Download - Wiley Online Library

The Plant Journal (2011) 38, 433–442
doi: 10.1111/j.1365-313X.2011.04698.x
OsIAA23-mediated auxin signaling defines postembryonic
maintenance of QC in rice
Ni Jun, Wang Gaohang, Zhu Zhenxing, Zhang Huanhuan, Wu Yunrong and Wu Ping*
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058,
China
Received 3 April 2011; revised 21 June 2011; accepted 2 July 2011; published online 18 August 2011.
*
For correspondence (fax +86 571 88206613; e-mail [email protected]).
SUMMARY
Although the quiescent center (QC) is crucial to root development, the molecular mechanisms that regulate its
postembryonic maintenance remain obscure. In this study, a semi-dominant mutant that exhibits pleiotropic
defects in root tissues, which includes the root cap, lateral and crown roots, was isolated. The mutant is
characterized by a loss of QC identity during postembryonic development, and the displayed defects result
from a stabilizing mutation in domain II of OsIAA23 (Os06g39590). Expression of OsIAA23 is specific to the QC
of the root tip during the development of primary, lateral and crown roots. Consistent with OsIAA23
expression in the QC, the auxin signaling marked by DR5p::GUS (ß-glucuronidase) was absent in the QC region
of Osiaa23. Transgenic rice plants harboring Osiaa23 under the control of the QHB promoter mimic partially the
defects of Osiaa23. These results indicate that the maintenance of the QC is dependent on OsIAA23-mediated
auxin signaling in the QC. These findings provide insight into Aux/IAA-based auxin signaling during
postembryonic maintenance of the QC in plants.
Keywords: Oryza sativa L., root development, quiescent center, OsIAA23, auxin signaling.
INTRODUCTION
The importance of the quiescent center (QC) to root development has been well documented in Arabidopsis. In
Arabidopsis, nearly all of the cells in the root are derived
from four types of stem cells. These stem cells are controlled
by the QC, which is composed of a small number of mitotically inactive central cells (Benfey and Scheres, 2000). The
QC acts to maintain the surrounding stem cells (SC) in an
undifferentiated state, and laser ablation of the QC has been
shown to cause differentiation of stem cells (van den Berg
et al., 1997).
Differences in the root structure of rice and Arabidopsis
have been noted. In contrast with the single layer epidermis–
endodermis structure in Arabidopsis, rice roots undergo
eight successive asymmetrical periclinal cell divisions following the first anticlinal division. This generates the
epidermis–endodermis, sclerenchyma layer, exodermis
and five layers of cortex (Rebouillat et al., 2009). In Arabidopsis, the epidermis and lateral root cap cell files are derived
from the same stem cell (Dolan et al., 1993). In rice, the
epidermis and lateral root cap cell files derive from independent initials (Coudert et al., 2010). In spite of these
differences, the basic developmental model of cell types in
rice roots is similar to Arabidopsis (Kamiya et al., 2003a,b;
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd
Cui et al., 2007). The molecular mechanism of QC/SC
maintenance and the development of different cell files in
Arabidopsis have been well studied (reviewed by IyerPascuzzi and Benfey, 2009), while knowledge of these
processes in rice, the monocot model plant, remains limited.
Auxin is crucial to many aspects of plant development,
including lateral and crown root formation, embryonic
development, and stem cell maintenance (Inukai et al.,
2005; Liu et al., 2005; Woodward and Bartel, 2005; Galinha
et al., 2007). Inhibition of auxin transport by N-1-naphthylphthalamic acid (NPA) causes abnormal auxin accumulation, resulting in changes in root tip cell identities (Sabatini
et al., 1999). Accordingly, the disruption of auxin transport
around the QC in one of the Arabidopsis PIN (PIN-FORMED)
mutants, Atpin4, causes a defect in the maintenance of
endogenous auxin gradients and leads to abnormal cell
division (Friml et al., 2002). PLETHORA (PLT) protein expression patterns are thought to be determined by auxin
gradients. The dosage can be translated into distinct cellular
responses, and high levels of PLT activity promote stem cell
identity and maintenance (Galinha et al., 2007). High-resolution measurements of endogenous indole-3-acetic acid
(IAA) concentrations revealed a distinct maximum in the QC
433
434 Ni Jun et al.
of the root apex (Petersson et al., 2009). These results
indicate that auxin in the QC may play an important role,
although direct evidence is not available.
Auxin signaling controlled by Aux/IAA in plants has been
well reviewed. Briefly, intracellular auxin is perceived by the
TIR1/AFB1-3 receptors. This triggers the degradation of
AUX/IAA proteins by the ubiquitin–proteasome pathway,
prompted by higher intracellular auxin concentrations.
Under low intracellular auxin concentrations, Aux/IAA proteins interact and inhibit the activity of AUXIN RESPONSE
FACTOR (ARF), thereby blocking the activity of ARFs (Lau
et al., 2008). The Aux/IAA comprises four highly conserved
domains, designated as domain I, II, III and IV (Abel et al.,
1994). A highly conserved core sequence, GWPPV in domain
II, is responsible for the rapid degradation of Aux/IAA
proteins (Ramos et al., 2001). Mutations in the core
sequence block the degradation of AUX/IAAs, which results
in constitutive suppression of ARF activities and ultimately
causes stabilized mutants due to the interruption of the
auxin signaling pathway (Ouellet et al., 2001). Many stabilized mutants of iaa have been isolated. These mutants
illustrate the importance of auxin signaling via IAAs in many
aspects of root development (reviewed by Overvoorde et al.,
2010).
