The Basic Helix-Loop-Helix Transcription Factor MYC2
Directly Represses PLETHORA Expression during
Jasmonate-Mediated Modulation of the Root Stem Cell
Niche in Arabidopsis
Qian Chen,a,1Jiaqiang Sun,a,b,1Qingzhe Zhai,a,bWenkun Zhou,aLinlin Qi,aLi Xu,aBao Wang,aRong Chen,a
Hongling Jiang,a,bJing Qi,b,cXugang Li,b,cKlaus Palme,b,cand Chuanyou Lia,b,2
aState Key Laboratory of Plant Genomics, National Centre for Plant Gene Research, Institute of Genetics and Developmental
Biology, Chinese Academy of Sciences, Beijing 100101, China
bChinese–German Joint Group for Plant Hormone Research, Institute of Genetics and Developmental Biology, Chinese
Academy of Sciences, Beijing 100101, China
cInstitute for Biology II/Botany and Freiburg Institute of Advanced Sciences, Faculty of Biology, University of Freiburg, D-79104
The root stem cell niche, which in the Arabidopsis thaliana root meristem is an area of four mitotically inactive quiescent
cells (QCs) and the surrounding mitotically active stem cells, is critical for root development and growth. We report here that
during jasmonate-induced inhibition of primary root growth, jasmonate reduces root meristem activity and leads to irregular
QC division and columella stem cell differentiation. Consistently, jasmonate reduces the expression levels of the AP2-
domain transcription factors PLETHORA1 (PLT1) and PLT2, which form a developmentally instructive protein gradient and
mediate auxin-induced regulation of stem cell niche maintenance. Not surprisingly, the effects of jasmonate on root stem
cell niche maintenance and PLT expression require the functioning of MYC2/JASMONATE INSENSITIVE1, a basic helix-
loop-helix transcription factor that involves versatile aspects of jasmonate-regulated gene expression. Gel shift and
chromatin immunoprecipitation experiments reveal that MYC2 directly binds the promoters of PLT1 and PLT2 and represses
their expression. We propose that MYC2-mediated repression of PLT expression integrates jasmonate action into the auxin
pathway in regulating root meristem activity and stem cell niche maintenance. This study illustrates a molecular framework
for jasmonate-induced inhibition of root growth through interaction with the growth regulator auxin.
Postembryonic root growth of higher plants is maintained by the
root meristem, in which stem cells, including the mitotically
inactive quiescent center (QC) and its surrounding stem cells,
reside in a specialized microenvironment called the stem cell
niche (Dolan et al., 1993; Watt and Hogan, 2000; Weigel and
Ju ¨rgens, 2002; Laux, 2003; Aida et al., 2004). In Arabidopsis
thaliana, the stem cell niche serves as the source of all differen-
tiated cell types during root development (van den Berg et al.,
meristematic activity can be altered by versatile developmental
and environmental cues during postembryonic growth.
Several hormonal pathways are involved in the regulation of
root growth, with auxin being the key player (Benjamins and
Scheres, 2008; Benkova ´ and Heja ´tko, 2009; Santner et al., 2009;
Vanneste and Friml, 2009). The whole process of root organo-
genesis, including initiation of the root pole (Friml et al., 2003),
formation of the root stem cell niche (Sabatini et al., 1999; Blilou
et al., 2005), maintenance of mitotic activity of the root meristem
(Beemster and Baskin, 2000; Dello Ioio et al., 2007; Stepanova
root meristem (Rahman et al., 2007), has been demonstrated to
be under the control of auxin, especially its featured gradient
distribution (Tanaka et al., 2006; Benkova ´ and Heja ´tko, 2009;
Petra ´sek and Friml, 2009). The PIN-FORMED (PIN) genes, which
encode components of the auxin efflux machinery mediating
polar auxin transport, are critical for the formation of a proper
auxin distribution gradient and therefore direct root pattern
formation and outgrowth (Blilou et al., 2005). It was proposed
that the instructive auxin gradient should be translated into
developmental information by molecular components. The
auxin-inducible PLETHORA (PLT) genes, which encode the AP2
class of transcription factors that are essential for root stem
cell niche patterning (Aida et al., 2004; Galinha et al., 2007), are
good candidates for performing this translation (Benjamins and
1These authors contributed equally to this work.
2Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Chuanyou Li
WOnline version contains Web-only data.
OAOpen Access articles can be viewed online without a subscription.
The Plant Cell, Vol. 23: 3335–3352, September 2011, www.plantcell.org ã 2011 American Society of Plant Biologists. All rights reserved.
Scheres, 2008). Interestingly, the expression domain of PLT
genes overlaps with the auxin maximum in the root. Recent work
reveals an elegant interaction network of PINs and PLTs in
regulating auxin-mediated root patterning and outgrowth; PIN
proteins restrict PLT expression in the basal embryo region to
initiate the root primordium. In turn, PLT genes maintain PIN
transcription, which stabilizes the position of the root stem cell
niche (Blilou et al., 2005; Grieneisen et al., 2007; Dinneny and
Significant progress in our understanding of auxin signaling
an auxin receptor (Dharmasiri et al., 2005; Kepinski and Leyser,
2005). Auxin binds to TIR1 and promotes the interaction of TIR1
with its auxin/indole-3-acetic acid (IAA) substrates. This interac-
tion leads to the degradation of the auxin/IAA repressors by
the 26S proteasome and therefore releases the transcriptional
regulation activity of the AUXIN RESPONSE FACTOR proteins
(Mockaitis and Estelle, 2008).
The jasmonate family of oxylipins, including jasmonic acid (JA)
and its metabolites, play a well-established role in mediating
defense responses and a wide range of developmental pro-
cesses (Creelman and Mullet, 1997; Turner et al., 2002; Browse,
2005; Wasternack, 2007; Howe and Jander, 2008; Kazan and
Manners, 2008). Among the first characterized physiological
effects of jasmonate is growth inhibition (Dathe et al., 1981). It is
proposed that JA-induced growth inhibition involves phase-
specific disruption of cell cycle progression. Indeed, application
of JA arrests synchronized tobacco (Nicotiana tabacum) BY-2
cells in both G1- and G2-phases (Swiatek et al., 2004). Whole-
genome gene expression profiling of Arabidopsis cell cultures
indicates that MeJA, the methyl ester of JA, represses the
activation of M-phase genes and arrests the cell cycle in the
G2-phase (Pauwels et al., 2008). Consistently, recent evidence
suggests that wound-induced jasmonates stunt plant growth by
inhibiting mitosis (Zhang and Turner, 2008). Even though the
action mechanism governing JA-induced inhibition of plant
growth remains elusive, characterization of JA-related mutants
defective in JA-induced inhibition of root growth has significantly
advanced our understanding of the molecular mechanism of JA-
regulated gene expression (Browse, 2009). The coronatine in-
sensitive1 (coi1) mutant is fully insensitive to JA both in terms of
root growth inhibition and defense gene expression (Feys et al.,
1994). COI1 encodes an F-box protein that interacts with SKP1
and CULLIN1 to assemble a functional SCFCOI1ubiquitin ligase
complex in vivo (Xie et al., 1998; Devoto et al., 2002; Xu et al.,
2002). COI1, which shows high sequence homology with the
auxin receptor TIR1, was shown recently to be the jasmonate
receptor (Yan et al., 2009; Sheard et al., 2010). Compared with
coi1, the jasmonate insensitive1 (jin1) (Berger et al., 1996;
Lorenzo etal., 2004) mutantexhibits a relatively weak phenotype
in JA-induced inhibition of root growth. JIN1 encodes a nuclear-
localized basic helix-loop-helix–type transcription factor known
as MYC2 (Boter et al., 2004; Lorenzo et al., 2004), which acts as
both an activator and a repressor to regulate diverse aspects of
JA-mediated gene expression (Dombrecht et al., 2007). A family
of jasmonate ZIM domain (JAZ) proteins was identified as the
target of the SCFCOI1ubiquitin ligase complex in JA signaling
(Chini et al., 2007; Thines et al., 2007). Recent structure-function
complex consisting of COI1, JAZ transcriptional repressors, and
inositol pentakisphosphate (Sheard et al., 2010). These studies
together revealed that jasmonate and auxin show a similar signal
perception and transduction paradigm in which F-box proteins
(receptors) mediate the degradation of negative regulators
(Mockaitis and Estelle, 2008). Jasmonoyl Ile, an active form of
JA, promotes the degradation of JAZ proteins and, in turn, frees
the transcriptional regulation activity of MYC2, the major tran-
scription factor of jasmonate-mediated gene expression.
