OsAUX1 controls lateral root initiation in rice (Oryza sativa L.)1

Accepted Article 1 2 3

HEMING ZHAO, TENGFEI MA, XIN WANG, YINGTIAN DENG, HAOLI MA, RONGSHENG

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ZHANG & JIE ZHAO*

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State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China

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* For correspondence (fax +86 27 68756010; e-mail [email protected]).

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pce.12467 1

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ABSTRACT

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Polar auxin transport, mediated by influx and efflux transporters, controls many aspects of

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plant growth and development. The auxin influx carriers in Arabidopsis have been shown to

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control lateral root development and gravitropism, but little is known about these proteins in

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rice. This paper reports on the functional characterization of OsAUX1. Three OsAUX1

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T-DNA insertion mutants and RNAi knockdown transgenic plants reduced lateral root

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initiation compared to WT plants. OsAUX1 overexpression plants exhibited increased lateral

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root initiation and OsAUX1 was highly expressed in lateral roots and lateral root primordia.

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Similarly, the auxin reporter, DR5-GUS, was expressed at lower levels in osaux1 than in the

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WT plants, which indicated that the auxin levels in the mutant roots had decreased.

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Exogenous NAA treatment rescued the defective phenotype in osaux1-1 plants, whereas IAA

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and 2,4-D could not, which suggested that OsAUX1 was a putative auxin influx carrier. The

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transcript levels of several auxin-signaling genes and cell cycle genes significantly declined in

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osaux1, hinting that the regulatory role of OsAUX1 may be mediated by auxin-signaling and

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cell cycle genes. Overall, our results indicated that OsAUX1 was involved in polar auxin

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transport and functioned to control auxin-mediated lateral root initiation in rice.

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Keywords: OsAUX1; lateral root; rice.

Accepted Article 1

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INTRODUCTION

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Lateral roots are a major component of the root system architecture in plants and play crucial

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roles in water and nutrient uptake and in the interaction between plants and the environment

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(Varney and Canny 1993; Hochholdinger et al. 2004; Coudert et al. 2010). Lateral root

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development is controlled by various endogenous and exogenous factors, such as hormones

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and metal ions (Benková et al. 2003; Malamy 2005; Dello Ioio et al. 2008; Marcon et al.

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2013). The auxin, indole-3-acetic acid (IAA), is an essential plant hormone, and can be

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synthesized in young apical tissues, such as shoots, leaves and roots (Swarup and Bennett

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2003; Ljung et al. 2005; Swarup and Péret 2012). Auxin is redistributed from where it is

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synthesized to all parts of the plant through two physiologically distinct and spatially

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separated transport pathways: a non-polar transport system in the phloem and cell-to-cell

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polar auxin transport in various other tissues (Michniewicz et al. 2007). Evidence from many

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studies has demonstrated that polar auxin transport plays a vital role in regulating lateral root

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development, including the specification of founder cells, the first anticlinal division and the

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development and emergence of lateral root primordia (Benková et al. 2003; Laskowski et al.

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2008; Overvoorde et al. 2010; De Smet 2012; Saini et al. 2013).

Accepted Article 1

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The polar transport of auxin from cell to cell is accomplished by the auxin influx and efflux

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transporters located in the plasma membrane (Zazímalová et al. 2010). It has been reported

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that AUX1/LAX subfamily proteins are auxin influx carriers (Marchant et al. 2002; Swarup et

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al. 2008; Péret et al. 2012) and that most PIN-FORMED (PIN) family proteins are auxin

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efflux carriers (Petrásek et al. 2006). There are also intracellular PIN proteins that seem to

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transport auxin between cell compartments (Mravec et al. 2009; Dal Bosco et al. 2012; Ding 3

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et al. 2012; Bender et al. 2013; Sawchuk et al. 2013; Cazzonelli et al. 2013).

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P-GLYCOPROTEIN (PGP) proteins that belong to a subfamily B of the ATP-binding

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cassette (ABC) transporters have also been shown to function as auxin uptake and/or efflux

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transporters and to mediate the cellular and long-distance transport of auxin (Geisler et al.

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2003; Santelia et al. 2005; Shen et al. 2010; Kaneda et al. 2011). The activity and polar

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subcellular localization of these transporters serve to regulate the direction of the auxin flow

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and thus define the establishment of auxin gradients (Kleine-Vehn et al. 2006; Kerr and

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Bennett 2007; Wisniewska et al. 2006; Petrášek and Friml 2009). Many PIN genes have been

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characterized in detail, including AtPIN1-AtPIN4 and AtPIN7 in Arabidopsis (Okada et al.

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1991; Friml et al. 2002a; Friml et al. 2002b; Ganguly et al. 2010), ZmPIN1a and ZmPIN1b in

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maize (Carraro et al. 2006), and OsPIN1 and OsPIN3t in rice (Xu et al. 2005; Zhang et al.

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2012). AtPGP1 and AtPGP19 function in auxin export, while AtPGP4 (AtABCB4) does not

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function only in auxin export and its dual role was reported (Geisler et al. 2005; Terasaka et

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al. 2005; Wu et al. 2007; Kubeš et al. 2012).

