Kai Robert Konrad

Reactive oxygen species (ROS) have been implicated in multiple signaling processes in plants, but the underlying mechanisms and roles remain enigmatic. In this study, we developed a method of live imaging of apoplastic ROS at the root surface. Distinct signals, including auxin, extracellular adenosine triphosphate, and rapid alkalinization factor 1 peptide, induce cytosolic calcium transients and apoplastic ROS bursts. Genetic and optogenetic manipulations of Arabidopsis identified calcium transients as necessary and sufficient for ROS bursts through activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases RBOHC and RBOHF. Apoplastic ROS bursts are not required, but they do limit gravity-induced root bending. Root bending is sensed by the stretch-activated calcium channel MCA1, leading to NADPH oxidase activation. The resulting ROS production stiffens cell walls to facilitate soil penetration. Apoplastic ROS thus provides a means to balance tissue flexibility and stiffness to navigate soil.

Reactive oxygen species (ROS) have been implicated repeatedly in multiple signaling processes in plants but the underlying mechanisms and roles remain enigmatic. Here, we developed live imaging of apoplastic ROS at the root surface. Different signals, including auxin, extracellular ATP and RALF1 peptide, all induce cytosolic calcium transients and apoplastic ROS bursts. Genetic and optogenetic manipulations identified calcium transients as necessary and sufficient for ROS bursts via activation of NADPH oxidases RBOHC and RBOHF. Apoplastic ROS bursts are not required but rather limit the gravity-induced root bending. Root bending is sensed by stretch-activated calcium channel MCA1 leading to NADPH oxidase activation at the stretched side. The resulting ROS production stiffens cell wall for better soil penetration. Apoplastic ROS thus provides a means to balance tissue flexibility and stiffness to efficiently navigate soil.

Early plant responses to different stress situations often encompass cytosolic Ca²⁺ increases, plasma membrane depolarization and the generation of reactive oxygen species^1–3. 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). We developed a genetically engineered channelrhodopsin variant called XXM 2.0 with high Ca²⁺ conductance that enabled triggering cytosolic Ca²⁺ elevations in planta. Plant responses to light-induced Ca²⁺ influx through XXM 2.0 were studied side by side with effects caused by an anion efflux through the light-gated anion channelrhodopsin ACR1 2.0⁴. Although both tools triggered membrane depolarizations, their activation led to distinct plant stress responses: XXM 2.0-induced Ca²⁺ signals stimulated production of reactive oxygen species and defence mechanisms; ACR1 2.0-mediated anion efflux triggered drought stress responses. Our findings imply that discrete Ca²⁺ signals and anion efflux serve as triggers for specific metabolic and transcriptional reprogramming enabling plants to adapt to particular stress situations. Our optogenetics approach unveiled that within plant leaves, distinct physiological responses are triggered by specific ion fluxes, which are accompanied by similar electrical signals.

Summary: 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 Ca²⁺ 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. K⁺ and anion fluxes at both, PM and VM, affected H⁺ and Ca²⁺, as changes in extracellular KCl or KNO₃ concentrations were accompanied by cytosolic and vacuolar pH shifts and changes in [Ca²⁺]_cyt and the membrane potential. At both membranes, the K⁺ transporter networks mediated an antiport of K⁺ and H⁺. By contrast, net transport of anions was accompanied by parallel H⁺ transport, with differences in transport capacity for chloride and nitrate. Guard and mesophyll cells exhibited similarities in K⁺/H⁺ transport but cell type‐specific differences in [H⁺]_cyt and pH‐dependent [Ca²⁺]_cyt signals. Computational cell biology models explained mechanistically the properties of transporter networks and the coupling of transport across the PM and VM. Our integrated approach indicates fundamental principles of coupled ion transport at membrane sandwiches to control H⁺/K⁺ homeostasis and points to transceptor‐like Ca²⁺/H⁺‐based ion signaling in plant cells.

Summary: 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. Here, we found that the additional loss of GAD1 , GAD4 , and GAD5 in gad2 leaves increased GABA deficiency but abolished the more open stomatal pore phenotype of gad2 , which we link to increased cytosolic calcium (Ca ²⁺ ) and ROS accumulation in gad1/2/4/5 guard cells. Compared with wild‐type and gad2 plants, glutamate was ineffective in closing gad1/2/4/5 stomatal pores, whereas lowering apoplastic calcium, applying ROS inhibitors or complementation with GAD2 reduced gad1/2/4/5 guard‐cell ROS, restored the gad2 ‐like greater stomatal apertures of gad1/2/4/5 beyond that of wild‐type. We conclude that GADs are important contributors to ROS homeostasis in guard cells likely via a Ca ²⁺ ‐mediated pathway. As such, this study reveals greater complexity in GABA's role as a guard‐cell signal and the interactions it has with other established signals.

