TY - JOUR
AU1 - Krapp, Anne
AU2 - David, Laure C.
AU3 - Chardin, Camille
AU4 - Girin, Thomas
AU5 - Marmagne, Anne
AU6 - Leprince, Anne-Sophie
AU7 - Chaillou, Sylvain
AU8 - Ferrario-Méry, Sylvie
AU9 - Meyer, Christian
AU1 - Daniel-Vedele, Françoise
AB - Abstract Plants have developed adaptive responses allowing them to cope with nitrogen (N) fluctuation in the soil and maintain growth despite changes in external N availability. Nitrate is the most important N form in temperate soils. Nitrate uptake by roots and its transport at the whole-plant level involves a large panoply of transporters and impacts plant performance. Four families of nitrate-transporting proteins have been identified so far: nitrate transporter 1/peptide transporter family (NPF), nitrate transporter 2 family (NRT2), the chloride channel family (CLC), and slow anion channel-associated homologues (SLAC/SLAH). Nitrate transporters are also involved in the sensing of nitrate. It is now well established that plants are able to sense external nitrate availability, and hence that nitrate also acts as a signal molecule that regulates many aspects of plant intake, metabolism, and gene expression. This review will focus on a global picture of the nitrate transporters so far identified and the recent advances in the molecular knowledge of the so-called primary nitrate response, the rapid regulation of gene expression in response to nitrate. The recent discovery of the NIN-like proteins as master regulators for nitrate signalling has led to a new understanding of the regulation cascade. Arabidopsis, nitrate transport, nitrate signalling, nitrogen, primary nitrate response, transport. Introduction Nitrogen (N) absorption at the root level determines plant growth and productivity for most plants. N can be found in soils under the form of nitrate, ammonium, amino acids, proteins, and other N-containing substances. In well-aerated soils, nitrification is rapid, and nitrate is the primary N source (Crawford and Forde, 2002). Therefore, this review focuses on that particular form. In natural habitats, soil nitrate concentrations are often <1mM, whereas in fertilized agricultural soils concentrations up to 70mM can be found (Reisenauer, 1966). Nitrate is readily dissolved in the soil solution and this mobility leads to rapidly changing concentrations across arable fields, for example, where nitrate concentrations can vary 100-fold (Lark et al., 2004). Clearly, plants need to acclimate to these changing supplies to optimize nitrate acquisition. This can be achieved through modifications of root architecture, adjustment of uptake and transport, and optimized metabolism. These acclimation processes are triggered by signals from the shoot according to its N-satiety status via systemic signalling (Ruffel et al., 2011) and also by direct nitrate signalling (Miller et al., 2007). Indeed, nitrate, besides being a nutrient, is a major signalling molecule. In this review, we focus on nitrate transport and nitrate signalling. Nitrate transport Under various environmental conditions, plants need to acquire nitrate efficiently from the soil, distribute it between source and sink organs, and adjust nitrate homeostasis at the cellular level. To do so, plants use a combination of transporters and channels with diverse ranges of affinity and specificity. Most of the functional studies of these proteins have been performed on Arabidopsis thaliana. Proteins encoded by four gene families have been shown to function as nitrate transporters in Arabidopsis: NRT1/PTR (NPF; nitrate transporter 1/peptide transporter family, 53 members), NRT2 (seven members), CLC (chloride channels, seven members), and SLAC1/SLAH (slow anion channel-associated 1 homologues, five members). Altogether these four families contain 73 genes, but only 35 have been characterized in detail and, among these, 24 encode nitrate transporters. The nitrate transporters characterized so far play diverse roles in nitrate transport on the cellular and whole-plant level and thus under various developmental and environmental conditions. In this review, we will use the new nomenclature for the NRT1/PTR family members as proposed by Léran et al. (2014). The old names are given in parentheses to facilitate the transition to this new nomenclature. Molecular structure and transport mechanism NRT1/PTR and NRT2 families The first nitrate transporter that was identified as early as 1993, NPF6.3 (NRT1.1/CHL1), belongs to the large NRT1/PTR family (NPF; Tsay et al., 1993). The common predicted structure displays 12 transmembrane domains connected by short peptide loops and, in the case of plant NPFs, a large hydrophilic loop between transmembrane domains 6 and 7 (Tsay et al., 2007). To date, nitrate transport activity has been demonstrated for 16 out of 53 NPF proteins in Arabidopsis (Hsu and Tsay, 2013; Léran et al., 2014). Besides nitrate, NPF proteins can transport amino acids, peptides, glucosinolates, auxin, and abscisic acid (ABA) (reviewed in Léran et al., 2014). Several NPF proteins transport more than one substrate. The nitrate transporters of this family have low affinity for nitrate (KM >1mM), except NPF6.3 (NRT1.1/CHL1). NPF6.3 not only displays dual affinity for nitrate in the high and low affinity ranges (Wang et al., 1998; Liu et al., 1999; Liu and Tsay, 2003), but also transports auxin (Krouk et al., 2010a) and is involved in nitrate sensing (Ho and Tsay, 2010; see below). The NRT2 gene family belongs to the