CRL6, a member of the CHD protein family, is required for crown root development in rice

Plant Physiology and Biochemistry 105 (2016) 185e194

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Research article

CRL6, a member of the CHD protein family, is required for crown root development in rice Yihua Wang a, 1, Di Wang a, 1, Ting Gan a, Linglong Liu a, Wuhua Long a, Yunlong Wang a, Mei Niu a, Xiaohui Li a, Ming Zheng a, Ling Jiang a, Jianmin Wan a, b, * a

State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 January 2016 Received in revised form 29 March 2016 Accepted 12 April 2016 Available online 13 April 2016

The root system in monocotyledonous plants is largely composed of postembryonic shoot-borne roots named crown roots, which are important for nutrients and water uptake. The molecular mechanism underlying regulation of crown root development is not fully explored. In this study, we characterized a rice (Oryza sativa) mutant defective in crown root formation, designated as crown rootless6 (crl6). Histological analysis showed that CRL6 influences crown root formation by regulating primordial initiation and development. Map-based cloning and subsequent complementation tests verified that the CRL6 gene encodes a member of the large chromodomain, helicase/ATPase, and DNA-binding domain (CHD) family protein. Realtime RT-PCR analysis showed that CRL6 was most highly expressed in the stem base region where crown roots initiated. In addition, auxin-action inhibited phenotype was observed during crl6 development. The expressions of OsIAA genes were down-regulated in crl6. Our results provide evidence that CRL6 plays an important role in crown root development in rice via auxin-related signaling pathway. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Auxin CHD family proteins Crown root primordia Oryza sativa

1. Introduction Roots are important organs for nutrient and water uptake and utilization (Bryan, 1955; Qi et al., 2012). Optimization of root architecture can avoid yield limitations caused by either water or nutrient poverty (Werner et al., 2010). Studies in a number of Arabidopsis mutants with defective root development have identified molecular mechanisms involved in root development in dicotyledonous plants (Liu et al., 2005; Liu et al., 2009). As a model species for monocotyledonous plants and one of the most important crops, rice is a staple foodstuff for over one-half of the

Abbreviations: ARF, auxin response factor; Aux⁄IAA, auxin⁄indole-3-acetic acid transcriptional repressor; CHD, chromodomain, helicase/ATPase, and DNA-binding domain family protein; LR, lateral root; ORF, open reading frame; PAT, polar auxin transport; QRT-PCR, quantitative real-time PCR; RACE, rapid amplification of cDNA ends. * Corresponding author. State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. E-mail address: [email protected] (J. Wan). 1 Y.H.W and D.W contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2016.04.022 0981-9428/© 2016 Elsevier Masson SAS. All rights reserved.

world population. Unlike the tap root system typical of Arabidopsis, rice has a fibrous root system that includes numerous crown roots. A healthy field-grown rice plant has several hundred crown roots which make up a major portion of the root system (Kawata et al., 1978). Hence the crown root system is an important determinant of nutrient and water use efficiency in rice (Liu et al., 2009). Crown root formation is a special case of adventitious root formation which is highly dependent on auxin action (Bellini et al., 2014). Crown root formation is a special case of adventitious root formation which is highly dependent on auxin action. The developmental process of crown roots in rice can be divided into seven stages (Itoh et al., 2005). At the first stage, the initial primordial cells form in a few layers by one or two periclinal divisions of the innermost ground-level meristem cells, which are adjacent to the peripheral cylinder of vascular bundles in the stem. Secondly, the initial cells divide anticlinally and periclinally to form the epidermaleendodermal initial, central cylinder initial and root cap initial cells. The epidermaleendodermal initials differentiate into the epidermis, and endodermal and endodermal cells begin to form cortical cells during the third and

