Plant Cell Physiol. 48(5): 678–688 (2007)
doi:10.1093/pcp/pcm042, available FREE online at www.pcp.oxfordjournals.org
ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Rapid paper
Identification of the Gravitropism-Related Rice Gene LAZY1 and Elucidation
of LAZY1-Dependent and -Independent Gravity Signaling Pathways
Takeshi Yoshihara and Moritoshi Iino *
much attention has been focused on two major hypotheses:
the starch–statolith hypothesis and the Cholodny–Went
theory of tropisms. In the former hypothesis, sedimentable
starch-filled amyloplasts are thought to play a key role in
sensing the direction of gravity (Němec 1901, Haberlandt
1902). The latter hypothesis proposes that tropisms,
including gravitropism, are mediated by lateral translocation of the plant hormone auxin (Went and Thimann 1937).
Recent studies with mutants of Arabidopsis thaliana have
provided strong genetic evidence for the applicability of
these hypotheses in gravitropism of both shoot organs and
roots, and have uncovered some cellular components that
are thought to be associated with amyloplast-based gravity
sensing or lateral auxin translocation (for reviews, see
Masson et al. 2002, Morita and Tasaka 2004).
The following basic questions should be answered,
however, before we can successfully model gravity sensing
and signaling in plants. First, it is not clear whether the
amyloplast-based gravity sensing is responsible for the
entire gravitropic response. In fact, hypocotyls and roots of
starchless Arabidopsis mutants that do not have any
sedimentable amyloplasts show some gravitropic response
(e.g. Fitzelle and Kiss 2001). Secondly, it has not been
resolved whether asymmetric distribution of auxin accounts
for the entire gravitropic response (Haga and Iino 2006).
There is a possibility that a part of the response is mediated
by other plant hormones or growth-regulating substances,
as in fact suggested by recent work (Aloni et al. 2004,
Gutjahr et al. 2005).
A number of rice mutants showing a ‘lazy’ phenotype
or prostrate growth habit have been isolated since one such
mutant was reported by Jones and Adair (1938). The best
characterized lazy mutants are the tester strain H79
maintained in Hokkaido University and near-isogenic
lines produced by crossing this strain with the japonica
cultivar Kamenoo (Goto 1978) or Taichung 69 (Yu et al.
1995). Suge and co-workers (Abe and Suge 1993, Abe et al.
1994a) used the lazy mutant in the Kamenoo background to
resolve that leaf sheath pulvini and coleoptiles of the mutant
have reduced gravitropic responsiveness. Extended work
with the same mutant line indicated that coleoptiles and leaf
We identified the gene responsible for three allelic lazy1
mutations of Japonica rice (Oryza sativa L.) by map-based
cloning, complementation and RNA interference. Sequence
analysis and database searches indicated that the wild-type
gene (LAZY1) encodes a novel and unique protein (LAZY1)
and that rice has no homologous gene. Two lazy1 mutants
were LAZY1 null. Confirming and advancing the previously
reported results on lazy1 mutants, we found the following.
(i) Gravitropism is impaired, but only partially, in lazy1
coleoptiles. (ii) Circumnutation, observed in dark-grown
coleoptiles, is totally absent from lazy1 coleoptiles.
(iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissuespecific manner in gravity-sensitive shoot tissues
(i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is
little expressed in roots. (v) The gravitropic response of lazy1
coleoptiles is kinetically separable from that absent from
lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of
auxin, found in wild-type coleoptiles, does not occur in lazy1
coleoptiles. Based on the genetic and physiological evidence
obtained, it is concluded that LAZY1 is specifically
involved in shoot gravitropism and that LAZY1-dependent
and -independent signaling pathways occur in coleoptiles.
It is further concluded that, in coleoptiles, only the
LAZY1-dependent gravity signaling involves asymmetric
distribution of auxin between the two lateral halves and is
required for circumnutation.
Keywords: Auxin — Circumnutation — Gravitropism —
lazy mutants — LAZY1 — Oryza sativa.
Abbreviations:
sequence; RNAi,
transcription–PCR.
CAPS, cleaved amplified polymorphic
RNA interference; RT–PCR, reverse
Introduction
Gravitropism plays a central role, in association with
other tropisms, in regulating the spatial orientation of plant
organs (Iino 2006). In studies of higher plant gravitropism,
*Corresponding author: E-mail, [email protected]; Fax, þ81-72-891-7199.
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Editor-in-Chief’s choice
Botanical Gardens, Graduate School of Science, Osaka City University, Kisaichi, Katano-shi, Osaka, 576-0004 Japan
Rice LAZY1 and gravity signaling
sheath pulvini of the mutant have normal amyloplasts
(Abe et al. 1994b) but are impaired in asymmetric
distribution of auxin (Godbolé et al. 1999). Furthermore,
lazy coleoptiles were found to lack circumnutation, which is
observed in dark-grown wild-type coleoptiles (Yoshihara
and Iino 2005, Yoshihara and Iino 2006).
The gene responsible for the above lazy mutation,
symbolized as la (Kinoshita 1998), was mapped to a 1.4 cM
region of chromosome 11 (Miura et al. 2003). Peijin et al.
