activation of mkk9-mpk3/mpk6 enhances phosphate acquisition in arabidopsis thaliana

Activation of MKK9-MPK3/MPK6 enhances phosphateacquisition in Arabidopsis thaliana

Lei Lei, Yuan Li, Qian Wang, Juan Xu, Yifang Chen, Hailian Yang and Dongtao Ren

State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China

Author for correspondence:Dongtao RenTel: +86 10 62733794

Email: [email protected]

Received: 27 March 2014

Accepted: 29 April 2014

New Phytologist (2014) 203: 1146–1160doi: 10.1111/nph.12872

Key words: Arabidopsis thaliana, MKK9,MPK3, MPK6, phosphate, Pi responses,signal transduction pathway.

Summary

� Despite the abundance of phosphorus in soil, very little is available as phosphate (Pi) for

plants. Plants often experience low Pi (LP) stress. Intensive studies have been conducted to

reveal the mechanism used by plants to deal with LP; however, Pi sensing and signal transduc-

tion pathways are not fully understood.� Using in-gel kinase assays, we determined the activities of MPK3 and MPK6 in Arabidopsis

thaliana seedlings under both LP and Pi-sufficient (Murashige and Skoog, MS) conditions.

Using MKK9 mutant transgenic and crossed mutants, we analyzed the functions of MPK3

and MPK6 in regulating Pi responses of seedlings. The regulation of Pi responses by down-

stream components of MKK9-MPK3/MPK6 was also screened.� LP treatment activated MPK3 and MPK6. Under both LP and MS conditions, mpk3 and

mpk6 seedlings took up and accumulated less Pi than the wild-type; activation of MKK9-

MPK3/MPK6 in transgenic seedlings induced the transcription of Pi acquisition-related genes

and enhanced Pi uptake and accumulation, whereas its activation suppressed the transcription

of anthocyanin biosynthetic genes and anthocyanin accumulation; WRKY75 was downstream

of MKK9-MPK3/MPK6 when regulating the accumulation of Pi and anthocyanin, and the

transcription of Pi acquisition-related and anthocyanin biosynthetic genes.� These results suggest that the MKK9-MPK3/MPK6 cascade is part of the Pi signaling path-

way in plants.

Introduction

Phosphorus is an essential macronutrient for plant growth, devel-opment and reproduction. It is a structural constituent of manymacromolecules (e.g. nucleic acids, adenosine-50-triphosphateand phospholipids) and is indispensable during energy transfer,metabolic regulation and signal transduction (Marschner, 1995;Chiou & Lin, 2011). Phosphate (H2PO4

� and HPO42�, hereaf-

ter Pi) is the major form of phosphorus taken up by plants fromthe soil (Bieleski, 1973; Holford, 1997; Vance et al., 2003).Despite the abundance of phosphorus in the soil, very little ispresent as Pi; therefore, plants often experience low Pi (LP) con-ditions (Marschner, 1995; Shen et al., 2011). To deal with thelimited availability of Pi, plants have evolved a variety of adaptivestrategies to facilitate Pi acquisition, usage and remobilization,such as by increasing root absorption surfaces, inducing Pi trans-porter gene expression, elevating phosphatase and ribonucleaseactivities, increasing the secretion of organic acids and reducingPi requirements via alerting the metabolism (Misson et al., 2005;Yuan & Liu, 2008; Lin et al., 2009; Yang & Finnegan, 2010;Chiou & Lin, 2011).

Experiments with either Phi or methylphosphate, non-metab-olized analogs of Pi, reveal that Pi itself is functional as an initialsignal (Carswell et al., 1996; Ticconi et al., 2001; Varadarajan

et al., 2002; Pratt et al., 2004). External Pi can either be trans-ported across the plasma membrane and sensed by an intracellu-lar sensor, or sensed directly by an unknown sensor(s) that islocalized in the plasma membranes of plant cells (Svistoonoffet al., 2007; Chiou & Lin, 2011). The signal is transduced viasignaling transduction pathway(s) and subsequently regulates anextensive array of responses (Yuan & Liu, 2008; Chiou & Lin,2011). Phytohormones, such as ethylene, gibberellic acid, cytoki-nins and auxin, have been determined to be important mediatorsin the integration of Pi signaling with Pi responses (Mart�ın et al.,2000; L�opez-Bucio et al., 2002; Franco-Zorrilla et al., 2005;Devaiah et al., 2009; Nagarajan & Smith, 2012). Sugar signalinghas been reported to regulate root system architecture (RSA) andthe expression of Pi starvation-induced genes (Hammond &White, 2008, 2011). MicroRNAs can either be a systemic signalin the transduction of Pi deficiency signaling or can target anddirect the cleavage of some Pi-responsive genes (Lin et al., 2008;Pant et al., 2008; Kuo & Chiou, 2011). Transcription factors,such as MYB family members (PHR1, PHL1, MYB62) andWRKY family members (WRKY75), have also been shown to beimportant signaling components in the regulation of Pi responses(Devaiah et al., 2007, 2009; Bustos et al., 2010). Some membersof the SPX domain-containing protein families have recentlybeen shown to be involved in Pi signaling (Kant et al., 2011;

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Secco et al., 2012; Wang et al., 2012). Our knowledge of the Pisignaling components in plants has been greatly improved duringthe past few years; however, to fully understand the mechanismof plant responses to Pi, research on the interaction between thesecomponents and the identification of the novel signaling machin-ery for the regulation of Pi responses is still needed (Chiou &Lin, 2011).

Mitogen-activated protein kinase (MAPK) cascades, consistingof MAPKKK, MAPKK and MAPK, are highly conserved signal-ing modules in eukaryotes. These cascades have been demon-strated to be modules that link upstream receptors or sensors todownstream processes in various ways. MAPK cascades haveimportant functions in regulating plant stress responses, growthand developmental processes (Colcombet & Hirt, 2008; Pit-zschke et al., 2009; Andreasson & Ellis, 2010; Rodriguez et al.,2010; Komis et al., 2011; Tena et al., 2011). The Arabidopsisgenome contains 60 MAPKKKs, 10 MAPKKs and 20 MAPKs(namely MKKK, MKK and MPK) (MAPK-Group, 2002). Pre-vious reports have shown that MPK3 and/or MPK6 can be acti-vated by different MKKs and participate in specific signalingpathways: MKK3 activates MPK6 to regulate jasmonic acid sig-naling (Takahashi et al., 2007); MKK2 activates MPK4/MPK6to mediate cold and salt stress tolerance (Teige et al., 2004);MKK4/MKK5 activate MPK3/MPK6 to regulate H2O2 produc-tion (Ren et al., 2002), H2O2-induced NO production (Wang Pet al., 2010), stomata and ovule development (Wang et al., 2007,2008) and defense responses (Asai et al., 2002; Wang Y et al.,2010); MKK4/MKK5/MKK9 activate MPK3/MPK6 to induceethylene and camalexin biosynthesis (Liu et al., 2008; Xu et al.,2008); and MKK9 activates MPK3/MPK6 to regulate leaf senes-cence (Zhou et al., 2009) and ethylene signaling (Yoo et al.,2008). Because Pi is involved in many cellular processes, LP con-ditions cause severe nutrient stress during plant growth anddevelopment. Genome-scale transcriptional profiling has revealedthat multiple genes in these MAPK modules are induced by LP(Misson et al., 2005; Graham et al., 2006). However, it remainsunknown whether MAPK signaling cascades are involved directlyin the regulation of plant responses to LP.

