Uptake, allocation and signaling of nitrate

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Uptake, allocation and signaling ofnitrateYa-Yun Wang, Po-Kai Hsu and Yi-Fang Tsay

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

Plants need to acquire nitrogen (N) efficiently from thesoil for growth. Nitrate is one of the major N sources forhigher plants. Therefore, nitrate uptake and allocationare key factors in efficient N utilization. Membrane-bound transporters are required for nitrate uptake fromthe soil and for the inter- and intracellular movement ofnitrate inside the plants. Four gene families, nitratetransporter 1/peptide transporter (NRT1/PTR), NRT2,chloride channel (CLC), and slow anion channel-associated 1 homolog 3 (SLAC1/SLAH), are involvedin nitrate uptake, allocation, and storage in higherplants. Recent studies of these transporters or channelshave provided new insights into the molecular mecha-nisms of nitrate uptake and allocation. Interestingly,several of these transporters also play versatile roles innitrate sensing, plant development, pathogen defense,and/or stress response.

Nitrogen for plant growthN, a building block of fundamental biological molecules,such as nucleotides, amino acids, and proteins, is a keynutrient for plant growth and development. Because N is amajor limiting factor in plant growth, considerableamounts of N fertilizers are applied each year to increasecrop or fruit yields. However, excess use of N fertilizer hascaused pollution of rivers and oceans, leading to eutrophi-cation and an increase in marine dead zones [1,2]. Toreduce the consumption of N fertilizer and increase Nutilization efficiency (NUE) in plants, an understandingof how plants take up, store, and metabolize N has becomean urgent issue.

Most terrestrial plants absorb N from the soil. Bothinorganic and organic forms of N can be directly taken upby plants. The concentrations of these two types of N in thesoil vary depending on soil type, temperature, and theactivities of microorganisms. Inorganic forms of N, suchas nitrate, ammonium, and urea, predominate in cropland,and are often supplied in fertilizers; by contrast, organicforms of N, such as amino acids, peptides, and proteins, aredominant in the soils of boreal forests [3]. Being immobileorganisms, plants cannot choose their surrounding envir-onments but only adapt to them. Therefore, they haveevolved several uptake and transport systems to optimizeand support their growth in response to rapidly changingconditions. Using Arabidopsis (Arabidopsis thaliana) as amodel plant, several N uptake and transport systems havebeen identified [4–11]. Nitrate is one of the major N sources

for higher plants. In this review, we focus on the physio-logical functions of NRT1, NRT2, CLCa/b in the CLCfamily, and SLAH3 in the uptake, allocation, and sensingof nitrate (Figure 1).

Nitrate transporter and channels in higher plantsChlorate resistant 1 (CHL1) was the first NRT1 transport-er to be identified. It was identified as a nitrate uptakemutant using chlorate selection [12]. The family to whichCHL1 belongs was named the NRT1/PTR family becauseNRT1/PTR members in animals, fungi, and bacteria trans-port dipeptides. In Arabidopsis, there are 53 NRT1/PTRtransporters. Of the 16 members characterized so far, sometransport nitrate and some transport dipeptides [13–25].With the exception of CHL1 (AtNRT1.1) and MtNRT1.3,which are dual-affinity nitrate transporters [26–28], mostof the NRT1 nitrate transporters characterized are low-affinity nitrate transporters. Given that nitrate transportactivity has only been found in plant members of the PTR/NRT1 family, it is likely that the nitrate transport activityof this family evolved from an ancient dipeptide transport-er. Most nitrate and peptide transporters characterized inthe NRT1/PTR family are proton-coupled transporters[14,16,18,19,22,25,27].

In contrast to NRT1/PTR transporters, which havediverse substrate specificity, all NRT2 transporters isolat-ed from Aspergillus, Chlamydomonas, and higher plantstransport nitrate [5,9,29,30]. In Chlamydomonas andhigher plants, a membrane protein, NAR2, is requiredfor the nitrate transport activity of NRT2 transporters(Figure 2) [31–36]. It is believed that the NRT2s are alsoproton-coupled transporters. In Arabidopsis, there areseven NRT2 transporters, four of which show nitrate-related phenotypes when mutated [37–41].

The third type of nitrate transporters belongs to theCLC family, some members of which function as anionchannels, and others as anion–proton exchangers [8]. InArabidopsis, there are seven CLC genes. CLCa and CLCbfunction as proton–nitrate exchangers, and have a higherselectivity for nitrate over chloride [42–44]. For CLCa, theproline residue at 160, which is also found at the corre-sponding position in CLCb, is important for nitrate selec-tivity [45–47].

Studies of an Arabidopsis mutant defective in stomatalclosure has led to the identification of the anion channelSLAC1 [48,49]. SLAC1 contains ten transmembranedomains and shares sequence similarity with bacterialand fungal dicarboxylate transporters. In Arabidopsis,

Review

Corresponding author: Tsay, Y.-F. ([email protected])

458 1360-1385/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2012.04.006 Trends in Plant Science, August 2012, Vol. 17, No. 8


there are five SLAC genes: SLAC1 and SLAH1–SLAH4(SLAC1 homologs). When expressed in oocytes, SLAC1 andSLAH3 show nitrate transport activity, although kinasesneed to be co-injected to activate their channel activity[50,51].

