RNAi knockdown of Oryza sativa root meander

Plant, Cell and Environment (2007) 30, 690–699
doi: 10.1111/j.1365-3040.2007.01663.x
RNAi knockdown of Oryza sativa root meander curling
gene led to altered root development and coiling which
were mediated by jasmonic acid signalling in rice
JIAFU JIANG1,3*, JUNHUA LI1,3*, YUNYUAN XU1, YE HAN1, YUE BAI1, GUOXIN ZHOU4, YONGGEN LOU4,
ZHIHONG XU1,2, & KANG CHONG1,2
1
Research Center for Molecular Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular
Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, 2National Centre for Plant Gene Research,
Beijing 100093, 3Graduate School of the Chinese Academy of Sciences, Beijing 100049 and 4Institute of Applied Entomology,
Zhejiang University, Hangzhou, 310029, China
ABSTRACT
Jasmonic acid (JA) is a well-known defence hormone, but
its biological function and mechanism in rice root development are less understood. Here, we describe a JA-induced
putative receptor-like protein (OsRLK, AAL87185) functioning in root development in rice. RNA in situ hybridization revealed that the gene was expressed largely in roots,
and a fusion protein showed its localization on the plasma
membrane. The primary roots in RNAi transgenic rice
plants meandered and curled more easily than wild-type
(WT) roots under JA treatment. Thus, this gene was
renamed Oryza sativa root meander curling (OsRMC).
The transgenic primary roots were shorter, the number of
adventitious roots increased and the number of lateral roots
decreased as compared to the WT. As well, the second
sheath was reduced in length. Growth of both primary roots
and second sheaths was sensitive to JA treatment. No significant change of JA level appeared in the roots between
the transgenic rice line and WT. Expression of RSOsPR10,
involved in the JA signalling pathway, was induced in transgenic rice. Western blotting revealed OsRMC induced by
JA. Our results suggest that OsRMC of the DUF26 subfamily involved in JA signal transduction mediates root
development and negatively regulates root curling in rice.
Key-words: DUF26; RSOsPR10; OsOPR.
low concentrations can induce the formation of adventitious roots (Moons et al. 1997) and lateral roots (Wang et al.
2002). However, little is known about JA’s role and its
mechanism in rice root development.
Receptor protein kinases play important functions in signalling processes regulating growth and development, such
as disease resistance, perceiving hormone signal transduction and response to internal and external cues (Torii 2000).
The receptor kinases have been grouped into 15 subfamilies
on the basis of extracellular domains (Shiu & Bleecker
2001, 2003). One of these subfamilies is the domain
unknown function 26 (DUF26) [cysteine-rich repeat
(CRR)] family. Several genes of the DUF26 family from
Arabidopsis were induced by pathogen infection, reactive
oxygen species and salicylic acid (Czernic et al. 1999; Du &
Chen 2000; Ohtake, Takahashi & Komeda 2000). These
studies revealed that at least some of the CRR protein
subfamily genes are involved in plant perception and
response to biotic and/or abiotic stress signalling, including
JA signal transduction. But less is known about the biological function of the CRR proteins.
Here, we used a reverse genetics approach to study the
functions of a JA-induced DUF26 protein in rice root development. The gene was designated Oryza sativa root
meander curling (OsRMC), because RNAi transgenic
plants showed a phenotype of root meander curling on
germination. On the basis of its response to JA, OsRMC
could be a negative regulator in JA signalling.
INTRODUCTION
Jasmonic acid (JA) plays an essential role in the plant
defence response, but is also relevant to plant growth and
development for seed germination, senescence, fruit development, root growth, pollen gestation, bulb formation and
tendril coiling (Browse 2005). Physiology assays in rice
showed that JA can reduce elongation of primary roots and
second sheaths (Yamane et al. 1981; Moons et al. 1997), and
Correspondence: K. Chong. Fax: 86 10 8 259 4821; e-mail:
chongk@ibcas.ac.cn
*Equal contributors to this work.