This report describes the isolation and characterization of
an Aux/IAA gene in rice, OsIAA23. A stabilizing mutation
of OsIAA23 exhibited defects in postembryonic maintenance
of the QC that caused the disintegration of the root cap and
termination of root growth. The initiation of lateral root and
crown root primordia was also blocked. Here, the specific
expression pattern of OsIAA23 in the root tip QC during postembryogenesis is described, and the impact of OsIAA23 on
root tissue development is discussed. These results provide
direct evidence of the importance of Aux/IAA-mediated
auxin signaling in the maintenance of the QC.
RESULTS
Isolation and characterization of the mutant
A mutant with lateral root (LR) defects was isolated from an
ethyl methane sulfonate-mutagenized population of rice
‘Nipponbare’. Mutant plants were self-pollinated and three
root phenotypes were distinguished in the offspring. These
phenotypes, wild type (WT), reduced number of lateral and
crown roots, and defects in both lateral and crown roots,
occurred in a 1:2:1 ratio (24:53:23, P = 0.857) (Figure 1a and
Table S1). Based on these data, the pleiotropic defects in the
root tissues of the mutant are controlled by a semi-dominant
single gene. This was confirmed by the trait segregation in a
backcross population between WT and the mutant (24:26,
P = 0.843).
Although the homozygous mutants were able to complete
their life cycle, plant growth was inhibited and fertility was
greatly affected. The heterozygous mutants were inter-
mediate in phenotype and exhibited normal fertility (Figure 1e,f). In addition, neither auxin nor ethylene treatment
was able to rescue the defects (Figure S1).
To examine the lateral and crown root primordia in the
mutant, the GUS reporter gene driven by the OsCYCB1;1
promoter was transformed into the heterozygous mutants
and the segregating offspring were analyzed. The GUS
staining and cross-sections of WT, the heterozygous mutant
and the homozygous mutant plants revealed that the lateral
and crown root primordia were absent in the homozygous
mutant and the LR primordia were decreased in the heterozygous mutant (Figure 1b–d,g–i).
The root gravitropic response in the mutant was also
reduced. Wild type, heterozygous mutants and homozygous
mutants were grown for 5 days in the g1 direction, followed
by growth for 2 days in the g2 direction. The homozygous
mutants showed a loss of gravitropic response when
compared to the WT, and the heterozygous mutants showed
a partial loss of gravitropic response (Figure 1j).
Root QC identity is lost in the mutant during postembryonic
development
To determine whether the root cap, the sensor of gravity,
was structurally disrupted in the mutant, the root tips of WT,
heterozygous mutants and homozygous mutants were
observed at 1, 5 and 10 days after germination (DAG) using a
stereomicroscope. The results revealed the presence of
normal root caps in both the heterozygous and homozygous
mutants at 1 DAG (Figure 2a1–a3). However, at 5 DAG, the
root cap was completely absent in the homozygous mutants
and the size of the root cap was reduced in the heterozygous
mutants (Figure 2b1–b3). At 10 DAG, root hairs in the root tip
were observed, indicating the degradation of the root meristem and the termination of root growth in the homozygous
mutant (Figure 2c1–c3).
To further investigate the root tip cell organization in the
mutant, longitudinal sections of root tips were examined. At
5 DAG, the root cap of the homozygous mutant was lost.
Additionally, the cell pattern in the meristem was abnormal
when compared with WT. An abnormal cell arrangement in
the QC region was also observed (Figure 2h1–i2). To determine the QC identity of the mutant, the GUS reporter gene
controlled by the QHB promoter (Kamiya et al., 2003b) was
transformed into the heterozygous mutants, and the GUS
staining was examined in WT, heterozygous mutants and
homozygous mutants. Consistent with the previously
reported QHB expression in the QC (Kamiya et al., 2003b),
GUS staining of WT revealed GUS activity in this region
(Figure 2l). In the homozygous mutant at 5 DAG, no staining
was detected in the same region (Figure 2m). At 1 DAG, GUS
staining as detected in both WT and mutants (Figure 2j,k).
However, a detailed section analysis revealed that abnormal
transverse cell divisions had occurred in the mutant when
compared to the WT (Figure 2f1–g2). Considering that
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442
Postembryonic maintenance of QC in rice 435
Figure 1. Characterization of the mutant.
(a) Root growth of 5-day-old seedlings of the wild
type (WT) (left) and the heterozygous (middle)
and homozygous mutant (right). Bar = 2 cm.
(b–d) Cross-sections at the stem base of the WT
(b), heterozygous mutant (c), and homozygous
mutant (d). Bar = 0.5 mm.
(e) Plants at the grain filling stage. From left to
right: WT, the heterozygous mutant, and the
homozygous mutant.
(f) The panicle of three genotypes. From left to
right: WT, the heterozygous mutant, and the
homozygous mutant. Bar = 2 cm.
(g–i)
Stereomicroscopic
images
of
OsCYCB1;1p::GUS in the roots of the three
genotypes. From left to right: WT, the heterozygous mutant, and the homozygous mutant.
Bar = 0.5 mm.
(j) Gravitropic response of three genotypes.
Seedlings were grown for 5 days under g1
gravitropic conditions and then moved to g2
where they were grown for 2 additional days.
From top to bottom: WT, the heterozygous
mutant, and the homozygous mutant.
Bar = 2 cm.
Figure 2. Time-lapse characterization of the root
tip in the mutant.
(a1–c3), Stereomicroscope images of the root
apex of three genotypes at 1 DAG (a1–a3), 5 DAG
(b1–b3), and 10 DAG (c1–c3). From left to right:
the wild type (WT), the heterozygous mutant, and
the homozygous mutant. Bars = 500 lm.
(d1–i2), Vertical sections of the root tip of the WT
(left) and the homozygous mutant (right) at 0
DAG (d1–e2), 1 DAG (f1–g2), and 5 DAG (h1–i2).