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In higher plants, the AUX1/LAX subfamily belongs to the auxin/amino acid permease

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(AAAP) family (Saier et al. 2009; Zhao et al. 2012). The Arabidopsis AUX1/LAX subfamily

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genes encode the four highly conserved transmembrane proteins AUX1, LAX1, LAX2 and

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LAX3 (Marchant et al. 2002; Swarup et al. 2004; Yang et al. 2006; Swarup et al. 2008; Péret

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et al. 2012). Auxin transport activity assays in heterologous expression systems have

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confirmed that all four members of the AUX1/LAX subfamily in Arabidopsis are

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high-affinity auxin influx transporters (Yang et al. 2006; Carrier et al. 2008; Swarup et al.

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2008; Péret et al. 2012). Furthermore, the aux1 and lax3 mutants display auxin-related 4

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developmental defects in Arabidopsis. The AUX1 mutation resulted in the loss of root

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gravitropism and reduced the number of lateral roots. In comparison, a loss-of-function

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mutation of the LAX3 gene led to reduced lateral root emergence. Previous studies have

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demonstrated that AUX1 and LAX3 influence lateral root development by regulating the

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primordia initiation and primordia emergence steps, respectively (Marchant et al. 2002;

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Swarup et al. 2008). Unlike AUX1 and LAX3, mutations in LAX1 and LAX2 did not disturb

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root system development. The lax2 loss-of-function mutants had vascular breaks in their

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cotyledons, which indicated that LAX2 regulated vascular patterning in cotyledons (Péret et

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al. 2012). In wild cherry, Prunus avium, PaLAX1 promoted the importation of auxin into cells

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and affected the level and distribution of free endogenous auxin (Hoyerová et al. 2008).

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CsAUX1 from cucumber was involved in responses to gravity and the seedling peg formation

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on the lower side of the transition zone between the hypocotyl and the root (Fujii and

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Takahashi 2003). CgAUX1 expression was also observed in Casuarina glauca cells infected

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by Frankia bacteria during actinorhizal nodule formation (Péret et al. 2007).

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Lateral roots in Arabidopsis are derived from pericycle cells adjacent to the xylem pole

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(Dubrovsky et al. 2001; Laskowski 2013). These lateral root founder cells undergo a well

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defined program of oriented cell division and expansion to form a primordium. The

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developing primordia must pass through several intervening parental root cell layers prior to

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emergence (Malamy et al. 1997; Dubrovsky et al. 2006; Hochholdinger and Zimmermann

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2008; Péret et al. 2009b). Auxin initially regulates the division of lateral root founder cells in

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the pericycle tissue (Dubrovsky et al. 2008; De Smet 2012). Subsequent patterning of tissues

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within a given lateral root primordium also requires the establishment of an auxin response 5

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gradient, which reaches a maximum at the tip (Malamy et al. 1997; Benková and Bielach

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2010). Mutations that disturb auxin transport and signaling are known to reduce the formation

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of lateral roots (Péret et al. 2009a; De Smet 2012).

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Rice is a model plant for monocotyledon species. Systematic sequence analysis has shown

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that there are five members in the AUX subfamily in rice (Zhao et al. 2012). In a previous

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study, we made expression profiling analysis of the AUX subfamily of rice (OsAUXs) at

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different developmental stages (Zhao et al. 2012). This study extended our previous rice AUX

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subfamily research and detailed the identification and functional analysis of OsAUX1 in rice

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by evaluating three T-DNA mutant lines, RNAi lines and overexpression lines. The mutant

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and RNAi plants showed abnormal distributions of free endogenous auxin, had a reduced

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number of primordia and lateral roots, lost their gravitropism responses and their growth were

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stunted. In contrast, the overexpression lines had an increased number of primordia and lateral

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roots. Here, we showed that OsAUX1 controlled lateral root initiation in rice and mediated

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auxin influx into cells.

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MATERIALS AND METHODS

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Plant material and growth condition

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The OsAUX1 T-DNA insertion mutants and the wild-type Oryza sativa japonica cv. Dongjin

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were obtained from Kyung Hee University, South Korea. Rice seeds were surface-sterilized

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and germinated in 1/2 MS medium. The 14-day-old seedlings were transplanted into soils and

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were grown under normal conditions.

Accepted Article 1

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For phenotypic characterization of 7-day-old seedlings of the mutants and transgenic lines,

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seeds previously immersed in water at 37°C for 30h were sown on a plastic net that was

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floating on a nutrient solution (Yoshida 1976; Ni et al. 2011) in a growth chamber with a

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14:10 light:dark photoperiod at 28°C. For auxin treatments, the culture solutions were

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supplemented

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2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) or 2-naphthoxyacetic acid

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(2-NOA). The root phenotypes were photographed using a digital camera (Nikon D5000 with

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a Micro NIKKOR 60mm lens, Japan) or a stereomicroscope (LEICA MZFLIII, Germany).

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The abiotic stress treatments with 7-day-old seedlings were performed as described by Zhao

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et al. (2012).

with

various

concentrations

of

1-naphthylacetic

acid

(NAA),

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Identification of OsAUX1 T-DNA insertion mutants

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Three independent T-DNA insertion mutants in the OsAUX1 locus, osaux1-1 (3A-51110),

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osaux1-2 (1A-20543) and osaux1–3 (3A-01770) were identified in the SIGnAL database and

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obtained

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polymerase chain reaction (PCR) using OsAUX1-specific primers (Figs. 1A; Table S1) and

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T-DNA border primers (Ngus-RB or LB). The flanking sequences of the T-DNA insertion

(http://signal.salk.edu/cgi-bin/RiceGE).