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fourth stages. During stages 5 and 6 the fundamental organization of the root is formed with establishment of columella from the root cap initial cells, and cells in the basal region commence of cell elongation and vacuolation. Finally the cells of all tissues elongate concurrently with emergence of the crown roots (Itoh et al., 2005). The architecture of the root system is regulated by phytohormones (Gao et al., 2014) among which auxin is a major factor. Many genes essential for crown root development in rice are involved in the auxin-signaling pathway. ADVENTITIOUS ROOTLESS1/CROWN ROOTLESS1 (ARL1/CRL1) encodes an ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) transcriptional factor which is a positive regulator for crown and lateral root formation. Its expression is induced by exogenous auxin treatment. The CRL1 promoter contains two putative auxin response elements (AuxREs). The proximal one specifically interacts with a rice auxin response factor (ARF). The arl1/crl1 mutant exhibits a few crown roots (Inukai et al., 2005; Liu et al., 2005). CRL4/OsGNOM1 plays an important role in crown root emergence by its influence on the polar localization of the auxin efflux carrier PIN1 (Liu et al., 2009; Xu et al., 2005). CROWN ROOTLESS5 functions in different genetic pathways for crown root initiation. Its expression can also be induced by exogenous auxin treatment and may be a direct target of an ARF (Kitomi et al., 2011). Furthermore, OsCAND1 is required for correct auxin signaling and is essential for G2/M cell cycle transition in crown root primordia (Wang et al., 2011). These various studies show that the auxin-signaling pathway is essential for crown root development in rice.

CHD (chromodomain helicase DNA-binding) family proteins are important regulators of transcription and play critical roles in developmental processes. This family is defined by two tandem chromodomains located in the N-terminal region. One SNF2-like ATPase/Helicase domain is located in the central region, and some members still have a DNA-binding domain located in the Cterminal region of the protein (Delmas et al., 1993; Hall and Georgel, 2007; Marfella and Imbalzano, 2007; Woodage et al., 1997). Several CHD members have been identified in a variety of higher eukaryotic organisms. For example, Mi-2, a member of the CHD family, was shown to be a central component of human nucleosome remodeling and the histone deacetylase complex NuRD (Nilasena et al., 1995; Xue et al., 1998). In Arabidopsis, PICKLE (PKL) encodes a putative ATP-dependent chromatin remodeler that is a member of the CHD subfamily. Mutation of PKL leads to phenotypes such as pickle roots and affects several different hormone signaling pathways including GA, IAA, and ABA (Aichinger et al., 2009; Aichinger et al., 2011; Dean et al., 2003; Fukaki et al., 2006; Furuta et al., 2011; Henderson et al., 2004; Li et al., 2005; Ogas et al., 1997; Ogas et al., 1999). In the Arabidopsis gain-of-function slr-1 mutant, stabilized mutant SOLITARY-ROOT (SLR)/IAA14 (mIAA14) protein inactivates ARF7/19 functions, thereby completely blocking lateral root (LR) initiation. The ssl2 (suppressor of slr2)/pkl mutant specifically restores LR formation in the slr-1 mutant. These mutants suggest that PKL/SSL2-mediated chromatin remodeling negatively regulates the auxin-induced pericycle cell divisions required for LR initiation (Fukaki et al., 2006). To study the mechanism of root formation, we previously

Fig. 1. Phenotypic characterization of the crl6 mutant. a Plant architectures of wild type (WT, left) and crl6 (right) at the booting stage. Bar ¼ 20 cm b The flag leaves of WT (left) and crl6 mutant (right). Bar ¼ 2 cm c Flag leaf cross sections of WT (left) and crl6 (right). Bar ¼ 100 mm d Panicles of WT (left) and crl6 (right). Bar ¼ 2 cm e Crown roots of WT (left) and crl6 (right) at 21-days-old seedlings. Seedlings were grown in a 12 h light (30  C)/12 h darkness (22  C) period. Bar ¼ 20 mm f Comparative analysis of crown root numbers during seedling development. Each datapoint is the mean ± SD, n ¼ 20. **, Significantly different at P < 0.01 (t-test).