In this study, we found that MPK3 and MPK6 could beactivated in wild-type (WT) seedlings under LP conditions.Constitutive activation of MPK3 and MPK6 via upstreamMKK9 in MKK9 transgenic seedlings suppresses LP-inducedanthocyanin production. After MKK9-MPK3/MPK6 activation,the transcription of several LP-responsive genes was inducedunder Pi-sufficient (Murashige and Skoog, MS) conditions andenhanced under LP conditions, and Pi uptake and accumulationwere increased under both LP and MS conditions. Further analy-sis showed that the MAPK module regulated Pi responsesthrough WRKY75.

Materials and Methods

Plant materials and growth conditions

WT and mutant Arabidopsis thaliana (L.) Heynh (ecotype,Columbia-0) seeds were surface sterilized and sown on 1% agar

plates containing 0.59MS and 1% sucrose, pH 5.7. After coldtreatment at 4°C for 2 d, the seeds were germinated and grownon plates at 22°C in a growth room with a 16-h photoperiod at aphoton flux density of 100 lmol m�2 s�1 for 7 d.

The MS and LP media used for the Pi treatments wereprepared as described by Chen et al. (2009). The Pi concentra-tion in MS medium was 1.26 mM and in LP medium was10 lM. For Pi response experiments, 7-d-old seedlings of similarsizes were transferred onto new plates containing either MS orLP medium, and grown for the additional days as indicated. ForMKK9 mutant protein induction, 0.02 lM dexamethasone(DEX) was pre-added to the media. Photographs were taken, andseedlings were harvested and used for anthocyanin and Pi contentmeasurements.

For soil growth, seedlings were transferred from 0.59MSmedium to soil and grown in a growth room with a 12-h photo-period at a photon flux density of 100 lmol m�2 s�1 for 7 d.Soil-grown plants were used for genetic crossing and seed setting.

T-DNA insertion mutants and genetic crosses

Vector, MKK9KR, MKK9DD, MKK9DD/mpk3, MKK9DD/mpk6,MKK9DD/ein2, mpk3, mpk6 and mkk9 plants were generated asdescribed previously (Xu et al., 2008). The T-DNA insertionmutants, including wrky33 (SALK_006603), wrky75(SALK_101367) and phr1 (SALK_067629), were obtained fromthe Arabidopsis Biological Resource Center. The homozygousphr1 and wrky75 mutants were screened using genomic PCR andconfirmed by reverse transcription-polymerase chain reaction(RT-PCR) using gene-specific primers. Homozygous wrky33 wasidentified according to Zheng et al. (2006). All mutants werecrossed into theMKK9DD background and the homozygous dou-ble mutants were used for the experiments. The sequences of theprimers used are listed in Supporting Information Table S1.

RNA isolation, RT-PCR and real-time quantitative RT-PCR(Q-PCR)

Total RNA was isolated from samples using Trizol reagent (Invi-trogen). First-strand cDNA was synthesized by M-MLV reversetranscriptase (Promega) using oligo dT(16) as the primer andtotal RNA as the template. The RT-PCR experiments wereconducted to confirm gene knockouts using WRKY33- andWRKY75-specific primers. Real-time Q-PCR was performed inthe presence of SYBR Green Mix (Takara, Dalian, China) tomonitor Pi-responsive gene expression. Amplification was con-ducted in real time with a 7500 real-time PCR system (AppliedBiosystems). Expression levels of Ubiquitin5 were used as theinternal control. The sequences of the primers are listed inTable S1.

Measurement of anthocyanin and total Pi content

For the anthocyanin content assay, the seedlings were harvestedand quickly frozen in liquid nitrogen. Anthocyanin content insamples was measured as described by Teng et al. (2005). For the

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measurement of Pi content, the shoots and roots of seedlingswere separately collected and dried at 80°C for 48 h. Afterrecording their dry weight, these samples were flamed to ash anddissolved in 0.1M HCl. Pi in the samples was measured asdescribed by Ames (1966).

Pi uptake assay

For pretreatment, 7-d-old seedlings that had germinated on0.59MS medium were transferred to either MS or LP mediumwith or without DEX for an additional 1 or 3 d. Groups of 10seedlings were weighed and used as one biological sample. Piuptake assay was performed as described by Devaiah et al. (2007)and with modifications. In this study, 0.02 lM DEX was addedto both the pretreatment and uptake solutions, and 0.3 lCi[32Pi]orthophosphate was used for each sample. Radioactivitywas measured using a Hidex 300SL Automatic Liquid Scintilla-tion Counter (Hidex, Turku, Finland).

Immunoblot and in-gel kinase assays

Protein extraction, separation, immunoblot and in-gel kinaseassays were performed as described previously (Xu et al., 2008).An anti-Flag M2 monoclonal antibody (1 : 10 000) was used asthe primary antibody, and a horseradish peroxidase-conjugatedgoat anti-mouse antibody was used as the secondary antibody(1 : 10 000). The protein membranes were visualized using anEnhanced Chemiluminescence Kit (Roche) and exposed to X-rayfilm. For in-gel kinase assays, myelin basic protein was embeddedin the separating gel as a substrate for the kinases.

Microarray analysis

WT and MKK9DD seedlings were grown on 0.59MS mediumfor 7 d, and WT seedlings were then transferred to either MS orLP medium and grown for an additional 3 d. MKK9DD seedlingswere then transferred to MS medium with or without DEX andgrown for an additional day. Samples were harvested and totalRNA was isolated. RNA purification and chip hybridization wereperformed using Affymetrix Microarray Services (CapitalBio Co.,Beijing, China). Microarray data were deposited at the ArrayEx-press database (www.ebi.ac.uk/arrayexpress) under accessionnumber E-MTAB-2553. The identification of differentiallyexpressed genes was performed using Significance Analysis ofMicroarrays software 2.10 with a fold change of 2.0 or 1.5 andP < 0.01 as cut-off values.

Accession numbers

Sequence data from this article can be found in the ArabidopsisGenome Initiative or GenBank/EMBL databases under thefollowing accession numbers: MKK9, At1g73500; MPK3,At3g45640; MPK6, At2g43790; WRKY75, At5g13080;WRKY33, At2g38470; Pht1;1, At5g43350; Pht1;4, At2g38940;At4, At5g03545; miR399d, At2g34202; MYB62, At1g68320;IPS1, At3g09922; DFR, At5g42800; LDOX, At4g22880;

PHR1, At4g28610; UBQ5, At3g62250. T-DNA insertion linesused here are: mpk3 (SALK_100651), mpk6 (SALK_127507),wrky75 (SALK_101367), phr1 (SALK_067629) and wrky33(SALK_006603).

Results

Pi deficiency activates MPK6 and MPK3

To determine whether MAPKs or other kinases are involved inthe response of Arabidopsis plants to different Pi conditions, wegrew Col-0 WT seedlings on 0.59MS medium for 7 d, trans-ferred them to new plates with either MS or LP medium, andtook samples at various times. Kinase activities in these seedlingswere analyzed using in-gel kinase activity assays. The resultsshowed that WT seedlings grown on 0.59MS medium beforetransfer to new medium contained a lower basal level activity of a46-kDa kinase (Fig. 1a, 0 d); the 46-kDa kinase activity wasincreased slightly in WT seedlings after transfer to MS mediumand was increased greatly in WT seedlings after transfer to LPmedium. A 40-kDa kinase was newly induced, and its activityreached similar levels in WT seedlings transferred to either MS orLP medium; however, a 43-kDa kinase was newly activated inWT seedlings only after transfer to LP medium. The activationof these 43- and 46-kDa kinases specifically on LP treatment sug-gests that their activities might be involved in seedling responsesto LP conditions.