The

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insertions

were

confirmed

by

sites were sequenced by Genescript (www.genescript.com.cn). Genotyping of the

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osaux1-segregating population was performed by PCR (Figs. 1G-I). The expression levels of

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OsAUX1 in osaux1 mutants were determined by reverse transcription PCR (RT-PCR) using

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the primers presented in Fig. 1A. Transcripts of RAc1 were amplified as a control, using the

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primer set RAc1-FP and RAc1-RP. All primer sequences for the PCR and RT-PCR

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experiments were listed in supplemental Table S1.

Accepted Article

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Vector constructions and rice transformation.

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To construct the RNA interference vector, a 252-bp cDNA fragment of OsAUX1 was

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amplified from the cDNA of 20cm rice panicles using the primer set OsAUX1 Ri-FP and

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OsAUX1 Ri-RP (Table S1). This fragment was inserted into the BamHI and XbaI sites (for the

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reverse insert) and the SalI and SacI sites (for the forward insert) in the pCGI vector (Fig.

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2A).

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For the overexpression construct, the full-length cDNA of OsAUX1 was amplified with the

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primer set OsAUX1 OE-FP and OsAUX1 OE-RP (Table S1) and was inserted into the BamHI

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and XbaI sites in the pCUN vector (Fig. 3A). For the fusion construct of the OsAUX1

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promoter and the GUS coding sequence (pOsAUX1-GUS), the 2.48-kb OsAUX1 promoter

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region was amplified from Nipponbare genomic DNA using the primers OsAUX1 Gus-FP and

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OsAUX1 Gus-RP (Table S1) and inserted into the pCAMBIA1381Xb vector at the XbaI and

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HindⅢ sites. These constructs were introduced into the Agrobacterium tumefaciens strain

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EHA105 and transformed into callus derived from mature seeds of rice var. Nipponbare, as

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previously described (Chen et al. 2003).

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For complementation of the osaux1-1 mutation, an 8.6 kb DNA fragment containing 8

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2475 bp 5’ of the OsAUX1 ATG codon, the entire OsAUX1 coding region, and 1236 bp 3’ of

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the OsAUX1 stop codon was amplified from wild type genomic DNA by PCR using the

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primers OsAUX1 Com-FP and OsAUX1 Com-RP (Table S1). The PCR product was digested

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with BamHI and XbaI and subsequently inserted into the pCAMBIA2301 vector. The

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pCAMBIA2301-OsAUX1 plasmid was transformed into Agrobacterium tumefaciens strain

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EHA105 which was then used to transform the osaux1-1 mutant.

Accepted Article

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GUS staining

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GUS staining of transgenic plants harvested at different developmental stages was performed

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using 100 mM sodium phosphate buffer (pH 7.0) with 0.5% v ⁄ v Triton X-100 and 2 mM

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X-Gluc (Biobasic Inc., Sangon, Shanghai, China) overnight at 37℃ (Jefferson et al. 1987).

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After staining, the samples were observed under an Olympus RX71 stereomicroscope

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(http://www.olympus-global.com/en/) and photographed with a digital camera (CoolSNAP,

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RS Photometric, http://www.photometrics.com/products/ccdcams).

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IAA concentration and distribution

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Indole-3-acetic acid concentration from leaves and roots of 7-day-old seedlings in WT and

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osaux1-1 plants was measured by selected reaction monitoring analysis with a

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gas-chromatography mass-spectrometer (GC-SRM-MS) (Ribnicky et al. 2002). A

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SHIMADZU GCMS-QP2010 Plus instrument equipped with an HP-5MS column (30 m long;

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0.25 mm i.d.; 0.25μm film, Agilent, USA) was used to quantify the IAA level of each sample,

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with five independent biological replicates, as described by Chen et al. (2010).

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The rice auxin-inducible reporter DR5-GUS line was obtained from Leiden University 9

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(Scarpella et al. 2003). Genetic crosses were performed to introduce the DR5- GUS reporter

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gene into both the WT Dongjin and the osaux1-1 mutant backgrounds. The auxin distribution

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in WT and osaux1-1 roots was analyzed by GUS staining, and photographed using an

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Olympus RX71 stereomicroscope.

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Semi- and real-time quantitative RT-PCR

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Total RNA was isolated from various rice organs (root, stem, leaf, panicle, and seed). The

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methods of RNA extraction, reverse transcription, and RT-PCR were performed according to

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Ren et al. (2012). The sequences of the primers used for qRT-PCR are listed in Table S2.

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Transcriptome analysis by RNA sequencing

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For the RNA sequencing experiment, total RNA was extracted from freshly harvested roots of

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7-day-old seedlings of WT and osaux1-1. The RNA samples were sequenced by BGI

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(www.genomics.cn) in Shenzhen, China. The differentially expressed genes were defined as

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the FDR (False Discovery Rate) ≤0.001 and log2 absolute value ≥1. The transcriptional levels

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of the genes related to auxin responses or the cell cycle which were identified by the RNA

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sequencing experiment were subsequently validated by qRT-PCR using the primer sets listed

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in Table S2.