Acquisition of nitrate from soilNitrate concentrations in the soil can fluctuate by morethan four orders of magnitude [52,53]. To survive, plantshave evolved two nitrate uptake systems, the high-affinitytransport system (HATS), and the low-affinity transport

NRT2.1 NRT2.2 NRT2.4 NRT1.1(CHL1) NRT1.2

NO3–NO

NO3– H+ (?)

HATS

LATS

NO H (?)

NR

T1.5

Embryo cell

Mesophyll cell

NAXT1

Xylem

NRT1.8 NRT1.9

Guard cell

NRT1.1

SLAH3

Epidermis Cortex Pericycle/ Parenchyma

Xylem

Endodermis

Casparian band

NO3–

NO3–

NO3–

NO3–

NO3–

NO3–

NO3–

NO3–

NO3–

NO3–

NO3–NO

(b)

(a)

3–

NO3–

NRT1.1(CHL1)

NRT2.1 NRT2.4 NRT1.5

NRT1.8 NAXT1 NRT1.2 Key:

Uptake

Petiole sto

re

NRT1.4

NR

T1:6

Seed

NRT1:7

Remobilize

Efflux

Vacuole

NRT2.7

Vacuole H+

CLCa,b

TRENDS in Plant Science

Figure 1. Physiological functions of Arabidopsis nitrate transporters. (a) Contribution of nitrate transporter 1 (NRT1), NRT2, chloride channel (CLC) a/b, and slow anion

channel-associated 1 homolog 3 (SLAH3) to different steps of nitrate uptake and allocation. Nitrate is taken up by roots from soils and transported to shoots and seeds for

storage and/or further assimilation. Abbreviations: HATS, high-affinity transport system; LATS, low-affinity transport system. (b) Detailed illustration of nitrate uptake and

movement in roots. NRT1.1 (CHL1), NRT1.2, NRT2.1, NRT2.2, and NRT2.4 are involved in nitrate uptake from soils. Nitrate excretion transporter 1 (NAXT1), a transporter in

the NRT1 family, mediates nitrate efflux under acid load. NRT1.5, NRT1.8, and NRT1.9 play a role in regulating root-to-shoot xylem transport of nitrate. The illustration does

not mean that these transporters have a polarized distribution in the root cells.

Review Trends in Plant Science August 2012, Vol. 17, No. 8

459


system (LATS). Thermodynamic calculations as well as theassociation of both the HATS and the LATS with mem-brane depolarization suggest that both systems requireenergy potentially provided by proton gradients [54–56]. InArabidopsis, CHL1 is a dual-affinity nitrate transporterinvolved in both high- and low-affinity nitrate uptake[26,27]. In addition to CHL1, NRT1.2, in the NRT1 family,also participates in low-affinity nitrate uptake [20]. In theNRT2 family, NRT2.1, NRT2.2, and NRT2.4 participate in

high-affinity nitrate uptake [37,40,41], with NRT2.4 hav-ing a very high-affinity range [41]. The relative contribu-tion of these transporters to nitrate uptake is dependent onthe developmental stages of the root and the N status of theplant.

The Km values of plants with an HATS are in the micro-molar range. Consistently, the Km value of HvNRT2.1 andthe high-affinity Km value of CHL1 (AtNRT1.1) measuredwhen expressed in Xenopus oocytes have been found to beapproximately 30 mM and approximately 50 mM, respec-tively [27,57]. The Km value of AtNRT1.2, as well as thelow-affinity Km value of CHL1 (AtNRT1.1), are both approx-imately 5 mM when expressed in Xenopus oocytes[20,27,58]. Consistent with these measurements in oocytes,some plant nitrate uptake studies showed that the LATSexhibits a saturable response with a Km value in the mMrange [26,59,60]. However, in other studies, the plant LATSshowed a linear response to concentration [61–63]. Accord-ing to thermodynamic calculations, it is unlikely that plantLATS is mediated by the passive transport of a channel. Thelinear response might need to be reevaluated with widerranges of concentrations. Nevertheless, it might be possiblethat a channel facilitates nitrate uptake when the mem-brane potential is depolarized and/or the cytosolic nitrateconcentration is low.

Nitrate uptake is regulated at both the transcriptionaland post-transcriptional level. As shown in Table 1, ex-pression of CHL1 (NRT1.1), NRT1.2, NRT2.1, NRT2.2,and/or NRT2.4 is regulated at the transcriptional levelby nitrate, nitrite, ammonium, glutamine, N starvation,light, sucrose, diurnal rhythm, and/or pH. A strong corre-lation between the transcript abundance of these nitratetransporter genes and nitrate uptake activities suggests

NO3–

H+(?)

NO3–

H+

NRT1.5

NO3– H

+

CIPK23

NAXT1

NA

R2

NO3–

H +

NRT1 NRT2

NO3–

CPK21

Cytosol NO3

–

H+

Vacuole

SLAH3

PO4–

NO3–

NRT2.7

CLCa,b

H+(?)