It has been shown that the jasmonate and auxin signaling
For example, the first characterized auxin-resistant mutant, axr1
(Lincoln et al., 1990; Leyser et al., 1993), was later shown to be
also resistant to JA in root growth (Tiryaki and Staswick, 2002).
AXR1 encodes a subunit of the heterodimeric RUB-E1 enzyme,
which is important for the modification of CUL1, a shared
component of SCFTIR1and SCFCOI1, demonstrating that the
auxin and jasmonate proteasome pathways are directly con-
nected through AXR1 (Tiryaki and Staswick, 2002). Recently, it
was reported that JAZs interact with Novel Interactor of JAZ to
recruit TOPLESS as a corepressor to repress JAZ-targeted
transcription factors (Pauwels et al., 2010). Because TOPLESS
also mediates auxin-dependent transcriptional repression
(Szemenyei et al., 2008), these results provide another line of
evidence for jasmonate/auxin interaction at the level of tran-
In this study, we investigate the cellular and molecular mech-
anisms of jasmonate action on the inhibition of primary root
growth. We show that JA modulates the cellular organization of
the stem cell niche by promoting QC division and columella stem
cell (CSC) differentiation. We also show that JA represses the
direct binding of MYC2 to the promoters of the PLT genes. Our
is involved in the long-standing observation that jasmonate
inhibits primary root growth in Arabidopsis. PLT1 and PLT2 are
key interaction nodes between jasmonate and auxin in the
regulation of root stem cell niche maintenance and meristem
JA-Induced Inhibition of Primary Root Growth Involves a
Reduction of Root Meristem Activity
primary root length of Arabidopsis seedlings by germinating
seeds in medium containing different concentrations of JA. As
dose-dependent manner, while root growth of the JA-insensitive
We then investigated the JA-induced cellular changes in the
three morphologically distinguishable developmental zones
along the longitudinal axis of the root: the differentiation zone
(DZ), the elongation zone (EZ), and the meristem zone (MZ). As
shown in Figure 1C, JA decreased the final cell length of the DZ
3336 The Plant Cell
and reduced the size of the EZ as well as the size of the MZ.
Closer observation of the DZ and EZ of JA-treated roots indi-
cated that JA reduced both cell number and cell length of these
regions (see Supplemental Figure 1 online), indicating that JA
reduces both cell proliferation and cell elongation, two basic
cellular processes affecting primary root growth (Scheres et al.,
We then focused on the cellular and molecular mechanisms
underlying the JA-induced reduction of root meristem size. The
effect of JA on root meristem size was evaluated by determining
the number of cortical cells in the region from the QC to the first
elongated cell (Casamitjana-Martı ´nez et al., 2003; Dello Ioio
substantially reduced in coi1-2 (Xu et al., 2002) and myc2-2
(Boter et al., 2004), indicating that JA reduces the number of the
transitamplifying cellsinaCOI1-andMYC2-dependent manner.