The sizes of the activated 43- and 46-kDa kinases are similarto MPK3 and MPK6, two MAPKs that have been reported to beactivated in response to multiple stresses in Arabidopsis. Thempk3 and mpk6 mutants, MPK3 and MPK6 T-DNA insert nullmutants which have been shown to lack MPK3 and MPK6 activ-ities (Xu et al., 2008), were used in our Pi treatment experiments.As shown in Fig. 1(a), LP treatment failed to activate either the43-kDa kinase in the mpk3 seedlings or the 46-kDa kinase in thempk6 seedlings. These results confirm that the 43- and 46-kDakinases are indeed MPK3 and MPK6, respectively. In addition tothe MPK3 and MPK6 activities, the transcription levels of thetwo kinase genes in WT seedlings treated with LP for 24 h werealso assessed. The results showed that transcription of both geneswas induced by LP treatment (Fig. 1b). The activation of MPK3and MPK6 kinase activities and the induction of their genetranscription by LP treatment indicate that MPK3- and MPK6-mediated MAPK cascades may be needed for Arabidopsis seedlingresponses to Pi conditions.

Activities of both MPK3 and MPK6 are needed for Piacquisition by seedlings

To explore the roles of MPK3 and MPK6 in the responses ofseedlings to Pi, Pi concentrations in shoots and roots of WT,mpk3 and mpk6 seedlings were further measured. The resultsshowed that Pi concentrations (lmol Pi g�1 DW) in both rootsand shoots of mpk6 seedlings and in roots of mpk3 seedlingsunder MS conditions were reduced significantly relative to thosein WT seedlings; Pi concentrations in roots of mpk3 and mpk6

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seedlings under LP conditions were reduced significantly relativeto those in WT seedlings (Fig. 1c). Because of the embryoniclethal phenotype of the mpk3/mpk6 double-knockout mutant, wecould not include it in our experiments. The reduction in Pi lev-els in mpk3 and mpk6 seedlings suggests that the activities ofMPK3 and MPK6 are needed for Pi accumulation in seedlings.

Total Pi content in plants is primarily dependent on theuptake of Pi. Because the Pi content in WT seedlings was higherthan that in mpk3 and mpk6 seedlings, Pi uptake by seedlings ofWT and the two mutants were further measured. Seven-day-oldseedlings, which were initially grown on 0.59MS medium, weretransferred to either MS or LP medium and pretreated for anadditional 3 d. Seedlings were then transferred into a Pi uptakesolution with 32Pi, and Pi uptake by seedlings over a 2-h periodwas measured. Figure 2 shows the Pi uptake results. After incuba-tion of the seedlings in the Pi uptake solution for 1 h, Pi uptakeamounts in seedlings of WT, mpk3 and mpk6 pretreated withMS conditions did not show significant differences; however, Piuptake in seedlings of WT pretreated with LP conditions was sig-nificantly higher than that in mpk3 and mpk6 seedlings. Afterincubation of the seedlings in the Pi uptake solution for 2 h, Piuptake amounts in seedlings of WT pretreated with either MS orLP conditions were significantly higher than those in mpk3 andmpk6 seedlings. These data suggest that the loss of MPK3 andMPK6 activities in mpk3 and mpk6 seedlings reduces Pi uptakeand thereby may cause the lower Pi accumulation.

Plants grown under lower Pi availability have evolved a seriesof responses to Pi deficiency. The accumulation of anthocyaninin the aerial portion of the plants (visualized as a purple-coloredaerial portion) is a common response to lower Pi and otherstresses (Dixon & Paiva, 1995; Marschner, 1995). As shown inFig. S1(a), the aerial portions of WT, mpk3 and mpk6 seedlingsturned purple under LP conditions, creating unrecognizable dif-ferences between WT and the two mutants. Alterations of theRSA, such as the inhibition of primary root elongation andincrease (under moderate Pi deficiency, 50 lM Pi) or decrease(under severe Pi deficiency, 6 lM Pi) of lateral root formation, inplants grown under lower Pi conditions, are adaptive responses toPi deficiency (P�eret et al., 2011; Gruber et al., 2013). The LPconditions used in this study represent severe Pi deficiency condi-tions (LP medium contains 10 lM Pi). Figure S1 shows thatWT, mpk3 and mpk6 seedlings grown under LP conditions havea shorter primary root length relative to seedlings under MS con-ditions; LP treatment decreases the lateral root density in WTand mpk3 seedlings, and increases the lateral root density inmpk6 seedlings. However, when comparing mpk6 and mpk3 withWT, the two mutant seedlings show shorter primary root lengthand higher lateral root density under either MS or LP conditions.Previously, M€uller et al. (2010) have shown that mpk6 seedlingsgrown on 0.59MS-phytogel medium have a shorter primaryroot length and slightly decreased lateral root number, whereasL�opez-Bucio et al. (2014) have reported that mpk6 seedlingsgrown on 0.29MS-agar medium have longer primary rootlength and higher lateral root density. The different resultsobtained by different groups may be a result of the use of differ-ent experimental conditions. The similar phenotypes in mpk3

(a)

(b)

(c)

Fig. 1 Low phosphate (Pi; H2PO4� and HPO4

2�) stress activates MPK3and MPK6, and loss of their activities reduces seedling Pi accumulation.Seven-day-old Arabidopsis Columbia-0 wild-type (WT),mpk3 andmpk6

seedlings germinated on 0.59Murashige and Skoog (MS) medium weretransferred to Pi-sufficient (MS) or low Pi (LP) medium, and the sampleswere taken at the indicated times. (a) Kinase activity in WT,mpk3 andmpk6 seedlings was detected by an in-gel kinase assay with myelin basicprotein (MBP) as the substrate. Each lane was loaded with 10 lg totalprotein. Coomassie-stained Rubisc L (large subunit of ribulose-1,5-biphosphate carboxylase) was used as the loading control. CCB, colloidalCoomassie blue. (b) Transcription levels ofMPK3 andMPK6 in WTseedlings were monitored by real-time quantitative reverse transcription-polymerase chain reaction (Q-PCR). (c) Pi content in shoots and roots ofseedlings after transfer into MS or LP medium for an additional 7 d. Datarepresent the means� SD of three biological replicates of each treatment.Statistically significant differences between WT and eithermpk3 ormpk6

seedlings (paired t-test): **, P < 0.01.





seedlings may also be explained in the same way. However, theprimary root and lateral root phenotypes shown in mpk3 andmpk6 seedlings under both MS and LP conditions suggest thatthe loss of MPK3 and MPK6 causes RSA changes independentof Pi status in the medium.

Activation of MPK3 and MPK6 by MKK9 enhances Piaccumulation

The activation of MAPKs requires dual phosphorylation of thre-onine (Thr) and tyrosine (Tyr) residues in the TXY motif by acti-vated upstream MAPKKs (Cobb & Goldsmith, 1995). Manyreports have shown that multiple MKKs, such as MKK4, MKK5and MKK9, activate both MPK3 and MPK6 in Arabidopsis (Renet al., 2002; Xu et al., 2008). Previously, Xu et al. (2008) gener-ated transgenic plants carrying mutated MKK9 genes (eitherMKK9DD or MKK9KR) under the control of a steroid-induciblepromoter. MKK9DD is a constitutively active MKK9 kinasemutant, and MKK9KR is an inactive MKK9 kinase mutant. InMKK9DD plants, induction of MKK9DD, by application of DEX,led to long-lasting activation of MPK3 and MPK6, whereas, inthe control (MKK9KR plants), induction of MKK9KR could notactivate either MPK3 or MPK6.

