Planta (2013) 237:1227–1239 DOI 10.1007/s00425-013-1838-8
ORIGINAL ARTICLE
Formin homology 1 (OsFH1) regulates root-hair elongation in rice (Oryza sativa) Jin Huang • Chul Min Kim • Yuan-hu Xuan • Jingmiao Liu • Tae Ho Kim • Bo-Kyeong Kim Chang-deok Han
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Received: 23 September 2012 / Accepted: 2 January 2013 / Published online: 22 January 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract The outgrowth of root hairs from the epidermal cell layer is regulated by a strict genetic regulatory system and external growth conditions. Rice plants cultivated in water-logged paddy land are exposed to a soil ecology that differs from the environment surrounding upland plants, such as Arabidopsis and maize. To identify genes that play important roles in root-hair growth, a forward genetics approach was used to screen for shortroot-hair mutants. A short-root-hair mutant was identified, and the gene was isolated using map-based cloning and
sequencing. The mutant harbored a point mutation at a splicing acceptor site, which led to truncation of OsFH1 (rice formin homology 1). Subsequent analysis of two additional T-DNA mutants verified that OsFH1 is important for root-hair elongation. Further studies revealed that the action of OsFH1 on root-hair growth is dependent on growth conditions. The mutant Osfh1 exhibited root-hair defects when roots were grown submerged in solution, and mutant roots produced normal root hairs in the air. However, root-hair phenotypes of mutants were not influenced by the external supply of hormones or carbohydrates, a deficiency of nutrients, such as Fe or Pi, or aeration. This study shows that OsFH1 plays a significant role in root-hair elongation in a growth condition-dependent manner.
Electronic supplementary material The online version of this article (doi:10.1007/s00425-013-1838-8) contains supplementary material, which is available to authorized users.
Keywords Map-based cloning Root hair Formin homology 1 Tip elongation Submergence
J. Huang Y. Xuan J. Liu C. Han (&) Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Gyeongsang National University, Jinju 660-701, Korea e-mail:
[email protected]
Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid ACC 1-Aminocyclopropane-1-carboxylic acid Ac/Ds Activator/dissociation Bnip BUD NECK INVOLVED PROTEIN CPC CAPRICE Cryo-SEM Cryo-scanning electron microscope FH1 Formin homology 1 GL2 GLABRA 2 LRR Leucine-rich repeat NAA Naphthalene-1-acetic acid PAC P1-based artificial chromosome PI Propidium iodine PRR Proline-rich region qRT-PCR Quantitative reverse transcriptase PCR sGFP Synthetic green fluorescent protein
C. M. Kim Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK T. H. Kim Genomics Division, Department of Agricultural Biotechnology, National Academy of Agricultural Science (NAAS), RDA, Suwon, Korea B.-K. Kim Rice Breeding and Cultivation Division, Department of Rice and Winter Cereal Crop (NICS), RDA, Iksan, Korea
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Introduction Rice (Oryza sativa) is one of the most important crops in the world because it provides food resources for more than half of the world’s population. Culture conditions of rice are different from those of other major grain crops such as wheat and corn. Rice is grown in water-logged paddy fields. This indicates that rice root hairs are exposed to different physical and chemical conditions from upland plants. Genetic regulatory mechanisms that determine root-hair formation have been extensively studied in Arabidopsis. There is a central transcriptional regulatory complex whose activity and composition is differently modulated in non-root hair (N) and root-hair (H) cells by an LRR receptor-like kinase SCRAMBLED (Grebe 2012). Roothair differentiation is dependent on intercellular communication between N and H cells. The transport of CPC (CAPRICE) from N to H cells leads to the repression of GL2 (GLABRA2) that eventually triggers cells to develop into trichoblasts (Grebe 2012). In many plants, root-hair cells develop from specialized epidermal cells called trichoblasts. In Arabidopsis, trichoblasts and atrichoblasts develop in separate longitudinal files. Root hairs are generated from epidermal cells lying over two epidermal cells; whereas non-hair cells lying over a single epidermal cell do not generate root hairs (Dolan et al. 1993; Galway et al. 1994). In rice plants, root-hair cells are shorter than nonhair cells along longitudinal epidermal cell files (Kim and Dolan 2011). The differences in root-hair patterns between Arabidopsis and rice suggest that root-hair cell fate determination is regulated differently in these two plants. In addition to strict genetic control mechanisms, external conditions, such as hormones, nutrients, or physical treatments significantly influence the formation and elongation of root hairs (Cho and Cosgrove 2002; Pitts et al. 1998; Muller and Schmidt 2004). The initiation of root hairs is influenced by treatments with ethylene or ethylene synthesis inhibitors, and auxin application can modulate root-hair development (Cho and Cosgrove 2002). To elucidate the relationship between exogenous nutrients and root-hair development, extensive analyses of genetic mutants have been conducted (Masucci and Schiefelbein 1994, 1996; Cernac et al. 1997; Cho and Cosgrove 2002; Muller and Schmidt 2004). The results indicate that Fe and Pi exert significant effects on the density and length of root hairs (Muller and Schmidt 2004). The physical separation of roots from media affects root-hair formation in wildtype plants (Cho and Cosgrove 2002). In addition, the Arabidopsis rhl (root hairless) and rhd6 (root-hair defective 6) mutants form normal root hairs when roots are in contact with air (Cho and Cosgrove 2002; Muller and Schmidt 2004).
