is an evolutionarily conserved mechanism determining the photosensitivity and photoreactivity of plant CRYs.

Regulation of Photomorphogenic Development by Plant Phytochromes.

Photomorphogenesis and skotomorphogenesis are two key events that control plant development, from seed germination to flowering and senescence. A group of wavelength-specific photoreceptors, E3 ubiquitin ligases, and various transcription factors work together to regulate these two critical processes. Phytochromes are the main photoreceptors in plants for perceiving red/far-red light and transducing the light signals to downstream factors that regulate the gene expression network for photomorphogenic development. In this review, we highlight key developmental stages in the life cycle of plants and how phytochromes and other components in the phytochrome signaling pathway play roles in plant growth and development.

Structural Basis of Design and Engineering for Advanced Plant Optogenetics.

In optogenetics, light-sensitive proteins are specifically expressed in target cells and light is used to precisely control the activity of these proteins at high spatiotemporal resolution. Optogenetics initially used naturally occurring photoreceptors to control neural circuits, but has expanded to include carefully designed and engineered photoreceptors. Several optogenetic constructs are based on plant photoreceptors, but their application to plant systems has been limited. Here, we present perspectives on the development of plant optogenetics, considering different levels of design complexity. We discuss how general principles of light-driven signal transduction can be coupled with approaches for engineering protein folding to develop novel optogenetic tools. Finally, we explore how the use of computation, networks, circular permutation, and directed evolution could enrich optogenetics.

Molecular shape under far‐red light and red light‐induced association of Arabidopsis phytochrome B

Phytochrome B (phyB) is a plant photoreceptor protein that regulates various photomorphogenic responses to optimize plant growth and development. PhyB exists in two photoconvertible forms: a red light-absorbing (Pr) and a far-red light-absorbing (Pfr) form. Therefore, to understand the mechanism of phototransformation, the structural characterization of full-length phyB in these two forms is necessary. Here, we report the molecular structure of Arabidopsisthaliana phyB in Pr form and the molecular properties of the Pfr form determined by small-angle X-ray scattering coupled with size-exclusion chromatography. In solution, the Pr form associated as a dimer with a radius of gyration of 50Å. The molecular shape was a crossed shape, in which the orientation of the photosensory modules differed from that in the crystal structure of dimeric photosensory module. PhyB exhibited structural reversibility in the Pfr-to-Pr phototransformation and thermal reversion from Pfr to Pr in the dark. In addition, Pfr only exhibited nonspecific association, which distinguished molecular properties of Pfr form from those of the inactive Pr form.

The Universally Conserved Residues Are Not Universally Required for Stable Protein Expression or Functions of Cryptochromes.

Universally conserved residues (UCRs) are invariable amino acids evolutionarily conserved among members of a protein family across diverse kingdoms of life. UCRs are considered important for stability and/or function of protein families, but it has not been experimentally examined systematically. Cryptochromes are photoreceptors in plants or light-independent components of the circadian clocks in mammals. We experimentally analyzed 51 UCRs of Arabidopsis cryptochrome 2 (CRY2) that are universally conserved in eukaryotic cryptochromes from Arabidopsis to human. Surprisingly, we found that UCRs required for stable protein expression of CRY2 in plants are not similarly required for stable protein expression of human hCRY1 in human cells. Moreover, 74% of the stably expressed CRY2 proteins mutated in UCRs retained wild-type-like activities for at least one photoresponses analyzed. Our finding suggests that the evolutionary mechanisms underlying conservation of UCRs or that distinguish UCRs from non-UCRs determining the same functions of individual cryptochromes remain to be investigated.

Production of Phytochromes by High-Cell-Density E. coli Fermentation

Phytochromes are important photoreceptors of plants, bacteria, and fungi responsive to light in the red and far-red spectrum. For increasing applications in basic research, synthetic biology, and materials sciences, it is required to recombinantly produce and purify phytochromes in high amounts. An ideal host organism for this purpose is E. coli due to its widespread use, fast growth, and ability for high-cell-density fermentation. Here, we describe the development of a generic platform for the production of phytochromes in E. coli that is compatible with high-cell-density fermentation. We exemplify our approach by the production of the photosensory domains of phytochrome B (PhyB) from A. thaliana and of the cyanobacterial phytochrome 1 (Cph1) from Synechocystis PCC 6803 in the multigram scale per 10 L fermentation run.