690
MATERIALS AND METHODS
OsRMC isolation and vector construction
Experiments of RNA extraction from young roots
in rice, RT–PCR, were as described previously (Jiang et al.
2006). The open reading frame (ORF) of OsRMC
(AAL87185) was amplified with a pair of primers:
5′-cgggatccatggcgcggtgcactttg-3′ containing a BamH I restriction site (underlined) and 5′-cggaattcctactcacgcagca
ccacc-3′ containing an EcoR I restriction site (underlined).
The products digested with BamH I and EcoR I were
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
OsRMC involved in JA signal transduction 691
inserted into the pGEX4T-1 plasmid to create an expression vector. Similarity, amplified products of ORF without a
stop codon were digested with Xba I and Kpn I and inserted
into the pBI121GFP plasmid to generate a green fluorescent protein (GFP) fusion gene expression construct for
subcellular localization study.
The specific fragment of OsRMC was amplified with a
forward primer (5′-ggggtaccactagtacacacatcgatcgctaatc-3′)
with Kpn I and Spe I restriction sites (underlined) and a
reverse primer (5′-ggggatccgagctcttattctagcttcacgcatg-3′)
with BamH I and Sac I restriction sites (underlined), then
inserted into the (PCR)-based RNAi vector pTCK303
(Wang et al. 2004).
Western blot analysis
The recombined plasmid pGEX4T-1 OsRMC was transformed into Escherichia coli BL21 (DE3) cells. The recombined protein was induced as described (Jiang et al. 2006).
Inclusion bodies of the recombined protein were washed
five times with lysis buffer [100 mM tris(hydroxymethyl)
aminomethane (Tris)–HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM NaCl, 1 mM phenylmethanesulphonyl fluoride (PMSF)] supplemented with
4 m urea and 1.0% Triton X-100. The washed inclusions
were analysed by 12% sodium dodecyl sulphate–
polyacrylamide gel electrophoresis (SDS–PAGE). The goal
proteins were cut from the gel, then ground in liquid nitrogen. Polyclonal antisera were raised by subcutaneously
inoculating New Zealand white rabbits with about 2.5 mg
recombinant proteins emulsified in an equal volume of Freund’s adjuvant. The animals were given four total booster
doses at 1 week intervals. Serum from the immunized
rabbits was harvested 10 d after the last inoculation.
Western blotting was performed as described (Han et al.
2005).
In situ hybridization
Specific fragments of OsRMC for probes (303 bp) were
amplified with a pair of primers, 5′-agtacgcggcgggggacat-3′
and 5′-ttattctagcttcacgcatggcc-3′, and inserted into T-easy
vector. A template containing the specific fragments of
OsRMC and T7 and SP6 was amplified with the primers
5′-cgccagggttttcccagtca-3′ and 5′-caacatacgagccggaagc-3′
from the combined vector. The sense and antisense probes
were synthesized with the template according to the manual
of the DIG RNA labelling kit (Roche, Indianapolis, IN,
USA). In situ hybridization was performed as described
(Ge et al. 2004).
a specific fragment (93 bp) from the cDNA sequence of
OsRMC. A master mix of sufficient cDNA and 2¥ SYBR
green reagent was prepared prior to dispersal into individual wells to reduce pipette errors and to ensure that each
reaction contained an equal amount of cDNA. Every reaction in a final volume of 10 mL contained 5 mL 2¥ SYBR
green master mix reagent (Applied Biosystems, Foster City,
CA, USA), 10 or 2.5 ng cDNA and 4 mm tubulin- and
OsRMC-specific primers. The standard thermal profile was
50 °C for 2 min, 95 °C for 10 min, 30 cycles of 95 °C for 15 s
and 60 °C for 1 min. Data were analysed using SDS 2.0
software (Applied Biosystems). PCR was performed in an
optical 96-well plate with the use of an ABI 7900 HT
Sequence Detection System (Applied Biosystems), with
SYBR green used to monitor dsDNA synthesis.
