the s-domain receptor kinase arabidopsis …...the s-domain receptor kinase arabidopsis receptor...

The S-Domain Receptor Kinase Arabidopsis ReceptorKinase2 and the U Box/Armadillo Repeat-ContainingE3 Ubiquitin Ligase9 Module MediatesLateral Root Development under PhosphateStarvation in Arabidopsis1[C][W][OPEN]

Srijani Deb2, Subramanian Sankaranarayanan2, Gayathri Wewala, Ellen Widdup, and Marcus A. Samuel*

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4

ORCID ID: 0000-0002-3936-5563 (M.A.S.).

When plants encounter nutrient-limiting conditions in the soil, the root architecture is redesigned to generate numerous lateral roots(LRs) that increase the surface area of roots, promoting efficient uptake of these deficient nutrients. Of the many essential nutrients,reduced availability of inorganic phosphate has a major impact on plant growth because of the requirement of inorganic phosphate forsynthesis of organic molecules, such as nucleic acids, ATP, and phospholipids, that function in various crucial metabolic activities. Inour screens to identify a potential role for the S-domain receptor kinase1-6 and its interacting downstream signaling partner, theArabidopsis (Arabidopsis thaliana) plant U box/armadillo repeat-containing E3 ligase9 (AtPUB9), we identified a role for this modulein regulating LR development under phosphate-starved conditions. Our results show that Arabidopsis double mutant plantslacking AtPUB9 and Arabidopsis Receptor Kinase2 (AtARK2; ark2-1/pub9-1) display severely reduced LRs when grownunder phosphate-starved conditions. Under these starvation conditions, these plants accumulated very low to no auxin intheir primary root and LR tips as observed through expression of the auxin reporter DR5::uidA transgene. Exogenous auxin wassufficient to rescue the LR developmental defects in the ark2-1/pub9-1 lines, indicating a requirement of auxin accumulation for thisprocess. Our subcellular localization studies with tobacco (Nicotiana tabacum) suspension-cultured cells indicate that interactionbetween ARK2 and AtPUB9 results in accumulation of AtPUB9 in the autophagosomes. Inhibition of autophagy in wild-type plantsresulted in reduction of LR development and auxin accumulation under phosphate-starved conditions, suggesting a role forautophagy in regulating LR development. Thus, our study has uncovered a previously unknown signaling module (ARK2-PUB9) that is required for auxin-mediated LR development under phosphate-starved conditions.

Roots are one of the most important adaptations ofland plants, because they provide anchorage, facilitateabsorption of water and minerals, and also, aid inspecialty functions, such as storage of food and waterand vegetative reproduction in some plant species.Plants have the unique ability to alter their root mor-phology depending on resource availability in the soil.Under nutrient-starved conditions, this allows them tomodify the roots to efficiently explore the heteroge-neous soil environment for nutrients. Phosphorous in

its inorganic form is often present at low concentra-tions and hence, heavily supplemented through fer-tilizers in agriculture. Inorganic phosphate (Pi) is themain form of plant-assimilated phosphorous presentin the soil. Pi starvation is an increasing global prob-lem in agriculture, and even in fertile soils, Pi con-centration rarely exceeds 10 mM (Bieleski, 1973). Tocope with these chronically low Pi levels, plants havedeveloped highly specialized physiological and bio-chemical mechanisms to efficiently acquire and use Pifrom the environment. This involves changes in theroot architecture, increase in root-shoot ratio, and roothair development (Williamson et al., 2001; López-Bucio et al., 2002; Al-Ghazi et al., 2003). When plantsperceive Pi starvation, the most common response isthe induction of numerous lateral roots (LRs), inhibi-tion of primary roots (PRs), and formation of denserroot hairs, thus restructuring the roots for exhaustiveexploration of the topsoil for Pi (Raghothama, 1999;Lynch and Brown, 2001). During Pi deficiency, plantroot hairs account for 90% of the total Pi uptake by theplants (Raghothama, 1999). Phosphate is acquired byhigh-affinity Pi transporters and loaded into the xylemin roots. Pi moves freely through both xylem andphloem. When Pi is not limiting, Pi efflux by anionchannels ensures Pi homeostasis. The high-affinity and

1 This work was supported by the Natural Sciences and Engineer-ing Research Council of Canada (funding to M.A.S.), and the Univer-sity Research Grants Committee from the University of Calgary(grants to M.A.S.).

2 These authors contributed equally to this work.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Marcus A. Samuel ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OPEN] Articles can be viewed online without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.114.244376

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low-affinity Pi transporters also mediate phosphatetransport across the plasma membrane and tonoplast,which is powered by the membrane proton ATPase(Raghothama, 2000).

