Rice actin-binding protein RMD is a key link in theauxin–actin regulatory loop that controls cell growthGang Lia, Wanqi Lianga, Xiaoqing Zhanga, Haiyun Renb, Jianping Huc, Malcolm J. Bennettd,e, and Dabing Zhanga,1

aState Key Laboratory of Hybrid Rice, Shanghai Jiao Tong University–University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciencesand Biotechnology, Shanghai Jiao Tong University, Shanghai 20040, China; bKey Laboratory of Cell Proliferation and Regulation Biology of the Ministryof Education, College of Life Science, Beijing Normal University, Beijing 100875, China; cDepartment of Energy Plant Research Laboratory, MichiganState University, East Lansing, MI 48824; dCentre for Plant Integrative Biology, University of Nottingham, Nottingham LE12 5RD, United Kingdom;and eCollege of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia

Edited by Jiayang Li, Chinese Academy of Sciences, Beijing, China, and approved June 6, 2014 (received for review January 27, 2014)

The plant hormone auxin plays a central role in plant growth anddevelopment. Auxin transport and signaling depend on actinorganization. Despite its functional importance, the mechanisticlink between actin filaments (F-actin) and auxin intracellularsignaling remains unclear. Here, we report that the actin-organiz-ing protein Rice Morphology Determinant (RMD), a type II forminfrom rice (Oryza sativa), provides a key link. Mutants lacking RMDdisplay abnormal cell growth and altered configuration of F-actinarray direction. The rmd mutants also exhibit an inhibition ofauxin-mediated cell elongation, decreased polar auxin transport,altered auxin distribution gradients in root tips, and suppressionof plasma membrane localization of auxin transporters O. sativaPIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demon-strate that RMD is required for endocytosis, exocytosis, andauxin-mediated OsPIN2 recycling to the plasma membrane. More-over, RMD expression is directly regulated by heterodimerized O.sativa auxin response factor 23 (OsARF23) and OsARF24, providingevidence that auxin modulates the orientation of F-actin arraysthrough RMD. In support of this regulatory loop, osarf23 and lineswith reduced expression of both OsARF23 and OsARF24 displayreduced RMD expression, disrupted F-actin organization and cellgrowth, less sensitivity to auxin response, and altered auxin dis-tribution and OsPIN localization. Our findings establish RMD asa crucial component of the auxin–actin self-organizing regula-tory loop from the nucleus to cytoplasm that controls rice cellgrowth and morphogenesis.

auxin signaling | actin cytoskeleton | rice morphogenesis

The plant hormone auxin plays a critical role in regulatingplant developmental programs by controlling cell expansion(1) and polarity (2–4), as well as organ patterning (5, 6). Auxinaction relies on its polar transport, which is mediated by specificinflux and efflux transporters (7, 8). Auxin efflux depends onpolar localization of PIN-FORMED (PIN) transporters (9, 10)that cycle between the plasma membrane and endosomalcompartments by means of vesicle trafficking (11, 12). Auxin per-ception by its receptor, TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB), promotes theproteolysis of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA)proteins, thereby activating auxin-responsive gene expression byderepressing AUXIN RESPONSE FACTOR (ARF) transcrip-tion factors (13).Auxin affects patterning and organization of the actin cyto-

skeleton during cell growth (14, 15). For example, the auxin IAAinduces actin bundling in Arabidopsis thaliana root cells (15).Pharmacological studies suggest that the actin cytoskeleton par-tially affects the directional transport of auxin by modulating cycl-ing of auxin efflux carriers (16, 17). In Arabidopsis root cells, theactin inhibitor cytochalasin D inhibits brefeldin A (BFA)-mediatedPIN1 internalization (11), whereas latrunculin B impairs thepolar localization of PIN1 in protophloem cells (18). Theseobservations suggest that a regulatory loop exists between auxinand the actin cytoskeleton during root development. Recently,

a positive feedback loop of auxin–Rho-like GTPases 2 fromplants (ROP2)–actin–PIN1–auxin, which is mediated by theauxin-binding protein 1/transmembrane kinase (ABP1/TMK)–dependent nontranscriptional auxin response pathway, has beenrevealed in Arabidopsis (3, 4, 19–21). However, the underlyingmolecular mechanism(s) of intracellular regulation betweenTIR1/AFB-mediated transcriptional auxin responses and actincytoskeleton is currently unclear.The type II formin protein, RICE MORPHOLOGY DETER-