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Vesicle trafficking, cytoskeleton reorganization, and cell wall loosening and synthesis are major driving forces for root-hair elongation (for review, Libault et al. 2010). Formins are a large family of multi-domain proteins that exist in yeast, plants, and animals, and they participate in linear actin polymer assembly (Paul and Pollard 2009). Formins are identified by the highly conserved formin homology 2 (FH2) domain. Most plant formins contain a more variable formin homology 1 (FH1) domain (Cvrckova et al. 2004; Blanchoin and Staiger 2010). To date, plant formins are reported to regulate tip growth, cell division, and cell expansion (Cheung and Wu 2004; Favery et al. 2004; Deeks et al. 2005; Fitz Gerald et al. 2009; Li et al. 2010; Xue et al. 2011; Yang et al. 2011; Zhang et al. 2011). Although a few rice genes for root-hair development have been reported (Kim et al. 2007; Ding et al. 2009; Yuo 2009; ZhiMing et al. 2011), many orthologous genes involved in the regulation of rice root-hair formation have not been identified. To isolate genes that play important roles in root-hair growth, short-root-hair mutants were screened via a forward genetics approach. A short-root-hair mutant was identified and was isolated by map-based cloning and sequencing methods. This study shows that rice formin 1 (OsFH1) is important for root-hair development. The significance of OsFH1 in root-hair elongation was verified by using an additional two T-DNA insertional mutants. Further studies revealed that the Osfh1 mutant developed root-hair defects when roots were grown submerged in solutions, whereas air-borne mutant roots produced normal root hairs. However, the treatment with exogenous hormones or a deficiency of nutrients such as iron (Fe) or inorganic phosphate (Pi) could not rescue defective root-hair phenotypes of mutants. This study shows that OsFH1 plays a significant role in the elongation of root hairs in the submergence condition of rice roots.
Materials and methods Plant materials and growth conditions Oryza sativa spp. Japonica cultivar ‘Dongjin’ was used in this study. The mutant Ds24567 was isolated from a largescale screen of an Ac/Ds transposon mutant population regenerated by gene trap Ds starter lines carrying Ac via tissue culture. Ds lines were cultured in 1/2 MS, pH 5.8, 16 h light/8 h dark conditions. To grow roots in solutions, germinating seeds were placed on one layer of vinyl mesh that touched the surface of the solutions. Roots easily penetrated the mesh layer and were submerged in the solutions. For roots growing in air, germinating seeds were placed on the solid supports of phyto-agar and agarose in vertically oriented plates. When
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paper towels were used as solid supports, they were kept wet in vertically oriented plates. To examine hormone effects on root-hair growth, 1/2 MS solutions were supplemented with NAA (5 or 50 lM) or ACC (50 or 100 lM). Kimura B solution (Ma et al. 2001) was utilized to test the effects of phosphorus or iron deficiency on roothair growth. To test the effect of aeration on root-hair growth, roots were grown in 2 L of 1/2 MS solution aerated with three bubble generators (A-250, KETI, South Korea). The phenotype and length of root hairs were determined in 5-dayold plants.
inserting the ubiquitin promoter and removing the GUS fragment from the pCAMBIA1381 binary vector. For construction of UBI::OsFH1::sGFP, the 2.8 kb OsFH1 cDNA was amplified by PCR with the following primers: forward 50 -GTTAACATGCCGTCTCTCCGGCG-30 and reverse 50 -GGTACCAGGTGATGATGCTGAAG-30 . The cDNA was inserted into the pCAMBIA1381-sGFP vector that is a pCAMBIA1381 plasmid modified by removing the GUS fragment and inserting the ubiquitin promoter and as GFP fragment. Rice transformation was mediated by Agrobacterium LBA4404, as previously described (Toki et al. 2006). Transgenic plants were confirmed by PCR and southern blot analyses.
