Integration of Nitrogen and Potassium Signaling

PP62CH09-Tsay ARI 16 April 2011 7:45

Integration of Nitrogenand Potassium SignalingYi-Fang Tsay, Cheng-Hsun Ho, Hui-Yu Chen,and Shan-Hua LinInstitute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529;email: [email protected]

Annu. Rev. Plant Biol. 2011. 62:207–26

First published online as a Review in Advance onApril 12, 2011

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev-arplant-042110-103837

Copyright c© 2011 by Annual Reviews.All rights reserved

1543-5008/11/0602-0207$20.00

Keywords

nitrate, ammonium, K+, nutrient signaling, ion sensor

Abstract

Sensing and responding to soil nutrient fluctuations are vital for thesurvival of higher plants. Over the past few years, great progress hasbeen made in our understanding of nitrogen and potassium signaling.Key components of the signaling pathways including sensors, kinases,miRNA, ubiquitin ligases, and transcriptional factors. These compo-nents mediate the transcriptional responses, root-architecture changes,and uptake-activity modulation induced by nitrate, ammonium, andpotassium in the soil solution. Integration of these responses allowsplants to compete for limited nutrients and to survive under nutrientdeficiency or toxic nutrient excess. A future challenge is to extend thepresent fragmented sets of data to a comprehensive signaling network.Then, such knowledge and the accompanying molecular tools can beapplied to improve the efficiency of nutrient utilization in crops.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 208TRANSCRIPTIONAL

RESPONSES . . . . . . . . . . . . . . . . . . . . . 208Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . 208Potassium . . . . . . . . . . . . . . . . . . . . . . . . . 214

ROOT-ARCHITECTURECHANGES . . . . . . . . . . . . . . . . . . . . . . . 215Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215Ammonium. . . . . . . . . . . . . . . . . . . . . . . . 216Nitrogen Metabolites . . . . . . . . . . . . . . 217Potassium . . . . . . . . . . . . . . . . . . . . . . . . . 218

MODULATION OF UPTAKEACTIVITY BY EXTERNALSUBSTRATES . . . . . . . . . . . . . . . . . . . . 218Nitrate Transporter CHL1

(AtNRT1.1) . . . . . . . . . . . . . . . . . . . . 218Ammonium Transporter AMT1 . . . . 219Potassium Channel AKT1 . . . . . . . . . . 219

INTERPLAY BETWEENNITROGEN ANDPOTASSIUM . . . . . . . . . . . . . . . . . . . . . 220

CONCLUSIONS AND FUTURECHALLENGES. . . . . . . . . . . . . . . . . . . 221

INTRODUCTION

There is an old Taiwanese saying that uses rape-seed as a metaphor to describe the fate of amarried woman, stating that wherever the seedsgerminate (i.e., the family into which a tradi-tional woman marries), she is compelled to liveout her life. In the same way as these resolutetraditional women, plants must adapt to theirown irrecusable environments. They have de-veloped various acclimation processes for thetask. With the exception of carbon, hydrogen,and oxygen, plants acquire their essential nu-trient elements from the soil. Sensing nutri-ent changes in the soil and responding to thesechanges to better cope with the fluctuation ofnutrients in the environment and to competefor limited nutrients are vital for plants to sus-tain vigorous growth. The root is the initial siteof nutrient perception. This review focuses onthe early sensing and signaling events that occur

in the root, including transcriptional responses,root-architecture changes, and modulation ofuptake activity. We use the examples of nitro-gen (N) and potassium (K) signaling to comparethe similarity and specificity of different nutri-ents and to illustrate how different responses tonutrients are integrated to ensure the survivalof the plants under various nutrient conditions.

TRANSCRIPTIONAL RESPONSES

To compete for limited nutrients in the soil,and prevent the overaccumulation of nutrients,genes encoding the transporters/channels re-sponsible for nutrient acquisition and the en-zymes required for assimilation are stringentlyand specifically regulated by the correspond-ing nutrients in both positive and negativemanners. One of the primary challenges whenstudying transcriptional responses in nutrientsignaling is to ascertain whether the specificnutrient itself, the downstream metabolites, orsome global change caused by nutrient assim-ilation (e.g., pH or redox) is the signal that isresponsible for a transcriptional response.

Nitrogen

In nonlegume plants, the two major N sourcesare nitrate and ammonium. In Arabidopsis,four nitrate transporters, including NRT1.1(CHL1), NRT1.2, NRT2.1, and NRT2.2, takeup nitrate. Nitrate is converted to nitrite bynitrate reductase encoded by NIA1 and NIA2,and nitrite is converted to ammonium by nitritereductase encoded by NIR. At least four am-monium transporters (AMT1.1, 1.2, 1.3, and1.5) can take ammonium from soil (93). Am-monium, taken directly from soil or convertedfrom nitrate, is then incorporated into aminoacids by the glutamine oxoglutarate amino-transferase (GOGAT) pathway. Expression ofseveral genes related to N-metabolism is reg-ulated by nitrate, N metabolites, as well as Ndeficiency.

Nitrate induction. In addition to being a nu-trient source, nitrate also functions as a signal

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Nit

rate

up

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Nitrate concentration (mM)

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Nit

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Figure 1Two-phase pattern of plant nitrate uptake (a), primary nitrate response (b), and CHL1 nitrate uptake activity (c). Dashed lines representhypothetic response if there is no low-affinity phase. (a) Nitrate uptake activity of Arabidopsis at different concentrations. The figure wasredrawn using the information presented in figure 5 of Wang et al. (85) and reproduced with kind permission from the NationalAcademy of Sciences. (b) Maximal levels of NRT2.1 expression induced by nitrate for 30 min. The figure was redrawn using theinformation presented in figure 2b of Ho et al. (27) and reproduced with kind permission from Elsevier. (c) Nitrate uptake activity ofnitrate transporter CHL1 expressed in Xenopus oocytes. The figure was redrawn using the information presented in figure 5a of Liu &Tsay (47).

Nitrate assimilation:nitrate is reduced tonitrite by nitratereductase in cytosol,and nitrite is reducedto ammonium bynitrite reductase inplastid/chloroplast.Ammonium isincorporated intoamino acids by theglutamine oxoglutarateaminotransferase(GOGAT) pathway

molecule, regulating gene expression. Toprepare plants for nitrate assimilation, nitrateinduces the expression of genes for nitrate up-take (NRT ), nitrate assimilation (NIA and NIR),and the generation of reducing equivalents andcarbon skeletons for N assimilation (74, 86).Because it happens quickly, within 5 to 10 minof nitrate exposure, and responds to nitrateat concentrations as low as 10 μM with norequirement for de novo protein synthesis, thistranscriptional response is referred to as theprimary nitrate response (66). Nitrate reduc-tase mutants show no defect in the primarynitrate response (61, 87), indicating that nitrateitself is responsible for this transcriptionalresponse. Three-fourths of the primary nitrateresponse genes, however, are also upregulatedby nitrite, even at low concentrations, suggest-ing that nitrite might also be recognized by thenitrate-sensing system (88).

