lateral organ boundaries domain16 and 18 act downstream ......lateral organ boundaries domain16and...

Lateral Organ Boundaries Domain16 and 18 ActDownstream of the AUXIN1 and LIKE-AUXIN3Auxin Influx Carriers to Control Lateral RootDevelopment in Arabidopsis1

Han Woo Lee, Chuloh Cho, and Jungmook Kim*

Department of Bioenergy Science and Technology and Kumho Life Science Laboratory, Chonnam NationalUniversity, Gwangju 500–757, Korea

ORCID IDs: 0000-0001-7997-1544 (C.C.); 0000-0003-1735-5564 (J.K.).

Several members of the Lateral Organ Boundaries Domain (LBD)/Asymmetric Leaves2-Like (ASL) gene family have been identified toplay important roles in Arabidopsis (Arabidopsis thaliana) lateral root (LR) development during auxin response, but theirfunctional relationship with auxin transporters has not been established yet. Here, we show that the AUXIN1 (AUX1) andLIKE-AUXIN3 (LAX3) auxin influx carriers are required for auxin signaling that activates LBD16/ASL18 and LBD18/ASL20 tocontrol LR development. The lax3 mutant phenotype was not significantly enhanced when combined with lbd16 or lbd18.However, LBD18 overexpression could rescue the defects in LR emergence in lax3 with concomitant expression of the LBD18target genes. Genetic and gene expression analyses indicated that LBD16 and LBD18 act with AUX1 to regulate LR initiation andLR primordium development, and that AUX1 and LAX3 are needed for auxin-responsive expression of LBD16 and LBD18.LBD18:SUPERMAN REPRESSIVE DOMAIN X in the lbd18 mutant inhibited LR initiation and LR primordium development inresponse to a gravitropic stimulus and suppressed promoter activities of the cell cycle genes Cyclin-Dependent Kinase A1;1 andCYCLINB1;1. Taken together, these results suggest that LBD16 and LBD18 are important regulators of LR initiation anddevelopment downstream of AUX1 and LAX3.

Root branching, such as lateral root (LR) formation,is critical for the absorption of water and nutrients, andfor the anchorage of plants in soil (Hochholdinger andZimmermann, 2008; Dastidar et al., 2012). In recentyears, intensive research on the molecular mechanismsof LR formation has been conducted with Arabidopsis(Arabidopsis thaliana; Lavenus et al., 2013). The devel-opmental events of LR formation include priming,initiation, primordium development, and the emer-gence of LRs, which are primarily regulated by auxin(Dubrovsky et al., 2008, 2011; De Smet, 2012; Lavenuset al., 2013). The pericycle cells associated with a xylem

pole are primed by auxin in the basal meristem tobecome pericycle founder cells, and the founder cellsdivide asymmetrically for LR initiation, generatingsmall daughter cells flanked by long daughter cells(Parizot et al., 2008; Péret et al., 2009a,b; Lavenuset al., 2013). These cells undergo a series of anticlinaldivisions to produce a few initial cells. Further anti-clinal, periclinal, and tangential divisions of lateralroot primordium (LRP) cells generate a dome-shapedLRP that continues to grow and emerge through thecortex and epidermal layers of the primary root viacell separation.

In the Arabidopsis root, auxin is transportedacropetally through the central cylinder toward theroot tip and columella cells (Friml et al., 2003). Basip-etal auxin transport from the root tip toward the basalregion plays an important role in the control of auxinhomeostasis in the root meristem (Band et al., 2014).The AUXIN1 (AUX1)/LIKE-AUXIN1 (LAX1) gene familyencoding major auxin influx carriers comprise fourhighly conserved genes: AUX1, LAX1, LAX2, andLAX3 (Péret et al., 2012; Swarup and Péret, 2012).AUX1 regulates the initiation of LR by basipetal auxintransport (Swarup et al., 2001; Marchant et al., 2002;De Smet et al., 2007). LAX2 regulates vascular pat-terning in cotyledons (Péret et al., 2012). LAX3 pro-motes LR emergence by affecting auxin influx of outerendodermis and cortex cells (Swarup et al., 2008). Theaux1 lax3 double mutations effectively blocked LR

1 This work was supported by the Next-Generation BioGreen 21Program (grant no. PJ01104701), Rural Development Administration,Republic of Korea (grant to J.K.), Basic Science Research Programsthrough the National Research Foundation of Korea funded by theMinistry of Education (grant no. 2013R1A6A3A01062668), and Chon-nam National University, 2013.

* 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:Jungmook Kim ([email protected]).

H.W.L. conductedmost of the experiments, analyzed the data, andwrote the article; C.C. conducted specific experiments and analyzedthe data; J.K. conceived the project, designed the experiments, ana-lyzed the data, and wrote and edited the article.

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1792 Plant Physiology�, August 2015, Vol. 168, pp. 1792–1806, www.plantphysiol.org � 2015 American Society of Plant Biologists. All Rights Reserved.

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http://orcid.org/0000-0001-7997-1544

http://orcid.org/0000-0003-1735-5564

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http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.15.00578

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formation, indicating that these two auxin influx car-riers are critical for LR formation (Swarup et al., 2008).Auxin efflux carriers, PIN-FORMED (PIN) proteins,have also been demonstrated to play a role in LR for-mation (Benková et al., 2003; Laskowski et al., 2008;Marhavý et al., 2013; Péret et al., 2013).Auxin-responsive transcriptional regulatory mod-

ules for LR formation have been identified in Arabi-dopsis. The GATA transcription factor23 specifies LRfounder cell identity in an INDOLE-3-ACETIC ACIDINDUCIBLE28 (IAA28)-dependent auxin signalingfor LR priming (De Rybel et al., 2010). SOLITARY-ROOT (SLR)/IAA14-Auxin Response Factor7 (ARF7)-ARF19 and others regulate nuclear polarization ofLR founder cells (De Rybel et al., 2010; Goh et al.,2012a). Two Aux/IAA-ARF modules, SLR/IAA14-ARF7-ARF19 and BODENLOS/IAA12-ARF5, controlthe LR initiation and patterning process (Fukaki et al.,2002; Vanneste et al., 2005; De Smet et al., 2010). LRemergence is distinctively modulated in the endo-dermis and the cortex and epidermis by two differentAux/IAA-ARF modules. SHORT HYPOCOTYL2/IAA3-ARF signaling operates in the endodermis, whereasSLR/IAA14-ARF7-ARF19 signaling functions in thecortex and epidermis (Goh et al., 2012b; Lavenuset al., 2013).Several transcription factors, in particular, Lat-