Multiple signals and molecular mechanisms are ini-tiated when plants are exposed to Pi starvation. Theyinclude transcriptional regulation of gene expression,gene silencing by microRNAs, and posttranslationalmodification, like sumoylation and ubiquitination (forreview, see Rojas-Triana et al., 2013). Although thecomplete signaling network through which plants copewith Pi limitation has not been deciphered, there isprecedence for the role of hormones in altering theroot system architecture during Pi starvation. Studiessuggest both auxin-dependent and independent mech-anisms during adaptive response to Pi limitation(Williamson et al., 2001; López-Bucio et al., 2002; Al-Ghazi et al., 2003; Nacry et al., 2005). However, cyto-kinins are known to negatively regulate this response(Martín et al., 2000; Wang et al., 2006). In addition, crosstalk between hormone signaling and sugar signalingpathways during response to Pi starvation has alsobeen reported (Hammond and White, 2011).

Several ubiquitin (Ub)-conjugating and -deconjugatingenzymes are known to function in controlling adaptiveresponse to Pi starvation. F-Box Protein2 (FBX2) pro-teins, which contain both WD40 and F-box motifs,negatively regulate responses, like root hair formation,expression of phosphoenolpyruvate carboxylase ki-nase (PPCK; PPCK1 and PPCK2), and anthocyaninaccumulation during Pi starvation (Chen et al., 2008).FBX2 also interacts with basic helix-loop-helix32 invitro, another negative regulator of Pi starvation re-sponses (Chen et al., 2008). Recently, a rice (Oryzasativa) U box containing E3 Ub ligase, OsUPS, that isinduced by Pi starvation has also been isolated (Huret al., 2012).

One common response of plant cells to nutrientstarvation is the induction of autophagy or self-eating,in which autophagosomes are used for bulk degrada-tion of cellular organelles to maintain the cell at a lowmetabolic state (Bassham, 2007). Depending on thesize of the cytoplasmic material engulfed for degra-dation, plant autophagy can be classified as eithermicroautophagy or macroautophagy (Bassham et al.,2006). Autophagy-specific gene 18a (AtATG18a) inArabidopsis (Arabidopsis thaliana) is required for auto-phagic response during Suc/nitrogen starvation andsenescence (Xiong et al., 2005). RNA interference lineswith reduced expression of AtATG18a are hypersen-sitive to Suc and nitrogen starvation (Xiong et al.,2005). Autophagy is also known to play a role in nu-trient remobilization. Autophagy mutants of Arabi-dopsis are reduced in their nitrogen remobilizationefficiency, leading to lower biomass and yield (Guiboileauet al., 2012). Similarly, disruption of ArabidopsisATG5 prevents formation of autophagosomes andcauses sensitivity to nitrogen starvation (Thompsonet al., 2005). Under Pi starvation, Ub-like proteinATG8 has been shown to be up-regulated in root tips,

and disruption of ATG5 leads to early consumption ofroot meristem (Sakhonwasee and Abel, 2009). Despiteall this evidence for the requirement of autophagy formaintaining root architecture, the mechanisms by whichautophagy regulates this process during Pi starvationremain unclear.

Arabidopsis plant U box/armadillo repeat protein9(AtPUB9) belongs to the armadillo repeat-containingproteins with a U box that is commonly present inmany E3 ligases (Mudgil et al., 2004). Although thebiological functions of most of these proteins are stillunknown, the roles of other PUB family proteins rangefrom self-incompatibility to hormone responses todefense and abiotic stress responses (for review, seeYee and Goring, 2009). S-domain receptor kinases areknown to function as upstream activators of PUBs(Samuel et al., 2008). Previously, it has been shownthat one of the S-domain receptor kinase1-6 Arabi-dopsis Receptor Kinase2 (ARK2) could efficientlyphosphorylate AtPUB9 in vitro (Samuel et al., 2008),and when coexpressed in tobacco (Nicotiana tabacum)suspension-cultured cells, this interaction led to local-ization of AtPUB9 in punctate structures (Samuel et al.,2008). In this report, we show that these punctatestructures are lytic compartments or autophagosomes.Because autophagosomes are induced under nutrient-starved conditions, we investigated pub9-1 and ark2-1mutant phenotypes under nutrient-limiting conditions.We found out that, under Pi starvation, ark2-1/pub9-1double mutants were deficient in their ability to de-velop LRs. Our additional investigation revealed thatthis defect is likely caused by lack of auxin accumu-lation at the LR initiation sites. The LR defects in thedouble mutants could be rescued through comple-mentation with either ARK2 or PUB9 as well as ap-plication of exogenous auxin.