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RESULTS

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Identification and morphological characterization of three osaux1 mutants

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A search of the SALK database (http://signal.salk.edu/cgi-bin/RiceGE) (Jeong et al. 2002; An

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et al. 2003) led to the identification of three rice T-DNA insertion lines (3A-51110, 1A-20543

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and 3A-01770 from the Kyung Hee databases) for the OsAUX1 locus (LOC_Os01g63770) in

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the Dongjin background. These lines were renamed: osaux1-1, osaux1-2 and osaux1-3,

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respectively (Fig. 1A). Homozygous plants for the three T-DNA insertions were identified by

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PCR (Figs. 1A, G–I). The RT-PCR analysis, using a primer set corresponding to the insertion

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sites, did not detect any OsAUX1 transcripts in the three mutants (Fig. 1B). Characterization

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of homozygous seedlings from the three insertion lines showed that the number of emerged

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lateral roots from the primary roots fell by about 51.8% in comparison with the wild-type

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(WT) plants. Furthermore, the lengths of the lateral roots in the mutants were also

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significantly shorter than those of the WT plants (Figs. 1C-F). The three mutants also

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displayed a reduced gravitropic response (Supporting Information Fig. S1). The plant heights

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and tiller numbers of the mutants were also noticeably reduced and the heading dates of the

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mutants were delayed compared to the WT plants (Supporting Information Figs. S2A-C).

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Additionally, the lengths of the main stems and internodes had dramatically decreased at the

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mature stage (Supporting Information Figs. S2 D, E). However, the lengths of the primary

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roots and the number of adventitious roots at the seedling stage (Fig. 1C) and the 1000-grain

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weight (Supporting Information Fig. S2F) did not differ significantly between the mutants and

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the WT plants.

Accepted Article 1

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To test whether these T-DNA insertions were responsible for the mutant phenotypes, twenty 11

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T2 plants were assayed for co-segregation between the lateral root phenotype and the three

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respective T-DNA insertions by PCR, using the primers listed in Figure 1A (Figs. 1 G-I). All

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of the plants homozygous for T-DNA insertions had fewer lateral roots. Heterozygous or

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homozygous WT plants showed no difference in the number of lateral roots as compared with

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the WT, indicating that the T-DNA insertion co-segregated with the lateral root phenotype in

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the T2 generation. The co-segregation result showed that the osaux1 mutant phenotype was

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caused by the T-DNA insertions.

Accepted Article

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To further verify that the lateral root phenotype resulted from the loss of OsAUX1 gene

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function, we constructed an RNA interfering vector harboring a 252 bp cDNA fragment from

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the 3′ region of OsAUX1, and generated transgenic plants (Figs. 2 A-C). Expression analysis

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of the transgenic plants identified ten lines with clearly reduced OsAUX1 mRNA levels (Fig.

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2B). Two of these RNA interference (RNAi) lines (Ri-2 and Ri-5) were selected for

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subsequent analysis. The 7-day-old RNAi seedlings had a reduced number of lateral roots,

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which was a similar phenotype to the osaux1 mutants (Figs. 2 C-F). Additionally,

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retransformation of the osaux1-1 line with a G418 selection-based binary vector harboring a

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8.6 kb genomic DNA fragment of the OsAUX1 locus phenocopied WT plants, while all of the

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transgene-negative plants showed the osaux1-1 phenotype (Supporting Information Fig. S3).

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These results indicated that OsAUX1 was required for lateral root development in rice.

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Overexpression of OsAUX1 induces the ectopic formation of lateral roots

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To further study the function of OsAUX1, we generated transgenic plants that overexpressed

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the OsAUX1 coding region that was under the control of the maize ubiquitin promoter (Figs. 12

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3A-C). Expression analysis of the transgenic plants identified eight lines that overexpressed

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OsAUX1 (Fig. 3B). One of these transgenic lines (OE3) was selected for further analysis. The

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primary roots of the 7-day-old OE3 seedlings had more lateral roots than the WT seedlings,

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but the shoots and primary roots of the overexpression line were shorter than those of the WT

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seedlings (Figs. 3C-G). Therefore, these results strongly indicated that OsAUX1 facilitated

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lateral root formation in rice.

Accepted Article

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The osaux1 mutant plants has a reduced number of lateral root primordia

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It has been proposed that lateral root formation can be divided into two phases: an initiation

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phase and an emergence phase (Laskowski et al. 1995; Bhalerao et al. 2002; Marchant et al.

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2002). We counted the total number of lateral root primordia in WT and mutant roots to gain

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further insight into the developmental basis for the reduced number of emerged lateral roots

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observed in the osaux1 mutants. Methylene blue stained primary roots from the 7-day-old

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seedlings showed that the number of lateral root primordia had significantly decreased in the

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osaux1-1 mutant (Figs. 4A, B). Furthermore, the transgenic seedlings with the OsAUX1 RNAi

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construct had a reduced number of lateral root primordia. However, the number of lateral root

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primordia in the OsAUX1 overexpression seedlings had significantly increased compared to

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the WT seedlings (Figs. 4C, D). Taken together, these results indicated that OsAUX1 was

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essential in controlling lateral root initiation in rice.

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OsAUX1 expression patterns

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To test whether OsAUX1 acts solely in roots, or whether it also functions in elsewhere in rice

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plants, we analyzed the expression pattern of OsAUX1 in various organs. Quantitative 13

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RT-PCR analysis, using total RNA extracted from vegetative and reproductive organs,

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revealed that OsAUX1 was expressed in a large number of organs, including young roots,

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stems, leaves and panicles. The strongest expression signals were observed in young roots and

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in 20 cm panicles (P5) (Fig. 5I). To more precisely determine the spatial and temporal

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expression patterns of OsAUX1, we cloned the 2475bp sequence upstream of the OsAUX1

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ATG start codon, constructed an OsAUX1 promoter β-glucuronidase (GUS) construct and

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transformed rice variety, Nipponbare, with this construct. We obtained fifteen positive

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transgenic lines containing this OsAUX1 promoter-GUS construct. Three of these fifteen lines

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were used for subsequent analysis. Consistent with the qRT-PCR results, we observed the

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GUS signal in various organs, including young roots, maturing stems and panicles, and the

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strongest GUS signals were found in young roots, nodes and spikelets (Figs. 5A-H). In young

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roots, the GUS signal was largely limited to the root stele and tip, the lateral root primordia

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and the lateral roots (Fig. 5A-D). The qRT-PCR and OsAUX1 promoter-GUS construct results

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indicated that OsAUX1 was broadly expressed in various tissues and organs, though the

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highest expression was found in young roots, particularly during the development of lateral

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root primordia.