eral Organ Boundaries Domain (LBD)/AsymmetricLeaves2-Like (ASL) proteins, are regulated down-stream of Aux/IAA-ARF modules during LR devel-opment (Okushima et al., 2007; Lee et al., 2009a;Berckmans et al., 2011; Goh et al., 2012a; Lee et al.,2013a; Lee and Kim, 2013). ARF7 and ARF19 directlyactivate LBD16/ASL18 and LBD29/ASL16 (Okushimaet al., 2007). LBD16 and related LBDs are involved inthe symmetry breaking of LR founder cells for LRinitiation (Goh et al., 2012b). LBD18/ASL20 regulatesLR formation in conjunction with LBD16 downstreamof ARF7 and ARF19 (Lee et al., 2009a,b) and con-tributes to both the initiation and emergence of LRs(Berckmans et al., 2011; Lee et al., 2013a; Lee andKim, 2013). LBD18 transcriptionally activates the E2Fatranscription factor, which regulates the asymmetriccell division for LR initiation (Berckmans et al., 2011).LBD18 promotes LR emergence by acting as a specificDNA-binding transcriptional activator that directlyup-regulates EXPANSIN14 (EXP14) encoding a cellwall-loosening protein (Lee et al., 2013a). LBD18 in-directly up-regulates EXP17, which promotes LRemergence during the auxin response (Lee and Kim,2013). Although the auxin-responsive transcriptionalcascade of the Aux/IAA-ARFs and LBD genes has beenwell characterized, no functional evidence has beenprovided yet on how auxin transport proteins arelinked to LBD gene expression to control LR devel-opment during the auxin response, and on the role ofLBD18 in LR initiation and LRP development.In this study, we investigated the connection be-

tween auxin influx carriers, LAX3 and AUX1, and twoauxin-responsive LBD genes, LBD16 and LBD18,

during LR development by conducting genetic analy-sis with various multiple mutants derived from lbd16,lbd18, lax3, and aux1 and gene expression analysis. Ourmolecular genetic analysis results suggested thatLBD16 and LBD18 are linked to auxin signaling viaAUX1 for LR initiation and LRP development, in partvia LAX3 for LRP development, and that LBD18 actsdownstream of LAX3 to control LR emergence inArabidopsis. To confirm that LBD18 is involved in LRinitiation and LRP development, we expressed LBD18:SUPERMAN REPRESSIVE DOMAIN X (SRDX), adominant repressor of LBD18, under the control of itsown promoter in wild-type or lbd18 mutant back-grounds and showed that LBD18:SRDX suppressedLR initiation events, periclinal divisions of primor-dium after LR initiation, and later stages of LRP de-velopment in response to a gravitropic stimulus. Inaddition, we identified a link between LBD18 and theregulation of cell cycle genes during LR initiationthrough the analyses of GUS expression under the cellcycle gene promoter Cyclin-Dependent Kinase A1;1(CDKA1;1), CDKB1;1, or CYCLINB1;1 (CYCB1;1), amarker gene for LR initiation, in transgenic Arabi-dopsis expressing LBD18:SRDX, and of expression ofthese cell cycle genes by LBD18:GLUCOCORTICOIDRECEPTOR (GR).

RESULTS

LR Phenotypes of Multiple Mutants Derived from lax3,lbd16, and lbd18 and Gene Expression Analysis

To investigate whether LBD16 and LBD18 are ge-netically linked to LAX3 in controlling LR develop-ment, we generated single, double, and triple mutantsand analyzed LR phenotypes of these mutants. Asshown in Figure 1, A and B, the mean 6 SE numbers ofemerged LRs in lax3 lbd16 (1.83 6 0.06) and lax3 lbd18(1.86 6 0.07) double mutants were slightly lower thanin single mutants (lax3, 2.03 6 0.08; lbd16, 2.1 6 0.09;lbd18, 2.03 6 0.08). The number of emerged LRs in lax3lbd16 lbd18 triple mutants (0.65 6 0.09) was consider-ably lower than that in lax3 lbd16 (1.836 0.06) and lax3lbd18 (1.86 6 0.07) double mutants, but to a lesserdegree than that of lbd16 lbd18 double mutants (1.07 60.09). The process of LRP development is divided byeight stages defined by specific anatomical character-istics and cell divisions (Malamy and Benfey, 1997). Ithas been previously shown that the lax3 mutationsignificantly increases the LRP number at stage I(Swarup et al., 2008), whereas the numbers at all LRPdevelopmental stages in lbd16 or lbd18 single anddouble mutants were similar to those in the wild type(Lee et al., 2009b). The LRP numbers of lax3 at stage I(2.63 6 0.12) were mitigated in lax3 lbd16 (1.80 6 0.06)and lax3 lbd18 (1.77 6 0.08) double mutants, and inlax3 lbd16 lbd18 (1.58 6 0.06) triple mutants (Fig. 1C).These results indicated that LR development in lax3mutants is dependent on LBD16 and LBD18 function.

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Networks of LBD16 and LBD18 with AUX1 and LAX3



Quantitative reverse transcription (qRT)-PCR anal-yses showed that the lax3 mutation significantly re-duced expression of LBD16 and LBD18 in response toauxin after a 4- or 8-h treatment (Fig. 2A). Moreover,auxin-induced expression of the LBD18 target genes,EXP14 and EXP17 (Lee et al., 2013a; Lee and Kim,2013), in the lax3 mutant decreased compared withthat in the wild type (Fig. 2B). However, LAX3 ex-pression with auxin was not affected by lbd16 or lbd18single or double mutations (Fig. 2C). These results in-dicated that LAX3 acts upstream of LBD16 and LBD18during the auxin response. GUS staining of ProLBD16:GUS, ProLBD18:GUS (Lee et al., 2009b), and ProLAX3:GUS(Swarup et al., 2008) transgenic Arabidopsis dem-onstrated that LBD18 and LAX3 were coexpressed inthe cortex and epidermis from stages I to VII duringLRP development and after the emergence of LRs(Supplemental Fig. S1). Although strong GUS stain-ing was detected in the LRPs and emerged LRs ofProLBD16:GUS and ProLBD18:GUS Arabidopsis, we didnot detect GUS staining in the same tissues of ProLAX3:GUS Arabidopsis (Supplemental Fig. S1). The over-lapping expression pattern of LBD18 and LAX3 in theoverlaying tissues (Supplemental Fig. S1) and thedecrease in the LBD18 transcript levels by the lax3mutation (Fig. 2A) are consistent with the observa-tion that GUS expression under the control of theLBD18 promoter was reduced in lax3mutants comparedwith that in the wild type in the overlaying tissues aswell as in the LRP without or with auxin treatment(Fig. 2, D–G). In contrast, we only noted reduced GUS

expression under the control of the LBD16 promoterin lax3 mutants compared with that in the wild typein the LRP with auxin treatment (Fig. 2, H–K). Theseresults indicated that LAX3 promotes LBD18 expres-sion in the overlaying tissues.

LBD18 Induces LR Formation and Expression of the TargetGenes, EXP14 and EXP17, in lax3 Mutants

To demonstrate that LBD18 functions downstreamof LAX3 for LR emergence, we constructed dexa-methasone (DEX)-regulated LBD18-transgenic Arabi-dopsis in the lax3 mutant background (Pro35S:LBD18:GR/lax3) by crossing these transgenic plants and thelax3 mutant. As shown in Figure 3A, LR densities inPro35S:LBD18:GR/lax3 were rescued to the wild-typelevels by DEX treatment for both 7 and 10 d, ascompared with what was observed in the absence ofDEX and in both the presence and absence of DEX inthe lax3 mutants. EXP14 and EXP17 are up-regulatedby LBD18 during the auxin response (Lee et al., 2013a;Lee and Kim, 2013). Using qRT-PCR analysis, weshowed that EXP14 and EXP17 expression was greatlyinduced in the lax3 mutants by DEX treatment of Pro35S:LBD18:GR/lax3 plants for 2 and 8 h (Fig. 3B). These re-sults demonstrated that LBD18 controls LR formationdownstream of LAX3. However, LR formation byLBD18 in the lax3 mutants under the control of theLBD18 promoter requires exogenous auxin treatment(Fig. 3C; Supplemental Fig. S2), indicating that lax3mutation caused defects in LR formation, which could

