Rice actin-binding protein RMD is a key link in the auxin–actin regulatory loop that controls cell growth

Significance

The positive feedback loop between the auxin pathway and actin cytoskeleton is essential for auxin self-organizing responsive signaling during plant development; however, its underlying mechanism remains largely unknown. Here, we showed that an actin-binding protein, rice morphology determinant (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 Oryza sativa auxin response factor 23 (OsARF23) and OsARF24 heterodimers on the RMD promoter, triggering changes in F-actin organization. In turn, RMD-dependent F-actin arrays affect auxin intracellular signaling, including polar auxin transport, localization and recycling of auxin efflux carriers, and auxin distribution in 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.

Abstract

The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin–actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis.

The plant hormone auxin plays a critical role in regulating plant developmental programs by controlling cell expansion (1) and polarity (2–4), as well as organ patterning (5, 6). Auxin action relies on its polar transport, which is mediated by specific influx and efflux transporters (7, 8). Auxin efflux depends on polar localization of PIN-FORMED (PIN) transporters (9, 10) that cycle between the plasma membrane and endosomal compartments by means of vesicle trafficking (11, 12). Auxin perception by its receptor, TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB), promotes the proteolysis of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, thereby activating auxin-responsive gene expression by derepressing AUXIN RESPONSE FACTOR (ARF) transcription factors (13).

Auxin affects patterning and organization of the actin cytoskeleton during cell growth (14, 15). For example, the auxin IAA induces actin bundling in Arabidopsis thaliana root cells (15). Pharmacological studies suggest that the actin cytoskeleton partially affects the directional transport of auxin by modulating cycling of auxin efflux carriers (16, 17). In Arabidopsis root cells, the actin inhibitor cytochalasin D inhibits brefeldin A (BFA)-mediated PIN1 internalization (11), whereas latrunculin B impairs the polar localization of PIN1 in protophloem cells (18). These observations suggest that a regulatory loop exists between auxin and the actin cytoskeleton during root development. Recently, a positive feedback loop of auxin–Rho-like GTPases 2 from plants (ROP2)–actin–PIN1–auxin, which is mediated by the auxin-binding protein 1/transmembrane kinase (ABP1/TMK)–dependent nontranscriptional auxin response pathway, has been revealed in Arabidopsis (3, 4, 19–21). However, the underlying molecular mechanism(s) of intracellular regulation between TIR1/AFB-mediated transcriptional auxin responses and actin cytoskeleton is currently unclear.

The type II formin protein, RICE MORPHOLOGY DETERMINANT (RMD; also called BENT UPPERMOST INTERNODE 1), plays a key role in regulating cytoskeleton organization by nucleating, capping, and bundling of actin. The rmd mutants exhibit abnormal microfilament and microtubule organization, causing altered plant morphology, including defective root and shoot growth as well as aberrant inflorescence and seed shape (22, 23). Here, we show that auxin modulates actin filament (F-actin) array orientation by directly regulating RMD expression via Oryza sativa auxin response factor 23 (OsARF23) and OsARF24 heterodimers. Defective F-actin arrays in rmd mutants disrupt polar auxin transport (PAT), the localization of O. sativa PIN-FORMED (OsPIN) proteins, auxin distribution, and auxin-mediated cell growth during root development. Our study reveals that RMD is a key integrator of a regulatory circuit underpinning auxin self-organization properties and actin cytoskeleton dynamics in root cell growth and morphogenesis.

Results

RMD-Mediated F-Actin Organization Controls Cell Growth and Morphogenesis.

RMD plays a pivotal role in regulating morphogenesis by modulating cytoskeleton organization in rice (22, 23). To investigate the role of RMD during rice root growth, we used two null mutant alleles of RMD, rmd-1 and rmd-2 (22). From 3 to 7 d after germination, both rmd mutants displayed slowed primary root growth (Fig. 1A). The elongation zones of rmd-1 and rmd-2 were 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 or liquid medium, rmd-1 and rmd-2 primary roots exhibited striking wavy growth in contrast to the straight growth of WT (Fig. 1D and Fig. S1 C–H). Consistent with previous observation (22), cells in the root elongation zone displayed predominantly longitudinally oriented microfilaments in WT vs. more abundant transverse F-actin arrays in the rmd mutants (Fig. S2 A and C).

