PHOSPHATASE 2A dephosphorylates PHYTOCHROME INTERACTING FACTOR3 to modulate photomorphogenesis in Arabidopsis.

The phytochrome (phy) family of sensory photoreceptors modulates developmental programs in response to ambient light. Phys also control gene expression in part by directly interacting with the bHLH class of transcription factors, PHYTOCHROME-INTERACTING FACTORS (PIFs), and inducing their rapid phosphorylation and degradation. Several kinases have been shown to phosphorylate PIFs and promote their degradation. However, the phosphatases that dephosphorylate PIFs are less understood. Here, we describe four regulatory subunits of the Arabidopsis (Arabidopsis thaliana) protein PHOSPHATASE 2A (PP2A) family (B'α, B'ß, B''α and B''ß) that interact with PIF3 in yeast two-hybrid, in vitro and in vivo assays. The pp2ab''αß and b''αß/b'αß mutants displayed short hypocotyls, while the overexpression of the B subunits induced longer hypocotyls compared to the wild type under red light. The light-induced degradation of PIF3 was faster in the b''αß/b'αß quadruple mutant compared to in the wild type. Consistently, immunoprecipitated PP2A A and B subunits directly dephosphorylated PIF3-MYC in vitro. RNA-seq analyses showed that B''α and B''ß alter global gene expression in response to red light. PIFs (PIF1, PIF3, PIF4 and PIF5) are epistatic to these B subunits in regulating hypocotyl elongation under red light. Collectively, these data show an essential function of PP2A in dephosphorylating PIF3 to modulate photomorphogenesis in Arabidopsis.

Shining Light on Plant Growth: Recent Insights into Phytochrome Interacting Factors.

Light serves as a pivotal environmental cue regulating various aspects of plant growth and development, including seed germination, seedling de-etiolation, and shade avoidance. Within this regulatory framework, the basic helix-loop-helix transcription factors known as PHYTOCHROME INTERACTING FACTORS (PIFs) play an essential role in orchestrating responses to light stimuli. Phytochromes, acting as red/far-red light receptors, initiate a cascade leading to the degradation of PIFs (except PIF7), thereby triggering transcriptional reprogramming to facilitate photomorphogenesis. Recent research has unveiled multiple post-translational modifications that regulate the abundance and/or activity of PIFs, including phosphorylation, dephosphorylation, ubiquitination, deubiquitination and SUMOylation. Moreover, intriguing findings indicate that PIFs can influence chromatin modifications. These include modulation of Histone 3 Lysine-9 acetylation (H3K9ac), as well as occupancy of histone variants such as H2A.Z (associated with gene repression) and H3.3 (associated with gene activation), thereby intricately regulating downstream gene expression in response to environmental cues. This review summarizes recent advances in understanding PIFs' role in regulating various signaling pathways with a major focus on photomorphogenesis.

Phytochrome phosphorylation in plant light signaling.

Plant phytochromes, renowned phosphoproteins, are red and far-red photoreceptors that regulate growth and development in response to light signals. Studies on phytochrome phosphorylation postulate that the N-terminal extension (NTE) and hinge region between N- and C-domains are sites of phosphorylation. Further studies have demonstrated that phosphorylation in the hinge region is important for regulating protein-protein interactions with downstream signaling partners, and phosphorylation in the NTE partakes in controlling phytochrome activity for signal attenuation and nuclear import. Moreover, phytochrome-associated protein phosphatases have been reported, indicating a role of reversible phosphorylation in phytochrome regulation. Furthermore, phytochromes exhibit serine/threonine kinase activity with autophosphorylation, and studies on phytochrome mutants with impaired or increased kinase activity corroborate that they are functional protein kinases in plants. In addition to the autophosphorylation, phytochromes negatively regulate PHYTOCHROME-INTERACTING FACTORs (PIFs) in a light-dependent manner by phosphorylating them as kinase substrates. Very recently, a few protein kinases have also been reported to phosphorylate phytochromes, suggesting new views on the regulation of phytochrome via phosphorylation. Using these recent advances, this review details phytochrome regulation through phosphorylation and highlights their significance as protein kinases in plant light signaling.

OptoREACT: Optogenetic Receptor Activation on Nonengineered Human T Cells.

Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.

The crosstalk of far-red energy and signaling defines the regulation of photosynthesis, growth, and flowering in tomatoes.