MINANT (RMD; also called BENTUPPERMOST INTERNODE1), plays a key role in regulating cytoskeleton organization bynucleating, capping, and bundling of actin. The rmd mutantsexhibit abnormal microfilament and microtubule organiza-tion, causing altered plant morphology, including defectiveroot and shoot growth as well as aberrant inflorescence andseed shape (22, 23). Here, we show that auxin modulates actinfilament (F-actin) array orientation by directly regulatingRMD expression via Oryza sativa auxin response factor 23(OsARF23) and OsARF24 heterodimers. Defective F-actin arraysin rmd mutants disrupt polar auxin transport (PAT), the localiza-tion of O. sativa PIN-FORMED (OsPIN) proteins, auxin distribu-tion, and auxin-mediated cell growth during root development.

Significance

The positive feedback loop between the auxin pathway andactin cytoskeleton is essential for auxin self-organizing re-sponsive signaling during plant development; however, itsunderlying mechanism remains largely unknown. Here, weshowed that an actin-binding protein, rice morphology de-terminant (RMD), acts as a key component mediating the auxin–actin loop pathway, affecting cell growth and morphogenesis.Auxin directly promotes RMD expression via binding of Oryzasativa auxin response factor 23 (OsARF23) and OsARF24 hetero-dimers on the RMD promoter, triggering changes in F-actin or-ganization. In turn, RMD-dependent F-actin arrays affect auxinintracellular signaling, including polar auxin transport, localiza-tion and recycling of auxin efflux carriers, and auxin distributionin root cells. Our work identifies RMD as a key link in the auxin–actin self-organizing regulatory loop that is required for auxin-mediated cell growth.

Author contributions: W.L. and D.Z. designed research; G.L., X.Z., and H.R. performedresearch; G.L., W.L., H.R., J.H., M.J.B., and D.Z. analyzed data; and G.L., J.H., M.J.B., andD.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the Na-tional Center of Biotechnology Information (www.ncbi.nlm.nih.gov/) [RMD (LOC_Os07g40510/LOC_Os07g40520 or Os07g0596300), OsARF23 (LOC_Os11g32110 or Os11g0523800), OsARF24(LOC_Os12g29520 or Os12g0479400), OsPIN1b (LOC_Os02g50960 or Os02g0743400), OsPIN2(LOC_Os06g44970 or Os06g0660200), and UBQ (LOC_Os03g13170 or Os03g0234200)].1To whom correspondence should be addressed. E-mail: zhangdb@sjtu.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1401680111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1401680111 PNAS | July 15, 2014 | vol. 111 | no. 28 | 10377–10382

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Our study reveals that RMD is a key integrator of a regula-tory circuit underpinning auxin self-organization propertiesand actin cytoskeleton dynamics in root cell growth andmorphogenesis.

ResultsRMD-Mediated F-Actin Organization Controls Cell Growth andMorphogenesis. RMD plays a pivotal role in regulating morpho-genesis by modulating cytoskeleton organization in rice (22, 23).To investigate the role of RMD during rice root growth, we usedtwo null mutant alleles of RMD, rmd-1 and rmd-2 (22). From 3 to7 d after germination, both rmd mutants displayed slowed primaryroot growth (Fig. 1A). The elongation zones of rmd-1 and rmd-2were shorter than WT (Fig. 1 B and C), due to reduced cell length

rather than cell number (Fig. S1 A and B). When grown in agar orliquid medium, rmd-1 and rmd-2 primary roots exhibited strikingwavy growth in contrast to the straight growth of WT (Fig. 1D andFig. S1 C–H). Consistent with previous observation (22), cells inthe root elongation zone displayed predominantly longitudinallyoriented microfilaments in WT vs. more abundant transverseF-actin arrays in the rmd mutants (Fig. S2 A and C).To determine whether RMD is responsible for the altered cell

