Molecular mechanisms involved in plant adaptation to low K+ availability

Journal of Experimental Botany, Vol. 65, No. 3, pp. 833–848, 2014doi:10.1093/jxb/ert402 Advance Access publication 30 November, 2013

Review papeR

Molecular mechanisms involved in plant adaptation to low K+ availability

Isabelle Chérel*, Cécile Lefoulon†, Martin Boeglin and Hervé Sentenac

Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, UMR 5004 CNRS/UMR 0386 INRA/Montpellier SupAgro/Université Montpellier 2, F-34060 Montpellier Cedex 1, France

† Present address: Laboratory of Plant Physiology and Biophysics, Bower Building, University of Glasgow, Glasgow G12 8QQ, UK.* To whom correspondence should be addressed. E-mail: [email protected]

Received 4 September 2013; Revised 22 October 2013; Accepted 31 October 2013

Abstract

Potassium is a major inorganic constituent of the living cell and the most abundant cation in the cytosol. It plays a role in various functions at the cell level, such as electrical neutralization of anionic charges, protein synthesis, long- and short-term control of membrane polarization, and regulation of the osmotic potential. Through the latter function, K+ is involved at the whole-plant level in osmotically driven functions such as cell movements, regulation of stomatal aperture, or phloem transport. Thus, plant growth and development require that large amounts of K+ are taken up from the soil and translocated to the various organs. In most ecosystems, however, soil K+ availability is low and fluctuating, so plants have developed strategies to take up K+ more efficiently and preserve vital functions and growth when K+ availability is becoming limited. These strategies include increased capacity for high-affinity K+ uptake from the soil, K+ redistribution between the cytosolic and vacuolar pools, ensuring cytosolic homeostasis, and modification of root system development and architecture. Our knowledge about the mechanisms and signalling cascades involved in these different adaptive responses has been rapidly growing during the last decade, revealing a highly complex network of interacting processes. This review is focused on the different physiological responses induced by K+ deprivation, their underlying molecular events, and the present knowledge and hypotheses regarding the mechanisms responsible for K+ sensing and signalling.

Key words: Auxin, ethylene, K+ membrane transport, K+ starvation, plant K+ nutrition, reactive oxygen species (ROS), root.

Introduction

Potassium (K+) is present in high amounts in plant cells (up to 10% of plant dry weight) and is absolutely required for plant growth. It is the main inorganic cation in the cytosol, where its concentration lies in the 100–200 mM range (Leigh and Wyn Jones, 1984). It plays a major role in electrical neu-tralization of inorganic and organic anions and macromol-ecules (e.g. nitrate, organates, DNA, and phospholipids), pH homeostasis, control of membrane electrical potential, regulation of cell osmotic pressure, and in related functions such as stomatal movements (Clarkson and Hanson, 1980). It is also involved in activation of enzymes, and a high K+ concentration is required for optimal protein synthesis and photosynthesis (Szczerba et al., 2009).

The concentration of K+ in the soil solution is typically in the 0.1–1 mM range, and can be much lower at the root sur-face due to local depletion (Jungk and Claasen, 1986). In the absence of added fertilizers, K+ availability is limiting for opti-mal plant growth in most natural ecosystems. K+ deprivation leads to a strong decrease in total K+ content, which generally occurs more rapidly in roots than in shoots (Drew et al., 1984; Pilot et al., 2003; Gierth et al., 2005; Nieves-Cordones et al., 2007; Ma et al., 2012). In tomato and rice, despite a rapid decline in global K+ content, plant growth is maintained at the same level as in K+-sufficient plants even after several days of starvation (Nieves-Cordones et al., 2007; Ma et al., 2012). This indicates that plants have evolved mechanisms

© The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]

Abbreviations: ABA, abscisic acid; CK, cytokinin; ROS, reactive oxygen species.

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that protect vital functions and ensure sustained growth dur-ing periods of K+ shortage. However, prolonged starvation periods result in growth inhibition and chlorosis, which is first observed in older leaves (Armengaud et al., 2004).

The consequences of K+ deprivation depend on the pres-ence of other monovalent cations such as Na+ and NH4

+. The absence of K+ ions in the germination medium renders Arabidopsis seedlings more sensitive to high (millimolar) external NH4

+ concentrations (Cao et al., 1993). At high concentrations, the Na+ alkali cation competes with K+ for K+ uptake as well as K+ regulation of enzymatic activities, protein synthesis, and ribosome function. Plant growth under salinity constraint is believed to be crucially dependent on the plant’s capacity to control the K+/Na+ ratio in its tissues (Shabala and Cuin, 2007).

This review will focus on mechanisms leading to plant adaptation to changes in K+ availability in plant cell environ-ments, focusing in particular on situations of K+ shortage. After a brief overview of the widely documented physio-logical and developmental responses to K+ deprivation, we will examine the molecular mechanisms identified as being involved in adaptation to low K+ availability by exploring, one after the other, the different physiological functions known to be mobilized for maintaining plant growth under low K+ availability: membrane polarization, K+ uptake, K+ remobilization at the cell and whole-plant levels, root growth, and modification of root system architecture.

Physiological responses induced by K+ deprivation

Electrical polarization of the cell membrane

Whatever the K+ nutritional status of the plant, the mem-brane potential is immediately and reversibly affected by a change in the external concentration of K+, with a reduc-tion in the K+ concentration shifting the potential towards more negative values. This widely reported phenomenon (Maathuis and Sanders, 1993; Wang et al., 1998; Spalding et al., 1999) can be ascribed to the fact that K+ transport sys-tems strongly contribute to the cell membrane conductance, which shunts the hyperpolarizing H+ excretion mediated by H+-ATPase proton pumps (Maathuis et al., 1997). Moreover, low-K+ plants, upon prolonged K+ deprivation, display more negative membrane potential values than control K+-replete plants (Walker et al., 1996; Nieves-Cordones et al., 2008). As discussed at the end of this review, it has been proposed that the electrical polarization of the cell membrane could be involved in sensing of the external availability of K+ and/or signalling of the plant K+ nutrition status (see below).

Changes in K+ uptake kinetics

K+ uptake by roots classically displays biphasic kinetics, thought to reflect the involvement of two different transport mechanisms according to Epstein’s model, the first with a Km in the low concentration range (<100 μM) and the second with a Km in the high concentration range (Epstein et al.,

1963). Evidence has been obtained that high-affinity uptake is active and mediated by co-transporters (probably H+:K+ symporters), whereas low-affinity K+ uptake is passive and essentially mediated by channels (Kochian and Lucas, 1983; Maathuis and Sanders 1992). However, identification of molecular actors of K+ uptake has led to a reconsideration of this dogma, since the potassium channel subunit AKT1 has been shown to participate in K+ depletion from the exter-nal medium down to K+ concentrations far below 100 μM (Hirsch et al., 1998; Spalding et al., 1999; see the section ‘K+ transport systems involved in K+ uptake from the soil’).

K+ starvation increases a plant’s capacity for K+ uptake from low concentrations. Drew et al. (1984) observed that 1 d of K+ starvation, in K+ depletion experiments, resulted in a strong increase in the affinity of the transport mechanism for K+ (4-fold decrease in Km) without change in the maximum rate of K+ uptake. In Arabidopsis, a significant change in K+ (Rb+) uptake kinetics has been observed after only 6 h of K+ deprivation (Shin and Schachtman, 2004). Induction of a high-affinity K+ uptake component has also been reported in tomato in response to K+ deprivation (Nieves-Cordones et al., 2007).