Fig. 1.

RMD regulates cell growth, morphology, and auxin response in rice roots. (A) Primary root growth in different genetic backgrounds (n > 30 for each genotype). E, treatment with 10 μM estradiol. WT and rmd-2 were treated 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 after germination (DAG) plants. (Scale bar: 1 cm.) (C) Length of the elongation zone 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 was used as a mock treatment. (Scale bar: 1 cm.) (F) Statistical analysis of relative root growth in response to different auxin concentrations. Data were collected 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.

To determine whether RMD is responsible for the altered cell growth and morphogenesis phenotypes in the mutant roots, we fused RMD to red fluorescent protein (RFP) and expressed the fusion protein under an estrogen-inducible promoter in rmd-1 cells (Fig. S1 I and J). Estradiol-induced expression of promoter LexA (pLex)::RMD-RFP in rmd-1 rescued the growth defect and abnormal cell growth in primary roots (Fig. 1A), the wavy-root phenotype (Fig. S1 C–H), and the aberrant orientation of F-actin arrays (Fig. S2C). These results confirmed the importance of RMD in regulating cell growth, morphogenesis, and F-actin array in rice. Consistent with its role in regulating root growth, RMD was found to be ubiquitously expressed in primary roots expressing pRMD (RMD promoter)::glucuronidase (GUS) (22), especially the tips and maturation zone (Fig. S1 K–M).

RMD-Mediated F-Actin Organization Controls Auxin-Regulated Cell Growth.

Given that rmd roots have defective cell growth and wavy growth (Fig. 1 A–D and Fig. S1 A–H), we treated rmd roots with IAA to determine whether RMD was linked to the auxin pathway. Treatment with 10 nM IAA induced cell elongation, whereas 10 μM IAA inhibited cell elongation in WT roots (Fig. 1 E and F), consistent with its concentration-dependent mode of action (24). However, the effect of IAA treatments in rmd-1 roots 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 longitudinal bundling of F-actin in WT root cells yet only induced subtle changes of F-actin bundling in RMD RNAi lines, which had been characterized to have a moderate mutant phenotype (22), and did not cause any obvious changes of F-actin organization in rmd-1 (Fig. S2 A and B) unless the mutant was rescued by the pLex::RMD-RFP transgene after estradiol induction (Fig. S2 C and D). Hence, RMD appears to function downstream of auxin to regulate cell growth, and RMD is essential for the auxin-mediated rearrangement of F-actin arrays.

Auxin Promotes RMD Expression.

To probe the relationship between auxin signaling and RMD, we first tested the regulation of RMD expression by auxin. RMD mRNA increased fivefold in roots after a 6-h IAA treatment, as shown by quantitative real-time (qRT) PCR (Fig. 2A). Reporter activity in root tips of pRMD::GUS transgenic plants (22) was enhanced after IAA application (Fig. S3A). IAA treatment also strongly stimulated RMD protein accumulation, which was abrogated when the plants were cotreated with the proteasome inhibitor MG132 to block auxin-dependent degradation of Aux/IAA repressor proteins (13) (Fig. 2B). Conversely, treatment with the auxin transport inhibitor N-1-naphthylphthalamic acid gradually decreased the abundance of RMD mRNA in roots (Fig. S3B). Hence, RMD expression is positively regulated by auxin.