Nitrate uptake activity displays two sat-urable phases with Km approximately 50 μMfor the high-affinity phase and 5 mM forthe low-affinity phase (Figure 1a). Similarto nitrate uptake, maximal levels of the pri-mary nitrate response also display two saturablephases with Km values similar to those of uptake(Figure 1b) (29). Interestingly, similar kineticproperties have been revealed for the uptakeactivity of the dual-affinity nitrate transporterCHL1 (AtNRT1.1) (Figure 1c) (46, 47).

The first step in the nitrate signaling path-way is binding to a sensor. The first clues onthe identity of a sensor came from the findingthat several nitrate responses, including the pri-mary nitrate response, are defective in chl1 mu-tants (29, 53, 67, 89); however, it is difficult totell whether these altered nitrate responses arean indirect consequence of a nitrate-uptake de-fect or whether CHL1 participates directly innitrate sensing. A study of an uptake and sig-naling decoupled mutant, chl1–9, showed thatnitrate transport activity is not required for thesignaling function of CHL1 and that it is a ni-trate sensor (transceptor) for the primary ni-trate response (27) (Figure 2). The proline 492residue, which is converted into leucine in thechl1–9 mutant, is essential for nitrate transportacross the membrane but not for signal trans-mission. These findings indicate that plants usethe transporter for nitrate acquisition to detectfluctuating nitrate concentrations in the soil andregulate nitrate-related genes accordingly.

In addition to nitrate assimilation–relatedgenes, several signaling components, includ-ing two kinase genes, CIPK8 and CIPK23(calcineurin B-like interaction protein kinase8 and 23), are upregulated by nitrate with asimilar temporal and concentration-dependentpattern. Studies of CIPK8 and CIPK23 indicatethat high- and low-affinity responses aregenetically distinct, with CIPK8 a positive

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Nucleus

Cytoplasm

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Primary nitrate response genes

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Glutamatedehydrogenase

Glutaminesynthetase

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Structure gene

Transcriptional activation

Transcriptional inhibition

Activation by post-transcriptional orunknown mechanism

Inhibition by post-transcriptional orunknown mechanism

Figure 2Schematic representation of signaling pathways for nitrogen-regulated transcriptional responses in Arabidopsis. Low external nitratestimulates phosphorylation of the nitrate transceptor CHL1, which induces a low level of primary nitrate response gene expression;high external nitrate, probably by binding to a low-affinity site, represses CHL1 phosphorylation and leads to high levels of geneexpression. Under low-nitrate conditions, CIPK23 phosphorylates CHL1, and CIPK8 is involved in high-nitrate responses. NLP7, thelateral organ boundary domains (LBDs), and CCA1 are transcription factors regulating nitrogen-related genes.

regulator specifically for the low-affinityresponse and CIPK23 a negative regula-tor for the high-affinity response (27, 29)(Figure 2). Both CIPK8 and CIPK23 areprimary nitrate response genes, indicating thatthey are upregulated by the signaling pathwayin which they participate. De novo proteinsynthesis is not required for the primary nitrateresponse, suggesting that the basal levels ofCIPK8 and CIPK23 are sufficient for theprimary nitrate response and that a nitrate-induced increase of these two proteins mightbe required for some additional response.At low external nitrate concentrations (inranges of the high-affinity phase), CIPK23

will phosphorylate CHL1 at threonine residue101 (T101). The phosphorylation status ofT101 may regulate the transport modes ofCHL1 and the primary nitrate response level.CHL1 phosphorylated at T101 is a high-affinity nitrate transporter and confers a low-level primary nitrate response, whereas T101-unphosphorylated CHL1 is a low-affinitytransporter that confers a high-level response.These findings for CHL1 raise several interest-ing questions, such as whether other channelsand transporters also function as nutrient sen-sors (transceptors) in higher plants, whetherthere are other types of nitrate sensors that de-tect internal or external nitrate-concentration

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NO3– NH4

+

NIT2

NIA1 AMT1.1

Nitrate reductase Ammonium transporter

Activationby binding

Figure 3Schematic representation of NIT2-mediatednitrogen-signaling network in Chlamydomonas.Expression of NIT2 is repressed by ammonium, butthe activity of NIT2 is activated by nitrate via directbinding. The active form of NIT2 inhibits theexpression of the gene encoding an ammoniumtransporter AMT1.1 but induces the expression ofthe gene encoding nitrate reductase NIA1.

Nitrogen-starvationresponse: plantsexperiencing Ndeficiency showeddecreased shoot-to-root biomass ratio,increased primary rootlength, increasedlateral root density,increased anthocyaninaccumulation, as wellas N-starvation-modulatedtranscriptional changes

changes under different conditions, and whatdownstream targets of CIPK8 and CIPK23regulate transcription levels.

In Chlamydomonas, the transcription factor(or coactivator) NIT2, which can bind to thepromoter of the nitrate reductase gene NIA1,is required for nitrate-induced expression ofnitrate assimilation genes (5). Internal nitrateis required for NIT2 to activate the NIA1 pro-moter, but expression of NIT2 is not regulatedby nitrate (Figure 3). Instead, expression ofNIT2 is repressed by ammonium, ensuring thatcells preferentially use ammonium rather thannitrate. NIT2 contains a putative DNA bind-ing and dimerization domain, RWP-RW. InArabidopsis, an RWP-RK domain–containingprotein, NLP7, has also been shown to bea positive regulator for nitrate-induced geneexpression (7) (Figure 2). In addition to the pri-mary nitrate response, NLP7 also participatesin responses to N starvation (Figure 4) be-cause, even in the presence of sufficient nitrate,the nlp7 mutant displays several N-starvationresponses, including decreased shoot-to-rootbiomass ratio, increased primary root length,

LBD37/38/39

PAP1/2

GL3

MYBL2

NLP7

DFRDFR ANS

AGT AAT

Anthocyaninaccumulation

Developmental adaptationof N limitation:

Primary root Lateral root

Shoot / Root ratio

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Proteinbinding

Key

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Structure gene

Transcriptionalactivation

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Figure 4Schematic representation of nitrogen (N)-starvation signaling in Arabidopsis.DFR (dihydroflavonol-4-reductase), ANS (anthocyanidin synthase), AGT(anthocyanin glycosyltransferase), and AAT (anthocyanin acyltransferase) aregenes encoding enzymes in the late steps of flavonoid biosynthesis. In responseto N deficiency, expression of these genes is upregulated by transcriptionfactors of GL3, PAP1/2, and lateral organ boundary domains (LBDs) toenhance anthocyanin accumulation. NLP7 and LBD37/38/39 are positive andnegative regulators of the primary nitrate response, respectively (Figure 2).Both nlp7 and lbd37/38/39 mutants show different aspects of constitutiveN-starvation phenotypes: anthocyanin accumulation in lbd mutants andincreased root growth as well as decreased shoot-to-root biomass ratio in thenlp7 mutant. NLA encodes a ubiquitin ligase, and the nla mutant shows reducedanthocyanin accumulation in response to N starvation.