RT–PCR of expression of OsRMC and its
related genes
Total RNA for RT–PCR was extracted from various tissues
(14 days old) with the use of TRIZOL reagent (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer’s
instructions. The cDNA synthesis was performed with the
use of avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA) according to the
manufacturer’s protocol. OsRMC was amplified with a
pair of primers, 5′-acacacatcgatcgctaatc-3′ and 5′-ttattctag
cttcacgcatg-3′. The RSOsPR10 (AB127580) fragment was
amplified with the primers 5′-gatggggggtcattcaaaac-3′ and
5′-tcaggaagcagcaatacgga-3′ and the 12-oxophytodienoic
acid reductases (OsOPR) (AB040743) fragment with the
primers 5′- atcagattatcgccgttcg-3′ and 5′-cagccaccaccttgttcc3′. The PCR reaction involved the following conditions:
cDNA was denatured at 94 °C for 2 min, followed by 25 and
28 cycles of amplification (94 °C for 15 s, 57 °C for 15 s and
72 °C for 20 s) for RSOsPR10 and OsOPR, respectively,
then 72 °C for 10 min. The forward primer 5′-tcagatgccc
agtgacagga-3′ and reverse primer 5′-ttggtgatctcggcaacaga-3′
of rice tubulin were used for amplification as an internal
standard for RT–PCR.
Southern blot analysis
Genomic DNA was isolated from 10-day-old seedlings and
digested with EcoR I and Hind III respectively. The fractioned DNA was electrophoresed on 0.7% agarose gel and
blotted on a nylon membrane. GUS gene used as a probe
was labelled with a-32 P-dCTP. Hybridization was as
described (Xu et al. 2005).
Real-time quantitative PCR (Q-PCR)
The forward primer 5′-catgatctgccagtgtggagtt-3′ and
reverse primer 5′-gcccattaagccccaaacat-3′ of rice tubulin
gene were used to amplify for an internal standard. The
forward primer 5′-cggaggtgtacccgttctaca-3′ and reverse
primer 5′-atttgtgccattttattctagcttca-3′ were used to amplify
Transformation in rice and Arabidopsis thaliana
The combined pTCK303 plasmid was transformed into
Agrobacterium tumefaciens EHA105, as described (Xu et al.
2005). For the OsRMC:GFP fusion study, A. tumefaciens
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699
692 J. Jiang et al.
C58 containing the combined pBI121GFP plasmid was
used for transforming into A. thaliana as described (Zhuang
et al. 2005).
Subcellular localization analysis of OsRMC
The epidermal cells of hypocotyls of transgenic A. thaliana
harbouring 35S::OsRMC:GFP were used to visualize the
subcellular localization of OsRMC after identification by
genomic PCR (Supplementary Fig. S1). Fluorescence dye
FM4-64 (Molecular Probes, Eugene, OR, USA) was used as
a marker for plasma membrane (Bolte et al. 2004; Grandjean et al. 2004). Four-day-old seedlings germinated in
Murashige and Skoog (MS) medium were incubated with
FM4-64 at a final concentration of 5 mg ml-1 for 10 min; for
plasmolysis, treatment with 0.8 m mannitol followed for
20 min. GFP and FM4-64 fluorescence were excited at
488 nm by the use of CLSM (Leica TCS SP2; Leica Microsystems, Heidelberg, Germany), and fluorescence was
detected with 510–550 and 635–680 nm filters, respectively.
Images were taken and merged by the use of the software
accompanying the instrument.
Treatment with JA
Seeds of transgenic rice were surface sterilized with 0.1%
HgCl2 and washed five times with distilled sterilized water.
Seeds were germinated in half-strength MS medium (pH
5.8) supplemented with different concentrations of JA at
28 °C. After 7 d, the differentiation of root length between
control and treatment was counted, then inhibition ratio
was calculated between the difference and control. For
Western blotting, 10-day-old seedlings of the wild type
(WT) were treated with 10 mm JA.
JA measurement in roots
Roots of 10-day-old plants germinated in half-strength MS
medium were pooled and frozen immediately in liquid
nitrogen. JA extraction and measurement by gas
chromatography–mass spectrometry (GC–MS) were done
as described by Heidel & Baldwin (2004); 262 ng of 1,313
C-JA (kindly provided by Ian T. Baldwin, Max-Planck
Institute of Chemical Ecology, Jena, Germany) was used as
internal standards.