(2003) used an allelic mutant in an indica background to
map the gene to a 0.4 cM region. Because it is unlikely that
only one gene is responsible for all the reported lazy
mutants, the lazy lines originated from the tester strain H79
and the lazy mutants shown to be allelic to these lines will
be called lazy1. According to commonly used nomenclature,
the mutant gene is then called lazy1 and the corresponding
wild-type gene and its product, LAZY1 and LAZY1,
respectively.
We identified LAZY1 as a novel and unique gene and
analyzed physiological functions of LAZY1. It has become
apparent that the gravitropism of coleoptiles involves
LAZY1-dependent and -independent signaling pathways
and that only the LAZY1-dependent pathway involves
asymmetric distribution of auxin across the organ and is
required for coleoptile circumnutation. Here we report these
and related findings.
Results
Identification and analysis of LAZY1
We used the lazy1 mutant in the Kamenoo background, hereafter named lazy1-1, to identify the LAZY1
locus by map-based cloning. A mapping population of
F2 caryopses was generated by crossing the lazy1-1 mutant
to the indica cultivar Kasalath. F2 seedlings, grown under
red light, were assayed for shoot gravitropism at the secondleaf stage and those showing clearly reduced shoot
gravitropism were selected. By using the genomic DNA
extracted from 884 selected individuals, the LAZY1 locus
was mapped between markers RM287 and RM7275 in
chromosome 11, which are located inside the previously
identified LAZY1-containing region (Miura et al. 2003,
Peijin et al. 2003). With the cleaved amplified polymorphic
sequence (CAPS) markers developed in this study, the
LAZY1 locus was further delimited to a 39.8 kb region,
which was predicted to contain 14 genes (Fig. 1A). By
sequencing PCR-amplified genomic fragments, the lazy1-1
mutant was found to have a deletion of 8.0 kb in this region
and an insertion of 0.76 kb at the deletion site (Fig. 1B and
Supplementary Fig. S1).
Some retrotransposon Tos17 insertion lines of
Nipponbare were recorded to show a lazy phenotype
(http://tos.nias.affrc.go.jp/miyao/pub/tos17/phenotype/
63.html; Miyao et al. 2003). We found that three of
A
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Markers RM287
Recombinants
P1 P2 P3
4
3
0
RM7275
1
BAC1
30 kb
B
BAC2
BAC3
LAZY1
4 kb
lazy1-1
lazy1-2
lazy1-3
C
ATG
TAA
Fig. 1 Identification of LAZY1. (A) Physical map of the genomic
region containing LAZY1. The LAZY1 locus was mapped between
markers P1 and P3 on chromosome 11. Molecular markers (P1–3,
CAPS markers designed in this study) are indicated with the
number of recombinants. Bacterial artificial chromosome (BAC)
clones: BAC1, OSJNBb0047D03; BAC2, OSJNBa0052B22; BAC3,
OSJNBb0089M05. (B) Genes predicted in the identified region and
mutations found in lazy1 mutants. The LAZY1 gene identified in
this study is indicated. lazy1-1: the lazy1 mutant in the Kamenoo
background. lazy1-2 and lazy1-3: Tos17 insertion lines (NC0873
and NF1106, respectively) in the Nipponbare background. lazy1-1
and lazy1-2 had a deletion at the position shown by a gray bar
(8,028 and 20,103 bp, respectively) and an insertion at the deletion
site (755 and 10,892 bp, respectively). lazy1-3 had a small deletion
(52 bp) at the position indicated by an arrowhead. (C) Genomic
structure of LAZY1. Exons (boxes), introns (lines), and start and stop
codons are shown.
them (NC0873, NF1106 and NF1128), confirmed to
show a lazy phenotype similarly to lazy1-1, are allelic to
lazy1-1. These lines had no Tos17 insertion but a deletion in
the LAZY1-linked region (Fig. 1B and Supplementary
Fig. S1). The line NC0873 had a deletion of 20.1 kb and, in
addition, an insertion of 10.9 kb. The line NF1106 had a
small deletion (52 bp). The deletion in NF1128 matched
exactly with that in NF1106, suggesting that the two lines
originated from the same one. The lines NC0873 and
NF1106 are hereafter named lazy1-2 and lazy1-3,
respectively.
The genomic deletions found in the three lazy1 mutants
strongly suggested that the LAZY1 locus corresponds to the
gene labeled ‘LAZY1’ in Fig. 1B. The full-length rice cDNA
collection (Kikuchi et al. 2003) contained a cDNA of this
putative LAZY1 gene (GenBank accession No. AK067664;
Supplementary Fig. S2A). We transformed the lazy1-1
mutant with this cDNA fused with the cauliflower mosaic
virus 35S promoter. Some of the transformants showed no
apparent lazy phenotype at the T1 generation (Fig. 2A)
and those were selected for further analysis. Expression of
the introduced cDNA could be confirmed in T2 coleoptiles;
1)
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(li
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ip
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Rice LAZY1 and gravity signaling
(li
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680
100
11.6
±1.4
7.9
±1.0
LAZY1
Complemented
transformant
lazy1-1
Kamenoo
RNAi
transformant
Nipponbare
C
Nipponbare
lazy1-2
lazy1-3
RNAi (line 1)
RNAi (line 2)
Kamenoo
lazy1-1
Complemented (line 1)
Complemented (line 2)
OsUBQ
1740
±245
810
±256
D
Nipponbare
Kamenoo
30
0
175 min (±2)
23.4° (±0.4)
lazy1-1
lazy1-2
Complemented (line1)
lazy1-3
−60
−90
Tip orientation
Tip orientation (degrees)
−30
153 min (±2)
20.0° (±1.1)
Coleoptiles
Coleoptiles
90
RNAi (line 1)
204 min (±2)
15.2° (±1.0)
60
Complemented (line2)
RNAi (line 2)
30
0
Roots
−30
0
1
2
3
4
5
Roots
6
0
1
2
3
4
5
Time (h)
30°
199 min (±3)
20.8° (±0.7)
3h
6
Time
Fig. 2 Complementation of lazy1 and RNAi of LAZY1. For the complementation test, the lazy1-1 mutant was transformed with the
putative LAZY1 cDNA fused with the 35S promoter. For RNAi, Nipponbare was transformed with an RNAi construct designed for the
putative LAZY1. (A) Examples of complemented and RNAi T1 transformants exhibiting wild-type and lazy phenotypes, respectively.