Accepted Article

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To test whether OsAUX1 expression was regulated by plant hormone and/or abiotic stresses,

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we used qRT-PCR to quantify the expression of OsAUX1 transcripts in 7-day-old WT

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seedlings treated with several hormones or subjected to various abiotic stresses. These

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analyses revealed that OsAUX1 expression was significantly up-regulated by IAA, NAA, and

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6-BA, and down-regulated by GA3, drought, and salt stresses (Fig. 5J, K).

22

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IAA content and distribution are altered in the osaux1 mutant

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We harvested 7-day-old primary roots of osaux1-1 and WT seedlings and measured the

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amount of IAA by GC-SRM-MS analysis to determine whether the reduction of the number

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of lateral roots in the osaux1 mutant was accompanied by an alteration in the auxin content.

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The auxin content in the primary roots of mutant osaux1-1 was significantly reduced

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compared to the WT seedlings (Fig. 6A). DR5-GUS can reflect auxin distribution at the

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cellular level because it is the auxin-responsive reporter system (Scarpella et al. 2003). We

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observed the DR5-GUS staining signals in transgenic WT and osaux1-1 seedlings. DR5-GUS

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expression was clearly seen in wild type roots, whereas the expression was very weak in the

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osaux1-1 roots (Fig. 6B). More precisely, DR5-GUS expressions in the root tip, stele, lateral

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root primordia and lateral roots of osaux1-1 were significantly lower than in the WT plants

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(Figs. 6C-H). DR5-GUS expression was barely detectable in the root tips or stele of osaux1-1

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plants (Fig. 6D). In lateral roots, DR5-GUS expression was restricted to the stele and root

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apical meristem in osaux1-1 plants (Fig. 6H), whereas DR5-GUS expression was additionally

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detected in the cortex cell layers in WT plants (Fig. 6G). In conclusion, the lack of DR5-GUS

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expression in lateral roots and root tips indicated that the distribution of auxin had

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dramatically decreased in the osaux1-1 mutant. These results suggested that the mutation of

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OsAUX1 gene clearly affected auxin levels and distribution in lateral roots and root tips.

Accepted Article

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OsAUX1 encodes a putative auxin influx carrier

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AUX1/LAX proteins in plants belong to the auxin and amino acid permease (AAAP) family

22

(Saier et al. 2009; Zhao et al. 2012). The sequences of these proteins have been reported to be 15

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highly similar at the amino acid level (Hoyerová et al. 2008). The major differences between

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these protein sequences occur at both the amino and carboxyl termini (Parry et al. 2001b;

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Hoyerová et al. 2008). Based on multiple alignments, we generated a phylogenetic tree of

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AUX-LAX proteins from rice and Arabidopsis using a neighbor-joining distance algorithm

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(Fig. 7A). Two subclasses could be defined among the AUX-LAX proteins. AtAUX1 and

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OsAUX1 were found to be members of the same subclass (Fig. 7A). The amino acid sequence

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identity of OsAUX1 and AtAUX1 was 83% (Fig. 7B). A comparison of these two protein

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sequences showed that the N and C terminus sequences were the most divergent regions,

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while the central sequence was highly conserved. The amino acids that have been shown to be

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important for AtAUX1 activity (Swarup et al. 2004) were found to be completely conserved

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in OsAUX1 (Fig. 7C). Additionally, the OsAUX1 protein had ten transmembrane domains,

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which was similar to AtAUX1 (Supporting Information Fig. S4), and their gene structures

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were also found to be similar (Supporting Information Fig. S5). These similarities in their

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protein sequences and gene structures implied that the function of OsAUX1 during polar

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auxin transport might be similar to that of AtAUX1.

Accepted Article

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It has been reported that NAA enters into cells by passive diffusion, that 2,4-D uptake

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mostly relies on the auxin influx carriers and that IAA is imported by influx carriers or by

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passive diffusion (Delbarre et al. 1996; Marchant et al. 1999). 2-naphthoxyacetic acid

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(2-NOA) has been shown to be a specific inhibitor of the auxin influx carriers, but has

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minimal effects on auxin efflux (Imhoff et al. 2000; Parry et al. 2001a; Stieger et al. 2002).

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We applied exogenous auxin to WT and osaux1-1 seedlings to determine whether OsAUX1

22

was involved in polar auxin transport and acted as an auxin influx carrier. WT and osaux1-1 16

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seedlings were grown in the presence of NAA, 2,4-D, or IAA for 7 days (Fig. 8A), after

2

which the number of lateral roots per centimeter of primary roots were counted. The lengths

3

of the primary roots were also measured. Although the untreated osaux1-1 control seedlings

4

had 51.8% fewer lateral roots compared with the WT, the osaux1-1 mutant roots treated with

5

0.01 μM NAA produced the same number of lateral roots as the WT plants. However,

6

osaux1-1 mutant roots treated with 2,4-D or IAA had fewer lateral roots than the WT

7

plants(Fig. 8B). Additionally, exogenous NAA applied to the osaux1 plants was able to rescue

8

the gravitropic responses of the mutant roots (Supporting Information Figs. S1C, D).