This study investigated the effect of light intensity and signaling on the regulation of far-red (FR)-induced alteration in photosynthesis. The low (LL: 440 µmol m-2 s-1) and high (HL: 1135 µmol m-2 s-1) intensity of white light with or without FR (LLFR: 545 µmol m-2 s-1 including 115 µmol m-2 s-1; HLFR: 1254 µmol m-2 s-1 + 140 µmol m-2 s-1) was applied on the tomato cultivar (Solanum Lycopersicon cv. Moneymaker) and mutants of phytochrome A (phyA) and phytochrome B (phyB1, and phyB2). Both light intensity and FR affected plant morphological traits, leaf biomass, and flowering time. Irrespective of genotype, flowering was delayed by LLFR and accelerated by HLFR compared to the corresponding light intensity without FR. In LLFR, a reduced energy flux through the electron transfer chain along with a reduced energy dissipation per reaction center improved the maximum quantum yield of PSII, irrespective of genotype. HLFR increased net photosynthesis and gas exchange properties in a genotype-dependent manner. FR-dependent regulation of hormones was affected by light signaling. It appeared that PHYB affected the levels of abscisic acid and salicylic acid while PHYA took part in the regulation of CK in FR-exposed plants. Overall, light intensity and signaling of FR influenced plants' photosynthesis and growth by altering electron transport, gas exchange, and changes in the level of endogenous hormones.

Bioproduction of porphyrins, phycobilins, and their proteins using microbial cell factories: engineering, metabolic regulations, challenges, and perspectives.

Porphyrins, phycobilins, and their proteins have abundant π-electrons and strongly absorb visible light, some of which bind a metal ion in the center. Because of the structural and optical properties, they not only play critical roles as an essential component in natural systems but also have attracted much attention as a high value specialty chemical in various fields, including renewable energy, cosmetics, medicines, and foods. However, their commercial application seems to be still limited because the market price of porphyrins and phycobilins is generally expensive to apply them easily. Furthermore, their petroleum-based chemical synthesis is energy-intensive and emits a pollutant. Recently, to replace petroleum-based production, many studies on the bioproduction of metalloporphyrins, including Zn-porphyrin, Co-porphyrin, and heme, porphyrin derivatives including chlorophyll, biliverdin, and phycobilins, and their proteins including hemoproteins, phycobiliproteins, and phytochromes from renewable carbon sources using microbial cell factories have been reported. This review outlines recent advances in the bioproduction of porphyrins, phycobilins, and their proteins using microbial cell factories developed by various microbial biotechnology techniques, provides well-organized information on metabolic regulations of the porphyrin metabolism, and then critically discusses challenges and future perspectives. Through these, it is expected to be able to achieve possible solutions and insights and to develop an outstanding platform to be applied to the industry in future research.

Conserved tyrosine in phytochromes controls the photodynamics through steric demand and hydrogen bonding capabilities.

Using ultrafast spectroscopy and site-specific mutagenesis, we demonstrate the central role of a conserved tyrosine within the chromophore binding pocket in the forward (Pr â†’ Pfr) photoconversion of phytochromes. Taking GAF1 of the knotless phytochrome All2699g1 from Nostoc as representative member of phytochromes, it was found that the mutations have no influence on the early (<30 ps) dynamics associated with conformational changes of the chromophore in the excited state. Conversely, they drastically impact the extended protein-controlled excited state decay (>100 ps). Thus, the steric demand, position and H-bonding capabilities of the identified tyrosine control the chromophore photoisomerization while leaving the excited state chromophore dynamics unaffected. In effect, this residue operates as an isomerization-steric-gate that tunes the excited state lifetime and the photoreaction efficiency by modulating the available space of the chromophore and by stabilizing the primary intermediate Lumi-R. Understanding the role of such a conserved structural element sheds light on a key aspect of phytochrome functionality and provides a basis for rational design of optimized photoreceptors for biotechnological applications.

Molecular mechanisms of shade tolerance in plants.

Shade tolerance is an ecological concept used in a wide range of disciplines, from plant physiology to landscaping or gardening. It refers to the strategy of some plants to persist and even thrive in environments with low light levels because of the shade produced by the vegetation proximity (e.g. in the understory). Shade tolerance influences the organization, structure, functioning, and dynamics of plant communities. However, little is known about its molecular and genetic basis. By contrast, there is a good understanding on how plants deal with the proximity of other plants, a divergent strategy used by most crops to respond to vegetation proximity. While generally shade-avoiding species strongly elongate in response to the proximity of other plants, shade-tolerant species do not. Here we review the molecular mechanisms that control the regulation of hypocotyl elongation in shade-avoiding species as a reference framework to understand shade tolerance. Comparative studies indicate that shade tolerance is implemented by components also known to regulate hypocotyl elongation in shade-avoiding species. These components, however, show differential molecular properties that explain how, in response to the same stimulus, shade-avoiding species elongate while shade-tolerant ones do not.