and increased lateral root density, as well as N-starvation-modulated transcriptional changes.Under low-N conditions, no growth differencescould be found between wild type and nlp7mutants. Therefore, the constitutive N-starvedphenotype of nlp7 mutants suggests that in thepresence of sufficient nitrate, NLP7 is requiredto suppress the N-starvation response. Similarto Chlamydomonas NIT2, the expression ofNLP7 is not upregulated by nitrate, implyingthat nitrate has to activate the function ofNLP7 by an unknown post-transcriptional


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Split-rootexperiment:half of the root wassubmerged inN-sufficient solutionand the other half inN-deficient solution

Nitrate inductionresponse: nitrate willinduce the expressionof several N-relatedgenes

mechanism. As both NLP7 and CIPK8 arepositive regulators in the nitrate response (7,29), it will be interesting to discover the po-tential connection between NLP7 and CIPK8in regulating the primary nitrate response.

The nitrate-responsive cis-acting element–containing region of the genes NRT2.1, NIR1,and NIA1 encoding, respectively, the Arabidop-sis high-affinity nitrate transporter (21), nitritereductase (34), and nitrate reductase (84) werenarrowed down to 150 base pairs (bp), 43 bp,and 180 bp, respectively, by testing them fornitrate-responsive activity in the context of the35S minimal promoter. The regions contain-ing nitrate-responsive cis-acting elements inNRT2.1 and NIR1 are very close to the tran-scription initiation site, for example, −40 bpfor NIR1. In a study of the NIA1 promoter,by including a 130-bp fragment from NIR1 inthe synthetic promoter, investigators found thatmultiple elements located ∼1.8 kb away fromthe transcription initiation site were importantfor the nitrate-responsive activity of NIA1. Al-though a core consensus sequence was not iden-tified from analyses of the promoters of thesethree genes, the candidate transcription factorbinding sites identified in the three promoterfragments were found to be over-representedin promoters of genes highly induced by ni-trate. This suggests that each gene might useindividual motifs in various arrangements fornitrate-responsive activity. Another possibilityis that different assay conditions, for example,different concentrations of nitrate and differentexposure times, account for the distinct motifsidentified in these studies. The properties ofCIPK8 show that high- and low-affinity pri-mary nitrate responses are genetically distinct(29); therefore, different cis-elements might beresponsible for high- and low-affinity nitrateresponses.

Repression by nitrogen metabolites. In ad-dition to the external signal (the local nitratesupply in the soil), nitrate uptake as well as sev-eral N assimilation steps are also regulated byinternal systemic signals that inform the root ofthe N status of the whole plant. Because nitrate

is assimilated into amino acids, it has been sug-gested that internal pools of amino acids mayprovide a systemic feedback signal of the N sta-tus of the plant to regulate nitrate uptake andassimilation (8, 52). Indeed, by adding aminoacids or inhibitors of N assimilation, investiga-tors have suggested that N metabolites, mostlikely glutamine and glutamate, inhibit the ex-pression of genes of N uptake and assimilation(56, 65, 81, 96). Using the pNRT2.1::LUC re-porter gene for genetic screening, researchersisolated three hni (high nitrogen insensitive)mutants that showed reduced downregulationof NRT2.1 expression by high N provision.Split-root experiments demonstrated that theyare defective in systemic feedback repression ofNRT2.1 by the high N status of the whole plant(Figure 2) (20). All three mutants showed nor-mal nitrate induction response, indicating thatthe local nitrate response and systemic N re-pression response are mediated by distinct sig-naling pathways.

Transcription factors LBD37/38/39 (lateralorgan boundary domain) are negative regu-lators of nitrate-responsive genes, includingnitrate-uptake and assimilation genes, and ex-pression of LBD37/38/39 is upregulated by ni-trate and ammonium, as well as glutamine (70),suggesting that LBD37 and its two close ho-mologs might participate in feedback repres-sion of nitrate-related genes by organic N.In addition to regulating nitrate-related genes,LBD37/38/39 could also suppress a typical N-starvation response: anthocyanin biosynthesisin the presence of sufficient N/NO3

−. There-fore, NLP7 (see the section Nitrate Induction,above) and LBD37/38/39 play opposite roles inregulating nitrate-related genes, being a posi-tive regulator and negative regulators, respec-tively (7, 70). They are all, however, negativeregulators of N-starvation responses. The in-volvement of NLP7, as well as LBD37/38/39,in both the N regulation of N-related genesand N-starvation responses suggests that the ni-trate/N metabolite response and N-starvationresponses share common signaling compo-nents. The constitutive N-starvation responsesin the nlp7 mutant and lbd37 (lbd38 and lbd39)

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suggest that N-starvation responses are defaultprocesses, and various repressors need to be ac-tivated in the presence of sufficient nutrients toinhibit different sets of N-starvation responses(Figure 4).

Another transcription factor known to beinvolved in N-metabolite regulation is the mas-ter clock control gene CCA1 (24). Associa-tion of CCA1 with nitrogen regulation wasfirst revealed by a genome-wide search fororganic-nitrogen-responsive genes and inte-grated network modeling for molecular inter-actions. To validate the predicted interaction,researchers found that pulses of glutamic acidand glutamine treatment advance and delay,respectively, the peak of the CCA1 expres-sion in the circadian cycle, and overexpres-sion of CCA1 represses the expression of ASN1(asparagine synthetase 1) and GDH1 (glutamatedehydrogenase 1) and induces the expression ofGLN1.3 (glutamine synthetase) through directbinding to their promoters. This study indi-cated that organic N serves as an input affectingplant circadian clock function by modulatingCCA expression, and in turn the master clockcontrol gene CCA1 participates in the regula-tory network of N assimilation.

Nitrogen-starvation response. Althoughboth AtNRT2.1 and CHL1 are primary nitrateresponse genes that respond to nitrate withidentical patterns, they are differentially regu-lated by N starvation. N deficiency upregulatesthe expression of AtNRT2.1 but downregulatesCHL1 (39). Upregulation of AtNRT2.1 by Nstarvation could result from relief of repressionby an N metabolite. N starvation could be alocal or systemic signal. For example, althoughexpression of the Arabidopsis high-affinitynitrate transporter NRT2.1 and expression ofthe ammonium transporter (AMT) AMT1.1are both upregulated by N deficiency, split-root experiments suggested that AtNRT2.1expression is regulated by systemic signalsfrom another portion of the root systemexperiencing N deprivation or from N demandfrom the shoot, whereas AtAMT1.1 expressionis mainly modulated by the local N status

of the roots (16). These studies suggest thatN-related genes can be differentially or coor-dinately regulated by N deficiency, and eventhe coordinated regulation may be mediatedby different types of mechanisms.