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isolated a rice mutant, designated as crown rootless 6 (crl6), which showed defects in crown root initiation and development. Through a map-based cloning strategy, we identified the CRL6 gene product as a CHD family protein, recently reported to function in early chloroplast development and methylation of histone H3 lysines 4 and 27 (Hu et al., 2012; Zhao et al., 2012). However, the mechanism underlying CRL6 in crown root development still needs to be addressed. Our results provide new evidence supporting roles of CHD family proteins in crown root development in rice. 2. Materials and methods 2.1. Plant materials and growth conditions The crl6 mutant was selected as a g-irradiation-induced mutant of indica rice cultivar 9311. All plants were grown in paddy fields during normal growing seasons, or in a greenhouse with a 12 h lights (30  C)/12 h darkness (22  C) photoperiod at Nanjing Agricultural University. For root phenotypic and crown root characterization, hydroponically cultured plants were grown in Yoshida nutrient solution (Yoshida et al., 1976), in a growth chamber with a 12 h light (30  C)/12 h darkness (22  C) photoperiod at approximately 70% humidity.

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described in a previous report (Liu et al., 2005). Stem bases of 7and 14-day-old seedlings were fixed in FAA solution at 4  C for at least 48 h, then dehydrated and embedded in paraffin. Sections (10 mm thickness) were cut with a microtome and stained with safranin-fast green to observe crown root primodia (Wang et al., 2011). Images were obtained with a light microscope. 2.4. Plasmid construction and plant transformation Rapid amplification of cDNA ends (RACE) was performed using a SMARTer™ RACE cDNA Amplification Kit (Clontech, Cat. No. 634923). Due to difficulties in performing transformation in cv. 9311, crl6 was backcrossed three times to cv. Nipponbare to develop a new crl6 mutant with a japonica background. For complementation of the crl6 mutant, a 6780 bp ORF was PCR-amplified from WT plants and cloned into pCUbi1390 using a ClonExpressTM II One

2.2. Gene cloning and phylogenetic analysis To map CRL6, an F2 population derived from a cross was made between crl6 mutant and 02428 (a wide compatibility japonica line). Five hundred F2 individuals with the recessive phenotype were selected for preliminary mapping. Subsequently, 3000 recessive segregants were obtained from F2:3 families for fine mapping. New molecular markers were developed according to sequence differences between japonica cv. Nipponbare and indica cv. 9311. Complementary DNA (cDNA) sequences of candidate genes were amplified from both WT and the crl6 mutant using gene-specific primers (Supplementary information, Table S1) and cloned into the T-vector (TaKaRa, Code No. 6011) for sequencing. A BLAST search of CRL6 homologues was performed against the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment of CRL6 homologues was performed using ClustalX (http://www. clustal.org). A phylogenic tree was constructed using MEGA 5.05 based on the neighbor-joining method. Bootstrap analysis was performed with 1000 trials. 2.3. Histological observations Rice tissue sections were prepared following procedures

Table 1 Mean measures and standard deviations of agronomic traits of wild type (WT) and crl6 mutant plants grown in the field. Trait

a

Plant height(cm) Number of tillers Number of panicles Length of flag leaf (cm) Length of panicle (cm) Grains per panicle Number of crown rootsb a

Wild type 105.0 7.8 6.6 25.6 22.5 94.3 9.5

± ± ± ± ± ± ±

2.8 7.8 0.9 4.4 0.4 3.0 1.9

crl6 73.9 4.4 3.5 20.5 17.8 64.7 4.8

± ± ± ± ± ± ±

9.8c 1.6c 1.3c 5.5c 0.5c 6.0c 1.0c

20 independent plants were measured. Number of crown roots were measured in 2-week-old seedlings. Seedlings were grown a 12 h light (30  C)/12 h darkness (22  C) photoperiod at approximately 70% humidity in rice culture solution. c Significantly different at P < 0.01 (t-test). b

Fig. 2. Morphological and histological analysis of crown root primordial initiation in crl6. a and b Crown roots of wild type (WT, left) and crl6 (right) at 7- (a) and 14-dayold (b) seedlings. Bars ¼ 5 cm c-f Cross sections of WT (c and d) and crl6 (e and f) stem bases of seedlings at 7- (c, e) and 14-day-old (d, f) seedlings. Bars ¼ 200 mm.