Root-hair length measurement
RNA extraction and qRT-PCR
Root hairs were observed and images were captured with an Olympus SZX12 stereo-microscope system. Images were processed with ImageJ (http://www.rsbweb.nih. gov/ij/). Root-hair length measurements were performed with ImageJ.
Whole seedlings of 5-day-old plants were used for RNA extraction with RiboEx (GeneAll) or the RNeasy Plant Mini Kit (QIAGEN), according to the manufacturer’s instructions. RQ1 DNase (Promega)-treated RNA was purified with phenol/chloroform. Then, 2 lg RNA was used as a template for first-strand synthesis with ReverTra Ace reverse transcriptase and oligodT (TOYOBO), according to the manufacturer’s instructions. Next, 20 lL of the reverse transcription products was diluted to 100, and 5 lL was used as a template for qRT-PCR. qRT-PCR was performed with the Eco real-time PCR system (Illumina). Primers for qRT-PCR were as follows: RT-F: 50 -CCCAATGATGCCACAGTTG-30 , RT-R 50 -TCAAGG TGATGATGCTGAAG-30 , RT2-F: 50 -GTTGTGTGTGGT GTCAATGG-30 and RT2-R: 50 -AAGAAGAACGCGATG GACAG-30 .
Map-based cloning Ds24567 (Japonica cultivar) was crossed with Milyang23 (Indica cultivar). F1 plants were self-crossed to produce F2 progeny. Individual F2 plants were inspected under a microscope for the phenotype of root hairs. F2 plants exhibiting short root hairs were selected for mapping Ds24567. Genomic DNA was extracted from short-roothair F2 plants. To isolate polymorphic markers, simple sequence repeats (SSR) were searched using the Rice DNA polymorphism database (http://www.shenghuan.shnu.edu. cn/Default.aspx?tabid=5641). Sequences of primer sets that were used to detect polymorphic markers are listed in supplemental Table 1. After fine mapping, the Ds24567 mutant, candidate genes between markers that were flanked on both sides of the mutant locus were cloned by PCR and sequenced. The sequencing results were compared with the rice sequence available in public databases (NCBI, http://www.ncbi.nlm.nih.gov/; Rice Genome Annotation Project, http://www.rice.plantbiology.msu.edu/). Genes with nucleotide differences were taken as candidates for further genetic analysis. Construction and transformation of UBI::OsFH1 and UBI::OsFH1::sGFP For the construction of UBI::OsFH1, the 2.8 kb cDNA of OsFH1 was amplified by RT-PCR with the following OsFH1-specific primers from RNA samples: forward 50 -GTTAACATGCCGTCTCTCCGGCG-30 and reverse 50 -GGTACCTCAAGGTGATGATGCTGAAG-30 . The OsFH1 cDNA was inserted into pUBI-1381 that was modified by
Confocal microscopy UBI::OsFH1::sGFP transgenic rice plants and tobacco (Nicotiana tabacum) leaves were used to observe the subcellular localization of the OsFH1::sGFP fusion protein. Transient expression of OsFH1::sGFP in tobacco leaves was performed by using the Agrobacterium-mediated transformation method, as described previously (Sparkes et al. 2006). The plasmolysis assay was performed by incubating tissues in 1 M mannitol for 15 min, as previously described (Hermans et al. 2011). Lateral roots of transgenic rice plants or infected tobacco leaves were inspected with an FV1000 confocal microscope (Olympus).
