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.

GMOCU: Digital Documentation, Management, and Biological Risk Assessment of Genetic Parts.

The continuous evolution of molecular biology and gene synthesis methods paired with an ever-increasing potential of synthetic biology approaches and genome engineering toolkits enables the rapid design of genetic bioparts and genetically modified organisms. Although various software solutions assist with specific design tasks and challenges, lab internal documentation and ensuring compliance with governmental regulations on biosafety assessment of the generated organisms remain the responsibility of individual academic researchers. This results in inconsistent and redundant documentation regimes and a significant time and labor burden. GMOCU (GMO documentation) is a standardized semi-automatic user-oriented software approach -written in Python and freely available- that unifies lab internal data documentation on genetic parts and genetically modified organisms (GMOs). It automatizes biological risk evaluations and maintains a shared up-to-date inventory of bioparts for team-wide data navigation and sharing. GMOCU further enables data export into customizable formats suitable for scientific publications, official biosafety documents, and the research community.

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems.

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

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.

Optogenetic Methods in Plant Biology.

Optogenetics is a technique employing natural or genetically engineered photoreceptors in transgene organisms to manipulate biological activities with light. Light can be turned on or off, and adjusting its intensity and duration allows optogenetic fine-tuning of cellular processes in a noninvasive and spatiotemporally resolved manner. Since the introduction of Channelrhodopsin-2 and phytochrome-based switches nearly 20 years ago, optogenetic tools have been applied in a variety of model organisms with enormous success, but rarely in plants. For a long time, the dependence of plant growth on light and the absence of retinal, the rhodopsin chromophore, prevented the establishment of plant optogenetics until recent progress overcame these difficulties. We summarize the recent results of work in the field to control plant growth and cellular motion via green light-gated ion channels and present successful applications to light-control gene expression with single or combined photoswitches in plants. Furthermore, we highlight the technical requirements and options for future plant optogenetic research.

Engineering and Implementation of Synthetic Molecular Tools in the Basidiomycete Fungus Ustilago maydis.

The basidiomycete Ustilago maydis is a well-characterized model organism for studying pathogen-host interactions and of great interest for a broad spectrum of biotechnological applications. To facilitate research and enable applications, in this study, three luminescence-based and one enzymatic quantitative reporter were implemented and characterized. Several dual-reporter constructs were generated for ratiometric normalization that can be used as a fast-screening platform for reporter gene expression, applicable to in vitro and in vivo detection. Furthermore, synthetic bidirectional promoters that enable bicisitronic expression for gene expression studies and engineering strategies were constructed and implemented. These noninvasive, quantitative reporters and expression tools will significantly widen the application range of biotechnology in U. maydis and enable the in planta detection of fungal infection.

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.

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.

Canonical strigolactones are not the major determinant of tillering but important rhizospheric signals in rice.

Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga-resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed-germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibitor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.

bHLH heterodimer complex variations regulate cell proliferation activity in the meristems of Arabidopsis thaliana.

Root, shoot, and lateral meristems are the main regions of cell proliferation in plants. It has been proposed that meristems might have evolved dedicated transcriptional networks to balance cell proliferation. Here, we show that basic helix-loop-helix (bHLH) transcription factor heterodimers formed by members of the TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) subclades are general regulators of cell proliferation in all meristems. Yet, genetics and expression analyses suggest specific functions of these transcription factors in distinct meristems, possibly due to their expression domains determining heterodimer complex variations within meristems, and to a certain extent to the absence of some of them in a given meristem. Target gene specificity analysis for heterodimer complexes focusing on the LONELY GUY gene targets further suggests differences in transcriptional responses through heterodimer diversification that could allow a common bHLH heterodimer complex module to contribute to cell proliferation control in multiple meristems.

Evaluation of existing guidelines for their adequacy for the food and feed risk assessment of genetically modified plants obtained through synthetic biology.