major facilitator superfamily (MFS) of transporters and was first identified in higher plants (including Arabidopsis) based on the identification of high-affinity nitrate transporters in Aspergillus nidulans and Chlamydomonas reinhardtii (Truemann et al., 1996; Filleur et al., 2001). Similarly to the NPF (NRT1/PTR family), but without sequence homology, these membrane proteins contain 12 predicted α-helical transmembrane spans, a central cytoplasmic loop, which is relatively short in Arabidopsis compared with A. nidulans (Forde, 2000), and hydrophilic N- and C-termini of various lengths. All the Arabidopsis NRT2 family members characterized to date are high-affinity nitrate transporters, and no substrate other than nitrate has been identified so far. Unlike A. nidulans NRT2, the nitrate transport function of C. reinhardtii and most higher plant NRT2s depends on the presence of a small protein, NAR2 (NRT3.1; Okamoto et al., 2006; Orsel et al., 2006) which interacts directly with several NRT2 proteins (Kotur et al., 2012). In Arabidopsis, two NAR2 genes are present, but only NAR2.1 seems to be crucial for the two-component high-affinity nitrate uptake system (Orsel et al., 2006; Li et al., 2007). The functional unit for high-affinity nitrate influx was recently proposed to be a complex composed of two subunits of AtNRT2.1 bound to two subunits of AtNAR2.1 (Yong et al., 2010). This contradicts a previous report by Wirth et al. (2007), who suggested that monomeric AtNRT2.1 could be the active form for nitrate transport as it was absent in the plasma membrane of the high-affinity transport system-deficient nar2.1-1 mutant. All Arabidopsis NRT2s except NRT2.7 interact with NAR2.1 in yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) studies (Kotur et al., 2012). Using functional studies in oocytes, Kotur et al. (2012) showed that nitrate uptake was stimulated by the co-expression of NAR2.1 by >2-fold only for NRT2.1, NRT2.2, and NRT2.5. However, slight but statistically significant increases in nitrate transport in oocytes have been observed for all NRT2 co-injections with NAR2.1 (Kotur et al., 2012). Several NRT2 proteins from C. reinhardtii and Hansenula polymorpha are nitrate/nitrite-bispecific transporters (Machin et al., 2004; Fernandez and Galvan, 2007). In higher plants, NPF3.1 (NITR) has been suggested as a chloroplastic nitrite transporter (Sugiura et al., 2007). Further studies have confirmed the nitrite transport activity of AtNPF3.1. However, in Arabidopsis and grapevine, NPF3.1 is localized at the plasma membrane and transports nitrite and nitrate (Pike et al., 2014). Proteins from both families, NPF and NRT2, are known to transport nitrate together with a proton (H+) in a symport mechanism that is driven by the pH gradients across membranes. Electrophysiological studies on Xenopus oocytes expressing either NPFs or NRT2s showed that high- and low-affinity transport systems (HATS and LATS) are accompanied by membrane depolarization and that the nitrate-induced inward currents are pH dependent, both consistent with a proton-coupled mechanism. This is in agreement with thermodynamic calculations indicating that both systems depend on energy potentially provided by proton gradients (Meharg and Blatt, 1995; Wang and Crawford, 1996). More recently it has been shown that members of the NPF facilitate not only nitrate influx but also nitrate efflux (see below). This efflux is probably a membrane potential-driven uniport of one nitrate per proton pumped by the H+-ATPase (Segonzac et al., 2007). SLAC/SLAH and CLC families Patch-clamp experiments on stomata guard cells demonstrated the existence of slow-type anion channels (SLACs), which showed strong preference for nitrate (Linder and Raschke, 1992; Schroeder and Keller, 1992; Schmidt and Schroeder, 1994). Proteins involved in these slow-type anion fluxes which are associated with CO2- and abscisic acid-dependent stomatal closure belong to the small SLAC/SLAH gene family (Negi et al., 2008; Vahisalu et al., 2008). This family contains five genes, SLAC1 and SLAH1–SLAH4 (SLAC1 homologues), that display a common predicted structure of 10 transmembrane α-helices. SLAC1, together with SLAH3, carries out S-type anion channel activity in guard cells (Geiger et al., 2011), whereas in mesophyll cells only SLAH3 has as yet been described to function as a slow-type anion channel. Despite their homology to bacterial and fungal tellurite resistance/C(4)-dicarboxylate transporters, SLAC1 and SLAH3 display nitrate transport activity when expressed in oocytes with NO3–/Cl– permeability ratios of ~10 and 20 for SLAC1 and SLAH3, respectively (Geiger et al., 2009, 2011). CLC proteins can be found in all kingdoms. Initially thought to be specifically involved in chloride transport, either as channels or as 2Cl−/1H+ antiporters (Jentsch, 2008; Lisal and Maduke, 2009), De Angeli et al. (2006) showed that the A. thaliana CLCa protein is a tonoplast-located 2NO3−/1H+ antiporter. Loss-of-function clca mutants accumulate up to 50% less nitrate compared with wild-type plants. Thus CLCa is a major component that drives nitrate accumulation in the vacuoles (Geelen et al., 2000; Monachello et al., 2009). The Arabidopsis CLC family contains seven members (a–f) and the difference in selectivity between CLCa and the other CLCs is mainly due to the presence of a proline residue in the selectivity motif (Bergsdorf et al., 2009; Wege et al., 2010; Zifarelli and Pusch, 2010). CLCb has