9

Moreover, the WT roots treated with the auxin influx inhibitor 2-NOA formed fewer lateral

10

roots, which was similar to the osaux1-1 mutant phenotype (Figs. 8A, B). These data

11

suggested that the uptake of auxin was blocked in the osaux1-1 mutant, indicating that

12

OsAUX1 was involved in polar auxin transport and functions as a likely auxin influx carrier.

Accepted Article

1

13

A root elongation bioassay showed that NAA, 2,4-D, and IAA inhibited wild-type and

14

osaux1-1 root elongation at the concentrations used in this study (Fig. 8A). Dose-response

15

curve results showed that osaux1-1 root growth had dramatically reduced sensitivity to 2,4-D

16

as compared with the WT (Fig. 8C). The selective responses of osaux1-1 roots to the

17

synthetic auxin, 2,4-D, versus NAA confirmed that auxin uptake was impaired in the osaux1

18

mutant. Therefore, these results provided further evidence that OsAUX1 was involved in

19

polar auxin transport as a likely auxin influx transporter.

20 21

Expressions of several auxin-responsive and cell cycle-related genes are up- and down-

22

regulated in the osaux1 mutant

23

Transcriptome analysis of 7-day-old seedling roots from osaux1-1 and the WT was performed 17

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using RNA-sequencing (RNA-seq) in order to define the key genes that regulate lateral root

2

development in osaux1. The 302 and 1047 genes were significantly up-regulated and

3

down-regulated in osaux1-1, respectively, compared to the WT (Fig. 9A). Many of the

4

differentially expressed genes were involved in plant hormone signaling, the cell cycle,

5

transcriptional regulation, amino acid transport and energy metabolism (Figs. 9B, C).

6

Interestingly, the expression levels of five genes related to auxin signaling and five genes

7

involved in the regulation of the cell cycles were significantly down-regulated in osaux1-1

8

(Fig. 9B, asterisk, Supporting Information Table S3), whereas several other genes related to

9

auxin signaling and the cell cycle were up-regulated (Fig. 9C, asterisk).

Accepted Article

1

10

There is abundant evidence that auxin signaling and cell cycle regulation play important

11

roles in the control of plant lateral root initiation and development in Arabidopsis (De Smet et

12

al. 2010). By using qRT-PCR analysis, we verified that the expression levels of six genes in

13

7-day-old osaux1-1 seedling roots were indeed significantly down-regulated. Three of these

14

genes (OsTIR1, OsIAA19 and OsIAA23) were related to auxin signaling and other three genes

15

(OsCYCD4;1, OsCYCD5;2 and OsCDK2) were related to cell cycles regulation (Fig. 9D).

16

When the osaux1-1 lateral root phenotype in 7-day-old seedling roots was rescued by 0.01μM

17

NAA, the expression levels of these six genes were not significantly different between the

18

mutant and the WT plants (Fig. 9D). Therefore, we suggested that these genes could be

19

involved in lateral root formation and played important roles during lateral root initiation.

20

18

This article is protected by copyright. All rights reserved.

DISCUSSION

2

OsAUX1 controls lateral root initiation

3

Lateral roots are very important organs in monocotyledonous rice. During their formation, at

4

least four recognizable developmental stages can be distinguished: priming, initiating,

5

patterning and emergencing (Malamy 1997; Peret et al. 2009a). In Arabidopsis, AUX1 and

6

LAX3 encoded high affinity auxin influx carriers and the number of lateral roots on their

7

mutants, aux1 and lax3, fell (Marchant et al. 2002; Swarup et al. 2008). To investigate the

8

functions of auxin influx carriers in rice, the members of the AUX1/LAX subfamily and its

9

features were investigated in our previous study (Zhao et al. 2012). In this study, we

Accepted Article 1

10

identified and characterized the biological function of OsAUX1 in rice.

11

The Arabidopsis gene, AUX1, is expressed during the initiation of lateral root primordia

12

and has been proved essential for lateral root initiation (Marchant et al. 2002; Swarup et al.

13

2008). In our study, the loss-of-function mutants and the transgenic plants that

14

underexpressed OsAUX1 showed a significant decrease in the number and the lengths of their

15

lateral roots, which indicated that OsAUX1 was required for lateral roots development in rice.

16

Interestingly, in the transgenic plants that overexpressed OsAUX1, the number of lateral roots

17

significantly increased, which suggested that OsAUX1 may be able to activate lateral roots

18

formation. Changes in the number of lateral roots in the OsAUX1 mutants, the RNAi plants

19

and the overexpression plants were caused by the arrest or activation of primordia initiation.

20

These data suggested that OsAUX1 controlled lateral root primordia formation, as the

21

expression of OsAUX1 was highly detected in emerging lateral root primordia and lateral root

22

meristems (Figs. 5A-D). Our results indicated that OsAUX1 functioned in controlling the 19

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initiation of lateral roots in rice.