Cytosolic K+ homeostasis and remobilization of K+ vacuolar stores

Measurements of pH, K+ activity, and membrane potential with triple-barrelled microelectrodes have allowed the decline in K+ activity in the cytoplasm and in the vacuole of epi-dermal and cortical barley root cells to be followed simul-taneously upon K+ deprivation. The results revealed that, whereas K+ activity in the vacuole declined following the decrease in global plant K+ contents, K+ activity in the cyto-sol remained constant over a wide range of whole-root K+ concentrations, up to a threshold of ~25 mM (expressed on a root fresh weight basis), below which the cytoplasmic activity began to decrease (Walker et al., 1996). These results indicate that priority is given to cytosolic K+ homeostasis, support-ing cell metabolism, at the expense of K+ vacuolar contents. This could explain why dry matter production is maintained during the first days of K+ starvation. In this study, in par-allel with the cytosolic K+ decline, a simultaneous decrease in cytosolic pH was also observed, revealing a physiological relationship between cytosolic K+ and pH homeostasis.

Plant growth and morphology

K+ deficiency affects shoot and root development (Cakmak et al., 1994; Jung et al., 2009). In roots, it impairs both lat-eral root initiation and development (Armengaud et al., 2004; Shin and Schachtman, 2004). It also promotes root hair elon-gation (Desbrosses et al., 2003; Jung et al., 2009) and seems to have a depressive effect on primary root growth (Jung et al., 2009; Kim et al., 2010), although this has not been observed systematically (Shin and Schachtman, 2004). A mild agravit-ropic behaviour has also been reported (Vicente-Agullo et al., 2004). In Arabidopsis, in response to low K+, there seems to be an antagonism between lateral root and main root growth,

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with the resulting root architecture depending on accessions (Kellermeier et al., 2013).

Pioneering experiments aimed at describing the effect of localized supply of nutrients on the barley root system archi-tecture have revealed that phosphate, nitrate, or ammonium promote localized growth of the root system in the enriched zone. This was not observed in the case of K+, which triggered lateral root development in the whole root system, whether it was supplied locally or homogeneously (Drew, 1975). Thus, the availability of K+ in the soil affects root development in a specific way, different from what is observed with other macronutrients, Pi, NO3

–, and NH4+.

K+ transport systems involved in K+ uptake from the soil

An impressive set of transport systems contribute to K+ transport in plants

In the model plant Arabidopsis thaliana, it has been pro-posed that >30 membrane proteins, grouped into five families (Shaker, TPK, Kir-like, HAK/KUP/KT, and KEA) are dedi-cated to K+ transport (Mäser et al., 2001; Gierth and Mäser, 2007; Sharma et al., 2013). Reverse genetics approaches indicate that members from two of these families, namely the Shaker channel family (Fig. 1A) and the HAK/KUP/KT transporter family (Fig. 1B), which comprise nine and 13 members in A. thaliana, respectively, and nine and 27 members in rice, play major roles in K+ uptake from the soil and long-distance transport in plants (Grabov, 2007; Sharma et al., 2013) (Fig. 2).

The Shaker and HAK/KUP/KT families seem to be pre-sent in all dicotylodenous and monocotylodenous species. In monocots, in addition to these two families, a further group of transporters active at the cell membrane and permeable to K+ might contribute to transport of this cation into/from the cells. These transporters belong to subfamily 2 of the so-called HKT transporter family (Fig. 1C). The HKT family, which is present in both monocots and dicots, comprises two phylogenetic subfamilies. HKT subfamily 1 includes trans-porters permeable to Na+ only [based on functional studies in heterologous systems (oocytes and/or yeast)] and is pre-sent in both dicots and monocots. In contrast, subfamily 2 comprises transporters permeable to both Na+ and K+ and is present in monocots only (Corratgé-Faillie et al., 2010). HKT transporters permeable only to Na+ have been shown to con-tribute to plant adaptation to salinity constraints. The role of HKT transporters permeable to both Na+ and K+ is still poorly understood and, thus, the physiological significance of the fact that such transporters are present only in mono-cots is unclear. The hypothesis that these systems could con-tribute to K+ transport in monocots is controversial (Haro et al., 2005), but still deserves to be considered since a HKT transporter from rice, OsHKT2;4, permeable to both K+ and Na+ and present at the cell membrane in root periphery cells (Horie et al., 2011; Lan et al., 2010), has been found to dis-play preferential permeability to K+ (Sassi et al., 2012).

Which transport systems mediate root K+ uptake from the soil under low-K+ conditions?

In Arabidopsis, the K+ transport systems that are present at the plasma membrane in root outer cell layers (epidermis, root hairs, and cortex) are the Shaker subunits AKT1 and AtKC1, which form inwardly rectifying channels (Dreyer et al., 1997; Hirsch et al., 1998; Reintanz et al., 2002), the Shaker subunit GORK, which mediates the outwardly rec-tifying current in root hairs (Ivashikina et al., 2001), and members of the HAK/KUP/KT family (Ahn et al., 2004; Qi et al., 2008) endowed with high-affinity K+ transport activ-ity (Santa-Maria et al., 1997; Fu and Luan, 1998; Kim et al., 1998; Gierth et al., 2005; Grabov, 2007). Using athak5 and atakt1 mutant plants to investigate the relative contributions of HAK5 and AKT1 to K+ uptake from low K+ concentra-tions has provided evidence that AKT1 and HAK5 are the main players in K+ uptake when the external concentration of this cation is decreased below ~100 μM (Rubio et al., 2008).

Amongst the genes encoding the channels and transporters expressed in root outer cell layers, AtHAK5 appears to be the most highly regulated. Up-regulation of the AtHAK5 gene has been observed repeatedly, both in reverse transcription–PCR (RT–PCR) experiments (Rubio et al., 2000; Ahn et al., 2004) and in transcriptomics studies (Armengault et al., 2004; Gierth et al., 2005). In experiments carried out by Gierth et al. (2005), AtHAK5 was even the only gene (in microarrays of 8300 and 23 000 genes) that displayed a >2-fold up-regula-tion in response to 48 h of K+ deprivation. Nieves-Cordones et al. (2010) measured a 600-fold induction after 14 d of K+ starvation. It should be noted that up-regulation of another gene (AtKUP3) from the same family can occur upon K+ starvation, but after a longer period (2–3 weeks) of K+ dep-rivation (Kim et al., 1998). In agreement with these results in Arabidopsis, a barley homologue of AtHAK5, HvHAK1, is expressed in outer layers of the root (Fulgenzi et al., 2008), displays up-regulation (increased transcript levels) upon K+ starvation (Fulgenzi et al., 2008), and encodes a transporter able to mediate high-affinity K+ uptake (Santa-Maria et al., 1997). Homologues of AtHAK5 in other plant species, such as pepper (CaHAK5, Martinez-Cordero et al., 2005) and tomato (LeHAK5, Nieves-Cordones et al., 2007), are also up-regulated upon K+ deprivation, the transcript increase being correlated with the acquisition of high-affinity uptake capacity. Like AtHAK5, CaHAK5, LeHAK5, and HvHAK1 belong to cluster 1 of the HAK/KUP/KT family. As mem-bers of this cluster are high-affinity transport systems (Rubio et al., 2000; Grabov, 2007), up-regulation of their expression is likely to play a major role in the increase in root capacity for high-affinity K+ uptake upon K+ deprivation.

AtHAK5 has been shown to be highly efficient in deplet-ing Rb+ (used as a tracer of K+) from the medium, having the capacity to decrease the Rb+ concentration below 1 μM (Rubio et al., 2008). However, AtHAK5 cannot contribute to K+ uptake from low concentrations in all situations of K+ shortage since it is inhibited by millimolar concentrations of NH4

+ (Spalding et al., 1999; Ashley and Grabov, 2006; Qi et al., 2008), as is HvHAK1 (Santa-Maria et al., 1997).