Statistical analysis
Data are expressed as means ⫾ SEM. Analysis involved
repeat-measures analysis of variance (anova) (two-sample
Student’s t-test) conducted with the use of Origin 6.0.
P < 0.05 was considered significant.
RESULTS
Characterization of OsRMC from rice
OsRMC (AAL87185) located at chromosome 4 was isolated by RT–PCR. Its predicted ORF encoded a 258-amino
acid peptide. Sequence analysis showed that the protein
belonged to the DUF26 group of receptor kinase subfamilies. The family contains two kinds of proteins: those with
receptor and kinase domains and those with only a receptor
domain (Chen 2001). All these proteins contain two copies
of the C-X8-C-X2-C motif (CRR) in their receptor
domains. A position of the fourth cysteine residue varies
slightly among repeats at the C-terminal side of the C-X8C-X2-C motif (Chen 2001). OsRMC shares a homolog
sequence with several proteins in plants. It has only a receptor domain – one signal peptide and two CRR motifs – but
no transmembrane or kinase domain (Fig. 1c). Phylogenetic
analysis showed that OsRMC has 51 and 30% homology
with protein of rice and Arabidopsis, respectively
(Fig. 1d).
Purification of OsRMC recombined protein and
preparation of antibodies
Glutathione-S-transferase (GST)-tagged recombinant
OsRMC was expressed in E. coli. We purified recombinant
OsRMC (about 54 kDa) by solubilizing the inclusion
bodies involving the protein (Fig. 2a). The recombinant
protein from SDS–PAGE gel was used to immunize rabbit
for antibodies. The other homologs of OsRMC in rice,
XP_478602, XP_478603, XP_478598, XP_478599 and
CAE05988, have molecular weights of 40.2, 68.9, 66.7, 72.0,
71.1 and 48.2 kDa, respectively. Western blotting showed
the antibody with specificity to a 27.9 kDa protein in WT
rice (Fig. 2b), which is consistent with the predicted
protein.
Expression pattern of OsRMC
RT–PCR showed the OsRMC expression higher in young
roots, young leaves and young shoots than in stems
(Fig. 3a). A more distinct expression pattern in primary
roots was revealed by RNA in situ hybridization. Antisense
probe revealed OsRMC expressed in dividing cells, cortex
and pericycle of the root apex; no obvious signal was
detected in parenchymal cells of root areas where cells are
vacuolated (Fig. 3b). No distinct signal could be detected by
the sense probe (Fig. 3c). The result suggested that OsRMC
plays a role in the development of roots in rice.
Subcellular localization of OsRMC:GFP
fusion protein
OsRMC:GFP fusion gene, driven by CaMV 35S, was transformed into A. thaliana (Supplementary Fig. S1). Results of
subcellular localization assay showed RMC:GFP located
out of the cytoplasm in transgenic plants (Fig. 4a), a pattern
different from that with GFP alone (Fig. 4g). OsRMC:GFP
signal on the plasma membrane was shown in the cell wall
after the hypocotyls were incubated with 0.8 m mannitol
for plasmolysis (Fig. 4d). Because kinetic studies of the
membrane-selective dye FM4-64 show instantaneous and
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699
OsRMC involved in JA signal transduction 693
Figure 1. Sequence conservation of
(a)
(b)
(c)
(d)
strong labelling of the plasma membrane, the dye is sometimes used to mark the plasma membrane (Bolte et al. 2004;
Grandjean et al. 2004). In this study, plasma membrane
localization was further confirmed by the use of FM4-64
dye. Under either normal or plasmolysed conditions, red
staining with FM4-64 overlapped with the green fluorescence of OsRMC:GFP in the plasma membrane of transgenic plants (Fig. 4a–f), whereas the green signals of GFP
alone appeared in the whole cell in the control emptyvector transgenic plant (Fig. 4g–l).