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 the accumulation of RMD protein in response to auxin treatment. Roots were treated 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 above the blot was calculated with ImageJ (National Institutes of Health) and normalized to tubulin. (C) OsARF23 and OsARF24 markedly enhance pRMD activity, 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::LUC and 35S::REN (Renilla) were used as reporters. LUC activity was normalized to REN. Student t test: **P < 0.01. (D) ChIP analysis showing the binding of OsARF23 and OsARF24 to pRMD fragments. (Upper) Diagram of pRMD with indications of the five promoter fragments (pRMD-A to pRMD-E) used for the ChIP assays. Asterisks indicate AuxRE-like motifs not bound (black) or bound (red) by OsARF23 and OsARF24. (Lower) qRT-PCR analysis of pRMD fragments after ChIP. Preimmune serum (IgG) was used as a control. (E and F) OsARF23 and OsARF24 interact with each other in vivo. Rice seedling extracts were immunoprecipitated by α-OsARF23 antibody (E), α-OsARF24 antibody (F), or IgG (G). The OsARF24/OsARF23 complex enhances pRMD activity in tobacco leaves, as revealed by dual-LUC assays. Student t test: **P < 0.01 (between expressed OsARF23 and coexpressed OsARF23/24). All error bars are ±SD. IP, immunoprecipitation; WB, Western blot.

RMD Expression Is Directly Regulated by OsARF23 and OsARF24.

The pRMD contains 15 auxin response element-like (AuxRE-like: TGTC) motifs within the 3,000-bp region upstream of the start codon (Fig. 2D, Upper and Fig. S3D), suggesting that RMD may be directly regulated by ARFs that bind to its promoter (25). To test this, dual-luciferase (dual-LUC) assays were performed in Nicotiana benthamiana leaves to coexpress pRMD::LUC transiently with each of the 18 rice ARFs that showed overlapping expression patterns with RMD according to online microarray data (http://ricexpro.dna.affrc.go.jp/). Only OsARF23 and OsARF24 activated the pRMD::LUC reporter (Fig. 2C and Fig. S3C). Chromatin immunoprecipitation (ChIP)-PCR assays confirmed that OsARF23 and OsARF24 directly bound to the same regions (D and E regions) in the pRMD and that OsARF23-binding activity increased after auxin treatment (Fig. 2D, Lower). Coexpression assays using truncated pRMDs confirmed that the OsARF23/OsARF24-bound regions were essential for the regulation of pRMD activity by these two proteins (Fig. S3 D and E). Furthermore, analysis of mutated pRMDs showed that at least two AuxRE-like motifs in the D or E region of pRMD were required for OsARF23/OsARF24-stimulated RMD expression (Fig. S3 F and G). Moreover, qRT-PCR and in situ analysis detected 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 accumulation of OsARF23 or OsARF24 in young roots (Fig. S4G), confirming the regulatory role of OsARF23 and OsARF24 on RMD expression.

RMD transcript and protein levels also increased in rice lines expressing an estradiol-inducible OsARF23 or OsARF24 transgene after estradiol treatment, even under conditions in which the auxin response pathway was blocked by the proteasome inhibitor MG132 (13) (Fig. S3 H and I), suggesting direct transcriptional regulation of RMD by OsARF23 and OsARF24. RMD protein accumulation was dramatically enhanced in lines expressing both OsARF23 and OsARF24 (Fig. S3I), suggesting they activate RMD expression synergistically.

Heterodimers of OsARF23 and OsARF24 Activate RMD Expression.

To test how OsARF23 and OsARF24 synergistically regulate RMD expression, we first conducted yeast two-hybrid assays, which demonstrated interaction between OsARF23 and OsARF24 via the carboxyl-terminal dimerization domain (CTD) (Fig. S5 A and B). This is consistent with a previous report demonstrating that the formation of ARF–ARF and Aux/IAA–ARF heterodimers was dependent on the CTD motif (25). Furthermore, pull-down assays demonstrated that OsARF23 interacted with OsARF24 in a CTD-dependent manner in vitro (Fig. S5 C–E). Coimmunoprecipitation (co-IP) assays showed that OsARF23 and OsARF24 could form a protein complex in rice (Fig. 2 E and F), as well as in tobacco leaves transiently expressing both proteins (Fig. S5F).

