Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters.

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets.

Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells' ozone response.

Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1.

The Cell Fate Controlling CLE40 Peptide Requires CNGCs to Trigger Highly Localized Ca2+ Transients in Arabidopsis thaliana Root Meristems.

Communication between plant cells and their biotic environment largely depends on the function of plasma membrane localized receptor-like kinases (RLKs). Major players in this communication within root meristems are secreted peptides, including CLAVATA3/EMBRYO SURROUNDING REGION40 (CLE40). In the distal root meristem, CLE40 acts through the RLK ARABIDOPSIS CRINKLY4 (ACR4) and the leucine-rich repeat (LRR) RLK CLAVATA1 (CLV1) to promote cell differentiation.

Plant Cyclic Nucleotide-Gated Channels: New Insights on Their Functions and Regulation.

Recent advances of plant cyclic nucleotide-gated channels give new insight into their molecular functions focusing on regulation, subunit assembly, and phosphorylation.

Durotropic Growth of Pollen Tubes.

To reach the female gametophyte, growing pollen tubes must penetrate different tissues within the pistil, the female reproductive organ of a flower. Past research has identified various chemotropic cues that guide pollen tubes through the transmitting tract of the pistil, which represents the longest segment of its growth path. In addition, physical mechanisms also play a role in pollen tube guidance; however, these processes remain poorly understood.

A unified multi-kingdom Golden Gate cloning platform.

Assembling composite DNA modules from custom DNA parts has become routine due to recent technological breakthroughs such as Golden Gate modular cloning. Using Golden Gate, one can efficiently assemble custom transcription units and piece units together to generate higher-order assemblies. Although Golden Gate cloning systems have been developed to assemble DNA plasmids required for experimental work in model species, they are not typically applicable to organisms from other kingdoms.

Multiple cyclic nucleotide-gated channels coordinate calcium oscillations and polar growth of root hairs.

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip-focused Ca -gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide-gated channel family are required to maintain cytosolic Ca oscillations and the normal growth of root hairs.

Protoplast-Esculin Assay as a New Method to Assay Plant Sucrose Transporters: Characterization of AtSUC6 and AtSUC7 Sucrose Uptake Activity in Arabidopsis Col-0 Ecotype.

The best characterized function of sucrose transporters of the SUC family in plants is the uptake of sucrose into the phloem for long-distance transport of photoassimilates. This important step is usually performed by one specific SUC in every species. However, plants possess small families of several different SUCs which are less well understood.

Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2.

Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P-insensitive TPC1 from the model plant Arabidopsis thaliana.

AUX1-mediated root hair auxin influx governs SCF-type Ca signaling.

Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner.

A quantitative hypermorphic allele confers ectopic calcium flux and impairs cellular development.

The coordinated control of Ca signaling is essential for development in eukaryotes. Cyclic nucleotide-gated channel (CNGC) family members mediate Ca influx from cellular stores in plants (Charpentier et al., 2016; Gao et al.

BiFC Assay to Detect Calmodulin Binding to Plant Receptor Kinases.

Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain.

Calmodulin as a Ca2+-Sensing Subunit of Arabidopsis Cyclic Nucleotide-Gated Channel Complexes.

Ca2+ serves as a universal second messenger in eukaryotic signaling pathways, and the spatial and temporal patterns of Ca2+ concentration changes are determined by feedback and feed-forward regulation of the involved transport proteins. Cyclic nucleotide-gated channels (CNGCs) are Ca2+-permeable channels that interact with the ubiquitous Ca2+ sensor calmodulin (CaM). CNGCs interact with CaMs via diverse CaM-binding sites, including an IQ-motif, which has been identified in the C-termini of CNGC20 and CNGC12.

The Xanthomonas campestris pv. vesicatoria Type-3 Effector XopB Inhibits Plant Defence Responses by Interfering with ROS Production.

The bacterial pathogen Xanthomonas campestris pv. vesicatoria 85-10 (Xcv) translocates about 30 type-3 effector proteins (T3Es) into pepper plants (Capsicum annuum) to suppress plant immune responses. Among them is XopB which interferes with PTI, ETI and sugar-mediated defence responses, but the underlying molecular mechanisms and direct targets are unknown so far.

The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix.

Two-pore channels (TPCs) constitute a family of intracellular cation channels with diverse permeation properties and functions in animals and plants. In the model plant Arabidopsis, the vacuolar cation channel TPC1 is involved in propagation of calcium waves and in cation homeostasis. Here, we discovered that the dimerization of a predicted helix within the carboxyl-terminus (CTH) is essential for the activity of TPC1.

The phosphoinositide PI(3,5)P₂ mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells.

Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions.

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1.

The cell growth-promoting peptide phytosulfokine (PSK) is perceived by leucine-rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca(2+) sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto- and transphosphorylation.

An IQ domain mediates the interaction with calmodulin in a plant cyclic nucleotide-gated channel.

Cyclic nucleotide-gated channels (CNGCs) form non-selective cation entry pathways regulated by calmodulin (CaM), a universal Ca(2+) sensor in eukaryotes. Although CaM binding has been shown to be important for Ca(2+)-dependent feedback regulation of CNGC activity, the CaM-binding properties of these channels have been investigated in a few cases only. We show that CNGC20 from Arabidopsis thaliana binds CaM in a Ca(2+)-dependent manner and interacts with all AtCaM isoforms but not with the CaM-like proteins CML8 and CML9.

An N-terminal dileucine motif directs two-pore channels to the tonoplast of plant cells.

Two-pore channels (TPCs) constitute a family of endolysosomal cation channels with functions in Ca²⁺ signaling. We used a mutational analysis to investigate the role of channel domains for the trafficking of the Arabidopsis TPC1 to the tonoplast, a process that is generally not well understood in plants. The results show that the soluble C-terminus was not essential for targeting but for channel function, while further C-terminal truncations of two or more transmembrane domains impaired protein trafficking.

Differential contribution of EF-hands to the Ca²⁺-dependent activation in the plant two-pore channel TPC1.

Two-pore channels (TPC) have been established as components of calcium signalling networks in plants and animals. In plants, TPC1 in the vacuolar membrane is gated open upon binding of calcium in a voltage-dependent manner. Here, we analyzed the molecular mechanism of the Ca²⁺-dependent activity of TPC1 from Arabidopsis thaliana, using site-directed mutagenesis of its two canonical EF-hands.

Novel PSI domains in plant and animal H+-inositol symporters.

Inositols are indispensable components of cellular signaling molecules, and impaired cytoplasmic inositol concentrations affect cellular development. Although most cells can synthesize inositol de novo, plasma membrane-localized inositol uptake systems are indispensable for normal development. Here, we present in-depth functional analyses of plasma membrane-localized H(+)-inositol symporters from human and from the higher plant Arabidopsis thaliana.

Salt-dependent regulation of a CNG channel subfamily in Arabidopsis.

Background: In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction.

Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses.

The putative two-pore Ca(2+) channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co-localizes with the K(+)-selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca(2+)-activated slow vacuolar (SV) currents, which were increased in AtTPC1-over-expressing Arabidopsis compared to the wild-type.

Arabidopsis INOSITOL TRANSPORTER2 mediates H+ symport of different inositol epimers and derivatives across the plasma membrane.

Of the four genes of the Arabidopsis (Arabidopsis thaliana) INOSITOL TRANSPORTER family (AtINT family) so far only AtINT4 has been described. Here we present the characterization of AtINT2 and AtINT3. cDNA sequencing revealed that the AtINT3 gene is incorrectly spliced and encodes a truncated protein of only 182 amino acids with four transmembrane helices.

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.