Oryza sativa root meander curling
(OsRMC). (a) Alignment of the deduced
amino acid sequence for OsRMC and
sequences encoded by related genes of
rice and Arabidopsis. Multiple sequence
alignment was constructed with the use of
the software DNAMAN. Residues
identical in the seven sequences are
highlighted in gray and invariants in
black. The three amino acid cysteine
positions indicated by black triangles are
conserved in the C-X8-C-X2-C motif. The
fourth cysteine residue position varies
slightly, as indicated by a black circle. (b)
Putative conserved domains of AtRLK3,
AtRLK4, XP_478602, XP_478603,
XP_478598 and XP_478599. (c) Putative
conserved domains of XP_478605,
CAE05988 and OsRMC. (d) Dendrogram
showing the phylogenetic relations of
OsRMC with related proteins constructed
by the alignment from DNAMAN
analysis. CRR1, cysteine-rich repeat 1
domain; CRR2, cysteine-rich repeat 2
domain; SP, signal peptide sites; TM,
transmembrane helix.
Phenotype of OsRMC RNAi in transgenic rice
Southern blot analysis showed differential expression of
OsRMC in various transgenic lines (Fig. 5a), which suggests
that the transgenic lines are independent. The expression of
OsRMC was decreased significantly in transgenic rice
(Fig. 5b), which agrees with the results of real-time Q-PCR
(Fig. 5c). The primary roots of transgenic seeds germinated
in the dark for 4 d meandered before they touched the
bottom of the culture bottle (Fig. 5d). They were shorter
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699
694 J. Jiang et al.
(a)
(b)
(a)
(b)
(c)
Figure 2. Purification of glutathione-S-transferase–Oryza sativa
root meander curling (GST–OsRMC) recombinant protein and
the specificity test of its antibody. (a) Purification of
GST–OsRMC recombinant protein. Samples subjected to 12%
sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) were stained with Coomassie blue. Lane 1 shows
molecular weight markers; lane 2 supernatants of culture lysates
of pGEX4T-1 OsRMC vector with IPTG induction for 0 h; lanes
3 and 4 pellets of culture lysates of recombinant plasmid
pGEX4T-1 OsRMC with IPTG induction for 1 and 3 h,
respectively; and lane 5 purified GST–OsRMC recombinant
protein. (b) Western blot analysis of the specificity of antibody to
OsRMC to a protein in wild-type (WT) rice roots. The top and
bottom panels show the results of Western blotting, and samples
were subjected to 12% SDS–PAGE stained with Coomassie blue,
respectively.
than those of the WT, and the numbers of adventitious roots
were increased and that of lateral roots decreased as compared with those of the WT (Fig. 5e). The second sheath in
transgenic rice was also shorter than that of the WT
(Fig. 5f). The growth of roots and second sheath in transgenic plants differed significantly from that of WT plants
(Table 1). The root phenotypes in various transgenic lines
coincided with the expression levels of OsRMC (Fig. 5b).
The results suggest that OsRMC is involved in the establishment of the rice root system.
Figure 3. Expression pattern of Oryza sativa root meander
curling (OsRMC). (a) RT–PCR results show OsRMC expressed
in roots, leaves, shoots, spikes and stems. (b) In situ localization of
OsRMC transcript in young roots of wild-type (WT) rice by
antisense probe. Bar = 200 mm. (c) In situ localization of OsRMC
transcript in young roots of WT rice by sense probe as a control.
Bars = 200 mm.
Response of transgenic rice to JA
JA suppresses the elongation of primary roots and second
sheath (Yamane et al. 1981; Moons et al. 1997) and at low
concentrations, stimulates the formation of adventitious
roots and lateral roots, but at high concentrations, suppresses their formation (Moons et al. 1997; Wang et al.