The images shown on the right are magnifications of the images on the left. Bars = 200 lm.
(j-m), QHBp::GUS expression in the root tips of
the WT at 1 DAG (j) and 5 DAG (l), and of the
homozygous mutant at 1 DAG (k) and 5 DAG (m).
Bars = 200 lm.
(a)
(b)
(c)
(e)
(g)
(a1)
(b1)
(c1)
(j)
(h)
(j)
(a3)
(b2)
(d1)
(d2)
(e1)
(e2)
(f1)
(f2)
(g1)
(g2)
(h1)
(h2)
(i1)
(i2)
(b3)
(c2)
(k)
(f)
(i)
(a2)
(d)
(c3)
(l)
(m)
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442
436 Ni Jun et al.
normal converged cell files were observed in the mutant at 1
DAG, it is suspected that the abnormal transverse cell
divisions occurred in the QC during the 24 h after germination. To verify this hypothesis, germinated mutant seeds
without apparent roots were used (designated 0 DAG) for
further investigation. The root tips that had not emerged
from the seeds were separated for section analysis. No
difference in the QC structure was observed between WT
and mutant roots (Figure 2d1–e2). Taken together, the
results suggest that the maintenance of QC structure in the
mutant is impaired during postembryonic root development.
(a)
Cloning of the OsIAA23 gene
A map-based positional approach was used to identify the
gene from an F2 population derived from a cross between
the heterozygous mutants and indica rice (Kasalath). The
mutated gene responsible for the root tissue defects was
mapped between two simple sequence repeat (SSR) markers, RM1340 and RM20328, on chromosome 6. This region
includes an Aux/IAA family gene, OsIAA23 (Os06g39590).
The OsIAA23 gene, sequenced from homozygous mutant
genomic DNA, was found to contain a G to A base pair
substitution causing a glycine (G) to glutamate (E) change
(Gly64Glu) in domain II of OsIAA23 (Figure 3a). To confirm
this result, a dCAPS marker was developed. Using this
marker, all of the homozygous mutants produced higher
mobility bands than WT, and all of the heterozygous
mutants produced double bands (Figure 3b).
To investigate whether the root tissue defects in the
mutant were caused by the mutation of OsIAA23, WT plants
were transformed with the mutated gene under the control
of its native promoter. The majority of the transgenic plants
exhibited phenotypes characteristic of heterozygous
Osiaa23, including fewer LRs and smaller root caps. A few
transgenic plants also exhibited phenotypes characteristic of
the homozygous Osiaa23 (Figure 3c). Sequencing analysis
found an allelic mutant of Osiaa23-2. The second allele
uncovered a point mutation in the domain II of OsIAA23,
resulting in a change of proline (P) to leucine (L) (Pro67Leu).
The phenotypic traits of Osiaa23-2 roots were similar to
those of Osiaa23 (Figure S2). These results confirm that the
root tissue defects in the mutant are caused by the point
mutation in domain II of OsIAA23.
Response to auxin is reduced in Osiaa23
To investigate the auxin response of the Osiaa23 mutant, WT
and Osiaa23 mutants were treated with a-naphthalene acetic
acid (NAA). The reduction of root length in WT was more
significant than that of the Osiaa23 mutant (Figures 4(a) and
S1b). Moreover, the root hairs of WT were notably induced
by NAA, while the development of root hairs in Osiaa23
mutant was not affected by NAA treatment (Figure 4b).
These results indicated a reduced sensitivity to auxin in the
(b)
(c)
Figure 3. Map-based cloning and complementation test of OsIAA23.
(a) A point mutation was detected in the mutant, which resulted in a G to A
base pair change of an Aux/IAA gene (OsIAA23), causing a change of glycine
(G) to glutamate (E) in the conserved core sequence of domain II. The exons of
OsIAA23 are indicated by thick rectangles, and introns are indicated by thin
lines. The four domain positions are indicated by black boxes.
(b) Confirmation of the point mutation in Osiaa23 using the dCAPS marker.
From left to right: the WT, the heterozygous Osiaa23, and the homozygous
Osiaa23, respectively.
(c) Two transgenic plants containing the Osiaa23 gene exhibited phenotypes
characteristic of the heterozygous and homozygous Osiaa23.
Osiaa23 mutant. When the Osiaa23 mutant was treated with
a higher concentration of NAA (1 lM), a few LRs were
induced in the Osiaa23, although other defects were not
rescued (Figure 4a).
To examine the auxin response of the Osiaa23 mutant at
the molecular level, the expression of OsIAA23 and two
other early auxin-responsive genes, OsIAA20 (Jain et al.,
2006) and ARL1/CRL1 (Inukai et al., 2005; Liu et al., 2005),
were investigated in the roots of the Osiaa23 mutant using
real-time reverse transcription polymerase chain reaction
(RT-PCR) analysis. The results showed that the induced
expression of these genes was suppressed significantly in
Osiaa23. Additionally, the results showed that OsIAA23 is
induced by exogenous auxin (Figure S2).
To further investigate the influence of the stabilizing
mutation of OsIAA23 on auxin signaling, the expression
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442
Postembryonic maintenance of QC in rice 437
Figure 4. Alteration of auxin response in the
Osiaa23 mutant.
(a) Root growth performance of the wild type
(WT; left) and Osiaa23 (right) plants grown in a
solution culture with 1 lM NAA for 7 days.
Bar = 1 cm.
(b) Proliferation of root hairs at the root apex of
WT (left) and Osiaa23 (right) induced by 0.1 lM
NAA treatment. Bar = 0.5 mm.
(c, d) Cross-sections indicate DR5p::GUS expression in the root zone with the initiation of lateral
root (LR) primordia of WT (c) and Osiaa23 (d).