RESULTS

PUB9 Localizes to Punctate Structures and Colocalizes withthe Autophagosomal Marker ATG8 in the Presenceof ARK2

AtPUBs have been shown to interact with the Ara-bidopsis S-domain receptor kinases (Samuel et al., 2008)and likely function as downstream signaling moleculesto these kinases. This interaction is known to lead toboth phosphorylation of the AtPUBs and alterationin their cellular localization (Samuel et al., 2008). Intobacco Bright Yellow2 (BY2) cells, PUB9 localizationcould be influenced by its interaction with the cytosolickinase domains of ARK1 and ARK2; whereas ARK1redistributes PUB9 from the nucleus to the plasmamembrane, ARK2 interaction leads to localization ofPUB9 in punctate structures in 40% of the cells (Samuelet al., 2008). To identify these subcellular structures, weperformed colocalization studies in the same systemwith fluorescent marker proteins or dyes that localizeon intracellular punctate structures. When PUB9 and

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ARK2 were cotransformed with red fluorescent protein(RFP)-tagged oleosin (the major structural protein as-sociated with oil bodies), they largely localized inde-pendent of each other (Fig. 1A). When PUB9 and ARK2were coexpressed with either lysotracker Red, an acid-otropic fluorescent dye that labels acidic compartmentslike vacuoles, or RFP-Syntaxin of Plants21 (SYP21),which is predominantly located on the membranes ofprevacuolar compartments, colocalization could be ob-served in the punctate structures (Fig. 1, B and C). Thissuggested to us that these compartments could be lyticvacuoles that are destined to the central vacuole. WhenPUB9 and ARK2 were coexpressed with ATG8, anautophagosome marker, complete colocalization ofPUB9 and ATG8 could be observed on the punctatesubcellular compartments (Fig. 1D).

Plants Lacking Both AtPUB9 and ARK2 Are Defective inLR Formation under Phosphate Starvation

The localization pattern of AtPUB9 in the presenceof ARK2 to autophagic compartments prompted us toinvestigate the in vivo role of these proteins under

conditions that induced autophagosomes. For this,homozygous transfer DNA (T-DNA) insertional linesof PUB9 (pub9-1) and ARK2 (ark2-1) and the homo-zygous double mutant, ark2-1/pub9-1, carrying boththe mutations were used (Fig. 2, A and B). When roottissue was examined for expression of these genes, thedouble mutant line ark2-1/pub9-1 lacked PUB9 orARK2 expression compared with the wild type (eco-type Columbia-0 of Arabidopsis [Col-0]) as revealedby reverse transcription (RT)-PCR (Fig. 2C). When3-d-old seedlings of the single as well as the doublemutants were transferred to nutrient-rich (+Pi/+Suc)medium and allowed to grow vertically for 7 d, therewas no observable difference in LR density (numberof LRs per centimeter of PR length; Fig. 2D). In con-trast, when 3-d-old seedlings were grown in mediumlacking Pi and Suc (2Pi/2Suc), ark2-1/pub9-1 hadseverely reduced numbers of LRs compared withpub9-1, ark2-1, or Col-0. The PR length was alsoinhibited in the double mutant relative to Col-0 andthe single mutants (Fig. 2E). To verify that the ob-served LR developmental defect in the double mutantis caused by a lack of these respective genes, com-plementation of ark2-1/pub9-1 with either PUB9 or

Figure 1. Subcellular localization oftransiently coexpressed glutathioneS-transferase::ARK2 (kinase domain)and GFP::PUB9 with oil body markerRFP::Oleosin (A), vacuolar marker Lyso-tracker Red (B), prevacuolar markerRFP::SYP21 (C), and autophagy markerRFP-ATG8 (D); 5 to 10 mg of plasmidswere biolistically transformed into tobaccoBY2 cells, and images were capturedthrough epifluorescence microscopy.To determine the colocalization pattern,the green and red channels were merged.

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ARK-PUB9 Module Regulates Lateral Root Development



ARK2 was performed. Overexpression (35S::PUB9) ofPUB9 in ark2-1/pub9-1 led to the rescue of the LRdefect phenotype observed under 2Pi/2Suc condi-tions (Supplemental Fig. S1). However, the PR lengthwas reduced as a result of PUB9 overexpression inark2-1/pub9-1 independent of the nutrient status(Supplemental Fig. S1). Expression of ARK2 similarlyrescued the LR defect observed in ark2-1/pub9-1 under2Pi/2Suc condition. (Supplemental Fig. S1). Theseresults indicated that both ARK2 and PUB9 are requiredfor induction of LR under Pi starvation. These two di-verse interacting partners likely play a redundant role

in regulating LR development under phosphate-starvedconditions.

Phosphate Starvation Leads to PUB9 Localization onPunctate Subcellular Structures in Planta

To determine if PUB9 has a role in autophagy, theroots of pub9-1 constitutively expressing GFP-taggedPUB9 (pub9-1/GFP-PUB9) were analyzed by confocalmicroscopy. Under normal nutrient-rich conditions(+Pi/+Suc), GFP-PUB9 is distributed throughout thecytosol in root epidermal cells (Fig. 3A). When GFP-

Figure 2. Absence of ARK2-PUB9 module leads to LR defect under Pi/Suc-deficient conditions. Schematic representation of thelocation of T-DNA inserts in pub9-1 and ark2-1 mutant lines (A). Homozygous double mutant ark2-1/pub9-1 line obtained bycrossing pub9-1 and ark2-1 was confirmed by PCR genotyping (B). RT-PCR analysis of AtPUB9 and AtARK2 expression inark2-1/pub9-1 roots relative to Col-0 (C). The LR density (calculated as the number of LRs per centimeter of length of PR) ofArabidopsis seedlings under nutrient-rich (+Pi/+Suc; D) and Pi/Suc-starved (2Pi/2Suc; E) conditions of the various genotypes;3-d-old seedlings were allowed to grow vertically in either +Pi/+Suc or 2Pi/2Suc media for 7 d before being photographed.The values represent mean 6 SE (n = 15). Bars = 1 cm.