PO4–


Figure 2. Directions of nitrate movement mediated by nitrate transporter 1 (NRT1),

NRT2, chloride channel (CLC) a,b, and slow anion channel-associated 1 homolog 3

(SLAH3) in plasma membrane or tonoplast. Most NRT1 and NRT2 transporters

characterized are H+-coupled symporters. NRT1.1 (CHL1), a dual-affinity nitrate

transporter, is phosphorylated by calcineurin B-like-interacting protein kinase 23

(CIPK23) to change the affinity. NAR2 is required for NRT2 nitrate transport. CLCa,b

localized in the tonoplast are H+/NO3� antiporters. Anion channel SLAH3 mediates

nitrate release from guard cells and its function is regulated by CPK21.

Abbreviations: CPK21, calcium-dependant protein kinase 21; NAXT1, nitrate

excretion transporter 1.

Table 1. Summary of the physiological functions and regulations of identified nitrate transporters in Arabidopsis thaliana

Gene Function Nitrate response N starvation Other regulations Refs

CHL1 (NRT1.1) Nitrate sensing Induction Repression Nitrite (long-term) and high

pH repression

[12,27,54,58,66,

70,103–108]

High- and low-affinity nitrate uptake Auxin, light, sugar, and nitrite

(short-term) induction

NRT1.2 Low-affinity nitrate uptake Constitutive Not known Not known [27,109]

NRT1.3 Not known Induction (shoot) Not known Light induction [108,109]

NRT1.4 Leaf nitrate homeostasis Constitutive Not known Not known [18,109]

NRT1.5 Root xylem loading Induction Not known High pH and potassium

limitation repression

[14,108]

Sugar induction

NRT1.6 Delivery of nitrate to developing embryos Not known Induction Not known [16]

NRT1.7 Nitrate remobilization from old to

young leaves

Not known Induction Sucrose induction [25]

NRT1.8 Xylem unloading and cadmium resistance Induction Not known Cadmium induction [13]

NRT1.9 Nitrate loading into root phloem Constitutive Not known Not known [22]

NAXT1 Root nitrate efflux Not known Not known Acidic pH induced at

protein level

[76]

NRT2.1 High-affinity nitrate uptake Induction Induction Ammonium and

glutamine repression

[37,40,65,66,

105,106,108–112]

Light and sugar induction

NRT2.2 High-affinity nitrate uptake Induction Not known Not known [37,40,109,112]

NRT2.4 High-affinity nitrate uptake at low

nitrate concentration

Repression Induction Ammonium repression [41,108,109]

Light induction

NRT2.7 Nitrate storage in mature embryos Constitutive Not known Not known [39,109]

CLCa Nitrate accumulation in vacuoles Induction Not known Not known [43,44]

CLCb Nitrate accumulation in vacuoles Not known Not known Not known [42]


460


that transcriptional regulation plays a key role in modu-lating nitrate uptake activities. Transcript abundance ofCHL1 (NRT1.1) and/or NRT2.1 has been particularlylinked to response to nitrate induction, N-starvation upre-gulation, ammonium repression, and/or diurnal changes[36,40,64–66].

Studies of 35S–NRT2.1 transgenic Arabidopsis andNicotiana have revealed post-transcriptional repressionof nitrate uptake in response to ammonium or dark treat-ments [36,67]. In wild type, both the AtNRT2.1 mRNAlevel and the HATS are downregulated by prolonged dark.In the Arabidopsis 35S–NRT2.1 plant, the mRNA levelremained unchanged in response to prolonged dark, butthe HATS and NRT2.1 protein levels were reduced. Theseresults indicate that, in addition to transcriptional control,protein stability also provides a path through which theHATS is repressed by prolonged (24-h) dark treatments[36]. Another example of post-transcriptional regulationwas revealed in a study of NAR2. In Arabidopsis andChlamydomonas, an oocyte functional assay as well asmutant phenotypes indicated that the two-transmem-brane-domain protein NAR2 is required for the nitratetransport activity of the NRT2 transporter. In the Arabi-dopsis nar2.1 mutant, the disappearance of NRT2.1protein in the membrane fraction suggested thatNAR2.1 is required for the plasma membrane targeting,and/or the protein stability, of NRT2.1 [68].

Protein phosphorylation provides another regulatoryroute for nitrate uptake. CHL1 (NRT1.1), expressed inroot epidermis, cortex, and endodermis, is a dual-affinitynitrate transporter mediating both the HATS and theLATS [26,27,58]. The switch between the two affinitiesis controlled by phosphorylation at the T101 residue be-tween the second and third transmembrane domains [69],and this phosphorylation is regulated by the calcineurin B-like-interacting protein kinase CIPK23 [70]. Dual-affinitytransport activity is not unique to CHL1, as it is alsoexhibited by the potassium transporter KUP and thenitrate transporter MtNRT1.3 [28,71,72]. Furthermore,some of the transporters characterized to date might nothave been examined with wide enough ranges of substrateconcentrations to eliminate completely the possibility thatthey are dual-affinity transporters. It is not known wheth-er there are two nitrate binding sites with different affini-ties in CHL1, or one binding site for both affinities that isaltered by conformational change. The crystal structure ofPepTso, a prokaryotic homolog of CHL1, revealed twohydrophilic cavities, an extracellular cavity and a centralcavity [73]. The peptide binding site is proposed to be in thecentral cavity. The leucine/sodium symporter LeuT, aprokaryotic ortholog of neurotransmitter/sodium sympor-ters (NSSs), also contains two hydrophilic cavities, and ithas been reported that it may have two leucine bindingsites, with one in each cavity [74]. It will be interesting todetermine whether there are also two substrate bindingsites in CHL1, and whether or how phosphorylation in thecytosolic loop affects the accessibility and/or affinity ofthese binding sites.