Results Identification of a short-root-hair mutant To identify genes regulating root-hair development in rice, Ds transposon lines (Kim et al. 2004) were screened for
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short-root-hair- or root-hairless phenotypes. These Ds lines were grown in 1/2 MS solution for 5 days after germination, and their roots were observed for root-hair phenotypes. Ds24567 was isolated from 706 Ac/Ds transposon rice lines. Ds24567 mutants exhibited short-root hairs in 1/2 MS solution (Fig. 1; Table 1). The length of root hairs was compared between mutants and wild-type plants under the microscope. Mutant root hairs were 85 % shorter than those of wild-type plants. Southern genomic DNA hybridization revealed that Ds24567 did not carry a Ds transposon that co-segregated with the root-hair phenotype (data not shown). Therefore, it was suspected that mutants might arise from a spontaneous mutation, or were induced during tissue culture to remobilize Ds (Kim et al. 2004). Map-based cloning of a root-hair mutant allele Map-based cloning was performed to isolate the mutant allele of a gene responsible for root-hair development. Ds24567 of Dongjin (Japonica cultivar) background was crossed with Milang23 (Indica cultivar). F2 plants were inspected for root-hair phenotypes, and F2 mutants were used for mapping analysis with molecular markers. Among a total of 2,591 F2 plants, 639 were classified as short-roothair mutants. The Chi square test indicated that the shortroot-hair phenotype of Ds24567 resulted from a single recessive mutation. Genomic DNA was extracted from short-root-hair F2 plants. To detect polymorphic markers, SSR were searched using the databases. The primer sets used for map-based cloning are listed in online resource 1. After fine-mapping, the Ds24567 mutant, the mutant locus was located on a P1-based artificial chromosome (PAC) clone P0674H90 on the rice chromosome 1. A 56 kb fragment between RM154.6-2 and RM154.8-8 was determined to contain the mutant locus (Fig. 2a). There were 13 ORFs within this fragment. These 13 ORFs in Ds24567 were cloned by PCR and sequenced. Sequence data were compared with data from a public database (Rice Genome Annotation Project, http://www.rice.plantbiology.msu.edu/ Fig. 1 Short-root-hair phenotype of Ds24567 mutants. a Root-hair images of wild-type (WT) and mutant (Mutant) plants. Root hairs from 5-dayold wild-type and mutant plants were observed under the stereo microscope. b Root-hair length measurements of WT and mutant (Mutant) plants of Ds24567. Error bars represent standard deviations. **P \ 0.01, Student’s t test, n = 50. Bar 500 lm
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Table 1 Chi square test for Mendelian segregation of short-root-hair phenotype of Ds24567 Wild-type plants
Mutant plants
Total
Observed
1,952
639
2,591
Expected
1,943.3
647.8
v2 test
v2 = 0.159 \ v2 (0.05)(1) = 3.841
A total of 2,591 5-day-old F2 plants from the cross of Ds24567 with Milyang23 were inspected under the stereo-microscope, and 639 plants exhibited a short-root-hair phenotype. The Mendelian segregation ratio was examined using the Chi square test
index.shtml). Genomic sequence analysis showed that only one locus (Os01g67240) encoding formin-like protein 1 (OsFH1) carried one-base change that was a one-nucleotide transition from G to A (Fig. 2b). This transition was located at the splicing acceptor site of the 30 -end of the first intron of the gene. It was likely that the mutation from G to A demolished the original splicing acceptor site and created a new acceptor site with the adjacent nucleotide G. To examine whether the base change could lead to an alteration in the splicing acceptor site of the first intron, the cDNA of Os01g67240 was cloned from the total RNAs of both wildtype and mutant 5-day-old seedlings. The cDNA was amplified by PCR with gene-specific primers. The sequencing results indicated that the single nucleotide replacement of G with A resulted in a one-base shift of the splicing acceptor site (Fig. 2b). The splicing alteration generated a frame shift of the coding region of Os01g67240 and resulted in premature termination of the protein. Ds24567 produces a truncated protein of 534 amino acid residues (Fig. 2c). Two additional Osfh1 mutant alleles showed similar root-hair phenotype defects To verify that the functional defect of Os01g67240 encoding OsFH1 leads to the short-root-hair phenotype of Ds24567, the public rice mutant database RiceGE (http:// www.signal.salk.edu/cgi-bin/RiceGE) was searched for additional mutant alleles at the locus Os01g67240. Two
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Fig. 2 Map-based cloning and analysis of a root-hair mutant allele. a The mutant locus was mapped with SSR markers and was located between 154.6 and 154.8 cM on chromosome 1. Of 13 genes in this interval, Os01g67240 (white arrow), encoding a rice formin 1 protein (OsFH1), contained one point mutation. b Wild type and mutant OsFH1 genomic and cDNA sequences around the point mutation were presented below the whole genomic structure. Exon sequences are in large characters, whereas intron sequences are shown in small
characters. The G-to-T transition created a stop codon in place of a valine codon. c Functional domains of OsFH1 are presented. Signal peptide (SP), proline-rich region (PRR), transmembrane domain (TMD), formin homology 1 (FH1), and formin homology 2 (FH2) domains are marked on the map. The numbers indicate the first and last amino acid numbers of each domain. The stop codon of the mutant allele was indicated with a white triangle
independent T-DNA insertion mutants were identified from the PFG T-DNA mutant collections of Kyeong Hee University. These mutants carried T-DNA insertions in the second intron and fourth exon, and were designated as Osfh1-2 and Osfh1-3, respectively (Fig. 3a). qRT-PCR confirmed the reduction of the level of OsFH1 mRNA in both mutants (Fig. 3d). Both mutants exhibited the same root-hair phenotypes and produced very short-root hairs (Fig. 3b, c). The root-hair phenotypes of the two T-DNA lines were almost identical to the root-hair phenotype
observed in Osfh1-1. The consistent root-hair phenotypes of these three mutant alleles demonstrate that Os01g67240 plays a role in root-hair development in rice. Partial OsFH1 RNAs that were truncated at T-DNA insertion sites were detected in the Osfh1-2 and Osfh1-3 lines (online resource 2). Since both insertion sites are located close to the junction of FH1 and FH2 domains, the possibility that the FH1 domain alone can affect root-hair growth was examined by generating transgenic rice plants overexpressing OsFH1 cDNA lacking FH2 (OsFH1DFH2).