2002). Our Western blot analysis showed increased
OsRMC expression with 10 mm JA treatment within 72 h
(Fig. 6a). Treatment of JA up to 5 mm suppressed the elongation of primary roots and second sheath growth
(Fig. 6b,c). The expression of RSOsPR10, a root-specific
rice PR10 gene involved in the JA signalling pathway
(Hashimoto et al. 2004), was enhanced in transgenic rice
(Fig. 6d), which suggests that OsRMC is a negative regulator of RSOsPR10 suppression. The expression of
OsOPR, an early gene of JA synthesis, in transgenic rice
Table 1. Root phenotype characteristics of five independent transgenic lines expressing RNAiOsRMC
Line
Primary root (cm)
Adventitious root (cm)
Number of adventitious
roots
Number of lateral roots
Second sheath (cm)
WT
L1
L2
L3
L4
L5
7.21 ⫾ 0.17
6.86 ⫾ 0.25
6.36 ⫾ 0.52*
7.05 ⫾ 0.63
6.31 ⫾ 0.62*
6.37 ⫾ 0.46*
3.30 ⫾ 0.19
3.42 ⫾ 0.80
3.63 ⫾ 0.42
4.27 ⫾ 0.22*
3.96 ⫾ 0.31*
3.96 ⫾ 0.22*
3.80 ⫾ 0.70
4.83 ⫾ 0.16*
4.75 ⫾ 0.21*
4.75 ⫾ 0.21*
4.63 ⫾ 0.27*
4.75 ⫾ 0.79*
67.43 ⫾ 7.06
65.50 ⫾ 8.88
53.29 ⫾ 6.82*
51.50 ⫾ 13.8*
44.17 ⫾ 4.50*
53.60 ⫾ 7.12*
6.14 ⫾ 0.25
5.47 ⫾ 0.30*
5.20 ⫾ 0.40*
5.30 ⫾ 0.22*
5.25 ⫾ 0.52*
4.94 ⫾ 0.45*
*Significant difference between wild-type (WT) and transgenic plants at P < 0.05 (n = 10).
30 °C, darkness, half-strength Murashige and Skoog (MS) (0.7% agar, pH 5.8), 7 d.
Data are means ⫾ SE.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699
OsRMC involved in JA signal transduction 695
JA concentration (mg g–1
FW)
WT
L1
L4
L5
0.96 ⫾ 0.36
0.82 ⫾ 0.29
0.73 ⫾ 0.33
0.92 ⫾ 0.26
Table 2. Jasmonic acid (JA) concentration
in roots in wild-type (WT) and Oryza sativa
root meander curling (OsRMC) RNAi
transgenic rice
No significant difference was found between WT and OsRMC RNAi transgenic rice at
P < 0.05 (n = 3).
Data are means ⫾ SE.
FW, fresh weight.
Line
Primary root coiling (%)
0.1 mm JA (Adventitious
root coiling %)
WT
L1
L4
L5
40 ⫾ 5
70 ⫾ 5
80 ⫾ 5
85 ⫾ 5
5⫾5
80 ⫾ 5
85 ⫾ 5
90 ⫾ 5
0.5 mm JA (All adventitious
root coiling %)
10 ⫾ 5
75 ⫾ 5
85 ⫾ 5
85 ⫾ 5
Table 3. Percentage of root coiling in
wild-type (WT) and Oryza sativa root
meander curling (OsRMC) RNAi transgenic
rice
Data are percentage ⫾ SE.
The experiment was repeated twice at least, with 20 seedlings used in each experiment.
JA, jasmonic acid.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699
Figure 4. Subcellular localization of
Oryza sativa root meander curling:green
fluorescent protein (OsRMC:GFP). (a–c)
Epidermal cells of hypocotyls from
transgenic plant harbouring
35S::OsRMC:GFP. (d–f) Treatment with
mannitol (0.8 m) for plasmolysis in
OsRMC:GFP transgenic Arabidopsis.
(g–i) Epidermal cells of hypocotyls from
transgenic Arabidopsis of 35S::GFP used
as a control. (j–l) Treatment with
mannitol (0.8 m) for plasmolysis in
35S::GFP transgenic Arabidopsis. Green
and red images are GFP and FM4-64
signals, respectively. Arrows indicate
fluorescent signals at the plasma
membrane. Bars = 50 mm.