Heterogeneous Nuclear Ribonucleoprotein H1 Coordinates with Phytochrome and the U1 snRNP Complex to Regulate Alternative Splicing in Physcomitrella patens.

Plant photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although gene expression is modulated by photoreceptors at various levels, the regulatory mechanism at the pre-mRNA splicing step remains unclear. Alternative splicing, a widespread mechanism in eukaryotes that generates two or more mRNAs from the same pre-mRNA, is largely controlled by splicing regulators, which recruit spliceosomal components to initiate pre-mRNA splicing. The red/far-red light photoreceptor phytochrome participates in light-mediated splicing regulation, but the detailed mechanism remains unclear. Here, using protein-protein interaction analysis, we demonstrate that in the moss Physcomitrella patens, phytochrome4 physically interacts with the splicing regulator heterogeneous nuclear ribonucleoprotein H1 (PphnRNP-H1) in the nucleus, a process dependent on red light. We show that PphnRNP-H1 is involved in red light-mediated phototropic responses in P. patens and that it binds with higher affinity to the splicing factor pre-mRNA-processing factor39-1 (PpPRP39-1) in the presence of red light-activated phytochromes. Furthermore, PpPRP39-1 associates with the core component of U1 small nuclear RNP in P. patens Genome-wide analyses demonstrated the involvement of both PphnRNP-H1 and PpPRP39-1 in light-mediated splicing regulation. Our results suggest that phytochromes target the early step of spliceosome assembly via a cascade of protein-protein interactions to control pre-mRNA splicing and photomorphogenic responses.

Transmission of ultraviolet, visible and near-infrared solar radiation to plants within a seasonal snow pack

Sunlight is strongly attenuated by the snowpack, causing irradiance to decrease exponentially with depth. The strength of attenuation is wavelength dependent across the spectrum. Changes in received irradiance and its spectral composition are used by plants as cues for the timing of phenology, and it is known that at shallow depths in the snowpack there is sufficient light for plants to photosynthesize if conditions are otherwise favourable. The spectral composition of solar radiation under snow in the visible region was already determined in the 1970s using scanning spectroradiometers, but spectral attenuation within the ultraviolet region (UV-B 280–315 nm, UV-A 315–400 nm) has not been well characterised because it is difficult to measure. We measured vertical transects of spectral irradiance (290–900 nm) transmitted through a settled seasonal snowpack. The peak transmission of radiation was in the UV-A region in the upper centimetres of the snowpack and transmittance generally declined at longer wavelengths. Given the known action spectra of plant photoreceptors, these results illustrate the possibility that changing UV-A : visible and red : far-red radiation ratios under the snowpack may serve as spectral cues for plants; potentially priming plants for the less stable environment they experience following snowmelt. Array spectrometers open opportunities for rapid and continuous measurement of irradiance in challenging environments, e.g. beneath the snowpack, and capturing changing light conditions for plants. Future research is needed to couple the spectral transmittance of snowpacks differing in their longevity and crystal structure with measurements of the perception and response to radiation by plants under snow.

Plant photoreceptors and their signaling components compete for COP1 binding via VP peptide motifs

Plants sense different parts of the sun's light spectrum using distinct photoreceptors, which signal through the E3 ubiquitin ligase COP1. Here, we analyze why many COP1‐interacting transcription factors and photoreceptors harbor sequence‐divergent Val‐Pro (VP) motifs that bind COP1 with different binding affinities. Crystal structures of the VP motifs of the UV‐B photoreceptor UVR8 and the transcription factor HY5 in complex with COP1, quantitative binding assays, and reverse genetic experiments together suggest that UVR8 and HY5 compete for COP1. Photoactivation of UVR8 leads to high‐affinity cooperative binding of its VP motif and its photosensing core to COP1, preventing COP1 binding to its substrate HY5. UVR8–VP motif chimeras suggest that UV‐B signaling specificity resides in the UVR8 photoreceptor core. Different COP1–VP peptide motif complexes highlight sequence fingerprints required for COP1 targeting. The blue‐light photoreceptors CRY1 and CRY2 also compete with transcription factors for COP1 binding using similar VP motifs. Thus, our work reveals that different photoreceptors and their signaling components compete for COP1 via a conserved mechanism to control different light signaling cascades.