the same selectivity filter as CLCa and is also located at the tonoplast level. In agreement with this, CLCb transports nitrate in exchange for protons in Xenopus oocytes. Whether CLCb is involved in vacuolar nitrate loading in planta is unknown (von der Fecht-Bartenbach et al., 2010). The selectivity and the mechanism of anion transport for the other five CLC proteins are unknown (Barbier-Brygoo et al., 2011). In planta function of nitrate transporters Nitrate accumulation within a plant varies widely according to the organ, the development stage, and environmental conditions. The root is obviously the predominant organ where large exchanges with the soil solution occur. Nitrate can then either be assimilated in the root or be transported to the shoots via the xylem. The percentage of root assimilation via shoot assimilation depends on the plant species. At high soil nitrate, nitrate assimilation occurs mainly in the shoot in most plants, probably because the shoot is more energy efficient (Andrews, 1986). This is also the case for Arabidopsis (Krapp et al., 2011). More recently, phloem nitrate transport, which ensures nitrate remobilization to sink tissues, has been evidenced (Fan et al., 2009; Kiba et al., 2012). In addition, nitrate transfer between xylem and phloem has been revealed as an important step for whole-plant nitrate management (Wang and Tsay, 2011; Hsu and Tsay, 2013). Nitrate uptake from the soil Physiological studies have led to the conclusion that at least three nitrate uptake systems are responsible for the influx of nitrate into roots (for reviews, see Crawford and Glass, 1998; Daniel-Vedele et al., 1998; Forde, 2000). Two HATS take up nitrate at low concentrations in the external medium, and both of them display saturable kinetics as a function of the external nitrate concentration, with saturation ranging between 0.2mM and 0.5mM. The first one, constitutive HATS, is active even in plants that have never been supplied with nitrate, whereas the second one is induced by nitrate supply. In addition to these systems, there is a LATS whose uptake activity was initially reported to be linear as a function of the external nitrate concentration (Siddiqi et al., 1990; Touraine and Glass, 1997; Kotur and Glass, 2013). However, other studies recorded saturable kinetics (Wang et al., 1998). This discrepancy needs to be revisited. For the time being, three NRT2 transporters (NRT2.1, NRT2.2, and NRT2.4) and two NRT1 transporters [NPF6.3 (NRT1.1/CHL1) and NPF4.6 (NRT1.2)] have been demonstrated as molecular components of root nitrate uptake (Tsay et al., 1993; Huang et al., 1999; Filleur et al., 2001; Kiba et al., 2012). All five proteins are localized at the plasma membrane level and their relative contribution to nitrate uptake depends on the developmental stage and the N status of the plant. NPF6.3 (NRT1.1/CHL1) and NPF4.6 (NRT1.2) are involved in LATS, with NPF6.3 as a dual-affinity transporter (Wang and Crawford, 1996; Liu et al., 1999, 2003). All three NRT2 proteins are involved in HATS. NRT2.1 is the main component of HATS in many conditions (Li et al., 2007). NRT2.4 expression is increased during long-term N starvation, while NRT2.1 and NRT2.2 expression is transiently increased by N starvation. The polarized epidermal localization of NRT2.4, facing the surrounding soil solution, and its affinity for nitrate at very low concentration seem to be perfectly suited for a nitrate transporter acting to scavenge even low amounts of nitrate available in the surrounding medium (Kiba et al., 2012). Whereas NRT2.4 loss-of-function mutants show no growth phenotype under N-sufficient and N-limiting growth conditions, triple mutants of NRT2.1, NRT2.2, and NRT2.4 produce less biomass under N limitation (Kiba et al., 2012). Thus, the interplay of these different transporters in the root seems essential for an efficient uptake of nitrate: in this way, the root adapts to various stimuli using the specific properties and expression patterns of each transporter (Kiba et al., 2012). Nitrate can also be excreted by the root. NPF2.7 (NAXT1) is localized at the plasma membrane of cortical root cells, and loss-of-function mutants are affected in nitrate excretion after acidification of the medium (Segonzac et al., 2007). Long-distance nitrate transport For nitrate to be transferred to the aerial parts of the plant, it has to be loaded into the xylem vessels of the root. NPF7.3 (NRT1.5), expressed in pericycle cells surrounding the protoxylem, is a low-affinity bidirectional (influx–efflux) nitrate transporter involved in this step by loading nitrate into the xylem (Lin et al., 2008). Recently, Léran et al. (2013) speculated that bidirectionality might be a general feature of the NRT1/PTR transporters, as not only NPF7.3 (NRT1.5) but also NPF6.3 (NRT1.1) and NPF6.2 (NRT1.4) facilitated nitrate influx and efflux in Xenopus oocytes. In addition, it was shown that root-supplied nitrate is more slowly transferred to the shoot in npf6.3 (chl1-5) mutants (Léran et al., 2013). However, the function of NPF6.3 (NRT1.1/CHL1) for root to shoot transport requires further investigations with regard to its impact on whole-plant nitrate assimilation and growth. Two other low-affinity nitrate transporters, NPF7.2 (NRT1.8) and NPF2.9 (NRT1.9), expressed predominantly in xylem parenchyma cells and root companion cells, respectively, negatively impact on root to shoot nitrate transport (Li et al., 2010; Wang and Tsay, 2011). Thus, the