The reduction of root meristem cell number by JA could result
from its negative effect on cell division. To test this idea, we
monitored the expression of the transcriptional fusion reporter
CYCB1;1pro:GUS (for b-glucuronidase), a widely used marker to
indicate G2/M-phase of the cell cycle (Colo ´n-Carmona et al.,
1999). WithoutJA treatment, CYCB1;1pro:GUS was expressed in
the actively dividing cells of the root meristem (Figures 2H and
2J). JA treatment markedly reduced the expression of CY-
CB1;1pro:GUS in the wild type (Figure 2I) but not in coi1-1 (Figure
2K), suggesting that the JA signaling pathway represses the cell
the effect of JA on CYCB1;1pro:GUS expression was different
from that of ethylene, which exerted little effect on CYCB1;1pro:
GUS expression (Ru ˚zicka et al., 2007). Consistent with JA-
induced reduction of CYCB1;1pro:GUS expression, our quanti-
tative RT-PCR (qRT-PCR) assays revealed that exogenous JA
markedly reduces the expression levels of several cell cycle–
related genes, including CYCB1;1 (Fuerst et al., 1996), CDC2A
(Martinez et al., 1992), KRP1 (Wang et al., 1997), and PCNA1
(Egelkrout et al., 2002) (Figure 2L). It is noteworthy that JA failed
to repress the expression of cell cycle–related genes in coi1-2
and myc2-2 (Figure 2L), indicating that JA negatively regulates
cell division activity of the root meristem through COI1 and
MYC2. Consistently, using transgenic plants containing gene
promoter and GUS fusions, we found that COI1pro:GUS and
MYC2pro:GUS are richly expressed in the root meristem (see
Supplemental Figures 2A to 2D online).
Figure 1. JA Represses Root Growth through Inhibition of both Cell Proliferation and Cell Elongation.
(A) Six-day-old seedlings of the wild type (Col-0) and coi1-1 were grown on medium without (MS) or with 20 mM JA.
(B) JA-mediated inhibition of root growth in Col-0 and coi1-1. Col-0 and coi1-1 seeds were germinated on medium containing different concentrations
of JA, and seedling root length was measured at 6 d after germination. The effects of JA on Col-0 and coi1-1 were significantly different. Data shown are
average and SD (n > 20) and are representative of at least three independent experiments.
(C) JA reduces the MZ, the EZ, and epidermal cell length of the DZ. A representative image of 6-d-old Col-0 seedlings grown in the absence or presence
of JA. Insets show that JA reduces cell length (marked with red lines) of Col-0 epidermal cells in the DZ. Bars = 50 mm.
Jasmonic Acid Modulates Root Stem Cell Niche3337
Figure 2. JA Reduces Cell Division Activity of the Root Meristem in the Wild Type but Not in coi1-2 and myc2-2.
(A) to (F) Root meristems of 6-d-old Col-0, coi1-2, and myc2-2 seedlings grown on medium without (MS) or with 20 mM JA. The meristem zone was
marked with red line. Bars = 50 mm.
(G) JA-induced reduction of root meristem cell number in Col-0, coi1-2, and myc2-2. Seedlings of the indicated genotypes were grown on medium
without (MS) or with 20 mM JA, and cell number in the root meristem was examined at a 2-d interval. Data shown are average and SD (n = 20). Asterisks
denote Student’s t test significance compared with untreated plants: *P < 0.05.
(H) to (K) JA-induced reduction of CYCB1;1pro:GUS expression in Col-0 and coi1-1. Five-day-old seedlings germinated on control medium were
transferred to medium without (MS) or with 20 mM JA for 1 d, and the CYCB1;1pro:GUS expression was monitored. Bars = 50 mm.
3338 The Plant Cell
JA Promotes QC Division in a COI1- and MYC2-
Our finding that JA alters root meristem activity prompted us to
investigate its possible effect on the cellular organization of QC
and its surrounding stem cells. In normal-grown wild-type roots,
the well-defined QC cells rarely divide (Figures 3A, 3G, 3I, 3K,
3M, and 3O). However, obvious QC division was observed in a
significant proportion of JA-treated roots (Figures 3B, 3H, 3J, 3L,
3N, and 3P). Using the QC-specific markers QC25 and WOX5pro:
GFP (for green fluorescent protein; Blilou et al., 2005; Sarkar
et al., 2007), we confirmed that the dividing cells were QC cells
(Figures 3Gto3J).To testwhetherJA inducesQCdifferentiation,
we performed Lugol staining experiments using the QC25
marker line. As shown in Figure 3H, no starch accumulation
was detected in the irregularly divided QC cells, suggesting that
JA promotes QC division but does not induce QC differentiation.
