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Nitrate, ammonium, and potassium sensing and signaling
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Nitrate, ammonium, and potassium sensing and signaling Cheng-Hsun Ho and Yi-Fang Tsay Plants acquire numerous nutrients from the soil. In addition, nutrients elicit many physiological and morphological responses especially in roots. Recently, there has been significant progress in identifying the sensing and regulatory mechanisms of several essential nutrients. In this review, we describe the newly identified signaling components of nitrate, ammonium, and potassium, focusing specifically on the initial sensing steps.
ment, flowering time, leaf expansion, and seed dormancy in order to balance nitrogen and carbon metabolism and tune plant growth to its availability levels. In recent years, great progress has been made in revealing the molecular mechanism of nitrate perception and signaling, particularly the regulatory network mediating transcriptional and root developmental responses.
Nitrate perception Address Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan Corresponding author: Tsay, Yi-Fang ([email protected])
Current Opinion in Plant Biology 2010, 13:604–610 This review comes from a themed issue on Cell signalling and gene regulation Edited by Zhiyong Wang and Giltsu Choi Available online 15th September 2010 1369-5266/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2010.08.005
Introduction Plants require various essential nutrients, which are present in soils over a wide range of concentrations depending on location, seasonal variation, and daily to yearly changes in climate [1,2]. To overcome such nutrientfluctuating environments, plants have undergone physiological adaptations such as the development of flexible mechanisms in their genetic architecture and gene expression. Recent studies of ions have begun to delineate the signaling mechanisms that underlie the complicated interactions among various responses. Several nutrient-specific transporter/channels as well as nutrient responses have been known for many years; however, at a more basic level, important mechanisms such as how ion sensing occurs and what initiation signals trigger the downstream cascade responses in plants are still not fully understood. Here, using nitrate, ammonium and potassium as examples, we describe recently identified signaling components with emphasis on advances in the understanding of ion sensing mechanisms.
Nitrate responses In aerobic soil conditions, nitrate is the major nitrogen source for most plants. In addition to being a nutrient source, nitrate is also a signaling molecule that regulates the expression of nitrate-related genes, root developCurrent Opinion in Plant Biology 2010, 13:604–610
CHL1 is a dual-affinity nitrate transporter involved in both high-affinity and low-affinity nitrate uptake. CHL1 phosphorylated at threonine residue 101 is a high-affinity nitrate transporter and dephosphorylated CHL1 is a lowaffinity nitrate transporter [3]. In addition to defective nitrate uptake, the chl1 mutant is also defective in several nitrate responses, including repression of high-affinity nitrate transporter NRT2.1 by high nitrate concentration [4], stimulation of lateral root elongation by locally applied nitrate [5], alienation of the inhibitory effect of external glutamate on primary root growth by nitrate [6], and nitrate-regulated expression of nitrate-related genes [7]. These defects suggest that CHL1 participates in nitrate signaling, but because of its transport activity, it is difficult to conclude whether CHL1 is directly or indirectly involved. However, a decoupled mutant with a mutation of P492L between putative 10th and 11th transmembrane domains showed that uptake activity is not required for the sensing function of CHL1, demonstrating that CHL1 is a nitrate sensor (transceptor) [8] (Figure 1). CHL1 is the first transceptor (i.e. transporter and receptor) identified in higher plants; however, several transceptors with or without transport activity have been documented in yeast and mammals for ammonium [9], amino acids [10,11], and glucose [12,13]. Soil nitrate concentration can vary by four orders of magnitude. In contrast with other transceptors, CHL1 with its dual-affinity binding properties can detect a wide range of nitrate concentration changes. To prepare the plant to assimilate nitrate, the expression of nitrate-related genes is induced, a response referred to as the primary nitrate response. Similar to nitrate uptake, the maximum levels of the nitrate-induced transcriptional responses also display two saturable phases, with Km about 40 mM for the highaffinity phase and 1 mM for the low-affinity phase. CHL1 is the sensor for primary nitrate response. In response to low concentrations of nitrate, CIPK23 (a CBL-interacting protein kinase) will phosphorylate CHL1 at T101, and T101-phosphorylated CHL1 will induce a low-level primary response [8] (Figure 1). When exposed to high concentrations of nitrate, T101 phosphorylation is prohibited and dephosphorylated-CHL1 leads to a high-level www.sciencedirect.com
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Figure 1
Overview of recent advances in nitrate sensing and signaling mechanisms. External nitrate ions (NO3 ) can be detected by sensors in the plasma membrane, and then regulate nitrate-related gene expression and root growth in plants. By changing the phosphorylation status of T101 residue, nitrate sensor CHL can detect a wide range of nitrate concentrations in soil. Under low nitrate conditions, CIPK23 phosphorylates CHL1 at T101. CIPK8, NLP7 and LBD37/38/39 are signaling components in the primary nitrate response. The target of CIPK8 remains to be identified. For root development, CHL1 acts upstream of MADS-box transcription factor ANR1. NRT2.1 or another nitrate sensing system may exist for nitrate response.