distribution of a root-derived nutrient to the shoot mainly depends on the transpiration stream of the xylem, but can be modified by the interaction between xylem and phloem. Nitrate remobilization from source to sink organs involves several nitrate transporters. In mature leaves, the low-affinity nitrate transporter NPF2.13 (NRT1.7) is involved in nitrate remobilization from older leaves to younger leaves by loading nitrate into minor veins (Fan et al., 2009). NPF2.13 loss of function reduces plant growth under N starvation conditions when nitrate remobilization from source tissues is required to support growth. Under N starvation, loss of function of NRT2.4, a high-affinity nitrate transporter that is expressed in phloem parenchyma cells, also decreased the nitrate concentration in the phloem sap (Kiba et al., 2012). The interplay of the function of these two nitrate transporters, one working in the high- and the other in the low-affinity range, has not been investigated so far. Recently, NPF1.1 (NRT1.12) and NPF1.2 (NRT1.11) have been found to be involved in the transfer of xylem-borne nitrate to the phloem in the petiole (Hsu and Tsay, 2013). Thus a similar physiological function is performed by different proteins in roots (NPF2.9/NRT1.9) and in shoots. In nrt1.11 nrt1.12 double mutants, partitioning of root-derived nitrate between source and sink leaves was modified compared with the wild type, with less nitrate transferred to the sink and more nitrate accumulated in the source leaves. Interestingly, the growth-promoting effect of high nitrate supply was reduced for the sink leaves of nrt1.11 nrt1.12 double mutants. Thus xylem to phloem nitrate transfer seems to be a critical step for optimal sink organ growth under high external nitrate supply. Although NPF1.1/NPF1.2 (NRT1.11/NRT1.12) and NPF2.13 (NRT1.7) participate in allocating nitrate from source leaves into developing tissues via the phloem, they remobilize nitrate from different sources. The interplay of these four transporters—and possibly more to be discovered—may ensure proper nitrate remobilization from source to sink tissues by a sophisticated spatio-temporal expression pattern. Concerning seeds, the ultimate sink tissue, NPF2.12 (NRT1.6), another low-affinity plasma membrane-localized nitrate transporter, is expressed in the vascular tissue of siliques and in the funiculus, ensuring nitrate supply to developing seeds (Almagro et al., 2008). Loss of function of NPF2.12 leads to higher seed abortion rates caused by the collapse of the suspensor cells at the one- or two-cell stage, indicating that nitrate is important for early embryonic development (Almagro et al., 2008). A high-affinity nitrate transporter, NRT2.7, localized in the tonoplast, is highly expressed in dry seeds and affects their nitrate content, thus probably being involved in the loading of nitrate into the vacuole (Chopin et al., 2007). In addition, proanthocyanidin accumulation is altered in seeds of nrt2.7 mutants, but the causal relationship with its nitrate transport function has not yet been revealed (David et al., 2014). Further challenges for investigations on nitrate transport The advances made during the last years led to the view that nitrate transport processes are essential at many different levels and that specialized transporters are involved. An important additional complexity layer is the extensive regulation of nitrate transporters at the levels of gene expression and of protein modification (for a review, see Wang et al., 2012). More members of the four gene families of nitrate-transporting proteins are still awaiting characterization. Besides these proteins, other so far unknown protein families may contribute to nitrate transport, and further system-wide studies and co-expression analyses should allow for the identification of membrane proteins with expression profiles predicted for such a function (Bordych et al., 2013). Furthermore, thanks to the advances in sequencing techniques, well-designed mutant screening approaches now promise rapid access to the underlying mutation. Useful information on as yet unknown (and known) transporters should equally arise from the wealth of genomic data from plant species other than Arabidopsis. In the same manner, it is rather astonishing that only three out of the 24 known nitrate transporters are localized at the tonoplast and only two of them have an impact on the nitrate content of plants. Whether more nitrate transporters are needed for loading and exporting nitrate from the vacuole is not obvious. The large number of transporters located at the plasma membrane might reflect the diverse and specific cell-type requirements for taking up or exporting nitrate at the soil–root interface or between neighbouring cells. We do not know either if nitrate, once inside the cell, is partitioned between the storage vacuole and the cytosol by only two types of transporters, CLCa(b) and NRT2.7 (De Angeli et al., 2006; Chopin et al., 2007; von der Fecht-Bartenbach et al., 2010). A functional role for NRT2.7 has been evidenced for seeds, where the gene is highly expressed (Chopin et al., 2007), but no consequences of the loss of function of NRT2.7 has yet been observed in other organs. Maybe we are still lacking information on vacuolar nitrate transporters in particular. A major difficulty in studying nitrate transporters is still the characterization of their transport properties. Heterologous expression in oocytes