Considering that WOX5 plays an important role in regulating the
maintenance of the root stem cell niche (Sarkar et al., 2007; Ding
and Friml, 2010), we examined whether JA treatment affects
WOX5 expression. RNA in situ assays indicated that JA treat-
ment led to a slight expansion of the WOX5 expression domain
(cf. Figures 3L and 3K), which could have resulted from the
irregular QC division. However, as revealed by our qRT-PCR
assays, JA treatment did not substantially alter the WOX5
transcript levels (Figure 3Q). To check whether cell cycle activity
is altered in the QC of JA-treated roots, we cultured 2-d-old
seedlings for 24 h in the presence of JA and EdU, a nucleoside
analog of thymidine. EdU has been used to mark cell division in
the root meristem because incorporation of this chemical in the
nuclei is indicative of S-phase progression (Vanstraelen et al.,
2009). In this assay, mitotically active cells show red fluorescent
nuclei after coupling of the EdU with the Alexa Fluor 647 sub-
strate. In untreated wild-type root meristems, most meristematic
cells, including those QC-associated stem cells, incorporated
EdU with red fluorescent nuclei, but only occasionally in the QC
cells (marked with WOX5pro:GFP), indicating their low mitotic
activity (Figures 3M and 3O). In JA-treated root meristems,
however, the QC cells were able to incorporate EdU (Figures 3N
15), indicating that JA application induces high mitotic activity of
the QC cells.
Similar to JA-induced root growth inhibition, we found that JA-
induced QC division was dose dependent (Figure 3R). We
followed the time course of the onset of the JA-induced QC
phenotype by quantifying extra QC cells for 10 d after germina-
tion on medium containing 20 mM JA. Quantification of extra
QC cells has been successfully used to characterize ethylene-
induced QC division (Ortega-Martı ´nez et al., 2007). In the wild
type, obvious QC division was observed as early as 2 d after
germination, and the number of extra QC cells increased grad-
ually with time (Figure 3T). Importantly, our parallel experiments
3C to 3F, 3S, and 3T), indicating that the promotional effect of
JA on QC division requires the function of COI1 and MYC2.
To test whether the above-described effect of JA on the
promotion of QC division is independent of that of ethylene,
which has also been shown to modulate stem cell division in
Arabidopsis roots (Ortega-Martı ´nez et al., 2007), we examined
the effect of JA on QC division in the previously described
ethylene signaling mutants ein2-5 and ein3-1 eil1-1 (Guo and
Ecker, 2003). As shown in Supplemental Figure 3 online, the
ein2-5 and ein3-1 eil1-1 mutants did not affect the promotional
effect of JA on QC division. Similarly, the addition of AgNO3,
which effectively blocks the ethylene signaling pathway, did not
alter the effect of JA on the promotion of QC division (see
Supplemental Figure 3 online). These results confirmed that the
effect of JA on the promotion of QC division is independent of
that of ethylene.
JA Induces Deregulated Differentiation of CSCs
In the root stem cell niche, the mitotically inactive QC cells act to
maintain the identity and function of the surrounding stem cells.
To assess the possible effect of JA-induced QC division on stem
cell maintenance, we examined the cell layer immediately below
the QC, at the position of the CSCs. The J2341 marker, which is
specifically expressed in the single layer of CSCs (Figure 4A),
expanded to more than one cell tier upon JA treatment (cf.
Figures 4A and 4B). We then asked whether the distal stem cells
might have undergone premature differentiation in JA-treated
roots, which in the columella can be visualized by the accumu-
lation of starch granule-containing organelles. In the absence of
JA, 6-d-old wild-type roots do not show starch granule accu-
mulation in the CSCs (Figure 4C, white arrow), which are distinct
from the underlying well-organized four tiers of columella cells
that accumulate starch granules (Figures 4C, white dashed line).