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Fig. 3 Short-root-hair phenotypes of two T-DNA insertion OsFH1 alleles. a The locations of T-DNA insertion sites in two mutants, Osfh1-2 and Osfh1-3, were shown along with the mutation site of Osfh1-1. Arrows indicate the primer set for qRT-PCR. b Images of short-root-hair phenotypes of the two Osfh1 mutant alleles. c Root-
hair lengths compared between wild-type and mutant plants of two T-DNA alleles. Bar 500 lm. d The expression levels of Osfh1-2 and Osfh1-3 measured with qRT-PCR. Gene expression was normalized with ubiquitin. Error bars represent standard deviations. **P \ 0.01, Student’s t test, n = 50
Four independent OsFH1DFH2 transgenic lines were generated and their root-hair morphologies were examined (online resource 3). The data indicate that the FH1 domain alone does not affect root-hair growth in rice plants.
formins (Cvrckova et al. 2004). Taken together, OsFH1 is a putative type I formin that carries all the conserved amino acid residues and motifs that are required for formin biochemical activity. Osfh1-1 is predicted to produce a truncated protein, which contains SP and TM at the N-terminal and a FH1 domain at the C-terminal (Fig. 2c).
A putative type I Formin (OsFH1) encoded by Os01g67240 Os01g67240 has been annotated as a formin-like protein 1 in public databases. Further sequence analysis indicated that it belongs to the rice formin family that contains FH1 and FH2 domains. Domain scanning analyses were performed using Motif Scan (http://www.myhits.isb-sib.ch/ cgi-bin/motif_scan) and PSORT (http://www.psort.hgc.jp/). The analyses revealed that OsFH1 contains secretory signal peptide (SP) and transmembrane (TM) domains at the N-terminus, and FH1 and FH2 domains at the C-terminus (Fig. 2c and online resource 4). OsFH1 also contains a proline-rich region (PRR) between the SP and TM domains. OsFH1 contains five proline stretches in the FH1 domain, which implies a putative function to bind globular actin or the actin/profilin complex as shown for other formin proteins (Grunt et al. 2008). Phylogenetic analyses and sequence alignments revealed that OsFH1 also contains two conserved residues, Ile-1431 and Lys-1601, which have been demonstrated to be critical for BUD NECK INVOLVED PROTEIN (Bni1p) to perform actin nucleation and barbed-end capping. The FH2 domain of OsFH1 contains the core sequence/motif that is conserved among type I
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Conditional effect of Osfh1 mutants on elongation of root hairs Phenotypic observations for screening mutants and subsequent studies were made with roots grown in 1/2 MS solution. To employ cryoscanning electron microscopy (SEM) for the observation of root hairs at higher magnification, wild type and mutant plants of the three OsFH1 alleles were vertically cultured on 1/2 MS solid media containing 10 g/L phytoagar. In solutions, mutant root-hair lengths were approximately 12–15 % of those of wild-type root hairs (Fig. 4a, b). However, when roots were grown on vertically oriented phytoagar plates, mutant root hairs showed similar lengths as wild-type root hairs (Fig. 4a, b). Root hair morphologies from wild type and mutant roots grown in solution and on solid media were examined using light microscopy and cryo-SEM (Fig. 5). Root hair initiation and maturation zones were compared in 3-day-old roots (online resource 5). Root hair bulges were formed the same distance from root tips in mutant and wild-type roots. On solid media, there was no significant difference in the root hair morphology of wild-type and mutant plants.