In this study, anthocyanin and Pi concentrations in MKK9mutant transgenic seedlings were compared. Seven-day-old seed-lings, initially grown on 0.59MS medium, were transferred toeither MS or LP medium in the presence or absence of DEX, andgrown for an additional 7 d. Two independent transgenic linesfor MKK9KR and MKK9DD were used in the experiments, and anempty vector transgenic line (hereby referred to as Vector) wasused as the vector control. Color observation showed that the aer-ial portions of the Vector, MKK9KR and MKK9DD seedlings onMS medium, with or without DEX, were all green, whereasVector, MKK9KR and MKK9DD seedlings on LP medium withoutDEX and Vector and MKK9KR seedlings on LP medium withDEX were all purple, but MKK9DD seedlings on LP mediumwith DEX were green (Fig. 3a). When we further measured theanthocyanin content in seedlings, seedlings grown on MSmedium with or without DEX produced lower but comparable

levels of anthocyanin; compared with seedlings grown on MSmedium with DEX, the anthocyanin content in Vector, MKK9KR

and MKK9DD seedlings grown on LP medium with DEXincreased. However, the anthocyanin content in MKK9DD seed-lings differed significantly from that in Vector and MKK9KR, withVector and MKK9KR seedlings accumulating over 200% anthocy-anin relative to MKK9DD seedlings (Fig. 3b). Pi content resultsshowed that MKK9DD seedlings grown on either MS or LPmedium with DEX accumulated significantly higher levels of Pithan Vector and MKK9KR seedlings (Fig. 3c,d). RSA changes indifferent transgenic plants were compared and did not show obvi-ous differences between the genotypes (Fig. S2). DEX treatmentsdid not alter Pi and anthocyanin levels in WT seedlings (Fig. S3).These results suggest that the activation of MKK9 suppressesanthocyanin accumulation under LP conditions and enhances Piaccumulation under both MS and LP conditions.

As the activity of MKK9 was shown to be involved in the regu-lation of anthocyanin and Pi contents, mkk9, a previously identi-fied MKK9 T-DNA insertion mutant (Xu et al., 2008), wasfurther analyzed in this study (Fig. S4). Significantly, inductionofMKK9 transcription in WT seedlings by LP treatment suggeststhe involvement of MKK9 in LP responses; however, anthocya-nin and Pi contents in WT and mkk9 seedlings under both MSand LP conditions did not show significant differences. Possibly,other closely related MKKs, such as MKK4 and MKK5, whichhave been shown previously to regulate ethylene and camalexinproduction, similar to MKK9, are functionally redundant withMKK9 in the regulation of the seedling response to Pi (Liu et al.,2008; Ren et al., 2008; Xu et al., 2008). Upregulation of MKK4and MKK5 mRNA expression in the mkk9 mutant, as shown byXu et al. (2008), supports this hypothesis.

To explore the roles of MPK3 and MPK6 in the MKK9-induced changes in anthocyanin and Pi concentrations, we fur-ther compared MKK9KR, MKK9DD, MKK9DD/mpk3 andMKK9DD/mpk6 seedlings. On MS medium with DEX, seedlingsof MKK9KR, MKK9DD, MKK9DD/mpk3 and MKK9DD/mpk6were all green, whereas, on LP medium with DEX, seedlings ofMKK9KR, MKK9DD/mpk3 and MKK9DD/mpk6 were purple, butonly MKK9DD seedlings were green (Figs 4a, S5). The four

Fig. 2 Loss of MPK3 or MPK6 activity in Arabidopsis seedlings reduces phosphate (Pi; H2PO4� and HPO4

2�) uptake. Seven-day-old wild-type (WT, closedcircles),mpk3 (open squares) andmpk6 (open circles) seedlings germinated on 0.59Murashige and Skoog (MS) medium were transferred and pretreatedwith either Pi-sufficient (MS) or low Pi (LP) medium for an additional 3 d. Seedlings were incubated in Pi uptake solution, and Pi uptake was monitoredover a 2-h period. Data represent the means� SD of three biological replicates for each treatment. Statistically significant differences betweenWTseedlings and eithermpk3 ormpk6 seedlings (paired t-test): *, P < 0.05; **, P < 0.01.



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genotype seedlings grown on MS medium with DEX producedlower levels of anthocyanin than seedlings grown on LP mediumwith DEX. However, MKK9KR, MKK9DD/mpk3 and MKK9DD/mpk6 seedlings grown on LP medium with DEX accumulatedover 190%, 150% and 160%, respectively, of the anthocyaninlevels of MKK9DD seedlings (Fig. 4b). Pi content measurementsshowed that Pi accumulation in MKK9DD/mpk3 and MKK9DD/mpk6 seedlings was significantly reduced compared with that inMKK9DD seedlings and reached the levels seen in MKK9KR seed-lings (Fig. 4c). These results suggest that full activation of MPK3and MPK6 by MKK9 is required for both the suppression ofanthocyanin production under LP conditions and the elevationof Pi accumulation under either MS or LP conditions.

Activation of MKK9-MPK3/MPK6 enhances Pi uptake

To further understand how the MKK9-MPK3/MPK6 cascadeinduces Pi accumulation, we analyzed Pi uptake by MKK9KR,MKK9DD,MKK9DD/mpk3 andMKK9DD/mpk6 seedlings. Seven-day-old seedlings, which were grown initially on 0.59MSmedium, were transferred and pretreated with either MS or LP

medium plus DEX for an additional day. Seedlings were thentransferred into the Pi uptake solution containing 32Pi plus DEX.Pi uptake by seedlings over a 2-h period was measured. Pi uptakein both MKK9DD and MKK9KR seedlings pretreated with MSplus DEX was similar in the first hour; however, Pi uptake byMKK9DD seedlings was 165% of the uptake by MKK9KR seed-lings by the second hour (Fig. 5a). Pi uptake by MKK9DD seed-lings pretreated with LP plus DEX was significantly higher thanthat by MKK9KR seedlings at both tested time points, with Piuptake by MKK9DD seedlings being 159% and 143% of theuptake by MKK9KR seedlings (Fig. 5b). Pi uptake by MKK9DD/mpk3 and MKK9DD/mpk6 seedlings was significantly reducedcompared with that byMKK9DD seedlings (Fig. 5c,d). At the firstand second time points, MKK9DD/mpk3 seedlings retained 81%and 67%, respectively, of the Pi taken up by MKK9DD seedlingspretreated with MS plus DEX, and 60% and 66%, respectively,of the Pi taken up by MKK9DD seedlings pretreated with LP plusDEX; MKK9DD/mpk6 seedlings retained 68% and 72%, respec-tively, of the Pi taken up by MKK9DD seedlings pretreated withMS plus DEX, and 69% and 86%, respectively, of the Pi takenup by MKK9DD seedlings pretreated with LP plus DEX. These

(a) (b)

(c) (d)

Fig. 3 Induction of activeMKK9mutant(MKK9DD) expression in transgenicArabidopsis plants enhances phosphate (Pi;H2PO4

