Potassium (K +)channel function is fundamental to many physiological processes. However, components and mechanisms regulating the activity of plant K + channels remain poorly understood. Here, we show that the calcium (Ca 2+ ) sensor CBL4 together with the interacting protein kinase CIPK6 modulates the activity and plasma membrane (PM) targeting of the K + channel AKT2 from Arabidopsis thaliana by mediating translocation of AKT2 to the PM in plant cells and enhancing AKT2 activity in oocytes. Accordingly, akt2, cbl4 and cipk6 mutants share similar developmental and delayed flowering phenotypes. Moreover, the isolated regulatory C-terminal domain of CIPK6 is sufficient for mediating CBL4-and Ca 2+-dependent channel translocation from the endoplasmic reticulum membrane to the PM by a novel targeting pathway that is dependent on dual lipid modifications of CBL4 by myristoylation and palmitoylation. Thus, we describe a critical mechanism of ion-channel regulation where a Ca 2+ sensor modulates K + channel activity by promoting a kinase interaction-dependent but phosphorylation-independent translocation of the channel to the PM.

The plant hormone auxin (indole-3-acetic acid, IAA) has a crucial role in plant development. Its spatiotemporal distribution is controlled by a combination of biosynthetic, metabolic and transport mechanisms. Four families of auxin transporters have been identified that mediate transport across the plasma or endoplasmic reticulum membrane. Here we report the discovery and the functional characterization of the first vacuolar auxin transporter. We demonstrate that WALLS ARE THIN1 (WAT1), a plant-specific protein that dictates secondary cell wall thickness of wood fibres, facilitates auxin export from isolated Arabidopsis vacuoles in yeast and in Xenopus oocytes. We unambiguously identify IAA and related metabolites in isolated Arabidopsis vacuoles, suggesting a key role for the vacuole in intracellular auxin homoeostasis. Moreover, local auxin application onto wat1 mutant stems restores fibre cell wall thickness. Our study provides new insight into the complexity of auxin transport in plants and a means to dissect auxin function during fibre differentiation.

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Bacterial Trk and Ktr, fungal Trk and plant HKT form a family of membrane transporters permeable to K(+) and/or Na(+) and characterized by a common structure probably derived from an ancestral K(+) channel subunit. This transporter family, specific of non-animal cells, displays a large diversity in terms of ionic permeability, affinity and energetic coupling (H(+)-K(+) or Na(+)-K(+) symport, K(+) or Na(+) uniport), which might reflect a high need for adaptation in organisms living in fluctuating or dilute environments. Trk/Ktr/HKT transporters are involved in diverse functions, from K(+) or Na(+) uptake to membrane potential control, adaptation to osmotic or salt stress, or Na(+) recirculation from shoots to roots in plants. Structural analyses of bacterial Ktr point to multimeric structures physically interacting with regulatory subunits. Elucidation of Trk/Ktr/HKT protein structures along with characterization of mutated transporters could highlight functional and evolutionary relationships between ion channels and transporters displaying channel-like features.

Living organisms sense and respond to changes in nutrient availability to cope with diverse environmental conditions. Nitrate (NO3-) is the main source of nitrogen for plants and is a major component in fertilizer. Unraveling the molecular basis of nitrate sensing and regulation of nitrate uptake should enable the development of strategies to increase the efficiency of nitrogen use and maximize nitrate uptake by plants, which would aid in reducing nitrate pollution. NPF6.3 (also known as NRT1.1), which functions as a nitrate sensor and transporter; the kinase CIPK23; and the calcium sensor CBL9 form a complex that is crucial for nitrate sensing in Arabidopsis thaliana. We identified two additional components that regulate nitrate transport, sensing, and signaling: the calcium sensor CBL1 and protein phosphatase 2C family member ABI2, which is inhibited by the stress-response hormone abscisic acid. Bimolecular fluorescence complementation assays and in vitro kinase assays revealed that ABI2 interacted with and dephosphorylated CIPK23 and CBL1. Coexpression studies in Xenopus oocytes and analysis of plants deficient in ABI2 indicated that ABI2 enhanced NPF6.3-dependent nitrate transport, nitrate sensing, and nitrate signaling. These findings suggest that ABI2 may functionally link stress-regulated control of growth and nitrate uptake and utilization, which are energy-expensive processes.

The conventional approach to categorizing transporters has been to class them according to their sequence homology, defining a 'family' (or a 'superfamily' if they are numerous), and according to their substrate specificity or selectivity. This general view is still relevant for some transporters, but it is being increasingly challenged. Here, we take the NRT1/PTR FAMILY (NPF) as one such example. NPF members do indeed display sequence and structural homologies with peptide transporter (PTR) proteins involved in the uptake of di- and tri-peptides in most other organisms. And in plants they were initially characterized as nitrate or peptide transporters. However, in recent years several other substrates have been identified, namely nitrite, chloride, glucosinolates, auxin (IAA), abscisic acid (ABA), jasmonates (JAs), and gibberellins (GAs). Some of these transporters are even capable of transporting more than one different substrate (e.g. nitrate/auxin, nitrate/ABA, nitrate/glucosinolates, or GA/JA). In this review, we give an overview of the substrate-specificity of the Arabidopsis NPF.

Ca2+ -dependent protein kinases (CPKs) form a large family of 34 genes in Arabidopsis (Arabidopsis thaliana). Based on their dependence on Ca 2+ , CPKs can be sorted into three types: strictly Ca 2+ -dependent CPKs, Ca 2+ -stimulated CPKs (with a significant basal activity in the absence of Ca 2+ ), and essentially calcium-insensitive CPKs. Here, we report on the third type of CPK, CPK13, which is expressed in guard cells but whose role is still unknown. We confirm the expression of CPK13 in Arabidopsis guard cells, and we show that its overexpression inhibits light-induced stomatal opening. We combine several approaches to identify a guard cell-expressed target. We provide evidence that CPK13 (1) specifically phosphorylates peptide arrays featuring Arabidopsis K + Channel KAT2 and KAT1 polypeptides, (2) inhibits KAT2 and/or KAT1 when expressed in Xenopus laevis oocytes, and (3) closely interacts in plant cells with KAT2 channels (Förster resonance energy transfer-fluorescence lifetime imaging microscopy). We propose that CPK13 reduces stomatal aperture through its inhibition of the guard cellexpressed KAT2 and KAT1 channels.

Calcium (Ca2+) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca2+ signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca2+ sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca2+-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca2+ signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca2+ dynamics in intact plants. We describe a simple method to image Ca2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca2+ signals. It is shown that Ca2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.