Subsequent analyses were conducted with homozygous T2 seedlings. (B) The levels of putative LAZY1 transcripts in dark-grown
coleoptiles of seven genotypes including two complemented and RNAi lines. The agarose gel represents RT–PCR products (OsUBQ,
internal standard). Numbers shown below the gel are the mean transcript levels SE determined by real-time RT–PCR from three
independent samples (wild type ¼ 100). (C) Gravitropic curvatures in red light-grown seedlings of the indicated genotypes. Seedlings were
displaced by 908 and monitored for the orientation of the coleoptile tip and primary root tip. The vertical position is represented by 08 and
the horizontal position, by 908 (coleoptiles) or 908 (roots). The length of coleoptiles at the onset of displacement ranged between 3.5 and
4.5 mm and that of primary roots ranged between 12 and 23 mm. Data shown are the means SE of 19–33 seedlings. (D) Coleoptile
circumnutation in dark-grown seedlings of the indicated genotypes. Tip orientation in the coleoptile’s front view was monitored.
The dotted line represents the vertical position. The length of coleoptiles ranged between 6 and 10 mm at the start of the experiments. For
the genotypes showing circumnutation, the mean period and amplitude obtained from 15–18 seedlings are indicated with SE.
the transcript level was substantially higher in all the
investigated lines than in the wild type (Fig. 2B).
Gravitropic responses of complemented lines were compared with those of the wild type and lazy1-1 mutant. This
comparison was made using red light-grown seedlings and
inducing gravitropism by 908 displacement. As shown in
Fig. 2C, the complemented T2 coleoptiles recovered
gravitropism partially or close to the wild-type level.
The circumnutation observed in dark-grown wild-type
coleoptiles was also recovered in the complemented lines,
although the period and amplitude were somewhat different
from those of wild-type coleoptiles (Fig. 2D).
We further performed RNA interference (RNAi)
experiments against the putative LAZY1 gene. The japonica
cultivar Nipponbare was transformed with an RNAi
construct (see Materials and Methods). Plants showing a
lazy phenotype segregated at the T1 generation (Fig. 2A).
The level of putative LAZY1 transcripts was 512% of the
Rice LAZY1 and gravity signaling
wild-type level in T2 coleoptiles (Fig. 2B). These coleoptiles
reduced their gravitropic response to the lazy1 mutant level
(Fig. 2C) and showed no coleoptile circumnutation
(Fig. 2D). Taken together, the results from complementation and RNAi experiments demonstrated that the investigated gene indeed corresponds to LAZY1.
The results shown in Fig. 2 deserve more detailed
attention. First, a full recovery of gravitropism was
achieved in the complemented line 2 which had an eight
times higher level of LAZY1 transcripts than in the wild
type, whereas the recovery was partial in the complemented
line 1 that had a 17 times higher level of LAZY1 transcripts
(Fig. 2B). Another complemented line (line 3) showed
results that were similar to those from line 1 with respect to
the level of LAZY1 transcripts and the extent of gravitropism recovery (data not shown). Therefore, the partial
recovery of gravitropism in complemented lines was
correlated with a considerably high expression of LAZY1,
suggesting that LAZY1 is inhibitory on gravitropism when
it is expressed well above the wild-type level. Secondly, the
lazy1-2 mutant is apparently LAZY1 null (Fig. 1B) whereas
the lazy1-3 mutant is expected to produce a mutated
LAZY1 that lacks a part of the C-terminus (Supplementary
Fig. S1). The gravitropic response remaining in lazy1-3
coleoptiles was not greater than that remaining in lazy1-2
coleoptiles, suggesting that the mutated LAZY1 is not
functional. Thirdly, the gravitropic response remaining in
LAZY1-RNAi lines was not more than that remaining in
the LAZY1-null lazy1-2 mutant (Fig. 2C), although some
LAZY1 transcripts could be detected in these lines.
At present, we have no clear explanation for this result.
In the experiments shown in Fig. 2C, the gravitropism of
primary roots was also investigated. The results of these
experiments will be described below along with other
responses in roots.