� and HPO42�) accumulation and

suppresses anthocyanin accumulation.Transgenic plants carrying the activeMKK9

mutant (MKK9DD) transgene, the inactivemutant (MKK9KR) transgene or the emptyvector (Vector) were analyzed. Twoindependent homozygous transgenic linesforMKK9DD andMKK9KR were included forPi and anthocynin measurement. Seven-day-old seedlings germinated on 0.59Murashigeand Skoog (MS) medium were transferred toeither Pi-sufficient (MS) or low Pi (LP)medium with or without dexamethasone(DEX) for an additional 7 d. (a) Phenotypecomparison of Vector,MKK9DD andMKK9KR

seedlings. (b) Anthocyanin content in Vector,MKK9DD andMKK9KR seedlings transferredto either MS or LP medium with or withoutDEX. (c) Pi content in shoots and roots ofVector,MKK9DD andMKK9KR seedlingstransferred to MS medium with or withoutDEX. (d) Pi content in shoots and roots ofVector,MKK9DD andMKK9KR seedlingstransferred to LP medium with or withoutDEX. Data represent the means� SD of threebiological replicates for each treatment.Statistically significant differences betweenMKK9DD seedlings and either Vector orMKK9KR seedlings (paired t-test):**, P < 0.01.





data demonstrate that activation of MPK3/MPK6 by MKK9 suf-ficiently enhances seedling Pi uptake and suggest that increasedPi uptake may be a major contributor to MKK9-MPK3/MPK6activation-enhanced Pi accumulation in seedlings.

Activation of MKK9-MPK3/MPK6 regulates some Pi-responsive gene transcription

Genome-wide transcriptional analyses have revealed that hun-dreds of genes in plants are either induced or suppressed by Pideficiency (Hammond et al., 2003; Wu et al., 2003; Missonet al., 2005). Such genes encode functional proteins to act asenzymes in metabolic pathways, transporters for Pi and otherions, transcription factors, components of signaling pathwaysand regulators of growth and developmental processes. To fullyunderstand how the activation of MKK9-MPK3/MPK6 regulatesPi responses, we performed a transcriptomics analysis ofMKK9DD seedlings using Affymetrix Arabidopsis Gene Chips,and compared it with that of LP-treated WT seedlings. As shownin Fig. 6, when using a two-fold cut-off value (P < 0.01), 398genes were upregulated and 69 genes were downregulated inMKK9DD seedlings after MKK9DD induction, whereas 434 geneswere upregulated and 80 genes were downregulated in WT seed-lings treated with LP. Among the regulated genes, 125 of theupregulated genes and 12 of the downregulated genes overlappedin both MKK9DD and WT seedlings. When the cut-off value wasrelaxed to 1.5-fold (P < 0.01), the up- and downregulated genesreached 699 and 155, respectively, in MKK9DD seedlings, and

855 and 157, respectively, in WT seedlings; 301 of the upregulat-ed genes and 25 of the downregulated genes overlapped in bothMKK9DD and WT seedlings. Changes in some previouslyreported Pi-responsive genes are shown in Table 1. These datasuggest that the activation of MKK9-MPK3/MPK6 regulatespart of the Pi-responsive gene transcription.

The transcription levels of several previously reportedPi-responsive genes in MKK9KR, MKK9DD, MKK9DD/mpk3 andMKK9DD/mpk6 seedlings were also tested using Q-PCR (Fig. 7).Pht1;1 and Pht1;4, two high-affinity Pi transporters, are involvedin Pi acquisition (Mitsukawa et al., 1997; Misson et al., 2004;Shin et al., 2004). As shown in Fig. 7, the transcription levels ofPht1;1 and Pht1;4 were significantly induced in MKK9DD seed-lings under LP conditions and slightly induced inMKK9DD seed-lings under MS conditions by MKK9DD expression (+DEX)compared with MKK9KR seedlings. WRKY75, encoding a WRKYfamily transcription factor, is induced by LP and positively regu-lates plant Pi uptake (Devaiah et al., 2007). Under both MS andLP conditions, WRKY75 transcription was significantly inducedin MKK9DD seedlings by MKK9DD expression (+DEX) com-pared with MKK9KR seedlings. MYB62, which encodes an R2R3MYB family transcription factor, has been shown to be inducedby LP and positively regulates Pi uptake (Devaiah et al., 2009).Here, transcription of MYB62 was strongly induced in MKK9DD

seedlings under both MS and LP conditions by MKK9DD expres-sion (+DEX). miR399d, which encodes a microRNA that targetsand directs the cleavage of PHO2 mRNA (PHO2 encodes aubiquitin-conjugating E2 enzyme), is highly induced by LP

(a) (b)

(c)

Fig. 4 Activation of endogenous MPK3 andMPK6 is required for MKK9-enhancedphosphate (Pi; H2PO4

� and HPO42�)

accumulation and MKK9-suppressedanthocyanin accumulation in Arabidopsis

seedlings. Seven-day-oldMKK9KR,MKK9DD,MKK9DD/mpk3 andMKK9DD/mpk6seedlings, germinated on 0.59Murashigeand Skoog (MS) medium, were transferred toeither Pi-sufficient (MS) or low Pi (LP)medium with dexamethasone (DEX) for anadditional 7 d. (a) Phenotype comparison ofMKK9mutant transgenic seedlings andcrossed seedlings. (b) Anthocyanin content inMKK9mutant transgenic seedlings andcrossed seedlings transferred to either MS orLP medium with DEX. (c) Pi content in shootsand roots of MKK9 mutant transgenicseedlings and crossed seedlings transferred toeither MS or LP medium with DEX. Datarepresent the means� SD of three biologicalreplicates for each treatment. Statisticallysignificant differences betweenMKK9DD

seedlings andMKK9KR,MKK9DD/mpk3 orMKK9DD/mpk6 seedlings (paired t-test):*, P < 0.05; **, P < 0.01.



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(Aung et al., 2006; Bari et al., 2006; Chiou et al., 2006). IPS1(Induced by Phosphate Starvation 1), a member of the Mt4/TPSI1gene family that encodes a non-coding RNA that sequestersmiR399 and inhibits miR399 cleavage activity, has beenreported to be strongly induced by LP (Devaiah et al., 2007;Franco-Zorrilla et al., 2007). At4, another member of the Mt4/TPSI1 gene family, has been shown to regulate Pi distributionbetween roots and shoots (Shin et al., 2006). In contrast with thegenes induced by MKK9DD expression, transcription levels ofIPS1, miR399 and At4 in this study were strongly suppressed inMKK9DD seedlings under LP conditions by MKK9DD expression(+DEX) compared with MKK9KR seedlings. The induction ofPht1;1, Pht1;4, WRKY75 and MYB62 by MKK9DD expressionwas compromised in MKK9DD/mpk3 and MKK9DD/mpk6 seed-lings, whereas the suppression of IPS1, miR399 and At4 tran-scription was partially or fully rescued in MKK9DD/mpk3 andMKK9DD/mpk6 seedlings. The data suggest that the regulation ofthese genes by MKK9 activation required both MPK3 andMPK6 activities, and the MKK9-MPK3/MPK6 cascade plays animportant role in regulating Pi-responsive gene transcription.

Because the accumulation of anthocyanin was suppressed afteractivation of MPK3/MPK6 by MKK9 (Figs 3, 4), the transcrip-tion of DFR and LDOX (also known as ANS), which encode

dihydroflavonol 4-reductase and leucoanthocyanidin dioxygen-ase, respectively, major enzymes in the anthocyanin biosyntheticpathway (Holton & Cornish, 1995; Martens et al., 2010), wasanalyzed by Q-PCR (Fig. 7). Our results show that the transcrip-tion levels of DFR and LDOX were suppressed in MKK9DD seed-lings under LP conditions by MKK9DD expression (+DEX)compared with their induction in MKK9KR, MKK9DD/mpk3 andMKK9DD/mpk6 seedlings. The gene transcription results agreedwell with our color observations and anthocyanin measurementresults, suggesting that the suppression of LP-induced anthocya-nin biosynthetic gene transcription and anthocyanin accumula-tion by MKK9 activation requires both MPK3 and MPK6activities.

