Probing plant signal processing optogenetically by two channelrhodopsins.

Early plant responses to different stress situations often encompass cytosolic Ca increases, plasma membrane depolarization and the generation of reactive oxygen species. However, the mechanisms by which these signalling elements are translated into defined physiological outcomes are poorly understood. Here, to study the basis for encoding of specificity in plant signal processing, we used light-gated ion channels (channelrhodopsins).

K and pH homeostasis in plant cells is controlled by a synchronized K /H antiport at the plasma and vacuolar membrane.

Stomatal movement involves ion transport across the plasma membrane (PM) and vacuolar membrane (VM) of guard cells. However, the coupling mechanisms of ion transporters in both membranes and their interplay with Ca and pH changes are largely unclear. Here, we investigated transporter networks in tobacco guard cells and mesophyll cells using multiparametric live-cell ion imaging and computational simulations.

The GABA shunt contributes to ROS homeostasis in guard cells of Arabidopsis.

γ-Aminobutyric acid (GABA) accumulates rapidly under stress via the GABA shunt pathway, which has been implicated in reducing the accumulation of stress-induced reactive oxygen species (ROS) in plants. γ-Aminobutyric acid has been demonstrated to act as a guard-cell signal in Arabidopsis thaliana, modulating stomatal opening. Knockout of the major GABA synthesis enzyme Glutamate Decarboxylase 2 (GAD2) increases the aperture of gad2 mutants, which results in greater stomatal conductance and reduces water-use efficiency compared with wild-type plants.

Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta.

Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs).

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.

Tobacco leaf tissue rapidly detoxifies direct salt loads without activation of calcium and SOS signaling.

Salt stress is a major abiotic stress, responsible for declining agricultural productivity. Roots are regarded as hubs for salt detoxification, however, leaf salt concentrations may exceed those of roots. How mature leaves manage acute sodium chloride (NaCl) stress is mostly unknown.

Transporter networks can serve plant cells as nutrient sensors and mimic transceptor-like behavior.

Sensing of external mineral nutrient concentrations is essential for plants to colonize environments with a large spectrum of nutrient availability. Here, we analyzed transporter networks in computational cell biology simulations to understand better the initial steps of this sensing process. The networks analyzed were capable of translating the information of changing external nutrient concentrations into cytosolic H and Ca signals, two of the most ubiquitous cellular second messengers.

Advances and prospects of rhodopsin-based optogenetics in plant research.

Microbial rhodopsins have advanced optogenetics since the discovery of channelrhodopsins almost two decades ago. During this time an abundance of microbial rhodopsins has been discovered, engineered, and improved for studies in neuroscience and other animal research fields. Optogenetic applications in plant research, however, lagged largely behind.

Optogenetics for light control of biological systems.

Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space.

Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel ACR1.

Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (ACR1). In tobacco guard cells that express ACR1, blue- and green-light pulses elicit Cl and NO currents of -1 to -2 nA.

Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis.

Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function.

Extending the Anion Channelrhodopsin-Based Toolbox for Plant Optogenetics.

Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin ACR1 from the green alga . The growth of pollen tube can then be manipulated by localized green light illumination.

Optogenetic control of plant growth by a microbial rhodopsin.

While rhodopsin-based optogenetics has revolutionized neuroscience, poor expression of opsins and the absence of the essential cofactor all-trans-retinal has complicated the application of rhodopsins in plants. Here, we demonstrate retinal production in plants and improved rhodopsin targeting for green light manipulation of plant cells using the Guillardia theta light-gated anion channelrhodopsin GtACR1. Green light induces a massive increase in anion permeability and pronounced membrane potential changes when GtACR1 is expressed, enabling non-invasive manipulation of plant growth and leaf development.

An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca and H reveals new insights into ion signaling in plants.

Whereas the role of calcium ions (Ca ) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca - and pH-dynamics revealed interconnections previously not described.

Molecular and electrophysiological characterization of anion transport in Arabidopsis thaliana pollen reveals regulatory roles for pH, Ca and GABA.

We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth in Arabidopsis thaliana (Col-0). Patch-clamp whole-cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca ] ). We investigated the pollen-expressed proteins AtSLAH3, AtALMT12, AtTMEM16 and AtCCC as the putative anion transporters responsible for these currents.

An interconnection between tip-focused Ca and anion homeostasis controls pollen tube growth.

Plant reproduction is the basis for economically relevant food production. It relies on pollen tube (PTs) growth into the female flower organs for successful fertilization. The high cytosolic Ca concentration ([Ca]) at the PT tip is sensed by Ca-dependent protein kinases (CPKs) that in turn activate R- and S-type anion channels to control polar growth.

High V-PPase activity is beneficial under high salt loads, but detrimental without salinity.

The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H -ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PP hydrolysis and proton-pumping functions has yet to be dissected. For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging.

Tip-localized Ca -permeable channels control pollen tube growth via kinase-dependent R- and S-type anion channel regulation.

Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca ) concentration ([Ca ] ) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca ] orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca and anion imaging.

Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth.

Reactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress responses and development. To better understand the function of NAD(P)H oxidases in plant development, we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip.

Pollen tube growth regulation by free anions depends on the interaction between the anion channel SLAH3 and calcium-dependent protein kinases CPK2 and CPK20.

Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca(2+) concentration.

Global analysis of FRET-FLIM data in live plant cells.

This chapter describes the procedure for globally analyzing fluorescence lifetime imaging (FLIM) data for the observation and quantification of Förster resonance energy transfer (FRET) in live plant cells. The procedure is illustrated by means of a case study, for which plant protoplasts were transfected with different visible fluorescent proteins and subsequently imaged using two-photon excitation FLIM. Spatially resolved fluorescence lifetime images were obtained by application of global analysis using the program Glotaran, which is open-source and freely available software.

Calcium regulation of tip growth: new genes for old mechanisms.

We review the recent advances on Ca(2+) in tip-growing cells, with a special focus on pollen tubes. New genes for Ca(2+) pumps, channels and sensing proteins have been recently described, with special emphasis on cyclic nucleotide gated channels (CNGCs) and glutamate receptor-like channels (GLRs). We also review the current state of knowledge in what concerns Ca(2+) sensor and relay proteins, where the knowledge of the cell models is less advanced.

Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTERS 1 and 2: fructose and xylitol/H+ symporters in pollen and young xylem cells.

The genome of Arabidopsis thaliana contains six genes, AtPMT1 to AtPMT6 (Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTER 1-6), which form a distinct subfamily within the large family of more than 50 monosaccharide transporter-like (MST-like) genes. So far, only AtPMT5 [formerly named AtPLT5 (At3g18830)] has been characterized and was shown to be a plasma membrane-localized H(+)-symporter with broad substrate specificity. The characterization of AtPMT1 (At2g16120) and AtPMT2 (At2g16130), two other, almost identical, members of this transporter subfamily, are presented here.

The use of voltage-sensitive dyes to monitor signal-induced changes in membrane potential-ABA triggered membrane depolarization in guard cells.

The plant membrane potential reports on the activity of electrogenic plasma membrane transport processes. The membrane potential is widely used to report for early events associated with changes in light regime, hormone action or pathogen attacks. The membrane potentials of guard cells can be precisely measured with microelectrodes, but this technique is not well suited for rapid screens with large sample numbers.

Ca2+-dependent and -independent abscisic acid activation of plasma membrane anion channels in guard cells of Nicotiana tabacum.

Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca(2+) concentration. In Vicia faba, however, guard cell anion channels activate in a Ca(2+)-independent manner.