Accepted Article

1 2

Additionally, in OsAUXs subfamily, OsAUX1 and OsAUX2 were segmentally duplicated

3

genes, have similar expression pattern in various organs at different developmental stages,

4

highly expressed in 20-25cm panicles (Zhao et al. 2012), and their gene structures and protein

5

sequences were also similar (Fig. 7, Supporting Information Fig. S5), hinting that there might

6

be functional redundancy between OsAUX1 and OsAUX2 during panicle development in rice.

7

But, at present, there was no experiment to verify the functional redundancy of the other three

8

OsAUX3/4/5 genes.

9

10

OsAUX1 is involved in various developmental processes

11

In general, the expression profile of a gene may be correlated with its function. The tissue

12

expression pattern of OsAUX1 showed that in primary roots and lateral roots, OsAUX1 was

13

abundantly expressed in the root tips, the stele and the elongation zone (Figs. 5A, D). The

14

highest expression level was observed in the lateral root primordia of primary roots (Fig. 5B).

15

This expression pattern was similar to that of AtAUX1 in Arabidopsis. AtAUX1 was thought to

16

be involved in the control of lateral roots initiation and in gravitropism. Loss-of-function of

17

AtAUX1 resulted in insensitivity to auxin and caused a reduction in the number of lateral roots

18

(Marchant et al. 1999; Marchant et al. 2002). As with Arabidopsis, auxin resistance and

19

reduced lateral roots were also observed in the osaux1 mutant (Fig. 1), which indicated that

20

AUX1 acts in response to auxin and seems to be conserved between Arabidopsis and rice with

21

regards to lateral root development. This suggestion was supported by the close evolutionary

22

relationship between AtAUX1 and OsAUX1 (Fig. 7). 20

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OsAUX1 was also highly expressed in the stem node and spikelet (Fig. 5). The osaux1

2

mutants had shortened stems, a decreased number of tillers and delayed flowering

3

(Supporting Information Fig. S2), which indicated that OsAUX1 was involved in various

4

developmental processes in rice. This may not be the case with AtAUX1 in Arabidopsis. These

5

data could be used to support the supposition that the OsAUX1 gene might have acquired

6

more biological functions than AtAUX1 during the evolutionary.

Accepted Article

1

7

By using a reflux loop model of auxin signaling in Arabidopsis root tips, it has been

8

demonstrated that auxin signals in basal meristem primers are adjacent to pericycle cells,

9

resulting in founder cells that determine lateral root initiation (De Smet et al. 2007). Therefore,

10

based on the OsAUX1 expression pattern and the phenotype of the osaux1 mutants, we

11

suggested that the auxin response of pericycle cells in the basal meristem determined founder

12

cell identity and was dependent on OsAUX1 in rice.

13 14

OsAUX1 might encode an auxin influx carrier in rice

15

The phylogenetic analysis showed that the auxin influx carriers were encoded by a highly

16

conserved gene family, which might play a key role in polar auxin transport (Friml and Palme

17

2002; Péret et al. 2012). The auxin import activity of all four AUX1/LAX proteins in

18

Arabidopsis has been well confirmed in heterologous expression systems (Yang et al. 2006;

19

Swarup et al. 2008; Péret et al. 2012). The alignments of the amino acid sequences illustrated

20

that the OsAUX1 protein had an approximately similarity of 80% to AtAUX1/LAXs and that

21

the amino acid residues, known to be involved in auxin influx carrier activities, were

22

completely conserved, which showed that OsAUX1 was evolutionarily close to the AUX1/LAX 21

This article is protected by copyright. All rights reserved.

family (Luschnig et al. 1998; Péret, et al. 2012). Moreover, our expression studies showed

2

that the OsAUX1 expression pattern was similar to AtAUX1 (Fig. 2). These data indicated that

3

OsAUX1 could have auxin import activities similar to AUX1/LAX proteins in Arabidopsis.

Accepted Article

1

4

GUS staining of transgenic plants harboring the OsAUX1p::GUS fusion construction was

5

detected in WT root tips, lateral roots and lateral root primordia. These results overlapped

6

with GUS staining controlled by the DR5 promoter in roots. The GUS staining in the roots of

7

osaux1 harboring DR5::GUS was notably weakened compared to WT, indicating that the

8

auxin level had dramatically decreased in osaux1 roots. This was consistent with the IAA

9

content measurements (Fig. 6A, B). These results indicated that OsAUX1 was involved in

10

polar auxin transport and auxin influx into rice cells.

11

That OsAUX1 acted as an auxin uptake transporter was further verified by the similar

12

phenotypes shown by the 2-NOA-treated wild-type and OsAUX1 suppression transgenic

13

plants. 2-NOA is a well-characterized inhibitor of polar auxin transport and strongly inhibits

14

auxin influx into cells (Parry et al. 2001a; Lanková et al. 2010). In our study, 2-NOA

15

treatment of wild-type rice plants produced the phenotype in roots that were similar to that in

16

the mutant osaux1, indicating that the effect of OsAUX1 suppression on the development of

17

lateral root primordia was similar to the 2-NOA-treatment. The application of exogenous

18

NAA rescued the defective phenotype in osaux1-1 mutant plants and insensitivity to 2,4-D

19

was also observed in osaux1-1 roots (Fig. 8B, C), which suggested that auxin import into cells

20

had been suppressed in the osaux1-1 mutant. Our data strongly supported the assertion that, as

21

with AUX1 in Arabidopsis, OsAUX1 could encode a putative auxin influx carrier in rice.

22

22

This article is protected by copyright. All rights reserved.