WRKY75 is required for MKK9-MPK3/MPK6-inducedalteration of Pi responses

Q-PCR and Genechip results revealed that the transcription of aset of LP-responsive genes is regulated by the activation ofMKK9-MPK3/MPK6. Among these genes, MYB62 andWRKY75 have been shown previously to positively regulate Piuptake in plants (Devaiah et al., 2007, 2009). WRKY33 encodesa WRKY transcription factor that can be phosphorylated by

(a) (b)

(c) (d)

Fig. 5 Activation of MKK9 enhances phosphate (Pi; H2PO4� and HPO4

2�) uptake through endogenous MPK3 and MPK6 in Arabidopsis seedlings. Seven-day-oldMKK9KR,MKK9DD,MKK9DD/mpk3 andMKK9DD/mpk6 seedlings germinated on 0.59Murashige and Skoog (MS) medium were transferred andpretreated with either Pi-sufficient (MS) or low Pi (LP) medium with dexamethasone (DEX) for an additional day. Seedlings were incubated in Pi uptakesolution with DEX, and Pi uptake was monitored over a 2-h period. (a) Comparison of Pi uptake betweenMKK9DD andMKK9KR seedlings from MSmedium with DEX. (b) Comparison of Pi uptake betweenMKK9DD andMKK9KR seedlings from LP medium with DEX. (c) Comparison of Pi uptake amongMKK9DD,MKK9DD/mpk3 andMKK9DD/mpk6 seedlings from MS medium with DEX. (d) Comparison of Pi uptake amongMKK9DD,MKK9DD/mpk3 andMKK9DD/mpk6 seedlings from LP medium with DEX. Data represent the means� SD of three biological replicates for each treatment. Statisticallysignificant differences betweenMKK9DD seedlings andMKK9KR,MKK9DD/mpk3 orMKK9DD/mpk6 seedlings (paired t-test): *, P < 0.05; **, P < 0.01.





MPK3 and MPK6 (Mao et al., 2011). To determine whetherMYB62, WRKY33 and WRKY75 are involved in MKK9-MPK3/MPK6-regulated Pi responses, we initially attempted to generateand analyze MKK9DD/myb62, MKK9DD/wrky75 and MKK9DD/wrky33 mutants. Because we failed to obtain T-DNA insertionhomozygous mutants of MYB62, we could not generate theMKK9DD/myb62 mutant; therefore, MKK9DD/wrky33 andMKK9DD/wrky75 seedlings were used in the Pi treatment experi-ments (Figs S5, S6).

As shown in Fig. 8(a), the aerial portions of MKK9DD, wrky75and MKK9DD/wrky75 seedlings were all purple after transfer toLP medium without DEX for an additional 7 d, whereas the aer-ial portions of MKK9DD seedlings were green after transfer to LPmedium with DEX, but the aerial portions of MKK9DD/wrky75and wrky75 seedlings were still purple. The anthocyanin contentin seedlings on LP medium with DEX showed that MKK9DD/wrky75 seedlings produced over 169% of the anthocyanin levelthat accumulated in MKK9DD seedlings (Fig. 8b). The anthocya-nin content results agreed well with the color observations. Mea-surements of Pi content showed that the Pi accumulation ofMKK9DD and MKK9DD/wrky75 seedlings was significantly dif-ferent; shoots and roots of MKK9DD/wrky75 seedlings accumu-lated c. 24% and 22% less Pi, respectively, under MS plus DEXconditions, and 34% and 24% less Pi, respectively, under LP plusDEX conditions than the levels seen in MKK9DD seedlings(Fig. 8c). The accumulation of Pi and anthocyanin in MKK9DD/wrky33 seedlings did not show a significant difference comparedwith that in MKK9DD seedlings under both MS and LP condi-tions plus DEX (Fig. S7). Loss of WRKY75 function inMKK9DD/wrky75 seedlings reduced MKK9DD-enhanced Piaccumulation and rescued anthocyanin production, suggestingthat WRKY75 is a downstream component of MKK9-MPK3/MPK6 in the regulation of anthocyanin and Pi accumulation.

To determine the role of WRKY75 in MKK9-MPK3/MPK6-regulated Pi-responsive gene transcription, gene transcription (seegenes in Fig. 7 with the exception of WRKY75) in MKK9DD/wrky75 seedlings was analyzed by Q-PCR and further compared

with MKK9DD seedlings (Fig. 9). The results revealed that thetranscription levels of Pht1;1, Pht1;4 and MYB62, which wereinduced inMKK9DD seedlings under both LP and MS conditionsby MKK9DD expression (+DEX), were reduced to varyingdegrees in MKK9DD/wrky75 seedlings; IPS1, DFR and LDOXtranscription, which was suppressed in MKK9DD seedlings underLP conditions by MKK9DD expression (+DEX), was either par-tially or fully rescued in MKK9DD/wrky75 seedlings, whereasIPS1 and LDOX transcription levels in MKK9DD/wrky75 seed-lings under MS with DEX conditions did not show any obviouschange, and DFR transcription was reduced in MKK9DD/wrky75seedlings compared with MKK9DD seedlings. miR399d

Fig. 6 Number of genes upregulated or downregulated in Arabidopsis

wild-type (WT) seedlings treated with low phosphate (Pi; H2PO4� and

HPO42�) (LP) and inMKK9DD seedlings treated with dexamethasone

(DEX).

Table 1 Comparison of the selected phosphate (Pi; H2PO4� and HPO4

2�)-responsive gene expression in Arabidopsis wild-type (WT) seedlings afterPi starvation andMKK9DD seedlings after MKK9DD induction

Functionalcategories Gene ID

Genesymbol

Relative expression(fold) P < 0.01

WTLP/MS

MKK9DD

+DEX/�DEX

Sensing andsignaling

At5g20150 AtSPX1 7.1674 1.0023At2g45130 AtSPX3 29.1861 2.3398

Transport At5g43350 Pht1:1 2.0227 0.8016At2g38940 Pht1:4 4.9968 1.5661At2g32830 Pht1:5 8.2338 2.4971At1g76430 Pht1:9 3.1698 **At3g52190 PHF1 2.7280 1.0046At1g73220 AtOCT1 7.2432 2.1828

Transcription factor At1g71130 AtERF070 3.1152 0.996At1g68320 MYB62 2.4767 3.6145At5g04340 ZAT6 1.9114 2.7880At5g13080 WRKY75 1.7483 1.9326At1g13300 HRS1 1.4148 1.9135At1g62300 WRKY6 1.3856 2.6625

Others At1g68740 PHO1;H1 2.7093 1.1055At5g09470 DIC3 7.6837 1.6997At1g08650 AtPPCK1 3.7556 0.7556

Scavenging/mobilizationof Pi

At1g56650 AtPAP1 5.7152 0.3168At1g52940 AtPAP5 8.0963 **At2g01880 AtPAP7 2.9542 1.1206At2g16430 AtPAP10 2.1150 0.9096At1g08310 AtPAP11 12.2583 1.2411At2g27190 AtPAP12 2.8961 1.2594At2g46880 AtPAP14 21.5065 0.6546At3g17790 AtPAP17 4.0488 1.2950At4g13700 AtPAP23 6.3252 **At4g24890 AtPAP24 8.5700 0.9377At4g36350 AtPAP25 5.5855 **At1g73010 AtPS2 22.6080 1.4196At1g17710 AtPECP1 22.5063 0.7784At2g02990 RNS1 16.0136 1.9051

Metabolism At4g15210 BAM5 21.2347 0.3812At5g42800 DFR 6.2305 0.2478At1g56650 CHS 5.7152 0.3047At5g54060 UF3GT 5.5309 0.2515At3g51240 F3H 1.9314 0.7817

Seven-day-old WT seedlings were transferred to Murashige and Skoog(MS) or low Pi (LP) medium for 3 d, andMKK9DD seedlings were trans-ferred to MS medium with or without dexamethasone (DEX) for 1 d.**, P > 0.01.