Bars = 20 lm.
(e, f) GUS staining of cross-sections at the shoot
base with the initiation of crown root primordia
in WT (e) and Osiaa23 (f). Bar = 0.5 mm.
(g, h) GUS staining of the root apex of 1 d-old
seedlings of WT and Osiaa23. Bars = 100 lm.
(a)
(b)
pattern of DR5p::GUS was determined. Although Osiaa23
failed to form lateral or crown roots, a significant difference
in the location of lateral and crown root primordia
formation was not detected between WT and Osiaa23,
respectively (Figure 4c–f). The DR5p::GUS pattern in the
Osiaa23 root tip was examined at 1 DAG to exclude the
interference of the irregular cell pattern in the mutant. At 1
DAG, GUS staining was observed in the columella, QC and
metaxylem in WT (Figure 4g). In contrast, no GUS staining
was observed in the QC region of the mutant (Figure 4h).
The DR5p::GUS expression pattern indicates that auxin
signaling is blocked in the QC region of the Osiaa23
mutant.
Expression of OsIAA23 is specific to the QC in the root tip
Using RT-PCR, OsIAA23 expression in the various organs of
WT plants was analyzed. OsIAA23 was found to be constitutively expressed in all of the examined tissues, although a
lower abundance was observed in the embryo (Figure S4d).
WT plants were transformed with a GUS (b-glucuronidase)
gene driven by the OsIAA23 promoter to characterize further
the expression pattern of OsIAA23. GUS staining was
observed in the vascular tissues of stems and leaves
(Figure S3e,f) and in the anthers (Figure S3b,cIn the maturation zone of the root, dense staining was noted in the stele
and epidermis (Figure 5d,e). In contrast, the GUS staining in
the root tip was exclusively located in the QC (Figure 5a,c).
(c)
(d)
(e)
(f)
(g)
(h)
The OsIAA23 expression pattern was similar in the LRs and
crown roots (Figure 5b).
Because the QC structure in the root was altered during
postembryonic development (Figure 2d1–i2), the temporal
and spatial expression pattern of OsIAA23 was further
investigated. Although OsIAA23 was expressed in the QC
during postembryonic development, a uniform expression
pattern was observed in the root apical meristem of the
embryo (Figure S4d). During the process of LR development, OsIAA23 was expressed initially uniformly in the
primordium during early stages, then the expression gradually converged until it was ultimately restricted to the QC of
the lateral roots (Figure 5f,g). A similar pattern was found
during the development of crown root primordia (Figure 5h–j).
The maintenance of the QC is dependent on OsIAA23mediated auxin signaling
The QC-specific expression of OsIAA23 in the root tip (Figure 5a,c) and the loss of auxin signaling in the QC region
(Figure 4h) indicated that OsIAA23-mediated auxin signaling in the QC may be crucial to the maintenance of the QC.
Five transgenic lines harboring Osiaa23 driven by the QHB
promoter (Kamiya et al., 2003b) were generated to investigate this hypothesis (Figure 6a). As expected, the transgenic
plants exhibited smaller root caps. Vertical sections of the
root tips showed abnormal divisions of QC cells in the
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442
438 Ni Jun et al.
(a)
(c)
(b)
(d)
(e)
(g)
(f)
(h)
Figure 5. Expression pattern of OsIAA23
revealed by a GUS reporter line.
(a, b) Stereomicroscope images showing GUS
staining in the primary root tip (a) and the lateral
root (LR) (b). Bar = 200 lm.
(c) Vertical section of the root tip of the transgenic plants.
(d, e) Cross section at the matured root zone with
the initiation of LR primordia of the transgenic
plants and the magnified view of the stele (e).
Bar = 20 lm.
(f, g) OsIAA23 (f) and QHB (g) expression during
the process of LR primordia development.
Bar = 20 lm.
(h–j) Cross-sections at the shoot base. (h), (i) and
(j) are different stages of crown root primordia.
Bar = 100 lm.
(i)
(j)
transgenic plants (Figure 6e–j). Additionally, the numbers of
lateral and crown roots were reduced in the transgenic
plants (Figure 6b–d). These results provided evidence that
OsIAA23-mediated auxin signaling regulated postembryonic
maintenance of the QC in rice.
DISCUSSION
This study reports the isolation of a stabilized rice mutant of
OsIAA23. This mutant, caused by a point mutation in the
conserved core sequence GWPPV of domain II of OsIAA23,
results in pleiotropic defects in the root tissues. These
defects include formation of the root cap, lateral and crown
roots, and the maintenance of QC identity during postembryonic development. Our report describes a stabilized
Aux/IAA mutation with pleiotropic effects on root development in rice. As a distal organizer in the root apex, the disappearance of QC identity results in the disintegration of the
root cap and the impairment of the root apical meristem.
Previous reports demonstrated that loss-of-function of IAAs
did not result in visible defects (Overvoorde et al., 2005).
Screening for revertants of Osiaa23 mutant plants identified
one plant that contained a mutation in domain IV of the
protein encoded by Osiaa23 that displayed a normal phenotype (Figure S6). Domain IV in IAA proteins is crucial for
the interaction between IAAs and ARFs (Ulmasov et al.,
1997). The rescue of the iaa23-dependent phenotype suggests that the point mutation that leads to a change in amino
acid in domain IV also leads to loss-of-function of the
stabilized OsIAA23. This result also indicates that loss-offunction of OsIAA23 does not affect root growth, due to
functional redundancy of IAA proteins.