An Anatomy of Fungal Eye: Fungal Photoreceptors and Signalling Mechanisms.

Organisms have developed different features to capture or sense sunlight. Vertebrates have evolved specialized organs (eyes) which contain a variety of photosensor cells that help them to see the light to aid orientation. Opsins are major photoreceptors found in the vertebrate eye. Fungi, with more than five million estimated members, represent an important clade of living organisms which have important functions for the sustainability of life on our planet. Light signalling regulates a range of developmental and metabolic processes including asexual sporulation, sexual fruit body formation, pigment and carotenoid production and even production of secondary metabolites. Fungi have adopted three groups of photoreceptors: (I) blue light receptors, White Collars, vivid, cryptochromes, blue F proteins and DNA photolyases, (II) red light sensors, phytochromes and (III) green light sensors and microbial rhodopsins. Most mechanistic data were elucidated on the roles of the White Collar Complex (WCC) and the phytochromes in the fungal kingdom. The WCC acts as both photoreceptor and transcription factor by binding to target genes, whereas the phytochrome initiates a cascade of signalling by using mitogen-activated protein kinases to elicit its cellular responses. Although the mechanism of photoreception has been studied in great detail, fungal photoreception has not been compared with vertebrate vision. Therefore, this review will mainly focus on mechanistic findings derived from two model organisms, namely Aspergillus nidulans and Neurospora crassa and comparison of some mechanisms with vertebrate vision. Our focus will be on the way light signalling is translated into changes in gene expression, which influences morphogenesis and metabolism in fungi.

Generation of High-Value Genomic Resource in Rice: A "Subgenomic Library" of Low-Light Tolerant Rice Cultivar Swarnaprabha.

Rice is the major staple food crop for more than 50% of the world's total population, and its production is of immense importance for global food security. As a photophilic plant, its yield is governed by the quality and duration of light. Like all photosynthesizing plants, rice perceives the changes in the intensity of environmental light using phytochromes as photoreceptors, and it initiates a morphological response that is termed as the shade-avoidance response (SAR). Phytochromes (PHYs) are the most important photoreceptor family, and they are primarily responsible for the absorption of the red (R) and far-red (FR) spectra of light. In our endeavor, we identified the morphological differences between two contrasting cultivars of rice: IR-64 (low-light susceptible) and Swarnaprabha (low-light tolerant), and we observed the phenological differences in their growth in response to the reduced light conditions. In order to create genomic resources for low-light tolerant rice, we constructed a subgenomic library of Swarnaprabha that expedited our efforts to isolate light-responsive photoreceptors. The titer of the library was found to be 3.22 × 105 cfu/mL, and the constructed library comprised clones of 4-9 kb in length. The library was found to be highly efficient as per the number of recombinant clones. The subgenomic library will serve as a genomic resource for the Gramineae community to isolate photoreceptors and other genes from rice.

Seed Priming with Nanoparticles and 24-Epibrassinolide Improved Seed Germination and Enzymatic Performance of Zea mays L. in Salt-Stressed Soil.

Saline stress is one of the most critical abiotic stress factors that can lessen crops' productivity. However, emerging nanotechnology, nano-fertilizers, and developing knowledge of phytochromes can potentially mitigate the negative effects of saline stress on seed germination. Therefore, the aim of this study was to investigate the effects of seed priming either with zinc oxide nanoparticles (ZnO-NPs; 50 and 100 mg L-1) or 24-epibrassinolide (EBL; 0.2 and 0.4 µM) and their combinations on maize (Zea mays L.) grains sown in salt-stressed soil (50 and 100 mM NaCl). Saline stress treatments significantly affected all germination traits and chemical analysis of seeds as well as α-amylase activity. Compared to un-primed seeds, seed priming with ZnO-NPs or EBL and their combinations significantly increased the cumulative germination percentage, germination energy, imbibition rate, increase in grain weight, K+ content, and α-amylase activity, and significantly reduced germination time, days to 50% emergence, Na+ uptake, and Na+/K+ ratio of maize sown in salt-stressed-soil (50 or 100 mM NaCl). The combination of 100 mg ZnO-NPs L-1 + 0.2 µM EBL resulted in the highest improvements for most of the studied traits of maize seeds sown in salt-stressed soil in comparison to all other individual and combined treatments.