Involvement of multiple transporters in nitrate uptaketogether with multiple routes of regulation would meanthat plants have a sophisticated uptake system fine-tuned

to optimize nitrate uptake capacity in response to internaldemand and external changes.

Nitrate effluxNet root nitrate uptake is the balance between nitrateinflux (uptake) and efflux. Although the physiological roleof nitrate efflux remains unclear [75,76], it is known that itincreases upon external environment and other stresses[77,78]. Using a functional biochemical approach, nitrateexcretion transporter 1 (NAXT1) was identified by massspectrometry in the plasma membrane of Arabidopsissuspension cells (Figures 1 and 2) [76]. Interestingly,NAXT1, a member of the NRT1/PTR family, is mainlyexpressed in the cortex of mature roots. Under normalconditions, nitrate efflux activity in wild type and naxt1mutants is similar. However, acidification of the cytosol byacid load stimulated nitrate efflux in wild type but not innaxt1 mutants, indicating that NAXT1 is responsible fornitrate excretion induced by lower pH in the cytosol [76].

The appearance of phenotypes of naxt1 under acid loadconditions is consistent with the fact that NAXT1 belongsto the proton-coupled symporter family. However, directevidence for the proton-coupling mechanism of NAXT1 isstill missing. Similar to NAXT1, NRT1.5, which is respon-sible for loading nitrate into the xylem, also mediatesnitrate efflux (see next section) [14]. It is generally believedthat nitrate influx is an active process, with energyobtained from proton coupling, whereas nitrate efflux isa passive process, which is more likely to be mediated by achannel. Therefore, it was surprising that two transportersin the proton-coupled transporter family, NAXT1 andNRT1.5, were found to be involved in nitrate efflux. Theefflux activity of NRT1.5 is pH dependent, suggesting thatefflux is also mediated by a proton-coupled mechanism. Itis worth noting that SLAH3 (a homolog of SLAC1), whencoexpressed with a calcium-dependent protein kinase(CPK21) in Xenopus oocytes, exhibits a nitrate-inducedanion current [50]. SLAH3 shows strong selectivity fornitrate over chloride. Given that no nitrate efflux differ-ence can be detected in the naxt1 mutant under normalconditions, it is possible that an unknown channel or ahomolog of SLAH3 is responsible for root nitrate effluxunder normal conditions. Moreover, the influence of nitrateexcretion on plant growth remains to be analyzed further.

Translocation from root to shootOnce inside plant cells, nitrate can be stored in vacuoles orassimilated to ammonium for amino acid synthesis. Undermany conditions, a significant proportion of nitrate assim-ilation takes place in shoots because the reducing powerrequired for the assimilation processes comes from photo-synthesis. For root-to-shoot nitrate translocation, the firststep is the loading of nitrate into xylem vessels. To date,only one nitrate transporter, NRT1.5, has been identifiedto mediate this process (Figures 1 and 3).

NRT1.5, a low-affinity nitrate transporter located in theplasma membrane, is expressed in the two pericycle cellsadjacent to the protoxylem (the xylem-pole pericycle) inArabidopsis roots (Figures 2 and 3) [14]. In nrt1.5 mutants,less nitrate is transported to the shoots and the nitratecontent in xylem sap is reduced [13,14], suggesting that


461


NRT1.5 is responsible for exporting nitrate out of pericyclecells for xylem loading. Interestingly, in the mutants, theamount of potassium transported to the shoot is alsoreduced in a nitrate-dependent manner [14], suggestingthat there is homeostatic balance between the major cat-ion, potassium, and the anion, nitrate, in the xylem stream.NRT1.5 is a bidirectional (influx–efflux) nitrate transport-er. Influx activity is higher when the external solution ismore acidic (pH 5.5), whereas efflux activity is more obvi-ous at neutral pH values (pH 7.4). Given that the pH valueof xylem sap often ranges from 5.7 to 6.2 [79–81], furtherstudy is required to determine how the two directions ofnitrate movement are controlled. Root-to-shoot nitratetransport is not completely abolished in the nrt1.5 mutant,and when tissue nitrate content is low, no nitrate translo-cation defect can be detected [14]; therefore, there must beother xylem loading mechanism(s) that are more activeunder low nitrate conditions.