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Fig. 4 MS-independent root-hair growth of OsFH1 and Osfh1-1 in solution and on solid supports. a Microscopic images of root hairs of OsFH1 and Osfh1-1 grown in 1/2 MS (left) and on the surface of 1/2 MS agar solid media (right). Bar 500 lm. b Comparison of root-hair lengths between wild type and mutant plants cultured in 1/2 MS (left graph) and on the surface of 1/2 MS agar solid media (right graph). Error bars represent standard deviations. **P \ 0.01, Student’s t test, n = 50. c Microscopic images of root hairs of OsFH1 and Osfh1-1
grown in dH2O (left) and on the surface of dH2O–agarose solid media (right). Bar 500 lm. d Comparison of root-hair length between wild type and mutant plants cultured in dH2O (left graph) and on the surface of dH2O agarose solid media (right graph). Error bars represent standard deviations. **P \ 0.01, Student’s t test, n = 50. The relative ratios of root-hair length were obtained by dividing roothair lengths of mutants by those of the wild type (b, d)
Mutant root hairs were shorter during growth in solutions; however, they displayed normal (although shorter) morphologies. These results implied that the Osfh1 mutation exerts the most significant effect on the elongation of root hairs during root growth in solutions. To understand the relationship between the root-hair phenotype and growth conditions, the possibility that MS media might alter the mutant phenotype was examined first. Wild type and mutant roots were grown in distilled water (dH2O) and on vertically oriented 1 % agarose melted in dH2O (Fig. 4c, d). Mutant roots produced shortroot hairs in dH2O, whereas wild-type and mutant roots developed similar lengths of root hairs on dH2O–agarose media. Therefore, MS media do not influence the root-hair phenotypes of Osfh1 mutants. The next examined possibility was that aerobic conditions (e.g., submergence or exposure to air) during root growth might influence root-hair elongation in Osfh1 mutants. Three culture conditions were compared with evaluate aerobic effects on root-hair growth of Osfh1
mutants: (1) air conditions, in which wild type and mutant seedlings were grown on paper towels soaked with 1/2 MS solution (Fig. 6a), (2) submergence conditions, in which wild type and mutant seedlings were cultured in 1/2 MS solution (Fig. 6b), and (3) alternating conditions, from submergence to air, in which wild type and mutant seedlings were cultured in 1/2 MS solution for 1.5 days and then transferred on to thin paper towels soaked with 1/2 MS solution for additional 1.5 days (Fig. 6c, d). In air conditions with roots grown on paper towels, the Osfh1 mutants generated root hairs with similar lengths to those of wild-type roots (Fig. 6 and online resource 6). Further, the use of paper towels, agar, or agarose as solid supports did not influence the phenotypes of mutant root hairs. During alternating conditions from submergence to air, after the first 1.5 day culture in 1/2 MS solution, mutants exhibited the short-root-hair phenotype. At the root-hair maturation zone of mutant roots, mutant root-hair lengths were approximately 12–15 % of those of wild-type root hairs. However, after the additional 1.5 day culture in the
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Fig. 5 Root-hair morphologies of OsFH1 and Osfh1-1. a Microscopic images of root-hair morphology of OsFH1 and Osfh1-1. Plants were cultured in 1/2 MS solution for 5 days. Mature root hairs (upper) and elongating roots (lower) were observed, respectively. Right panels are magnified images of white boxes in left panels. Bars 100 lm. b Cryo-
SEM images of root-hair morphology of OsFH1-1 and Osfh1-1. Plants were vertically cultured on the surface of 1/2 MS solid media for 5 days. Right panels are magnifications of images indicated as black boxes in left panels. Bars 100 lm
air condition, new root-hair maturation regions produced similar length of root hairs in both mutant and wild-type roots (Fig. 6d and online resource 6). Taken together, the results demonstrate that OsFH1 plays a significant role in root-hair elongation under the submergence condition. Previous reports indicated that the treatment with exogenous hormones or nutrients, such as phosphorus or iron affected root-hair development (Cho and Cosgrove 2002; Pitts et al. 1998; Muller and Schmidt 2004). To examine if auxin and ethylene could recover the root-hair defect of Osfh1 mutants, 3-day-old wild-type and mutant
plants were moved to 1/2 MS solution supplemented with various concentrations of NAA or ACC. However, there was no significant recovery of root-hair length in mutant plants (online resource 7). The possibility that phosphorus or iron deficiency could affect root-hair development of Osfh1 was examined by growing the three Osfh1 mutants in Kimura B (KB) solution, or modified KB solutions in which KH2PO4 was replaced with KCl or Fe-EDTA was omitted. Roots of 5-day-old seedlings were inspected in the root-hair maturation region (online resource 8). The results of these studies showed that neither hormone treatments