Synthetic biology (SynBio) is an interdisciplinary field at the interface of molecular engineering and biology aiming to develop new biological systems and impart new functions to living cells, tissues and organisms. EFSA has been asked by the European Commission to evaluate SynBio developments in agri-food with the aim of identifying the adequacy and sufficiency of existing guidelines for risk assessment and determine if updated guidance is needed. In this context, the GMO Panel has previously adopted an Opinion evaluating the SynBio developments in agri-food/feed and the adequacy and sufficiency of existing guidelines for the molecular characterisation and environmental risk assessment of genetically modified plants (GMPs) obtained through SynBio and reaching the market in the next decade. Complementing the above, in this Opinion, the GMO Panel evaluated the adequacy and sufficiency of existing guidelines for the food and feed risk assessment of GMPs obtained through SynBio. Using selected hypothetical case studies, the GMO Panel did not identify novel potential hazards and risks that could be posed by food and feed from GMPs obtained through current and near future SynBio approaches; considers that the existing guidelines are adequate and sufficient in some Synbio applications; in other cases, existing guidelines may be just adequate and hence need updating; areas needing updating include those related to the safety assessment of new proteins and the comparative analysis. The GMO Panel recommends that future guidance documents provide indications on how to integrate the knowledge available from the SynBio design and modelling in the food and feed risk assessment and encourages due consideration to be given to food and feed safety aspects throughout the SynBio design process as a way to facilitate the risk assessment of SynBio GMPs and reduce the amount of data required.

Organ-specific COP1 control of BES1 stability adjusts plant growth patterns under shade or warmth.

Under adverse conditions such as shade or elevated temperatures, cotyledon expansion is reduced and hypocotyl growth is promoted to optimize plant architecture. The mechanisms underlying the repression of cotyledon cell expansion remain unknown. Here, we report that the nuclear abundance of the BES1 transcription factor decreased in the cotyledons and increased in the hypocotyl in Arabidopsis thaliana under shade or warmth. Brassinosteroid levels did not follow the same trend. PIF4 and COP1 increased their nuclear abundance in both organs under shade or warmth. PIF4 directly bound the BES1 promoter to enhance its activity but indirectly reduced BES1 expression. COP1 physically interacted with the BES1 protein, promoting its proteasome degradation in the cotyledons. COP1 had the opposite effect in the hypocotyl, demonstrating organ-specific regulatory networks. Our work indicates that shade or warmth reduces BES1 activity by transcriptional and post-translational regulation to inhibit cotyledon cell expansion.

Genetically Encoded Biosensors for the Quantitative Analysis of Auxin Dynamics in Plant Cells.

Plants, as sessile organisms, possess complex and intertwined signaling networks to react and adapt their behavior toward different internal and external stimuli. Due to this high level of complexity, the implementation of quantitative molecular tools in planta remains challenging. Synthetic biology as an ever-growing interdisciplinary field applies basic engineering principles in life sciences. A plethora of synthetic switches, circuits, and even higher order networks has been implemented in different organisms, such as bacteria and mammalian cells, and facilitates the study of signaling and metabolic pathways. However, the application of such tools in plants lags behind, and thus only a few genetically encoded biosensors and switches have been engineered toward the quantitative investigation of plant signaling. Here, we present a protocol for the quantitative analysis of auxin signaling in Arabidopsis thaliana protoplasts. We implemented genetically encoded, ratiometric, degradation-based luminescent biosensors and applied them for studying auxin perception dynamics. For this, we utilized three different Aux/IAAs as sensor modules and analyzed their degradation behavior in response to auxin. Our experimental approach requires simple hardware and experimental reagents and can thus be implemented in every plant-related or cell culture laboratory. The system allows for the analysis of auxin perception and signaling aspects on various levels and can be easily expanded to other hormones, as for example strigolactones. In addition, the modular sensor design enables the implementation of sensor modules in a straightforward and time-saving approach.

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.

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.

Green Light-Controlled Gene Switch for Mammalian and Plant Cells.

The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.

Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors.

Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

A Protoplast-Based Bioassay to Quantify Strigolactone Activity in Arabidopsis Using StrigoQuant.

Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors may be also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, the genetically encoded, ratiometric sensor StrigoQuant was developed and allowed the quantification of the activity of a wide concentration range of SLs. StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL analogue GR24. We then show the recording of the luminescence signal and how the obtained data are processed and used to assess/determine SL perception.

The role of clock genes in sleep, stress and memory.

Circadian clock genes serve as the molecular basis for animals' ~24-h internal timekeeping. Clock gene expression inside and outside of the mammalian brain's circadian pacemaker (i.e. the SCN) integrates temporal information into a wealth of physiological processes. Ample data suggests that in addition to canonical cellular timekeeping functions, clock proteins also interact with proteins involved in cellular processes not related to timekeeping, including protein regulation and the interaction with other signaling mechanisms not directly linked to the regulation of circadian rhythms. Indeed, recent data suggests that clock genes outside the SCN are involved in fundamental brain processes such as sleep/wakefulness, stress and memory. The role of clock genes in these brain processes are complex and divers, influencing many molecular pathways and phenotypes. In this review, we will discuss recent work on the involvement of clock genes in sleep, stress, and memory. Moreover, we raise the controversial possibility that these functions may be under certain circumstances independent of their circadian timekeeping function.