Table 2 Number of crown root primordia in wild type and crl6.

Wild type crl6

7-day-old

14-day-old

3.88 ± 0.23 1.88 ± 0.35a

4.38 ± 0.32 1.88 ± 0.23a

The stem base samples of 8 WT and 8 crl6 plants were measured each sample quantify at least 10 cross sections. a Significantly different at P < 0.01 (t-test).

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Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China). The resulting plasmid was transformed into the new crl6 line as previously described (Hiei et al., 1994). Primers used in plasmid construction are listed in Table S1.

expression levels were calculated by the 2 eDDCt method (Wang et al., 2011). Primers used in quantitative RT-PCR of OsIAAs were according to Song et al. (2009a). Other primers used in quantitative RT-PCR are listed in Table S1.

2.5. Quantitative RTePCR analysis

3. Results

Total RNA was extracted from different tissues using an RNAprep pure Plant Kit (TIANGEN, Beijing, China). Quantitative RT-PCR was performed with Fast SYBR®Green Master Mix (Applied Biosystems, Foster, CA, USA) on an Applied Biosystems 7500 Real-Time PCR System following the manufacturer's instructions. Amplifications were performed at 95  C for 15 s, 60  C for 60 s. Three biological repetitions, each with three technical replicates, were performed. Rice ubiquitin (UBQ) was used as an internal control. Relative

3.1. Phenotypic characterization of the crl6 mutant The phenotype of the crl6 mutant is shown in Fig. 1. From the seedling stage, leaves of crl6 showed a very light green phenotype similar to an Oschr4 mutant described by (Zhao et al., 2012) (Fig. 1b). However, there was no significant difference in the leaf anatomical structure in 14-day-old seedlings between WT and the crl6 mutant (Fig. 1c). Adult crl6 plants exhibited a series of

Fig. 3. Crown root primordia development. a-g Developmental processes of crown root primordia in wild type (WT) as described by Itoh et al. (2005). g, enlargement of the region within the white box in f. s, stele (central cylinder); co, cortex; col, columella; Bars ¼ 100 mm (a-f); 20 mm (g). h-n Development of crown root primordia in crl6. n, enlargement of the region within the white box in m. Bars ¼ 100 mm (h - m); 20 mm (n).

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morphological differences from WT, including reduced plant height, shorter and narrower flag leaves, smaller panicle size (Fig. 1a, b, d), and lower seed setting rate (Table 1). Thus CRL6 seems to be important for plant development. Notably, the crl6 mutant has less crown root development compared to WT. Crown root number on 14-day-old seedlings of crl6 was about 50% of wild type (4.8 ± 1.0 versus 9.5 ± 1.9) (Fig. 1 e, f). Therefore, CRL6 shows a crucial role in controlling crown root formation. 3.2. CRL6 influences crown root primordium initiation and development The reduction of crown root number in the crl6 mutant became obvious on 3-day-old seedlings (Fig. 1 f). Cross sections stained with safranin-fast green an indicator of cell division (Bryan, 1955), revealed that the crl6 mutant formed less crown root primordia than WT, indicating impaired initiation of root primordia (Fig. 2 and Table 2). However, once formed, most of the primordia developed into mature crown roots, but with deformed cell structures (Fig. 3 a-f and h-m). WT primordia had clear stele (central cylinder), cortex, and root cap structures at stage 3 of crown root development and these fundamental organizations formed visible borders after stage 4 (Fig. 3 d, e, f). However, the borders were indistinguishable at the corresponding stage in the crl6 mutant (Fig. 3 k, l, and m). In contrast to most of the WT primordial cells that were quadrilateral in appearance and formed visible cell layers, the mutant had irregular polygon shaped cells and lacked the normal arrangement (Fig. 3 g and n). To further analyze the molecular mechanism of CRL6 in crown root formation, stem bases of WT and crl6 were sampled for quantitative RT-PCR. Five genes affecting the initiation of crown root primordia were assayed, including ARL1/CRL1 (Inukai et al., 2005; Liu et al., 2005), OsPIN1 (Xu et al., 2005), OsGNOM1/CRL4 (Kitomi et al., 2008; Liu et al., 2009), WOX11 (Zhao et al., 2009) and CRL5 (Kitomi et al., 2011), as well as gene OsCAND1 that influences crown root primordium development (Wang et al., 2011). There was significant down-regulation of all six genes in the mutant compared to WT (Fig. 4), consistent with the histological results. These results indicated that CRL6 influences crown root formation through control of both initiation and development of the primordia.