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In planta, inverse correlations between K+ and NH4+ uptake,

together with sensitivity of NH4+ uptake to K+ uptake inhibi-

tors, have led to the suggestion that NH4+ (which is similar

to K+ in charge and size) is a competitor of K+ (ten Hoopen et al., 2010). Heterologous expression in yeast cells has revealed that the sensitivity of AtHAK5 K+ transport activ-ity to NH4

+ is markedly more pronounced than that of the Shaker K+ channel AKT1 (ten Hoopen et al., 2010). As a K+ channel, AKT1 was thought to contribute to K+ uptake from the soil only in the high concentration range [i.e. at concentra-tions (>100 μM) corresponding to Epstein’s mechanism 2]. Surprisingly, in the presence of NH4

+, this channel provides a significant component of K+ uptake even at K+ concentra-tions in the medium as low as 10 μM (Hirsch et al., 1998; Spalding et al., 1999). This implies that the membrane poten-tial can be negative enough in K+-starved plants to allow channel-mediated (passive) K+ uptake even when the external concentration of K+ is reduced to ~10 μM. In the absence of NH4

+ in the uptake medium, AtHAK5 is the main contribu-tor to high-affinity K+ uptake, but it is completely inhibited in the presence of NH4

+, leaving AKT1 to contribute alone

to K+ absorption. However, when compared with AtHAK5, AKT1 is less efficient for K+ uptake from low concentrations [being inefficient below 25 μM under the experimental condi-tions used by Rubio et al. (2008)], and thus the presence of NH4

+ in the growth medium results in a decrease in the root capacity for high-affinity K+ uptake. It is also worth noting that the effect of NH4

+ on HAK5 activity and/or expression depends on the presence of Na+ in the external solution (see below, ‘Effect of Na+ and NH4

+ on K+ uptake’).In monocots, the hypothesis that some HKT transporters

could contribute to K+ uptake especially under low-K+ condi-tions, together with HAK5 and AKT1 homologues (Fig. 3), is supported by several lines of indirect evidence (see above), such as expression in root periphery cells and strong induction upon K+ starvation (Ma et al., 2012; Takehisa et al., 2013), or selectivity for K+ against Na+ (Sassi et al., 2012). So far, how-ever, this hypothesis has not received any direct support from in planta analysis of K+ transport activity (Corratgé-Faillie et al., 2010). This might be due to important redundancy in K+ transport systems in monocots. Physiological studies using multiple mutants could allow the investigation of this

pore domain

voltage sensor

N-ter

S1 S6

+ + +

CNBD

anky

cytoplasm

membrane

Shaker channels A

N-ter

KUP/HAK/KT transporters

B

N-ter

pore domain

HKT transporters C

Fig. 1. Main transporter families mediating K+ transport at the plasma membrane. (A) Shaker channels. The Shaker subunit (upper panel) displays six transmembrane segments (S1–S6, in yellow), the fourth one (S4) harbouring positively charged amino acids (+) that constitute a voltage sensor. A cyclic nucleotide-binding domain (CNBD) is located in the cytosolic C-terminal region. The ankyrin domain (anky), which interacts with regulatory proteins, is present in most, but not all, members of the Shaker family. Lower panels: side view and upper view (below) of four Shaker subunits assembled to form the tetrameric functional Shaker channel, with the four pore domains (light blue) forming the central pore of the channel. (B) Structure of KUP/HAK/KT transporters. (C) Proposed topology for HKT transporters. The four pore domains present in the polypeptide meet at the centre of the protein to form a central pore similar to that of Shaker channels (lower panel).

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redundancy and the contribution of the different transport system families to K+ uptake from the soil solution under dif-ferent K+, Na+, NH4

+, and pH conditions.

Regulation of AtHAK5 expression, trafficking of the encoded transporter, and induction of high-affinity K+ uptake from the soil

As stated above, K+ deprivation rapidly results in both increased capacity for high-affinity K+ uptake and up-regula-tion of AtHAK5 expression. The mechanisms underlying these responses (illustrated in Fig. 4) have thus to be sought in early signalling events. It should, however, be noted that up-regula-tion of AtHAK5 is not a sine qua non condition of increased capacity for high-affinity K+ uptake, and that post-transla-tional regulation of AtHAK5 activity is also likely to contrib-ute to the shift towards high-affinity transport (see below).

Membrane potential and regulation of HAK5 expression in tomato

Evidence has been obtained that hyperpolarization of the cell membrane, which is one of the earliest physiological responses that can be detected following a decrease in exter-nal K+ concentration (see above), is likely to play a role in development of high-affinity K+ uptake capacity. Nieves-Cordones et al. (2008) grew tomato plants under different conditions (the presence or absence of NH4

+, K+, and NaCl

AKT1 AtKC1/AKT1

xylem phloem

Root

SKOR AKT2 HAK5

AKT2

stem & leaf

Fig. 2. Transport systems involved in K+ uptake from the soil and distribution in the plant vasculature in Arabidopsis thaliana. In root periphery cells, K+ is taken up from the soil by HAK5 transporters and AKT1/AtKC1 inward Shaker channels. K+ ions then migrate via the symplasm to reach stelar tissues, where they are secreted into the xylem sap. The outward Shaker channel SKOR plays a major role in K+ secretion into the xylem sap. In the phloem vasculature, the weakly rectifying AKT2 Shaker channel has the capacity to allow K+ influx or efflux across the cell membrane, depending on the transmembrane K+ electrochemical gradient. It plays a role in the control of membrane potential and sugar transport, and probably also in K+ (re)circulation within the phloem vasculature from source to sink organs.

Dicots and monocots

A role in K+ uptake of HKT transporters?

"High affinity" active uptake

"Low affinity" passive uptake

A external K+

concentration

Threshold:

10-6 M 10-.4 M

Shaker channels HAK transporters

HAK

out in

Shaker

out in

Identified transporters Identified channels

AKT1 with AtKC1 in Arabidopsis OsAKT1 in rice

HAK5 in Arabidopsis LeHAK5 in tomato HAK1 in barley and rice

Monocots B

K+ K+

out in

HKT K+

Na+

OsHKT2;4 in rice OsHKT2;2 in rice TaHKT1 in wheat HvHKT1 in barley

Fig. 3. Transport systems involved in high- and low-affinity K+ uptake from the soil. Different types of transport systems are used for K+ uptake from the soil, depending on the concentration of this cation in the soil solution (upper dotted line). (A) Systems common to dicots and monocots. Transporters from the KUP/HAK/KT family (HAK5 in Arabidopsis) mediate ‘high-affinity’ active K+ uptake. Such systems, which are endowed with H+: K+ symport activity and thus energized by the transmembrane electrochemical gradient of H+, have the capacity to decrease the external concentration of K+ to <1 μM (Rubio et al., 2008). K+ channels from the Shaker family (encoded by the AKT1 and AtKC1 genes in Arabidopsis) are involved in passive ‘low-affinity’ K+ uptake. Membrane hyperpolarization can allow such channels to decrease the external concentration of K+ down to ~10 μM (Véry and Sentenac, 2003). (B) In monocots, members from the HKT transporter family displaying K+ selectivity (such as OsHKT2;4 in rice) might play a role in high-affinity K+ uptake (Corratgé-Faillie et al., 2010).

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in the external solution) that were shown to modulate strongly the membrane potential in root cortical cells. After several days of growth in these conditions, expression of LeHAK5 (the tomato orthologue of AtHAK5) was found to be cor-related to root cell membrane potential and not to root K+ contents. Interestingly, pharmacological agents that depolar-ized the membrane also induced a rapid reduction, within 2 h, of the tomato LeHAK5 transcript level. A direct role for K+ starvation-induced hyperpolarization of the cell membrane potential in LeHAK5 gene induction has therefore been pro-posed (Nieves-Cordones et al., 2008).