primary nitrate response. Therefore, the phosphorylation status of CHL1 T101 not only switches the transport modes but also induces different levels of primary nitrate response. How the phosphorylation status of T101 modulates the transcriptional levels, however, remains an open question. It is possible that CHL1 homologues of the NRT1/PTR family in Arabidopsis and other plants may also function as transceptors. By position cloning, transporters from the NRT1/PTR family were found to be responsible for the short-panicle phenotype in rice (SP1) and nodule development defect in Medicago truncatula (LATD/NIP) [14,15]. In M. truncatula, both nitrate and ammonium inhibit primary root growth. The latd/nip mutants responded normally to ammonium but had defects in responses of the root architecture to nitrate, suggesting that LATD/NIP participates in nitrate signaling. It will be interesting to determine whether nitrate perception or transport activity is responsible for the developmental defects in sp1 and latd/nip mutants. It is worth noting that consistent with the decoupled mutant CHL1 P492L, which has defective transport activity but normal sensing www.sciencedirect.com
function, SP1 does not have the conserved proline residue found in most NRT1 transporters and shows no nitrate transport activity when expressed in oocytes [14]. It is also possible that Arabidopsis could have some other type(s) of nitrate sensors. For example, another nitrate-transporter mutant nrt2.1 has also been shown to be defective in nitrate-regulated root development [16,17]. Since expression of CHL1 is downregulated by nitrogen starvation while expression of NRT2.1 is upregulated [18], NRT2.1 might be the dominant nitrate sensor under nitrogenlimited conditions. Moreover, cytosolic and vacuolar nitrate sensors, important for the coordination between assimilation and storage, remain to be uncovered.
Nitrate signaling pathways CIPK23 is a negative regulator of the high-affinity phase of the primary nitrate response [8]; however, CIPK8 is a positive regulator of the low-affinity primary response [19]. Studies of CIPK23 and CIPK8 indicate that different signaling networks are responsible for high-affinity and low-affinity nitrate responses. Similar to its algal homologue NIT2 (which regulates nitrate assimilation by binding to the nitrate reductase gene-promoter [20]), Current Opinion in Plant Biology 2010, 13:604–610
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nodule inception-like protein, NLP7 in Arabidopsis was shown to be a positive regulator of the primary nitrate response [21] (Figure 1). Members of a family of a different type of transcription factor, LBD37/38/39 (LATERAL ORGAN BOUNDARY DOMAIN), have also been shown to be negative regulators of nitrate-related genes as well as anthocyanin synthesis [22]. CIPK8, CIPK23 and LBD37/38/39 were first identified and found to be primary nitrate-response genes from transcriptome analyses [19,22], therefore some of the genes involved in primary nitrate response are coordinately regulated by their own signaling network. Another nitrate response which has been studied in detail is nitrate-modulated root architecture. A decade ago, it was shown that external nitrate stimulates lateral root growth via a signaling pathway involving the ANR1 MADS-box transcription factor [23]. Using a different assay method, a recent study showed that CHL1 could be the upstream sensor of ANR1 in regulating lateral root proliferation [5]. In addition, CHL1 also participates in regulating primary root growth. External glutamate inhibits primary root growth. Nitrate can antagonize the glutamate inhibitory effect in wild type and anr1 mutant but not in chl1 mutant, indicating that CHL1 but not ANR1 is involved in nitrate-regulated primary root growth [6]. Therefore, although CHL1 is involved in
nitrate-regulated transcriptional response, lateral root proliferation, and primary root growth, there may be differences in the downstream signaling networks.
Ammonium responses Under anoxic soil conditions, ammonium will be the primary nitrogen source, but its essentiality stands in contrast with its toxicity in excess. In tobacco, inhibition of leaf growth was shown to be an early response to ammonium treatment [24]. The mechanisms underlying ammonium toxicity are not fully understood, but acidification of the external environment, disruption of the acid/base balance and the energy lost exporting excess ammonium may be key factors. Moreover, study of an ammonium hypersensitive mutant hsn1, defective in GDP (guanosine diphosphate) mannose pyrophosphorylase suggested that, as in animals, ammonium sensitivity in plants is associated with reduced protein glycosylation [25].
