Anne-Aliénor Véry

Functional analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2‐1 and sas2‐2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2‐1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2‐1 mutation strongly decreased Na+ concentration in the phloem sap. It led to Na+ overaccumulation in every aerial organ (except the stem), but to Na+ underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na+ recirculation from shoots to roots, probably by mediating Na+ loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na+ from the shoot and playing a crucial role in plant tolerance to salt.

The Arabidopsis outward K+ channel GORK is involved in regulation of stomatal movements and plant transpiration

Microscopic pores present in the epidermis of plant aerial organs, called stomata, allow gas exchanges between the inner photosynthetic tissue and the atmosphere. Regulation of stomatal aperture, preventing excess transpirational vapor loss, relies on turgor changes of two highly differentiated epidermal cells surrounding the pore, the guard cells. Increased guard cell turgor due to increased solute accumulation results in stomatal opening, whereas decreased guard cell turgor due to decreased solute accumulation results in stomatal closing. Here we provide direct evidence, based on reverse genetics approaches, that the Arabidopsis GORK Shaker gene encodes the major voltage-gated outwardly rectifying K+ channel of the guard cell membrane. Expression of GORK dominant negative mutant polypeptides in transgenic Arabidopsis was found to strongly reduce outwardly rectifying K+ channel activity in the guard cell membrane, and disruption of the GORK gene (T-DNA insertion knockout mutant) fully suppressed this activity. Bioassays on epidermal peels revealed that disruption of GORK activity resulted in impaired stomatal closure in response to darkness or the stress hormone azobenzenearsonate. Transpiration measurements on excised rosettes and intact plants (grown in hydroponic conditions or submitted to water stress) revealed that absence of GORK activity resulted in increased water consumption. The whole set of data indicates that GORK is likely to play a crucial role in adaptation to drought in fluctuating environments.

K+ channel activity in plants: Genes, regulations and functions

Potassium (K+) is the most abundant cation in the cytosol, and plant growth requires that large amounts of K+ are transported from the soil to the growing organs. K+ uptake and fluxes within the plant are mediated by several families of transporters and channels. Here, we describe the different families of K+‐selective channels that have been identified in plants, the so‐called Shaker, TPK and Kir‐like channels, and what is known so far on their regulations and physiological functions in the plant.

Cation channels in the Arabidopsis plasma membrane

In vivo analyses have identified different functional types of ion channels in various plant tissues and cells. The Arabidopsis genome contains approximately 70 genes for ion channels, of which 57 might be cation-selective channels (K(+), Ca(2+) or poorly discriminating channels). Here, we describe the different families of (putative) cation channels: the Shakers, the two-P-domain and Kir K(+) channels (encoded by the KCO genes), the cyclic-nucleotide-gated channels, the glutamate receptors, and the Ca(2+) channel TPC1. We also compare molecular data with the data obtained in planta, which should lead to a better understanding of the identity of these channels and provide clues about their roles in plant nutrition and cell signalling.

Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family

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.

Over‐expression of an Na+‐ and K+‐permeable HKT transporter in barley improves salt tolerance

Soil salinity is an increasing menace that affects agriculture across the globe. Plant adaptation to high salt concentrations involves integrated functions, including control of Na+ uptake, translocation and compartmentalization. Na+ transporters belonging to the HKT family have been shown to be involved in tolerance to mild salt stress in glycophytes such as Arabidopsis, wheat and rice by contributing to Na+ exclusion from aerial tissues. Here, we have analysed the role of the HKT transporter HKT2;1, which is permeable to K+ and Na+, in barley, a relatively salt-tolerant crop that displays a salt-including behaviour. In Xenopus oocytes, HvHKT2;1 co-transports Na+ and K+ over a large range of concentrations, displaying low affinity for Na+, variable affinity for K+ depending on external Na+ concentration, and inhibition by K+ (K(i) approximately 5 mm). HvHKT2;1 is predominantly expressed in the root cortex. Transcript levels are up-regulated in both roots and shoots by low K+ growth conditions, and in shoots by high Na+ growth conditions. Over-expression of HvHKT2;1 led to enhanced Na+ uptake, higher Na+ concentrations in the xylem sap, and enhanced translocation of Na+ to leaves when plants were grown in the presence of 50 or 100 mm NaCl. Interestingly, these responses were correlated with increased barley salt tolerance. This suggests that one of the factors that limits barley salt tolerance is the capacity to translocate Na+ to the shoot rather than accumulation or compartmentalization of this cation in leaf tissues. Thus, over-expression of HvHKT2;1 leads to increased salt tolerance by reinforcing the salt-including behaviour of barley.