Figure 1. Genetic analysis of lax3, lbd16, andlbd18 in LR development. A, LR phenotypesof 7-d-old seedlings of the wild type (WT;Columbia-0 [Col-0], gray), lax3 (purple), lbd16(yellow), lbd18 (red), lax3 lbd16 (blue), lax3lbd18 (green), lbd16 lbd18 (lilac), and lax3lbd16 lbd18 (sky blue) are shown. B, Densityof emerged LRs (LR no. per unit primary rootlength; #/cm). Error bars = SE. n = 20. Asterisksdenote statistical significance (*, P , 0.05; **,P , 0.01; and ***, P , 0.001). C, Density ofprimordia at given stages. Stages I to VII ofprimordia were based on the classification byMalamy and Benfey (1997). Error bars = SE. n =10. Asterisks denote statistical significance (**,P , 0.01; and ***, P , 0.001).

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not be rescued by LBD18 normally expressed underits own promoter but could be complemented byectopic overexpression of LBD18. We further showedthat LBD18 expression under the control of theLBD18 promoter induced expression of EXP14 andEXP17 in lax3 mutants at levels similar to those thatcan be gained by LBD18 expression in the wild-typebackground (Fig. 3, D and E). These results togetherwith genetic and gene expression analysis data (Figs.1 and 2) demonstrated that LBD18 regulates expres-sion of EXP14 and EXP17 downstream of LAX3 dur-ing the auxin response.

LBD18 Up-Regulates POLYGALACTURONASE Expressionas a Secondary Response

POLYGALACTURONASE (PG) is induced byauxin, and this depends on proper auxin influx reg-ulated by LAX3 (Swarup et al., 2008). PG enzymescleave demethylated pectin, a substrate enriched inthe middle lamella of root cells overlaying LRP, andare important for cell separation during LR emer-gence (Hadfield and Bennett, 1998; Laskowski et al.,2006; González-Carranza et al., 2007). We investigated

whether PG is regulated by LBD16 and/or LBD18.We generated ProPG:GUS in the lbd16, lbd18, or lbd16lbd18 mutant backgrounds and conducted GUS ex-pression analyses (Fig. 4A). GUS staining detected inoverlaying tissues of the LRP was significantly re-duced in lbd18 compared with that in the wild type,but not in lbd16 (Fig. 4A). GUS staining in lbd16 lbd18double mutants was similar to that in lbd18 singlemutants. Consistent with these observations, qRT-PCR analysis of the GUS transcripts confirmed thatthe lbd18 mutation decreased GUS expression ofProPG:GUS, but the lbd16 mutation did not (Fig. 4B).DEX treatment of Pro35S:LBD18:GR plants resulted ina sustained increase in the PG transcript levels after 4h of treatment (Fig. 4C), whereas the same treatmentof Pro35S:LBD16:GR plants did not alter the PG tran-script levels until 48 h of treatment. PG expression inPro35S:LBD18:GR plants was not induced by DEXtreatment in the presence of cycloheximide after 8and 12 h of incubation (Fig. 4D), indicating that PG isa secondary response gene of LBD18. We furtherdemonstrated that LBD18 up-regulates PG expres-sion in tissues where LBD18 is normally expressed(Fig. 4E).

Figure 2. Expression analysis of LBD16,LBD18, EXP14, and EXP17 in lax3 andLAX3 in lbd16, lbd18, and lbd16 lbd18.A, LBD16 and LBD18 expression inlax3 in response to auxin. Seven-day-old seedlings were incubated with auxin(20 mM IAA) for 0, 4, or 8 h and subjectedto qRT-PCR. Data are the mean 6 SE

of three independent biological rep-lications. Asterisks denote statisticalsignificance (*, P , 0.05; and ***, P ,0.001). B, EXP14 and EXP17 expres-sion in lax3 in response to auxin. Treat-ment and analysis of the samples wereperformed as described in A. C, LAX3expression in the wild type (WT),lbd16, lbd18, and lbd16 lbd18 in re-sponse to auxin. Seven-day-old seed-lings were incubated with auxin (20 mM

IAA) for 8 h and subjected to qRT-PCR.Data are the mean 6 SE of three inde-pendent biological replications. Aster-isks denote statistical significance (***,P , 0.001). D to K, GUS expression ofProLBD18:GUS (D and F), ProLBD18:GUS/lax3 (E and G), ProLBD16:GUS (H and J),and ProLBD16:GUS/lax3 transgenic Arabi-dopsis during LR development (I and K).Six-day-old light-grown seedlings wereincubated without (D, E, H, and I) orwith (F, G, J, and K) 20 mM IAA for 4 hand subjected to GUS staining. Bars =50 mm.





Analysis of LR Phenotypes of Multiple Mutants Derivedfrom aux1, lax3, lbd16, and lbd18

AUX1 facilitates LR initiation by promoting auxinaccumulation in the root apex, and later LR emergencethrough the uptake in the LRP (Marchant et al., 2002;Swarup et al., 2008). Strong GUS expression in theLRPs and emerged LRs of ProLBD16:GUS and ProLBD18:GUS (Supplemental Fig. S1) overlaps with that ofProAux1:GUS (Marchant et al., 2002), indicating that apotential link may exist between LBD18 and AUX1during LR development. To investigate a functionallink between LBD16/LBD18 and AUX1 for LR devel-opment, we generated multiple mutants derived fromlbd16, lbd18, and aux1-22 and analyzed LR initiation,

LRP development, and LR emergence of these multiplemutants. As shown in Figure 5, A and B, the numbersof emerged LRs in aux1 lbd16 (1.08 6 0.09), aux1 lbd18(1.11 6 0.07) double mutants, and aux1 lbd16 lbd18(0.35 6 0.06) triple mutants decreased additivelycompared with that of corresponding single mutants(aux1, 1.8 6 0.06; lbd16, 1.61 6 0.12; and lbd18, 1.86 60.08) and double mutants (lbd16 lbd18, 0.65 6 0.08),indicating that LBD16/LBD18 and AUX1 combinato-rially contribute to LR emergence. In contrast, thenumbers of primordia at the initiation stage I in aux1lbd16 (0.86 6 0.04) and aux1 lbd18 (0.88 6 0.03) doublemutants increased compared with that in aux1 (0.57 60.07), but decreased compared with that in lbd16(1.17 6 0.07) or lbd18 (1.11 6 0.04; Fig. 5C), indicating