Ammonium perception Transcriptional and post-transcriptional regulation of the ammonium transporters (AMTs) can prevent ammonium toxicity. Four ammonium transporters AMT1.1, AMT1.2, AMT1.3, and AMT1.5 are involved in ammonium acquisition [26]. At the transcriptional level, AtAMT1.1 expression in Arabidopsis root is generally repressed by high
Figure 2
Overview of ammonium (NH4+) sensing and signaling mechanism. Expression of ammonium transporter AMT1.1 is repressed by glutamine and derepressed in nitrogen-deficient conditions. At the post-translational level, phosphorylation of a threonine residue in the C-terminal exhibits an allosteric effect leading to the cooperative closure of the trimer. In yeast, MEP2 in the same AMT family has been proposed to act as an ammonium sensor. The transport and sensing function of MEP2 is regulated by Npr1 kinase, which is activated by dephosphorylation upon nitrogen deprivation. Current Opinion in Plant Biology 2010, 13:604–610
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nitrogen, most likely by the internal pool of Gln, and derepressed under nitrogen deficiency [27,28]. At the post-transcriptional level, phosphorylation of a threonine residue (460 in AMT1.1 and 472 in AMT1.2) in the Cterminal of a single monomer exhibits an allosteric effect, leading to the cooperative closure of all three pores in the trimer [29,30]. More interestingly, phosphorylation of T460 is upregulated by rhizosphere ammonium in a concentration and time-dependent manner [31]. These findings suggest that either some unknown sensor or AMT1.1, functioning as a transceptor, is able to detect external ammonium status and downregulate the transport activity of AMT to prevent toxic accumulation of ammonium (Figure 2). Indeed, in yeast, an ammonium carrier MEP2 (methylammonium permease), a member of the same family as the AMTs, has been proposed to act as an ammonium sensor for ammonium limitation-induced diploid pseudohyphal growth and haploid invasive growth, and for ammonium-induced activation of PKA and trehalase [9,32] (Figure 2). In yeast, there are three ammonium carriers, MEP1, MEP2 and MEP3, involved in ammonium uptake, but only MEP2 is responsible for pseudohyphal growth induced by nitrogen limitation. The transport and sensing functions of MEP2 are
regulated by Npr1 (Nitrogen permease reactivator protein) kinase, which is activated by dephosphorylation upon nitrogen deprivation [33]. Decoupled mutants defective only in sensing or only in transport have been isolated [32,34], suggesting that sensing and transport might be two independent processes of a protein with no order of precedence. In Arabidopsis, AMT2.1, which shares high sequence similarity with yeast MEP transporters, showed no ammonium uptake defect when mutated [26]. When expressed in yeast, AtAMT2.1 could sustain both transport and trehalase activation but not haploid invasive growth [32], suggesting that AMT2.1 can sustain some parts of the sensing function of MEP2, and thus may exhibit similar activity in plants.
Potassium responses Potassium is the most abundant cation in plants. Unlike nitrate and ammonium, potassium, serving as a major osmoticum, is not assimilated into organic matter. Studies of potassium signaling focus on potassium starvation responses. Physiological consequences of potassium deficiency include growth arrest, impaired phloem transport of sucrose, redistribution of potassium from mature to developing tissues, reduced photosynthesis, reduced
Figure 3
Overview of potassium (K+)-dependent signaling cascades in response to potassium limitation. Low potassium activates ethylene and calcium signals to regulate ROS production and the CBL-CIPK23 network, respectively. CIPK23 phosphorylates and then activates the potassium uptake channel AKT1. ROS production stimulates root hair elongation and upregulates the expression of potassium transporter HAK5, to enhance potassium uptake and stress tolerance. Mutation of CIPK9 did not affect potassium uptake but resulted in a low potassium phenotype, suggesting a different signaling system in potassium deprivation [48]. www.sciencedirect.com
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water content and replacement of potassium with an alternative osmoticum [35]. To obtain better yield under potassium deficiency, high-affinity potassium uptake systems are activated at both the transcriptional and posttranscriptional levels [36,37,38].