Transcriptional regulation of HAK5 triggered by reactive oxygen species (ROS) and hormones

In Arabidopsis, after only 6 h of K+ deprivation, a strong increase in H2O2 production can be detected in a root area

of high uptake activity, upstream of the elongation zone (Shin and Schachtman, 2004). This is observed in parallel with the induction of enzymes involved in ROS synthesis or ROS detoxification such as the NADPH oxidase RHD2 (‘Root Hair Defective 2’, also named AtrbohC), peroxidases, and a glutathione S-transferase. In tomato, H2O2 production has been shown to reach a peak after 24 h of K+ starvation. During that time, H2O2 overaccumulated in roots tips and spread to other root areas such as the epidermis of the elon-gation zone and mature parts of the roots. This coincided in time with changes in activities of ROS antioxidative enzymes (Hernandez et al., 2012).

The rhd2 Arabidopsis mutant displays a loss of induc-tion by K+ deprivation of most up-regulated genes, includ-ing AtHAK5 (Shin and Schachtman, 2004; Shin et al., 2005). Addition of H2O2 into the external medium partially restored AtHAK5 induction in the mutant plants (Shin and

Fig. 4. Signalling pathways activated in Arabidopsis root cells in response to K+ deprivation and involved in increased K+ uptake capacity and altered root architecture. K+ deprivation leads to membrane hyperpolarization (1), allowing more efficient K+ uptake via AKT1–AtKC1 Shaker channels (2) and induction of the expression of the HAK5-encoding gene, via unknown mechanisms (3). AKT1 is itself, in turn, a regulator of the membrane potential (4). It might also behave as a K+ sensor (5), recruiting the CIPK23 kinase under low K+ conditions. AKT1 is activated by CIPK–CBL complexes, which are themselves activated by increased cytosolic Ca2+ activity (6, 7). This Ca2+ signal is likely to result, at least in part, from increased Ca2+ influx through ANN1 calcium transport systems (8), which are themselves activated by ethylene/ROS signalling events (8, 9) triggered by K+ starvation (9). The ethylene/ROS signalling pathway is also involved in the regulation of many genes, including RAP2.11. This gene encodes a transcription factor involved in the induction of a set of K+ deprivation-related genes, including HAK5 (10). RAP2.11 also plays a role in root hair development (10). Ethylene inhibits root growth by up-regulating the PIN3 and PIN7 auxin transporter genes (11, 12), in an EIN2- and ETR1-dependent manner (11). In root hairs, the TRH1 K+ transporter is a putative K+ sensor (13). It is also involved in auxin transport (14) and relocalization of the auxin transporter PIN1 (13), affecting auxin-related processes such as root hair growth and gravitropism. 1, Maathuis and Sanders (1993); 2, Sharma et al. (2013, and references therein); 3, Nieves-Cordones et al. (2008); 4. Spalding et al. (1999); 5, Tsay et al. (2011); 6, Xu et al. (2006); 7, Li et al. (2006); 8, Laohavisit et al. (2012); 9, Jung et al. (2009); 10, Kim et al. (2012); 11, Lewis et al. (2011); 12, Billou et al. (2005); 13, Rigas et al. (2013); 14, Vicente-Agullo et al. (2004).

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Schachtman, 2004). Furthermore, exogenous H2O2 was also capable of inducing AtHAK5 expression in K+-sufficient rhd2 plants (Shin and Schachtman, 2004). All these data support that H2O2 plays a prominent role in transcriptional regula-tion of the AtHAK5 gene. Another gene, named RCI3, is also involved in ROS production and transcription of AtHAK5. Using a luciferase screen with the AtHAK5 promoter in order to find upstream regulators of AtHAK5 transcription, Kim et al. (2010) identified the RCI3 gene, which encodes a peroxidase located in the endoplasmic reticulum (ER). This gene is strongly up-regulated under K+-deficient conditions. Furthermore, RCI3 overexpression induces H2O2 produc-tion and AtHAK5 expression under K+-sufficient conditions, and the rci3 mutant displays a reduced induction of AtHAK5 under low-K+ conditions.

Ethylene is also involved in AtHAK5 induction (Jung et al., 2009). It is produced in response to K+ deprivation and this coincides with up-regulation of ethylene-related enzymes (Shin and Schachtman, 2004). Experiments performed with ‘ethylene-insensitive’ and ‘constitutively ethylene-responding’ mutants, and ethylene inhibitors or the ethephon precursor, allow the conclusion that ethylene acts upstream of ROS in the response to K+ deficiency, and that EIN2 is a strong, but not exclusive, regulator of AtHAK5 induction (Jung et al., 2009).

In contrast to ethylene and ROS, cytokinins (CKs) decrease during K+ starvation. Using genetic and pharma-cological approaches comparable with those for ethylene, Nam et al. (2012) found that CKs are negative regulators of ROS production under low-K+ conditions. After 7 d on K+-free medium, AtHAK5 induction was much reduced in the CK-overproducing plants, and enhanced in the mutant defec-tive in CK synthesis (Nam et al., 2012).

Additional elements of the signalling cascade have recently been identified with the discovery of transcription factors involved in regulation of AtHAK5 gene expression. Using a screening strategy based on the luciferase reporter gene under control of the AtHAK5 promoter region together with activation tagging, Kim et al. (2012) identified a transcrip-tion factor, named RAP2.11, whose overexpression in plants under K+-sufficient conditions leads to increased expression of AtHAH5 and many other genes that are induced by low K+ availability, notably genes involved in ROS production, and ethylene and calcium signalling. The expression of the RAP2.11 gene is induced by H2O2 and ethephon, thus plac-ing it downstream of the ethylene–ROS signalling cascade. RAP2.11 directly binds to the GCC box of the AtHAK5 pro-moter. Furthermore, ROS production in roots is increased in RAP2.11-overexpressing plants and decreased in rap2.11 mutant plants, in agreement with the fact that this transcrip-tion factor up-regulates the expression of genes encoding ROS-producing enzymes (Kim et al., 2012). Thus, RAP2.11 promotes ROS production and is induced by ROS, which results in increased AtHAK5 expression. Very recently, a luciferase-based screening with a ‘Transcription Factor-Over-eXpressor’ library has allowed the identification of additional transcription factors activating AtHAK5 expression under conditions of K+ limitation. Four of these transcription

factors have been further characterized and shown to stimu-late root growth, especially under low K+ availability, when overexpressed in transgenic Arabidopsis plants (Hong et al., 2013).

Post-translational regulation of AtHAK5 activity by trafficking

Development of high-affinity K+ uptake over a short period of K+ deprivation could involve post-translational control of AtHAK5 activity. Indeed, Qi et al. (2008) have provided evi-dence that K+ deprivation affects the trafficking of AtHAK5 from the ER to the cell membrane. In membrane preparations of plants grown under K+-sufficient conditions, AtHAK5 was found in the ER and possibly in tonoplast membrane fractions, while it was partially detectable in the plasma mem-brane fractions after 2 d of K+ starvation (Qi et al., 2008). Molecular determinants of this regulation are still unknown. Further studies would be helpful in order to determine whether this mechanism might account for the rapid adapta-tion of root K+ uptake kinetics to K+ deprivation.

Post-translational regulation of the AKT1 K+ channel

The Arabidopsis AKT1 gene, unlike its homologue TaAKT1 from wheat (Buschmann et al., 2000), is not induced by K+ deprivation (Pilot et al., 2003; Gierth et al., 2005), suggest-ing the involvement of post-translational regulation. So far, the major breakthrough in the analysis of such regulation has probably been the discovery of AKT1 regulation by CIPKs (CBL-interacting protein kinases) and their calcium-binding CBL (calcineurin B-like) partners (Xu et al., 2006). Since then, a large amount of work has been devoted to that aspect of K+ uptake regulation. Using a genetic approach, Xu et al. (2006) identified the CIPK23 kinase as a protein involved in K+ uptake under low-K conditions. CIPK23 is necessary for AKT1 channel activity in the oocyte expression system, and requires CBL1 or CBL9 for its own activation (Li et al., 2006; Xu et al., 2006) (Fig. 5). Whereas CBL1 and CBL9 seem to be constitutively expressed, their CIPK23 target is up-regulated after 2 d of growth on low-K medium (Cheong et al., 2007). CBL proteins possess four EF-hands that bind Ca2+, and thereby behave as sensors for Ca2+ signals (Li et al., 2009). Specificity exists in CIPK and CBL interactions, allowing multiple combinations of stress responses (Li et al., 2006; Xu et al., 2006; Li et al., 2009). The AKT1–CIPK–CBL com-plex is negatively regulated by PP2Cs (protein phosphatases 2C) belonging to the clade A involved in abscisic acid (ABA) signalling (Lee et al., 2007; Lan et al., 2011). CBLs can also interact directly with AKT1 (Grefen and Blatt, 2012; Ren et al., 2013).

