A Monochromatically Excitable Green-Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent Protein.

Genetically encoded sensors enable quantitative imaging of analytes in live cells. Sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP can be susceptible to artifacts caused by changes in sensor levels or distribution in vivo. To develop intensiometric sensors with the capacity for ratiometric quantification, dual-FP Matryoshka sensors were generated by using a single cassette with a large Stokes shift (LSS) reference FP nested within the reporter FP (cpEGFP). Here, we present a genetically encoded calcium sensor that employs green apple (GA) Matryoshka technology by incorporating a newly designed red LSSmApple fluorophore. LSSmApple matures faster and provides an optimized excitation spectrum overlap with cpEGFP, allowing for monochromatic coexcitation with blue light. The LSS of LSSmApple results in improved emission spectrum separation from cpEGFP, thereby minimizing fluorophore bleed-through and facilitating imaging using standard dichroic and red FP (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new FP, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.

OzTracs: Optical Osmolality Reporters Engineered from Mechanosensitive Ion Channels.

Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive osmotic stresses with the help of mechanically gated ion channels and osmolyte transporters. Plant mechanosensitive ion channels have been shown to function in defense signaling. Here, we engineered genetically encoded osmolality sensors (OzTracs) by fusing fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, the OzTrac sensors reported osmolality changes as a proportional change in the emission ratio of the two fluorescent protein domains. Live-cell imaging revealed an accumulation of fluorescent sensors in internal aggregates, presumably derived from the endomembrane system. Thus, OzTrac sensors serve as osmolality-dependent reporters through an indirect mechanism, such as effects on molecular crowding or fluorophore solvation.

Shaping up: Recent advances in the study of plant calcium channels.

Calcium has long been recognized as a preeminent signaling molecule in plants with staggeringly diverse functions. The central mystery has therefore been how a single ion species can fulfill distinct functions while maintaining specificity and fidelity. Part of the answer lies in calcium being the most heavily controlled element in the cytosol, with dedicated transporters for sequestration into the apoplasm and intracellular stores. Controlled release of calcium into the cytosol by ion channels is the initiating step in signal transduction. Calcium-permeable ion channels are therefore important research targets. Recent studies have identified previously unknown channels, revealed atomic structures, and pinpointed locations of channels to specific cells and membranes. Here, we highlight key findings, transformative technologies, and pathways for further discovery.

Stress-associated developmental reprogramming in moss protonemata by synthetic activation of the common symbiosis pathway.

Symbioses between angiosperms and rhizobia or arbuscular mycorrhizal fungi are controlled through a conserved signaling pathway. Microbe-derived, chitin-based elicitors activate plant cell surface receptors and trigger nuclear calcium oscillations, which are decoded by a calcium/calmodulin-dependent protein kinase (CCaMK) and its target transcription factor interacting protein of DMI3 (IPD3). Genes encoding CCaMK and IPD3 have been lost in multiple non-mycorrhizal plant lineages yet retained among non-mycorrhizal mosses. Here, we demonstrated that the moss Physcomitrium is equipped with a bona fide CCaMK that can functionally complement a Medicago loss-of-function mutant. Conservation of regulatory phosphosites allowed us to generate predicted hyperactive forms of Physcomitrium CCaMK and IPD3. Overexpression of synthetically activated CCaMK or IPD3 in Physcomitrium led to abscisic acid (ABA) accumulation and ectopic development of brood cells, which are asexual propagules that facilitate escape from local abiotic stresses. We therefore propose a functional role for Physcomitrium CCaMK-IPD3 in stress-associated developmental reprogramming.

Sponging of glutamate at the outer plasma membrane surface reveals roles for glutamate in development.

Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals, and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra- and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center, number of lateral roots, vegetative growth, and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes.

Interdependence of a mechanosensitive anion channel and glutamate receptors in distal wound signaling.

Glutamate has dual roles in metabolism and signaling; thus, signaling functions must be isolatable and distinct from metabolic fluctuations, as seen in low-glutamate domains at synapses. In plants, wounding triggers electrical and calcium (Ca2+) signaling, which involve homologs of mammalian glutamate receptors. The hydraulic dispersal and squeeze-cell hypotheses implicate pressure as a key component of systemic signaling. Here, we identify the stretch-activated anion channel MSL10 as necessary for proper wound-induced electrical and Ca2+ signaling. Wound gene induction, genetics, and Ca2+ imaging indicate that MSL10 acts in the same pathway as the glutamate receptor­like proteins (GLRs). Analogous to mammalian NMDA glutamate receptors, GLRs may serve as coincidence detectors gated by the combined requirement for ligand binding and membrane depolarization, here mediated by stretch activation of MSL10. This study provides a molecular genetic basis for a role of mechanical signal perception and the transmission of long-distance electrical and Ca2+ signals in plants.