provides information on KM values and substrate specificity, but the actual properties in planta might differ. For example, Glass and Kotur (2013) recently questioned the role of NPF6.3 (NRT1.1/CHL1) for high-affinity nitrate uptake. In addition, slightly modified protocols (incubation times, etc.) can lead to different results, for example in the case of the NAR2-independent transport activities for several NRT2 proteins (Kotur et al., 2012). Beside the usefulness of heterologous expression systems, these data need to be interpreted cautiously. Nitrate signalling In addition to being an essential nutrient, nitrate also serves as a signalling molecule to break seed dormancy (Alboresi et al., 2005), induce leaf expansion (Walch-Liu et al., 2000), regulate lateral root development (Zhang and Forde, 2000), and coordinate the expression of nitrate-related genes (Wang et al., 2000). Deciphering of the latter response, also called the primary nitrate response, has made important progress in these last years. In this review, we focus on molecular players involved in the primary nitrate response and refer to excellent recent reviews regarding the other aspects of nitrate signalling (Miller et al., 2007; Vidal et al., 2010; Castaings et al., 2011; Tsay et al., 2011; Alvarez et al., 2012; Bouguyon et al., 2012; Wang et al., 2012). Primary nitrate response Nitrate regulates the expression of many proteins required for its use by the plant, such as nitrate transporters and enzymes for nitrate assimilation. This so-called primary nitrate response includes rapid (within minutes) regulation by nitrate of the expression of up to 1000 genes (Wang et al., 2000; Scheible et al., 2004; Orsel et al., 2005; Gutiérrez et al., 2007; Krouk et al., 2010b, Marchive et al., 2013, Vidal et al., 2013). Several studies differentiate direct molecular responses to nitrate from responses to nitrite (Wang et al., 2007) and general responses to N supply, using nitrate reductase null mutants (Wang et al., 2004) and mutants of the nitrate sensor NPF6.3 (NRT1.1/CHL1) (Muños et al., 2004; Ho et al., 2009; Wang et al., 2009). Moreover, nitrate-inducible expression of NIA and NII genes (encoding nitrate reductase and nitrite reductase, respectively) is known to occur in the presence of an inhibitor of protein synthesis, suggesting that the components for nitrate signalling and nitrate-responsive transcription pre-exist in plant cells independently of the presence or absence of nitrate (Gowri et al., 1992). In addition to genes involved in nitrate uptake and assimilation, genes involved in amino acid and nucleic acid biosynthesis, transcription and RNA processing, ribosome and hormone biosynthesis, reductant supply, and trehalose metabolism respond within 3–360min after nitrate induction. Indeed, a detailed time course analysis of the early nitrate responses in roots of young seedlings demonstrated that the earliest responses (3–9min) involve genes and functions that are required to set up conditions that allow plants to use or reduce nitrate, such as ribosomes and the oxidative pentose phosphate (OPP) pathway (Krouk et al., 2010b), the latter providing reductants for nitrate assimilation. Comparison with hormone-regulated genes led to the hypothesis that after an early nitrate-specific response (up to 9min), interactions with other signals such as hormones occur (Krouk et al., 2010b). This very rapid gene expression response to the nitrate signal remains complex. Many of the early expression changes are transient. For example, during the first 20min after nitrate addition to N-starved Arabidopsis seedlings, the expression level of only three genes increased steadily over the 20min (Castaings et al., 2011). Many of the early induced genes are down-regulated later on and would not be detectable in samples taken at later time points after nitrate supply. Now that we are aware of these early responding genes, their role in nitrate signalling needs to be further studied. Identification of nitrate-responsive cis-elements One could think that this wealth of nitrate-regulated genes should allow us easily to identify promoter structures responsible for transcriptional regulation by nitrate. However, none of the global transcriptome analyses performed so far has identified nitrate-responsive promoter elements. This might be due to the fact that the nitrate response is a combination of a large number of molecular players, acting in cascades or in synergy, and in cross-talk with other signalling cascades, which makes it difficult to identify a consensus sequence involved in this massive reprogramming of transcription in response to a signal as important as N supply. However, individual promoters of nitrate-inducible genes have been analysed. The promoters of the Arabidopsis nitrate reductase-encoding genes (NIA1 and NIA2) conferred nitrate-inducible expression to reporter genes (Lin et al., 1994), and further analysis of these promoters via linker scanning mutagenesis led to the definition of a nitrate-responsive cis-element (Hwang et al., 1997). Another nitrate-responsive sequence (NRE) was identified in the Arabidopsis nitrite reductase (NII) promoter and found to be necessary and sufficient for nitrate-responsive transcription (Konishi and Yanagisawa, 2010). The NRE is a pseudopalindromic 43bp sequence composed of two half-sites separated by a 10bp non-conserved spacer sequence. Both sites are necessary for full nitrate induction of the expression of the