Another type of post-translational regulation of AKT1 relies on the tetrameric structure of Shaker channels, allow-ing interaction between channel subunits encoded by distinct Shaker genes. AKT1 forms heteromers with AtKC1, a Shaker subunit present in root hairs, root cortex, and epidermis

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(Ivashikina et al., 2001; Reintanz et al., 2002; Pilot et al., 2003) (Fig. 2). AtKC1 transcript is induced during the first hours of K+ deprivation, but decreases thereafter (Shin and Schachtman, 2004), which might account for the absence of a change in the level of AtKC1 transcripts observed after several days of K+ starvation (Pilot et al., 2003). AtKC1 is silent when expressed alone and it forms functional channels only when assembled with other Shaker subunits (Dreyer et al., 1997; Duby et al., 2008; Geiger et al., 2009; Jeanguenin et al., 2011). Upon co-expression, AKT1 and AtKC1 subu-nits preferentially form heterotetramers (Duby et al., 2008). In association with AKT1, AtKC1 modulates K+ influx in root hairs (Reintanz et al., 2002). When expressed in Xenopus oocytes bathed in low-K+ medium, AKT1 opens at mem-brane potentials less negative than the K+ equilibrium poten-tial EK, and thus mediates K+ efflux in the voltage range between its activation potential (close to –50 mV) and EK (Geiger et al., 2009). With a K+ external concentration from 10 μM to 100 μM, EK is expected to range from –240 mV to –180 mV, and thus strong and permanent hyperpolarization of the root cell membrane is required for K+ influx, and not K+ leakage, to occur through AKT1 (Geiger et al., 2009). In the presence of AtKC1, resulting in the formation of hetero-tetrameric channels, the activation potential of the hetero-meric AKT1–AtKC1 channels is shifted towards much more negative potentials (close to –100 mV; Geiger et al., 2009), when compared with the activation potential of the homo-tetrameric AKT1 channel. Furthermore, the conductance of the heteromeric AtKC1–AKT1 channel is reduced, thus pre-venting massive K+ leak (Geiger et al., 2009).

However, in planta, the role of AtKC1 in plant adaptation to low K+ is still unclear. When grown in low-K+ conditions, atkc1 mutant plants can display a growth phenotype either similar to that of the akt1 mutant (short roots: Geiger et al.,

2009; Honsbein et al., 2009) or opposite to it (longer roots than in wild-type plants: Wang et al., 2010), depending on the experimental conditions. In agreement with the latter phe-notype, K+ uptake and accumulation under low-K+ condi-tions were found to be more important in the atkc1 mutant than in wild-type plants (Wang et al., 2010). Such a behav-iour might be ascribed, in some environmental conditions, to the negative regulation of AKT1 current by AtKC1. AtKC1 is also the target of the SNARE protein SYP121, a traffick-ing protein also reported to be involved in AKT1 activity regulation (Honsbein et al., 2009). The syp121 mutant dis-played the same short root phenotype on low-K+ medium as the atkc1 and akt1 mutant. When co-expressed with AKT1 and AtKC1 in Xenopus oocytes, SYP121 allowed the channel to be activated at much less negative membrane potentials, compared with the activation potential observed in control oocytes co-expressing only AKT1 and AtKC1. This regula-tion by SYP121 resulted in a global increase in K+ current, and this current was found to be more similar to the cur-rent observed in planta than the current recorded through AKT1 expressed alone or co-expressed with AtKC1. AKT1, AtKC1, and SYP121 were all needed for measurement of a detectable current in root epidermal protoplasts. The roles of SYP121 and AtKC1 in K+ acquisition and adaptation to K+-deficiency conditions have to be further specified. A role in control of cell volume (and related functions such as cell growth/elongation or guard cell movements) by coordination of vesicle fusion and osmolarity via K+ uptake has been pro-posed (Honsbein et al., 2009).

Effect of Na+ and NH4+ on K+ uptake

High-affinity K+ uptake has been shown to be negatively impacted by NH4

+ and Na+ (supplied at 1.4 mM and 50 mM, respectively) in tomato (Nieves-Cordones et al., 2007).

The effects of Na+ on K+ nutrition and plant growth are complex and depend on the relative concentrations of these two cations in the external medium. Na+ supply at low con-centrations (in the 10 μM to 1 mM range) has a beneficial effect on Arabidopsis akt1 mutant growth at low external K+ by stimulating the non-AKT1 component of K+ uptake (Spalding et al., 1999). Also, in K+-deprived rice plants, Na+ uptake (mediated by OsHKT2;1) from submillimolar concentrations promotes plant growth (Horie et al., 2007). Conversely, at high concentrations, Na+ has been shown to inhibit K+ uptake into the cells, to induce K+ efflux, and thereby to decrease the cytoplasmic K+/Na+ ratio (Wu et al., 1996; Shabala and Cuin, 2007).

The interdependence of Na+ toxicity and K+ uptake abil-ity has been investigated using Arabidopsis wild-type, akt1, and hak5 mutant plants (Nieves-Cordones et al., 2010). The induction of the AtHAK5 gene by K+ starvation is strongly reduced in the presence of 50 mM Na+. Furthermore, in that situation of membrane depolarization by Na+, AKT1 medi-ates K+ efflux (Nieves-Cordones et al., 2010). This might explain why AKT1 does not play a role in adaptation to high Na+ concentrations (Spalding et al., 1999). In contrast, despite its low level of expression due to the presence of Na+,

PP2C

Ca2+ CBL CIPK AKT1

K+ AtKC1

SYP121

IN OUT

Fig. 5. Model for post-translational regulation of AKT1 upon low K+ availability in Arabidopsis. Cytosolic calcium signals evoked by increased ROS concentrations (Laohavisit et al., 2012) allow CBL proteins to be activated and to bind to their CIPK targets for AKT1 phosphorylation (Li et al., 2006; Xu et al., 2006). The resulting activation of AKT1 can be counteracted by protein phosphatases of the PP2C family (Lee et al., 2007; Lan et al., 2011). The AtKC1 gene encoding the Shaker regulatory subunit is up-regulated, leading to formation of AKT1–AtKC1 tetramers with decreased ability for K+ efflux (Geiger et al., 2009). In the presence of SYP121, the AKT1–AtKC1 complex activates at less negative potentials, which results in an increase in current (Honsbein et al., 2009).

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AtHAK5 plays a major role in K+ uptake under low K+ avail-ability and high Na+ concentration by strongly limiting K+ net efflux and allowing re-absorption of K+ during a long-term stress period (Nieves-Cordones et al., 2010).

Na+ transporters, by controlling Na+ fluxes, also affect K+ uptake and accumulation. Mutations in the SOS1 gene, which encodes an Na+/H+ antiporter preferentially expressed in cells around the vasculature (Shi et al., 2002) as well as mutations in genes encoding the SOS1 regulators SOS2 (CIPK24) and SOS3 (CBL4), result in both Na+ hypersen-sitivity and growth impairment on low-K+ medium (SOS1, Wu et al., 1996; SOS2, Zhu et al., 1998; SOS3, Liu and Zhu, 1997). The sos mutants plants have also been shown to dis-play lower K+ contents under salt stress than wild-type plants (Zhu et al., 1998). Furthermore, the sos1 mutant is defective in high-affinity K+ uptake (Wu et al., 1996). Finally, salt toler-ance of the three sos mutants was found to be correlated to the K+ but not to the Na+ tissue contents (Zhu et al., 1998), highlighting the interdependency of Na+ and K+ transport and accumulation in plants.