Potassium perception In Escherichia coli, kinase KdpD functions as a potassium sensor [39], but no such system was found in plants. Although it is not clear how plants detect soil potassium concentration changes, it has been proposed that hyperpolarization of plasma membrane, due to reduction of potassium influx and activation of H+-ATPase could initiate an immediate response to potassium deficiency [40]. In addition to participating in nitrate signaling, CIPK23 is also involved in potassium deficiency response [36,37]. AKT1, an influx potassium channel, and HAK5, a high affinity potassium (K) transporter, are responsible for potassium acquisition under low potassium conditions [41,42]. CIPK23, activated by calcium-binding proteins CBL1 and CBL9 (calcineurin B-like protein), were able to phosphorylate AKT1 and enhance AKT1 uptake activity [36,37]. Both the cipk23 mutant and the cbl1cbl9 double mutant showed a low potassium sensitive phenotype. These data suggest that low potassium detected by some unknown sensor may trigger calcium signaling, activate CBL1/9, and then activate CIPK23 to enhance the channel activity of AKT1 (Figure 3). Alternatively, as an extension of our knowledge of the CIPK23 regulation of nitrate sensor CHL1 [8], it is possible to postulate that AKT1 also functions as a potassium sensor, detecting the external potassium changes and leading to the activation of the CIPK23/CBL complex and then phosphorylating itself. The gating properties of AKT1 are known to be modified by forming the complex with KC (K+-selective ion channel) and SNARE (SNAP receptors) [43,44]; if this second hypothesis is correct these interactions could add dynamic modulation to the sensing process.
Conclusions and remarks The activities of several transporters/channels for nutrient acquisition are regulated by their substrate in the soil via modification of phosphorylation. This mechanism serves as a safeguard to prevent the toxic effects of overaccumulation, or to energize uptake rate when needed. As a further evolutionary adaptation, the regulatory mechanism can participate directly in ion sensing, and trigger downstream responses. Despite the recent identification of several new signaling components in nutrient sensing, there are still a lot of gaps to be filled between the perception of transcriptional responses and developmental responses and the readouts we are obtaining. Other unsolved issues are the connection between different ions and the crosstalk between nutrient sensing and hormone signaling. For example the question of how plant root systems are able to differentiate between the specific signal cues and then regulate the same component appropriately according to each different signaling response, e.g. CIPK23 is involved in both nitrate and potassium signaling. It is expected that the new molecular markers together with the development of recent new technologies will make the challenge of mapping the sequence of the events from nutrient perception through to expression changes and developmental/growth response a much more attainable goal.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Potassium signaling pathways Expression of high-affinity potassium transporter HAK5 is upregulated by potassium deprivation. Upregulation of HAK5 depends on ROS production mediated by a NADPH oxidase rhd2 and a type III peroxidase RCI3 [45,46]. Another important signal component involved in potassium deprivation is ethylene. In low potassium conditions, the promotion of root hairs, inhibition of primary root growth, and ROS production as well as the induction of HAK5 expression were eliminated in plants treated with ethylene inhibitors, and partially eliminated in ethylene insensitive mutants (e.g. ein2 and ctr1) [47]. These results indicate that ethylene acts upstream of ROS in response to potassium deprivation (Figure 3). Current Opinion in Plant Biology 2010, 13:604–610
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Walch-Liu P, Forde BG: Nitrate signalling mediated by the NRT1.1 nitrate transporter antagonises L-glutamate-induced changes in root architecture. Plant J 2008, 54:820-828. Plant root architecture is responsive to changes in nitrogen supply. Plants’ sensitivity to nitrate was restored in a chl1–5 mutant constitutively expressing NRT1.1. However, expression in chl1–5, a dephosphorylated form of NRT1.1, not only failed to restore nitrate sensitivity but also had a dominant-negative effect on Glu sensitivity suggesting that NRT1.1 has a nitrate-sensing role regulating primary root growth.
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Using a forward genetic approach, this study showed that CHL1 is involved in primary nitrate response. In addition, it also showed that the contribution of CHL1 in signaling depends on the nitrogen status of the plant. 8. Ho CH, Lin SH, Hu HC, Tsay YF: CHL1 functions as a nitrate sensor in plants. Cell 2009, 138:1184-1194. This study demonstrated that dual-affinity nitrate transporter CHL1 also functions as a nitrate sensor in plants. Using the sensing and uptake decoupled mutant chl1–9, it was shown that nitrate transport was not required for the sensing function of CHL1. In addition, this work identified a protein kinase CIPK23 involved in the phosphorylation regulation mechanism that gives CHL1 sensing capacity over a wide range of concentrations. 9.