growth and morphogenesis phenotypes in the mutant roots, wefused RMD to red fluorescent protein (RFP) and expressed thefusion protein under an estrogen-inducible promoter in rmd-1cells (Fig. S1 I and J). Estradiol-induced expression of promoterLexA (pLex)::RMD-RFP in rmd-1 rescued the growth defect andabnormal cell growth in primary roots (Fig. 1A), the wavy-rootphenotype (Fig. S1 C–H), and the aberrant orientation of F-actinarrays (Fig. S2C). These results confirmed the importance ofRMD in regulating cell growth, morphogenesis, and F-actin ar-ray in rice. Consistent with its role in regulating root growth,RMD was found to be ubiquitously expressed in primary rootsexpressing pRMD (RMD promoter)::glucuronidase (GUS) (22),especially the tips and maturation zone (Fig. S1 K–M).

RMD-Mediated F-Actin Organization Controls Auxin-Regulated CellGrowth. Given that rmd roots have defective cell growth andwavy growth (Fig. 1 A–D and Fig. S1 A–H), we treated rmd rootswith IAA to determine whether RMD was linked to the auxinpathway. Treatment with 10 nM IAA induced cell elongation,whereas 10 μM IAA inhibited cell elongation in WT roots (Fig. 1E and F), consistent with its concentration-dependent mode ofaction (24). However, the effect of IAA treatments in rmd-1roots was attenuated compared with WT (Fig. 1 E and F),suggesting that rmd-1 cells have reduced sensitivity to auxin.Moreover, treatment with 10 μM IAA enhanced longitudinalbundling of F-actin in WT root cells yet only induced subtlechanges of F-actin bundling in RMD RNAi lines, which hadbeen characterized to have a moderate mutant phenotype (22),and did not cause any obvious changes of F-actin organization inrmd-1 (Fig. S2 A and B) unless the mutant was rescued by thepLex::RMD-RFP transgene after estradiol induction (Fig. S2 Cand D). Hence, RMD appears to function downstream of auxinto regulate cell growth, and RMD is essential for the auxin-mediated rearrangement of F-actin arrays.

Auxin Promotes RMD Expression. To probe the relationship be-tween auxin signaling and RMD, we first tested the regulation ofRMD expression by auxin. RMD mRNA increased fivefold inroots after a 6-h IAA treatment, as shown by quantitative real-time (qRT) PCR (Fig. 2A). Reporter activity in root tips ofpRMD::GUS transgenic plants (22) was enhanced after IAAapplication (Fig. S3A). IAA treatment also strongly stimulatedRMD protein accumulation, which was abrogated when theplants were cotreated with the proteasome inhibitor MG132 toblock auxin-dependent degradation of Aux/IAA repressor pro-teins (13) (Fig. 2B). Conversely, treatment with the auxin trans-port inhibitor N-1-naphthylphthalamic acid gradually decreasedthe abundance of RMD mRNA in roots (Fig. S3B). Hence, RMDexpression is positively regulated by auxin.

RMD Expression Is Directly Regulated by OsARF23 and OsARF24. ThepRMD contains 15 auxin response element-like (AuxRE-like:TGTC) motifs within the 3,000-bp region upstream of the startcodon (Fig. 2D, Upper and Fig. S3D), suggesting that RMD maybe directly regulated by ARFs that bind to its promoter (25). Totest this, dual-luciferase (dual-LUC) assays were performed inNicotiana benthamiana leaves to coexpress pRMD::LUC tran-siently with each of the 18 rice ARFs that showed overlappingexpression patterns with RMD according to online microarraydata (http://ricexpro.dna.affrc.go.jp/). Only OsARF23 and OsARF24activated the pRMD::LUC reporter (Fig. 2C and Fig. S3C).Chromatin immunoprecipitation (ChIP)-PCR assays confirmedthat OsARF23 and OsARF24 directly bound to the same regions