As discussed above (‘Which transport systems mediate root K+ uptake from the soil under low-K+ conditions?’), the presence of NH4

+ in the external medium can result in inhibition of HAK5 activity. Besides this effect, NH4

+ can also act at the transcriptional level by affecting the induction of AtHAK5 gene expression resulting from K+ deprivation (Rubio et al., 2008). In tomato, however, whereas Na+ coun-teracts LeHAK5 induction by low-K+ availability, NH4

+ has a beneficial effect and allows LeHAK5 transcript to be accu-mulated even in the presence of Na+ (Nieves-Cordones et al., 2007) or K+ (Nieves-Cordones et al., 2008).

Vacuolar K+ release in epidermal and cortical cells

K+ transporters belonging to the TPK family, which com-prises six members in Arabidopsis, are all active at the tono-plast, except TPK4, which is present at the plasma membrane (Sharma et al., 2013). Expression of TPK genes has been detected in various tissues by PCR experiments (Sharma et al., 2013), but the expression patterns of these genes in root tissues have not been specified so far, except for TPK1 whose promoter region is active in the stele (Czempinski et al., 2002). Efflux systems mediating K+ release from vac-uoles in root cortex and epidermis upon K+ starvation are thus still unknown. However, other putative molecular com-ponents of K+ vacuolar efflux have recently been identified. A member of the CIPK family, CIPK9, is involved in plant adaptation to K+ starvation (Pandey et al., 2007). The CIPK9 gene is induced by K+ starvation (Pandey et al., 2007; Liu et al., 2013). Intriguingly, CIPK9 disruption has been found to result either in hypersensitivity (Pandey et al., 2007) or in tolerance (Liu et al., 2013) to K+ starvation, probably due to differences in experimental conditions. Unlike AtCIPK23, AtCIPK9 is not involved in regulation of K+ uptake from the medium, since Rb+ uptake and K+ content were unaffected in the cipk9 mutant compared with the wild type even when

plants were previously grown for 4 d in a low-K medium (Pandey et al., 2007). AtCIPK9 is expressed in all root layers, and the CIPK9 protein has been found to interact with CBL2 and CBL3 at the tonoplast (Liu et al., 2013). Furthermore, in experiments of Liu et al. (2013) the cbl3 (but not cbl2) mutant displayed the same tolerant phenotype on low-K+ medium as the cipk9 mutant. It has been hypothesized that the CIPK9–CBL3 complex might regulate an as yet unidentified vacuolar K+ transport system to maintain cytosolic K+ homeostasis under low-K conditions (Amtmann and Armengaud 2007; Liu et al., 2013).

K+ transport in vascular tissues

In Arabidopsis, two K+ channels from the Shaker family, SKOR and GORK, are endowed with outwardly rectify-ing gating properties allowing them to mediate K+ secretion from the cytosol into the apoplast (Véry and Sentenac, 2003). GORK displays expression in guard cells (Ache et al., 2000), where it plays a role in stomatal movements (Hosy et al., 2003). SKOR is essentially expressed in root stelar tissues where it contributes to K+ secretion into the xylem sap towards the shoots (Gaymard et al., 1998) (Fig. 2). Like GORK, SKOR is sensitive to the external concentration of K+: its opening probability decreases when the external concentration of K+ is increased. This regulation ensures that these channels can mediate K+ efflux only, whatever the external concentration of this cation (Johansson et al., 2006). The sensing mecha-nism has been shown to involve interactions between the so-called ‘gating domain’ present in the sixth transmembrane segment (S6) of the channel polypeptide and the base of the pore helix (Johansson et al., 2006). Besides this intrinsic regulation by external K+, SKOR displays post-translational activation by H2O2 and transcriptional regulation. Activation by H2O2 involves a cysteine residue present in the third trans-membrane segment (S3) of the channel polypeptide, and the H2O2-sensitive site is accessible only from the outside and when the channel is in the open conformation (Garcia-Mata et al., 2010). In agreement with its sensitivity to H2O2, SKOR’s contribution to K+ translocation and shoot/root partitioning is increased after H2O2 treatment (Garcia-Mata et al., 2010). Increased levels of SKOR transcripts can be observed upon salinity constraint (Maathuis, 2006), but not systematically (Pilot et al., 2003). Decreased SKOR transcript accumulation has also been reported in response to K+ deprivation (Pilot et al., 2003) and ABA treatments (Gaymard et al., 1998). This suggests that adaptation to K+ shortage, and maybe also to drought conditions, involves reduced K+ translocation to shoots, allowing this cation to be preferentially accumulated in young growing roots for osmotic adjustment (Sharp et al., 1990; Pritchard et al. 1991).

Phloem transport involves wholesale K+ re-circulation from shoots to roots, the descending K+ flux in the phloem vasculature representing up to >50% of the ascending flux in the xylem vasculature (Armstrong and Kirkby, 1979; Touraine et al., 1988). The Shaker channel AKT2, which is expressed in the phloem vasculature in both shoots and roots,

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displays unique gating properties within the plant Shaker family, resulting in weak rectification and thereby in effi-cient capacity to mediate both K+ influx and efflux, depend-ing on the transmembrane K+ electrochemical gradient, in source and sink organs (Lacombe et al., 2000). Evidence has been obtained that a major role for this channel is to regu-late phloem membrane potential rather than the concentra-tion of K+ in the phloem sap. This regulation would play a role in the control of membrane transport energization, particularly of sucrose transport into/from the phloem sap, leading to improved sugar allocation (Deeken et al., 2002; Gajdanowicz et al., 2011). AKT2 currents are reduced in the presence of the phosphatase AtPP2CA (Chérel et al., 2002) and enhanced by CBL4 (SOS3) and the kinase CIPK6 (Held et al., 2011), whose expression is increased upon K+ starva-tion (Armengaud et al., 2004). The activation of AKT2 at the plasma membrane by CIPK6–CBL complexes is not due to a phosphorylation event, as in the case of AKT1 regulation by CIPK23, but to a modification of channel targeting, which does not result from channel phosphorylation (Held et al., 2011). In Arabidopsis, K+ deprivation does not seem to affect the levels of AKT2 transcripts, and the akt2 mutant has been found to display a growth phenotype (shorter roots) only on K+-sufficient medium (Spalding et al., 1999; Gajdanowicz et al., 2011). In rice, the AKT2-like gene is however induced by K+ deficiency (Ma et al., 2012).

Molecular events involved in the modification of root architecture

As stated above, the concentration of K+ in the external medium has an impact on root growth, root hair develop-ment, and gravitropism. Evidence is available that auxin and ethylene play major roles in these morphological responses (Fig. 4), although the underlying mechanisms are still largely unknown.

Growth and development of the root system, gravitropism, and root hair development are dependent on auxin (Teale et al., 2005). Interplay between K+ and auxin signalling could involve the TRH1/AtKUP4 K+ transporter, which also behaves as an auxin efflux facilitator, and has been identi-fied as an essential component regulating root hair growth and root gravitropism (Vicente-Agullo et al., 2004). The trh1 mutant is more agravitropic on 20 mM K+ than on low-K medium, in contrast to the wild type that is mildly agravit-ropic only when starved for K+ (Vicente-Agullo et al., 2004). Recently, it was found that the trh1 mutation results in mislo-calization of the PIN1 auxin efflux carrier (Rigas et al., 2013). This leads to impaired auxin transport both acropetally towards the shoot tip and basipetally to the differentiation zone where root hairs are formed, and finally to altered root gravitropism and root hair growth. It has been suggested that K+ deficiency might modulate auxin distribution via changes in TRH1 activity, leading to a reduction of gravitropism in order to explore new soil areas (Rigas et al., 2013).