Phytochromes and flowering: legumes do it another way.

Phytochromes have a crucial role in the regulation of flowering in many plants, but the underlying molecular mechanisms vary among species. Recently, Lin et al. described a unique phytochrome A (phyA)-controlled photoperiodic flowering pathway in soybean (Glycine max), revealing a novel mechanism for photoperiodic regulation of flowering.

Evidence for evolutionary relationship between archaeplastidal and cyanobacterial phytochromes based on their chromophore pockets.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. In plants, phytochromes play a central role in growth and differentiation during the entire life cycle. Phytochromes of plants and other groups of archaeplastida have a common evolutionary origin in prokaryotes, but the exact prokaryotic origin is as yet uncertain. Two possibilities are presently discussed: either, archaeplastidal phytochromes arose from the last eukaryotic common ancestor (LECA) or they arose from the cyanobacterial endosymbiont that gave rise to plastids. We first constructed standard phylogenetic trees based on N-terminal protein sequences of the chromophore module. As usual, variation of algorithms and parameters led to different trees. A relationship between cyanobacteria and archaeplastida was observed in 7 out of 36 trees. The lack of consistency between results obtained from variation of parameters of tree constructions reflects the uncertainty of archaeplastidal origin. To gain more information about a possible cyanobacterial and archaeplastidal relationship, we performed phylogenetic studies based on the amino acids that line the chromophore pockets. These amino acids are highly conserved and could provide more accurate information about long evolutionary time scales, but the reduction of traits could also lead to insignificant results. From 30 selected chromophore-binding amino acids, 6 were invariant. The subsequent studies were thus based on the information dependent on 24 or fewer amino acid positions. Again, multiple trees were constructed to get information about the robustness of relationships. The very low number of information-containing traits resulted in low bootstrap values and many indistinguishable leaves. However, the major groups fungi, bacteria, cyanobacteria, and plants remained united. Without exception, cyanobacteria and archaeplastida were always closely linked. In this respect, the results were more robust than those of the classic approach, based on long contiguous sequences. We therefore consider cyanobacteria as the most likely origin of archaeplastidal phytochromes.

A red-green photochromic bacterial protein as a new contrast agent for improved photoacoustic imaging.

The GAF3 domain of the cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803, binding phycocyanobilin as a chromophore, shows photochromicity between two stable, green- and red-absorbing states, characterized by relatively high photoconversion yields. Using nanosecond-pulsed excitation by red or green light, respectively, and suitable cw photoconversion beams, we demonstrate that the light-modulatable photoacoustic waveforms arising from GAF3 can be easily distinguished from background signals originating from non-modulatable competitive absorbers and scattering media. It is demonstrated that this effect can be exploited to identify the position of the photochromic molecule by using as a phantom a cylindrical capillary tube filled with either a GAF3 solution or with an E.coli suspension overexpressing GAF3. These properties identify the high potential of GAF3 to be included in the palette of genetically encoded photochromic probes for photoacoustic imaging.

Benchmarking of Cph1 Mutants and DrBphP for Light-Responsive Phytochrome-Based Hydrogels with Reversibly Adjustable Mechanical Properties.

In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light-responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome-based hydrogels with an increased dynamic range for the storage modulus but a different light-response for the loss modulus compared to the original Cph1-based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1-R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems.

Upregulation of GLRs expression by light in Arabidopsis leaves.

BACKGROUND: Glutamate receptor-like (GLR) channels are plant homologs of iGluRs, animal ionotropic glutamate receptors which participate in neurotransmission. GLRs mediate plant adaptive processes and photomorphogenesis. Despite their contribution to light-dependent processes, signaling mechanisms that modulate GLR response to light remain unknown. Here we show that leaf expression of 7 out of 20 Arabidopsis GLRs is significantly up-regulated by monochromatic irradiation. RESULTS: Our data indicates that both red and blue light stimulate the expression of selected AtGLRs. Using a photosynthesis inhibitor and different irradiation regimes, we demonstrated that retrograde signaling from photosystem II is unlikely to be involved in light-dependent GLR up-regulation. Analysis of transcriptional patterns in mutants of key photoreceptors allowed us to observe that both phytochromes and cryptochromes are likely to be involved in the control of light-dependent up-regulation of AtGLR expression, with phytochromes playing a clearly dominating role in this process. CONCLUSIONS: In mature Arabidopsis leaves, phytochromes, assisted by cryptochromes, mediate light-driven transcriptional up-regulation of several genes encoding GLR proteins. Since GLRs are known to be involved in a wide range of plant developmental processes our results provide mechanistic insight into how light may influence plant growth and development.