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This study showed for the first time that a kinase in the CIPK family is involved in nutrient signaling. Using genetic, biochemical and electrophysiological analyses, it was demonstrated that AKT1, as a direct downstream target of CIPK23, is regulated by a CBL–CIPK network in response to potassium limitation. 38. Qi Z, Hampton CR, Shin R, Barkla BJ, White PJ, Schachtman DP: The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis. J Exp Bot 2008, 59:595-607. 39. Walderhaug MO, Polarek JW, Voelkner P, Daniel JM, Hesse JE, Altendorf K, Epstein W: KdpD and KdpE, proteins that control expression of the kdpABC operon, are members of the two-component sensor-effector class of regulators. J Bacteriol 1992, 174:2152-2159. 40. Amtmann A, Hammond JP, Armengaud P, White PJ: Nutrient sensing and signalling in plants: potassium and phosphorus. Adv Bot Res 2006, 43:209-257. 41. Rubio F, Nieves-Cordones M, Aleman F, Martinez V: Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high-affinity range of concentrations. Physiol Plantarum 2008, 134:598-608. 42. Gierth M, Maser P, Schroeder JI: The potassium transporter AtHAK5 functions in K+ deprivation-induced high-affinity K+ uptake and AKT1 K+ channel contribution to K+ uptake kinetics in Arabidopsis roots. Plant Physiol 2005, 137:1105-1114. 43. Honsbein A, Sokolovski S, Grefen C, Campanoni P, Pratelli R, Paneque M, Chen Z, Johansson I, Blatt MR: A tripartite
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Nitrate, ammonium, and potassium sensing and signaling Cheng-Hsun Ho and Yi-Fang Tsay Plants acquire numerous nutrients from the soil. In addition, nutrients elicit many physiological and morphological responses especially in roots. Recently, there has been significant progress in identifying the sensing and regulatory mechanisms of several essential nutrients. In this review, we describe the newly identified signaling components of nitrate, ammonium, and potassium, focusing specifically on the initial sensing steps.
ment, flowering time, leaf expansion, and seed dormancy in order to balance nitrogen and carbon metabolism and tune plant growth to its availability levels. In recent years, great progress has been made in revealing the molecular mechanism of nitrate perception and signaling, particularly the regulatory network mediating transcriptional and root developmental responses.
Nitrate perception Address Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan Corresponding author: Tsay, Yi-Fang ([email protected])
Current Opinion in Plant Biology 2010, 13:604–610 This review comes from a themed issue on Cell signalling and gene regulation Edited by Zhiyong Wang and Giltsu Choi Available online 15th September 2010 1369-5266/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2010.08.005
Introduction Plants require various essential nutrients, which are present in soils over a wide range of concentrations depending on location, seasonal variation, and daily to yearly changes in climate [1,2]. To overcome such nutrientfluctuating environments, plants have undergone physiological adaptations such as the development of flexible mechanisms in their genetic architecture and gene expression. Recent studies of ions have begun to delineate the signaling mechanisms that underlie the complicated interactions among various responses. Several nutrient-specific transporter/channels as well as nutrient responses have been known for many years; however, at a more basic level, important mechanisms such as how ion sensing occurs and what initiation signals trigger the downstream cascade responses in plants are still not fully understood. Here, using nitrate, ammonium and potassium as examples, we describe recently identified signaling components with emphasis on advances in the understanding of ion sensing mechanisms.