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PUB9 was observed under Pi/Suc starvation (2Pi/2Suc),a few punctate structures could be observed on theedge of the large central vacuole (Fig. 3B). To observethe autophagic compartments, the transgenic seedlingswere treated with concanamycin A (ConcA), a specificinhibitor of vacuolar ATPase that blocks vacuolardegradation and has been previously used to revealautophagosomes (Thompson et al., 2005; Chung et al.,2010). After ConcA treatment, PUB9 could be ob-served to localize on punctate structures undernutrient-rich conditions (Fig. 3C). Interestingly, thenumber of these punctate structures dramatically in-creased in ConcA-treated and Pi/Suc-starved root cells(Fig. 3D; Supplemental Fig. S2). The punctate struc-tures observed were 0.5 to 1.5 mm in diameter andappeared to be mobile and in the vacuolar lumen. Theincrease in the number of these structures after phos-phate starvation likely indicates the occurrence ofsome degree of basal autophagy under normal condi-tions, which gets elevated on starvation.

Inhibiting Autophagy Leads to Defective LR Formationunder Phosphate Starvation in Wild-Type Plants

To determine if autophagy has a role in regulatingLR development under phosphate starvation, we

transferred 3-d-old seedlings of wild-type Col-0 to ei-ther a nutrient-rich (+Pi/+Suc) or nutrient-starvedmedium (2Pi/2Suc) supplemented with 1 mM

3-methyladenine (3-MA), a known autophagy inhibitor,and allowed them to grow for 7 d. Interestingly, onlythe seedlings grown on2Pi/2Suc medium with 3-MAshowed severely reduced numbers of LRs phenocopyingthe ark2-1/pub9-1 double mutants (Figs. 2E and 3E) Theseedlings grown in nutrient-rich media did not displayany observable LR defects (Fig. 3E). These results indi-cate that autophagy is required for LR formation underphosphate starvation and that the LR defect in ark2-1/pub9-1 double mutants could be a result of a defectiveautophagy process in this mutant.

ark2-1/pub9-1 Plants Lack DR5 Promoter Activity in RootTips under Starvation

Because auxin accumulation is tightly associatedwith root development, we aimed at investigating anypossible role of auxin in ARK2-PUB9-mediated regu-lation of LR development. For this, pub9-1, ark2-1, andark2-1/pub9-1 lines expressing GUS under the controlof the auxin-responsive DR5 promoter (DR5::uidA)were generated. When 10-d-old seedlings grown ineither rich medium or 2Pi/2Suc medium were

Figure 3. Subcellular localization ofGFP::PUB9 in stably transformed Ara-bidopsis pub9-1 root epidermal cellsgrown under different nutrient condi-tions. Localization of GFP::PUB9 undernutrient-rich condition without ConcA(A), Pi/Suc-deficient condition withoutConcA (B), 2 mM ConcA-supplementednutrient-rich condition (C), and 2 mM

ConcA-supplemented Pi/Suc-deficientcondition (D). Effect of 3-MA on theLR formation of wild-type Col-0 undernutrient-rich (+Pi/+ Suc) and nutrient-deficient (2Pi/2Suc) conditions (E).






subjected to GUS assays, similar GUS staining could beobserved in the PR tip across all genotypes undernutrient-rich conditions (Fig. 4A, top). This indicatedthat auxin accumulation response was similar in allthese lines under unchallenged conditions. UnderPi/Suc-starved condition, the ark2-1/pub9-1 PRs didnot exhibit any GUS staining, whereas Col-0 and othersingle mutants displayed GUS staining (Fig. 4A, bot-tom). This indicates severely reduced levels of auxin inark2-1/pub9-1 PR tips under nutrient-starved condi-tions (Fig. 4A, bottom). A similar pattern was observedfor LR tips, with severely reduced GUS staining inark2-1/pub9-1 lines in the LR tips analyzed (Fig. 4B).

Autophagy Is Required for Sustained Auxin Accumulationin Root Tips under Starvation

To investigate whether autophagy has a role in con-trolling auxin accumulation under phosphate starvation,we carried out GUS assays with 10-d-old DR5::uidA-expressing Col-0 seedlings grown in either rich medium(+Pi/+Suc) with 1 mM 3-MA or2Pi/2Suc mediumwith1 mM 3-MA. We observed that GUS staining was re-duced in the primary and LR tips of plants grown in thepresence of 3-MA under 2Pi/2Suc-starved conditions(Supplemental Fig. S3) compared with plants grown inthe presence of 3-MA under nutrient-rich conditions(Supplemental Fig. S3). These results indicate that auxinaccumulation is controlled by autophagy under Pi star-vation, and reduced GUS staining observed in ark2-1/pub9-1 lines could be a result of the defective autophagy.