Monoseeding Increases Peanut (Arachis hypogaea L.) Yield by Regulating Shade-Avoidance Responses and Population Density.

We aimed to elucidate the possible yield-increasing mechanisms through regulation of shade-avoidance responses at both physiological and molecular levels under monoseeding. Our results revealed that monoseeding decreased the main stem height but increased the main stem diameter and the number of branches and nodes compared to the traditional double- and triple-seeding patterns. The chlorophyll contents were higher under monoseeding than that under double- and triple-seeding. Further analysis showed that this, in turn, increased the net photosynthetic rate and reallocated higher levels of assimilates to organs. Monoseeding induced the expression patterns of Phytochrome B (Phy B) gene but decreased the expression levels of Phytochrome A (Phy A) gene. Furthermore, the bHLH transcription factors (PIF 1 and PIF 4) that interact with the phytochromes were also decreased under monoseeding. The changes in the expression levels of these genes may regulate the shade-avoidance responses under monoseeding. In addition, monoseeding increased pod yield at the same population density through increasing the number of pods per plant and 100-pod weight than double- and triple-seeding patterns. Thus, we inferred that monoseeding is involved in the regulation of shade-avoidance responsive genes and reallocating assimilates at the same population density, which in turn increased the pod yield.

Ultrafast Photoconversion Dynamics of the Knotless Phytochrome SynCph2.

The family of phytochrome photoreceptors contains proteins with different domain architectures and spectral properties. Knotless phytochromes are one of the three main subgroups classified by their distinct lack of the PAS domain in their photosensory core module, which is in contrast to the canonical PAS-GAF-PHY array. Despite intensive research on the ultrafast photodynamics of phytochromes, little is known about the primary kinetics in knotless phytochromes. Here, we present the ultrafast Pr ⇆ Pfr photodynamics of SynCph2, the best-known knotless phytochrome. Our results show that the excited state lifetime of Pr* (~200 ps) is similar to bacteriophytochromes, but much longer than in most canonical phytochromes. We assign the slow Pr* kinetics to relaxation processes of the chromophore-binding pocket that controls the bilin chromophore's isomerization step. The Pfr photoconversion dynamics starts with a faster excited state relaxation than in canonical phytochromes, but, despite the differences in the respective domain architectures, proceeds via similar ground state intermediate steps up to Meta-F. Based on our observations, we propose that the kinetic features and overall dynamics of the ultrafast photoreaction are determined to a great extent by the geometrical context (i.e., available space and flexibility) within the binding pocket, while the general reaction steps following the photoexcitation are most likely conserved among the red/far-red phytochromes.

Protein Kinase Activity of Phytochrome A Positively Correlates With Photoresponses in Arabidopsis.

Plant phytochromes are known as autophosphorylating serine/threonine protein kinases. However, the functional importance of their kinase activity is not fully elucidated. Previously, the kinase activity is shown to be necessary for the function of Avena sativa phytochrome A (AsphyA) using transgenic plants with mutants displaying reduced kinase activity, such as K411L and T418D. In this study, we isolated and analyzed two AsphyA mutants, K411R and T418V, that showed increased kinase activity. Transgenic phyA-201 plants with these mutants showed hypersensitive responses to far-red (FR) light, such as shorter hypocotyls and more expanded cotyledons than those of control plant (i.e., transgenic phyA-201 plant with wild-type AsphyA). Contrary to the mutants with reduced kinase activity, these mutants accelerated FR-induced phosphorylation and subsequent degradation of phytochrome-interacting factor 3 (PIF3) in Arabidopsis. Moreover, elongated hypocotyl 5 (HY5), a critical positive regulator of photoresponses in plants, accumulated in higher amounts in the transgenic plants under FR light than in the control plant. In addition, PIF1 degradation was accelerated in the transgenic plants. Consequently, the transgenic plants exhibit higher germination frequencies than the control plant. Collectively, our results demonstrate that the AsphyA mutants with increased kinase activity are hyperactive in plants, supporting a positive relationship between the kinase activity of phytochromes and photoresponses in plants.