The genomic structure of LAZY1 was resolved by
comparing the genomic sequence, which was identical
between Kamenoo and Nipponbare, with the sequence of
Nipponbare LAZY1 cDNA (Fig. 1C). The deduced amino
acid sequence of LAZY1 revealed no apparent motif
(Supplementary Fig. S2B). Our BLAST searches indicated
that rice has no homologous gene and that Arabidopsis has
one LAZY1-like gene (GenBank accession No. AY735682).
The amino acid sequence predicted from this Arabidopsis
gene showed 20.5% identity to LAZY1.
LAZY1 transcript distribution and effects of gravity and red
light
Quantitative real-time PCR was used to investigate the
distribution of LAZY1 transcripts. In the coleoptile of darkgrown seedlings, the transcript level was highest in the most
apical zone and decreased basipetally (Fig. 3A). The level in
leaves was much lower than that in the coleoptile base.
681
In primary roots, the transcript level was extremely low in
the most apical 1 mm zone (0.07% of the level in the
coleoptile top zone) and increased acropetally. Even the
highest level found in the most basal zone was only 0.6% of
the level in the coleoptile base.
In mature green plants, the leaf-sheath pulvinus (P)
had the highest level of LAZY1 transcripts (Fig. 3B). About
a half of this level could be found in the lamina joint (LJ).
Other leaf tissues adjacent to the leaf-sheath pulvinus or
lamina joint (LS1–3, LB1 and LB2) as well as internode
tissues adjacent to the node and leaf-sheath pulvinus
(IN1–4) showed a much lower level. Separate experiments
indicated that the transcript level in the central region of
internodes or leaf blades was 3–5 or 50.1%, respectively, of
the level in leaf-sheath pulvini. The transcript level in
immature panicles was also low (2.6 0.3%, n ¼ 4).
In the above experiments, LAZY1 transcript levels
were determined relative to the level in either the coleoptile
top zone or leaf-sheath pulvinus. A direct comparison
between the two tissues indicated that the coleoptile top
zone had about three times more transcripts than the leafsheath pulvinus (relative transcript levels for the coleoptile
top zone and pulvinus were 100 3.2 and 28.4 1.9,
respectively). Therefore, the coleoptile had the highest
level of LAZY1 transcripts among all the tissues investigated. Tissue-specific expression of LAZY1 in coleoptiles,
pulvini and lamina joints agrees with the knowledge that
these parts of rice shoots are gravity sensitive (see below).
We investigated whether the expression of LAZY1 is
affected by gravitropic stimulation. The transcript levels in
the upper and lower halves of horizontally placed red lightgrown coleoptiles were determined at 30 and 60 min of
displacement. No significant change in transcript level was
found (Supplementary Fig. S3A). We further investigated,
in relation to our finding that red light eliminates
circumnutation (Yoshihara and Iino 2005), whether the
transcript level in dark-grown coleoptiles is affected by red
light. No significant effect of red light was observed within
the period in which circumnutation disappears
(Supplementary Fig. S3B).
Gravitropism, circumnutation and lateral root formation in
primary roots of lazy1 mutants and LAZY1-complemented
lines
As seen from the data in Fig. 2C, the primary roots of
lazy1 mutants showed time courses of gravitropism that are
almost identical to those of wild-type roots including the
phase of oscillation (for this oscillation, see Haga et al.
2005). Furthermore, lazy1-1 roots showed circumnutation
with a period and amplitude that were almost identical to
those of wild-type circumnutation (Fig. 4B). These results
indicated that LAZY1 is not required for the gravitropism
and circumnutation of primary roots.
682
Rice LAZY1 and gravity signaling
A
Shoots
Roots
Transcript level (relative)
100
1
0.5
2
3
80
0.4
4
4
60
0.3
40
0.2
20
0.1
0
1
2
3
4
Leaves
3
2
1
0.0
4
Zones
3
2
1
Zones
B
Transcript level (relative)
100
IN4
LB1
IN3
80
P
LJ
N
IN2
60
LB2
LS1
LS3
IN1
LS2
40
Nodal region
Lamina joint
region
20
0
P
N
IN1
IN2
IN3
IN4
LS1
LS2
LS3
Although LAZY1 is little expressed in wild-type primary
roots (Fig. 3A), LAZY1-complemented lines had a high
level of LAZY1 transcripts in primary roots (Fig. 4A).
Interestingly, these lines showed altered root gravitropism
(Fig. 2C). The major difference was noted in the phase of
oscillation. Furthermore, the complemented lines showed
root circumnutation with a period and amplitude that were
greater than those in wild-type roots (Fig. 4B). Therefore,
although LAZY1 is not required for root gravitropism and
circumnutation, it affects these processes if expressed in roots.
Elongation growth of primary roots and lateral root
formation in these roots were not significantly affected by
lazy1 mutation (Fig. 4C), indicating that LAZY1 is also not
required for these processes. However, lateral root formation was strongly suppressed in the complemented plants,
although primary root growth was almost normal (Fig. 4C).
Therefore, LAZY1 affects lateral root formation when
expressed in primary roots as it does root gravitropism and
circumnutation.
LJ
LB1
LB2
Fig. 3 Distribution of LAZY1 transcripts in rice plants (Kamenoo). The
transcript level was determined by
real-time RT–PCR. (A) Distribution
in 3-day-old dark-grown seedlings.
The coleoptile (9–12 mm) was
divided into four zones of equal
length as illustrated with a photograph; leaves inside the coleoptile
were harvested separately. The root
(15–20 mm) was divided into zones
1 (1 mm), 2 (3 mm) 3 (5 mm) and 4
(the remaining part) as illustrated.