Fig. 1. RMD regulates cell growth, morphology, and auxin response in riceroots. (A) Primary root growth in different genetic backgrounds (n > 30 foreach genotype). E, treatment with 10 μM estradiol. WT and rmd-2 weretreated with DMSO. (B) Root tip of WT and rmd-1. Red signals indicate FM4-64 staining. Arrowheads indicate borders of the elongation zone of 7-d aftergermination (DAG) plants. (Scale bar: 1 cm.) (C) Length of the elongationzone in 7-DAG WT (n > 30), rmd-1 (n > 35), and rmd-2 (n > 30) primary roots.(D) Vertically grown roots (10 DAG) in Murashige and Skoog solid medium.Arrows indicate primary root growth depolarization (wavy growth). (Scale bar:2 cm.) (E) Response of roots from 5-DAG seedlings to low auxin. DMSO wasused as a mock treatment. (Scale bar: 1 cm.) (F) Statistical analysis of relativeroot growth in response to different auxin concentrations. Data were col-lected from 5-DAG seedlings of WT (n > 30), rmd-1 (n > 25), and rmd-2 (n >36) primary roots. Student t test: *P < 0.05; **P < 0.01. All error bars are ±SD.

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(D and E regions) in the pRMD and that OsARF23-bindingactivity increased after auxin treatment (Fig. 2D, Lower). Coex-pression assays using truncated pRMDs confirmed that theOsARF23/OsARF24-bound regions were essential for the reg-ulation of pRMD activity by these two proteins (Fig. S3 D and E).

Furthermore, analysis of mutated pRMDs showed that at leasttwo AuxRE-like motifs in the D or E region of pRMD were re-quired for OsARF23/OsARF24-stimulated RMD expression(Fig. S3 F and G). Moreover, qRT-PCR and in situ analysisdetected transcripts of OsARF23 or OsARF24 in various tissues,including young roots (Fig. S4 A–F), overlapping with RMD (22).Furthermore, immunoblot analysis showed the protein accumula-tion of OsARF23 or OsARF24 in young roots (Fig. S4G), con-firming the regulatory role of OsARF23 and OsARF24 onRMD expression.RMD transcript and protein levels also increased in rice lines

expressing an estradiol-inducible OsARF23 or OsARF24 trans-gene after estradiol treatment, even under conditions in whichthe auxin response pathway was blocked by the proteasome in-hibitor MG132 (13) (Fig. S3 H and I), suggesting direct tran-scriptional regulation of RMD by OsARF23 and OsARF24. RMDprotein accumulation was dramatically enhanced in lines expressingboth OsARF23 and OsARF24 (Fig. S3I), suggesting they activateRMD expression synergistically.

Heterodimers of OsARF23 and OsARF24 Activate RMD Expression. Totest how OsARF23 and OsARF24 synergistically regulate RMDexpression, we first conducted yeast two-hybrid assays, whichdemonstrated interaction between OsARF23 and OsARF24 viathe carboxyl-terminal dimerization domain (CTD) (Fig. S5 A andB). This is consistent with a previous report demonstratingthat the formation of ARF–ARF and Aux/IAA–ARF heter-odimers was dependent on the CTD motif (25). Furthermore,pull-down assays demonstrated that OsARF23 interacted withOsARF24 in a CTD-dependent manner in vitro (Fig. S5 C–E).Coimmunoprecipitation (co-IP) assays showed that OsARF23and OsARF24 could form a protein complex in rice (Fig. 2 Eand F), as well as in tobacco leaves transiently expressing bothproteins (Fig. S5F).ChIP-PCR and dual-LUC analysis revealed that OsARF23 and

OsARF24 bind to the same regions of the pRMD (Fig. 2D andFig. S3 E and G). DNA-binding assays showed that incubation ofOsARF23 with OsARF24 markedly enhanced the ability ofOsARF23 to bind to the pRMD in a CTD-dependent manner (Fig.S5G). Furthermore, coexpression of OsARF23 and OsARF24 in N.benthamiana leaves effectively increased the expression of thepRMD::LUC reporter gene compared with OsARF23 or OsARF24alone (Fig. 2G and Fig. S3G). In contrast, pRMD activity hada similar level when only OsARF23 or OsARF24 was expressedafter coexpressing OsARF23 and OsARF24 without the CTDmotif(Fig. 2G). Thus, OsARF23 and OsARF24 form heterodimers invitro and in vivo to facilitate RMD transcription.