Arabidopsis NII gene in planta, although the distal half-site has the dominant role (Konishi and Yanagisawa, 2010). NRE-mediated nitrate-inducible expression of a β-glucuronidase (GUS) reporter gene construct is independent of protein synthesis. NRE-like sequences are present in various dicotyledonous and monocotyledonous NII promoters, but have not been detected in other nitrate-regulated genes. For example, a 150bp sequence of the NRT2.1 promoter was found to be sufficient to mediate induction by nitrate and repression by N metabolites (Girin et al., 2007) without homology to the NRE. Interestingly, nitrate-responsive sequence elements do not only occur in the 5′-flanking regions of nitrate-regulated genes. The 3′-flanking sequence downstream of the transcriptional terminator is required for nitrate-inducible expression of NIA1 (Konishi and Yanagisawa, 2011). Indeed, sequence homology between the NRE sequence at the NII promoter and the 3′-flanking sequence downstream of the NIA1 promoter has been evidenced. Two NRE-like sequences are located ~2kb and 3kb downstream of the NIA1-coding sequence (Konishi and Yanagisawa, 2013). Mutation of the first one reduces induction by nitrate of a reporter gene construct (Konishi and Yanagisawa, 2013). Whether the entire 43bp palindromic sequence is essential for this regulation was not investigated. The eight-nucleotide half-site may well be the nitrate-responsive element in this case. Despite these interesting data, a clear picture of nitrate-responsive cis-elements still needs to be established. Apparent disagreement between the fact that the 2.7kb and 1.9kb promoter sequences of NIA2 and NIA1, respectively, did not direct nitrate-inducible expression (Konishi and Yanagisawa, 2011) and the study of Hwang et al. (1997), mentioned before, might be explained by the conditions during the nitrate induction experiments. Transcription factors governing the primary nitrate response Transcription factors are expected to be molecular players involved in the massive changes in gene expression in response to a nitrate signal. During the last years, identification of transcription factors involved in nitrate signalling has made progress, and recently NIN-like proteins (NLPs) have turned out to be master regulators of nitrate signalling (Konoshi and Yanagisawa, 2013; Marchive et al., 2013). These RWP-RK transcription factors are homologous to the Lotus japonicus NIN (nodule inception) protein, involved in the early steps of the N-regulated symbiosis between rhizobia and legume roots (Schauser et al., 1999), and to the NIT2 protein which regulates nitrate reductase expression in Chlamydomonas (Camargo et al., 2007). NLP7, one of the nine members of the Arabidopsis NIN-like family, was reported as a factor involved in nitrate and N starvation responses, as nlp7 mutants displayed a constitutive N starvation phenotype possibly due to impaired N signalling (Castaings et al., 2009). Indeed, using a ChIP-chip approach, Marchive et al. (2013) showed that NLP7 binds to 851 genes in response to nitrate, with preferential binding near the transcriptional start site of the target genes. This gene set is enriched for genes involved in N metabolism and related metabolic pathways such as the OPP pathway, sulphur and carbon metabolism, and for regulatory proteins such as transcription factors. Among the bound genes were nearly all of those previously characterized as being involved in nitrate signalling, such as ANR1 (Zhang and Forde, 1998), LBD37/38 (Rubin et al., 2009), CIPK8 (Hu et al., 2009), and NPF6.3 (NRT1.1/CHL1; Ho et al., 2009). However, no obvious DNA-binding motifs were found to be clearly enriched among the NLP7-immunoprecipitated sequences. Among these 851 genes, 91 displayed an attenuated nitrate response in the nlp7 mutant background in the same experimental conditions as used for the ChIP. The deregulation of direct NLP7 targets has far-reaching consequences for genome-wide nitrate regulation. Indeed, the transcriptomic changes that result from NLP7 loss of function extend beyond the genes directly bound by NLP7. Altogether these results pinpoint the upper hierarchical role of NLP7 as an orchestrator of nitrate responses. An additional argument for the crucial role of NLP7 is its rapid activation by the nitrate signal. The NLP7 protein is located in the nucleus of many tissues involved in N transport (e.g. root hairs, emerging lateral roots, and stem vascular tissues). However, N starvation leads to the delocalization of nuclear NLP to the cytosol. Re-supplying nitrate after N starvation led, within minutes, to the relocation of NLP7 into the nucleus. This relocalization is specific for nitrate and independent of transcriptional regulation. Using leptomycin B, a drug that inhibits nuclear export, the effect of nitrate can be mimicked. Thus it was suggested that nitrate directly or indirectly inhibits, by an as yet unknown mechanism, the export of NLP7 from the nucleus, leading to a rapid nuclear accumulation in response to the signal (Marchive et al., 2013). Furthermore, important evidence for the major role of NLPs in nitrate signalling arose from a yeast one-hybrid (Y1H) screening using the 43bp NRE (Konishi and Yanasigawa, 2013). All nine NLPs were evidenced to bind to the NRE in yeast, suggesting that all NLPs may bind to this element in planta. Different NLPs have distinct binding specificity concerning the two parts of the palindromic NRE sequence. The role of NLPs