Sensors and controllers—for and from plants

A characteristic of plant life is the ability to rapidly acclimate tissue function and to change body plans in response to varying conditions. The remarkable degree of responsiveness of plants is underpinned by sophisticated signaling networks linked to pronounced dynamics in metabolism. The control and occurrence of those molecular changes are typically invisible and require nondestructive, live study methods to be understood in their context. Over the last decade in planta understanding of some of those events has been gained, and to a large proportion that progress was made through the development and deployment of in vivo fluorescent protein-based biosensing of plant signaling, physiology, metabolism, and development. More recently in vivo processes could even be rewired by optogenetic controllers, providing novel options to actively control plant cell and tissue functions. Strikingly, optogenetics, the research field focused on controlling cellular processes with light that has been revolutionizing bacterial, yeast, and animal research, has been largely relying on light switches that are derived from plant photoreceptors. Targeted engineering of optoswitches requires advanced insight into plant photobiology and photoreceptor mechanistic function. Both biosensors and optogenetic controllers rely on light and synthetic molecular switches for minimal invasive and specific monitoring or control of biological processes in vivo.

This focus issue aims to be a platform of reflection, looking back and ahead, on how biosensors and synthetic controllers are shaping plant research. While the issue has its emphasis on recent methodological advances, the individual articles paint a picture of how the technological innovations may be used to unlock a deeper understanding of how plants work. The articles jointly carry the vision of a quantitative, high-resolution understanding of plant processes, and of manipulating them in a specific manner across the scales in time and space.

Sadoine et al. provide an update on principles and engineering designs of fluorescent protein-based biosensors showing the coming-of-age of the field (Sadoine et al., 2021). Emerging mechanisms for detecting sensor readout, as well as remaining experimental constraints, such as silencing of sensor expression and optimization strategies, are highlighted.

Even though biosensors for Ca²⁺ were among the first fluorescent protein-based sensors to be constructed and used in plants, new designs, and sensor properties have continuously been developed. The current burst of activity in Ca²⁺ biosensing in plants reflects the importance of the roles that genetically encoded Ca²⁺ sensors have been playing in our current understanding of Ca²⁺ signaling. Grenzi et al. provide an update on recent innovations emphasizing strengths and limitations of ratiometric and intensiometric sensor variants to draw quantitative comparisons between free Ca²⁺ levels, and to characterize Ca²⁺ waves across tissues in long-distance signaling (Grenzi et al., 2021).

Sensing the activation of kinases in vivo is opening new avenues for an understanding of signaling cascades at the single-cell level. While in-gel kinase activity assays have been instrumental, their ability to resolve positional and temporal differences between cells is limited. Zhang et al. review approaches of kinase activity biosensing from its beginnings by different strategies, including activation- and translocation-based sensors (Zhang et al., 2021). Developments in the animal field and the recent introduction of MAP kinase and SnRK2 kinase biosensing in plant cells are highlighted as blueprints for dissecting the roles of the large diversity of plant kinases in the signaling networks of distinct plant cell types.

Analogous concepts as for Ca²⁺ signaling have been emerging for reactive oxygen species (ROS), not least since both messenger classes are linked through specific signaling proteins, such as the NADPH oxidases. Yet, dynamic and selective ROS biosensing remains challenging due to the diverse features of the different chemical species that strongly depend on the physiological context they are exposed to. While the first broadly used fluorescent protein-based biosensor for dynamic H₂O₂ monitoring, HyPer, was introduced 15 years ago (Belousov et al., 2006), a new variant, HyPer7, was recently constructed and applied in mammalian cells (Pak et al., 2020). In their Letter, Ugalde et al. contrast the properties of the new H₂O₂ sensor with the original HyPer variant and with roGFP2-Orp1 through empirical side-by-side comparisons in Arabidopsis (Ugalde et al., 2021). The data indicate the potential of HyPer7 as an addition to the repertoire of plant H₂O₂ sensors due to its ability to respond to particularly low H₂O₂ concentrations and its independence of the glutathione redox state.

An update on the current repertoire of biosensors for plant redox and energy physiology, including H₂O₂, glutathione redox potential, thioredoxin redox state, NAD⁺/NADH, NADPH and MgATP^2-, was recently included in the focus issue on Plant Redox Biology (Müller-Schüssele et al., 2021). A separate recent review focussed on biosensing of plant NAD- and NADP-based metabolism (Smith et al., 2021).