Figure 3. Induction of LRs in lax3 mutants by LBD18:GR with DEX treatment. A, LR densities of Pro35S:LBD18:GR/lax3 plants.Plants were grown vertically for 7 or 10 d in the absence or presence of 10 mM DEX, and LR numbers per unit root length (#/cm)measured were plotted. Asterisks denote statistical significance (**, P, 0.01). DAG, Days after germination. B, Expression analysisof EXP14 and EXP17 in the wild type (WT), lax3, and Pro35S:LBD18:GR/lax3 plants. Seven-day-old seedlings were incubated withDEX for 2 or 8 h in the light and subjected to qRT-PCR. 28 h, Samples that were incubated with mock treatment for 8 h. Data arethe mean6 SE of three independent biological replications. Asterisks denote statistical significance (*, P , 0.05; **, P , 0.01; and***, P , 0.001). C, The effects of auxin on LR densities of ProLBD18:LBD18:GR/lax3 plants. Plants were grown vertically for 7 d inthe absence or presence of 10 mM DEX and 0, 0.05, or 0.1 mM auxin 2,4-dichlorophenoxyacetic acid (2,4-D), and LR numbers perunit root length (#/cm) measured were plotted. Asterisks denote statistical significance (*, P , 0.05; and **, P , 0.01). D, Ex-pression analysis of EXP14 and EXP17 in the wild type, lax3, and Pro35S:LBD18:GR/lax3 plants. Plants were grown for 7 d in theabsence or presence of 10 mM DEX and subjected to qRT-PCR. Data are the mean6 SE of three independent biological replications.Asterisks denote statistical significance (*, P , 0.05). E, Expression analysis of EXP14 and EXP17 in Pro35S:LBD18:GR and Pro35S:LBD18:GR/lax3 plants. Plants were grown for 7 d in the absence or presence of 10 mM DEX and subjected to qRT-PCR. Data are themean 6 SE of three independent biological replications. Asterisks denote statistical significance (*, P , 0.05).


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that the aux1 phenotype requires LBD16 and LBD18gene function during LR initiation and at early stagesof the LRP development. The aux1 lbd16 lbd18 triplemutants (0.68 6 0.04) exhibited a further decreasedLRP number compared with that of aux1 lbd16 or aux1lbd18 mutants. At stage II, lbd16 and lbd18 mutationsin aux1 rescued the LRP number reduced by aux1mutation to the wild-type levels. Similar patternswere observed at stages II to VI, albeit the aux1 im-pact on the LRP numbers was small. These resultsindicated that LBD16 and LBD18 function together

with AUX1 to control LR initiation and early LRPdevelopment.

To define a combinatorial role of AUX1 and LAX3in LBD16 and LBD18 function during LR develop-ment, we further generated lbd16 and lbd18 muta-tions in aux1 lax3 double mutants. Double mutationsin AUX1 and LAX3 dramatically reduced numbers ofemerged LRs (0.06 6 0.11) compared with that ofsingle mutants (aux1, 1.8 6 0.06; and lax3, 2.03 6 0.08;Figs. 1B and 5, B and D), consistent with a previousreport (Swarup et al., 2008). The lbd16 (0.03 6 0.02 for

Figure 4. LBD18 activates PG expression. A, GUS expression analysis of ProPG:GUS in the wild type (WT), lbd16, lbd18, or lbd16lbd18 double mutant backgrounds. Seven-day-old light-grown seedlings were subjected to GUS staining. Scale bars = 50 mm. B,qRT-PCR analysis of ProPG:GUS in the wild type, lbd16, lbd18, or lbd16 lbd18. Seven-day-old seedlings were subjected to qRT-PCRfor GUS expression. Relative transcript levels are plotted. Data are the mean 6 SE of three independent biological replications.Asterisks denote statistical significance (**, P , 0.01). C, Time course expression analysis of PG in Pro35S:LBD16:GR and Pro35S:LBD18:GR plants. Seven-day-old plants were incubated without or with DEX for the indicated time and subjected to qRT-PCR for PGexpression. Relative transcript levels are plotted. Data are the mean6 SE of three independent biological replications. D, The effect ofcycloheximide (CHX) treatment on DEX-induced expression of PG. Bars represent SEs. Data are the mean6 SE of three independentbiological replications. Asterisks denote statistical significance (*, P , 0.05; and **, P , 0.01). E, Expression of PG followingtreatment with DEX in ProLBD18:LBD18:GR plants. ProLBD18:LBD18:GR seedlings were grown for 7 d in the absence (black squares)or presence (white squares) of DEX. Expression of PG was then analyzed by qRT-PCR and plotted as relative expression levels.Ubiquitin-Conjugating enzyme E2 (UBC) was used as a negative control. Asterisk denotes statistical significance (*, P , 0.05).





7 d; and 1.57 6 0.13 for 14 d) or lbd18 (0.20 6 0.09 for7 d; and 2.03 6 0.18 for 14 d) single mutation in aux1lax3 double mutants further reduced the emerged LRnumbers of aux1 lax3 mutants (0.60 6 0.11 for 7 d;and 2.64 6 0.18 for 14 d), and the lbd16 lbd18 (0.03 60.02 for 7 d; and 0.38 6 0.07 for 14 d) double muta-tions additively reduced the emerged LR number ofaux1 lax3 mutants, which is due to the independentroles of AUX1, LBD16, and LBD18 during LR emer-gence (Fig. 5D; Supplemental Fig. S3). Numbers ofprimordia of aux1 lax3 double mutants at stage I werenot significantly altered by lbd16 and lbd18 single ordouble mutations, whereas those numbers from stagesIII to VII were significantly reduced by the lbd16 muta-tion (Fig. 5E). The lbd18 or lbd16 lbd18 mutation greatlyreduced the LRP numbers from stages V to VII. Theseresults indicated that both LBD16 and LBD18 controlLRP development in combination with LAX3 and

AUX1 but at different stages after stage III and afterstage V, respectively.

AUX1 and LAX3 Are Involved in Auxin-ResponsiveExpression of LBD16 and LBD18

qRT-PCR analysis of auxin-responsive expression ofLBD16 or LBD18 in aux1 mutants showed that the aux1mutation did not alter LBD18 expression, but de-creased LBD16 expression slightly after 8 h of auxintreatment (Fig. 6A). However, aux1 lax3 double mu-tations reduced the expression of LBD16 and LBD18 byapproximately 50% in response to auxin after a 4- or8-h treatment compared with those of wild-type andaux1 or lax3 mutants (Figs. 2A and 6B). These resultsshow that both LAX3 and AUX1 are involved in auxin-responsive expression of LBD16 and LBD18. These

Figure 5. Genetic analysis of aux1,lax3, lbd16, and lbd18 in LR develop-ment. A, LR phenotypes of 7-d-oldseedlings of the wild type (WT; Col-0,gray), aux1 (ocean blue), lbd16 (yel-low), lbd18 (red), aux1 lbd16 (orange),aux1 lbd18 (pastel green), lbd16 lbd18(lilac), and aux1 lbd16 lbd18 (indigoblue) are shown. B, Density of emergedLRs (LR no. per unit primary rootlength; #/cm). Error bars = SE. n = 20.Asterisks denote statistical significance(**, P , 0.01; and ***, P , 0.001). C,Density of primordia at given stages.Stages I to VII of primordia were basedon the classification by Malamy andBenfey (1997). Error bars = SE. n = 10.Asterisks denote statistical significance(*, P , 0.05; **, P , 0.01; and ***, P ,0.001). D, LR densities (LR no. per unitprimary root length; #/cm) of 7- or14-d-old seedlings of the wild type,aux1 lax3, aux1 lax3 lbd16, aux1 lax3lbd18, and aux1 lax3 lbd16 lbd18. Er-ror bars = SE. n = 20. Asterisks denotestatistical significance (**, P , 0.01;and ***, P , 0.001). DAG, Days aftergermination. E, Density of primordia atgiven stages of 7-d-old seedlings of thewild type, aux1 lax3, aux1 lax3 lbd16,aux1 lax3 lbd18, and aux1 lax3 lbd16lbd18. Error bars = SE. n = 10. Asterisksdenote statistical significance (*, P ,0.05; **, P, 0.01; and ***, P, 0.001).