The transcript levels were determined relative to the level in zone
1 of the coleoptile. Data shown are
the means SE of three independent
samples (413 seedlings each).
(B) Distribution in mature plants
(a few days before heading).
Tissues around the first node below
the flag leaf node and the lamina
joint region of the leaf attached to
the former node were divided as
illustrated with photographs (tissue
contour is marked with lines in the
vertical section of the nodal region).
P, leaf-sheath pulvinus; N, nodal
region; IN1–4, internode 5 mm
zones; LS1–3, leaf-sheath 5 mm
zones; LJ, lamina joint; LB1 and
LB2, leaf-blade 5 mm zones.
The transcript levels were determined relative to the level in
leaf-sheath pulvini. Data shown are
the means SE of three independent
samples.
Resolving LAZY1-dependent and -independent gravitropic
responses
Investigation was extended to address the question of
why LAZY1 null mutants, which do not have any LAZY1
homolog, have only partially reduced gravitropism. We first
examined gravitropic curvature induced by a brief stimulation. The time course results shown in Fig. 5A were
obtained by displacing red light-grown wild-type and
lazy1-1 coleoptiles by 30 or 908 for 25 min and returning
them to the vertical position. At either displacement angle,
wild-type and lazy1 coleoptiles initially bent similarly, but
the latter coleoptiles reached the peak earlier and returned
to the vertical position earlier (Fig. 5A). A possible
explanation for the observed kinetic difference is that
LAZY1 simply delays the response peak while enhancing
the extent of response. However, as shown in Fig. 5B, the
complemented lazy1-1 lines that recovered the gravitropic
response to different extents showed a response peak at a
similar time. Furthermore, the time courses for wild-type
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683
2)
Rice LAZY1 and gravity signaling
Kamenoo
LAZY1
OsUBQ
103 min (±4)
26.2° (±1.2)
C
lazy1-1
Kamenoo
lazy1-1
80
Complemented
(line 1)
60
100 min (±3)
23.5° (±0.8)
Tip orientation
Length of primary roots (mm)
100
40
20
Complemented (line 1)
0
213 min (±15)
38.8° (±4.0)
Number of lateral roots
Kamenoo
lazy1-1
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Complemented (line 2)
Complemented
(line 1)
40
20
163 min (±9)
41.2° (±2.8)
30°
1h
0
2
4
6
8
Time
Days after sowing
Fig. 4 Effects of LAZY1 overexpression on root circumnutation and lateral root formation. (A) LAZY1 expression in primary roots. Primary
roots of the indicated genotypes were harvested from dark-grown seedlings and LAZY1 transcripts were analyzed by RT–PCR. Details were
as described for Fig. 2B. (B) Primary root circumnutation. The circumnutation observed in the tip of primary roots was analyzed from the
seedling’s front view. Red light-grown seedlings were used for this analysis (the material as described for Fig. 2C). A typical example is
shown for each genotype with the mean period and amplitude (SE) obtained from 12–15 seedlings. (C) Primary root growth and lateral
root formation. Experiments were conducted with white light-grown seedlings. Primary roots were grown in deionized water. Data shown
are the means SE of 16–18 seedlings. Each point represents a separate group of seedlings.
and complemented lines tended to curve outward at around
45–50 min before reaching the peak (Fig. 5A, B), suggesting
that the LAZY1-independent response, which peaked at
45–50 min, accompanied the LAZY1-dependent response,
which peaked at 65–70 min. Together, these results indicated that the LAZY1-dependent and -independent
responses are kinetically separable, the latter being induced
more transiently than the former after brief stimulation.
In dark-grown coleoptiles of the lazy1-1 mutant,
gravitropic curvature was more confined to the upper part
as compared with wild-type coleoptiles (Yoshihara and Iino
2006). This feature was not apparent in red light-grown
684
Rice LAZY1 and gravity signaling
A
30°
Curvature (degrees)
30
Kamenoo
90°
Kamenoo
20
lazy1-1
10
lazy1-1
0
0
60
30
90
120 0
30
60
90
120
Time (min)
B
30°
Curvature (degrees)
30
Complemented
line 2
20
10
line 3
0
0
30
60
90
120
Time (min)
C
75
3H-IAA distribution (%)
Upper side
Lower side
lazy1-1
Kamenoo
50
75
75
0
30
90
0
30
90
Displacement angle (degrees)
Fig. 5 Curvature kinetics and lateral auxin translocation. (A) Curvature kinetics: comparison between wild-type and lazy1 coleoptiles.
Red light-grown wild-type and lazy1-1 seedlings were displaced by 30 or 908 at time zero and returned to the vertical position at 25 min
(dashed line). Coleoptile curvature was monitored from the onset of displacement. Data shown are the means SE of 37 (308, Kamenoo),
34 (308, lazy1-1) and 21 (908) seedlings. (B) Curvature kinetics in complemented lazy1-1 lines. Data shown are the means SE of 14
(line 2) and 12 (line 3) seedlings. Complemented line 1 showed results nearly identical to those from line 3. (C) Lateral auxin translocation:
comparison between wild-type and lazy1 coleoptiles. Wild-type and lazy1-1 seedlings were stimulated for gravitropism as described
above. Lanolin containing [3H]IAA (3.14 MBq g1) was applied to the coleoptile tip 2 h before the displacement treatment. After
displacement, the coleoptile was excised from its base and placed on a pair of agar blocks for 30 min to obtain [3H]IAA diffusing out of the
basal halves which had been the upper and lower sides during displacement. A razor blade separated the two agar blocks and divided the
coleoptile base into two halves. The amount of [3H]IAA obtained from either side was calculated as a percentage of the total amount
obtained from both sides. Data shown are the means SE of three independent samples (12 coleoptiles each).
