Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s.

Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

Blue light promotes ascorbate synthesis by deactivating the PAS/LOV photoreceptor that inhibits GDP-L-galactose phosphorylase.

Ascorbate (vitamin C) is an essential antioxidant in fresh fruits and vegetables. To gain insight into the regulation of ascorbate metabolism in plants, we studied mutant tomato plants (Solanum lycopersicum) that produce ascorbate-enriched fruits. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a class of photoreceptor named PAS/LOV protein (PLP), which acts as a negative regulator of ascorbate biosynthesis. This trait was confirmed by CRISPR/Cas9 gene editing and further found in all plant organs, including fruit that accumulated 2 to 3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the 2 isoforms of GDP-L-galactose phosphorylase (GGP), known as the controlling step of the L-galactose pathway of ascorbate synthesis. The interaction with GGP occurred in the cytoplasm and the nucleus, but was abolished when PLP was truncated. These results were confirmed by a synthetic approach using an animal cell system, which additionally demonstrated that blue light modulated the PLP-GGP interaction. Assays performed in vitro with heterologously expressed GGP and PLP showed that PLP is a noncompetitive inhibitor of GGP that is inactivated after blue light exposure. This discovery provides a greater understanding of the light-dependent regulation of ascorbate metabolism in plants.

Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

Optogenetic control of gene expression in plants in the presence of ambient white light.

Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.

Lighting the light reactions of photosynthesis by means of redox-responsive genetically encoded biosensors for photosynthetic intermediates.

Oxygenic photosynthesis involves light and dark phases. In the light phase, photosynthetic electron transport provides reducing power and energy to support the carbon assimilation process. It also contributes signals to defensive, repair, and metabolic pathways critical for plant growth and survival. The redox state of components of the photosynthetic machinery and associated routes determines the extent and direction of plant responses to environmental and developmental stimuli, and therefore, their space- and time-resolved detection in planta becomes critical to understand and engineer plant metabolism. Until recently, studies in living systems have been hampered by the inadequacy of disruptive analytical methods. Genetically encoded indicators based on fluorescent proteins provide new opportunities to illuminate these important issues. We summarize here information about available biosensors designed to monitor the levels and redox state of various components of the light reactions, including NADP(H), glutathione, thioredoxin, and reactive oxygen species. Comparatively few probes have been used in plants, and their application to chloroplasts poses still additional challenges. We discuss advantages and limitations of biosensors based on different principles and propose rationales for the design of novel probes to estimate the NADP(H) and ferredoxin/flavodoxin redox poise, as examples of the exciting questions that could be addressed by further development of these tools. Genetically encoded fluorescent biosensors are remarkable tools to monitor the levels and/or redox state of components of the photosynthetic light reactions and accessory pathways. Reducing equivalents generated at the photosynthetic electron transport chain in the form of NADPH and reduced ferredoxin (FD) are used in central metabolism, regulation, and detoxification of reactive oxygen species (ROS). Redox components of these pathways whose levels and/or redox status have been imaged in plants using biosensors are highlighted in green (NADPH, glutathione, H2O2, thioredoxins). Analytes with available biosensors not tried in plants are shown in pink (NADP+). Finally, redox shuttles with no existing biosensors are circled in light blue. APX, ASC peroxidase; ASC, ascorbate; DHA, dehydroascorbate; DHAR, DHA reductase; FNR, FD-NADP+ reductase; FTR, FD-TRX reductase; GPX, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; MDA, monodehydroascorbate; MDAR, MDA reductase; NTRC, NADPH-TRX reductase C; OAA, oxaloacetate; PRX, peroxiredoxin; PSI, photosystem I; PSII: photosystem II; SOD, superoxide dismutase; TRX, thioredoxin.

The Red Edge: Bilin-Binding Photoreceptors as Optogenetic Tools and Fluorescence Reporters.

This review adds the bilin-binding phytochromes to the Chemical Reviews thematic issue "Optogenetics and Photopharmacology". The work is structured into two parts. We first outline the photochemistry of the covalently bound tetrapyrrole chromophore and summarize relevant spectroscopic, kinetic, biochemical, and physiological properties of the different families of phytochromes. Based on this knowledge, we then describe the engineering of phytochromes to further improve these chromoproteins as photoswitches and review their employment in an ever-growing number of different optogenetic applications. Most applications rely on the light-controlled complex formation between the plant photoreceptor PhyB and phytochrome-interacting factors (PIFs) or C-terminal light-regulated domains with enzymatic functions present in many bacterial and algal phytochromes. Phytochrome-based optogenetic tools are currently implemented in bacteria, yeast, plants, and animals to achieve light control of a wide range of biological activities. These cover the regulation of gene expression, protein transport into cell organelles, and the recruitment of phytochrome- or PIF-tagged proteins to membranes and other cellular compartments. This compilation illustrates the intrinsic advantages of phytochromes compared to other photoreceptor classes, e.g., their bidirectional dual-wavelength control enabling instant ON and OFF regulation. In particular, the long wavelength range of absorption and fluorescence within the "transparent window" makes phytochromes attractive for complex applications requiring deep tissue penetration or dual-wavelength control in combination with blue and UV light-sensing photoreceptors. In addition to the wide variability of applications employing natural and engineered phytochromes, we also discuss recent progress in the development of bilin-based fluorescent proteins.