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3.3. CRL6 encodes a CHD family member protein To genetically map the mutation, an F2 population derived from a cross of mutant crl6 and rice line 02428 was examined. Five hundred F2 individuals showing the mutant phenotypes were selected for linkage analysis and primary mapping. The CRL6 locus was initially mapped on the long arm of chromosome 7 between markers M3 and M6. The subsequent analysis of 3000 mutant individuals selected from F2:3 lines restricted CRL6 between markers M23 and M28 in a region of 35 kb on PAC clone P0005E02 (Fig. 5 a). This region contained 6 predicted open reading frames (ORFs). Sequencing analysis only revealed a “T” deletion in ORF5 (LOC_Os07g31450) that encodes a putative chromatin remodeling factor. Through RACE we obtained the 6780 bp full length ORF (Fig. 5 b). The “T” deletion resulted in a frameshift and premature termination of protein synthesis from 2259aa to 1682aa. The encoded protein of ORF5, which consists of a PHD zinc finger domain, two tandem chromodomains, and an SNF2-like ATPase/helicase domain, showed high amino acid similarity with other CHD family members, especially in the Helicase C domain (Supplemental Fig. S1). To determine whether functional loss of ORF5 was responsible for the mutant phenotype, the 6780 bp WT full length ORF was cloned into the pCUbi1390 vector under the control of an Ubiquitin promoter. Following Agrobacterium tumefaciens-mediated transformation, twelve positive independent transgenic lines developed from the crl6 mutant were obtained and all the lines regained WT phenotype (Fig. 5 c, d, e and f), confirming that the mutant phenotype was caused by functional disruption of the ORF5 gene. Phylogenetic analysis using the full length amino acid sequence showed that CHD family members can be separated into three groups. CLR6 is a member of subfamily 2, which is evolutionarily close to CHR4 (At5g44800, PKR1) (Fig. 6a). Quantitative RT-PCR revealed that CRL6 is expressed in various tissues, including seedlings, flag leaves, panicles, roots, leaf sheaths and stem bases (Fig. 6b), but the highest expression level of CRL6 was in the stem base region, which is supposed to be the major growth area for crown roots (Itoh et al., 2005).

Fig. 4. Expression levels of crown root development-related genes qRT-PCR analysis of six crown root development-related genes in stem bases of 7-day-old seedlings of wild type (WT) and crl6. Bars are mean ± SD (n ¼ 3).