Microarray analyses showed that, in additionto modulating the expression of genes involvedin metabolism, N limitation upregulates genesinvolved in protein degradation, anthocyaninand phenylpropanoid biosynthesis, and repro-gramming of mitochondrial electron transportbut downregulates genes associated with pho-tosynthesis, chlorophyll synthesis, and plastidprotein synthesis (58, 74). Among these pro-cesses, upregulation of anthocyanin biosynthe-sis by N deprivation has been studied in themost detail (Figure 4). Because N limitationreduces photosynthesis and therefore generatesexcessive light-induced oxidative damage, an-thocyanin synthesized during low N conditionsmay function as a sunscreen and scavenger of re-active oxygen species (ROS) to protect the plant(10).

In Arabidopsis, six regulatory proteins inthree different families—the WD-repeat fam-ily protein TTG1 (transparent testa glabra1);bHLH family members GL3 (glabra3), EGL3(enhancer of glabra 3), and TT8 (transparenttesta 8); and the MYB family PAP1 (productionof anthocyanin pigment 1 [AtMYB75]) andPAP2 (AtMYB90) transcription factors—areknown to positively regulate the synthesis offlavonoids, including anthocyanins, in a hierar-chical network (22). TTG1, bHLH, and MYBproteins can form an activating complex to reg-ulate the expression of several structure genesin flavonoid metabolism. Expression of PAP1/2and GL3 is upregulated by N deficiency (38).The increase in leaves was 9 × 102-fold forPAP2 and sixfold for PAP1 and GL3. The roleof GL3 in anthocyanin synthesis in responseto N deficiency was further confirmed usinga genetic approach (13). After 3 to 7 days ofN deprivation, the wild type, but not the gl3mutant, accumulated anthocyanins. The defectin anthocyanin accumulation in the gl3 mu-tant resulted from activation of DFR (encod-ing dihydroflavonol-4-reductase) expression in


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response to N deprivation, whereas the increaseof ANS (encoding anthocyanidin synthase) ex-pression under the same conditions was not af-fected in the gl3 mutant. However, under fullnutrient conditions, GL3 was shown to be ableto regulate both DFR and ANS. MYBL2, a GL3interacting protein, is able to bind to the DFRpromoter to inhibit its expression (51); there-fore, it has been proposed that the specific effectof GL3 on DFR under N-deficient conditionsresults from the release of the inhibiting com-plex of MYBL2 from the DFR promoter (13)(Figure 4).

In addition, there is some evidence thatto some extent, activation of PAP1/2 expres-sion in response to N limitation probably re-sults from the alleviation of suppression byLBD37/38/39. Suppression of PAP1 and PAP2in LBD37, 38, or 39 overexpression lines indi-cated that LBD37 and its two close homologsare negative regulators of PAP1/2 expression(70). N starvation downregulates the expres-sion of LBD37/38/39 to relieve the suppressionof PAP1 and PAP2 by LBDs and then stim-ulates the expression of structure genes DFR,ANS, AGT (anthocyanin glycosyltransferase),and AAT (anthocyanin acyltransferase) in thelate steps of flavonoid biosynthesis (Figure 4).

In addition to transcriptional control, in-creased anthocyanin synthesis in response to Ndeficiency can also be regulated by the NLA(nitrogen limitation adaptation) gene encod-ing a ubiquitin ligase (59). Upon N limita-tion, the nla mutant failed to develop severalessential acclimation responses, including an-thocyanin accumulation, N remobilization, adecrease in photosynthesis, and early senes-cence. Consistent with the lack of acclimationresponses, the majority of the 629 N-limitation-modulated genes failed to respond to N lim-itation in the nla mutant (58). In addition tothe typical RING domain found in RING-typeubiquitin ligase, NLA contains an SPX domain,which is also found in several regulatory pro-teins that control P metabolism. Interestingly,although the nla mutation does not affect Plimitation–induced anthocyanin synthesis, theearly senescence phenotype of the nla mutants

induced by N limitation can be rescued whensupplied with both low N and low P (60), indi-cating that NLA plays some role in the cross talkbetween N limitation and P limitation, whichis probably mediated by the SPX domain.

In addition to the potential protein degra-dation mediated by NLA, RNA degradationalso plays an important role in N-starvationadaptation. Transgenic Arabidopsis express-ing 35S:AtAMT1;1 showed N-deficiency-dependent accumulation of AtAMT1;1 mRNAin shoots but not in roots (93). In con-trast, 35SAtAMT1;3 plants did not display N-dependent accumulation of AMT1;3 mRNA,indicating that determination of RNA half-life is a specific regulatory mechanism forAtAMT1;1 in N-deficiency acclimation.

Potassium

In contrast to nitrate and ammonium, K isnot assimilated into organic matter. Beingthe most-abundant cation in higher plants,K serves as a major osmoticum, regulatingturgor pressure and membrane potentials. Inaddition, K is also required for the activity ofcertain enzymes (73). K deficiency leads togrowth arrest, impaired phloem transport ofsucrose, redistribution of K from mature todeveloping tissues, reduced photosynthesis,reduced water content, and replacement of Kwith an alternative osmoticum (91).

To better cope with conditions of K de-ficiency, K-uptake systems, particularly thehigh-affinity uptake system, are enhanced bytranscriptional and post-transcriptional con-trol. Three types of transport proteins areknown to be involved in high-affinity K+ up-take: K+/H+ symporters of the KT/HAK/KUPfamily (AtHAK5 in Arabidopsis and HvHAK1in barley) (14, 19, 63, 72), inward-rectifierK+ channels of the Shaker-type channel fam-ily (AtAKT1 in Arabidopsis) (40, 62, 71,92), and K+/H+ symporters of the CPA1(cation/protein-antiporter) family (AtCHX13in Arabidopsis) (95). Among them, the trans-porters AtHAT5, HvHAK1, and AtCHX13 aretranscriptionally upregulated by K deficiency,

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whereas the channel AtAKT1 is constitu-tively expressed but upregulated by a post-transcriptional mechanism.

For the transcriptional regulation, themolecular mechanism of AtHAK5 upregula-tion by K deficiency has been studied in themost detail. As shown in Figure 5, in responseto K deficiency, upregulation of AtHAK5 de-pends on ROS production mediated by theNADPH oxidase RHD2 and the type III per-oxidase RCI3 (33, 75). Moreover, ethyleneacts upstream of ROS to induce the expres-sion of HAK5. K deficiency stimulates ethyleneproduction and upregulates genes involved inethylene production and signaling (75). Thestimulation of ROS production as well as theinduction of HAK5 expression in response to Kdeficiency are eliminated in plants treated withethylene inhibitors and are partially eliminatedin ethylene-insensitive mutants (e.g., ein2, ctr1)(31).