Two NRT1 transporters, NRT1.8 and NRT1.9, are in-volved in regulating root-to-shoot nitrate translocation(Figure 1). NRT1.8, a low-affinity nitrate transporter lo-cated in the plasma membrane, is expressed dominantly inthe xylem parenchyma cells of roots (Figure 3) [13]. nrt1.8mutants show increased nitrate content in xylem sap andincreased root-to-shoot nitrate translocation, suggestingthat NRT1.8 functions in removing nitrate from the xylemsap back into the root cells [13]. Studies of NRT1.8 haverevealed an interesting interplay between nitrate andcadmium. In wild type, expression of NRT1.8 is upregu-lated by cadmium, and cadmium treatment reduces theamount of nitrate translocated to the shoot. The nrt1.8mutant shows a nitrate-dependent cadmium-sensitivephenotype and, compared with the wild type, an increasedamount of cadmium is transported to the shoot. Therefore,although the detailed mechanism remains to be deter-mined, it seems that the function of NRT1.8 in removing

nitrate from xylem sap also allows Cd2+ to stay in the roots,and consequently enhances Cd2+ tolerance [13].

In a similar manner to nrt1.8 mutants, nrt1.9 mutantsalso show enhanced root-to-shoot nitrate transport [22](Figure 1). However, unlike NRT1.8, which is expressedin xylem parenchyma cells, NRT1.9 is expressed in thecompanion cells of the phloem (Figure 3), indicating thatboth NRT1.8 and NRT1.9 are negative regulators of root-to-shoot nitrate transport but through different mecha-nisms. In nrt1.9 mutants, downward transport of nitratethrough the phloem is reduced, resulting in increasedupward (root-to-shoot xylem) transport of nitrate [22].Moreover, nrt1.9 mutants show enhanced plant growthunder high nitrate conditions. These results suggest thatNRT1.9 mediates the downward phloem transport of ni-trate in roots and that, under high nitrate conditions,phloem transfer of nitrate in the root serves as an ‘overflowroute’ to prevent excess amounts of nitrate being trans-ported to the shoot. Under laboratory growth conditions,nrt1.9 mutants gain a growth advantage under high ni-trate conditions, but the lack of this additional regulatorypath in nrt1.9 mutants may result in a loss of flexibility toadapt to certain environmental changes in the wild.

Studies of NRT1.5, NRT1.8, and NRT1.9 indicate thatthe root-to-shoot xylem transport of nitrate is a key step inregulating nitrate distribution and so needs to be finetuned by several accessory pathways. Root-to-shoot nitratetransport is also crucial for the homeostatic interplaybetween nitrate and other cations.

Allocation in vegetative tissuesAfter transportation to leaves, nitrate can undergo assimi-lation in cytosol or storage in vacuoles. NRT1.4, a low-affinity nitrate transporter, plays a role in regulating leafnitrate homeostasis and leaf development (Figure 1) [18].NRT1.4 is dominantly expressed in the leaf petioles. Thenitrate content of the petiole is reduced but the nitratecontent in the leaf lamina is slightly increased in nrt1.4mutants, indicating that NRT1.4 regulates nitrate distri-bution in leaves. Interestingly, the leaves of nrt1.4 mutantsare wider than those of the wild type, suggesting that thenitrate distribution in leaves influences leaf expansion.These studies indicate that the petiole, a nitrate storagesite, serves as a checkpoint for regulating nitrate distribu-tion in leaves. Interestingly, in some crop species, farmersuse petiole nitrate content to monitor the N fertilizerdemands of the crops [82,83]. Moreover, NRT1.4 expressedin the petiole could affect lamina nitrate content andlamina growth, suggesting that there are multiple layersof interplay between the lamina and petiole for nitratedistribution and assimilation. For example, it would beinteresting to find out when and how N demand in thelamina sends a signal to the petiole to trigger the remobi-lization of stored nitrate.

It is well known that sucrose can be allocated fromsource (mature) leaves to sink (young) leaves to supportthe growth of sink tissues with low levels of photosynthesisactivity. A study of NRT1.7 has shown that nitrate can alsobe allocated from older leaves to younger leaves (Figure 1)[25]. NRT1.7 is expressed in the phloem of minor veins inolder leaves (Figure 4). In nrt1.7 mutants, more nitrate

Root-to shoot xylem transport of nitrate

Ph

Downward phloem transport of nitrate

Nitrate removal from xylem

Pe

En

Co

Xy


Figure 3. Regulation of root-to-shoot nitrate transport. Nitrate transporter 1.5

(NRT1.5), expressed in the xylem pole pericycle, mediates xylem-loading of nitrate

(red arrow) and facilitates root-to-shoot xylem transport of nitrate (blue arrow).

NRT1.9, expressed in companion cells of phloem, mediates phloem loading (dark-

purple arrow) for downward phloem transport of nitrate (dark blue arrow), which

may facilitate xylem-to-phloem transfer of nitrate (broken purple arrow), and then

reduce root-to-shoot nitrate transport. NRT1.8, expressed in xylem parenchyma,

mediates nitrate removal from xylem (green arrow), and could also reduce root-to-

shoot nitrate transport. Abbreviations: Co, cortex; En, endodermis; Pe, pericycle;

Ph, phloem; Xy, xylem.