for the primary nitrate response was addressed by expressing an NLP6–EAR fusion construct that transforms an activator into a dominant chimeric repressor (Hiratsu et al., 2003) and is therefore well suited to study gene families with putative redundant functions. These NLP6–EAR-expressing transgenic lines displayed reduced nitrate induction for several genes (Konishi and Yanasigawa, 2013). Whether this modification of the primary nitrate response is due to a modified function of NLP6 or to the impaired function of other NLPs such as NLP7 still remains unclear. In addition, in agreement with the suggested regulation of NLP7 by nuclear retention, evidence has been found that the N-terminal region flanking the RWP-RK domain is responsible for the activation of NLP6 in response to nitrate signalling (Konishi and Yanasigawa, 2013). Indeed, the predicted nuclear export sequence of NLP7 is located at the N-terminal end of the protein, and a fairly homologous sequence exists in NLP6. NLPs are certainly not the only transcription factors involved in the primary nitrate response. Using a systems approach, squamosa promoter-binding-like protein 9 (SPL9) was predicted to be a player of the primary nitrate response. SPL9 overexpression led to modified expression of nitrate-regulated sentinel genes during a nitrate resupply kinetic (Krouk et al., 2010b). No further data on the impact of SPL9 are yet available. Other transcription factors involved in nitrate signalling such as the MADS box transcription factor ANR1 (Zhang and Forde, 1998) or the LOB domain-binding proteins LDB37/38/39 (Rubin et al., 2009) are involved in the regulation of nitrate-related traits, but have not yet been shown to be players in the primary nitrate response. Many studies use several well-known nitrate-induced genes as so-called sentinel genes for the primary nitrate response. It is certainly important to keep in mind that the primary nitrate response represents a complex change of global gene expression, which comprises many different response patterns, such as very early induction, transient induction, repression, etc. (compare Krouk et al., 2010b; Marchive et al., 2013). It is likely that the chosen sentinel gene might not reflect the overall primary nitrate response. In addition, nitrate supply to plants might influence the transcriptome, depending on the experimental condition, the age of the plants, etc., and a given sentinel gene could be under the control of other factors. Nitrate sensors Similarly to animal cells, plant cells sense and respond to signals in their environment through receptors that relay biochemical information via protein–protein interactions to the nucleus where changes in gene expression occur. Thus, nitrate as a signal molecule needs to be identified by the plant at the cellular level. Several scenarios are possible. First, the external presence of nitrate may be sensed most probably by a membrane-bound protein. On the other hand, the intracellular nitrate level might be the parameter that is sensed, either in the cytosol or in other cellular compartments such as the storage vacuole. Also a third possibility could be that nitrate fluxes are sensed, for example, by nitrate-transporting/metabolizing proteins, as proposed for hexokinase in the case of sugar sensing (Hanson and Smeekens, 2009). In addition, a combination of these options might occur in reality. To date, nitrate transporters have been evidenced as playing a role not only as transporters but also as nitrate sensors. NRT2.1, a high-affinity nitrate transporter, was suggested as a nitrate sensor involved in the regulation of lateral root formation (Little et al., 2005). The first convincing evidence for a nitrate transporter also playing the role of a sensor involved in the primary nitrate response arose from a forward genetic screen based on a synthetic nitrate-inducible promoter fused to the yellow fluorescent protein (YFP). One of the isolated mutants with reduced YFP expression after nitrate supply carried a mutation at the NPF6.3 (NRT1.1/CHL1) locus (nrg1). Nitrate regulation of 113 genes was affected in the nrg1 mutant, including genes involved in nitrate assimilation, energy metabolism, and the pentose phosphate pathway (Wang et al., 2009). The role of NPF6.3 as a so-called nitrate transceptor (transporter–receptor) is supported by the exciting identification of an npf6.3 mutant (chl1-9) with a P492L mutation between the 10th and 11th transmembrane domains, which is impaired for nitrate transport, but functional for transducing the nitrate signal (Ho et al., 2009). NPF6.3, which is a dual-affinity nitrate transporter, can thus sense a wide range of nitrate concentrations changing in the soil and trigger different levels of transcriptional responses. Two nitrate-inducible protein kinases belonging to the family of CBL-interacting protein kinases (CIPK8 and CIPK23) were identified in the npf6.3 (nrt1.1/chl1-5) mutant by transcriptome studies. In response to low nitrate availability, CIPK23 phosphorylates NPF6.3 at T101 which triggers the primary response at a low level (Ho et al., 2009). In cipk23 mutants, NPF6.3 is not phosphorylated, leading to high levels of primary transcriptional response in the presence of low nitrate. This indicates that CIPK23 is a negative regulator of the high-affinity response. In contrast, CIPK8 is a positive regulator of the low-affinity response, but its targets are unknown (Hu et al., 2009). Future exciting aspects