Growth of root hairs and primary root is also regulated by ethylene (Jung et al., 2009). Ethylene inhibitors suppress

the root hair elongation increase induced by low-K+ treat-ment. Looking for genes involved in the signalling pathway, Jung et al. revealed that the decrease in primary root growth under low K+ is not observed in ethylene-insensitive ein2 and in ethylene receptor etr1 mutant plants, while these mutants display the same phenotype as wild-type plants regarding (increased) ROS production and root hair growth. This sug-gests that different ethylene-mediated signalling pathways are involved in root development under low-K conditions. Ethylene is involved in auxin synthesis and transport, lead-ing to modulation of root growth (Ruzicka et al., 2007). It has been shown to inhibit lateral root formation, via the induction of auxin efflux carrier genes PIN3 and PIN7 in an EIN2- and ETR1-dependent manner (Lewis et al., 2011). In a recent study, mutant plants affected in CK synthesis or defec-tive in CK receptors have enabled the analysis of the complex pathways controlling primary and lateral root growth after a long period of K+ deprivation to be carried out. CK synthe-sis mutants displayed an enhanced response to K+ shortage, including reduction in primary root length and in the number of lateral roots, as well as increased ROS production and root hair growth, whereas some CK receptor mutants displayed reduced responses. These results have highlighted the role of AHK2 and AHK3 cytokinin receptors in the transduction pathway (Nam et al., 2012).

Sensing and signalling of low K+ availability: current issues and prospects

Despite some recent advances (Wang and Wu, 2013), the mechanisms responsible for low external K+ sensing and K+ deficiency signalling are still poorly deciphered. The present knowledge in this domain points to several major questions that have to be investigated further.

Hypotheses about the sensing mechanisms

Different levels of K+ sensing can be expected (Figs 4, 6), allowing roots to perceive not only the availability of K+ in the soil but also the cytosolic concentration of this cation in order to control, for instance, the exchanges of K+ between the cytosolic and vacuolar pools and K+ translocation towards the shoots.

Sensing of external K+ probably involves the cell mem-brane. Three different hypotheses, that are not mutually exclusive, are frequently proposed in the literature, involving the electrical polarization of the cell membrane (Fig. 6A), the Shaker K+ channel GORK in root periphery cells (and SKOR in root stelar tissues), or the Shaker K+ channel AKT1 (Fig. 6B). Regarding the former hypothesis, changes in exter-nal K+ concentration immediately affect the cell membrane electrical potential, as indicated above. A decrease in K+ avail-ability results in a decrease in the depolarizing inward K+ cur-rent and thereby in membrane hyperpolarization, which, in turn, activates voltage-gated inward channels such as AKT1 (Fig. 6A). The mechanism by which the membrane potential would trigger specific responses such as increased expression

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of high-affinity K+ uptake component remains to be eluci-dated. The second hypothesis (Ache et al., 2000; Johansson et al., 2006) proposes that K+ sensing relies on the sensitivity of the outwardly rectifying Shaker channels GORK and SKOR to external K+ concentration (see above). GORK, which is expressed in root hairs, could thereby play a role in sensing of the soil K+ availability (Fig. 6B). In the stele, the K+ sensitiv-ity of the SKOR channel would allow adjustment of the flux of K+ excreted to the xylem according to the apoplastic K+ concentration (Johansson et al., 2006). The third hypothesis, proposing a role for AKT1 in K+ sensing, is based on the fact that AKT1 and the NO3

– transceptor NRT1.1 (CHL1) are regulated by the same set of CIPK–CBL partners. As dis-cussed above, AKT1 is phosphorylated and activated by the CIPK23 kinase, which has also been shown to inhibit CHL1. In addition to its contribution to NO3

– uptake from the soil, CHL1 plays a role in sensing the availability of NO3

– in the external medium (Ho et al., 2009), resulting in either low or high levels of nitrate-dependent transcriptional response. It has been proposed that CHL1 senses nitrate availability via specific sites for nitrate, and that CIPK23 phosphorylates CHL1, triggering a low-level response, only when the exter-nal concentration of nitrate sensed by CHL1 is low (Ho et al., 2009; Tsay et al., 2011). By analogy, the regulation of AKT1 by CIPK23 has led to the assumtion that this channel would behave as a K+ sensor, triggering mobilization of CIPK–CBL complexes that, in turn, would lead to its activation (Tsay et al., 2011). Providing direct support for this hypothesis and placing AKT1 upstream of the membrane polarization response, experimental data indicate that sustained long-term changes in membrane polarization in response to altered K+ availability in the soil are not observed in the akt1 mutant when NH4

+ is present in the medium (Hirsch et al., 1998; Spalding et al., 1999). Thus, the hypothesis that AKT1 plays a role in K+ sensing via functional interactions with CIPK23 is very attractive and clearly needs to be investigated fur-ther. This could open a wide field of investigations aiming, for instance, at identifying the AKT1 residues that are phos-phorylated by CIPK23 and their roles in K+ signalling using mutations mimicking phosphorylated and dephosphorylated states.

After a long period of K+ starvation, K+ cytosolic activ-ity declines (Walker et al., 1996). It has been proposed that the cytosolic K+ concentration is sensed by K+-sensitive cytosolic enzymes or by plasma membrane proton pumps (Fig. 6). Regarding the former hypothesis, pyruvate kinases might fulfil the role of internal K+ sensors since they are among the most sensitive enzymes to low K+ concentrations (Armengaud et al., 2009) and, depending on experimental conditions, pyruvate kinase activity is either greatly enhanced (in leaves of wheat plants: Sugiyama et al., 1968) or reduced (in Arabidopsis roots: Armengaud et al., 2009) by K+ starva-tion. Plasma membrane ATPases are also sensitive to internal K+. K+ binds to an aspartate residue in the P-domain and can stimulate dephosphorylation of the E1 (ATP-hydrolysing) form of the ATPase, which prevents conversion to the E2 form and H+ extrusion, thus uncoupling ATP hydrolysis and H+ extrusion (Buch-Pedersen et al., 2006). De-activation of

cytosol

high external K+

plasma membrane

H+ -

pu

mp

g plasma

membrane

low external K+

cytosol

A A role of membrane potential in K+ sensing

C Sensing of internal K+

B Sensing of external K+ by Shaker K+ channels

K+

GORK

K+ sensitive channel gating

CBLs CIPK23

AKT1

K+ K+

NRT1.1

NO3-

H+

AHA2 H+-pump

K+

K+ binding site (D617)

K+

K+-sensitive enzymes

e.g. pyruvate kinase

K+

Fig. 6. Current hypotheses for the mechanisms involved in K+ sensing in the external solution or the cytosol. (A) Role of the membrane potential. K+-permeable conductances (g) short-circuit the electrogenic H+ pumps active at the cell membrane. A decrease or an increase in external K+ concentration affects the flow of K+ through these conductances, resulting in immediate membrane hyperpolarization or depolarization, respectively. Such sensitivity of the membrane potential to the external concentration of K+ can be assumed to play a role in external K+ sensing. It is also worth noting that a long period of K+ deprivation results in sustained membrane hyperpolarization, which might be due to increased activity of the H+ pumps. (B) A role for Shaker channels in external K+ sensing. The hypothesis that the outwardly rectifying GORK channel is involved in external K+ sensing (left panel) is based on the fact that this channel is expressed in root hairs, and displays a gating mechanism dependent on external K+. The inward K+ channel AKT1, which is expressed in root periphery cells, has been hypothesized to play a role in external K+ sensing by analogy with the nitrate transceptor NRT1.1 (CHL1), which mediates both external NO3

– sensing and NO3– uptake

in Arabidopsis roots and is regulated by the kinase CIPK23 like AKT1 (right panel). (C) Putative internal K+-sensing mechanisms. ATP hydrolysis activity of plasma membrane H+-pumps (Buch-Pedersen et al., 2006), and enzymatic activity of pyruvate kinases (Sugiyama et al., 1968; Armengaud et al., 2009), are sensitive to internal K+. This sensitivity to cytosolic K+ has led to the hypothesis that these proteins play a role in internal K+ sensing.