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 FRET (Förster resonance energy transfer)-based reporter that visualizes the conformational changes in Ca²⁺-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca²⁺-sensitivities, highly Ca²⁺-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca²⁺-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca²⁺ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca²⁺ changes, pointing to the isoform-specific Ca²⁺-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca²⁺ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca²⁺ decoding in a multitude of plant developmental and stress responses.

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.

Changes in cytosolic calcium concentration are among the earliest reactions to a multitude of stress cues. Whereas a plethora of calcium-permeable channels may generate distinct calcium signatures and contribute to response specificities, the mechanisms by which calcium signatures are decoded is poorly understood. Here we develop a genetically encoded, FRET-based reporter that visualizes the conformational change of calcium-dependent protein kinases (CDPKs/CPKs), preceding kinase activation, for calcium-dependent AtCPK21 and calcium-independent AtCPK23. In pollen tubes, naturally displaying a physiological calcium range, CPK21-FRET, but not CPK23-FRET, report activity oscillations with similar features to cytosolic calcium, suggesting an isoform-specific calcium dependency and reversibility of the conformational change. In guard cells CPK21-FRET identifies CPK21 as a decoder of signal-specific calcium signatures in response to ABA and flg22. Based on this data, CDPK-FRET stands as a novel approach for tackling real-time live-cell calcium decoding in a multitude of plant developmental and stress responses.

Polar growth and guidance of pollen tubes (PTs) is regulated by chemical and electrical signaling cues. Central regulators controlling cellular protrusion of PTs tip-focused Ca ²⁺ and H ⁺ cytosolic gradients and ion fluxes depend, but also feedback, on membrane voltage ( V _m ). However, the functional relationship between V _m , Ca ²⁺ , and H ⁺ dynamics is largely unclear. We carried out parallel live-cell Ca ²⁺ and H ⁺ -imaging together with V _m recordings followed by cross-correlation analysis to study their interconnection in tobacco PTs. Upon electrode impalement at the tip or shank, PT growth ceased but sustained oscillatory behavior of all three signals continued and remained mostly in phase, although shifted in time. The Ca ²⁺ oscillations were largely restricted to the apex, temporally leading the H ⁺ increases by ∼18 s, whereas H ⁺ tip-focused peaks spread into the shank. Voltage recordings in the shank (50–80 μm behind the tip) revealed transient bell-shaped depolarizations that correlated strongly with H ⁺ but not Ca ²⁺ oscillations. In contrast, V _m oscillations readout at the tip correlated equally with tip Ca ²⁺ - and H ⁺ -signals, having action potential-like appearances that were approximately five times faster compared with those in the shank. Our multiparameter readout approach identified distinct relationships of chemical and electric signals along PTs, where at the tip changes in both Ca ²⁺ and H ⁺ correlated with V _m dynamics, whereas at the shank only H ⁺ correlated with V _m . We propose that spatially confined chemo-electric coupling support the manifestation of propagating voltage and pH waves along nongrowing PTs, which are preceded by short-lasting but high amplitude tip Ca ²⁺ spikes.

Summary: 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. To analyze the mechanisms for NaCl redistribution in leaves, salt was infiltrated into intact tobacco leaves. It initiated pronounced osmotically‐driven leaf movements. Leaf downward movement caused by hydro‐passive turgor loss reached a maximum within 2 h. Salt‐driven cellular water release was accompanied by a transient change in membrane depolarization but not an increase in cytosolic calcium ion (Ca²⁺) level. Nonetheless, only half an hour later, the leaves had completely regained turgor. This recovery phase was characterized by an increase in mesophyll cell plasma membrane hydrogen ion (H⁺) pumping, a salt uptake‐dependent cytosolic alkalization, and a return of the apoplast osmolality to pre‐stress levels. Although, transcript numbers of abscisic acid‐ and Salt Overly Sensitive pathway elements remained unchanged, salt adaptation depended on the vacuolar H⁺/Na⁺‐exchanger NHX1. Altogether, tobacco leaves can detoxify sodium ions (Na⁺) rapidly even under massive salt loads, based on pre‐established posttranslational settings and NHX1 cation/H⁺ antiport activity. Unlike roots, signaling and processing of salt stress in tobacco leaves does not depend on Ca²⁺ signaling.