OsIAA23-mediated auxin signaling is crucial to the identity
of the QC during postembryonic root development
The QC controls the undifferentiated states of the nearby SC,
and loss of the QC results in the differentiation of these stem
cells (van den Berg et al., 1997). Two types of SC are
responsible for the production of the root cap, the first type
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442
Postembryonic maintenance of QC in rice 439
(a)
(c)
(b)
(d)
(e)
(f)
(g)
et al., 2003). It is notable that no clear difference in the GUS
staining in the root cap and stele, except for the QC region,
was found between the WT and Osiaa23 mutant
(Figure 4c–f). This finding suggests that the QC may be the
only tissue in which there is no functional redundancy of
Aux/IAA-mediated auxin signaling.
It has been proposed that auxin signaling is crucial for
root stem cell niche patterning during both embryonic and
postembryonic development (Aida et al., 2004). The specification of the QC in embryonic development requires auxin
signaling (Berleth and Jurgens, 1993; Hardtke and Berleth,
1998; Hamann et al., 1999, 2002). The data presented here
suggest that another OsIAA (or other OsIAAs) may mediate
the auxin signaling of the QC during embryonic development. This hypothesis is supported by the spatio-temporal
expression of OsIAA23 in both the primary root (Figure 5a)
and the lateral or crown root primordial development
(Figure 5f–j).
QC identity during postembryonic development determines
lateral root development in rice
(h)
(i)
(j)
Figure 6. Phenotype and histological characterization of the QHBp::Osiaa23
transgenic rice.
(a) The construct of QHBp::Osiaa23.
(b) Root phenotype of the wild type (WT) and two lines of transgenic rice.
Bar = 2 cm.
(c, d) Number of lateral roots (LR) (c) and crown roots (d) of 7 d-old WT and
transgenic rice. The data represents a mean of five plants and the bar indicates
the SD.
(e–j) Vertical sections of the root tip of the WT (e) and two transgenic lines
(f, g). (h), (i) and (j) are magnifications of (e), (f) and (g). Bar = 200 lm.
is the columella initial cell and the second is the LR cap initial
cell. These cells generate the columella cells and LR cap
cells, respectively (Coudert et al., 2010). With the loss of QC
identity, the root cap SC differentiate and stop supply of new
root cap cells. Up to 1 DAG, no change in the root cap
structure of Osiaa23 was observed when compared with the
root cap of WT. However, at 5 DAG, the root cap had disappeared in Osiaa23 (Figure 2). This situation indicates that
OsIAA23-mediated auxin signaling is crucial to the identity
of the QC during postembryonic root development. This
conclusion is supported by other evidence: the QC identity of
Osiaa23 was lost gradually during postembryonic development (Figure 2), and the auxin signaling indicated by the
DR5p::GUS reporter gene is blocked in the QC region of the
root tip (Figure 4h). DR5, a synthetic promoter, has been
widely used to monitor auxin distribution in vivo and auxin
responses at the cellular level (Sabatini et al., 1999; Benkova
The development of lateral and crown roots is a postembryonic event (Rebouillat et al., 2009). We investigated
the earlier stage of root on the LR primordium at 1 day after
germination and did not found lateral root primordium. In
roots, auxin is transported acropetally through the central
cylinder toward the root tip. There, it is redistributed into a
basipetal stream through the LR cap and the epidermis, and
then refluxes upward along the meristem back into the
central downward flow to complete the auxin loop (reviewed
by Kepinski and Leyser, 2005). Pericycle cells in the basal
meristem require increased auxin levels to attain founder
cell identity for the development of LR primordia (Dubrovsky
et al., 2008). The Osiaa23 mutant exhibits defects in both the
stele and root cap, which may disturb the auxin loop in the
root tip and ultimately result in LR defects. Acropetal to
basipetal auxin signaling loop from LR cap is important for
lateral root development. Thus impairment of root cap
development during postembryonic growth in Osiaa23
mutant may disturb auxin signaling for LR primordium
initiation.
Up to 1 DAG, the changed central cylinder structure of
Osiaa23 was observed compared with WT, although the root
cap structure was still maintained (Figure 2f2,g2). In this
case, the mechanism of pericycle differentiation is unclear.
In Arabidopsis, it was previously reported that the roots of
the ahp6 (histidine phosphotransfer protein 6) mutant
(Mahonen et al., 2006) and transgenic plants harboring
Pro35S-VND7:SRDX (vascular-related NAC-Domain7 fused
to the strong repression domain SRDX) (Kubo et al., 2005)
showed perturbations in protoxylem differentiation. However, neither pericycle differentiation nor LR initiation was
disturbed in these plants, which suggested that protoxylem
is dispensable for these morphogenetic events (Parizot
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442
440 Ni Jun et al.
et al., 2008). Based on the data presented here, it is
hypothesized that the impairment of the acropetal and
basipetal auxin signaling loop through the central cylinder
and the LR cap in Osiaa23 results mainly in the impairment
of lateral root primordia initiation at an earlier stage, while
at a later stage, the abnormal central cylinder structure
may affect the development of lateral root primordia initiation.
Recent studies present evidence that a process involving
oscillations in gene expression is the first step in positioning
new LRs (Moreno-Risueno et al., 2010). It is possible that
some oscillating transcriptional regulators may be disturbed
in Osiaa23.
It is proposed that OsIAA23 does not act only in the QC; it
is expressed in various tissues and the reduced sensitivity to
exogenous auxin(reduced root hairs numbers in response to
exogenous NAA (Figure 4, 5). Transgenic rice harboring a
constitutive expressed mutant Osiaa23 (QHBp::Osiaa23)
resulted in a phenocopy of Osiaa23 under its native
promoter, especially in the root tip (Figure 6), while did not
show the reduced sensitivity to exogenous auxin (Figure S5). These results suggest that OsIAA23 does not act
only in the QC, but auxin signaling mediated by OsIAA23 in
the QC plays a unique role in QC maintenance.