462


accumulated in older leaves, less 15NO3– dropped on the

older leaves could be transported to younger leaves, andless nitrate was detected in the phloem sap of the olderleaves. These findings indicate that phloem loading medi-ated by NRT1.7 in the minor veins of older leaves couldremove excess nitrate from older leaves to feed youngerleaves. In addition to NRT1.7, NRT2.4 also participates inphloem nitrate transport in shoots, particularly under Nstarvation conditions [41]. NRT2.4 is expressed in thephloem parenchyma of leaves, and the expression is highlyinduced by N starvation. Consistent with expression pat-terns, the nitrate content in the phloem exudates of matureleaves in nrt2.4 mutants is decreased under N starvationconditions but not under normal conditions, suggesting aspecial role for NRT2.4 in phloem-mediated nitrate remo-bilization. [41]. Intriguing questions are raised, particular-ly relating to the temporal and spatial differences betweenNRT1.7 and NRT2.4 in their contribution to nitrate remo-bilization under normal and starvation conditions.

For some time, it was generally believed that nitratecould only be transported via the xylem, whereas organicN, such as amino acids, could be transported via both thexylem and phloem. Therefore, it was surprising to find thatphloem nitrate transport mediated by NRT1.7 and NRT1.9plays a crucial role in controlling nitrate distribution.Based on this new information, the understanding of ni-trate allocation has been modified: it is now thought thatroot-to-shoot xylem transport driven by transpiration isthe primary route of long-distance nitrate transport andthat phloem nitrate transport driven by osmotic gradientsprovides a secondary route by which to modulate localnitrate redistribution.

NRT1.7 expression is diurnally regulated, with levelspeaking at dusk. This is the opposite situation to that of themajor nitrate reductase gene NIA2, suggesting that nitratein older leaves is assimilated during the light period andexported during the dark period. Expression of NRT1.7 isupregulated by N starvation and, under N starvation

conditions, nrt1.7 mutants show growth retardation, sug-gesting that remobilization of nitrate among leaves isimportant to sustain plant growth under such conditions[25]. Because excess nitrate is stored in the vacuoles toawait export from the leaf, nitrate has to be transportedacross the tonoplast and the plasma membrane of themesophyll cells before it can be loaded into the phloem.How N starvation and/or N demand from the youngerleaves could coordinately regulate these steps is an inter-esting question.

The genes responsible for exporting nitrate out ofvacuoles have not yet been identified. However, the genesresponsible for transporting nitrate into vacuoles are welldocumented. CLCa and CLCb, proton–nitrate exchangerslocated in the tonoplast, mediate nitrate import into thevacuole (Figures 1 and 2) [42,43]. CLCa expressed in meso-phyll cells accounts for up to 50% of nitrate storage [44]. Theexpression level of CLCb is much lower than that of CLCa. Incontrast to the clca mutant, the clcb mutant shows nodifference in nitrate content compared with the wild type[42]. Both CLCa and CLCb are diurnally regulated andupregulated by nitrate. CLCa activity can be inhibited bythe binding of ATP to the cystathionine-synthetase (CBS)domains at its cytosolic C-terminus [84]. This suggests thatnitrate storage activity is regulated by the energy andphotosynthesis status of the mesophyll cells, and thatnitrate storage activity consequently affects nitrate assimi-lation rate as this is determined by nitrate reductase activityand cytosolic nitrate concentration.

Nitrate in guard cellsNitrate is one of the major anionic osmotica in higherplants. Stomatal closure and opening are driven bychanges in turgor pressure, and several nitrate transpor-ters and channels are associated with stomatal pheno-types. CHL1 is expressed not only in roots, but also inthe guard cells in leaves (Figure 1) [85]. In the presence ofnitrate, CHL1 expressed in guard cells promotes stomatalopening. In chl1 mutants, less nitrate is accumulated inguard cells, and stomatal opening and transpiration ratesare reduced in the presence of nitrate. Therefore, chl1mutants show more drought tolerance than does the wildtype in the presence of nitrate [85]. Interestingly, thetranscription factor NIN-like protein 7 (NLP7), a positiveregulator of nitrate-induced expression of N-related genes,including CHL1, also shows drought tolerance when mu-tated [86]. These results further strengthen a linkagebetween nitrate and water loss.

Anion efflux is a key player in stomatal closure. SLAC1,identified by stomatal closure mutants, exhibits similarpermeability for nitrate and chloride [51]. In contrast toSLAC1, SLAH3 shows a strong preference for nitrate overchloride (Figures 1 and 2) [50]. Even though SLAH3 showssimilar anion selectivity to the S-type anion current foundin guard cell protoplasts, no stomatal phenotype has beenseen in slah3 mutants under the conditions tested [50].These findings suggest that, under normal conditions,SLAC1 is more dominant in regulating stomatal closure.In addition to CPK, high external nitrate is required toactivate SLAH3 with K0.5 of 8.3 mM [50]. Therefore, undernitrate-rich conditions, the nitrate-preferred channel can

Younger leaves

NO3–

NO3– Xy

Ph

Older leaves

NOOO333–––

NONONO333– Xy

Ph


Source-to-sink remobilization of nitrate

Figure 4. Function of nitrate transporter 1.7 (NRT1.7) in nitrate remobilization from

older to younger leaves. Root-to-shoot xylem transport of nitrate (red arrows) is

driven by transpiration, so less nitrate is transported to the smaller younger leaves

via the xylem. NRT1.7 (yellow circles), expressed in the phloem of the minor veins

in older leaves, mediates phloem-loading of nitrate in older leaves (short blue

arrows) and facilitates remobilization of excess nitrate stored in older leaves into

younger leaves (long blue arrow). Abbreviations: Ph, phloem; Xy, xylem.