Rice LAZY1 and gravity signaling
short coleoptiles. The photographs of seedlings obtained in
the above experiments as well as those obtained for Fig. 2C
indicated that the lazy1 coleoptile bent along its length.
Therefore, in red light-grown coleoptiles, the kinetic
difference between LAZY1-dependent and -independent
responses was not associated with a clear spatial difference
in the location of curvature.
The 25 min displacement protocol used above and the
[3H]IAA tracer method described in Haga et al. (2005) were
next used to examine auxin distribution in gravitropically
stimulated coleoptiles. In brief, lanolin containing [3H]IAA
was applied to the coleoptile tip. After 25 min displacement,
the radioactivity diffusing out of the cut surface of the
basal halves was collected for 30 min and determined.
Most of the collected radioactivity corresponded to
[3H]IAA (Haga et al. 2005). As shown in Fig. 5C,
[3H]IAA was asymmetrically distributed in wild-type
coleoptiles. This asymmetry was greater at 908 than at
308, as was the case for curvature response. In sharp
contrast, however, no significant asymmetry could be
detected in lazy1 coleoptiles at either displacement angle.
In separate experiments, diffusible [3H]IAA was collected
for a shorter period (20 min) after 25 min displacement.
In this case too, no significant asymmetry could be detected
in lazy1-1 coleoptiles (data not shown). Two important
conclusions are reached from these results. First, auxin
asymmetry occurs downstream of LAZY1, accounting most
probably for LAZY1-dependent curvature. Secondly,
LAZY1-independent curvature does not involve auxin
asymmetry between the two halves. Discussion on
LAZY1-dependent and -independent gravity signaling will
be elaborated below.
Discussion
The lazy1 mutant is characterized by partial impairment of gravitropisms in coleoptiles (Abe et al. 1994a,
Yoshihara and Iino 2006, Fig. 2C) and leaf-sheath pulvini
(Abe and Suge 1993) as well as by total absence of the
circumnutation in coleoptiles (Yoshihara and Iino 2006,
Fig. 2D). In this study, we have additionally found that
primary roots of lazy1 mutants show normal gravitropism
(Fig. 2C) and circumnutation (Fig. 4B). These results
indicate that LAZY1 is specifically involved in shoot
gravitropism and coleoptile circumnutation.
Any possible function of LAZY1 could not be
predicted from the deduced amino acid sequence. Because
transcriptional expression of LAZY1 was not affected in the
upper and lower halves of displaced coleoptiles and also in
red light-treated coleoptiles (see Results), it is likely that
LAZY1 is constitutively involved in gravity signaling.
Godbolé et al. (1999) used a unique tracer method
(i.e. collecting [3H]IAA from the intact coleoptile surface
685
into agar blocks) to show that auxin asymmetry is severely
impaired in lazy1-1 coleoptiles. By using a more classical
method (collecting basipetally transported [3H]IAA from
the basal cut end into agar blocks), we were able to
demonstrate with a high resolution that auxin asymmetry
does not occur in lazy1-1 coleoptiles (Fig. 5C). Clearly
lateral auxin translocation occurs downstream of LAZY1 in
the LAZY1-dependent gravity signaling. Coleoptiles of a
maize mutant having a defect in sedimentable amyloplasts
were shown to be impaired in gravity-induced auxin
asymmetry (Hertel et al. 1969). Although plants may have
gravity-sensing mechanisms that do not depend on amyloplasts (see below), it is most likely that the amyloplast-based
gravity sensing is the one linked to the induction of auxin
asymmetry. The lazy1 mutant at least has normal sedimentable amyloplasts in coleoptiles and leaf sheath pulvini
(Abe et al. 1994b). Taking these results into considerations
along with the above-mentioned results, it is concluded that
LAZY1 functions between amyloplast sedimentation and
lateral auxin translocation in gravity signal transduction. It is
possible that LAZY1 interacts directly or indirectly with
the plasma membrane-associated PIN proteins to regulate
lateral translocation of auxin (Friml et al. 2002, Noh et al
2003). If so, however, LAZY1 must be a gravitropismspecific PIN regulator, because phototropism of coleoptiles is
not impaired in lazy1 mutants (Yoshihara and Iino 2006).
This study provides genetic as well as kinetic evidence
for the occurrence of LAZY1-independent gravity signaling. Although gravity-induced auxin asymmetry does not
occur in lazy1 coleoptiles, gravitropism is only partially
impaired in lazy1 coleoptiles. This partial impairment
cannot be attributed to gene redundancy because rice has
no homologous gene. Also, two of the three lazy1 mutants
are apparently LAZY1 null (Fig. 1B). The most straightforward conclusion is that rice has a LAZY1-independent
signaling pathway that does not involve auxin asymmetry.