ROOT-ARCHITECTURECHANGES

The net nutrient uptake of a plant dependson two factors: uptake activity and rootarchitecture. Therefore, modulation of rootarchitecture in response to changes in externalnutrient concentration is a critical adaptationprocess that enables plants to survive duringperiods of severe nutrient deficiency. Theplasticity of root architecture as regulated bynutrients allows the root system to be optimizedto the distribution of nutrients in the soil.

Nitrate

Many reports have shown that nitrate affectsprimary root growth and root branching, andtwo of the nitrate acquisition transporters,NRT2.1 and CHL1, have been found to par-ticipate in nitrate-regulated root development.However, probably due to different environ-mental factors [e.g., hydroponic conditions oragar plates (30), light intensity, pH, and othernutrient concentrations], the same concentra-tion of nitrate was reported to have opposing

K deficiency

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rhd2 RCF3

NADPHoxidase

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?

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Figure 5Schematic representation of potassium-starvation signaling in Arabidopsis.Ethylene acts upstream of H2O2 accumulation to induce the expression of thehigh-affinity potassium transporter gene HAK5 in response to potassiumdeficiency. Mediated through ethylene accumulation, potassium deficiency willstimulate root-hair growth and inhibit primary root growth.

effects on root development. The same genewas even found to have an opposing effect onroot-system architecture in different reports.

For example, studies by two differentgroups showed that the high-affinity nitratetransporter NRT2.1 plays a central role in reg-ulating root morphological changes in responseto N limitation (44, 68). However, two oppo-site phenotypes were found to be associatedwith the nrt2.1 mutation. When plants weregrown under low-N (0.01- to 0.5-mM nitrate)as well as high-sucrose (7.5%) conditions, lowconcentration of nitrate (together with highsucrose) repressed lateral root initiation, andmutation of NRT2.1 led to increases (cessationof repression) in lateral root initiation (44)(Figure 6). But, in another study, when plantswere transferred from high concentrationof nitrate to low concentration of nitrate,moderate N limitation (1 or 0.5 mM) increasedvisible lateral root development, and mutationof NRT2.1 abolished the stimulating effect ofN limitation and led to a decreased number


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High NO3–Low NO3

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Figure 6Schematic representation of nitrogen (N)-regulated root-system architecture.CHL1 and NRT2.1 are nitrate transporters, and AMT1 is an ammoniumtransporter. All three transporters are involved N acquisition and N-regulatedchanges in root-system architecture. The F-box protein AFG3 and miR393module integrate internal N-metabolite signaling and external nitrate signalingto modulate the root-system architecture. The MADS box transcription factorANR1 functions downstream of CHLl in lateral root proliferation stimulatedby high external nitrate. HSN1 (hypersensitive to NH4

+) encodes GDP-mannose pyrophosphorylase, and the hsn1 mutant, showing the ammonium-dependent short-root phenotype, is defective in N-glycosylation of proteins.

of lateral roots (68). In one of the studies,plants were grown continuously at the nitrateconcentrations tested (44), whereas in theother, plants were shifted from a high-nitratemedium to a low-nitrate medium (68). Anotherdifference in growth conditions between thetwo studies was the sucrose content in themedium. It is not clear whether the externalsucrose concentration, dramatic changes in theexternal nitrate concentration, or some otherfactor is responsible for the opposing effects ofNRT2.1 mutation on lateral root number.

In addition to NRT2.1, the dual-affinitynitrate transporter CHL1 has also been shown

to be involved in nitrate-regulated root-systemarchitecture. When the root was split on asegmented plate, there was a dramatic prolifer-ation and an increased number of lateral rootswith increased elongation on the high-nitrateside (67). This high-nitrate-stimulated rootproliferation was found only in wild type butwas eliminated in chl1 mutants. Similarly, un-derexpression of a MADS box gene, ANR1, alsoprevented the stimulation of lateral root elon-gation by local high nitrate (94). Expression ofANR1 in the root-tip region was found to bedownregulated in the chl1 mutant (67), suggest-ing that CHL1 acts upstream of ANR1 regulat-ing lateral root proliferation stimulated by highnitrate (Figure 6). At high-nitrate concentra-tions, chl1 mutants showed reduced lateral rootproliferation (67), but, at low concentrations orin the absence of nitrate, chl1 mutants displayedan opposite phenotype: a higher density of lat-eral roots (35). It was proposed that CHL1 mayfacilitate auxin transport, and at low-nitrateconcentrations, CHL1 may repress lateralroot growth by promoting basipetal auxintransport and reducing auxin concentration inthe root tip (35) (Figure 6). This suggests aninteresting interaction between nutrients andhormone signaling. In heterologous expressionsystems, the auxin transport activity facili-tated by CHL1 can be completely inhibitedby high nitrate (�1mM). Therefore, underhigh-nitrate conditions, the promoting effectof CHL1 on lateral root proliferation cannotbe explained by auxin transport.

Ammonium

Studies of rice, Arabidopsis, and Lotus japonicushave shown that high ammonium levels ingrowth media will inhibit root growth (25,41, 69). The question is whether root-growthinhibition is caused by ammonium itself ordownstream metabolites. Most studies, withthe notable exception of those using rice, sug-gest that ammonium itself, and probably thedirect contact of ammonium with the root tip,is responsible for the root-growth inhibition(41). In rice, the application of methionine

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sulfoximine, an inhibitor of glutamine syn-thetase, is able to relieve the ammoniumrepression of primary root elongation, whilethe elongation of lateral roots remains inhibited(25). This suggests that in rice, downstreammetabolites, rather than ammonium, areresponsible for the inhibition of seminalroot elongation, but a different mechanism,probably mediated by ammonium itself, is re-sponsible for repressing lateral root elongation.

The short-root phenotype (as well as thereduced-shoot-growth phenotype) of theArabidopsis hsn1 (hypersensitive to ammonium)mutant is ammonium dependent. HSN1encodes GDP mannose pyrophosphorylase(GMPase) (64). Further studies suggestedthat defective N-glycosylation of proteinsin the hsn1 mutant is associated with theammonium-hypersensitive phenotype (3, 64).It will be interesting to find out which of thedownstream targets of GMPase is responsiblefor the ammonium-sensitivity response. Is itthe AMT/channel responsible for ammoniumefflux or uptake? (41) AMT was found to par-ticipate in ammonium-regulated root growth.Although multiple AMT transporters aresynergistically involved in ammonium uptake(9, 93), only one AMT transporter showed amarked effect on the regulation of root growth(42, 69). Overexpression of LjAMT1;3 but notLjAMT1;1 produced a short-root phenotypeeven in the absence of ammonium supply.Similarly, in yeast, three ammonium carriers,MEP1, MEP2, and MEP3 (which are classifiedin the same family as plant AMT transporters),are involved in ammonium uptake, but onlyMEP2 acts as an ammonium sensor (tran-sceptor) regulating N-limitation-inducedpseudohyphal growth (50). In addition to N-limitation responses, MEP2 is also responsiblefor ammonium-induced activation of PKA andtrehalase (50, 79), suggesting that transceptorscan elicit differential responses in the presenceand absence of the substrate.

