Molecular mechanisms involved in plant adaptation to low K(+) availability.

Journal of Experimental Botany, Vol. 65, No. 3, pp. 833–848, 2014 doi: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 neutralization of inorganic and organic anions and macromolecules (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 surface due to local depletion (Jungk and Claasen, 1986). In the absence of added fertilizers, K+ availability is limiting for optimal 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

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

Downloaded from http://jxb.oxfordjournals.org/ at Univ Massachusetts Healey Library on September 3, 2014

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834 | Chérel et al.

Physiological responses induced by K+ deprivation Electrical polarization of the cell membrane Whatever the K+ nutritional status of the plant, the membrane potential is immediately and reversibly affected by a change in the external concentration of K+, with a reduction 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 systems 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 (30 membrane proteins, grouped into five families (Shaker, TPK, Kir-like, HAK/KUP/KT, and KEA) are dedicated 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 present 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 socalled HKT transporter family (Fig. 1C). The HKT family, which is present in both monocots and dicots, comprises two phylogenetic subfamilies. HKT subfamily 1 includes transporters permeable to Na+ only [based on functional studies in heterologous systems (oocytes and/or yeast)] and is present 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 contribute 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 monocots is unclear. The hypothesis that these systems could contribute 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 display preferential permeability to K+ (Sassi et al., 2012).

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 rectifying 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 activity (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+ concentrations 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-regulation 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+ deprivation (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 members 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 depleting 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).


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

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


A

B

Shaker channels

KUP/HAK/KT transporters

voltage pore sensor domain S1 membrane

C

HKT transporters pore domain

S6 + + +

cytoplasm N-ter

CNBD

N-ter

N-ter

anky

In planta, inverse correlations between K+ and NH4+ uptake, together with sensitivity of NH4+ uptake to K+ uptake inhibitors, 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 activity 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 concentrations (>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+ concentrations in the medium as low as 10 μM (Hirsch et al., 1998; Spalding et al., 1999). This implies that the membrane potential 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 contributor 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 conditions 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+ conditions, 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, however, 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


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).

Plant response to K+ shortage | 837 stem & leaf

A

Dicots and monocots

AKT2 xylem

HAK5

10-6 M

phloem

10-.4 M

external K+ concentration

Threshold:

SKOR AKT2

"High affinity" active uptake

"Low affinity" passive uptake

HAK transporters

Shaker channels

AKT1 AtKC1/AKT1 Root

out

redundancy and the contribution of the different transport system families to K+ uptake from the soil solution under different 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-regulation 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-regulation of AtHAK5 is not a sine qua non condition of increased capacity for high-affinity K+ uptake, and that post-translational regulation of AtHAK5 activity is also likely to contribute 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 external K+ concentration (see above), is likely to play a role in development of high-affinity K+ uptake capacity. NievesCordones et al. (2008) grew tomato plants under different conditions (the presence or absence of NH4+, K+, and NaCl

K+

in

out

K+

HAK

in

Shaker

Identified transporters

Identified channels

HAK5 in Arabidopsis LeHAK5 in tomato HAK1 in barley and rice

AKT1 with AtKC1 in Arabidopsis OsAKT1 in rice

B

Monocots out

K+ Na+

in

HKT

A role in K+ uptake of HKT transporters? 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 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,


AtHAK5 plays a major role in K+ uptake under low K+ availability and high Na+ concentration by strongly limiting K+ net efflux and allowing re-absorption of K+ during a longterm 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+ hypersensitivity 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 display 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 tolerance 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+ counteracts LeHAK5 induction by low-K+ availability, NH4+ has a beneficial effect and allows LeHAK5 transcript to be accumulated even in the presence of Na+ (Nieves-Cordones et al., 2007) or K+ (Nieves-Cordones et al., 2008).


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 development, 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 identified 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 agravitropic only when starved for K+ (Vicente-Agullo et al., 2004). Recently, it was found that the trh1 mutation results in mislocalization 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+ treatment. 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 suggests that different ethylene-mediated signalling pathways are involved in root development under low-K conditions. Ethylene is involved in auxin synthesis and transport, leading 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 defective 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 synthesis 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 membrane. 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 external K+ concentration immediately affect the cell membrane electrical potential, as indicated above. A decrease in K+ availability results in a decrease in the depolarizing inward K+ current 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


displays unique gating properties within the plant Shaker family, resulting in weak rectification and thereby in efficient capacity to mediate both K+ influx and efflux, depending 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 regulate phloem membrane potential rather than the concentration 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+ starvation (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).