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transcription, which was suppressed in MKK9DD seedlings underboth LP and MS conditions by MKK9DD expression (+DEX),could not be rescued in MKK9DD/wrky75 seedlings; At4 tran-scription, which was suppressed in MKK9DD seedlings underboth LP and MS conditions by MKK9DD expression (+DEX),was decreased in MKK9DD/wrky75 seedlings under LP with DEXconditions and increased in MKK9DD/wrky75 seedlings underMS with DEX conditions compared with MKK9DD seedlings.These data suggest that the regulation of the Pi-responsive tran-scription of certain genes by MKK9-MPK3/MPK6 requiresWRKY75.

Discussion

On limited Pi availability in soil, plants have evolved adaptivemechanisms to sense Pi availability, transduce the signal and sub-sequently promote a series of responses that maximize Pi acquisi-tion, alter Pi utilization and facilitate survival under Pi-limitedconditions (Marschner, 1995; Raghothama, 1999; Poirier &Bucher, 2002; Yuan & Liu, 2008; Lin et al., 2009; Rouachedet al., 2010; Yang & Finnegan, 2010; Chiou & Lin, 2011; Kuo& Chiou, 2011; P�eret et al., 2011). The transcriptional and/orpost-transcriptional regulation of many Pi starvation-responsive(PSR) genes and the post-translational regulation of proteins havebeen well documented in many plant species in response to Piavailability; however, the Pi sensor(s)/receptor(s) and the signal-ing pathway(s) in plants are not fully understood. In this study,our results reveal that the MAPK signaling cascade, MKK9-MPK3/MPK6, plays an important role in the regulation of Piresponses in Arabidopsis.

MAPK cascades have been reported to be important modulesfor the perception and transduction of signals from receptors orsensors for the induction of intracellular responses (Colcombet &Hirt, 2008; Pitzschke et al., 2009; Yoo et al., 2009; Andreasson& Ellis, 2010; Rodriguez et al., 2010). In Arabidopsis, activation

of MPK3 and/or MPK6 is involved in the regulation of plantresponses to multiple stresses, such as pathogen infection (Asaiet al., 2002; Ren et al., 2008; Beckers et al., 2009; Galletti et al.,2011), dehydration (Xu & Chua, 2012), salt (Yu et al., 2010),cold (Teige et al., 2004), heat shock (Li et al., 2012), UV-B(Gonz�alez Besteiro et al., 2011) and oxidative stress (Lee & Ellis,2007; Wang P et al., 2010). The increase in MPK3 and MPK6activities and their gene transcription in seedlings induced by LP(Fig. 1) suggest that these MAPKs may also mediate the regula-tion of seedling responses to LP conditions. This finding was fur-ther supported by the following evidence: compared with WTseedlings, mpk3 and mpk6 mutants accumulated less Pi under LPconditions and had lower levels of Pi uptake under both MS andLP conditions (Figs 1, 2). After transgene induction, MKK9DD

seedlings with overly activated MPK3 and MPK6 accumulatedless anthocyanin under LP conditions, and accumulated more Piunder both LP and MS conditions, relative to MKK9KR seedlings(Fig. 3). Activation of MPK3 and MPK6 in MKK9DD seedlingsenhanced Pi uptake, whereas loss of MPK activity in eitherMKK9DD/mpk3 or MKK9DD/mpk6 seedlings reduced Pi uptake(Fig. 5). Activation of MPK3 and MPK6 by MKK9DD regulatedthe transcription of a set of LP-responsive genes in seedlingsunder either LP or MS conditions (Figs 6, 7). Furthermore, pre-vious genome-wide transcriptional analyses, which have reportedthe induction of MKKK14, MKKK15, Raf43 and Raf35 inArabidopsis (Misson et al., 2005), and MAPKK2 and WIPK incommon bean (Phaseolus vulgaris L.) (Graham et al., 2006), alsosupport the involvement of MAPK cascades in the Pi stressresponse.

Previous studies have demonstrated that activation of MKK9-MPK3/MPK6 induces camalexin production (Xu et al., 2008)and WRKY33 is required for camalexin induction (Mao et al.,2011). Currently, there is no evidence for a correlation betweencamalexin biosynthesis and Pi responses; thus, we detected antho-cyanin and Pi contents in MKK9DD/wrky33 seedlings, which

Fig. 7 Transcription detection of multiplephosphate (Pi; H2PO4

� and HPO42�)-

responsive genes inMKK9mutant transgenicand crossed Arabidopsis seedlings. Seven-day-oldMKK9mutant transgenic seedlingsand crossed seedlings, germinated on0.59Murashige and Skoog (MS) medium,were transferred to either Pi-sufficient (MS;open bars) or low Pi (LP; closed bars)medium with dexamethasone (DEX) for anadditional 3 d. Transcription levels of Pht1;1,Pht1;4,WRKY75,MYB62, IPS1, At4,miR399d, DFR and LDOX in seedlings weremonitored by real-time quantitative reversetranscription-polymerase chain reaction(Q-PCR). Data are the means� SD of threebiological replicates for each treatment, andrepresent fold changes normalized to thetranscription levels of theMKK9mutanttransgenic seedlings or crossed seedlings inMS medium.





produce a rare camalexin after MPK3/MPK6 activation (Renet al., 2008; Xu et al., 2008; Mao et al., 2011). However, thecomparable anthocyanin and Pi contents in MKK9DD andMKK9DD/wrky33 seedlings suggest that MKK9-MPK3/MPK6-induced Pi responses are independent of camalexin production(Fig. S7).