ChIP-PCR and dual-LUC analysis revealed that OsARF23 and OsARF24 bind to the same regions of the pRMD (Fig. 2D and Fig. S3 E and G). DNA-binding assays showed that incubation of OsARF23 with OsARF24 markedly enhanced the ability of OsARF23 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 the pRMD::LUC reporter gene compared with OsARF23 or OsARF24 alone (Fig. 2G and Fig. S3G). In contrast, pRMD activity had a similar level when only OsARF23 or OsARF24 was expressed after coexpressing OsARF23 and OsARF24 without the CTD motif (Fig. 2G). Thus, OsARF23 and OsARF24 form heterodimers in vitro and in vivo to facilitate RMD transcription.

RMD Acts Downstream of OsARF23 and OsARF24.

Consistent with OsARF23 and OsARF24 coregulating RMD expression, RMD mRNA level (Fig. S6A), promoter activity (Fig. S6B), and protein abundance (Fig. S6C) decreased in the osarf23 mutant. The osarf23 mutant phenotype mimicked rmd, displaying delayed root growth and reduced cell elongation after auxin treatment, as well as reduced cell growth (Fig. 3 A and B and Fig. S6 E–H). Additionally, osarf23 root cells had more transversal F-actin arrays, whereas overexpression of RMD (osarf23/35S::RMD) in osarf23 could rescue F-actin array and root growth defects (Fig. 3 A–E). Further, the osarf23/35S::RMD plants exhibited stronger longitudinal F-actin bundles with higher density compared with those of the WT (Fig. 3 C–G), consistent with a role for RMD in actin bundling organization (22, 23). In response to IAA, osarf23 also displayed less sensitivity with respect to auxin-regulated root cell growth and RMD expression and the rearrangement of F-actin arrays (Fig. S6 F and I–K). Finally, transgenic plants expressing a Double RNAi (DRi) construct to repress both OsARF23 and OsARF24 had reduced levels of RMD transcripts (Fig. S6D), as well as abnormal F-actin arrays (Fig. 3H) and wavy root growth (Fig. 3 A and B) similar to rmd. We conclude that RMD functions downstream of OsARF23 and OsARF24 to regulate directional arrangement of F-actin arrays and cell growth in rice.

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, and OsARF23/24 double-RNAi (line 3; DRi-L3). (Scale bar: 1 cm.) (B) Statistical analysis of average primary root length from the same plants shown in A (n > 30 roots for each genotype). Student t test: **P < 0.01. F-actin arrays 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 on skewness (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.

RMD Modulates PAT, OsPIN Localization, and Auxin Distribution.

Auxin distribution is regulated by PAT, which relies on fine cytoskeleton organization (5, 6, 16–18, 26, 27). To test whether RMD-mediated actin cytoskeleton dynamics regulate PAT in rice, we measured shootward and rootward IAA transport in roots. In rmd-1, both rootward and shootward PAT were significantly reduced compared with WT (Fig. 4A). Consistent with this finding, auxin accumulation assays revealed significantly reduced 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 of OsPIN1b and OsPIN2 in plasma membranes of WT root cells (Fig. 4C). In contrast, rmd-1 and rmd-2 exhibited enhanced OsPIN1b and OsPIN2 internalization and reduced localization on the plasma membrane (Fig. 4C). Consistent with this observation, plants expressing the auxin response reporter pDR5::GUS revealed, in contrast to a strong reporter signal in the root tip and the maturation zone of WT roots, that GUS activity was weaker in the root tip and ubiquitously distributed throughout the root of rmd-1 (Fig. S7 A–C). Similarly, confocal imaging of WT roots expressing pDR5 fused in the CaMV minimal 35S promoter (pDR5rev)::3xVenus-N7 revealed expression in stele cells and an auxin maximum in the quiescent center (Fig. 4D), whereas the reporter failed to form an auxin maximum in rmd-2 or osarf23 root tip (Fig. 4E and Fig. S7 D and E). Hence, RMD plays a positive role in maintaining root auxin response gradients, especially in the root tip.