463


be activated to substitute for the nonselective one in regu-lating stomatal closure.

Movement in reproductive tissuesIn addition to organic N, nitrate also accumulates in seedsand affects seed dormancy [87]. NRT1.6, a plasma-mem-brane localized, low-affinity nitrate transporter, is onlyexpressed in the vascular bundles of the siliques and thefuniculi, suggesting that it is involved in delivering nitrateto developing seeds (Figures 1 and 5). Indeed, in nrt1.6mutants, the amount of nitrate in mature seeds is reducedand the seed abortion rate is increased [16]. The abnormalembryos are caused by the collapse of the suspensor cells atthe one- or two-cell stage, indicating that nitrate is impor-tant for early embryo development.

In the NRT2 family, NRT2.7 is expressed in the matureseeds and located in the tonoplast (Figures 2 and 5) [39].NRT2.7 alone without NAR2 can mediate nitrate uptake inXenopus oocytes and less nitrate is detected in nrt2.7 seeds,indicating that NRT2.7 plays a role in nitrate accumula-tion in seeds. If, as expected, NRT2.7 functions as a proton-coupled nitrate transporter, one would expect that NRT2.7located in the tonoplast is involved in transporting nitrateout of the vacuole. However, reduced seed nitrate contentsuggests that NRT2.7 is involved in importing nitrate intothe vacuole (Figure 2). The magnitude of the reduction inseed nitrate content in the mutants is ecotype dependent[39], indicating that different ecotypes might adapt differ-ent mechanisms for nitrate storage in seeds.

Versatile roles of nitrate transceptor CHL1In addition to being a transporter involved in nitrateuptake, CHL1 also functions as a nitrate sensor, regulatinga transcriptional response called the primary nitrate re-sponse [70,88]. In the primary response, several nitrate-related genes, including NRT1.1, NRT2.1, the nitrate re-ductase-encoding genes NIA1 and NIA2, and the nitritereductase-encoding gene NIR, are induced by nitrate with-in 10 min. A study of the uptake- and sensing-decoupledmutant chl1-9 (P492L) showed that nitrate does not needto be transported across the membrane when CHL1 exerts

its function as a nitrate sensor to trigger the signal [70]. Bychanging the phosphorylation status at the T101 residue,CHL1 can sense the concentration changes in the externalenvironment, leading to different levels of primary re-sponse. At a low nitrate concentration, T101 is phosphory-lated by CIPK23, and phosphorylated CHL1 triggers a low-level of primary response [70].

CHL1 also plays a role in regulating root architecture.The growth of the primary root in wild type is inhibited byexogenous L-glutamate (Glu). This inhibition effect is an-tagonized by nitrate. However, the antagonism by nitrateis lost in chl1 mutants, suggesting that CHL1 senses thepresence of nitrate and mediates the effect [89].

In addition to primary root growth, CHL1 (NRT1.1) alsoparticipates in promoting lateral root elongation in nitrate-rich patches of the external medium [90]. A study hasshown that, in the absence of nitrate, CHL1 (NRT1.1)facilitates the uptake of the phytohormone auxin, whereasin the presence of nitrate, CHL1-dependent auxin trans-port is inhibited [91]. This study also showed that thisnitrate-regulated CHL1-dependent auxin transport is re-sponsible for nitrate-promoted lateral root elongation. Inaddition, ethylene is involved in repressing lateral rootdevelopment under high nitrate conditions, and this re-pression is alleviated in ethylene-insensitive mutants(etr1-3 and ein2-1) and in chl1 mutants [92]. The expres-sion of CHL1 is also affected by ethylene signaling, sug-gesting that CHL1 (NRT1.1) is also involved in theethylene-mediated inhibition of lateral root development.

As well as having nitrate-related functions, severalreports have shown that CHL1 also has nitrate-indepen-dent functions [93–96]. The chl1 mutant has an acidicintracellular pH and reduced primary root growth in theabsence of nitrate [95,96]. Mutation of CHL1 (NRT1.1) alsoenhances ammonium and/or low pH tolerance in Arabi-dopsis and real-time PCR analysis suggests that thistolerance results from the upregulation of aliphatic gluco-sinolate biosynthesis through a nitrate-independent signalfrom CHL1 [93].

Effect of NRT2.1 on lateral root development andpathogen defenseThe high-affinity uptake complex NRT2.1–NAR2.1 alsoparticipates in regulating lateral root development. Undernitrate-limiting conditions, both genes are positive regu-lators of lateral root initiation even in nitrate-free condi-tions, suggesting that the regulation of lateral root growthby NRT2.1 and NAR2.1 is independent of their uptakefunction [33,97]. However, under high sucrose and lownitrate conditions, NRT2.1 and NAR2.1 function as repres-sors [33,98]. These results indicate that the NRT2.1–NAR2.1 complex has dual effects on lateral root develop-ment. Another surprising phenotype seen in nrt2.1mutants is reduced susceptibility to the bacterial pathogenPseudomonas syringae pv tomato DC3000 (Pst) [99]. Thisphenotype probably has reduced sensitivity to the bacterialphytotoxin coronatine because no difference could be foundbetween the wild type and nrt2.1 mutant when challengedwith a P. syringae strain defective in coronatine synthesis.