Exogenous Auxin Rescues Phosphate Starvation-InducedLR Defect in ark2-1/pub9-1

Based on the lack of auxin accumulation in root tipsof ark2-1/pub9-1, after phosphate starvation, we hy-pothesized that the interaction between these two

proteins could mediate auxin accumulation in the roottips. If a defect in auxin accumulation is the sole reasonfor the observed LR developmental phenotypes inthese lines, then supplementation of auxin shouldrescue this phenotype. To test this, 3-d-old seedlings ofCol-0 and ark2-1/pub9-1 initially germinated on 0.53Murashige and Skoog (MS) medium with 1% (w/v)Suc were transferred to either +Pi/+Suc or 2Pi/2Sucmedium with or without the addition of various con-centrations of naphthylacetic acid (NAA; 0.001–1 mM;Fig. 5, A and B). Addition of 0.001 mM NAA was suf-ficient to rescue the ark2-1/pub9-1 LR defect in2Pi/2Sucmedium (Fig. 5C). Both 0.001 and 0.01 mM NAA werethe optimal concentrations for the rescue of LR phe-notype in ark2-1/pub9-1 lines without much effect onthe PR length in both NAA concentrations in Col-0(Fig. 5D). Treatment with 0.1 and 1 mM NAA resultedin a significant increase in the number of LRs in bothnutrient-rich and nutrient-starved conditions, and bothCol-0 and ark2-1/pub9-1 produced a similar number ofLRs under these conditions but had an inhibitory effecton the PR elongation (Fig. 5, C and D; SupplementalFig. S4). The rescue of LR defect in ark2-1/pub9-1 byauxin supplementation suggests that auxin is requiredfor LR development under nutrient-starved conditionsand that these two proteins control the accumulationof auxin under phosphate-starved conditions.

Gene Expression Analysis in LRs Suggests anAuxin-Dependent Mechanism

To further confirm our hypothesis that the ARK2-PUB9 module regulates auxin accumulation, quan-titative RT-PCR was performed using key genesinvolved in auxin signaling and transport. For this,3-d-old seedlings were transferred to either +Pi/+Sucor 2Pi/2Suc condition and allowed to grow verticallyfor 4 d. The LR initiation zone, 2 to 2.5 cm away fromthe PR region, was used as the tissue for expression

Figure 4. DR5::uidA expression in PR (A) and LR tips (B). GUS staining was performed to detect the level of auxin accumulationin the PR and LR tips of seedlings of the various genotypes grown on medium with or without phosphate and Suc. Bars = 16 mm.


Deb et al.







analysis. The expression of indole-3-acetic acid28(IAA28), an AUXIN (AUX) /IAA repressor of auxin-responsive genes, was found to be up-regulated inPi/Suc-starved ark2-1/pub9-1 compared with Col-0(Fig. 6). However, the auxin transport genes PIN-

FORMED1 (PIN1), PIN3, PIN5, PIN6, and PIN7 weresignificantly down regulated several fold in Pi/Suc-starved ark2-1/pub9-1 compared with Col-0 (Fig. 6),with PIN5 showing the lowest level of expression,being reduced by 3.03-fold, and PIN2 displaying a mod-erately enhanced expression (Fig. 6). Under nutrient-richconditions, the expression of these genes in ark2-1/pub9-1was similar to that of Col-0 (Supplemental Fig. S5).These observations are indicative of the requirement ofthe ARK2-PUB9 module in maintaining the expressionof the PIN transport proteins and the IAA28 repressorunder nutrient-starved conditions.

DISCUSSION

Despite the identification of 417 receptor kinases withan extracellular domain in Arabidopsis, functions ofmost of these kinases and their downstream interactingpartners remain unknown (Shiu and Bleecker 2001a,2001b). Previously, it was shown that the ArabidopsisS domain1 receptor kinase subfamily could potentiatesignaling through the PUBs (Samuel et al., 2008). Inparticular, PUB9 is a short E3 Ub ligase containing aU-box domain and armadillo repeats and is expressed innearly all tissue types, except leaves (Mudgil et al.,2004). ARK1, ARK2, and M-locus protein kinase arecapable of phosphorylating PUB9 based on in vitrophosphorylation assays (Samuel et al., 2008). Phosphor-ylation of AtPUB9 by ARK2 led to its relocalizationfrom the nucleus to the cytosol, and 40% of the cellsformed punctate structures (Samuel et al., 2008). Inthis study, we have identified these compartments asautophagic bodies based on its colocalization withmarkers for lytic compartments and autophagosomemarker ATG8 (Fig. 1). AtPUB9 in root epidermal cellsof transgenic pub9-1::GFP-PUB9 plants localized topunctate structures in the vacuole under starvation and

Figure 5. Rescue of the LR defect phenotype by exogenous auxinsupplementation. Three-day-old Col-0 (A) and ark2-1/pub9-1 (B)seedlings were allowed to grow vertically for 7 d in either +Pi/+Suc or2Pi/2Suc media with or without 0.1 mM NAA. Graphical represen-tation of the LR density (C) and PR length (D) for Col-0 and ark2-1/pub9-1 grown on 2Pi/2Suc media in the presence of various con-centrations of NAA. The values represent mean 6 SE (n = 6). [Seeonline article for color version of this figure.]