Fig. 4.

RMD controls auxin transport, polar localization of OsPIN1b and OsPIN2, and auxin distribution in roots. (A) PAT assays in roots (n > 20 for each genotype). N-1-naphthylphthalamic acid (10 μM) was used to inhibit IAA transport. (B) Cellular IAA export analysis using protoplasts from rice roots. (C) Immunolocalization of OsPIN1b and OsPIN2 using α-OsPIN1b and α-OsPIN2 antibodies. Each image (Right) is the magnification of the boxed area (Left). (Scale bars: original images, 50 μm; magnified regions, 10 μm.) Confocal images show pDR5rev::3xVenus-N7 expression in WT (D) and rmd-2 (E) root tips. (Scale bars: 100 μm.) Student t test: *P < 0.05. All error bars are ±SD.

Like rmd, the osarf23 mutant and OsARF23/24 DRi lines also exhibited increased levels of OsPIN1b and OsPIN2 in the cytoplasm and less polarized distribution of these transporters on plasma membranes (Fig. S8 A–F), whereas the mislocalization of OsPIN2 was largely rescued in osarf23/35S::RMD plants (Fig. S8 C and D). Our results suggest that OsARF-RMD–mediated auxin–actin cytoskeleton dynamics play an essential role in PAT and auxin distribution gradients by regulating the polarized localization of OsPIN1b and OsPIN2 in rice roots.

RMD Regulates Vesicle Trafficking.

Compared with the WT, both rmd-1 and rmd-2 contained reduced OsPIN1b and OsPIN2 protein abundance 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 by modifying endocytosis and exocytosis (3, 4, 20, 28–30), to reveal whether the OsARF23/24-RMD pathway controls endocytosis, we examined root cells using FM4-64, a tracer for endocytosis and vesicle trafficking (20, 21, 30). Treatment with FM4-64 for 30 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 transgenic plants. Long-term treatment with FM4-64 (1.5 h) resulted in increased signal internalization in rmd cells, mimicking the accumulation of internalized bodies in WT cells after treatment with the exocytosis inhibitor BFA (21) (Fig. S9E). FM4-64–labeled bodies almost completely disappeared in WT cells treated with BFA, followed by washing out. In contrast, strong BFA-induced FM4-64–labeled bodies persisted in the rmd mutant after BFA removal, suggesting that exocytosis is defective in rmd (Fig. S9E). Moreover, BFA increased OsPIN2 internalization into BFA compartments in rmd-1 cells compared with WT, whereas treatment with the auxin 1-naphthaleneacetic acid restored OsPIN2 localization at the plasma membrane in WT cells but was more compromised in rmd-1, suggesting the mutant is defective for OsPIN2 recycling (Fig. 5 A–C). Hence, RMD appears to play an essential role in vesicle trafficking of endocytosis and exocytosis, especially in auxin-mediated OsPIN2 polar recycling to the plasma membrane.

Fig. 5.

RMD modulates OsPIN recycling in root cells. (A) Immunostaining analysis of OsPIN2 recycling to the plasma membrane. Seven-day-old roots of WT and rmd-1 were treated with DMSO and 50 μM BFA, and cotreated with 50 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 plasma membrane (PM) (B) and cytoplasm (C) of root cells mentioned in A. At least 60 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 activity of OsARF23 and OsARF24, which together bind to the promoter and induce the expression of the gene encoding the actin-binding protein RMD. RMD promotes the polarized localization of OsPIN1b and OsPIN2 through vesicle trafficking to ensure their function in transporting auxin to the site of action.