Designs, applications, and limitations of genetically encoded fluorescent sensors to explore plant biology.

The understanding of signaling and metabolic processes in multicellular organisms requires knowledge of the spatial dynamics of small molecules and the activities of enzymes, transporters, and other proteins in vivo, as well as biophysical parameters inside cells and across tissues. The cellular distribution of receptors, ligands, and activation state must be integrated with information about the cellular distribution of metabolites in relation to metabolic fluxes and signaling dynamics in order to achieve the promise of in vivo biochemistry. Genetically encoded sensors are engineered fluorescent proteins that have been developed for a wide range of small molecules, such as ions and metabolites, or to report biophysical processes, such as transmembrane voltage or tension. First steps have been taken to monitor the activity of transporters in vivo. Advancements in imaging technologies and specimen handling and stimulation have enabled researchers in plant sciences to implement sensor technologies in intact plants. Here, we provide a brief history of the development of genetically encoded sensors and an overview of the types of sensors available for quantifying and visualizing ion and metabolite distribution and dynamics. We further discuss the pros and cons of specific sensor designs, imaging systems, and sample manipulations, provide advice on the choice of technology, and give an outlook into future developments.

Using Genetically Encoded Fluorescent Biosensors for Quantitative In Vivo Imaging.

Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)-based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca2+ in Arabidopsis thaliana using a ratiometric genetically encoded Ca2+ indicator, MatryoshCaMP6s.

Author Correction: A calcium signalling network activates vacuolar K+ remobilization to enable plant adaptation to low-K environments.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A calcium signalling network activates vacuolar K+ remobilization to enable plant adaptation to low-K environments.

Potassium (K) is an essential nutrient, but levels of the free K ions (K+) in soil are often limiting, imposing a constant stress on plants. We have discovered a calcium (Ca2+)-dependent signalling network, consisting of two calcineurin B-like (CBL) Ca2+ sensors and a quartet of CBL-interacting protein kinases (CIPKs), which plays a key role in plant response to K+ starvation. The mutant plants lacking two CBLs (CBL2 and CBL3) were severely stunted under low-K conditions. Interestingly, the cbl2 cbl3 mutant was normal in K+ uptake but impaired in K+ remobilization from vacuoles. Four CIPKs-CIPK3, 9, 23 and 26-were identified as partners of CBL2 and CBL3 that together regulate K+ homeostasis through activating vacuolar K+ efflux to the cytoplasm. The vacuolar two-pore K+ (TPK) channels were directly activated by the vacuolar CBL-CIPK modules in a Ca2+-dependent manner, presenting a mechanism for the activation of vacuolar K+ remobilization that plays an important role in plant adaptation to K+ deficiency.

Phylogeny, historical biogeography, and diversification of angiosperm order Ericales suggest ancient Neotropical and East Asian connections.

Inferring interfamilial relationships within the eudicot order Ericales has remained one of the more recalcitrant problems in angiosperm phylogenetics, likely due to a rapid, ancient radiation. As a result, no comprehensive time-calibrated tree or biogeographical analysis of the order has been published. Here, we elucidate phylogenetic relationships within the order and then conduct time-dependent biogeographical and diversification analyses by using a taxon and locus-rich supermatrix approach on one-third of the extant species diversity calibrated with 23 macrofossils and two secondary calibration points. Our results corroborate previous studies and also suggest several new but poorly supported relationships. Newly suggested relationships are: (1) holoparasitic Mitrastemonaceae is sister to Lecythidaceae, (2) the clade formed by Mitrastemonaceae + Lecythidaceae is sister to Ericales excluding balsaminoids, (3) Theaceae is sister to the styracoids + sarracenioids + ericoids, and (4) subfamilial relationships with Ericaceae suggest that Arbutoideae is sister to Monotropoideae and Pyroloideae is sister to all subfamilies excluding Arbutoideae, Enkianthoideae, and Monotropoideae. Our results indicate Ericales began to diversify 110 Mya, within Indo-Malaysia and the Neotropics, with exchange between the two areas and expansion out of Indo-Malaysia becoming an important area in shaping the extant diversity of many families. Rapid cladogenesis occurred along the backbone of the order between 104 and 106 Mya. Jump dispersal is important within the order in the last 30 My, but vicariance is the most important cladogenetic driver of disjunctions at deeper levels of the phylogeny. We detect between 69 and 81 shifts in speciation rate throughout the order, the vast majority of which occurred within the last 30 My. We propose that range shifting may be responsible for older shifts in speciation rate, but more recent shifts may be better explained by morphological innovation.

Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.

Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

Constant change: dynamic regulation of membrane transport by calcium signalling networks keeps plants in tune with their environment.

Despite substantial variation and irregularities in their environment, plants must conform to spatiotemporal demands on the molecular composition of their cytosol. Cell membranes are the major interface between organisms and their environment and the basis for controlling the contents and intracellular organization of the cell. Membrane transport proteins (MTPs) govern the flow of molecules across membranes, and their activities are closely monitored and regulated by cell signalling networks. By continuously adjusting MTP activities, plants can mitigate the effects of environmental perturbations, but effective implementation of this strategy is reliant on precise coordination among transport systems that reside in distinct cell types and membranes. Here, we examine the role of calcium signalling in the coordination of membrane transport, with an emphasis on potassium transport. Potassium is an exceptionally abundant and mobile ion in plants, and plant potassium transport has been intensively studied for decades. Classic and recent studies have underscored the importance of calcium in plant environmental responses and membrane transport regulation. In reviewing recent advances in our understanding of the coding and decoding of calcium signals, we highlight established and emerging roles of calcium signalling in coordinating membrane transport among multiple subcellular locations and distinct transport systems in plants, drawing examples from the CBL-CIPK signalling network. By synthesizing classical studies and recent findings, we aim to provide timely insights on the role of calcium signalling networks in the modulation of membrane transport and its importance in plant environmental responses.

Tonoplast CBL-CIPK calcium signaling network regulates magnesium homeostasis in Arabidopsis.

Although Mg(2+) is essential for a myriad of cellular processes, high levels of Mg(2+) in the environment, such as those found in serpentine soils, become toxic to plants. In this study, we identified two calcineurin B-like (CBL) proteins, CBL2 and CBL3, as key regulators for plant growth under high-Mg conditions. The Arabidopsis mutant lacking both CBL2 and CBL3 displayed severe growth retardation in the presence of excess Mg(2+), implying elevated Mg(2+) toxicity in these plants. Unexpectedly, the cbl2 cbl3 mutant plants retained lower Mg content than wild-type plants under either normal or high-Mg conditions, suggesting that CBL2 and CBL3 may be required for vacuolar Mg(2+) sequestration. Indeed, patch-clamp analysis showed that the cbl2 cbl3 mutant exhibited reduced Mg(2+) influx into the vacuole. We further identified four CBL-interacting protein kinases (CIPKs), CIPK3, -9, -23, and -26, as functionally overlapping components downstream of CBL2/3 in the signaling pathway that facilitates Mg(2+) homeostasis. The cipk3 cipk9 cipk23 cipk26 quadruple mutant, like the cbl2 cbl3 double mutant, was hypersensitive to high-Mg conditions; furthermore, CIPK3/9/23/26 physically interacted with CBL2/3 at the vacuolar membrane. Our results thus provide evidence that CBL2/3 and CIPK3/9/23/26 constitute a multivalent interacting network that regulates the vacuolar sequestration of Mg(2+), thereby protecting plants from Mg(2+) toxicity.

Comparative phylogenomics of the CBL-CIPK calcium-decoding network in the moss Physcomitrella, Arabidopsis, and other green lineages.

Land plants have evolved a host of anatomical and molecular adaptations for terrestrial growth. Many of these adaptations are believed to be elaborations of features that were present in their algal-like progenitors. In the model plant Arabidopsis, 10 Calcineurin B-Like proteins (CBLs) function as calcium sensors and modulate the activity of 26 CBL-Interacting Protein Kinases (CIPKs). The CBL-CIPK network coordinates environmental responses and helps maintain proper ion balances, especially during abiotic stress. We identified and analyzed CBL and CIPK homologs in green lineages, including CBLs and CIPKs from charophyte green algae, the closest living relatives of land plants. Phylogenomic evidence suggests that the network expanded from a small module, likely a single CBL-CIPK pair, present in the ancestor of modern plants and algae. Extreme conservation of the NAF motif, which mediates CBL-CIPK physical interactions, among all identified CIPKs supports the interpretation of CBL and CIPK homologs in green algae and early diverging land plants as functionally linked network components. We identified the full complement of CBL and CIPK loci in the genome of Physcomitrella, a model moss. These analyses demonstrate the strong effects of a recent moss whole genome duplication: CBL and CIPK loci appear in cognate pairs, some of which appear to be pseudogenes, with high sequence similarity. We cloned all full-length transcripts from these loci and performed yeast two-hybrid analyses to demonstrate CBL-CIPK interactions and identify specific connections within the network. Using phylogenomics, we have identified three ancient types of CBLs that are discernible by N-terminal localization motifs and a "green algal-type" clade of CIPKs with members from Physcomitrella and Arabidopsis.