RMD Acts Downstream of OsARF23 and OsARF24. Consistent withOsARF23 and OsARF24 coregulating RMD expression, RMDmRNA level (Fig. S6A), promoter activity (Fig. S6B), and pro-tein abundance (Fig. S6C) decreased in the osarf23 mutant. Theosarf23 mutant phenotype mimicked rmd, displaying delayed rootgrowth and reduced cell elongation after auxin treatment, as wellas reduced cell growth (Fig. 3 A and B and Fig. S6 E–H). Ad-ditionally, osarf23 root cells had more transversal F-actin arrays,whereas overexpression of RMD (osarf23/35S::RMD) in osarf23could rescue F-actin array and root growth defects (Fig. 3 A–E).Further, the osarf23/35S::RMD plants exhibited stronger longi-tudinal F-actin bundles with higher density compared with thoseof the WT (Fig. 3 C–G), consistent with a role for RMD in actinbundling organization (22, 23). In response to IAA, osarf23 alsodisplayed less sensitivity with respect to auxin-regulated rootcell growth and RMD expression and the rearrangement ofF-actin arrays (Fig. S6 F and I–K). Finally, transgenic plantsexpressing a Double RNAi (DRi) construct to repress bothOsARF23 and OsARF24 had reduced levels of RMD transcripts(Fig. S6D), as well as abnormal F-actin arrays (Fig. 3H) and wavyroot growth (Fig. 3 A and B) similar to rmd. We conclude thatRMD functions downstream of OsARF23 and OsARF24 to regu-late directional arrangement of F-actin arrays and cell growth in rice.

Fig. 2. Auxin promotes RMD expression via OsARF23 and OsARF24. (A)RMD mRNA accumulation in response to 10 μM IAA in 7-DAG WT roots. (B)Immunoblot analysis using the anti-RMD antibody (α-RMD) to show theaccumulation of RMD protein in response to auxin treatment. Roots weretreated with 10 μM IAA or cotreated with 10 μM IAA and 100 μM MG132.Tubulin was used as a loading control. The signal intensity indicated abovethe blot was calculated with ImageJ (National Institutes of Health) andnormalized to tubulin. (C) OsARF23 and OsARF24 markedly enhance pRMDactivity, as revealed by dual-LUC reporter gene assays in tobacco leaf cells.The 35S::OsARFs served as effectors, 35S::GFP was the control, and pRMD::LUCand 35S::REN (Renilla) were used as reporters. LUC activity was normalized toREN. Student t test: **P < 0.01. (D) ChIP analysis showing the binding ofOsARF23 and OsARF24 to pRMD fragments. (Upper) Diagram of pRMD withindications of the five promoter fragments (pRMD-A to pRMD-E) used forthe ChIP assays. Asterisks indicate AuxRE-like motifs not bound (black) orbound (red) by OsARF23 and OsARF24. (Lower) qRT-PCR analysis of pRMDfragments after ChIP. Preimmune serum (IgG) was used as a control. (E andF) OsARF23 and OsARF24 interact with each other in vivo. Rice seedlingextracts were immunoprecipitated by α-OsARF23 antibody (E), α-OsARF24antibody (F), or IgG (G). The OsARF24/OsARF23 complex enhances pRMDactivity in tobacco leaves, as revealed by dual-LUC assays. Student t test:**P < 0.01 (between expressed OsARF23 and coexpressed OsARF23/24). Allerror bars are ±SD. IP, immunoprecipitation; WB, Western blot.

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RMD Modulates PAT, OsPIN Localization, and Auxin Distribution.Auxin distribution is regulated by PAT, which relies on fine cy-toskeleton organization (5, 6, 16–18, 26, 27). To test whetherRMD-mediated actin cytoskeleton dynamics regulate PAT inrice, we measured shootward and rootward IAA transport inroots. In rmd-1, both rootward and shootward PAT were sig-nificantly reduced compared with WT (Fig. 4A). Consistent withthis finding, auxin accumulation assays revealed significantly re-duced auxin export in rmd-1 and rmd-2 protoplasts (Fig. 4B).Because auxin efflux relies on PIN proteins (9, 10, 26),

immunolocalization studies revealed polar distribution ofOsPIN1b and OsPIN2 in plasma membranes of WT root cells(Fig. 4C). In contrast, rmd-1 and rmd-2 exhibited enhancedOsPIN1b and OsPIN2 internalization and reduced localizationon the plasma membrane (Fig. 4C). Consistent with this obser-vation, plants expressing the auxin response reporter pDR5::GUSrevealed, in contrast to a strong reporter signal in the root tipand the maturation zone of WT roots, that GUS activity wasweaker in the root tip and ubiquitously distributed throughoutthe root of rmd-1 (Fig. S7 A–C). Similarly, confocal imaging of