Furthermore, circumnutation is totally absent from lazy1
coleoptiles, strongly suggesting that circumnutation is
controlled only by the LAZY1-dependent pathway.
The above conclusion on the presence of a LAZY1independent signaling pathway is related to the following
long-discussed issues. First, it was once reported that the
lack of sedimentable amyloplasts only partially reduces
gravitropic response in Arabidopsis hypocotyls (Caspar and
Pickard 1989). The gravitropic response remaining in such
mutants can become smaller under certain growth conditions, but is hard to eliminate (Fitzelle and Kiss 2001).
Thus, there is no strict evidence that amyloplast-based
gravity sensing accounts for the entire gravitropic response.
This argument does not indicate that LAZY1-independent
gravity signaling is based on a different sensing mechanism,
but nevertheless would warrant investigation of alternative
mechanisms such as the one proposed by Wayne and
686
Rice LAZY1 and gravity signaling
co-workers (Stevens et al. 1997 and references cited therein).
Secondly, it has not yet been clearly demonstrated if the
entire gravitropic response of any given organ is mediated
by lateral auxin translocation (Iino 2001, Haga and Iino
2006). A recent investigation suggested that jasmonic acid
mediates, at least in part, the gravitropism of rice coleoptiles
(Gutjahr et al. 2005). Our results do not necessarily
demonstrate that the control of auxin distribution is not
involved in LAZY1-independent gravitropism, because it is
possible that auxin translocation within peripheral cell
layers instead of across the organ (Macdonald and Hart
1987, Haga and Iino 2006) is responsible for this
gravitropism. Nevertheless, our results would warrant
investigation of gravity signaling that does not involve the
control of auxin distribution.
This study provided a few other results that are worth
mentioning. The leaf-sheath pulvinus is the common and
major gravity-responding part of mature Gramineae plants
(Kaufman et al. 1987). In rice, it has additionally been
shown that the lamina joint contains sedimentable amyloplasts and undertakes a gravity-induced nastic movement
(Maeda 1965, Nakano and Maeda 1978). Indeed, LAZY1 is
highly expressed in the lamina joint (Fig. 3B). It is probable
that the LAZY1-mediated gravity signaling also takes
place in the lamina joint, contributing to the maintenance
and control of the upright orientation of leaf blades.
Another interesting set of results came from the analysis of
LAZY1-complemented lines. LAZY1 is little expressed in
wild-type primary roots (Fig. 3A), and mutant analysis
indicated that LAZY1 is not required for gravitropism and
circumnutation of primary roots (Figs. 2C, 4B). However,
LAZY1 affected these processes when expressed in primary
roots (Figs. 2C, 4). It has also become apparent that
overexpressed LAZY1 inhibits lateral root formation
although it is not required in this process of the wild type.
Auxin is known to be involved in root gravitropism and
lateral root formation (for a review, see Woodward and
Bartel 2005). In light of the conclusion that LAZY1
functions upstream of lateral auxin translocation in gravity
signaling, it may be suggested that LAZY1 affects the
auxin-mediated responses in roots by affecting the distribution of auxin.
The model shown in Fig. 6 summarizes the two
gravity signaling pathways concluded to operate in rice
shoots, or at least in coleoptiles. In this model, LAZY1 is
considered to regulate coleoptile circumnutation, in principle, independently of gravitropism. This follows our
previous conclusion that, although gravity sensing is
required for circumnutation, circumnutation is not
based on gravitropism itself (Yoshihara and Iino 2006).
The model only represents genetic evidence and does not
explain how oscillatory movement of circumnutation is
created. It is anticipated that investigation of the
LAZY1-independent
pathway
Gravitropism
Gravity
LAZY1
Lateral auxin
translocation
Circumnutation
Fig. 6 A model for gravity signaling in rice shoots. The presented
model summarizes the LAZY1-dependent and -independent gravity
signaling pathways discussed in the text. In addition to those
described in the model, the gravity sensing for the LAZY1dependent pathway is considered to be based on amyloplast
displacement. We do not conclude whether or not the gravity
sensing in the other pathway is also based on amyloplast
displacement, leaving the possibility that a distinct sensing
mechanism participates in this pathway.
molecular function of LAZY1 provides a clue to our
understanding of the mechanism of circumnutation, a long
studied subject (Darwin 1880, Brown 1991, Kitazawa et al.
2005, Yoshihara and Iino 2006), as well as of shoot
gravitropism.
Materials and Methods
Plant growth conditions
The dark-grown and red light-grown seedlings of rice were
prepared as described in Yoshihara and Iino (2005). In brief,
seedlings were raised, unless otherwise specified, in acrylic cuvettes
filled with 0.7% agar. The fluence rate of the red light used to
prepare red light-grown seedlings was 2.5–3.0 mmol m2 s1.
An infrared viewer was used to handle dark-grown seedlings for
experiments. Mature plants were grown in a soil mixture in a
greenhouse or growth cabinets as described in Haga et al. (2005).
Map-based cloning
Map-based cloning of the LAZY1 locus was conducted as
described in Haga et al. (2005) using the molecular markers
obtained from the DNA bank at the National Institute of
Agrobiological Sciences (http://www.dna.affrc.go.jp/). Public databases were used to design the CAPS markers for fine mapping
(http://shenghuan.shtu.edu.cn/genefunction/index.htm) and to
analyze the genes contained in the LAZY1-linked region
(http://ricegaas.dna.affrc.go.jp/).