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qRT-PCR data are consistent with the observation thataux1 mutation reduced GUS expression in the pri-mordium under the control of the LBD16 or LBD18promoter in response to auxin compared with that inthe wild type (Fig. 6, C–J).

Expression of LBD18:SRDX in lbd18 Mutant CausedInhibition of LR Initiations, Periclinal Divisions in theInitiated LRP, and Progression of LRP Development

The present genetic analyses with multiple mutantsconsisting of aux1, lax3, and lbd18 indicated that LBD18is involved in controlling LR initiation and LRP devel-opment. Our previous study showed that any altera-tions in the numbers of the LRPs from stages I to VIIwere not detected in lbd18 compared with those in thewild type (Lee et al., 2009b). To further investigate therole of LBD18 during LR initiation and LRP develop-ment, we constructed transgenic Arabidopsis express-ing LBD18 fused to an SRDX repression domain underits own promoter and analyzed LR phenotypes. TheSRDX chimeric repressors cause dominant repression oftarget genes, allowing the functional analysis of planttranscription factors with genetic redundancy (Hiratsuet al., 2003). This approach has been previously utilizedto reveal a unique function of LBD16 in the polar nu-clear migration prior to LR initiation (Goh et al., 2012a).A complete block of LR formation has been observedwith ProLBD16:LBD16:SRDX transgenic plants in thewild-type background (Goh et al., 2012a). We also ob-served the same phenotype with ProLBD16:LBD16:SRDX

transgenic plants in wild-type as well as in the lbd16mutant backgrounds (Supplemental Fig. S4). In con-trast, ProLBD18:LBD18:SRDX transgenic plants in thewild-type background displayed wild-type appearancewith normal LR formation (Fig. 7A), whereas ProLBD18:LBD18:SRDX transgenic plants in the lbd18 mutantbackground exhibited more reduced LR formationcompared with that of the lbd18mutant (Fig. 7, B and C).Consistent with these observations, a significantdominant repression of EXP14, a direct target of LBD18(Lee et al., 2013a), by LBD18:SRDX was found in boththe absence and presence of auxin in the lbd18 mutant(Fig. 7D). None of eight ProLBD18:LBD18:SRDX linesanalyzed showed a complete block of LR formation,unlike ProLBD16:LBD16:SRDX transgenic plants. Deter-mination of LRP numbers at all developmental stages ofProLBD18:LBD18:SRDX lines showed wild-type distribu-tion of LRPs (Supplemental Fig. S5). We then conductedan LR induction experiment, which allows the directanalysis of LR development kinetics as opposed to theanalysis of the LRPs and LRs at a given plant devel-opmental stage (Péret et al., 2012). We applied a grav-itropic stimulus inducing initiation of LRs to wild-typeand ProLBD18:LBD18:SRDX/lbd18 plants grown verti-cally for 30 h by rotating the agar plate through 90°. Wethen measured the number of newly developed LRPson the convex side of the curves after 18 or 42 h ofgravitropic induction and determined the relative dis-tribution of the LRPs at stages I to VIII. We found thatProLBD18:LBD18:SRDX lines displayed 40% block of LRinitiation events compared with those of the wild type

Figure 6. Expression of LBD16 orLBD18 in aux1 and aux1 lax3 com-pared with that in the wild type (WT) inresponse to auxin. A, LBD16 and LBD18expression in aux1 in response to auxin.Seven-day-old seedlings were incu-bated with auxin (20 mM IAA) for 0, 4,or 8 h and subjected to qRT-PCR. Dataare the mean 6 SE of three indepen-dent biological replications. Asterisksdenote statistical significance (**, P ,0.01). B, LBD16 and LBD18 expres-sion in aux1 lax3 double mutants inresponse to auxin. Treatment and anal-ysis of the samples were performedas described in the legend of Figure 7A.C to J, GUS expression of ProLBD16:GUS(C and E), ProLBD16:GUS/aux1 (D and F),ProLBD18:GUS (G and I), and ProLBD18:GUS/aux1 plants (H and J). Six-day-oldseedlings were incubated without(C, D, G, and H) or with (E, F, I, and J)20 mM IAA for 4 h. Bars = 50 mm.







and lbd18mutant 18 h postgravitropic induction (pgi) inLR induction experiments with a gravitropic stimulus(Fig. 7E). Moreover, distribution of the LRPs at stages Ito VIII showed that developmental transition fromstage I to II was significantly inhibited 18 h pgi and LRPdevelopment was significantly delayed 42 h pgi, com-pared with that of the wild type (Fig. 7F). These resultsshowed that LBD18:SRDX suppressed LR initiation

events, periclinal divisions of primordium after LRinitiation, and later stages of LRP development in re-sponse to a gravitropic stimulus. In contrast, LBD18:SRDX did not suppress auxin-induced LR initia-tions compared with the wild type and lbd18 mutant(Supplemental Fig. S6), indicating that exogenousauxin overrides dominant repression of LR initiationby LBD18:SRDX, activating LR initiation.

Figure 7. Analysis of LR formation of ProLBD18:LBD18:SRDX in wild-type (WT) and lbd18 mutant backgrounds. A, Seven-day-old seedlings of ProLBD18:LBD18:SRDX Arabidopsis and the wild type (Col-0). #, Line number of ProLBD18:LBD18:SRDX/Col-0 plants. B, Seven-day-old seedlings of ProLBD18:LBD18:SRDX/lbd18 plants and the lbd18 single mutant. #, Line numberof ProLBD18:LBD18:SRDX/lbd18 plants. C, LR densities of Col-0, lbd18, ProLBD18:LBD18:SRDX/Col-0, and ProLBD18:LBD18:SRDX/lbd18 plants. Error bars = SE. n = 20. Asterisks denote statistical significance (**, P , 0.01; and ***, P , 0.001). D,Expression of EXP14 in wild-type (Col-0), lbd18, ProLBD18:LBD18:SRDX/Col-0 (line no.), and ProLBD18:LBD18:SRDX/lbd18plants. Seven-day-old seedlings were incubated with mock or 20 mM IAA for 6 h, and subjected to qRT-PCR for EXP14expression. Relative transcript levels are plotted. Data are the mean 6 SE of three independent biological replications.Asterisks denote statistical significance (*, P , 0.05). LBD18:SRDX/Col-0 = ProLBD18:LBD18:SRDX/Col-0 (line#2–5); LBD18:SRDX/lbd18 = ProLBD18:LBD18:SRDX/lbd18 (line#1–5). E, Analyses of LR initiation events of Col-0, lbd18, and two differentlines of ProLBD18:LBD18:SRDX/lbd18 plants after synchronization with a gravitropic stimulus. Three-day-old Col-0, lbd18,and ProLBD18:LBD18:GR/lbd18 transgenic plants treated with DEX were subjected to a 90˚ gravitropic stimulus, and thenumbers of LR initiation events were determined at 18 h pgi. Error bars = SE. n . 85. #, Line number of transgenic plants. F,Analyses of LR development of the wild type and two different lines of ProLBD18:LBD18:SRDX/lbd18 plants after synchro-nization with a gravitropic stimulus.