Nitrogen Metabolites

Exogenous glutamate has two different effectson root development: inhibition of primary

root growth and stimulation of root branch-ing (83). For primary root growth, external ni-trate sensed by NRT1.1 was shown to able toantagonize the inhibitory effect of glutamate(82). Even though ANR1 is known to act down-stream of NRT1.1 to stimulate lateral rootgrowth in response to local high nitrate supply(67, 94), ANR1 is not involved in the NRT1.1-dependent antagonizing effect of nitrate on pri-mary root growth (82).

Another level of interaction between Nmetabolites and nitrate in affecting root de-velopment is mediated by the miR393/AFB3regulatory module (80). Expression of AFB3(auxin signaling F-box 3), induced by nitrateitself, reaches a maximal level in 1 h and thendeclines. The decline of AFB3 expression afterlonger nitrate treatment results from upregu-lation of miR393 by N metabolites generatedfrom nitrate assimilation and miR393-mediatedcleavage of the AFB3 transcript. The alteredroot-architecture response to nitrate in theafb3 mutant and plants overexpressing miR393indicated that AFB3 is responsible for nitrateinhibition of primary root elongation andfor nitrate stimulation of lateral root density.Therefore, this miR393/AFB3 module servesas a regulatory loop through which the positivesignals from external nitrate and the negativesignals from internal N metabolites converge toinhibit primary root growth but stimulate lat-eral root branching (Figure 6).

Study of the miR393/ABF3 module indi-cates that auxin also plays an important rolein N-regulated primary root growth. However,the effect of auxin in primary root growth isdifferent from that in lateral root growth. Anincrease in auxin activity in the primary roottip induced by nitrate treatment is associatedwith inhibition of root growth (80). In contrast,an increase in auxin activity in the lateral roottip exposed to high nitrate is associated withstimulation of lateral root growth (35). It willbe interesting to find out whether this resultsfrom different auxin-dosage sensitivity in pri-mary and lateral roots.

Changes in root architecture are a pow-erful tool by which to identify N signaling


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mechanisms. Interestingly, three transporters,the high-affinity nitrate transporter AtNRT2.1,dual-affinity nitrate transporter CHL1, andLjAMT1;3, have been found to be involved,directly or indirectly, in N-regulated root de-velopment. CIPK8, a positive regulator ofthe primary nitrate response, is also involvedin nitrate-modulated primary root growth,suggesting that short-term transcriptional re-sponses and long-term root-growth responsesshare some common signaling components(29).

In addition to root-architecture changes,the shoot-to-root biomass ratio is anotherdevelopmental characteristic regulated by Nmetabolites or N starvation. Characterizationof a Nicotiana sylvestris mutant defective in mito-chondrial respiratory pathways suggested thatthe amine-to-nitrate ratio interacts with gib-berellin signaling and the respiratory pathwayto regulate the shoot-to-root biomass ratio (57).

Potassium

K accumulation is required for turgor pressure-mediated cell expansion in the elongation zoneof roots (11). Therefore, plants under K-deficient conditions exhibit poorly developedroots. In addition to reduced primary rootgrowth, K starvation stimulates root-hair elon-gation but decreases lateral root length and lat-eral root number in Arabidopsis (31, 75). Similarto upregulated expression of the K transporterAtHAK5, inhibition of primary root growthand promotion of root-hair elongation byK deprivation are ethylene dependent (31).However, inhibition of primary root growthby K deprivation is not affected in the type IIIperoxidase mutant rci3, indicating that primaryroot inhibition is ROS independent (33). Thesteps in the signaling cascades concerned withtranscriptional responses and the primary rootinhibitory response to K deficiency thereforeare identical in the initiation stage up toethylene production and then diverge into anROS-dependent pathway for transcriptionalresponses and an ROS-independent pathwayfor root-development modulation.

MODULATION OF UPTAKEACTIVITY BY EXTERNALSUBSTRATES

The transport activities and properties ofseveral transporters and channels involved innutrient acquisition are modulated by theirsubstrates in the soil solution. In the case ofthe nitrate transporter CHL1, this kind ofmodulation has further evolved to participatedirectly in nitrate sensing. In this section, wediscuss the post-transcriptional regulation oftransporters and channels involved in N or Kacquisition by their substrates.

Nitrate Transporter CHL1(AtNRT1.1)

Soil nitrate concentration can vary by fourorders of magnitude. As shown in Figure 1,there are two nitrate-uptake systems, thehigh-affinity nitrate-uptake system, with Km inthe range of 10 to 50 μM, and the low-affinitysystem, with Km in the range of 1 to 10 mM.Two types of nitrate transporters, NRT1and NRT2, are involved in nitrate uptake.Most of the nitrate transporters characterizedin the NRT1 family are low-affinity nitratetransporters (43). CHL1 is an exception. Itis a dual-affinity nitrate transporter, which isinvolved in both high- and low-affinity nitrateuptake (46, 85) (Figure 1). When T101 is phos-phorylated, CHL1 functions as a high-affinitynitrate transporter; however, when T101 isdephosphorylated, it functions as a low-affinitynitrate transporter (Figure 7a) (47). Thisphosphorylation switch is regulated by changesin external nitrate concentration. In responseto low concentrations of nitrate, CIPK23 willphosphorylate CHL1 at T101 and thus convertit into a high-affinity transporter (27). Whenexposed to high concentrations of nitrate, T101phosphorylation is prohibited and dephos-phorylated CHL1 functions as a low-affinitynitrate transporter. This phosphorylationswitch mechanism modulates uptake proper-ties quickly in response to changes in soil nitrateconcentration.

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Ammonium Transporter AMT1

To prevent excess ammonium accumula-tion in plants, AMTs responsible for am-monium acquisition are transcriptionally andpost-transcriptionally regulated. Four AMTs(AMT1;1, AMT1;2, AMT1;3, and AMT1;5)are involved in ammonium acquisition (93).AtAMT1;1 expression in Arabidopsis roots isrepressed by high N, probably by the inter-nal pool of glutamine, and is derepressed byN deficiency (17, 49). In addition, two AMTsare also regulated at the post-transcriptionallevel. The crystal structures of two prokary-otic AMTs indicate that they function as timerswith a substrate-conducting channel in eachsubunit (1, 32). Phosphorylation of a threonineresidue (460 in AMT1;1 and 472 in AMT1;2)in the C terminus of a single monomer exhibitsan allosteric effect leading to the cooperativeclosure of all three pores in the trimer (48,54) (Figure 7c). Moreover, phosphorylation ofT460 is upregulated by external ammonium ina concentration- and time-dependent manner(37). Therefore, either an unknown sensor orAMT1;1, functioning as a transceptor, is ableto detect that status of external ammonium andshut down the transport activity of AMT. Thismechanism could prevent the toxic accumula-tion of ammonium.