Fig. 3. OsARF23 and OsARF24 regulate RMD-mediated cell growth and F-actin arrays in root. (A)Phenotype of 4-d-old roots of Zhonghua11 (ZH11),osarf23, osarf23/35S::RMD (line 8), rmd-1, andOsARF23/24 double-RNAi (line 3; DRi-L3). (Scale bar: 1 cm.) (B)Statistical analysis of average primary root lengthfrom the same plants shown in A (n > 30 roots foreach genotype). Student t test: **P < 0.01. F-actinarrays in ZH11 (C), osarf23 (D), and osarf23/35S::RMD (E) root cells (7 DAG) are shown. (Scale bars:10 μm.) (F) Quantitative analysis of the extent of F-actin bundling in ZH11 (n > 60), osarf23 (n > 50),and osarf23/35S::RMD (n > 50) root cells based onskewness (7 DAG). Student t test: **P < 0.01. (G)Quantitative analysis of F-actin density within ZH11,osarf23, and osarf23/35S::RMD root cells (7 DAG, n >50 for each genotype). (H) Confocal images show F-actin arrays in OsARF23/24 DRi root cells. (Scale bar:10 μm.) All error bars are ±SD.

Fig. 4. RMD controls auxin transport, polar locali-zation of OsPIN1b and OsPIN2, and auxin distribu-tion in roots. (A) PAT assays in roots (n > 20 for eachgenotype). N-1-naphthylphthalamic acid (10 μM) wasused to inhibit IAA transport. (B) Cellular IAA exportanalysis using protoplasts from rice roots. (C) Immu-nolocalization of OsPIN1b and OsPIN2 using α-OsPIN1band α-OsPIN2 antibodies. Each image (Right) is themagnification of the boxed area (Left). (Scale bars:original images, 50 μm; magnified regions, 10 μm.)Confocal images show pDR5rev::3xVenus-N7 expres-sion in WT (D) and rmd-2 (E) root tips. (Scale bars: 100μm.) Student t test: *P < 0.05. All error bars are ±SD.

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WT roots expressing pDR5 fused in the CaMV minimal 35Spromoter (pDR5rev)::3xVenus-N7 revealed expression in stelecells and an auxin maximum in the quiescent center (Fig. 4D),whereas the reporter failed to form an auxin maximum in rmd-2or osarf23 root tip (Fig. 4E and Fig. S7 D and E). Hence, RMDplays a positive role in maintaining root auxin response gra-dients, especially in the root tip.Like rmd, the osarf23 mutant and OsARF23/24 DRi lines also

exhibited increased levels of OsPIN1b and OsPIN2 in the cyto-plasm and less polarized distribution of these transporters onplasma membranes (Fig. S8 A–F), whereas the mislocalization ofOsPIN2 was largely rescued in osarf23/35S::RMD plants (Fig.S8 C and D). Our results suggest that OsARF-RMD–mediatedauxin–actin cytoskeleton dynamics play an essential role in PATand auxin distribution gradients by regulating the polarized lo-calization of OsPIN1b and OsPIN2 in rice roots.