Nitrate responses In aerobic soil conditions, nitrate is the major nitrogen source for most plants. In addition to being a nutrient source, nitrate is also a signaling molecule that regulates the expression of nitrate-related genes, root developCurrent Opinion in Plant Biology 2010, 13:604–610
CHL1 is a dual-affinity nitrate transporter involved in both high-affinity and low-affinity nitrate uptake. CHL1 phosphorylated at threonine residue 101 is a high-affinity nitrate transporter and dephosphorylated CHL1 is a lowaffinity nitrate transporter [3]. In addition to defective nitrate uptake, the chl1 mutant is also defective in several nitrate responses, including repression of high-affinity nitrate transporter NRT2.1 by high nitrate concentration [4], stimulation of lateral root elongation by locally applied nitrate [5], alienation of the inhibitory effect of external glutamate on primary root growth by nitrate [6], and nitrate-regulated expression of nitrate-related genes [7]. These defects suggest that CHL1 participates in nitrate signaling, but because of its transport activity, it is difficult to conclude whether CHL1 is directly or indirectly involved. However, a decoupled mutant with a mutation of P492L between putative 10th and 11th transmembrane domains showed that uptake activity is not required for the sensing function of CHL1, demonstrating that CHL1 is a nitrate sensor (transceptor) [8] (Figure 1). CHL1 is the first transceptor (i.e. transporter and receptor) identified in higher plants; however, several transceptors with or without transport activity have been documented in yeast and mammals for ammonium [9], amino acids [10,11], and glucose [12,13]. Soil nitrate concentration can vary by four orders of magnitude. In contrast with other transceptors, CHL1 with its dual-affinity binding properties can detect a wide range of nitrate concentration changes. To prepare the plant to assimilate nitrate, the expression of nitrate-related genes is induced, a response referred to as the primary nitrate response. Similar to nitrate uptake, the maximum levels of the nitrate-induced transcriptional responses also display two saturable phases, with Km about 40 mM for the highaffinity phase and 1 mM for the low-affinity phase. CHL1 is the sensor for primary nitrate response. In response to low concentrations of nitrate, CIPK23 (a CBL-interacting protein kinase) will phosphorylate CHL1 at T101, and T101-phosphorylated CHL1 will induce a low-level primary response [8] (Figure 1). When exposed to high concentrations of nitrate, T101 phosphorylation is prohibited and dephosphorylated-CHL1 leads to a high-level www.sciencedirect.com
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Figure 1
Overview of recent advances in nitrate sensing and signaling mechanisms. External nitrate ions (NO3 ) can be detected by sensors in the plasma membrane, and then regulate nitrate-related gene expression and root growth in plants. By changing the phosphorylation status of T101 residue, nitrate sensor CHL can detect a wide range of nitrate concentrations in soil. Under low nitrate conditions, CIPK23 phosphorylates CHL1 at T101. CIPK8, NLP7 and LBD37/38/39 are signaling components in the primary nitrate response. The target of CIPK8 remains to be identified. For root development, CHL1 acts upstream of MADS-box transcription factor ANR1. NRT2.1 or another nitrate sensing system may exist for nitrate response.
primary nitrate response. Therefore, the phosphorylation status of CHL1 T101 not only switches the transport modes but also induces different levels of primary nitrate response. How the phosphorylation status of T101 modulates the transcriptional levels, however, remains an open question. It is possible that CHL1 homologues of the NRT1/PTR family in Arabidopsis and other plants may also function as transceptors. By position cloning, transporters from the NRT1/PTR family were found to be responsible for the short-panicle phenotype in rice (SP1) and nodule development defect in Medicago truncatula (LATD/NIP) [14,15]. In M. truncatula, both nitrate and ammonium inhibit primary root growth. The latd/nip mutants responded normally to ammonium but had defects in responses of the root architecture to nitrate, suggesting that LATD/NIP participates in nitrate signaling. It will be interesting to determine whether nitrate perception or transport activity is responsible for the developmental defects in sp1 and latd/nip mutants. It is worth noting that consistent with the decoupled mutant CHL1 P492L, which has defective transport activity but normal sensing www.sciencedirect.com
function, SP1 does not have the conserved proline residue found in most NRT1 transporters and shows no nitrate transport activity when expressed in oocytes [14]. It is also possible that Arabidopsis could have some other type(s) of nitrate sensors. For example, another nitrate-transporter mutant nrt2.1 has also been shown to be defective in nitrate-regulated root development [16,17]. Since expression of CHL1 is downregulated by nitrogen starvation while expression of NRT2.1 is upregulated [18], NRT2.1 might be the dominant nitrate sensor under nitrogenlimited conditions. Moreover, cytosolic and vacuolar nitrate sensors, important for the coordination between assimilation and storage, remain to be uncovered.