Lee et al.




LBD18 Regulates Expression of Cell Cycle Genes CDKA1;1and CYCB1;1

Several lines of evidence suggested that the regulatedcell cycle is essential for asymmetric divisions of asubset of pericycle cells during LR initiation (De Smet,2012). For example, progression of pericycles throughthe cell cycle is prevented by the treatment of auxintransport inhibitor in Arabidopsis seedlings or in a gain-of-function slr mutant blocking early auxin response(Fukaki et al., 2002; Himanen et al., 2002; Vannesteet al., 2005). Auxin activates many cell cycle-relatedgenes, such as cyclins and CDKs, before pericycle di-vision (Himanen et al., 2002). Various cell cycle geneshave been identified to play a role in LR initiation(Himanen et al., 2002, 2004; Vanneste et al., 2005; Renet al., 2008; Nieuwland et al., 2009; Sanz et al., 2011). Toidentify a link between LBD18 function and the regu-lation of cell cycle during LR initiation and LR devel-opment, we first made multiple mutants, lbd16 and/orlbd18, crossed to transgenic Arabidopsis harboring thepromoter of cell cycle genes, CDKA1;1, CDKB1;1, orCYCB1;1, fused to the GUS reporter gene (Hemerlyet al., 1993; Doerner et al., 1996; de Almeida Engleret al., 1999). However, none of the mutations in LBD16,LBD18, or LBD16 and LBD18 altered GUS expression inthe LRPs due to the genetic redundancy with other LBDgene family members (Supplemental Fig. S7). We thenconstructed transgenic Arabidopsis expressing LBD18:SRDX under its own promoter and harboring CDKA1;1,CDKB1;1, or CYCB1;1-GUS in the lbd18 mutant back-ground, and found that LBD18:SRDX greatly repressedGUS expression in the CDKA1;1 promoter but slightlysuppressed GUS expression under the CDKB1;1 pro-moter (Fig. 8, A and B). LBD18:SRDX completely re-moved GUS expression under the CYCB1;1 promoter inthe primordium and greatly reduced GUS staining inthe root tip (Fig. 8C). LBD16:SRDX greatly reducedGUS expression under the CDKB1;1 promoter in theroot (Fig. 8B). However, LBD16:SRDX did not affectGUS expression under the CDKA1;1 promoter (Fig. 8A).These results showed that LBD18:SRDX repressed thepromoter activities of cell cycle genes, CDKA1;1 andCYCB1;1. We further showed that LBD18:GR up-regulates expression of CDKA1;1 and CYCB1;1, butnot that of CDKB1:1, under the control of the Cauliflowermosaic virus 35S promoter (Fig. 8D) and under thecontrol of the LBD18 promoter (Fig. 8E). These resultsindicated that LBD18 plays a role in the regulation ofcell cycle genes. Cycloheximide treatment of Pro35S:LBD18:GR transgenic plants in the presence of DEXprevented DEX-inducible expression of CDKA1;1 andCYCB1;1 (Supplemental Fig. S8), demonstrating thatinduction of these cell cycle genes by LBD18 is a sec-ondary response requiring new protein synthesis.

DISCUSSION

Emerged LR densities of lax3 lbd16 and lax3 lbd18were similar to those of lax3, lbd16, or lbd18 single

mutants (Fig. 1B). However, the lax3 mutation inlbd16 lbd18 double mutants significantly reduced theemerged LR density to some extent, but not in an ad-ditive manner (Fig. 1B). These results indicate that,although LAX3 and LBD16 or LBD18 function largelyin a linear pathway to control LR emergence, LAX3also acts in parallel with LBD16 and LBD18 to someextent during LR emergence. Auxin-inducible expres-sion of LBD16 and LBD18 and LBD18-regulated targetgenes, EXP14 and EXP17, significantly decreased inlax3 mutants as compared with the wild type (Fig. 2, Aand B). In contrast, double mutations in LBD16 andLBD18 did not affect the expression of LAX3 in re-sponse to auxin (Fig. 2C). Altogether, genetic and geneexpression analysis results indicate that LAX3 acts inpart upstream of LBD16 and LBD18 during the auxinresponse, whereas LBD16 and LBD18 are not criticallyinvolved in auxin-responsive LAX3 expression. Con-sistent with this, overexpression of LBD18 in lax3 fullyrestored the defect in LR emergence of lax3 to the wild-type levels with concomitant overexpression of LBD18target genes, EXP14 and EXP17 (Fig. 3, A and B).However, in the case of using the LBD18 promoter toexpress LBD18 in lax3, the defect in LR formation inlax3 was rescued to some extent, although only in thepresence of auxin. This result indicates that lax3 mu-tation produced some defects in LR formation that arecaused by other components and/or transcriptionfactors acting downstream of LAX3 as well as the de-fect in LBD18 function under the LBD18 promoter.Auxin activates the signaling pathway, inducing ex-pression of certain transcription factor genes alongwith which LBD18 acts to promote LR formation to acertain degree in the absence of lax3 function. Wefurther showed that LBD18 up-regulates expression ofEXP14 and EXP17 in lax3 mutants under the control ofthe LBD18 promoter (Fig. 3, D and E), indicating thatLBD18 can regulate its target gene expression in theabsence of LAX3 function in tissues where LBD18 isnormally expressed. Expression of PG, encoding a cellwall remodeling enzyme, whose expression is depen-dent upon LAX3 function (Swarup et al., 2008), wasdown-regulated in the overlaying tissues of the LRP inlbd18 and up-regulated by LBD18 expression under thecontrol of the Cauliflower mosaic virus 35S promoter andunder the control of the LBD18 promoter (Fig. 4).Taken together, these results demonstrated that LBD18modulates its target gene expression downstream ofLAX3 to promote LR emergence. Overlapping ex-pression patterns of LAX3 and LBD18 in the overlay-ing tissues of the LRP (Supplemental Fig. S1) andreduced GUS expression under the control of theLBD18 promoter by lax3 mutation in the same tissue(Fig. 2, D–G) are consistent with the notion that LAX3induces LBD18 expression by promoting auxin influxinto the cortex and epidermis to facilitate LR emer-gence. We noted significant auxin-inducible expressionof LBD16, LBD18, EXP14, and EXP17 in lax3, whichmay be due to involvement of other auxin trans-porters. Two additional LAX genes, LAX1 and LAX2








(Kerr and Bennett, 2007; Ugartechea-Chirino et al., 2010),could be involved redundantly in auxin-responsiveexpression of LBD16 and LBD18. Several studies havedemonstrated that PINs are involved in LR devel-opment (Benková et al., 2003; Laskowski et al., 2008;Marhavý et al., 2013; Péret et al., 2013). However, it

remains to be determined whether other LAXs andPINs are involved in auxin-responsive expression ofLBD16 and LBD18 to control LR development.