METHODS
Plant material, growth conditions and mutant screening
Reduction of auxin signaling mediated by OsIAA23 is
important for crown root primordia development
The initiation of crown root primordia, initiated from the
parenchyma cells adjacent to the peripheral vascular cylinder of the shoot base, is also controlled by auxin signaling
(Inukai et al., 2005; Liu et al., 2005). The data presented here
reveal that the expression of CRL1 (ARL1), a key regulator for
crown root primordia initiation, is not changed in Osiaa23
under normal growth conditions. However, the response of
the gene to exogenous auxin is reduced markedly (Figure S3c). This situation suggests that the reduction of local
auxin signaling caused by the stabilized mutation of
OsIAA23 may be important for crown root primordia
development. Furthermore, the OsIAA23-regulated factor(s)
for crown root primordia development may be downstream
of CRL1(ARL1).
The emergence of crown roots can be enhanced by
flooding and is mediated by ethylene action (Mergemann
and Sauter, 2000). In this study, ethylene was not able to
rescue the defect of crown roots in Osiaa23 (Figure S1c).
This outcome is reasonable because histological analysis
showed that the crown rootless phenotype of Osiaa23 was
caused by the defect of primordia initiation, not the emergence of the primordia (Figure 1d).
OsIAA23 does not act only in the QC, but auxin signaling
mediated by OsIAA23 in the QC plays a unique role
in QC maintenance.
Several indirect pieces of evidence have implicated auxin
signaling in QC maintenance. NPA treatment leads to the
abnormal auxin accumulation in the root tip and results in
unusual QC identities (Sabatini et al., 1999). AtPIN4 is
expressed around the QC region. Atpin4 mutants are
defective in the establishment and maintenance of endogenous auxin gradients, which results in abnormal cell divisions in the QC (Friml et al., 2002). High-resolution
measurements of endogenous IAA concentrations revealed
a distinct maximum in the QC of the root apex. However, the
function of this high auxin concentration was unknown
(Petersson et al., 2009).
The mutant was isolated in an EMS-mutagenized population (Nipponbare). Rice were grown in culture solution (Yoshida et al., 1976)
in a growth room with temperature regimes of 28C/22C (day/night)
and 70% humidity under a 12-h photoperiod. The mutant with
defects in LRs was isolated from 7-day-old seedlings.
Histological observation
The roots of 0 DAG were manually dissected under the stereomicroscope, and the procedures of staining, dehydration, clearing,
infiltration, and embedding were performed according to Liu et al.
(2005). The microtome sections were mounted on glass slides for
imaging (Zeiss Axioskop, http://www.zeiss.de/en).
Auxin treatment and quantitative RT-PCR analysis
For auxin treatment, germinated seeds were plated on the culture
solution containing 0.1 lM NAA (a-naphthalene acetic acid). After
7 days, the root hairs in the root tip were investigated using the
stereomicroscope (Leica DC 300). For higher auxin treatment, 1 lM
NAA was used. For quantitative RT-PCR analysis of auxin-responsive gene expression, 7-day-old seedlings were moved to the rice
culture solution containing 10 lM IAA. After 3 h, the roots were
harvested and total RNA was extracted using the RNeasy Plant Mini
Kit (Qiagen, http://www.qiagen.com) according to the manufacturer’s instructions. First-strand cDNA was synthesized using
SuperScript II reverse transcriptase (Invitrogen, http://www.invitro
gen.com). Real-time RT-PCR was performed using the FastStart
DNA Master SYBR Green I Kit (Roche, http://www/roche.com) on the
LightCycler480 machine (Roche), following the manufacturer’s
instructions.
Cloning of OsIAA23
Heterozygous Osiaa23 (japonica) was crossed with Kasalath
(indica). Approximately half of the F1 population that exhibited the
heterozygous mutant phenotype was selected and self-pollinated.
The derived F2 population was used to map OsIAA23. The gene was
mapped between RM1340 and RM20328 on chromosome
6. A dCAPS marker was developed and the PCR products were
digested using Hinf I. A CAPS marker was also developed to confirm
the mutated site of Osiaa23-2. These PCR products were digested by
MspI. To detect the gain of function allele of the mutant, the
homozygous mutant genomic sequence was amplified by PCR
using the primers OsIAA23-U and OsIAA23-L. A 5.2 kb PCR product
that contained the mutated gene was cloned into pUCm-T (Sangon,
China), followed by digestions with HindIII and EcoRV. The insert
was cloned into the HindIII and SmaI sites of pCAMBIA-1300 and
transformed into the wild-type plant (‘Nipponbare’) via Agrobacterium tumefaciens EHA105.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442
Postembryonic maintenance of QC in rice 441
Histochemical analysis and GUS assay
Histochemical GUS analysis was performed according to Jefferson
et al. (1987). Transgenic plant root samples were incubated with
X-gluc at 37C. After staining, the tissues were rinsed and fixed in
formalin-acetic-alcohol (FAA) for 24 h, embedded in Spurr resin,
and sectioned. Sections (5 lm) were mounted on slides and
photographed.
Construction of GUS fusion constructs
For the DR5p::GUS vector, the DR5 element (Ulmasov et al., 1997)
coupled to the CaMV 35S minimal promoter was amplified and
digested with SalI and BamHI. The construct was then inserted into
pBI101.3, which carries the structural gene for GUS and the terminator sequence of the nopaline synthase (NOS) gene. The primers
used in PCR amplification were DR5-U and DR5-L. For the
OsCYCB1;1p::GUS vector, the promoter of OsCYCB1;1 was amplified by PCR, digested with SalI and KpnI, and inserted into pBI101.3.