In the presence of JA, however, irregular cells containing starch
granules were observed immediately below the QC cells (Figure
4D, red arrow), indicating that the CSCs were in a state of differ-
entiation. Our time-course study revealed that, in JA-treated
roots, the appearance of QC division is generally earlier than
that of CSC differentiation (Figure 4K), suggesting that the JA-
induced irregular QC divisions may lead to a failure to maintain
the stem cell fate of CSCs. In addition, JA treatment led to the
formation of extra columella cell layers, and the general cellular
organization of columella tiers was disturbed (Figures 4D, red
dashed line, 4I, and 4J). The above-described effect of JA on
Figure 2. (continued).
(L) JA-regulated expression of cell cycle–related genes in Col-0, coi1-2, and myc2-2. Five-day-old Col-0 seedlings germinated on control medium were
transferred to liquid medium without (control) or with 20 mM JA for 6 h, and 2-mm root tips were harvested for RNA extraction and qRT-PCR analysis.
Transcript levels of cell cycle–related genes were normalized to ACTIN2 expression. The transcript levels of the indicated genes in Col-0 without JA
treatment were arbitrarily set to 1. Error bars represent the SD of triplicate reactions of independent RNA samples prepared from three batches of
Arabidopsis roots. Asterisks denote Student’s t test significance compared with untreated plants: *P < 0.05.
Jasmonic Acid Modulates Root Stem Cell Niche 3339
Figure 3. JA-mediated Promotion of QC Division in the Wild Type, coi1-2, and myc2-2.
(A) to (F) Representative confocal images showing that JA promotes QC division in Col-0 but not in coi1-2 and myc2-2. Five-day-old seedlings were
transferred to medium without (MS) or with 20 mM JA for another day before QC division was monitored. Bars = 20 mm.
(G) and (H) JA induces QC division and CSC differentiation as revealed by Lugol staining of the QC25 marker line. Five-day-old seedlings of the QC25
3340 The Plant Cell
CSC differentiation and extra columella layer formation was
substantially reduced in coi1-2 (Figures 4E, 4F, 4I, and 4J) and
myc2-2 (Figures 4G to 4J), suggesting that the effect of JA on
CSC differentiation and columella organization requires the
function of COI1 and MYC2.
To test whether endogenous JA has a similar effect as exog-
enous JA (i.e., to reduce root meristem activity and to disturb the
maintenance of the root stem cell niche), we sought to obtain
mutants with elevated endogenous JA levels or with increased
JA sensitivity. The constitutive expression of VSP1 (cev1) mutant
has increased production of both JA and ethylene and exhibits a
short root phenotype (Ellis and Turner, 2001; Ellis et al., 2002).
When grown on control medium, cev1 seedlings show reduced
meristem cell number (see Supplemental Figure 4A online),
increased QC division (see Supplemental Figures 4B and 4C
online), and CSC differentiation (see Supplemental Figures 4D to
4I online) than wild-type seedlings. When grown on medium
containing 2-aminoethoxyvinyl glycine or AgNO3, which can
effectively block ethylene biosynthesis and perception, respec-
tively, cev1 roots still exhibit reduced meristem size, exag-
gerated QC division, and CSC differentiation compared with
wild-type roots (seeSupplemental Figure 4online). These results
JA on stem cell niche maintenance.
Given that JAZ proteins serve as negative regulators of JA
of JAZ genes exhibit increased sensitivity to JA in root growth
inhibition. Indeed, it has been shown that RNA interference
(RNAi) lines of JAZ10 show a hypersensitive response to JA (Yan
et al., 2007). We found that, like the JAZ10 RNAi lines, a T-DNA
insertion line (CS872819, SAIL_92_D08) that disrupts the ex-
pression of JAZ10 also exhibits increased sensitivity to JA in root
growth inhibition (see Supplemental Figure 5A online). Consis-
tently, JA-induced meristem cell number reduction (see Supple-
mental Figure 5B online), QC division (see Supplemental Figure
5C online), CSC differentiation (see Supplemental Figures 5E to
5H online), and columella layer disorganization (see Supplemen-
tal Figure 5D online) are all increased in jaz10 relative to the wild
JA also modulates the cellular organization of the root stem cell
niche and reduces root meristem activity.