Reversible Shielding and Immobilization of Liposomes and Viral Vectors by Tailored Antibody-Ligand Interactions.

Controlling the time and dose of nanoparticulate drug delivery by administration of small molecule drugs holds promise for efficient and safer therapies. This study describes a versatile approach of exploiting antibody-ligand interactions for the design of small molecule-responsive nanocarrier and nanocomposite systems. For this purpose, antibody fragments (scFvs) specific for two distinct small molecule ligands are designed. Subsequently, the surface of nanoparticles (liposomes or adeno-associated viral vectors, AAVs) is modified with these ligands, serving as anchor points for scFv binding. By modifying the scFvs with polymer tails, they can act as a non-covalently bound shielding layer, which is recruited to the anchor points on the nanoparticle surface and prevents interactions with cultured mammalian cells. Administration of an excess of the respective ligand triggers competitive displacement of the shielding layer from the nanoparticle surface and restores nanoparticle-cell interactions. The same principle is applied for developing hydrogel depots that can release integrated AAVs or liposomes in response to small molecule ligands. The liberated nanoparticles subsequently deliver their cargoes to cells. In summary, the utilization of different antibody-ligand interactions, different nanoparticles, and different release systems validates the versatility of the design concept described herein.

COLD REGULATED 27 and 28 are targets of CONSTITUTIVELY PHOTOMORPHOGENIC 1 and negatively affect phytochrome B signalling.

Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development. Phytochromes can sense the light environment and contribute to measuring day length; thereby, they allow plants to respond and adapt to changes in the ambient environment. Two well-characterized signalling pathways act downstream of phytochromes and link light perception to the regulation of gene expression. The CONSTITUTIVELY PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA-105 (COP1/SPA) E3 ubiquitin ligase complex and the PHYTOCHROME INTERACTING FACTORs (PIFs) are key components of these pathways and repress light responses in the dark. In light-grown seedlings, phytochromes inhibit COP1/SPA and PIF activity and thereby promote light signalling. In a yeast-two-hybrid screen for proteins binding to light-activated phytochromes, we identified COLD-REGULATED GENE 27 (COR27). COR27 and its homologue COR28 bind to phyA and phyB, the two primary phytochromes in seed plants. COR27 and COR28 have been described previously with regard to a function in the regulation of freezing tolerance, flowering and the circadian clock. Here, we show that COR27 and COR28 repress early seedling development in blue, far-red and in particular red light. COR27 and COR28 contain a conserved Val-Pro (VP)-peptide motif, which mediates binding to the COP1/SPA complex. COR27 and COR28 are targeted for degradation by COP1/SPA and mutant versions with a VP to AA amino acid substitution in the VP-peptide motif are stabilized. Overall, our data suggest that COR27 and COR28 accumulate in light but act as negative regulators of light signalling during early seedling development, thereby preventing an exaggerated response to light.

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.

Auxin methylation is required for differential growth in Arabidopsis.

Asymmetric auxin distribution is instrumental for the differential growth that causes organ bending on tropic stimuli and curvatures during plant development. Local differences in auxin concentrations are achieved mainly by polarized cellular distribution of PIN auxin transporters, but whether other mechanisms involving auxin homeostasis are also relevant for the formation of auxin gradients is not clear. Here we show that auxin methylation is required for asymmetric auxin distribution across the hypocotyl, particularly during its response to gravity. We found that loss-of-function mutants in Arabidopsis IAA CARBOXYL METHYLTRANSFERASE1 (IAMT1) prematurely unfold the apical hook, and that their hypocotyls are impaired in gravitropic reorientation. This defect is linked to an auxin-dependent increase in PIN gene expression, leading to an increased polar auxin transport and lack of asymmetric distribution of PIN3 in the iamt1 mutant. Gravitropic reorientation in the iamt1 mutant could be restored with either endodermis-specific expression of IAMT1 or partial inhibition of polar auxin transport, which also results in normal PIN gene expression levels. We propose that IAA methylation is necessary in gravity-sensing cells to restrict polar auxin transport within the range of auxin levels that allow for differential responses.