It has been reported that the increased number ofprimordia at stage I in lax3 (Fig. 1C) was due to accu-mulation of leaf-derived auxin at supraoptimal levels

Figure 8. GUS expression of Arabidopsis plants harboring the promoter of cell cycle genes fused to GUS reporter and ProLBD16:LBD16:SRDX or ProLBD18:LBD18:SRDX in lbd18. A and B, GUS expression of CDKA1;1::GUS (A) or CDKB1;1::GUS (B) in Col-0, ProLBD16:LBD16:SRDX, and ProLBD18:LBD18:SRDX/lbd18 plants. The lower two rows show GUS staining of the LRP. C, GUSexpression of CYCB1;1::GUS in Col-0 and ProLBD18:LBD18:SRDX/lbd18 plants. GUS staining of the LRP at stage II and theprimary root tip is shown. Bars = 1 cm (in seedlings) or 50 mm (in LRPs and root tip). D, Time course expression analysis ofCDKA1;1, CDKB1;1, and CYCB1;1 genes following treatment with DEX in Pro35S:LBD18:GR plants. Seven-day-old Pro35S:LBD18:GR seedlings were incubated with 10 mM DEX for the indicated times, and subjected to qRT-PCR analysis for CDKA1;1,CDKB1;1, and CYCB1;1 gene expression;212 h, Samples incubated with mock for 12 h. E, Expression of CDKA1;1, CDKB1;1,and CYCB1;1 following treatment with DEX in ProLBD18:LBD18:GR plants. ProLBD18:LBD18:GR seedlings were grown for 7 d inthe absence (black squares) or presence (white squares) of DEX. Expression of CDKA1;1, CDKB1;1, and CYCB1;1 was thenanalyzed by qRT-PCR and plotted as relative expression levels. Asterisks denote statistical significance (*, P , 0.05).


Lee et al.



in the root pericycle, thus stimulating the ectopicinitiation of new primordia in lax3, but not due to thedefects in LR initiation (Fig. 5C; Swarup et al., 2008).We found that lbd18 mutation reduced the primor-dium number at stage I, which was exaggeratedby lax3 mutation (Fig. 1C). This result indicates in-volvement of LBD18 in LR initiation. A previousstudy showed that lbd18 mutation did not affect thedistribution of the LRPs at all developmental stages,but significantly reduced the number of emergedLRs as compared with the wild type, indicating thatthe emergence of LRs in the lbd18 mutant is a rate-limiting step in LR formation compared with thewild type (Lee et al., 2009b). The present geneticanalysis results with multiple mutants of aux1 or lax3provided the evidence that LBD18 is involved inLR initiation and LRP development in addition toplaying a role in LR emergence (Figs. 1 and 5). Thisnotion was further confirmed by the analysis of LRdevelopment kinetics using transgenic Arabidopsisexpressing LBD18:SRDX under its own promoterin the lbd18 mutant background (Fig. 7). We foundthat LBD18 target gene expression and LR formationby LBD18:SRDX were not affected in wild-type plantswhen compared with that in the lbd18 mutant back-ground, indicating that wild-type LBD18 homodimers(Lee et al., 2013b) might have higher DNA-bindingaffinity to the target promoter and/or higher tran-scriptional activity than LBD18:SRDX homodimers andthus can overcome dominant repression by LBD18:SRDX.

A model integrating the present data and previousreports (Marchant et al., 2002; Okushima et al., 2007;Swarup et al., 2008; Lee et al., 2009a,b, 2013a; Berckmanset al., 2011; Goh et al., 2012a; Lee and Kim, 2013) intoa gene regulatory pathway for LR development ispresented in Figure 9. LBD16 plays a role in the polarnuclear migration in the LR founder cell after LR prim-ing (Goh et al., 2012a), whereas LBD18 is involved inasymmetric anticlinal division during LR initiationand periclinal divisions of the LRP initiated in thexylem pole pericycle cells and in the progression ofLRP development through the regulation of cell cyclegenes such as CDKA1;1 and CYCB1;1 (Figs. 7 and 8).LBD18 also activates E2Fa expression for LR initiation(Berckmans et al., 2011). LBD16 and LBD18 functiontogether to regulate LRP development with AUX1(Figs. 5 and 6). LAX3 activates the SLR/IAA14-ARF7-ARF19 signaling module (Swarup et al., 2008), thusinducing LBD18 expression (Figs. 1–3). LBD18 in theendodermis and cortex in turn directly activatesEXP14 and indirectly activates EXP17 and other EXPs(Kim and Lee, 2013; Lee et al., 2013a; Lee and Kim,2013) for cell wall loosening, and induces PG expres-sion at a later time (Fig. 4) for cell wall remodeling,which facilitates local cell separation. PG enzymati-cally modifies the structures of the cell wall, renderingit more responsive to wall loosening mediated by ex-pansions (Cosgrove, 2000; Péret et al., 2009a, 2009b).These molecular events help promote the emergence ofLRP through the outer cell layers of the primary root.As ARF7 induces expression of ARF19 and LBD16 in

Figure 9. A model showing the molecular signaling network of LBD16 and LBD18 via the auxin influx carriers AUX1 and LAX3during LR development. The column shaded with lines indicates the xylem. The thick arrow and dotted arrow indicate directand indirect activation of target genes, respectively. P, Pericycle; En, endodermis; C, cortex; Ep, epidermis.





response to auxin signal, and subsequently ARF19induces LBD18 expression (Okushima et al., 2007; Leeet al., 2009a), the ARF7/ARF19-LBD16/LBD18 tran-scriptional network via the AUX1/LAX3 auxin influxcarriers plays a key role in controlling LR developmentfrom polar nuclear migration of LR founder cells toinitiation, primordium development, and emergenceof LR during the auxin response.

MATERIALS AND METHODS

Plant Growth and Tissue Treatment

Arabidopsis (Arabidopsis thaliana; Col-0) seedlings were grown and treatedas described previously (Park et al., 2002). For the treatment of hormone andchemicals, plants were grown in a 16-h photoperiod on 3MM Whatman filterpaper (GE Healthcare) on top of agar plates, and the filter paper with theseedlings was then transferred to a plate containing plant hormone orchemicals (20 mM for IAA, 10 mM for DEX, or 50 mM cycloheximide) and in-cubated for a given period of time with gentle shaking in the light at 23°C. Thelight intensity was approximately 120 mmol m22 s21 and was provided bythree wavelength daylight color fluorescent bulbs (Kumho Electric Co.).