Discussion

Auxin-dependent plant development is closely associated with directional cell-to-cell auxin transport (27, 31, 32). Genetic or pharmacological manipulation of PAT that disturbs the auxin maximum dramatically alters root patterning (1, 31). Numerous pharmacological investigations have confirmed the close correlation between PAT and the actin cytoskeleton (11, 16–18). In Arabidopsis, auxin transport inhibitors like 2,3,5-triiodobenzoic acid induce bundling of actin filaments and inhibit endocytosis (15, 18). Expressing the mouse actin-binding protein talin in rice caused actin bundling accompanied by decreased PAT but could be restored by auxin treatment (33). Moreover, in Arabidopsis, accumulation of cortical fine F-actin is induced by ROP via ABP1/TMK-dependent auxin plasma membrane signaling, thereby promoting the polarized distribution and dynamic trafficking 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 spatial coordination of auxin intracellular signaling, OsPIN localization, and auxin transport and distribution in rice as a result. The rmd roots display reduced PAT due to decreased intracellular auxin export, most likely caused by reconfiguration of F-actin. The altered 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 OsPIN2 and decreased exocytosis in rmd suggest that RMD-mediated F-actin organization is required for inhibition of endocytosis and activation of exocytosis, as well as OsPIN recycling.

Previous investigations in Arabidopsis and Zea mays show that 6-h treatment with the microtubule-depolymerizing chemical oryzalin causes no obvious effect on the polarity of PIN1 or PIN2 (11, 28), whereas prolonged treatment may induce a change of the polarity of PINs (28). On the other hand, a microtuble-associated protein, CLASP, was shown to connect microtubule arrays with auxin transport by affecting PIN2 trafficking in Arabidopsis (34). Given that RMD directly binds to and bundles microtubules and that rmd mutants display abnormal microtubule arrays (22), a future research direction would be to address whether RMD modulates auxin transport by changing microtubule 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). Previous investigations showed that auxin changes the level of F-actin bundles, stimulates cell elongation rapidly, and modulates actin organization and endocytosis via the ABP1/TMK-dependent pathway, thereby explaining how auxin induces rapid cellular changes within minutes (3, 4, 20, 21, 24, 29, 30). In rice coleoptiles, transient expression of an actin-binding protein led to the accumulation of more longitudinal actin bundles and arrested cell elongation, whereas fine cortical actin strands correspond to active cell elongation (35). In this study, we show that auxin-inhibited cell growth in roots is associated with enhanced bundling of F-actin and that F-actin debundling correlates with auxin-induced cell growth. Thus, auxin-mediated F-actin bundling or debundling explains the auxin-regulated cell responses. We also report that RMD-mediated F-actin organization is directly regulated by the auxin TIR1/AFB-dependent pathway via OsARF23-OsARF24 heterodimers. In agreement with this regulatory role, osarf23 or knockdown lines of both OsARF23 and OsARF24 show reduced expression of RMD and display defects similar to those of the rmd mutants, including more transverse F-actin arrays and suppression of cell elongation. According to the phenotype, osarf23 mutants only display shorter root growth, but OsARF23/24 DRi lines exhibit both reduced root growth and wavy growth in roots, indicating that OsARF23 and OsARF24 function redundantly in controlling RMD-dependent root development. Our work establishes a nuclear auxin-signaling pathway that controls F-actin arrays, which is required for auxin self-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 concentration sustains RMD expression by the OsARF23 and OsARF24 heterodimers, and, in turn, RMD mediates the formation and nucleation of the actin cytoskeleton. As such, RMD plays a pivotal role in regulating auxin intracellular signaling by functioning as a molecular switch to control F-actin array orientation in root cells (Fig. 5D). In summary, RMD is a crucial link between the hormone auxin and actin cytoskeleton in the coordination of cell growth and patterning of roots.

Materials and Methods

All rice plants used in this study, including mutants and transgenic plants, were in the japonica cultivar genetic background. Details on the following are provided in SI Materials and Methods: plant materials and growth conditions, treatments, dual-LUC assays, antibody preparation and purification, ChIP PCR assays, yeast two-hybrid assays, recombinant protein generation 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 in protoplasts, histochemical staining for GUS, in situ hybridization, RNA extraction, and mRNA expression analysis. The primers used in this study are listed in Table S1.