Figure 6. Altered expression pattern of auxin signaling and transportgenes under Pi/Suc-deficient condition. Quantitative RT-PCR analysiswas performed with RNA isolated from 7-d-old Arabidopsis seedlingsthat were subjected to Pi and Suc starvation for 4 d. Relative expres-sion levels were calculated by the D-DCt method, with Pi/Suc-starvedCol-0 as the calibrator, and all quantifications were normalized usingUbq10 mRNA as an internal control. Data represent mean6 SE (n = 4).






on ConcA treatment (Fig. 3D). This suggests thatPUB9 localizes to autophagic bodies after phosphatestarvation. Previous studies have shown that ATG8 lo-calizes to similar compartments on ConcA treatment(Thompson et al., 2005; Chung et al., 2010). ConcA is aspecific inhibitor of vacuolar type H+ATPase (Dröseet al., 2001) that increases the pH in vacuoles, preventingthe activity of vacuolar proteases and thus, enabling thevisualization of GFP in the vacuole (Tamura et al., 2003).

When examining a role for the ARK2-PUB9 modulein mediating stress responses under nutrient-starvedconditions, we identified that the LR developmentphenotype manifested only during phosphate-starvedconditions in the ark2-1/pub9-1 double mutants (Fig. 2).Our results with 3-MA using Col-0 seedlings alsoconfirmed the need for autophagy to mediate LR de-velopment under phosphate-starved conditions (Fig.3E). The developmental plasticity of the root systemallows it to respond and adapt to the external envi-ronment by modifying itself through a cascade ofhormone-mediated events. We observed that the lack ofLR formation is because of the inability of ark2-1/pub9-1plants to accumulate auxin in the root tips underphosphate starvation (Fig. 4). The phytohormone auxinand the associated polar auxin transport are the prin-cipal stimulators of LR initiation, primordium devel-opment, and emergence (for review, see Lavenus et al.,2013). Previous studies have shown that exogenousapplication of auxin promotes the production of nu-merous LRs (Malamy and Ryan, 2001; Himanen et al.,2002). Likewise, mutation or overexpression of genesinvolved in auxin biosynthesis, metabolism, transport,and signaling led to an altered number of LRs (Celenzaet al., 1995; Hobbie and Estelle, 1995; Rogg et al., 2001;Fukaki et al., 2002; Swarup et al., 2008; Mashiguchiet al., 2011). Interestingly, IAA28, a repressor of auxin-responsive genes, was up-regulated in ark2-1/pub9-1compared with the wild type under Pi starvation.IAA28 functions as a repressor of LR development,because gain-of-function mutant iaa28-1 is defectivein LR development, and GATA TRANSCRIPTIONFACTOR23 expression can rescue this phenotype (Rogget al., 2001; De Rybel et al., 2010). Although the re-pressor IAA28 can regulate auxin signaling, auxin gra-dients facilitated by PIN efflux carrier proteins usingboth root- and shoot-derived auxin are crucial for LRdevelopment (Benková et al., 2003). During Pi starva-tion, expression levels of PIN1, PIN3, PIN5, PIN6, andPIN7 were found to be down-regulated in ark2-1/pub9-1plants, providing additional evidence that both auxinefflux carriers and the signaling proteins are affected,which could lead to lack of auxin accumulation in theroot tips of ark2-1/pub9-1 mutants. Recent studies havealso unraveled a link between E3 ligases and the Ubproteasome system during LR development and areknown to function by modulating auxin accumulationand signaling under limited phosphate availability. Pistarvation increases the expression of the auxin receptorTRANSPORT INHIBITOR RESPONSE1, which triggersubiquitination and subsequent degradation of AUX/

IAA repressors, thus relieving the AUXIN RESPONSEFACTORs and regulating the expression of genes in-volved in LR formation (Pérez-Torres et al., 2008).

The lack of auxin accumulation in the root tips ofark2-1/pub9-1 seedlings under phosphate starvationand the rescue of the LR phenotype by exogenousauxin (Fig. 5) are indicative of the ARK2-PUB9 moduleregulating auxin accumulation in the root tips duringphosphate starvation. In accordance with this, 3-MAtreatment of Col-0 plants showed a reduction in auxinaccumulation in LRs after phosphate starvation(Supplemental Fig. S3), suggesting a role for auto-phagy in controlling auxin accumulation. Our resultssuggest that the lack of these two proteins could blockautophagy under phosphate-starved conditions andpossible degradation of repressors of auxin accumu-lation. Alternatively, activation of PUB9 by ARK2could lead to ubiquitination of repressors of auxinaccumulation, which are subsequently targeted to theautophagosomes through a selective autophagy pro-cess. In this scenario, ark2-1/pub9-1 would have normalformation of autophagosomes, except that selectivetargeting of proteins through the ARK2-PUB9 modulewould be absent. Identifying potential interactors ofPUB9 would provide clues to possible downstreamtargets of this module that could negatively regulateauxin accumulation.