The primers used for PCR were CYCB-U and CYCB-L. The
QHBp::GUS vector is described by Kamiya et al. (2003b). These
constructs were transformed into the heterozygous mutant of
Osiaa23 via Agrobacterium tumefaciens EHA105, and the offspring
were analyzed. For developing the OsIAA23p::GUS vector, the
promoter sequence was amplified from Nipponbare genomic DNA
and inserted into the HindIII and BamHI sites of pBI101.3. The
primers used in PCR amplification are listed in Table S2. The construct was transformed into the WT (‘Nipponbare’) via Agrobacterium tumefaciens EHA105.
Construction of the QHBp::Osiaa23 fusion construct
The QHB promoter and the coding region of Osiaa23 were amplified by PCR to obtain the QHBp::Osiaa23 fusion. The primers
QHBOsIAA23-1U and QHBOsIAA23-1L were used to amplify the
promoter of QHB, and the primers QHBOsIAA23-2U and QHBOsIAA23-2L were used to amplify Osiaa23. Overlap extension PCR,
using two partially overlapped PCR products as the template, was
performed. The overlap extension PCR product, obtained using the
primers listed in Table S2, was digested with KpnI and HindIII and
cloned into pGOFS1-g (provided by Professor Wu Changying,
Huazhong Agricultural University, China). The construct was
transformed into the WT (‘Nipponbare’) via Agrobacterium tumefaciens EHA105.
ACKNOWLEDGEMENTS
We thank Dr Philip N. Benfey (Duke University, USA) for reading
the manuscript critically. We also thank Drs Karen Champ and
Nicola Edwards for editing the manuscript. This research is supported by the National Basic Research and Development Program
of China (grant number 2005CB120900) and the Special Program
of Rice Functional Genomics of China (grant number
2006AA10A102).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Exogenous auxin and ethylene response of Osiaa23.
Figure S2. Phenotypes and molecular characterization of Osiaa23-2.
Figure S3. The expression of auxin response genes in the Osiaa23.
Figure S4. RT-PCR analysis of OsIAA23 in various tissues and
expression pattern of OsIAA23::GUS in the tissues above ground.
Figure S5. Phenotypic traits of roots and response to exogenous
auxin treatment (NAA at 0.1 lM) of the transgenic plants harboring
iaa23 driven by promoter of QHB.
Figure S6. A mutant with rescue of iaa23-dependent root traits
designated as mKiaa23.
Table S1. Root parameter of 7d-old seedlings of the wild type,
heterozygous and homozygous mutants.
Table S2. Primers used in this paper.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
REFERENCES
Abel, S., Oeller, P.W. and Theologis, A. (1994) Early auxin-induced genes
encode short-lived nuclear proteins. Proc. Natl. Acad. Sci. USA, 91, 326–330.
Aida, M., Beis, D., Heidstra, R., Willemsen, V., Blilou, I., Galinha, C., Nussaume, L., Noh, Y.S., Amasino, R. and Scheres, B. (2004) The PLETHORA
genes mediate patterning of the Arabidopsis root stem cell niche. Cell, 119,
109–120.
Benfey, P.N. and Scheres, B. (2000) Root development. Curr. Biol. 10, R813–
R815.
Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova, D., Jurgens, G. and Friml, J. (2003) Local, efflux-dependent auxin gradients as a
common module for plant organ formation. Cell, 115, 591–602.
van den Berg, C., Willemsen, V., Hendriks, G., Weisbeek, P. and Scheres, B.
(1997) Short-range control of cell differentiation in the Arabidopsis root
meristem. Nature, 390, 287–289.
Berleth, T. and Jurgens, G. (1993) The role of the monopteros gene in
organising the basal body region of the Arabidopsis embryo. Development, 118, 575–587.
Coudert, Y., Perin, C., Courtois, B., Khong, N.G. and Gantet, P. (2010) Genetic
control of root development in rice, the model cereal. Trends Plant Sci. 15,
219–226.
Cui, H., Levesque, M.P., Vernoux, T., Jung, J.W., Paquette, A.J., Gallagher,
K.L., Wang, J.Y., Blilou, I., Scheres, B. and Benfey, P.N. (2007) An evolutionarily conserved mechanism delimiting SHR movement defines a single
layer of endodermis in plants. Science, 316, 421–425.
Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K.
and Scheres, B. (1993) Cellular organisation of the Arabidopsis thaliana
root. Development, 119, 71–84.
Dubrovsky, J.G., Sauer, M., Napsucialy-Mendivil, S., Ivanchenko, M.G., Friml,
J., Shishkova, S., Celenza, J. and Benkova, E. (2008) Auxin acts as a local
morphogenetic trigger to specify lateral root founder cells. Proc. Natl.
Acad. Sci. USA, 105, 8790–8794.
Friml, J., Benkova, E., Blilou, I. et al. (2002) AtPIN4 mediates sink-driven auxin
gradients and root patterning in Arabidopsis. Cell, 108, 661–673.
Galinha, C., Hofhuis, H., Luijten, M., Willemsen, V., Blilou, I., Heidstra, R. and
Scheres, B. (2007) PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development. Nature, 449, 1053–1057.
Hamann, T., Mayer, U. and Jurgens, G. (1999) The auxin-insensitive bodenlos
mutation affects primary root formation and apical-basal patterning in the
Arabidopsis embryo. Development, 126, 1387–1395.
Hamann, T., Benkova, E., Baurle, I., Kientz, M. and Jurgens, G. (2002) The
Arabidopsis BODENLOS gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo patterning. Genes Dev. 16, 1610–
1615.
Hardtke, C.S. and Berleth, T. (1998) The Arabidopsis gene MONOPTEROS
encodes a transcription factor mediating embryo axis formation and vascular development. EMBO J. 17, 1405–1411.