OsAUX1 promotes lateral root formation through its effects on auxin signaling and the

2

cell cycle

3

Although there is a paucity of data on the molecular machinery responsible for generating a

4

lateral root, several observations have indicated that each of the lateral root developmental

5

phases is controlled or influenced by auxin (Benková et al. 2003; Péret et al. 2009b; De Smet

6

et al. 2010). It has been reported that various mutations and RNAi transgenic plants, which

7

display auxin transport and signaling obstruction, exhibit reduced lateral root formation. In

8

particular, the Arabidopsis slr-1/iaa14 mutant has no lateral roots because the anticlinal cell

9

divisions needed for lateral root initiation are inhibited in the protoxylem pericycle (Fukaki et

10

al. 2002; Vanneste et al. 2005). Moreover, many studies have suggested that auxin promotes

11

lateral root initiation by activating cell cycle-related genes such as cyclins and

12

cyclin-dependent kinases (CDKs). For example, CYCDs participate in lateral root initiation as

13

regulators of the G1- to -S transition. Arabidopsis cycd4;1 and cycd2;1 mutants have reduced

14

lateral root densities and low concentrations of exogenous auxin can rescue the defective

15

phenotype (Nieuwland et al. 2009; Sanz et al. 2011).

Accepted Article

1

16

Recent investigations have shown that mutations of OsIAA11, OsIAA13 and OsIAA23,

17

which are members of the AUX/IAA gene family in rice, led to less lateral roots and low

18

sensitivity to exogenous NAA (Zhu et al. 2012; Kitomi et al. 2012; Ni et al. 2011). Our

19

results showed that in the 7-day-old osaux1-1 seedling roots, the auxin signaling-related genes

20

(OsAIR1, OsIAA19 and OsIAA23) and the cell cycle-related genes (OsCYCD4;1, OsCYCD5;2

21

and OsCDK2) were significantly down-regulated compared with WT roots. Furthermore,

22

when the osaux1-1 lateral root phenotype was rescued by 0.01 μM NAA, the expression 23

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levels of these genes were not significantly different from the WT. In a phylogenetic tree of

2

the AUX/IAA gene family from the rice genome, the A clade included OsIAA11, OsIAA13,

3

OsIAA19 and OsIAA23. These four genes had high sequence similarities, and their expression

4

had been shown to be induced by auxin (Jain et al. 2006; Song et al. 2009). Moreover,

5

previous reports had strongly suggested that CYCD4;1 and CYCD2;1 in Arobidopsis affected

6

the lateral root initiation number (Nieuwland et al. 2009; Sanz et al. 2011). The close

7

evolutionary relationship between OsCYCD4;1 and CYCD4;1 and OsCYCD5;2 and

8

CYCD2;1 indicated that they might have similar functional roles during root development.

9

Our study showed that OsAUX1 might play an important role in the control of lateral root

10

initiation in rice, probably through its effects on bringing sufficient amount of auxin

11

molecules that can act on its signaling pathways and cell cycle regulation.

Accepted Article

1

12

24

This article is protected by copyright. All rights reserved.

ACKNOWLEDGMENTS

2

This research was supported by National Basic Research Program of China (2013CB126903,

3

2012CB944801) and National Natural Science Foundation of China (31370348).

Accepted Article 1

4

25

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SUPPORTING INFORMATION

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Figure S1. Gravitropism of OsAUX1 mutants.

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Figure S2. Morphology of OsAUX1 mutants in flowering date.

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Figure S3. Transgenic complementation of the osaux1-1 mutant.

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Figure S4. Putative transmembrane of OsAUX1 protein.

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Figure S5. Comparison for genes structure of AUX subfamily in rice and Arabidopsis.

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Table S1. Primers for identification of OsAUX1 mutants and transgenic lines.

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Table S2. Primers for quantitative RT-PCR.

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Table S3. Selected genes with different expression in osaux1-1 roots as identified by

Accepted Article 1

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RNA-Seq analysis.

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Figure Legends

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Figure 1. Identification of three T-DNA insertion lines in OsAUX1 gene.

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(A) Schematic representation of T-DNA insertion mutations in osaux1-1, osaux1-2 and osaux1-3 in

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OsAUX1 gene. The open arrow heads represent T-DNA insertion positions. Filled boxes, exons; open

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boxes, untranslated region; black lines, introns. The primers used for genotyping and RT-PCR are indicated

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by arrows. RB and LB indicate the T-DNA border primer in right and left, respectively.

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(B) RT-PCR detection of OsAUX1 transcripts in the seedlings of the WT (Dongjin) and mutant lines in

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homozygous states. OsAUX1 expression was detected by using the primers indicated RT-F1/RT-R1 in

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osaux1-1 in Fig 1A, as RT-F2/RT-R2 in osaux1-2 and osaux1-3. Rac1 transcripts were amplified as

Accepted Article 1

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controls.

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(C) Comparison of 7-day-old seedlings between the WT and the mutant lines. Bar, 2cm.

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(D) Comparison of lateral roots in primary roots between the WT and the mutant lines. Bar, 5mm.

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(E, F) Analysis of lateral roots density and length in primary roots between the WT and the mutant lines.

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Error bars indicate standard deviations of independent biological replicates (n = 20). Two asterisks (**, P

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OsAUX1 controls lateral root initiation in rice (Oryza sativa L.).

Polar auxin transport, mediated by influx and efflux transporters, controls many aspects of plant growth and development. The auxin influx carriers in...
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