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 7Schematic representation of modulating transportactivities of (a) the nitrate transporter CHL1, (b) thepotassium channel AKT1, and (c) an ammoniumtransporter (AMT) by their substrates in theexternal solutions. AKT1 is constitutively expressed,but expression of KC1 is induced by potassiumdeficiency. KC1, AKT1, and SYP121 form atripartite complex that modifies the gating propertyof AKT1. In response to low nitrate, CIPK23phosphorylates CHL1 to convert it into a high-affinity transporter, whereas in response topotassium deficiency, CIPK23 enhances AKT1channel activity. In response to high externalammonium, the C-terminal tail of AMT1.1 andAMT1.2 is phosphorylated, leading to cooperativeclosure of all three subunits in the trimer complex.

Potassium Channel AKT1

AKT1, an influx K channel, and HAK5, a high-affinity K transporter, are responsible for Kacquisition under low-K conditions (19, 72).High-affinity K-uptake activity of the athak5,atakt1 double-mutant was reduced to 17% ofthe wild-type level, indicating that AtHAK5

High-affinity nitrate transporter Low-affinity nitrate transporter

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+

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and AtAKT1 are the two major components ofthe high-affinity K-uptake system in Arabidopsis(62, 71). CIPK23, involved in nitrate signaling,is also implicated in K-deficiency response (40,92). CIPK23, activated by calcium binding pro-teins CBL1 and CBL9, is able to phosphorylateAKT1 and enhance AKT1-uptake activity (40,92). Similar to the akt1 mutant, the cipk23 mu-tant and cbl1cbl9 double-mutant showed a low-K-sensitive phenotype. These data suggest thatlow K detected by some unknown sensor orby AKT1 itself may trigger Ca2+ signaling andactivate CBL1/9 and CIPK23 to enhance thechannel activity of AKT1 (Figure 7b).

The gating properties of AKT1 are knownto be modified by forming a complex with KCor SNRE (18, 28). Although the expressionof AKT1 is not upregulated by K starvation,the expression of the silent K channel AtKC1is induced by K starvation (75). By forming aheterotetramer with AtAKT1, AtKC1 inhibitsthe AKT1-mediated inward K current and neg-atively shifts the gating potential of the AKTchannel (12, 18, 90), indicating that AtKC1acts as an inhibitory subunit to downregulatethe activity of AtAKT1 and, additionally,may prevent K leakage at low external Kconditions (Figure 7b). It is possible thatpositive regulation of AtAKT1 activity by theCIPK23/CBL1/9 module is an early adaptationresponse of K deficiency to enhance K uptake.After expression of AtKC1 is induced by Kstarvation, negative regulation of AtAKT1 ac-tivity and, more importantly, modulation of thegating property of AtAKT1 may function as asecond step in adaptation to prevent K leakage.

INTERPLAY BETWEENNITROGEN AND POTASSIUM

The interaction between N and K is compli-cated and occurs at multiple levels. NH4

+ andK+ are highly similar with respect to charge,size, and hydration energy, suggesting that theymight share some common membrane trans-port components. Indeed, ammonium blockssome K-uptake systems (63); therefore, the rel-ative contribution of Arabidopsis HAK5 and

AKT1 in K uptake is influenced by the presenceor absence of ammonium (26, 72). A study of thehak5 mutant and akt1 mutant suggested thatHAK5-mediated K uptake is ammonium sen-sitive, whereas AKT1-mediated uptake is not(72). Consistent with these in vivo behaviors,a recent study of yeast growth suggested thatwhen expressed in yeast, both HAK5 and AKT1may facilitate ammonium transport (78), andK+ uptake of HAK5 is inhibited by ammonium.In the ammonium-tolerant species of rice, am-monium exerts a more complicated effect onK+ uptake: It inhibits high-affinity K+ uptakebut activates low-affinity K+ uptake (76). Themolecular mechanisms for the ammonium ac-tivation of low-affinity K+ uptake remain to bedetermined.

Alternatively, K+ may also affect ammoniumtoxicity and uptake. It has long been known thatthe addition of K can alleviate ammonium tox-icity (6). Futile plasma membrane cycling ofammonium is proposed to be a critical factorfor ammonium toxicity (4). Studies of rice andbarley have shown that a high concentrationof K reduces both the influx and efflux of am-monium, therefore significantly reducing theefflux-to-influx ratio and alleviating futile am-monium cycling (76, 77). The genes respon-sible for low-affinity ammonium uptake havenot been identified. Studies of K effects sug-gest that low-affinity ammonium transport ismediated by a K-sensitive component and a K-independent component. The K-sensitive com-ponent may be mediated by nonselective cationchannels (77).

N and K also exert their effects on each otherat the transcriptional level. Hyperpolarizationof the root cell membrane is considered to bethe earliest event of K deficiency (55). The hy-perpolarization of the membrane potential mayenhance inward K+ channels, such as AtAKT1(15). Moreover, in tomato, membrane poten-tials were found to correlate well with the ex-pression levels of the high-affinity K transporterLeHAK5; it has therefore been proposed thatthe hyperpolarization of the membrane poten-tial trigged by K starvation might be respon-sible for inducing the expression of LeHAK5

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(55). Consistent with this model, growth in thepresence of ammonium also induces the hy-perpolarization of the root membrane poten-tial and is associated with enhanced expressionof LeHAK5. But, in Arabidopsis, the presence ofammonium in the medium inhibits the induc-tion of AtHAK5 by K starvation (63, 72), indi-cating that ammonium has opposite effects onHAK5 expression in different species.

Moreover, there are multiple steps of inter-action between N and K. K starvation inducesthe expression of AtNRT1.5, a gene encod-ing nitrate transporter responsible for nitrateloading into the xylem, and therefore facilitatesroot-to-shoot nitrate transport (43). In the latersteps of N assimilation, multilevel analyses ofprimary metabolism showed that K deficiencyincreases the N-to-carbon ratio and maintainscarbon flux into amino acids (2). In addition,CIPK23 is involved in regulating both K up-take and nitrate uptake (27, 40, 92). It will beinteresting to find out whether CIPK23 servesas a connecting node for K and nitrate at anearly stage of nutrient perception.