RMD Regulates Vesicle Trafficking. Compared with the WT, bothrmd-1 and rmd-2 contained reduced OsPIN1b and OsPIN2 proteinabundance but no change in mRNA levels (Fig. S9 A and B),suggesting that RMD may regulate OsPIN protein trafficking.Because the cytoskeleton modulates protein trafficking bymodifying endocytosis and exocytosis (3, 4, 20, 28–30), to revealwhether the OsARF23/24-RMD pathway controls endocytosis,we examined root cells using FM4-64, a tracer for endocytosisand vesicle trafficking (20, 21, 30). Treatment with FM4-64 for30 min resulted in more labeled endocytic vesicles in rmd-1,osarf23, and OsARF23/24 DRi plants than WT (Fig. S9 C and D),suggesting altered endocytosis in these mutants and transgenicplants. Long-term treatment with FM4-64 (1.5 h) resulted inincreased signal internalization in rmd cells, mimicking the ac-cumulation of internalized bodies in WT cells after treatmentwith the exocytosis inhibitor BFA (21) (Fig. S9E). FM4-64–labeled bodies almost completely disappeared in WT cells trea-ted with BFA, followed by washing out. In contrast, strong BFA-induced FM4-64–labeled bodies persisted in the rmd mutantafter BFA removal, suggesting that exocytosis is defective in rmd(Fig. S9E). Moreover, BFA increased OsPIN2 internalizationinto BFA compartments in rmd-1 cells compared with WT,whereas treatment with the auxin 1-naphthaleneacetic acid re-stored OsPIN2 localization at the plasma membrane in WT cellsbut was more compromised in rmd-1, suggesting the mutant isdefective for OsPIN2 recycling (Fig. 5 A–C). Hence, RMD appearsto play an essential role in vesicle trafficking of endocytosis andexocytosis, especially in auxin-mediated OsPIN2 polar recycling tothe plasma membrane.

DiscussionAuxin-dependent plant development is closely associated withdirectional cell-to-cell auxin transport (27, 31, 32). Genetic orpharmacological manipulation of PAT that disturbs the auxinmaximum dramatically alters root patterning (1, 31). Numerouspharmacological investigations have confirmed the close corre-lation between PAT and the actin cytoskeleton (11, 16–18). InArabidopsis, auxin transport inhibitors like 2,3,5-triiodobenzoicacid induce bundling of actin filaments and inhibit endocytosis (15,18). Expressing the mouse actin-binding protein talin in ricecaused actin bundling accompanied by decreased PAT but couldbe restored by auxin treatment (33). Moreover, in Arabidopsis,accumulation of cortical fine F-actin is induced by ROP viaABP1/TMK-dependent auxin plasma membrane signaling,thereby promoting the polarized distribution and dynamic traf-ficking of PIN1 and PIN2 by locally inhibiting clathrin-dependent endocytosis (3, 4, 19–21, 29, 30).In this study, we provide genetic and biochemical evidence

that RMD-dependent actin organization is pivotal for spatialcoordination of auxin intracellular signaling, OsPIN localization,and auxin transport and distribution in rice as a result. The rmdroots display reduced PAT due to decreased intracellular auxinexport, most likely caused by reconfiguration of F-actin. Thealtered actin array in rmd affects the activity of the auxin efflux

carriers OsPIN1b and OsPIN2, as well as vesicle trafficking.Moreover, increased internalization of OsPIN1b and OsPIN2and decreased exocytosis in rmd suggest that RMD-mediatedF-actin organization is required for inhibition of endocytosis andactivation of exocytosis, as well as OsPIN recycling.Previous investigations in Arabidopsis and Zea mays show that

6-h treatment with the microtubule-depolymerizing chemicaloryzalin causes no obvious effect on the polarity of PIN1 or PIN2(11, 28), whereas prolonged treatment may induce a change ofthe polarity of PINs (28). On the other hand, a microtuble-associated protein, CLASP, was shown to connect microtubulearrays with auxin transport by affecting PIN2 trafficking in Ara-bidopsis (34). Given that RMD directly binds to and bundlesmicrotubules and that rmd mutants display abnormal microtu-bule arrays (22), a future research direction would be to address

Fig. 5. RMD modulates OsPIN recycling in root cells. (A) Immunostaininganalysis of OsPIN2 recycling to the plasma membrane. Seven-day-old roots ofWT and rmd-1 were treated with DMSO and 50 μM BFA, and cotreated with50 μM BFA and 50 μM 1-naphthaleneacetic acid (NAA) for 1.5 h. (Scale bars:10 μm.) Quantification of OsPIN2 fluorescence intensity (FI) at the plasmamembrane (PM) (B) and cytoplasm (C) of root cells mentioned in A. At least60 cells from three independent experiments were analyzed for each assay.Student t test: **P < 0.01. Error bars are ±SD. (D) Working model for RMD-mediated auxin–actin feedback regulatory loop. Auxin activates the activityof OsARF23 and OsARF24, which together bind to the promoter and inducethe expression of the gene encoding the actin-binding protein RMD. RMDpromotes the polarized localization of OsPIN1b and OsPIN2 through vesicletrafficking to ensure their function in transporting auxin to the site of action.