Whether these two proteins play a redundant role inparallel pathways or whether they form one of the manyS domain1 kinase-PUB modules that are necessary forthis process is not known. At the kinase level, ARK2 hasthe ability to interact with and phosphorylate multiplePUBs (Samuel et al., 2008). Kinase activity of ARK1has been shown to be essential for ARK1-mediatedrelocalization of PUB9 from the nucleus to the plasmamembrane, whereas in our study, ARK2 interactionwith PUB9 results in localization of PUB9 to autopha-gosomes (Fig. 1). ARK1 and PUB9 function in a linearpathway, negatively regulating abscisic acid responses,because both the single and double ark1/pub9-1 mutantsexhibited similar hypersensitivity to abscisic acid duringseed germination (Samuel et al., 2008). In this study,only lack of both ARK2 and PUB9 resulted in the LRphenotypes, suggesting that genetic interaction betweenthese two protein products plays a functionally redun-dant role during phosphate starvation. Thus, it is likelythat PUB9 can be used by multiple upstream kinases tomediate unique tissue-specific responses. Nevertheless,our study has uncovered a previously unknown, re-dundant role for these highly diverse interacting partnersand provides additional evidence for PUB proteinsfunctioning as potential downstream signaling tar-gets for S-domain receptor kinases.

MATERIALS AND METHODS

Plant Materials and Genetic Analysis

Arabidopsis (Arabidopsis thaliana) Col-0 was used as the wild type for allexperiments. T-DNA insertion lines for AtPUB9 (SALK_020751) and AtARK2


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(SAIL_594_A06) were obtained from the Arabidopsis Biological ResourceCenter. Homozygous lines were generated by PCR genotyping using primerslisted in Supplemental Table S1. Double homozygotes (ark2-1/pub9-1) werecreated by crossing these single mutant lines.

Transient Expression Using BY2 Cells

The full-length AtPUB9 (At3g07360) and the kinase domain of AtARK2(At1g65800) were cloned into plant expression vector pRTL2 as GFP-taggedand glutathione S-transferase-tagged constructs under the control of Cauli-flower mosaic virus 35S promoter, respectively, as described in Samuel et al.,2008. RFP-Olesoin and SYP21 (At5g16830) were provided by Robert Mullen(University of Guelph, Guelph, Canada). Lysotracker Red (Invitrogen) stain-ing was performed as per the manufacturer’s instructions. Cultured tobacco(Nicotiana tabacum) BY2 cells were used to perform biolistic bombardment asdescribed previously (Stone et al., 2003). Cells were fixed in 4% (w/v) para-formaldehyde and visualized directly through a Leica DMR epifluorescencemicroscope for detecting GFP and RFP (Stone et al., 2003).

Cloning in Binary Vectors and Plant Transformation

AtPUB9 with GFP fused to its N terminus and ARK2 with yellow fluo-rescent protein fused to its C terminus were cloned into pCAMBIA binaryvectors pCAM-ter and pCAMBIA1301, respectively. The absence of a singlenucleotide in ARK2 (RAFL09-14-P09; RIKEN) was corrected by site-directedmutagenesis using the primers listed in Supplemental Table S1. These con-structs were mobilized into Agrobacterium tumefaciens strain GV3101 sepa-rately and used for transforming ark2-1/pub9-1 plants by the floral dip method(Clough and Bent, 1998). The transgenic plants were obtained by selecting theharvested seeds on MS medium (Murashige and Skoog, 1962) with kanamycinfor PUB9 and hygromycin for ARK2.

Phosphate Starvation and Root Growth Assay

Vapor-phase sterilization of seeds was carried out by adding 3 mL ofconcentrated HCl to 75 mL of commercial bleach and allowing it to sit for 4 h ina desiccator jar. Seeds were then plated on 0.53 MS medium supplementedwith 1% (w/v) Suc and 0.75% (w/v) agar. After stratifying for 3 d in the dark,the plates were oriented vertically and allowed to germinate under light. After3 d, the seedlings were transferred to either nutrient-rich (+Pi/+Suc) orphosphate starvation (2Pi/2Suc) medium.

The +Pi/+Suc was composed of 0.53 MS medium supplemented with 1.5%(w/v) Suc and 0.75% (w/v) agar, whereas the 2Pi/2Suc medium contained3 mM CaCl2, 3 mM MgSO40.7H2O, 5 mM KCl, 2 mM MES hydrate, 3 mM KNO3 13micronutrient salt (Sigma), and 13 vitamins (Sigma) supplemented with 0.75%(w/v) agar. The plates were placed vertically to allow root growth on the agarsurface at 22°C in a plant growth chamber with a photoperiod of 16 h of light and8 h of dark. The seedlings were photographed after 7 d; the number of LRs wascounted, and the PR lengths were measured using ImageJ software (Abramoffet al., 2004). For root growth assay with autophagy inhibitor 3-MA, seedlingswere grown in similar media composition as described above (+Pi/+Suc and2Pi/2Suc) with the addition of 1 mM 3-MA (Sigma) in the respective media.