of nitrate signalling We are far from fully grasping the molecular bases of the primary nitrate response. Despite the identification of the nitrate transceptor NPF6.3 (NRT1.1/CHL1) and the elucidation of its regulation by phosphorylation, other nitrate transporters are likely to have similar roles, and other types of membrane-localized or soluble receptor proteins may be involved in nitrate perception. Despite the identification of NLP7 as a master transcription factor for early nitrate signalling and awaiting more knowledge on the function of the other eight NLPs, further regulatory proteins or small RNAs need to be integrated into the multilayer nitrate response network. In addition, the molecular link between nitrate perception, for example by NPF6.3, and changes in gene expression, for example governed by the master regulator NLP7, are completely unknown. Figure 1 summarizes the current knowledge on the molecular events involved in the primary nitrate response. Fig. 1. View largeDownload slide Molecular players involved in nitrate signalling governing the primary nitrate response. Schematic presentation of players involved in the primary nitrate response. For simplicity, all players are presented in the same cell, which might be not the case in a plant. The dual-affinity nitrate transporter NRT1.1 has a double role for transport and signalling of nitrate. Other nitrate transporters are proposed to fulfil similar roles. Other nitrate receptors might exist. The CBL-interacting protein kinase CIPK23 regulates the affinity of NRT1.1 for nitrate. CIPK8 is involved in the nitrate-regulated mRNA accumulation of several nitrate-regulated genes. The master regulator NLP7 is regulated by nitrate-triggered nuclear retention. The targets of this high hierarchy transcription factor are enriched for regulatory genes and genes involved in N metabolism. The transcription factor SPL9 is involved in the regulation of N assimilation genes. Fig. 1. View largeDownload slide Molecular players involved in nitrate signalling governing the primary nitrate response. Schematic presentation of players involved in the primary nitrate response. For simplicity, all players are presented in the same cell, which might be not the case in a plant. The dual-affinity nitrate transporter NRT1.1 has a double role for transport and signalling of nitrate. Other nitrate transporters are proposed to fulfil similar roles. Other nitrate receptors might exist. The CBL-interacting protein kinase CIPK23 regulates the affinity of NRT1.1 for nitrate. CIPK8 is involved in the nitrate-regulated mRNA accumulation of several nitrate-regulated genes. The master regulator NLP7 is regulated by nitrate-triggered nuclear retention. The targets of this high hierarchy transcription factor are enriched for regulatory genes and genes involved in N metabolism. The transcription factor SPL9 is involved in the regulation of N assimilation genes. Similarly to the identification of new nitrate transporters, genetic screens in combination with next-generation sequencing (NGS) will rapidly provide more genes involved in nitrate signalling (Liu et al., 2012). A major point will be the use of adapted forward screens. Approaches using reporter genes under the control of nitrate-regulated promoter constructs have been partially successful, and more mutants identified in the approaches published so far are eagerly expected (Liu et al., 2012; Wang et al., 2012). Besides 5′ regions, 3′-untranslated regions are also involved in nitrate signalling (Konishi and Yanasagiwa, 2011) and should be used in reporter gene constructs when searching for nitrate-unresponsive mutants. System-wide approaches integrating not only transcriptome data, but also proteome and metabolic profiles, are promising tools for further disentangling the complex nitrate signalling cascade. Moreover, the exploitation of natural variation will certainly reveal the extent of the complexity of the plants’ responses to nitrate, as we far too often restrict our studies of nitrate signalling to a small number of Arabidopsis accessions. Exciting new discoveries are thus expected in the years to come. Acknowledgements This work was supported by grants from the Agence Nationale de la Recherche (ANR, NITRAPOOL, ANR08-Blan-008), the Institut National de la Recherche Agronomique (INRA, French-Brazilian INRA-FAPESP, and AAP ‘Biologie végetale’), and the LABEX Saclay Plant Sciences. We apologize to the authors of many publications which we were unable to cite due to space constraints. References Alboresi A Gestin C Leydecker MT Bedu M Meyer C Truong HN . 2005. Nitrate, a signal relieving seed dormancy in Arabidopsis. Plant, Cell and Environment  28, 500– 512. Google Scholar CrossRef Search ADS   Almagro A Lin SH Tsay YF . 2008. Characterization of the Arabidopsis nitrate transporter NRT1.6 reveals a role of nitrate in early embryo development. The Plant Cell  20, 3289– 3299. Google Scholar CrossRef Search ADS PubMed  Alvarez JM Vidal EA Gutiérrez RA . 2012. Integration of local and systemic signaling pathways for plant N responses. Current Opinion Plant Biology  15, 185– 191. 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TI - Nitrate transport and signalling in Arabidopsis
JF - Journal of Experimental Botany
DO - 10.1093/jxb/eru001
DA - 2014-02-13
UR - https://www.deepdyve.com/lp/oxford-university-press/nitrate-transport-and-signalling-in-arabidopsis-umLkAtqXb8
SP - 789
EP - 798
VL - 65
IS - 3
DP - DeepDyve
ER -