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Planta

, Volume 237, Issue 5, pp 1227–1239 | Cite as

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 HanEmail author
Original Article

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 short-root-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 P i , or aeration. This study shows that OsFH1 plays a significant role in root-hair elongation in a growth condition-dependent manner.

Keywords

Map-based cloning Root hair Formin homology 1 Tip elongation Submergence 

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

Notes

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.

Supplementary material

425_2013_1838_MOESM1_ESM.pdf (13 kb)
Online Resource 1 Primer sets used for map-based cloning. Primer sets such as S01054, R1M30, R1M471, RM6696, and RM1988 were obtained from public databases. The other primer sets were newly designed based on public rice sequence databases
425_2013_1838_MOESM2_ESM.pdf (24 kb)
Online Resource 2 Diagram of OsFH1 protein (a) and qRT-PCR analysis of truncated OsFH1 mRNA (b). The C-terminal truncations of Osfh1-2 and Osfh1-3 following insertion of T-DNA are shown by vertical arrows above the OsFH1 protein. The expression levels of truncated Osfh1-2 and Osfh1-3 were measured by qRT-PCR using the primer set denoted by horizontal arrows. Gene expression was normalized against the level of ubiquitin cDNA in the same samples. Error Bars represent standard deviations
425_2013_1838_MOESM3_ESM.pdf (88 kb)
Online Resource 3 Root-hair phenotype (a) and FH1ΔFH2 mRNA expression levels (b) of pUBI:FH1ΔFH2 transgenic plants. Four independent transgenic lines (pUBI:FH1ΔFH2) were generated in which amino acids 1–497 of OsFH1 (shown in ‘a’ of Online Resource 2) were expressed under the control of a ubiquitin promoter. The root-hair phenotypes of these four transgenic plant lines and that of the parental line Dongjin (DJ) were imaged by stereomicroscopy (upper panels). Bar, 500 μm. The root-hair lengths of these plants were measured (lower panels). b The expression levels of FH1ΔFH2 mRNA were measured by qRT-PCR. Gene expression was normalized against the level of ubiquitin cDNA in the same samples. Error Bars represent standard deviations
425_2013_1838_MOESM4_ESM.pdf (100 kb)
Online Resource 4 OsFH1 protein sequence, domain structure, and phylogenetic analysis with selected Arabidopsis and rice formins. a Protein sequence of OsFH1 analyzed using SMART and SignalP. Signal peptide (SP), proline-rich region (PRR), transmembrane domain (TMD), formin homology 1 (FH1) domain, and formin homology 2 (FH2) domain were shaded with different colors. Proline residues in PRR and FH1 domains, which are important for domain functions, were underlined in red. b Phylogenetic analysis of OsFH1 with selected Arabidopsis and rice formins. Protein sequences such as Bni1p (yeast), AtFH1, AtFH3, AtFH5, AtFH8, OsFH1, and OsFH5 were aligned. Two highly conserved motifs, which are important for formin functions, were marked with red and green colors, respectively
425_2013_1838_MOESM5_ESM.pdf (434 kb)
Online Resource 5 Comparison of the root-hair initiation zones of wild-type and Osfh1 mutant roots. a Seminal roots of 3-day-old plants were imaged by stereomicroscopy. Arrows indicate root-hair initiation sites. Bar, 500 μm. b Higher magnification views of arrowed regions are shown. Bar, 50 μm
425_2013_1838_MOESM6_ESM.pdf (151 kb)