Complementation
The full-length LAZY1 cDNA, obtained from the Rice
Genome Resource Center, was cloned into the binary vector
pIG121-Hm (Ohta et al. 1990) using XbaI and SacI restriction
sites. The resulting vector was introduced into Escherichia coli
(strain DH5a), transferred to Agrobacterium tumefaciens (strain
EHA101) and transformed into the lazy1-1 mutant by
Agrobacterium-mediated transformation (Hiei et al. 1994).
Control transformants were produced similarly using the empty
vector.
RNAi analysis
The RNAi construct was prepares as described in Miki and
Shimamoto (2004). In brief, the DNA fragment for RNAi analysis
Rice LAZY1 and gravity signaling
(Supplementary Fig. S2A) was amplified by PCR from LAZY1
cDNA. This fragment was cloned into the Gateway vector
pENTR/D-TOPO (Invitrogen, Carlsbad, CA, USA) and transferred into the two recombination sites of the pANDA vector using
LR Clonase (Invitrogen). The resulting vector was introduced
into A. tumefaciens and transformed into Nipponbare by
Agrobacterium-mediated transformation.
Physiological analyses
Gravitropism and circumnutation of coleoptiles were
analyzed as described in Yoshihara and Iino (2005). The tracer
experiments with [3H]IAA (3-[5(n)-3H]IAA, 925 GBq mmol1;
Amersham Pharmacia Biotech, Buckinghamshire, UK) were
conducted as described in Haga et al. (2005) using gravitropic
stimulation in place of phototropic stimulation.
To investigate primary root growth and lateral root
formation, each of the surface-sterilized caryopses was placed
in a hole made in a thin round sheet of silicon sponge
(diameter, 18 mm; autoclaved before use), floated on water in an
autoclaved and covered 25 mm test tube (20 cm long, 40 ml of
deionized water), and incubated at 25 0.58C under white light
(5 mmol m2 s1). A surface swelling of about 0.2 mm was counted
as a lateral root.
Transcript analysis
Tissues were harvested, stored, and extracted for total RNA
as described in Haga and Iino (2004). LAZY1 transcript
abundance was analyzed by reverse transcription–PCR
(RT–PCR) or real-time RT–PCR. RT–PCR was conducted as
described in He et al. (2005). Real-time RT–PCR was performed
on an ABI 7300 real-time PCR (Applied Biosystems, Foster City,
CA, USA) using the SuperScript III Platinum SYBR Green OneStep qRT-PCR kit (Invitrogen). Reverse transcription conditions
were 30 min at 558C and subsequent PCR conditions were 10 min
at 958C and 45 cycles of 15 s at 958C, 30 s at 608C and 30 s at 728C,
followed by 1 min at 408C and a melting cycle (60–958C). Samples
to be compared were run simultaneously in triplicate. Each run
included a concentration series of one of the analyzed samples for
calibration purposes. In either analysis, OsUBQ (GenBank
accession No. L31941) was amplified as an internal control.
The gene-specific primers used for RT–PCR were 50 -ACGGT
GACGAAGAGCAAGTT-30 (forward) and 50 -ACAGCACAT
TCAAGCCCTTC-30 (reverse) for LAZY1, and 50 -ATCACG
CTGGAGGTGGAGT-30 (forward) and 50 -AGGCCTTCTG
GTTGTAGACG-30 (reverse) for OsUBQ. These primers were
designed using Primer3 (http//www-genome.wi.mit.edu/cgi-bin/
primer/primer3_www.cgi). The gene-specific primers used for
real-time RT–PCR were 50 -ACGGTGACGAAGAGCAAGTT-30
(forward) and 50 -AGGAGTGTGTTCTCGGGGTA-30 (reverse)
for LAZY1, and 50 -CAAGCCAAGAAGATCAAGC-30 (forward)
(reverse)
for
and
50 -GTAGTGGCGATCGAAGTGGT-30
OsUBQ. These primers were designed using OligoPerfect
Designer (Invitrogen).
Supplementary material
Supplementary material mentioned in the article is available
to online subscribers at the journal website www.pcp.
oxfordjournals.org.
687
Acknowledgments
We thank Dr. Hideyuki Takahashi (Tohoku University)
for providing lazy1-1 seeds, Dr. Kenzo Nakamura
(Nagoya University) for providing the binary vector
pIG121-Hm, Dr. Ko Shimamoto (Nara Institute of Science and
Technology) for providing the pANDA vector, and Drs. Takefumi
Hattori and Keiichi Baba (Kyoto University) for allowing us to use
radioisotope research facilities. We also thank Dr. Rainer Hertel
for stimulating discussion, Dr. Ken Haga for technical advice, and
Ms. Yuki Mitsubayashi and Mr. Makoto Nakazono for technical
assistance. Tos17 insertion lines were provided by the Rice Genome
Resource Center, National Institute of Agrobiological Science,
Japan. This work was supported by a Grant-in-Aid for Special
Research (No. 17084007) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and a grant from
the Ministry of Agriculture, Forestry and Fisheries of Japan
(Green Technology Project IP1006).
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