ConcA Treatment of Arabidopsis Seedlings andConfocal Microscopy

Two different lines of Arabidopsis pub9-1 seedlings constitutivelyexpressing GFP-tagged PUB9 under the control of Cauliflower mosaic virus 35Spromoter were grown vertically for 3 d in 0.53 MS medium with 1% (w/v)Suc and 0.75% (w/v) agar. The seedlings were then transferred to either+Pi/+Suc or 2Pi/2Suc liquid media and incubated for 24 h at 22°C in light.Ten to twelve seedlings were then transferred to 24-well ELISA plates with+Pi/+Suc or 2Pi/2Suc liquid media either with or without 2 mM ConcA(Santa Cruz Biotechnology; 1 mM stock in dimethyl sulfoxide). In treatmentswithout ConcA, the same volume of dimethyl sulfoxide was added to theliquid media. The seedlings were then incubated for 12 to 16 h in the dark atroom temperature, mounted in an aqueous environment, and visualized usinga confocal microscopy. A Leica TCS SP5 confocal microscope system was usedin this study. Quantification was carried out by counting the number ofpunctate structures (0.5–1.5 mm) in three different 100-mm2 areas of at leasttwo different root epidermal cells for each treatment.

Histochemical GUS Assay

The mutant lines pub9-1, ark2-1, and ark2-1/pub9-1 containing the transgeneDR5::uidA andwild-type Col-0 expressing DR5::uidAwere grown under +Pi/+Sucor 2Pi/2Suc as described above and incubated overnight in GUS reaction buffer(0.5 mg mL21 of 5-bromo-4-chloro-3-indolyl-b-D-glucuronide, 0.1 mM K4[Fe(CN)6],0.1 mM K3[Fe(CN)6], and Triton X-100 in 100 mM sodium phosphate buffer, pH 7.2).The seedlings were then cleared in 70% (v/v) ethanol, mounted on 50% (v/v)glycerol, and visualized under Leica DMR epifluorescence microscope bright-fieldoptics. For GUS assay in the presence of autophagy inhibitor 3-MA, a similarprotocol was followed using wild-type plants (Col-0) expressing the transgeneDR5::uidA and grown under +Pi/+Suc or2Pi/2Sucwith 1mM 3-MA in the media.

Auxin Treatment of Arabidopsis Seedlings

For hormonal complementation of ark2-1/pub9-1 LR defect, 3-d-old seed-lings were transferred to +Pi/+Suc or 2Pi/2Suc medium supplemented with0.001 to 1 mM NAA. After 7 d, the seedlings were photographed and analyzedby ImageJ software.

Quantitative RT-PCR

RNA was isolated from the LR forming zone of Col-0 and ark2-1/pub9-1seedlings grown under either +Pi/+Suc or 2Pi/2Suc condition for 4 d usingTRIzol (Invitrogen). First-strand complementary DNA was synthesized from 800ng of DNase-treated total RNA using oligo(dT)12–18 primer and SuperScript IIReverse Transcriptase (Invitrogen) following the manufacturer’s instructions. Thequantitative PCR was performed using the StepOnePlus Real-Time PCR System(Applied Biosystems). Primer pairs are listed in Supplemental Table S1. EachPCR reaction contained 13 Fast SYBR Green Master Mix (Applied Biosystems),200 nM each primer, and 0.5 mL of complementary DNA in a final volume of20 mL. PCR amplification was performed for 40 cycles at 95°C for 3 s and 60°Cfor 30 s with a preceding initial enzyme activation of 20 s at 95°C. Relative ex-pression levels were calculated by the D-DCt method, and all quantificationswere normalized usingUbq10 mRNA as an internal control. For each target gene,the reactions were carried out in duplicate for two biological replicates.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. AtPUB9 or AtARK2 complementation rescues LRdefect of the pub9-1/ark2-1 mutant.

Supplemental Figure S2. Quantification of the punctate structures in Ara-bidopsis root epidermal cells.

Supplemental Figure S3. Autophagy inhibitor 3-MA reduces auxin accu-mulation under phosphate starvation.

Supplemental Figure S4. Exogenous application of 1 mM NAA inhibits PRgrowth in pub9-1/ark2-1 mutant.

Supplemental Figure S5. Root expression pattern of auxin signaling andtransport genes under nutrient-rich conditions.

Supplemental Table S1. Primers used in this study.

ACKNOWLEDGMENTS

We thank Dr. Robert Mullen (University of Guelph) for providing theconstructs for colocalization experiments.

Received June 2, 2014; accepted June 20, 2014; published June 25, 2014.

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the s-domain receptor kinase arabidopsis …...the s-domain receptor kinase arabidopsis receptor...

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