Phospho-mutant activity assays provide evidence for alternative phospho-regulation pathways of the transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR.

The key basic helix-loop-helix (bHLH) transcription factor in iron (Fe) uptake, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C-terminus, a regulatory domain of FIT, provides a trigger for FIT activation. Here, we use phospho-mutant activity assays and study phospho-mimicking and phospho-dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272. Phospho-mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT-activating partner bHLH039, promoter transactivation, and protein stability. Phospho-mimicking Tyr mutations of FIT disturb fit mutant plant complementation. Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.

Synthetic Switches and Regulatory Circuits in Plants.

Synthetic biology is an established but ever-growing interdisciplinary field of research currently revolutionizing biomedicine studies and the biotech industry. The engineering of synthetic circuitry in bacterial, yeast, and animal systems prompted considerable advances for the understanding and manipulation of genetic and metabolic networks; however, their implementation in the plant field lags behind. Here, we review theoretical-experimental approaches to the engineering of synthetic chemical- and light-regulated (optogenetic) switches for the targeted interrogation and control of cellular processes, including existing applications in the plant field. We highlight the strategies for the modular assembly of genetic parts into synthetic circuits of different complexity, ranging from Boolean logic gates and oscillatory devices up to semi- and fully synthetic open- and closed-loop molecular and cellular circuits. Finally, we explore potential applications of these approaches for the engineering of novel functionalities in plants, including understanding complex signaling networks, improving crop productivity, and the production of biopharmaceuticals.

Shade avoidance responses become more aggressive in warm environments.

When exposed to neighbour cues, competitive plants increase stem growth to reduce the degree of current or future shade. The aim of this work is to investigate the impact of weather conditions on the magnitude of shade avoidance responses in Arabidopsis thaliana. We first generated a growth rate database under controlled conditions and elaborated a model that predicts daytime hypocotyl growth as a function of the activity of the main photosensory receptors (phytochromes A and B, cryptochromes 1 and 2) in combination with light and temperature inputs. We then incorporated the action of thermal amplitude to account for its effect on selected genotypes, which correlates with the dynamics of the growth-promoting transcription factor PHYTOCHROME-INTERACTING FACTOR 4. The model predicted growth rate in the field with reasonable accuracy. Thus, we used the model in combination with a worldwide data set of current and future whether conditions. The analysis predicted enhanced shade avoidance responses as a result of higher temperatures due to the geographical location or global warming. Irradiance and thermal amplitude had no effects. These trends were also observed for our local growth rate measurements. We conclude that, if water and nutrients do not become limiting, warm environments enhance the shade avoidance response.

Transcriptional and Metabolic Profiling of Potato Plants Expressing a Plastid-Targeted Electron Shuttle Reveal Modulation of Genes Associated to Drought Tolerance by Chloroplast Redox Poise.

Water limitation represents the main environmental constraint affecting crop yield worldwide. Photosynthesis is a primary drought target, resulting in over-reduction of the photosynthetic electron transport chain and increased production of reactive oxygen species in plastids. Manipulation of chloroplast electron distribution by introducing alternative electron transport sinks has been shown to increase plant tolerance to multiple environmental challenges including hydric stress, suggesting that a similar strategy could be used to improve drought tolerance in crops. We show herein that the expression of the cyanobacterial electron shuttle flavodoxin in potato chloroplasts protected photosynthetic activities even at a pre-symptomatic stage of drought. Transcriptional and metabolic profiling revealed an attenuated response to the adverse condition in flavodoxin-expressing plants, correlating with their increased stress tolerance. Interestingly, 5-6% of leaf-expressed genes were affected by flavodoxin in the absence of drought, representing pathways modulated by chloroplast redox status during normal growth. About 300 of these genes potentially contribute to stress acclimation as their modulation by flavodoxin proceeds in the same direction as their drought response in wild-type plants. Tuber yield losses under chronic water limitation were mitigated in flavodoxin-expressing plants, indicating that the flavoprotein has the potential to improve major agronomic traits in potato.

Construction of a fusion enzyme for astaxanthin formation and its characterisation in microbial and plant hosts: A new tool for engineering ketocarotenoids.