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Planta (2013) 237:1227–1239 Fig. 6 Growth conditiondependent root-hair phenotypes of Osfh1. Germinating seeds of three mutants and their wildtype siblings were grown in three different conditions. In the first condition (a), plants were cultured on 1/2 MS soaked paper towels for 3 days (Air). In the second condition (b), plants were cultured in 1/2 MS solution for 3 days (Solution). In the third condition, plants were cultured in 1/2 MS for 1.5 days (c) and then cultured on 1/2 MS soaked paper towels for an additional 1.5 days (d). In all the conditions, root-hair lengths were measured at 0.5–0.8 cm from the tips of seminal roots. For the third condition, two measurements were made; the 1st measurement was done after initial 1.5 days (c) and the 2nd one was done after additional 1.5 days (d).The data are presented in the relative ratio of mutants to wild type. The relative ratios were obtained by dividing root-hair lengths of mutants by those of the wild type. Error bars represent standard deviations. **P \ 0.01, Student’s t test, n = 50
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nor phosphorus or iron deficiency affected the root-hair phenotype of Osfh1 mutants grown in the submergence condition. Expression pattern and protein localization of OsFH1 To characterize OsFH1 in rice plants further, the expression patterns of OsFH1 were examined by qRT-PCR. Total cellular RNAs were extracted from vegetative tissues of 10-day-old seedlings cultured in 1/2 MS solution, and from reproductive tissues of heading and flowering rice plants grown in the field. For qRT-PCR, ubiquitin was used to normalize the expression data. The results showed that OsFH1 mRNA is detectable in all tissues and at all stages, but relatively higher mRNA levels accumulate in roots at the vegetative stage and immature flowers at the reproductive stage (Fig. 7a). The qRT-PCR results are consistent with the data from ricearray (http://www.ricearray.org) (online resource 9). To determine the subcellular localization of OsFH1, a T-DNA vector was constructed that expressed OsFH1 fused to sGFP at the C-terminal under the control of the ubiquitin promoter. Roots of transgenic rice plants were
inspected for GFP fluorescence. Fusion proteins were detected at the cell perimeter (Fig. 7b). To determine whether OsFH1 is a cell-wall-localized or plasma-membrane-localized protein, plasmolysis analysis was performed in lateral roots expressing OsFH1::sGFP. After treatment with 1 M sorbitol for 15 min, GFP fluorescence was clearly separated from the propidium iodine (PI)labeled cell walls (Fig. 7b). Therefore, OsFH1::sGFP-fused proteins are localized in the plasmamembrane. The same results were obtained in tobacco leaves transiently expressing OsFH1::sGFP-fused proteins (data not shown).
Discussion Formin family members are defined by the presence of a FH2 domain. There are at least 21 putative formin genes in Arabidopsis, whereas rice genomes contain 16 putative formin genes (Cvrckova et al. 2004). Extensive biochemical data have demonstrated that formins participate in actin cytoskeleton organization by nucleating actin polymerization and elongation and bundling actin filaments. Several formins have been shown to bind and bundle
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1236 Fig. 7 Expression pattern and subcellular localization of OsFH1. a Tissue-specific mRNA expression determined with qRT-PCR. Whole seedlings, shoots, shoot apices, leaf blades, leaf sheaths, and roots of 10-day-old rice plants were used for total RNA extraction. Immature flowers (booting stage) and mature flowers (heading stage) were used for total RNA extraction. RNA was reverse-transcribed into cDNA and then quantified using qRT-PCR. Gene expression levels were normalized with ubiquitin. b Subcellular localization of OsFH1. Lateral roots from 5-day-old pUBI::OsFH1::sGFP transgenic plants were examined for sGFP-fused OsFH1 protein (upper panels). Plasmolysis was performed with 1 M mannitol. Plasmolyzed lateral roots of pUBI::OsFH1::sGFP transgenic plants were observed using a confocal microscope (lower panels). Left panels were confocal microscope images obtained with GFP filter (GFP); right panels were merged images obtained with GFP and PI filters. BFPL and AFPL denote before plasmolysis and after plasmolysis, respectively. Bars 10 lm
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microtubules (Young et al. 2008; Miki et al. 2009; Deeks et al. 2010). Therefore, formins play critical roles in the organization of microfilaments and microtubules. Biological functions of formins have been extensively described in cell division, tip growth of pollen, and cell expansion. Root-hair elongation is mediated by a number of cellular events, including formation of Ca2? concentration gradients, production of reactive oxygen species (ROS), expansion of the cell wall, reorganization of the cytoskeleton, and vesicle trafficking of membrane and cell wall components (Galway et al. 1997; Wymer et al. 1997). Cytoskeletal reorganization and vesicle trafficking are considered as the