Nitrate signaling pathways CIPK23 is a negative regulator of the high-affinity phase of the primary nitrate response [8]; however, CIPK8 is a positive regulator of the low-affinity primary response [19]. Studies of CIPK23 and CIPK8 indicate that different signaling networks are responsible for high-affinity and low-affinity nitrate responses. Similar to its algal homologue NIT2 (which regulates nitrate assimilation by binding to the nitrate reductase gene-promoter [20]), Current Opinion in Plant Biology 2010, 13:604–610
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nodule inception-like protein, NLP7 in Arabidopsis was shown to be a positive regulator of the primary nitrate response [21] (Figure 1). Members of a family of a different type of transcription factor, LBD37/38/39 (LATERAL ORGAN BOUNDARY DOMAIN), have also been shown to be negative regulators of nitrate-related genes as well as anthocyanin synthesis [22]. CIPK8, CIPK23 and LBD37/38/39 were first identified and found to be primary nitrate-response genes from transcriptome analyses [19,22], therefore some of the genes involved in primary nitrate response are coordinately regulated by their own signaling network. Another nitrate response which has been studied in detail is nitrate-modulated root architecture. A decade ago, it was shown that external nitrate stimulates lateral root growth via a signaling pathway involving the ANR1 MADS-box transcription factor [23]. Using a different assay method, a recent study showed that CHL1 could be the upstream sensor of ANR1 in regulating lateral root proliferation [5]. In addition, CHL1 also participates in regulating primary root growth. External glutamate inhibits primary root growth. Nitrate can antagonize the glutamate inhibitory effect in wild type and anr1 mutant but not in chl1 mutant, indicating that CHL1 but not ANR1 is involved in nitrate-regulated primary root growth [6]. Therefore, although CHL1 is involved in
nitrate-regulated transcriptional response, lateral root proliferation, and primary root growth, there may be differences in the downstream signaling networks.
Ammonium responses Under anoxic soil conditions, ammonium will be the primary nitrogen source, but its essentiality stands in contrast with its toxicity in excess. In tobacco, inhibition of leaf growth was shown to be an early response to ammonium treatment [24]. The mechanisms underlying ammonium toxicity are not fully understood, but acidification of the external environment, disruption of the acid/base balance and the energy lost exporting excess ammonium may be key factors. Moreover, study of an ammonium hypersensitive mutant hsn1, defective in GDP (guanosine diphosphate) mannose pyrophosphorylase suggested that, as in animals, ammonium sensitivity in plants is associated with reduced protein glycosylation [25].
Ammonium perception Transcriptional and post-transcriptional regulation of the ammonium transporters (AMTs) can prevent ammonium toxicity. Four ammonium transporters AMT1.1, AMT1.2, AMT1.3, and AMT1.5 are involved in ammonium acquisition [26]. At the transcriptional level, AtAMT1.1 expression in Arabidopsis root is generally repressed by high
Figure 2
Overview of ammonium (NH4+) sensing and signaling mechanism. Expression of ammonium transporter AMT1.1 is repressed by glutamine and derepressed in nitrogen-deficient conditions. At the post-translational level, phosphorylation of a threonine residue in the C-terminal exhibits an allosteric effect leading to the cooperative closure of the trimer. In yeast, MEP2 in the same AMT family has been proposed to act as an ammonium sensor. The transport and sensing function of MEP2 is regulated by Npr1 kinase, which is activated by dephosphorylation upon nitrogen deprivation. Current Opinion in Plant Biology 2010, 13:604–610
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nitrogen, most likely by the internal pool of Gln, and derepressed under nitrogen deficiency [27,28]. At the post-transcriptional level, phosphorylation of a threonine residue (460 in AMT1.1 and 472 in AMT1.2) in the Cterminal of a single monomer exhibits an allosteric effect, leading to the cooperative closure of all three pores in the trimer [29,30]. More interestingly, phosphorylation of T460 is upregulated by rhizosphere ammonium in a concentration and time-dependent manner [31]. These findings suggest that either some unknown sensor or AMT1.1, functioning as a transceptor, is able to detect external ammonium status and downregulate the transport activity of AMT to prevent toxic accumulation of ammonium (Figure 2). Indeed, in yeast, an ammonium carrier MEP2 (methylammonium permease), a member of the same family as the AMTs, has been proposed to act as an ammonium sensor for ammonium limitation-induced diploid pseudohyphal growth and haploid invasive growth, and for ammonium-induced activation of PKA and trehalase [9,32] (Figure 2). In yeast, there are three ammonium carriers, MEP1, MEP2 and MEP3, involved in ammonium uptake, but only MEP2 is responsible for pseudohyphal growth induced by nitrogen limitation. The transport and sensing functions of MEP2 are
regulated by Npr1 (Nitrogen permease reactivator protein) kinase, which is activated by dephosphorylation upon nitrogen deprivation [33]. Decoupled mutants defective only in sensing or only in transport have been isolated [32,34], suggesting that sensing and transport might be two independent processes of a protein with no order of precedence. In Arabidopsis, AMT2.1, which shares high sequence similarity with yeast MEP transporters, showed no ammonium uptake defect when mutated [26]. When expressed in yeast, AtAMT2.1 could sustain both transport and trehalase activation but not haploid invasive growth [32], suggesting that AMT2.1 can sustain some parts of the sensing function of MEP2, and thus may exhibit similar activity in plants.