The high-value ketocarotenoid astaxanthin, a natural red colorant with powerful antioxidant activity, is synthesised from ß-carotene by a hydroxylase and an oxygenase enzyme, which perform the addition of two hydroxyl and keto moieties, respectively. Several routes of intermediates, depending on the sequence of action of these enzymes, lead to the formation of astaxanthin. In the present study, the enzyme activities of 3, 3' ß-carotene hydroxylase (CRTZ) and 4, 4' ß-carotene oxygenase (CRTW) have been combined through the creation of "new to nature" enzyme fusions in order to overcome leakage of non-endogenous intermediates and pleotropic effects associated with their high levels in plants. The utility of flexible linker sequences of varying size has been assessed in the construction of pZ-W enzyme fusions. Frist, in vivo color complementation assays in Escherichia coli have been used to evaluate the potential of the fusion enzymes. Analysis of the carotenoid pigments present in strains generated indicated that the enzyme fusions only possess both catalytic activities when CRTZ is attached as the N-terminal module. Astaxanthin levels in E. coli cells were increased by 1.4-fold when the CRTZ and CRTW enzymes were fused compared to the individual enzymes. Transient expression in Nicotiana benthamiana was then performed in order to assess the potential of the fusions in a plant system. The production of valuable ketocarotenoids was achieved using this plant-based transient expression system. This revealed that CRTZ and CRTW, transiently expressed as a fusion, accumulated similar levels of astaxanthin compared to the expression of the individual enzymes whilst being associated with reduced ketocarotenoid intermediate levels (e.g. phoenicoxanthin, canthaxanthin and 3-OH-echinenone) and a reduced rate of leaf senescence after transformation. Therefore, the quality of the plant material producing the ketocarotenoids was enhanced due to a reduction in the stress induced by the accumulation of high levels of heterologous ketocarotenoid intermediates. The size of the linkers appeared to have no effect upon activity. The potential of the approach to production of valuable plant derived products is discussed.

Long-Day Photoperiod Enhances Jasmonic Acid-Related Plant Defense.

Agricultural crops are exposed to a range of daylengths, which act as important environmental cues for the control of developmental processes such as flowering. To explore the additional effects of daylength on plant function, we investigated the transcriptome of Arabidopsis (Arabidopsis thaliana) plants grown under short days (SD) and transferred to long days (LD). Compared with that under SD, the LD transcriptome was enriched in genes involved in jasmonic acid-dependent systemic resistance. Many of these genes exhibited impaired expression induction under LD in the phytochrome A (phyA), cryptochrome 1 (cry1), and cry2 triple photoreceptor mutant. Compared with that under SD, LD enhanced plant resistance to the necrotrophic fungus Botrytis cinerea This response was reduced in the phyA cry1 cry2 triple mutant, in the constitutive photomorphogenic1 (cop1) mutant, in the myc2 mutant, and in mutants impaired in DELLA function. Plants grown under SD had an increased nuclear abundance of COP1 and decreased DELLA abundance, the latter of which was dependent on COP1. We conclude that growth under LD enhances plant defense by reducing COP1 activity and enhancing DELLA abundance and MYC2 expression.

Multiple links between shade avoidance and auxin networks.

Auxin has emerged as a key player in the adjustment of plant morphology to the challenge imposed by variable environmental conditions. Shade-avoidance responses, including the promotion of stem and petiole growth, leaf hyponasty, and the inhibition of branching, involve an intimate connection between light and auxin signalling. Low activity of photo-sensory receptors caused by the presence of neighbouring vegetation enhances the activity of PHYTOCHROME INTERACTING FACTORs (PIFs), which directly promote the expression of genes involved in auxin biosynthesis, conjugation, transport, perception, and signalling. In seedlings, neighbour signals increase auxin levels in the foliage, which then moves to the stem, where it reaches epidermal tissues to promote growth. However, this model only partially accounts for shade-avoidance responses (which may also occur in the absence of increased auxin levels), and understanding the whole picture will require further insight into the functional significance of the multiple links between shade and auxin networks.

Synthetic strategies for plant signalling studies: molecular toolbox and orthogonal platforms.

Plants deploy a wide array of signalling networks integrating environmental cues with growth, defence and developmental responses. The high level of complexity, redundancy and connection between several pathways hampers a comprehensive understanding of involved functional and regulatory mechanisms. The implementation of synthetic biology approaches is revolutionizing experimental biology in prokaryotes, yeasts and animal systems and can likewise contribute to a new era in plant biology. This review gives an overview on synthetic biology approaches for the development and implementation of synthetic molecular tools and techniques to interrogate, understand and control signalling events in plants, ranging from strategies for the targeted manipulation of plant genomes up to the spatiotemporally resolved control of gene expression using optogenetic approaches. We also describe strategies based on the partial reconstruction of signalling pathways in orthogonal platforms, like yeast, animal and in vitro systems. This allows a targeted analysis of individual signalling hubs devoid of interconnectivity with endogenous interacting components. Implementation of the interdisciplinary synthetic biology tools and strategies is not exempt of challenges and hardships but simultaneously most rewarding in terms of the advances in basic and applied research. As witnessed in other areas, these original theoretical-experimental avenues will lead to a breakthrough in the ability to study and comprehend plant signalling networks.

Optogenetics for gene expression in mammalian cells.

Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.