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Fig. 5. Map-based cloning of CRL6. a Mapping of the CRL6 gene between markers M28 and M23 in PAC clone P0005E02. The numbers of recombinants are indicated below the map. Six ORFs were predicted in the mapped region. b Gene structure of CRL6 (LOC_Os07g31450). The mutation site is marked by an arrow. White boxes represent untranslated regions (UTRs) and black ones represent exons. Lines represent introns. c, d, e and f Genetic complementation of crl6 mutant. Wild-type (WT, left), crl6 mutant (middle), and representative transgenic plant(s) (right) are shown. c, mature stage, Bar ¼ 20 cm; d, 5-day-old seedlings, Bar ¼ 20 mm; f, flag leaves of mature plants, Bar ¼ 2 cm e, statistical analysis of crown root numbers of 7-day-old seedlings (C1eC6 are six independent transgenic lines developed from the crl6 mutant). Each datapoint is the average of 20 plants. Bars are means ± SD.

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Fig. 6. Phylogenetic analysis and expression patterns of CRL6. a Phylogenetic tree of CRL6 and homologous proteins from different organisms. The proteins cluster into 3 subfamilies. Os, Oryza sativa; At, Arabidopsis thaliana; h, Homo sapiens; m, Mus musculus; d, Drosophila melanogaster; Sc, Saccharomyces cerevisiae. ScCHD1 (NP_011091.1); dCHD1 (NP_001259975.1); mCHD1 (NP_031716.2); hCHD1 (NP_001261.2); mCHD2 (NP_001074814.2); hCHD2 (NP_001262.3); dCHD3 (NP_649154.2); hCHD3 (NP_001005271.2); dCHD4 (NP_001014591.1); mCHD4 (NP_666091.1); hCHD4 (NP_001264.2); mCHD5 (NP_001074845.1); hCHD5 (NP_056372.1); mCHD6 (NP_775544.2); hCHD6 (NP_115597.3); mCHD7 (NP_001264078.1); hCHD7 (NP_060250.2); mCHD8 (NP_963999.2); hCHD8 (NP_001164100.1); mCHD9 (NP_796198.1); hCHD9 (NP_079410.4); CRL6 (gbjEEC82090.1); CHR6 (NP_565587.1); CHR4 (NP_199293.3); CHR7 (NP_565587.1). b The expression levels of CRL6 in various organs of wild type plant. Bars are mean ± SD (n ¼ 3).

3.4. CRL6 may influence crown root formation through the IAAsignaling pathway Beside effects on plant architecture and crown root formation, auxin-action inhibited phenotype was observed during crl6 development according to previous studies (Song et al., 2009b; Zhao et al., 2010). We examined the root gravitropic response in crl6 by measuring the curvature after gravistimulation at 90 to the vertical (Fig. 7b). Wild-type roots responded sharply to the change in the gravity vector, whereas the response of crl6 roots was impaired (Fig. 7a). The root tip angles of wild-type and crl6 roots were compared. Approximately 80% of wild type roots had root tip angles of 61e80 , and no plants had an angle of <40 ; by contrast, only about 10% of crl6 plant roots had angles of >60 .

During the complementation analysis we also observed that callus induction from crl6 seeds was much more difficult to achieve. Compared to WT, crl6 showed significantly reduced callus induction rate in both indica and japonica backgrounds (Fig. 7c, d; Fig. S2). The crl6 mutant also showed significantly larger flag leaf angles than wild type during the booting stage. The average angles of the flag leaves of crl6 plants were >50 , whereas those for WT were <5 (Fig. 7 e, g, h and f). To test whether CRL6 influences the IAA-signaling pathway, 7day-old seedlings were sampled to perform quantitative RT-PCR analyses with 31 IAA/UAA-related genes (Song et al., 2009a). The results revealed that most IAA genes showed decreased expression compared to WT (Fig. 8). Therefore, CRL6 may regulate crown root formation through the IAA-signaling pathway.