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this inhibitory mechanism results in stimulation of H+ extru-sion. This could be involved in changes in membrane polari-zation observed after several days of K+ starvation. The implications of H+-ATPases and pyruvate kinases in internal K+ signalling have to be supported by physiological studies.

Integration and completion of the different K+-dependent signalling pathways

Plant adaptation to low K+ availability involves an intricate network of signalling pathways, as highlighted in the above paragraphs, and the current knowledge in this domain is sum-marized in Fig. 4. It is clear that the available pieces of the puzzle are still very far from each other and that major infor-mation is missing. Not only the sensing molecules, but also upstream and intermediate components leading to the acti-vation of ethylene, calcium, and ROS pathways are largely unknown. Many questions remain unresolved, such as the activation process leading to ethylene production upstream of RHD2, and the origin of the calcium signal triggering the activation of CIPK–CBL complexes. Regarding the lat-ter process, a first step was accomplished with the identifica-tion of the annexin ANN1 as a Ca2+ transporter. ANN1 is activated by ROS and hyperpolarized membrane potentials, mediates Ca2+ influx, and accounts for 23% of ROS-induced elevation of cytoplasmic calcium in root hairs and epidermal cells (Laohavisit et al., 2012). Other calcium transporters involved in that response remain to be identified.

The regulation scheme might be rendered more complex by cross-talk that is likely to occur between signalling path-ways. Cross-talk exists between ethylene and auxin (Růzicka et al., 2007; Lewis et al., 2011), and between Ca2+ and ROS (Mazars et al., 2010). Regarding this last point, NADPH oxi-dases possess EF-hand motifs that could bind Ca2+ ions, and are also activated by calcium-dependent kinases. Conversely, ROS regulate calcium oscillations (Mazars et al., 2010). Such interconnections are likely to play a role in adaptation to K+ nutritional status. For instance, it has recently been shown in Arabidopsis that CBL1 and CBL9, involved in the calcium-dependent regulation of AKT1, also stimulate ROS produc-tion by the NADPH oxidase AtrbohF (Drerup et al., 2013).

Interplay between K+ and other mineral nutrients

Analysis of the interactions between K+ deficiency and plant mineral nutrition constitutes another promising field of inter-est. A signalling network integrating plant responses to low nitrate availability and K+ deficiency is emerging (Wang and Wu, 2013), and comparison of the responses to N, P, or K deficiencies have revealed common features (Takehisa et al., 2013). Genes encoding membrane proteins carrying solutes other than K+ have been found to display regulation upon K+ shortage. In Arabidopsis, ABC transporter, PIP aquaporin, calcium pump, and high-affinity nitrate transporter genes were down-regulated (Maathuis et al., 2003; Armengaud et al., 2004), whereas genes encoding calmodulins or the cal-cium/cation exchanger CAX3, mediating Ca2+ influx into the vacuole, were up-regulated (Armengaud et al., 2004).

The AHA1 and AHA2 H+-ATPase-encoding genes were mod-erately down-regulated after K+ resupply (Armengaud et al., 2004). In rice, accumulation of HKT transcripts was observed after K+ starvation (Ma et al., 2012; Takehisa et al., 2013), especially that of OsHKT2;1, which displayed an 8-fold increase after a 3 d starvation (Ma et al., 2012). The physi-ological significance of these types of regulation, especially their impact on ion homeostasis and plant growth, clearly deserves to be investigated.

Identification of new signalling pathways and genes

Targeted and systematic studies aimed at identifying genes that are up-regulated after a few hours of K+ deprivation and/or down-regulated after K+ resupply have pinpointed new candidate genes and functions putatively involved in K+ sig-nalling (Maathuis et al., 2003; Armengaud et al., 2004; Shin and Schachtman, 2004; Gierth et al., 2005; Ma et al., 2012; Takehisa et al., 2013). These analyses have also revealed that, unlike nitrate or phosphate deficiency, K+ deficiency does not lead to major alterations in transcript levels (Maathuis et al., 2003; Gierth et al., 2005; Ma et al., 2012; Takehisa et al., 2013). In Arabidopsis, genes regulated after 6 h of K+ depri-vation were mostly up-regulated and therefore likely to play a role in early responses (Armengaud et al., 2004). In rice, only a few genes were regulated after this short time lapse, suggest-ing that most initial events were post-translational (Ma et al., 2012; Takehisa et al., 2013).

It can also be stated that many processes and functions are affected by changes in the plant K+ status, such as metabolic processes (as a long-term consequence of cyto-plasmic K+ decrease), ROS signalling, or protein phospho-rylation/dephosphorylation (by CIPKs and phosphatases for instance) (Shin and Schachtman, 2004; Ma et al., 2012). In Arabidopsis, transcriptomic studies have revealed a prominent role for genes involved in jasmonic acid synthesis or response in shoots (in agreement with the increased susceptibility to pathogens of K+-starved plants), and of stress-related and membrane transporter genes in roots (Armengaud et al., 2004). Changes in the activities of metabolic enzymes are also detectable following prolonged K+ starvation (Armengaud et al., 2009). In rice, many auxin-related (Ma et al., 2012) and jasmonic acid-induced genes (Takehisa et al., 2013) have been found to be regulated in roots in response to K+ deficiency. In Arabidopsis shoots, the Coronatine-Insensitive 1 (COI1) F-box protein has been identified as an essential intermediate of the jasmonic acid signalling pathway for transcriptional gene regulation in response to low K+ (Armengaud et al., 2010). The relationship between K+ deficiency and the jas-monic acid pathway is however still poorly characterized, and its investigation is likely to constitute a major objective in the future.

Very little is known about what happens in shoots upon K+ deficiency. It is however known that stomatal closing in response to water stress is impaired under K+ deficiency condi-tions (Benlloch-Gonzales et al., 2008). It has been proposed that K+, together with other nutrients, is relocated from old senescent leaves to the young photosynthetically active leaves

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to ensure proper stomatal functioning and photosynthetic ability (Amtmann and Armengaud, 2007; Cochrane and Cochrane, 2009), and that jasmonic acid could play a signifi-cant role in that process (Amtmann and Armengaud, 2007; Armengaud et al., 2010). Senescence signals may also move from shoots to roots (Wang et al., 2012). None of these physi-ological responses has been investigated at the molecular level so far. Information provided by transcriptomic studies of leaf responses to K+ deficiency can be expected to provide new clues for addressing the question of shoot adaptation to this stress.

Conclusion

Studies carried out during the last 10 years have allowed iden-tification of interconnected regulatory pathways involved in adaptive responses to potassium deficiency in plants. They have revealed the complexity of these responses, and raised questions that provide the bases for future investigations. Clearly, however, the identified signalling pathways have still to be deciphered further, and many signalling intermediates remain to be discovered. Sensing mechanisms would also warrant further investigation in order to test current hypoth-eses and identify downstream targets. Identification of cross-talk with other pathways such as those involved in nutritional adaptation, development, and pathogen responses will pave the way to integrative studies of plant K+ nutrition and adap-tation to abiotic and biotic environmental conditions.

Acknowledgements

CL was funded by the ANR (Agence Nationale de la Recherche), ‘PumpKin’ project (ANR-08-1 312133).

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Molecular mechanisms involved in plant adaptation to low K+ availability