696 J. Jiang et al.
(a)
(c)
(b)
(d)
(e)
(f)
was comparable to that in the WT (Fig. 6d). There was no
significant difference of JA level in statistics in the roots
between transgenic plant and WT (Table 2). It suggests
that response on root elongation and the second sheath to
JA was enhanced in the transgenic plant as JA signalling
pathway was enhanced. Auxin and gibberellin also affect
root–shoot development; the response to both auxin and
GA3 did not differ between OsRMC transgenic rice and
the WT (data not shown).
Figure 5. Phenotype of RNAiOsRMC
T2 transgenic rice and molecular
identification. (a) Southern blot analysis
of transgenic plants. Lanes 1 and 6,
non-transgenic plants; 2 and 7, L1
transgenic line; 3 and 8, L2 line; 4 and 9,
L3 line; 5 and 10, L5 line. Lanes 1–5,
genomic DNA digested by EcoR I, and
6-10, genomic DNA digested by Hind III.
GUS was used as a hybridization probe.
(b) Western blot analysis of Oryza sativa
root meander curling (OsRMC)
expression in transgenic plants. L1–5: five
independent transgenic lines (14-day-old
seedlings). (c) Real-time quantitative
PCR (Q-PCR) analysis of OsRMC
expression in transgenic plants. (d) Root
phenotype of T2 transgenic rice line of
RNAiOsRMC at 4 d (darkness); L5, line 5
transformed by RNAiOsRMC. The
curling of roots is shown by arrows.
(e) Root phenotype of T2 transgenic line
of RNAiOsRMC at 7 d (darkness). (f)
Second sheath of T2 transgenic line of
RNAiOsRMC at 10 d. Sheath is shown
with an arrow. WT, wild type.
Root coiling in OsRMC RNAi transgenic rice
The primary roots in transgenic rice meandered after germination in half-strength MS 0.7% agar medium in light
(Fig. 7a). Up to 30% of primary roots coiled easily on
low-concentration JA (0.1 mm) in darkness, with no change in
the WT (Fig. 7b). The same tendency of coiling was significant in light, although the proportion of coiling in both
transgenic and WT roots differed: if 40% of WT primary
(a)
(c)
(b)
(d)
Figure 6. Effect of jasmonic acid (JA)
on expression of Oryza sativa root
meander curling (OsRMC) and
elongation of primary roots and second
sheath growth. (a) Time course of
alteration in expression of OsRMC
induced by JA (10 mm) in rice seedlings.
(b) Response of growth of primary roots
to various concentrations of JA (n = 20).
(c) Response of growth of second sheath
to various concentrations of JA (n = 20).
(d) RT–PCR analysis of expression of
JA-related genes in transgenic rice roots.
Tubulin was used as an internal control.
WT, wild-type rice; L4 and L5, two
independent transgenic lines.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699
OsRMC involved in JA signal transduction 697
(a)
(b)
Figure 7. Phenotype analysis of root
coiling in T2 transgenic rice plants [25 °C,
half-strength MS (0.7% agar, pH 5.8)].
Plants were grown under 10 d of
treatment; arrows show root meander or
coiling on test of touching the bottom of
the bottle. (a) Root phenotype in the T2
line of RNAiOsRMC transgenic rice not
touching the bottom of the culture bottle
under 16 h light/8 h dark. (b) 0.1 mm
jasmonic acid (JA) in darkness. (c) and
(d) 0.1 mm JA (c) and 0.5 mm JA (d) on
root coiling under 16 h light/8 h dark. WT,
wild type; WT-JA, wild type treated with
JA; L5, transgenic line 5; L5-JA,
transgenic line 5 treated with JA.
(c)
(d)
roots coiled, up to 85% of the transgenic roots coiled
(Table 3). On 0.1 mm JA treatment, up to 90% of transgenic
adventitious roots coiled as compared to only 5% of WT
roots (Fig. 7c; Table 3). With 0.5 mm JA, 75–85% of transgenic adventitious roots coiled as compared to only 10% of
WT roots (Fig. 7d; Table 3). This observation suggested that
JA was involved in root coiling, and OsRMC is a negative
regulator of the process.