Inukai, Y., Sakamoto, T., Ueguchi-Tanaka, M., Shibata, Y., Gomi, K., Umemura, I., Hasegawa, Y., Ashikari, M., Kitano, H. and Matsuoka, M. (2005)
Crown rootless1, which is essential for crown root formation in rice, is a
target of an AUXIN RESPONSE FACTOR in auxin signaling. Plant Cell, 17,
1387–1396.
Iyer-Pascuzzi, A.S. and Benfey, P.N. (2009) Transcriptional networks in root
cell fate specification. Biochim. Biophys. Acta, 1789, 315–325.
Jain, M., Kaur, N., Garg, R., Thakur, J.K., Tyagi, A.K. and Khurana, J.P. (2006)
Structure and expression analysis of early auxin-responsive Aux/IAA gene
family in rice (Oryza sativa). Funct. Integr. Genomics, 6, 47–59.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442
442 Ni Jun et al.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher
plants. EMBO J. 6, 3901–3907.
Kamiya, N., Itoh, J., Morikami, A., Nagato, Y. and Matsuoka, M. (2003a) The
SCARECROW gene’s role in asymmetric cell divisions in rice plants. Plant J,
36, 45–54.
Kamiya, N., Nagasaki, H., Morikami, A., Sato, Y. and Matsuoka, M. (2003b)
Isolation and characterization of a rice WUSCHEL-type homeobox gene
that is specifically expressed in the central cells of a quiescent center in the
root apical meristem. Plant J, 35, 429–441.
Kepinski, S. and Leyser, O. (2005) Plant development: auxin in loops. Curr.
Biol. 15, R208–R210.
Kubo, M., Udagawa, M., Nishikubo, N., Horiguchi, G., Yamaguchi, M., Ito, J.,
Mimura, T., Fukuda, H. and Demura, T. (2005) Transcription switches for
protoxylem and metaxylem vessel formation. Genes Dev. 19, 1855–1860.
Lau, S., Jurgens, G. and De Smet, I. (2008) The evolving complexity of the
auxin pathway. Plant Cell, 20, 1738–1746.
Liu, H., Wang, S., Yu, X., Yu, J., He, X., Zhang, S., Shou, H. and Wu, P. (2005)
ARL1, a LOB-domain protein required for crown root formation in rice.
Plant J, 43, 47–56.
Mahonen, A.P., Bishopp, A., Higuchi, M., Nieminen, K.M., Kinoshita, K., Tormakangas, K., Ikeda, Y., Oka, A., Kakimoto, T. and Helariutta, Y. (2006)
Cytokinin signaling and its inhibitor AHP6 regulate cell fate during vascular
development. Science, 311, 94–98.
Mergemann, H. and Sauter, M. (2000) Ethylene induces epidermal cell
death at the site of crown root emergence in rice. Plant Physiol. 124, 609–
614.
Moreno-Risueno, M.A., Van Norman, J.M., Moreno, A., Zhang, J., Ahnert,
S.E. and Benfey, P.N. (2010) Oscillating gene expression determines
competence for periodic Arabidopsis root branching. Science, 329,
1306–1311.
Ouellet, F., Overvoorde, P.J. and Theologis, A. (2001) IAA17/AXR3: biochemical insight into an auxin mutant phenotype. Plant Cell, 13, 829–841.
Overvoorde, P.J., Okushima, Y., Alonso, J.M. et al. (2005) Functional Genomic Analysis of the AUXIN/INDOLE-3-ACETIC ACID Gene Family Members
in Arabidopsis thaliana. Plant Cell, 17, 3282–3300.
Overvoorde, P.J., Fukaki, H. and Beeckman, T. (2010) Auxin control of root
development. Cold Spring Harb Perspect Biol. 2, a001537.
Parizot, B., Laplaze, L., Ricaud, L. et al. (2008) Diarch symmetry of the vascular
bundle in Arabidopsis root encompasses the pericycle and is reflected in
distich lateral root initiation. Plant Physiol. 146, 140–148.
Petersson, S.V., Johansson, A.I., Kowalczyk, M., Makoveychuk, A., Wang,
J.Y., Moritz, T., Grebe, M., Benfey, P.N., Sandberg, G. and Ljung, K. (2009)
An auxin gradient and maximum in the Arabidopsis root apex shown by
high-resolution cell-specific analysis of IAA distribution and synthesis.
Plant Cell, 21, 1659–1668.
Ramos, J.A., Zenser, N., Leyser, O. and Callis, J. (2001) Rapid degradation of
auxin/indoleacetic acid proteins requires conserved amino acids of domain
II and is proteasome dependent. Plant Cell, 13, 2349–2360.
Rebouillat, J., Dievart, A., Verdeil, L., Escoute, J., Giese, G., Breitler, C.,
Gantet, P., Espeout, S., Guiderdoni, E. and Perin, C. (2009) Molecular
Genetics of Rice Root Development. Rice, 2, 15–34.
Sabatini, S., Beis, D., Wolkenfelt, H. et al. (1999) An auxin-dependent distal
organizer of pattern and polarity in the Arabidopsis root. Cell, 99, 463–472.
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T.J. (1997) Aux/IAA proteins
repress expression of reporter genes containing natural and highly active
synthetic auxin response elements. Plant Cell, 9, 1963–1971.
Woodward, A.W. and Bartel, B. (2005) Auxin: regulation, action, and interaction. Ann. Bot. 95, 707–735.
Yoshida, S., Forno, D.A., Cock, J.H. and Gomez, K.A. (1976) Laboratory
Manual for Physiological Studies of Rice, 3rd edn. Manila, The Philippines:
International Rice Research Institute.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442