CONCLUSIONS ANDFUTURE CHALLENGES

The integration of transcriptional responses,uptake-activity modulation, and root-architecture modulation provides versatileadaptations allowing plants to survive undernutrient deficiency and avoid the accumulation

of toxic levels of nutrients. Over the pastfew years, great progress has been made inunderstanding N and K signaling through theidentification of several key components suchas a sensor at the initial step and transcriptionfactors at the later steps. A future challenge willbe to build on these isolated studies to extendthe current fragmented picture into a completenetwork. Several of the signaling componentsidentified are involved in multiple responses ordifferent nutrient signaling. For example, thenitrate transceptor (sensor) CHL1 is associatedwith multiple aspects of transcriptional as wellas developmental responses (23, 27, 35, 36,53, 67, 82); the transcription factor NLP7is involved in nitrate-induced transcriptionalresponse as well as N-starvation-eliciteddevelopmental responses (7); and CIPK23is involved in both nitrate signaling and Ksignaling (27, 40, 92). Therefore, the currentimportant questions are how these compo-nents exert specific influences on each of theresponses, whether different responses aremediated by overlapping signaling cascades,and at what point signaling is diversified.Nevertheless, an ultimate challenge of nutrientsignaling will be to discover how to use theknowledge and molecular tools gained fromthe research to improve the nutrient utiliza-tion efficiency of crops so that the fertilizerdemand of agriculture can be reduced and theenvironment can be saved from eutrophicationand excess energy consumption (45).

SUMMARY POINTS

1. The nitrate transporter CHL1 (AtNRT1.1) functions as a nitrate sensor, and usingdual-affinity binding and phosphorylation switch, it can sense a wide range of nitrate-concentration changes and induce different levels of transcriptional responses.

2. Similar to CHL1, several transporters and channels are activated or inhibited via a phos-phorylation switch by external substrates, and these proteins might be potential sensors(transceptors).

3. The characterization of signaling components and transcriptional factors in N and Ksignaling reveals interesting cross-talk for different responses and different nutrients.

4. The interaction between N and K occurs at multiple levels.


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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

I thank Dr. Nigel Crawford at University of California, San Diego for critical reading of thismanuscript. Work in the Tsay laboratory is supported by Academia Sinica and Frontier ScienceResearch Program of NSC (99-2321-B-001-007).

LITERATURE CITED

1. Andrade SL, Dickmanns A, Ficner R, Einsle O. 2005. Crystal structure of the archaeal ammonium trans-porter Amt-1 from Archaeoglobus fulgidus. Proc. Natl. Acad. Sci. USA 102:14994–99

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PP62-FrontMatter ARI 15 April 2011 10:42

Annual Review ofPlant Biology

Volume 62, 2011

Contents

It Is a Long Way to GM AgricultureMarc Van Montagu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Anion Channels/Transporters in Plants: From Molecular Bases toRegulatory NetworksHelene Barbier-Brygoo, Alexis De Angeli, Sophie Filleur, Jean-Marie Frachisse,

Franco Gambale, Sebastien Thomine, and Stefanie Wege � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �25

Connecting the Plastid: Transporters of the Plastid Envelope andTheir Role in Linking Plastidial with Cytosolic MetabolismAndreas P.M. Weber and Nicole Linka � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �53

Organization and Regulation of Mitochondrial Respiration in PlantsA. Harvey Millar, James Whelan, Kathleen L. Soole, and David A. Day � � � � � � � � � � � � � � � � �79

Folate Biosynthesis, Turnover, and Transport in PlantsAndrew D. Hanson and Jesse F. Gregory III � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 105

Plant Nucleotide Sugar Formation, Interconversion, and Salvageby Sugar RecyclingMaor Bar-Peled and Malcolm A. O’Neill � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 127

Sulfur Assimilation in Photosynthetic Organisms: Molecular Functionsand Regulations of Transporters and Assimilatory EnzymesHideki Takahashi, Stanislav Kopriva, Mario Giordano, Kazuki Saito,

and Rudiger Hell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Signaling Network in Sensing Phosphate Availability in PlantsTzyy-Jen Chiou and Shu-I Lin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 185

Integration of Nitrogen and Potassium SignalingYi-Fang Tsay, Cheng-Hsun Ho, Hui-Yu Chen, and Shan-Hua Lin � � � � � � � � � � � � � � � � � � � � 207

Roles of Arbuscular Mycorrhizas in Plant Nutrition and Growth:New Paradigms from Cellular to Ecosystem ScalesSally E. Smith and F. Andrew Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 227

v

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PP62-FrontMatter ARI 15 April 2011 10:42

The BioCassava Plus Program: Biofortification of Cassava forSub-Saharan AfricaRichard Sayre, John R. Beeching, Edgar B. Cahoon, Chiedozie Egesi, Claude Fauquet,

John Fellman, Martin Fregene, Wilhelm Gruissem, Sally Mallowa, Mark Manary,Bussie Maziya-Dixon, Ada Mbanaso, Daniel P. Schachtman, Dimuth Siritunga,Nigel Taylor, Herve Vanderschuren, and Peng Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 251

In Vivo Imaging of Ca2+, pH, and Reactive Oxygen Species UsingFluorescent Probes in PlantsSarah J. Swanson, Won-Gyu Choi, Alexandra Chanoca, and Simon Gilroy � � � � � � � � � � � � 273

The Cullen-RING Ubiquitin-Protein LigasesZhihua Hua and Richard D. Vierstra � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 299

The Cryptochromes: Blue Light Photoreceptors in Plants and AnimalsInes Chaves, Richard Pokorny, Martin Byrdin, Nathalie Hoang, Thorsten Ritz,

Klaus Brettel, Lars-Oliver Essen, Gijsbertus T.J. van der Horst,Alfred Batschauer, and Margaret Ahmad � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 335

The Role of Mechanical Forces in Plant MorphogenesisVincent Mirabet, Pradeep Das, Arezki Boudaoud, and Olivier Hamant � � � � � � � � � � � � � � � � 365

Determination of Symmetric and Asymmetric Division Planesin Plant CellsCarolyn G. Rasmussen, John A. Humphries, and Laurie G. Smith � � � � � � � � � � � � � � � � � � � � � � 387

The Epigenome and Plant DevelopmentGuangming He, Axel A. Elling, and Xing Wang Deng � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 411

Genetic Regulation of Sporopollenin Synthesis and PollenExine DevelopmentTohru Ariizumi and Kinya Toriyama � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 437

Germline Specification and Function in PlantsFrederic Berger and David Twell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 461

Sex Chromosomes in Land PlantsRay Ming, Abdelhafid Bendahmane, and Susanne S. Renner � � � � � � � � � � � � � � � � � � � � � � � � � � � � 485

Evolution of PhotosynthesisMartin F. Hohmann-Marriott and Robert E. Blankenship � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 515

Convergent Evolution in Plant Specialized MetabolismEran Pichersky and Efraim Lewinsohn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 549

Evolution and Diversity of Plant Cell Walls: From Algaeto Flowering PlantsZoe Popper, Gurvan Michel, Cecile Herve, David S. Domozych,

William G.T. Willats, Maria G. Tuohy, Bernard Kloareg,and Dagmar B. Stengel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 567

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