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whether RMD modulates auxin transport by changing microtu-bule organization.Both nuclear TIR1/AFB-dependent and plasma membrane

ABP1-mediated auxin-signaling pathways are essential for auxin-related cell growth and patterning (1, 3, 4, 13, 27). Previousinvestigations showed that auxin changes the level of F-actinbundles, stimulates cell elongation rapidly, and modulates actinorganization and endocytosis via the ABP1/TMK-dependent path-way, thereby explaining how auxin induces rapid cellular changeswithin minutes (3, 4, 20, 21, 24, 29, 30). In rice coleoptiles,transient expression of an actin-binding protein led to the ac-cumulation of more longitudinal actin bundles and arrested cellelongation, whereas fine cortical actin strands correspond toactive cell elongation (35). In this study, we show that auxin-inhibited cell growth in roots is associated with enhanced bun-dling of F-actin and that F-actin debundling correlates withauxin-induced cell growth. Thus, auxin-mediated F-actin bun-dling or debundling explains the auxin-regulated cell responses.We also report that RMD-mediated F-actin organization is di-rectly regulated by the auxin TIR1/AFB-dependent pathway viaOsARF23-OsARF24 heterodimers. In agreement with this reg-ulatory role, osarf23 or knockdown lines of both OsARF23 andOsARF24 show reduced expression of RMD and display defectssimilar to those of the rmd mutants, including more transverseF-actin arrays and suppression of cell elongation. According tothe phenotype, osarf23 mutants only display shorter root growth,but OsARF23/24 DRi lines exhibit both reduced root growth andwavy growth in roots, indicating that OsARF23 and OsARF24function redundantly in controlling RMD-dependent rootdevelopment. Our work establishes a nuclear auxin-signalingpathway that controls F-actin arrays, which is required for auxinself-regulatory F-actin action and root development.On the basis of our findings, we propose a working model for

the auxin–RMD signaling loop underlying root cell growth and

morphogenesis in rice (Fig. 5D). In the loop, local auxin con-centration sustains RMD expression by the OsARF23 and OsARF24heterodimers, and, in turn, RMD mediates the formation andnucleation of the actin cytoskeleton. As such, RMD plays a piv-otal role in regulating auxin intracellular signaling by functioningas a molecular switch to control F-actin array orientation in rootcells (Fig. 5D). In summary, RMD is a crucial link between thehormone auxin and actin cytoskeleton in the coordination of cellgrowth and patterning of roots.

Materials and MethodsAll rice plants used in this study, including mutants and transgenic plants,were in the japonica cultivar genetic background. Details on the followingare provided in SI Materials and Methods: plant materials and growthconditions, treatments, dual-LUC assays, antibody preparation and purifi-cation, ChIP PCR assays, yeast two-hybrid assays, recombinant protein gen-eration and in vitro pull-down assays, immunoblotting, immunostaining,co-IP, DNA-binding assays, F-actin staining, quantitative analysis of the F-actin array bundling and density, microscopy, assays for PAT and efflux inprotoplasts, histochemical staining for GUS, in situ hybridization, RNA ex-traction, and mRNA expression analysis. The primers used in this study arelisted in Table S1.

ACKNOWLEDGMENTS. We thank Lizhong Xiong for providing rice osarf23mutant lines; Zhijing Luo and Mingjiao Chen for performing rmd mutantscreens and rice crosses; and Yutaka Tabei, Hao Yu, and Hongquan Yangfor providing pLex, pGreenII-0000, and pGreenII-0800 vectors. Especially, weappreciate Professor Ping Wu, for his help in transgenic plants, who passedaway on June 12, 2014. This work was supported by Grants 31230051,30971739, 31270222, and 31110103915 from the National Natural ScienceFoundation of China; Grants 2013CB126902 and 2011CB100101 from the Na-tional Key Basic Research Developments Program, Ministry of Science andTechnology, China; and Grants 2011AA10A101 and 2012AA10A302 fromthe 863 Hitech Project, Ministry of Science and Technology, China (to D.Z.).

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