Several fluorescent protein-based biosensing concepts have been developed in animal systems for another central and redox-active signaling molecule, namely NO. By contrast, experience with those systems in plants is still lacking. In her letter, Safavi-Rizi (Safavi-Rizi, 2021) explores the potential and limitations of the different available biosensor concepts, highlighting geNOps (Eroglu et al., 2016) as a particularly promising candidate for implementation in plants. The letter provides a theoretical fundament for sensor selection and explores where successful biosensing may take the understanding of NO signaling to.

While redox-sensitive GFP (roGFP) biosensors have hitherto been used to explore the redox status of subcellular glutathione pools extensively in Arabidopsis, a study by Hipsch et al. introduces roGFP2-based biosensing in potato plants (Hipsch et al., 2021). The authors make use of the scalability of the sensing approach to assess the impact of abiotic stress factors, as exemplified for light intensity and temperature, at the whole plant level. At the same time, genetic targeting of the sensor enables monitoring of the glutathione redox potential exclusively in the plastid stroma. This approach represents an important methodological innovation because it (1) allows monitoring a major redox stress marker in intact crop plants, (2) reveals differential behavior between the leaves of a single plant, and (3) bridges in vivo biosensing with whole-plant phenotyping for conditions of relevance in the field.

pH has remained one of the most difficult cell physiological parameters to assess in living plants but has universal impact on cellular functions. Acidic compartments, such as those of the secretory pathway as well as the apoplast, require specific methodological attention. Many fluorescent protein variants are quenched in their fluorescence at low pH. There is strong evidence that pH regulation in the apoplast is centrally involved in plasma membrane energization, cell growth, and biotic interactions, which has made the apoplast a frontier for in vivo pH sensing. Moreau et al. review recent progress in live monitoring of pH in acidic cellular environments exemplifying the pH regulation underpinning cell elongation in the primary root (Moreau et al., 2021).

Even though the use of biosensors can open an entirely new dimension of spatiotemporal dynamics for a given analyte, the exclusive use of a single biosensor comes with the risk of being blinded for other changes. Given that any single signal or metabolic change is unlikely to occur in isolation within the cell physiological network, simultaneous monitoring of several parameters has the potential to provide insights at the systems level. Multiparameter analysis by multiplexing of different biosensors in a given plant can be achieved by separating sensor signals through their excitation and/or emission wavelengths. Strategies are reviewed by Waadt et al. (2021) who sketch out the current toolset for sensor multiplexing along with a roadmap of how sensor multiplexing will aid the mechanistic dissection of plant signaling networks.

Resentini et al. illustrate the power of using two biosensors in parallel by expressing a green and a red Ca²⁺ sensor variant (GCaMP6-210 and R-Geco1) in the cytosol and the ER of the same Arabidopsis line (Resentini et al., 2021). Monitoring Ca²⁺ transients at the cellular level on the one hand, and Ca²⁺ waves at the level of whole adult rosettes on the other hand, reveal that ER Ca²⁺ dynamics follow that of the cytosol. Since the ER appears to act as a Ca²⁺ sink modulating the cytosolic transient, the study raises important questions as to the identity and management of Ca²⁺ stores in plant cells, which are clearly different from those in mammalian cells.

For phytohormone-mediated regulation of plant development and acclimation, a precise understanding of spatiotemporal dynamics in the living plant is key. In vivo biosensing is particularly well-suited to resolve hormonal regulation and complements other emerging single-cell approaches. Balcerowicz et al. provide an update on phytohormone biosensing concepts for ABA, GA, auxin, and ethylene, which have recently gained momentum (Balcerowicz et al., 2021). Those biosensors bind directly to plant hormone molecules reporting on their concentrations in cells and tissues. The sensor designs are contrasted with more traditional but complementary reporters for hormone responses, at the level of transcription, translation, posttranslational modification, and protein turnover.

Related to hormonal regulation, tracking the developmental status of cells within a given tissue is a key challenge in understanding plant development. Guiziou et al. review synthetic biological switches, effectors, and related technologies, focusing on the customization of CRISPR/Cas9-, recombinase- and integrase-based approaches (Guiziou et al., 2021). While several of those molecular tools have been successfully implemented in yeast and animal systems their future adaptation to plants will not only allow to trace and record cell differentiation and tissue development but also to monitor and manipulate cell fate by engineering cell signaling regulation.