Diversity in expression patterns and functional properties in the rice HKT transporter family.

Plant growth under low K+ availability or salt stress requires tight control of K+ and Na+ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K+ Transporters), permeable either to K+ and Na+ or to Na+ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na+ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na+ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na+-K+ symport, Na+ uniport, or inhibited states, depending on external Na+ and K+ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K+ and Na+ accumulation in monocots.

Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels

At least four genes encoding plasma membrane inward K+ channels (Kin channels) are expressed in Arabidopsis guard cells. A double mutant plant was engineered by disruption of a major Kin channel gene and expression of a dominant negative channel construct. Using the patch-clamp technique revealed that this mutant was totally deprived of guard cell Kin channel (GCKin) activity, providing a model to investigate the roles of this activity in the plant. GCKin activity was found to be an essential effector of stomatal opening triggered by membrane hyperpolarization and thereby of blue light-induced stomatal opening at dawn. It improved stomatal reactivity to external or internal signals (light, CO2 availability, and evaporative demand). It protected stomatal function against detrimental effects of Na+ when plants were grown in the presence of physiological concentrations of this cation, probably by enabling guard cells to selectively and rapidly take up K+ instead of Na+ during stomatal opening, thereby preventing deleterious effects of Na+ on stomatal closure. It was also shown to be a key component of the mechanisms that underlie the circadian rhythm of stomatal opening, which is known to gate stomatal responses to extracellular and intracellular signals. Finally, in a meteorological scenario with higher light intensity during the first hours of the photophase, GCKin activity was found to allow a strong increase (35%) in plant biomass production. Thus, a large diversity of approaches indicates that GCKin activity plays pleiotropic roles that crucially contribute to plant adaptation to fluctuating and stressing natural environments.

Molecular biology of K+ transport across the plant cell membrane: what do we learn from comparison between plant species?

Cloning and characterizations of plant K(+) transport systems aside from Arabidopsis have been increasing over the past decade, favored by the availability of more and more plant genome sequences. Information now available enables the comparison of some of these systems between species. In this review, we focus on three families of plant K(+) transport systems that are active at the plasma membrane: the Shaker K(+) channel family, comprised of voltage-gated channels that dominate the plasma membrane conductance to K(+) in most environmental conditions, and two families of transporters, the HAK/KUP/KT K(+) transporter family, which includes some high-affinity transporters, and the HKT K(+) and/or Na(+) transporter family, in which K(+)-permeable members seem to be present in monocots only. The three families are briefly described, giving insights into the structure of their members and on functional properties and their roles in Arabidopsis or rice. The structure of the three families is then compared between plant species through phylogenic analyses. Within clusters of ortologues/paralogues, similarities and differences in terms of expression pattern, functional properties and, when known, regulatory interacting partners, are highlighted. The question of the physiological significance of highlighted differences is also addressed.

Arabidopsis Annexin1 Mediates the Radical-Activated Plasma Membrane Ca2+- and K+-Permeable Conductance in Root Cells

The Arabidopsis thaliana root cell plasma membrane contains a calcium channel that is activated by oxidizing conditions and operates in cell growth. It was identified here as the most abundant member of the Arabidopsis annexins. These are soluble proteins that can undergo conditional attachment to or insertion into membranes. Plant cell growth and stress signaling require Ca2+ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca2+-permeable conductance that permits Ca2+ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca2+-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca2+- and K+-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca2+ in response to OH•. An OH•-activated Ca2+ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca2+-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca2+ in plants.