DISCUSSION
OsRMC, a member of the DUF26 family of proteins, has
only a receptor domain and no kinase zone as do other
receptor-like kinases. However, we found the signal peptide
with transmembrane character (5–27AA) (Fig. 1c) involved
in OsRMC, which may cause its localization on the plasma
membrane (www.cbs.dtu.dk/services/TMHMM). So far,
several genes of DUF26 have been isolated and characterized. AtRLK3 is activated by oxidative stress, salicylic acid
and pathogen attack (Czernic et al. 1999). AtRLK4~6,
located at the same chromosome IV, are also induced by
salicylic acid and pathogen attack (Du & Chen 2000), which
suggests that the proteins of DUF26 might be involved in
biotic and abiotic stress signalling. Our results showed that
OsRMC was induced by JA at the translation level (Fig. 6a).
OsRMC is expressed as well under pathogen inoculation
and JA treatment (Kim et al. 2003, 2004) and wound stimuli
(Shen, Jing & Kuang 2003). Our knockdown of OsRMC
enhanced sensitivity to JA in rice plants (Fig. 6b,c), so
OsRMC might be involved in JA signal transduction.
The gene RSOsPR10 is exclusively induced in rice roots
by stress such as salt, drought, blast fungus infection and JA,
but not abscisic acid and salicylic acid (Hashimoto et al.
2004). In the transgenic rice, we found RSOsPR10 accumulated to a higher level than in the WT (Fig. 6d), which
suggests that the OsRMC is involved in RSOsPR10mediated JA signalling.
In OsRMC transgenic rice, the primary roots coiled as
they touched the bottom of culture bottles in light
(Fig. 7c,d). The expression of rice OsOPR gene, an early
gene of JA synthesis, is induced by red light (Riemann
et al. 2003). More JA was synthesized in light, and primary
transgenic roots coiled easily (Fig. 7c,d). If the medium in
the bottle was deep enough so that the primary roots
could not touch the bottom, the roots were wavy rather
than coiled (Fig. 7a). In Bryonia dioica tendrils, touch
rapidly initiates the formation of JA from a-linolenic
acid, which is released as a messenger triggering the
coiling response (Weiler et al. 1993). Recently, 12oxophytodienoate-10,11-reductase (OPR3), an enzyme in
the JA biosynthetic pathway, was found induced in Arabidopsis by touch (Chotikacharoensuk, Arteca & Arteca
2006). Other evidence shows that the tip and peripheral
cells of root caps are sensitive to touch, which alters
growth (Legue et al. 1997), and brief touch stimulation of
root cap peripheral cells results in a transient reduction of
the gravitropic sensitivity of the root (Massa & Gilroy
2003). These findings point to our result of OsRMCinvolved JA signalling mediating the rice root coiling
response.
In short, our results showing enhanced JA-sensitive
response and increased root coiling in OsRMC knockdown transgenic rice plants suggest that the plasma membrane protein OsRMC with a signal peptide of the DUF26
subfamily involved in JA signal transduction mediates
root development and negatively regulates root curling
in rice.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699
698 J. Jiang et al.
ACKNOWLEDGMENT
This work was supported by the National Natural Science
Foundation of China (NSFC) (30270143) and the Outstanding Young Research Fund of NSFC (30525026).
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Received 15 December 2006; received in revised form 1 February
2007; accepted for publication 3 February 2007
SUPPLEMENTARY MATERIAL
The following supplementary material is available for this
article:
Figure S1. Identification of the transgenic OsRMC:GFP
Arabidopsis lines. M, molecular weight marker DL2000;
lanes 1–8, 35S::OsRMC:GFP transgenic lines; 9, wild-type
(WT) (negative control). Lanes 1–8 showed band of PCR
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699
OsRMC involved in JA signal transduction 699
product with the OsRMC primers (5′-cgtctagaatggcgcgg
tgcactttg-3′ and 5′-cgggtaccctcacgcagcaccacc-3′).
This material is available as part of the online article
from
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j.1365-3040.2007.01663x
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© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 690–699