The most broadly used endogenous fluorescent probe for in vivo monitoring in plants is chlorophyll. Chlorophyll fluorescence analysis has been instrumental in delivering deep insights into photosynthetic function. Several of the techniques, however, require dark-acclimation prior to the light treatment with a given intensity. Those requirements limit experimental flexibility and may give rise to inaccuracies. Nedbal and Lazár developed a theoretical-experimental strategy to overcome current limitations based on the use of sinusoidally/pulsative modulated light of varying frequencies in combination with an ad-hoc developed mathematical model (Nedbal and Lazár, 2021). Proof-of-principle in the green alga Chlorella sorokiniana indicates novel perspectives for photosynthetic research in greenhouses or even in the field.

The introduction of light-gated ion channels of the opsin family over 15 years ago revolutionized fundamental neurobiological research and has given rise to biomedical applications (Deisseroth and Hegemann, 2017). This development is often referred to as “Optogenetics 1.0.” A second set of optogenetic tools referred to as “Optogenetics 2.0” uses photosensory modules from bacteria and plant photoreceptors to allow unprecedented spatiotemporal control over signaling and metabolic pathways (Christie and Zurbriggen, 2021). Those tools have been engineered to control a wide palette of cellular processes, including gene expression, protein stability, membrane receptors, and subcellular localization of proteins and organelles. Since plants need light to grow, the approaches developed for bacteria, yeast, and animal systems cannot be straightforwardly adopted as the optogenetic switches would be constitutively active under normal growth conditions. A first set of optogenetic tools to control gene expression and stomatal control have initiated a slow but steady expansion in the plant field, including strategies to avoid activation under ambient light (Christie and Zurbriggen, 2021).

Recently, the first opsin-based optogenetic approaches have been successfully introduced into plants. Zhou et al. review the engineering of all-trans-retinal production, a key breakthrough overcoming the lack of cofactors for opsins in plants that will enable the broad application of the light-gated channels in plants (Zhou et al., 2021). There is a plethora of natural microbial rhodopsins as well as their engineered variants with different sensitivities, ion selectivity and specificity, and color tunability. Those rhodopsins may be implemented to manipulate chemo-electric dynamics and thereby to control signaling networks that regulate physiological and developmental responses.

Molecular tools of the Optogenetics 2.0 generation integrate the photosensory properties of a bacterial or plant photoreceptor with an output module that performs or regulates a specific cellular function. To fully exploit and improve the color tunability, sensitivity and functionality, and applicability of optoswitches there is a need to expand the repertoire of available photosensory and output modules. Blain-Hartung et al. assess a class of cyanobacterial cyanobacteriochromes (CBCRs), which comprise a photosensory domain regulating a diguanylate cyclase domain (GGDEF domain) that catalyzes the production of the cyclic dinucleotide messenger (cyclic di-GMP; Blain-Hartung et al., 2021). The authors compare a broad set of natural and synthetic, rationally designed chimeric variants in terms of their photobiology/spectral reactivity (from UV up to the near IR), structure, and functionality. The study lays the foundation for future engineering and application of new optogenetic tools in plants, using CBCRs as a source of orthogonal photoreceptor modules with expanded applicability.

The articles in this focus issue jointly illustrate that we have come to a stage of advanced molecular and synthetic biology that enables the engineering of a wide range of optical intervention and monitoring strategies. In parallel, the required optical technology and instrumentation have rapidly evolved and become accessible to standard plant research laboratories. While several of the molecular components and concepts have their origin in green organisms, their engineering and exploitation in different biological systems (predominantly yeast and animal systems) has taken them to a degree of technical maturity that facilitates their targted customization and implementation in plants.

Much future potential may be expected from the combination of both, control and monitoring, by optical and genetic means. Optogenetic controllers and fluorescent protein-based biosensors are both able to address processes (1) quantitatively, (2) nondestructively in vivo, and (3) at high resolution in all four dimensions. Inducing signaling or metabolic changes by optogenetic or synthetic means with simultaneous monitoring of the consequences will transform living plants into experimental platforms to establish specific cause-and-effect relationships in the context of the intact system. The control-monitoring approach works across scales, from the single organelle, single-cell, tissues, organs, whole plants, up to plant populations and interactions with other organisms. In the future “controllomics” and “sensomics” approaches appear in reach to rewire and understand the dynamic networks of signal transduction and metabolism at any organizational scale. With those, a genuine systems-level understanding of plant biology in its in vivo context may be achievable along with fresh perspectives to develop novel plant traits and a new generation of green biotechnological applications.