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Fig. 7. Auxin-action inhibited phenotype during crl6 development. a Root gravitropic responses of wild type (WT) and crl6. Root tip angles (Q) in (b) were measured 24 h after horizontal reorientation. c Calli of WT (left) and crl6 (right) grown on N6 media 30 days after induction (japonica background). Bar ¼ 2 cm d Calli induction rates of WT and crl6 in both japonica and indica backgrounds. Values are means ± SD from three independent experiments. **, significantly different at P < 0.01 (t-test). e The leaf angles of flag leaves of WT (left) and crl6 (right) at the booting stage. Bar ¼ 10 cm g and h are enlargements picture of the region within the white and red boxes in e, respectively. Bar ¼ 1 cm f Leaf angles of flag leaves of WT and crl6 at the booting stage. Each datapoint is an average of 20 plants. Bars are means ± SD. **, significantly different at P < 0.01 (t-test).

4. Discussion Several rice mutants with impaired crown root formation have been documented, including arl1/crl1, crl4/gnom1, wox11 and crl5 (Kitomi et al., 2008; Kitomi et al., 2011; Inukai et al., 2005; Liu et al., 2005; Liu et al., 2009; Zhao et al., 2009). However, the involvement of CHD family protein in crown root formation has not been reported yet. In this study, we isolated a mutant with defective crown roots. Through histological studies of cell structure we observed that CRL6 was essential for crown root primordia initiation and development. Map-based cloning and gene complementation test confirmed that CRL6 encodes a CHD family protein. Our study clearly indicated that the CHD family protein is essential for crown root development.

Phylogenetic analysis showed that Arabidopsis PKL is an ortholog of rice CRL6 (Fig. 6a). In Arabidopsis, the pkl mutant was dwarf and defective in root development (Aichinger et al., 2011; Fukaki et al., 2006; Ogas et al., 1997), which is very similar to the rice crl6 mutant, suggesting that this CHD family protein may have similar functions in plant development of both monocotyledons and dicotyledons. In Arabidopsis, PKL mutant lead to aberrant differentiation state in primary root meristem (Ogas et al., 1997). Later, Eshed reported that GYMNOS/PKL, controls primordia formation at the margins of the carpels (Eshed et al., 1999). Both studies suggest that PKL plays an important role in cell differentiation. In crl6 mutant, histological sections of the stem base indicated that primordia numbers were significantly reduced relative to WT (Fig. 2 and Table 2). Consistent

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Fig. 8. Expression of genes in the IAA-signaling pathway in WT and crl6. qRT-PCR analysis of 31 OsIAA genes in 7-day-old seedlings of wild type (WT) and crl6. Bars mean ± SD from three independent experiments.

with this result, the expressions of several genes that control crown root primordia initiation were repressed (Fig. 4a) (Inukai et al., 2005; Kitomi et al., 2011; Liu et al., 2005; Liu et al., 2009; Xu et al., 2005; Zhao et al., 2009). Meanwhile, histological studies of the primordia region showed the mutant lacked visible cellular organization as seen in WT (Fig. 3). Hence CRL6 influences cell differentiation in the rice crown root primordia initiation and development progress. Among these crown root development related genes, CRL1 and CRL5 are direct targets of ARF genes (Inukai et al., 2005; Kitomi et al., 2011). OsPIN1 and OsGNOM1 directly influence polar auxin transport (Liu et al., 2009; Xu et al., 2005). These genes all significantly down-regulated, suggesting that CRL6 may influences crown root development through the auxin-signaling pathway. Consistent with our hypothesis, auxin-action inhibited behavior was observed during crl6 development, such as increased flag leaf angle at the booting stage, low rate of callus induction, and aberrant gravitropic response (Fig. 7) (Vanneste and Friml, 2009). Degradation of auxin/ indole-3-acetic acid (Aux/IAA) transcriptional repressors is necessary for auxin response (Worley et al., 2000). In Petunia hybrid, the expression of Aux/IAA was strongly regulated during adventitious root formation, suggesting important control functions during the different phases (Druege et al., 2014). Disturbed expression of the auxin-related gene (OsIAA1) exhibits multiple morphological alterations including stunted growth, small panicle size, and low setting rate (Song et al., 2009b), which is similar to the crl6 mutant. Consistently with above result, most of the 31 IAA/UAA genes were down-regulated in crl6 mutant (Fig. 8a). In knock-down mutant of OsIAA6, the PIN1 expression was repressed (Jung et al., 2015). Moreover, the expression level and/or localization of PIN proteins also affect crown root formation. CRL4/OsGNOM1 plays an important role in crown root emergence by its influence on the polar localization of the auxin efflux carrier PIN1 (Liu et al., 2009). The impaired PIN1 localization in crl4 resulted in defective crown root formation and impaired root gravitropism (Kitomi et al., 2008). In arabidopsis, PIN protein is essential for auxin response (Bender et al., 2013). Moreover, transient disruption of polar auxin transport (PAT) results in ectopic auxin responses (Larsson et al., 2014). In this study, the expression of OsPIN1 was greatly down-regulated in the stem base of 7-days-old seedlings (Fig. 4). Thus, the auxin transport and auxin response may be affected in crl6. These results confirmed that mutation of CRL6 influences the crown root development through IAA-signal pathway. Except for defect in crown root formation, the crl6 mutant exhibited a series of morphological differences from WT, including reduced plant height, shorter and narrower flag leaves, smaller panicle size (Fig. 1a, b, d), and lower seed setting rate (Table 1). Previous study showed that CHR729/CRL6 regulates plant development through recognizing and modulating H3K4 and H3K27 methylation of repressed or tissue-specific genes. Meanwhile it was reported that the chr729 mutation preferentially affected the