The multiple effects of NRT1.1 and/or NRT2.1 on nitratesignaling, root development, and pathogen defense suggest

F

Nitrate storage in mature embryos

Nitrate supply for early embryo development em


Figure 5. Nitrate transport to embryos. Nitrate transporter 1.6 (NRT1.6), expressed

in the funiculus (F), provides nitrate for early embryo development (orange arrow).

NRT2.7, located in the tonoplast, mediates nitrate storage in mature embryos (blue

arrow). The GUS (blue) staining and the GFP (green) signals in this photo

represents NRT1.6 and NRT2.7 expression patterns, respectively. This figure was

produced by combining two micrographs reproduced, with permission, from

[20,44].


464


that, in addition to being a nutrient source, nitrate is asignaling molecule coordinating multiple cellular process-es. A challenge for future research will be to find outwhether these nitrate transporters are directly or indirect-ly involved in these responses, and to find out the details ofthe underling mechanism(s) and potential cross talkamong these responses.

Concluding remarksSince the identification of the first member of the NRT1/PTR family in 1993, considerable effort has been put intocharacterizing the transport activities and in vivo func-tions of other members in this family. Although someprogress has been made, the information available is stilljust the tip of the iceberg. For example, there are at least 80genes in the rice (Oryza sativa) NRT1 family [5,100] but todate only two of them have been functionally characterized[101,102]. Even in Arabidopsis, less than one third ofNRT1 and NRT2 genes have been characterized. A bigquestion that plant scientists have is why higher plantsneed so many nitrate transporters. Considering that ni-trate is a major N source and plants are multicellularorganisms with specialized cells and tissues, it is notsurprising that several nitrate transporters are requiredto optimize plant growth in response to the external envi-ronment. However, how those genes function and coordi-nate external stimuli with internal growth requires morestudy. As more details come to light, it might be possible tobuild a holistic picture of nitrate usage in plants; thisknowledge could then be used to improve crop yield inunfavorable environments or harsh climatic regions.

Conflict of interestThe authors declare that they have no conflicts of interest relevant to thispaper.

AcknowledgmentsThis work was supported by grants from the National Science Council(NSC 100-2321-B-001-013 and NSC 100-2311-B-001-004-MY3) and theAcademia Sinica Postdoctoral Fellows Program.

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Erratum to: ‘‘Uptake, allocation and signalingof nitrate’’[Trends in Plant Sciences 17 (2012) 458-467]

Ya-Yun Wang, Po-Kai Hsu and Yi-Fang Tsay

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

Erratum

In the Review article ‘Uptake, allocation and signaling ofnitrate’ by Ya-Yun Wang, Po-Kai Hsu, and Yi-Fang Tsay,which was published in the August 2012 issue of Trends inPlant Science, the direction of nitrate transport mediated byCLCa,b in Figure 2 was incorrectly shown as out of thevacuole rather than into the vacuole, whereas the transport

NRT1.5

H+(?)

NO3-

NO3-

NO3- NO3

-H+

H+ CIPK23

NAXT1

NRT1 NRT

NO3-

Cytosol

CLCa,b

PO4-

Figure 2. Directions of nitrate movement mediated by nitrate transporter 1 (NRT1), NR

(SLAH3) in plasma membrane or tonoplast. Most NRT1 and NRT2 transporters character

phosphorylated by calcineurin B-like-interacting protein kinase 23 (CIPK23) to change

tonoplast are H+/NO3� antiporters. Anion channel SLAH3 mediates nitrate release from

dependant protein kinase 21; NAXT1, nitrate excretion transporter 1.

DOI of original article: http://dx.doi.org/10.1016/j.tplants.2012.04.006.

624

direction of CLCa, b shown in Figure 1 is correct. Thischange in Figure 2 does not affect the conclusions mentionedin the paper. Below is the corrected version of Figure 2

1360-1385/$ – see front matter � 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.tplants.2012.08.007 Trends in Plant Science, October 2012,

Vol. 17, No. 10

H+

H+(?)

NO3-

NO3-

NA

R2

2

CPK21

H+

Vacuole

SLAH3

NRT2.7

PO4-


T2, chloride channel (CLC) a,b, and slow anion channel-associated 1 homolog 3

ized are H+-coupled symporters. NRT1.1 (CHL1), a dual-affinity nitrate transporter, is

the affinity. NAR2 is required for NRT2 nitrate transport. CLCa,b localized in the

guard cells and its function is regulated by CPK21. Abbreviations: CPK21, calcium-

http://dx.doi.org/10.1016/j.tplants.2012.08.007

http://dx.doi.org/10.1016/j.tplants.2012.04.006

Uptake, allocation and signaling of nitrate

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