Potassium responses Potassium is the most abundant cation in plants. Unlike nitrate and ammonium, potassium, serving as a major osmoticum, is not assimilated into organic matter. Studies of potassium signaling focus on potassium starvation responses. Physiological consequences of potassium deficiency include growth arrest, impaired phloem transport of sucrose, redistribution of potassium from mature to developing tissues, reduced photosynthesis, reduced
Figure 3
Overview of potassium (K+)-dependent signaling cascades in response to potassium limitation. Low potassium activates ethylene and calcium signals to regulate ROS production and the CBL-CIPK23 network, respectively. CIPK23 phosphorylates and then activates the potassium uptake channel AKT1. ROS production stimulates root hair elongation and upregulates the expression of potassium transporter HAK5, to enhance potassium uptake and stress tolerance. Mutation of CIPK9 did not affect potassium uptake but resulted in a low potassium phenotype, suggesting a different signaling system in potassium deprivation [48]. www.sciencedirect.com
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water content and replacement of potassium with an alternative osmoticum [35]. To obtain better yield under potassium deficiency, high-affinity potassium uptake systems are activated at both the transcriptional and posttranscriptional levels [36,37,38].
Potassium perception In Escherichia coli, kinase KdpD functions as a potassium sensor [39], but no such system was found in plants. Although it is not clear how plants detect soil potassium concentration changes, it has been proposed that hyperpolarization of plasma membrane, due to reduction of potassium influx and activation of H+-ATPase could initiate an immediate response to potassium deficiency [40]. In addition to participating in nitrate signaling, CIPK23 is also involved in potassium deficiency response [36,37]. AKT1, an influx potassium channel, and HAK5, a high affinity potassium (K) transporter, are responsible for potassium acquisition under low potassium conditions [41,42]. CIPK23, activated by calcium-binding proteins CBL1 and CBL9 (calcineurin B-like protein), were able to phosphorylate AKT1 and enhance AKT1 uptake activity [36,37]. Both the cipk23 mutant and the cbl1cbl9 double mutant showed a low potassium sensitive phenotype. These data suggest that low potassium detected by some unknown sensor may trigger calcium signaling, activate CBL1/9, and then activate CIPK23 to enhance the channel activity of AKT1 (Figure 3). Alternatively, as an extension of our knowledge of the CIPK23 regulation of nitrate sensor CHL1 [8], it is possible to postulate that AKT1 also functions as a potassium sensor, detecting the external potassium changes and leading to the activation of the CIPK23/CBL complex and then phosphorylating itself. The gating properties of AKT1 are known to be modified by forming the complex with KC (K+-selective ion channel) and SNARE (SNAP receptors) [43,44]; if this second hypothesis is correct these interactions could add dynamic modulation to the sensing process.
Conclusions and remarks The activities of several transporters/channels for nutrient acquisition are regulated by their substrate in the soil via modification of phosphorylation. This mechanism serves as a safeguard to prevent the toxic effects of overaccumulation, or to energize uptake rate when needed. As a further evolutionary adaptation, the regulatory mechanism can participate directly in ion sensing, and trigger downstream responses. Despite the recent identification of several new signaling components in nutrient sensing, there are still a lot of gaps to be filled between the perception of transcriptional responses and developmental responses and the readouts we are obtaining. Other unsolved issues are the connection between different ions and the crosstalk between nutrient sensing and hormone signaling. For example the question of how plant root systems are able to differentiate between the specific signal cues and then regulate the same component appropriately according to each different signaling response, e.g. CIPK23 is involved in both nitrate and potassium signaling. It is expected that the new molecular markers together with the development of recent new technologies will make the challenge of mapping the sequence of the events from nutrient perception through to expression changes and developmental/growth response a much more attainable goal.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Potassium signaling pathways Expression of high-affinity potassium transporter HAK5 is upregulated by potassium deprivation. Upregulation of HAK5 depends on ROS production mediated by a NADPH oxidase rhd2 and a type III peroxidase RCI3 [45,46]. Another important signal component involved in potassium deprivation is ethylene. In low potassium conditions, the promotion of root hairs, inhibition of primary root growth, and ROS production as well as the induction of HAK5 expression were eliminated in plants treated with ethylene inhibitors, and partially eliminated in ethylene insensitive mutants (e.g. ein2 and ctr1) [47]. These results indicate that ethylene acts upstream of ROS in response to potassium deprivation (Figure 3). Current Opinion in Plant Biology 2010, 13:604–610
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