Online Resource 6 Growth condition-dependent root-hair phenotypes of Osfh1. Germinating seeds of three mutants and their wild-type siblings were grown in three different conditions. In the first condition, plants were cultured on 1/2 MS soaked paper towels for 3 days (3 d Air). In the second condition, plants were cultured in 1/2 MS solution for 3 days (3 d Solution). In the third condition, plants were cultured in 1/2 MS for 1.5 days (1.5 d Solution) and then cultured on 1/2 MS soaked paper towels for an additional 1.5 days (1.5 d Solution + 1.5 d Air). After 3 days cultures, root hairs around 0.5-0.8 cm from the tips of seminal roots were photographed under the microscope. Bar, 500 μm
425_2013_1838_MOESM7_ESM.pdf (131 kb)
Online Resource 7 Effects of auxin and ACC on root-hair growth of Osfh1 mutants. Root hairs of Osfh1-1 and its wild-type siblings, which were cultured in 1/2 MS for 3 days and then moved to 1/2 MS supplemented with NAA (5 or 50 μM) or ACC (50, or 100 μM) for an additional 2 days, were inspected under the stereo-microscope. Bars, 500 μm
425_2013_1838_MOESM8_ESM.pdf (122 kb)
Online Resource 8 Effects of nutrients on short-root-hair phenotypes of Osfh1 mutants. Root hairs of OsFH1 and Osfh1 plants, which were cultured in Kimura B solution (one without KH2PO4 [Kimura B (–P)], and one without Fe-EDTA [Kimura B (-Fe)]) for 5 days, were inspected under the stereo-microscope. Bar, 500 μm
425_2013_1838_MOESM9_ESM.pdf (34 kb)
Online Resource 9 Gene expression pattern of OsFH1 analyzed using the rice array database (www.ricearray.org). Gene expression pattern of OsFH1 in whole plants, callus, suspension cell, dry seeds, germinating seeds, shoots, leaves, stems, whole internodes, roots, shoot apical meristems (SAM), flag leaves, ovaries, developing anthers, mature anthers, embryo sacs, embryos, and endosperm were examined using the rice Affymetrix expression database. The expression data were converted into the graph
425_2013_1838_MOESM10_ESM.pdf (112 kb)
Online Resource 10 Effects of aeration on root-hair growth of Osfh1 mutants. Osfh1-1, Osfh1-2 and Osfh1-3 mutants (Mut) and their corresponding wild-type siblings (WT) were cultured in 1/2 MS for 5 days without (a) or with (b) continuous aeration. Root hairs were imaged by stereomicroscopy. Error Bars represent standard deviations. **P < 0.01, Student’s t-test, n = 50. Bars, 500 μm
425_2013_1838_MOESM11_ESM.pdf (20 kb)
Online Resource 11 Effects of sucrose, glucose, and mannose on the root-hair growth of Osfh1 mutants. Wild-type and mutant plants were cultured for 5 days in 1/2 MS solution media (pH 4) supplemented with 50 mM (a) or 100 mM (b) sucrose, glucose or mannitol, and root-hair lengths were measured (c and d). Error Bars represent standard deviations. *P < 0.05, **P < 0.01, Student’s t-test, n = 50. Bars, 500 μm
425_2013_1838_MOESM12_ESM.pdf (98 kb)
Online Resource 12 Short statures of Osfh1-1, Osfh1-2, and Osfh1-3 mutant plants. a Wild-type (WT) and mutant plants of 5-day-old seedlings of Osfh1-1, Osfh1-2, and Osfh1-3. b Shoot length measurements of 5-day-old WT and mutant (Mutant) plants of Osfh1-1, Osfh1-2, and Osfh1-3. c Sixty-five-day-old WT and mutant plants grown in the field. Error Bars represent standard deviations. *P < 0.05, **P < 0.01, Student’s t test, n = 50. Bar, 10 cm (PDF 98 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Jin Huang
    • 1
  • Chul Min Kim
    • 2
  • Yuan-hu Xuan
    • 1
  • Jingmiao Liu
    • 1
  • Tae Ho Kim
    • 3
  • Bo-Kyeong Kim
    • 4
  • Chang-deok Han
    • 1
    Email author
  1. 1.Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center (PMBBRC)Gyeongsang National UniversityJinjuKorea
  2. 2.Department of Plant SciencesUniversity of OxfordOxfordUK
  3. 3.Genomics Division, Department of Agricultural BiotechnologyNational Academy of Agricultural Science (NAAS)SuwonKorea
  4. 4.Rice Breeding and Cultivation Division, Department of Rice and Winter Cereal Crop (NICS)RDAIksanKorea

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