WRKY75, which encodes a WRKY family transcriptionfactor, can be induced by LP, pathogen infection and senescence(Robatzek & Somssich, 2001, 2002; Guo et al., 2004; Devaiahet al., 2007). Under LP conditions, downregulation of WRKY75transcription in WRKY75 RNAi plants has been reported toreduce Pi uptake and PSR gene induction, and increase anthocya-nin accumulation (Devaiah et al., 2007). These results suggestthat WRKY75 is involved in the regulation of LP responsesin Arabidopsis (Devaiah et al., 2007). Here, we found that

LP-induced WRKY75 transcription was enhanced by MKK9-MPK3/MPK6 activation (Fig. 7; Table 1). To determine whetherWRKY75 is a functional downstream component of the MKK9-MPK3/MPK6 cascade in regulating LP responses, the MKK9DD/wrky75 mutant was analyzed. To avoid the effect of a lower levelof WRKY75 mRNA retained in the RNAi plants (Devaiah et al.,2007), a T-DNA insertion mutant of WRKY75 was crossed toMKK9DD. Interestingly, the loss of WRKY75 function in theMKK9DD background (MKK9DD/wrky75) reduced the transcrip-tion of Pi uptake-related genes (including Pht1;1, Pht1;4 andMYB62 in our experiments) and MKK9-MPK3/MPK6-enhanced Pi accumulation. In addition, the transcription ofanthocyanin biosynthetic genes (e.g. LDOX and DFR) andanthocyanin accumulation, which were suppressed by the activa-tion of MKK9-MPK3/MPK6, were rescued in MKK9DD/wrky75

(a)

(b)

(c) Fig. 8 MKK9-MPK3/MPK6 enhancedphosphate (Pi; H2PO4

� and HPO42�)

accumulation and suppressed anthocyaninproduction throughWRKY75 in Arabidopsis

seedlings. Seven-day-oldMKK9DD transgenicseedlings and crossed seedlings, germinatedon 0.59Murashige and Skoog (MS)medium, were transferred to low Pi (LP)medium either with or withoutdexamethasone (DEX) for an additional 7 d.(a) Phenotype comparison ofMKK9DD

seedlings and crossed seedlings transferred toLP medium either with or without DEX. (b)Anthocyanin content inMKK9DD seedlingsand crossed seedlings transferred to LPmedium with DEX. (c) Pi content in shootsand roots ofMKK9DD seedlings and crossedseedlings transferred to LP medium withDEX. Data represent the means� SD of threebiological replicates for each treatment.Statistically significant differences betweenMKK9DD/wrky75 seedlings and eitherMKK9DD orwrky75 seedlings (paired t-test):**, P < 0.01.



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NewPhytologist1156

seedlings. These results suggest that MKK9-MPK3/MPK6 regu-lates both anthocynin production and Pi accumulation throughWRKY75.

MAPK phosphorylation sites generally contain a P-X-S/T-Psequence (Alvarez et al., 1991; Clark-Lewis et al., 1991; Sorens-son et al., 2012). The putative WRKY75 protein, however, doesnot contain the P-X-S/T-P sequence, and MKK9-MPK3/MPK6cannot phosphorylate recombinant WRKY75 protein (data notshown), thereby excluding WRKY75 as a MAPK substrate, andfurther suggesting that WRKY75 is genetically downstream ofMKK9-MPK3/MPK6 during the regulation of Pi responses. AsWRKY75 is not a substrate of MAPK, but transcription ofWRKY75 can be induced by MKK9-MPK3/MPK6, we proposethat MKK9-MPK3/MPK6 may phosphorylate an unrevealedtranscription factor TFa, subsequently promote the transcriptionofWRKY75 and thereby regulate Pi responses.

As PHR1 has been reported to be a key component in the reg-ulation of transcription of a large proportion of Pi-responsivegenes and positively regulates Pi accumulation (Rubio et al.,2001; Bustos et al., 2010), we speculate that it may be the TFacandidate. However, the following reasons exclude PHR1 as thecandidate (Fig. S8). First, PHR1 did not interact with MPK3and MPK6 in a yeast two-hybrid system; second, PHR1 couldnot be successfully phosphorylated by MPK3 and MPK6 (onlyan extremely weak band was shown compared with the well-usedMAPK substrate MBP protein); third, loss of PHR1 function inMKK9DD/phr1 double mutant did not affect MKK9-MPK3/MPK6 activation-enhanced Pi accumulation under MS condi-tions.

Based on previous reports and our data, we propose a workingmodel for Pi responses in plants (Fig 10). When the availabilityof Pi in environment is altered, plants sense the varied Pi state byan unidentified sensor or receptor. The Pi transporter PHO84 inyeast (Giots et al., 2003) and nitrate transporter CHL1 in Ara-bidopsis (Ho et al., 2009) also function as Pi sensors (called

transceptors). Whether the Pi transporters in plants also act as‘transceptors’ awaits elucidation. The active sensor/receptor acti-vates either the MAPK cascade or a MAPK-independent signal-ing module. MKKKx-MKK9-MPK3/MPK6 was suggested asthe MAPK cascade in our study. The sensor/receptor can activatethe MKKKx-MKK9-MPK3/MPK6 cascade directly or throughan additional mediator. MPK3 and MPK6 phosphorylate anunknown transcription factor TFa, thereby promoting the tran-scription of the WRKY75 gene, and subsequently induce theadaptive responses to facilitate Pi acquisition. Identification ofTFa and MKKKx will facilitate the understanding of the mecha-nism by which MKK9-MPK3/MPK6 regulates Pi responses.

Acknowledgements

This work was supported by grants from the State Basic ResearchProgram (2012CB114200) and the National Natural ScienceFoundation of China (31125006 and 31030010) to D.R.; the

Fig. 9 Transcription detection of multiplephosphate (Pi; H2PO4

� and HPO42�)-

responsive genes inMKK9DD andMKK9DD/wrky75 Arabidopsis seedlings. Seven-day-oldMKK9DD andMKK9DD/wrky75seedlings, germinated on 0.59Murashigeand Skoog (MS) medium, were transferred toeither Pi-sufficient (MS; open bars) or low Pi(LP; closed bars) medium withdexamethasone (DEX) for an additional 3 d.Transcription levels of Pht1;1, Pht1;4,MYB62, IPS1, At4,miR399d, DFR andLDOX in seedlings were monitored by real-time quantitative reverse transcription-polymerase chain reaction (Q-PCR). Data arethe means� SD of three biological replicatesfor each treatment, and represent foldchanges normalized to the transcriptionlevels of theMKK9DD orMKK9DD/wrky75

seedlings in MS medium.

Fig. 10 Proposed model for phosphate (Pi; H2PO4� and HPO4

2�) sensing,signaling and responses in Arabidopsis plants.





National Natural Science Foundation of China (30771124) toH.Y.; and the National Natural Science Foundation of China(31000127) to Y.L.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Phenotypes of Arabidopsis wild-type (WT), mpk3 andmpk6 seedlings.

Fig. S2 Effect of MKK9 activity on primary root length andlateral root density of Arabidopsis.

Fig. S3 Anthocyanin and Pi (phosphate; H2PO4� and HPO4

2�)accumulation in Arabidopsis Col wild-type (WT) seedlings under

Murashige and Skoog (MS) and low Pi (LP) conditions with orwithout dexamethasone (DEX).

Fig. S4 Anthocyanin and Pi (phosphate; H2PO4� and HPO4

2�)contents in Arabidopsis wild-type (WT) and mkk9 seedlings aftertransfer to Murashige and Skoog (MS) or low Pi (LP) medium.

Fig. S5 Immunoblot analyses of MKK9 mutant transgene expres-sion, and MPK3 and MPK6 kinase activity assays of MKK9mutant transgenic and crossed Arabidopsis seedlings.

Fig. S6 T-DNA insertion of Arabidopsis WRKY33 and WRKY75mutants.

Fig. S7 Activation of MKK9-MPK3/MPK6 in Arabidopsis seed-lings enhances Pi (phosphate; H2PO4

� and HPO42�) accumula-

tion and suppresses anthocyanin production through acamalexin-independent pathway.

Fig. S8 Detection of the interaction between Arabidopsis PHR1and MPK3 or MPK6, phosphorylation by MPK3 and MPK6 onPHR1, and regulation of PHR1 on MKK9-MPK3/MPK6-enhanced Pi (phosphate; H2PO4

� and HPO42�) accumulation.

Table S1 Oligonucleotides used in this study

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activation of mkk9-mpk3/mpk6 enhances phosphate acquisition in arabidopsis thaliana

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