expression of transcription factor genes, suggesting that CHR729/ CRL6 may be a higher hierarchical regulator of transcriptional cascades in plant developmental regulation (Hu et al., 2012). Here we demonstrated that the mutation of CRL6 affected crown root formation through IAA-signaling pathway. Therefore, we speculate that CRL6 have pleiotropic effects on plant development and CRL6 may be a general factor controlling plant development. 5. Conclusion This study identified that CRL6 controls crown root primordial initiation and development through IAA-signal pathway. Except for crown root development, crl6 could have pleiotropic development defects. These results indicate CRL6 is essential for plant development, especially crown root development. Conflict of interest The authors declare that they have no conflicts of interest. Author contribution Yihua Wang, Di Wang, Ling Jiang and Jianmin Wan conceived and designed the experiments. Yihua Wang, Di Wang, Ting Gan, Wuhua Long, Yunlong Wang, Mei Niu, Xiaohui Li and Ming Zheng: performed the experiments. Yihua Wang, Di Wang, Ting Gan and Linglong Liu wrote the paper. Acknowledgments This work was supported by grants from the National Science and Technology Support Program (2011BAD35B02-02), Jiangsu Science and Technology Support Project (grants BE2012303 and BK2010016), and Jiangsu Province Self- Innovation Program (CX(12)1003). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2016.04.022. References Aichinger, E., Villar, C.B., Di Mambro, R., Sabatini, S., Kohler, C., 2011. The CHD3 chromatin remodeler PICKLE and polycomb group proteins antagonistically regulate meristem activity in the arabidopsis root. Plant Cell 23, 1047e1060. Aichinger, E., Villar, C.B., Farrona, S., Reyes, J.C., Hennig, L., Kohler, C., 2009. CHD3 proteins and polycomb group proteins antagonistically determine cell identity in Arabidopsis. Plos Genet. 5, e1000605. Bellini, C., Pacurar, D.I., Perrone, I., 2014. Adventitious roots and lateral roots: similarities and differences. Annu. Rev. Plant Biol. 65, 639e666. Bender, R.L., Fekete, M.L., Klinkenberg, P.M., Hampton, M., Bauer, B., Malecha, M., Lindgren, K.A, Maki, J., Perera, M.A.D.N., Nikolau, B.J., Carter, C.J., 2013. PIN6 is required for nectary auxin response and short stamen development. Plant J. 74,

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