Plant response to K+ shortage | 843

A A role of membrane potential in K

high external K+ low external K+

g

H+-pump

K+

plasma membrane

plasma membrane

cytosol

cytosol

B Sensing of external K

+

by Shaker K+ channels

K+

K+

NO3-

GORK

AKT1

NRT1.1

K+

CBLs CIPK23

C Sensing of internal K

+

H+

K+-sensitive enzymes e.g. pyruvate kinase

AHA2 H+-pump K+ binding site (D617)

K+

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) shortcircuit 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 (BuchPedersen 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.

of high-affinity K+ uptake component remains to be elucidated. 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+ sensitivity 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 discussed 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 external 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 further. This could open a wide field of investigations aiming, for instance, at identifying the AKT1 residues that are phosphorylated by CIPK23 and their roles in K+ signalling using mutations mimicking phosphorylated and dephosphorylated states. After a long period of K+ starvation, K+ cytosolic activity 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+ starvation. 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


+ Ksensitive channel gating

+ sensing

844 | Chérel et al. this inhibitory mechanism results in stimulation of H+ extrusion. This could be involved in changes in membrane polarization 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

Interplay between K+ and other mineral nutrients Analysis of the interactions between K+ deficiency and plant mineral nutrition constitutes another promising field of interest. 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 calcium/cation exchanger CAX3, mediating Ca2+ influx into the vacuole, were up-regulated (Armengaud et al., 2004).

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+ signalling (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+ deprivation 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, suggesting 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 cytoplasmic K+ decrease), ROS signalling, or protein phosphorylation/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 jasmonic 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 conditions (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


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 summarized in Fig. 4. It is clear that the available pieces of the puzzle are still very far from each other and that major information is missing. Not only the sensing molecules, but also upstream and intermediate components leading to the activation 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 latter process, a first step was accomplished with the identification 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 pathways. 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 oxidases 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 calciumdependent regulation of AKT1, also stimulate ROS production by the NADPH oxidase AtrbohF (Drerup et al., 2013).

The AHA1 and AHA2 H+-ATPase-encoding genes were moderately 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 physiological significance of these types of regulation, especially their impact on ion homeostasis and plant growth, clearly deserves to be investigated.

Plant response to K+ shortage | 845 to ensure proper stomatal functioning and photosynthetic ability (Amtmann and Armengaud, 2007; Cochrane and Cochrane, 2009), and that jasmonic acid could play a significant 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 physiological 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

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 varicocele-associated infertility.

Molecular mechanisms of genetic adaptation to xenobiotic compounds.

Molecular mechanisms of attachment of Rhizobium bacteria to plant roots.

Molecular mechanisms underlying phosphate sensing, signaling, and adaptation in plants.

Plant adaptation to drought stress.

Mechanisms of adaptation to cold.

Molecular mechanisms involved in initiation of the DNA damage response.

A Review of Molecular Mechanisms Involved in Toxicity of Nanoparticles.

Molecular and cellular mechanisms involved in leg joint morphogenesis.

Molecular mechanisms involved in tumor repopulation after radiotherapy.

Molecular mechanisms involved in the response to desiccation stress and persistence in Acinetobacter baumannii.

Adaptation to low protein intakes.

K+ uptake in plant roots. The systems involved, their regulation and parallels in other organisms.

Unraveling the early molecular and physiological mechanisms involved in response to phenanthrene exposure.

Hippocampal molecular mechanisms involved in the enhancement of fear extinction caused by exposure to novelty.

Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms.

Molecular Mechanisms of Adaptation of the Moderately Halophilic Bacterium Halobacillis halophilus to Its Environment.

Plant Resistance Inducers against Pathogens in Solanaceae Species-From Molecular Mechanisms to Field Application.

Molecular Mechanisms behind the Physiological Resistance to Intense Transient Warming in an Iconic Marine Plant.

Global plant-responding mechanisms to salt stress: physiological and molecular levels and implications in biotechnology.

Satellite monitoring of desert plant community response to moisture availability.

Transcription factors involved in prostate gland adaptation to androgen deprivation.

Retention of K at low temperature in mouse L cells before and after cold adaptation.

Plant Heat Adaptation: priming in response to heat stress.