Plant Materials

All plants used were the Arabidopsis Col-0 ecotype. The lax3 and aux1-21Arabidopsis mutants and the ProPG:GUS line were provided by Dr. MalcolmBennett (University of Nottingham) and confirmed via genotyping prior tousage (Swarup et al., 2008). The lbd16, lbd18, or lbd16 lbd18 mutants (female)were crossed with lax3, aux1-21, and aux1 lax3 mutants (male) to generate lax3lbd16, lax3 lbd18, lax3 lbd16 lbd18, aux1 lbd16, aux1 lbd18, aux1 lbd16 lbd18, aux1lax3 lbd16, aux1 lax3 lbd18, and aux1 lax3 lbd16 lbd18 mutants, and the ho-mozygous lines were isolated based on PCR genotyping and agrotropicphenotype of aux1-21 (Swarup et al., 2008; Lee et al., 2009b). Transgenicmutant plants, ProPG:GUS/lbd16, ProPG:GUS/lbd18, and ProPG:GUS/lbd16lbd18,were generated by crossing lbd16, lbd18, and lbd16 lbd18 mutants (female) withthe ProPG:GUS line (male), and the homozygous lines were isolated. The lbd16,lbd18, or lbd16 lbd18 mutants and ProLBD16:LBD16:SRDX or ProLBD18:LBD18:SRDX/lbd18 transgenic Arabidopsis (female) were crossed with the ProCDKA1;1:GUS, ProCDKB1;1:GUS, or ProCYCB1;1:GUS line (male) to generate ProCDKA1;1:GUS,ProCDKB1;1:GUS, and ProCYCB1;1:GUS reporters in lbd16, lbd18, and lbd16 lbd18mutant backgrounds and in ProLBD16:LBD16:SRDX and ProLBD18:LBD18:SRDX/lbd18 transgenic Arabidopsis. Homozygous lines were isolated by geno-typing for lbd16, lbd18, lbd16 lbd18, ProLBD16:LBD16:SRDX, and ProLBD18:LBD18:SRDX/lbd18 and by PCR detection of genomic DNA for theCDKA1;1::GUS, CDKB1;1::GUS, or CYCB1;1::GUS transgenes. Pro35S:LBD18:GR and ProLBD18:LBD18:GR in the lax3 mutant background (Pro35S:LBD18:GR/lax3 and ProLBD18:LBD18:GR/lax3) were generated by crossing Pro35S:LBD18:GR and ProLBD18:LBD18:GR (female) with lax3 (male), and the ho-mozygous lines were isolated. ProLBD16:GUS and ProLBD18:GUS transgenicArabidopsis in aux1 or lax3 mutant backgrounds (ProLBD16:GUS/aux1 andProLBD18:GUS/aux1 or ProLBD16:GUS/lax3 and ProLBD18:GUS/lax3) were gener-ated by crossing ProLBD16:GUS and ProLBD18:GUS (female) with aux1 or lax3(male). Homozygous lines were isolated by genotyping for ProLBD16:GUS,ProLBD18:GUS, and lax3 or agrotropic phenotype of aux1-21. Primer sequencesused in this study are shown in Supplemental Table S1.

Plasmid Construction and Arabidopsis Transformation

To generate ProLBD16:LBD16:SRDX, ProLBD16:LBD16:SRDX/lbd16, ProLBD18:LBD18:SRDX, and ProLBD18:LBD18:SRDX/lbd18 plants, the ProLBD16:LBD16and ProLBD18:LBD18 sequences were isolated by PCR from the existingProLBD16:LBD16:GR and ProLBD18:LBD18:GR plasmids (Lee et al., 2013a) usingthe primers with SRDX sequence (NH2-LDLDLELRLGFA-COOH) and sub-cloned into pDONR221 (Invitrogen) by the Gateway BP recombination re-action, yielding pDONR-ProLBD16:LBD16:SRDX and pDONR-ProLBD18:LBD18:SRDX, respectively. This construct was subcloned into pB7WG vector(destination vector, Vlaams Instituut voor Biotechnologie) by the Gateway LRrecombination reaction, yielding ProLBD16:LBD16:SRDX and ProLBD18:LBD18:

SRDX. ProLBD16:LBD16:SRDX and ProLBD18:LBD18:SRDX plasmids were thenintroduced into Arabidopsis, lbd16, or lbd18 mutants by Agrobacteriumtumefaciens-mediated transformation, respectively. T3 homozygous trans-formants were prepared and amplified. All constructs were confirmed viaDNA sequencing prior to plant transformation. Primer sequences used inthis study are shown in Supplemental Table S1.

RNA Isolation, Reverse Transcription-PCR, and qRT-PCR Analysis

Following treatment, Arabidopsis plants were immediately frozen in liquidnitrogen and stored at 280°C. Total RNA was isolated from frozen Arabi-dopsis using TRI Reagent (Molecular Research Center). For reverse tran-scription (RT)-PCR analysis, total RNA was isolated using an RNeasy plantmini kit (Qiagen) and subjected to RT-PCR analysis with the Access RT-PCRSystem (Promega) according to the manufacturer’s instructions. Real-time RT-PCR was carried out using a QuantiTect SYBR Green RT-PCR kit (Qiagen) in aCFX96 real-time system using a C1000 thermal cycler (Bio-Rad) as describedpreviously (Jeon et al., 2010). All real-time RT-PCR analyses were conducted intriplicate biological replications and subjected to statistical analysis. RT-PCRconditions and primer sequences are shown in Supplemental Table S1.

Microscopy and Histochemical GUS Assays

For whole-mount visualization, the seedlings were then cleared in 80% (v/v)ethanol for 24 h and mounted in 90% (v/v) glycerol and observed under theLeica DM2500 microscope with differential interference contrast according toMalamy and Benfey (1997). Histochemical assays for GUS activity were per-formed with 5-bromo-4-chloro-3-indolyl glucuronide, as described previously(Jefferson and Wilson, 1991). Samples were observed using a DM2500 mi-croscope (Leica) at 200- or 400-fold magnification with differential interferencecontrast.

Statistical Analysis

Quantitative data were subjected to statistical analysis for every pairwisecomparison, using the software for Student’s t test (Predictive AnalyticsSoftware for Windows, version 20.0).

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: LBD16 (At2g42430), LBD18(At2g45420), AUX1 (At2g38120), LAX3 (At1g77690), PG (At5g14650),CDKA1;1 (At3g48750), CDKB1;1 (At3g54180), CYCB1;1 (At4g37490), EXP14,(At5g56320), and EXP17 (At4g01630).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. GUS expression of ProLBD16:GUS, ProLBD18:GUS,and ProLAX3:GUS transgenic Arabidopsis during LR development.

Supplemental Figure S2. LR densities of wild-type, lax3, and ProLBD18:LBD18:GR/lax3 plants without or with DEX.

Supplemental Figure S3. LR phenotypes of aux1 lax3, aux1 lax3 lbd16, aux1lax3 lbd18, and aux1 lax3 lbd16 lbd18 mutants.

Supplemental Figure S4. Analysis of LR formation of ProLBD16:LBD16:SRDX transgenic plants in Col-0 and lbd16 mutant backgrounds.

Supplemental Figure S5. Analysis of the numbers of primordia at differentstages of LRP development in 7-d-old wild-type (Col-0), lbd18, ProLBD18:LBD18:SRDX/Col-0, and ProLBD18:LBD18:SRDX/lbd18 plants.

Supplemental Figure S6. Differential interference contrast images of LRinitiations of wild-type (Col-0), lbd18, and ProLBD18:LBD18:SRDX/lbd18plants without or with auxin.

Supplemental Figure S7. Analysis of GUS expression of transgenic Arabi-dopsis harboring the promoter of cell-cycle genes fused to GUS in lbd16,lbd18, or lbd16 lbd18 mutant backgrounds.


Lee et al.













Supplemental Figure S8. The effect of cycloheximide treatment on DEX-induced expression of CDKA1;1 or CycB1;1 in Pro35S:LBD18:GR trans-genic Arabidopsis.

Supplemental Table S1. Oligonucleotides and PCR conditions.

ACKNOWLEDGMENTS

We thank Dr. Bennett for lax3, aux1-21, LAX3:GUS, and PG:GUS seeds andDr. De Veylder for the CDKA1;1:GUS, CDKB1;1:GUS, and CYCB1;1:GUSseeds, and Arabidopsis Biological Resource Center for transfer DNA insertionmutants.

Received April 20, 2015; accepted June 8, 2015; published June 9, 2015.

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lateral organ boundaries domain16 and 18 act downstream ......lateral organ boundaries domain16and...

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