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major cellular processes that are involved in cytoplasmic streaming and tip growth (Shimmen and Yokota 2004). There are reports that the aberrant morphology of root hairs was detected in transgenic Arabidopsis plants that expressed a truncated version or over-expressed AtFH8 (Yi et al. 2005; Deeks et al. 2005). The overexpression of formins or truncated formin proteins exerted dominant negative effects on cytoskeletal organization during elongation of root hairs. However, the root-hair phenotype of the AtFH8-null mutant needs to be determined to clarify the role of AtFH8 in roothair elongation. This study reported that Osfh1 knock-out mutants showed a defect in root-hair elongation. The data
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demonstrated that OsFH1 is one of the major formin genes involved in elongation of root hairs in rice roots. This study revealed that the requirement of OsFH1 for root-hair elongation was dependent on growth conditions. There was no difference in mutant root-hair growth between MS and dH2O media. Neither supplementation of phytohormones (ACC and NAA) nor depletion of phosphorus or iron could relieve the defect in root-hair growth. The alternate growth experiment in which plants were first grown submerged in solution and then transferred to growth in air strongly suggests that aerobic growth rescues the genetic defect of OsFH1. Therefore, it is reasonable to speculate that cellular differences that are crucial for root-hair growth processes might exist between roots grown submerged in liquid versus roots grown in air. OsFH1 is most required for elongation processes when root hairs are grown under the submerged condition. Root cells are exposed to a series of metabolic and physiological stresses when grown in anoxia [for reviews, see Bailey-Serres and Voesenek (2008) and Colmer and Voesenek (2009)]. These include low availability of ATP due to limited respiration, depletion of carbohydrate stores, excessive formation of ROS, and water deficiency leading to wilting. In particular, ROS and water flux significantly influence the development and function of root hairs. Oxygen deprivation induces production of ROS and cytosolic Ca2? via a Rop signal transduction pathway (BaxterBurrell et al. 2002). Ca2? polarization and ROS production are essential cellular events for root-hair elongation. RhoGDP dissociation inhibitor (RhoGDI) regulates the growth of root-hair tips (Carol et al. 2005). Therefore, it is likely that oxygen deficiency may affect root-hair development. It has also been suggested that decreased root hydraulic conductivity is related to cytoplasmic acidification upon oxygen shortage (Holbrook and Zwieniecki 2003; Tournaire-Roux et al. 2003). Pratley and Rosene (1954) and Rosene and Bartlett (1950) reported that root hairs reduce water influx in anoxic conditions. Because mutants developed a short-root-hair phenotype in the submergence condition, this may indicate that the Osfh1 mutant is more sensitive to oxygen depletion or energy shortage than wild-type plants. To examine this possibility, mutants were cultured in an aerated solution or in solutions supplemented with sucrose, glucose, or mannitol. Continuous aeration was achieved by supplying air with an automatic air-pump. Sucrose, glucose, or mannitol were added to MS media (pH 4) at a concentration of 50 or 100 mM; in these conditions wild-type rice plants maintain normal root-hair growth (Narukawa et al. 2010). After 5 days of culture, the root-hair lengths of the mutants and their wild-type siblings were compared. These treatments did not significantly affect the growth of mutant root hairs in submergence. Supplementation of sucrose, glucose and
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mannitol increased the length of wild-type root hairs. The root-hair length of mutant and wild-type plants remained significantly different in each of the conditions. Taken together, these results indicate that neither aeration nor sugar supplementation are able to rescue the root-hair defect of Osfh1 mutant plants grown in submergence (online resources 10 and 11). Ubiquitous expression patterns of OsFH1 are expected for the involvement of various cellular activities and developmental processes. In fact, the Osfh1 mutant displayed is reduced plant stature (online resource 12). Although formins consist of a multi-gene family and can be expected to be functionally redundant, Osfh1 showed clear phenotypic expression in root hair and plant growth. This suggests that OsFH1 plays an important role in various development processes, possibly via the regulation of actin-dependent transport. Because rice is one of the most important crops in the world, OsFH1 might be an important genetic factor in determining crop yield. Acknowledgments This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ008215 and PJ008168), the Rural Development Administration, Republic of Korea. Jingmiao Liu is supported by a scholarship from the BK21 program. This research was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0009096). We are grateful to Dr. Liam Dolan (University of Oxford, UK) for helping us with cryo-SEM work.
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