JA Reduces the Expression of PLT1 and PLT2 in a MYC2-
The PLT family of transcription factors provides important pat-
terning information for the root stem cell niche and determines
the number of the transit-amplifying daughter cells that make up
the meristem (Aida et al., 2004; Galinha et al., 2007). We show
that Arabidopsis roots grown on JA-containing medium exhibit
ectopic cell division in the QC, irregular differentiation of CSCs,
and loss of transit-amplifying cells, defects that are also ob-
servedintheplt1-4plt2-2double mutantroots (Aidaetal.,2004).
The root phenotype similarity between JA-treated roots and
mutants of the PLT genes prompted us to test the possible link
We first examined JA-induced expression of PLT1 and PLT2 at
the transcription level. Using transgenic plants containing the
promoters of PLT1 and PLT2 fused to the cyan fluorescent
protein (CFP) gene (Galinha et al., 2007), we found that JA
Figure 3. (continued).
marker line were transferred to medium without (MS) or with 100 mM JA for 1 d before GUS (blue) and Lugol (dark brown) double stainings were
performed. White arrow indicates no starch accumulation in the irregular QC cells. Red arrow shows starch accumulation in the irregular CSCs. Bars =
(I) and (J) JA-induced QC division in the QC marker line WOX5pro:GFP. Five-day-old seedlings of WOX5pro:GFP were transferred to medium without
(MS) or with 20 mM JA for another day before QC division was monitored. White arrows mark the cell divisions in the QC. Bars = 20 mm.
(K) and (L) Whole-mount in situ hybridization with a WOX5-specific probe showing that JA causes supernumerary QC cells. Roots of seedlings 5 d after
germination grown on medium without (MS) or with 20 mM JA were used for whole-mount in situ hybridization. Bars = 20 mm.
(M) to (P) EdU incorporation assays showing that JA promotes QC division. Two-day-old WOX5pro:GFP seedlings grown on medium without (MS) ([M]
and [O]) or with 100 mM JA treatment ([N] and [P]) were cultured with EdU for 1 d before EdU incorporation in the QC was examined. (O) represents the
outlined area in (M), and (P) represents the outlined area in (N). Red fluorescence of EdU-positive nuclei in QC cells of JA-treated roots indicates that QC
is in a state of active division (white arrow). Bars = 20 mm.
(Q) JA-induced WOX5 expression revealed by qRT-PCR. Five-day-old Col-0 seedlings germinated on control medium were transferred to liquid
medium without (control) or with 20 mM JA for 6 h, and 2-mm root tips were harvested for RNA extraction and qRT-PCR analysis. WOX5, PLT1, and
PLT2 transcription levels without JA treatment were arbitrarily set to 1. Error bars represent the SD of triplicate reactions of independent RNA samples
prepared from three batches of Arabidopsis roots. Asterisks denote Student’s t test significance compared with untreated plants: ***P < 0.001.
(R) Dose-dependent effect of JA on QC division in Col-0 roots. Col-0 seedlings were grown on medium containing indicated concentrations of JA for 6 d
before QC division frequency (percentage of seedlings with obvious QC division) was determined. At least 50 seedlings were examined for each
concentration for each biological repeat. Data shown are average and SD and are representative of at least three independent experiments.
(S) JA-induced QC division frequency in Col-0, coi1-2, and myc2-2. Seedlings were grown on medium without (Control) or with 20 mM JA for 6 d before
QC division frequency was determined. At least 50 seedlings were examined for each biological repeat. Data shown are average and SD and are
representative of at least three independent experiments. Asterisks denote Student’s t test significance compared with untreated plants: *P < 0.05 and
***P < 0.001.
(T) Time course of JA-induced extra QC cells in Col-0, coi1-2, and myc2-2. Seeds of the indicated genotypes were germinated on medium without
(Control) or with 20 mM JA for 10 d, and extra QC cells were quantified at a 2-d interval. The effects of JA on Col-0 compared with coi1-2 and myc2-2
were significantly different. At least 50 seedlings were examined for each biological repeat. Data shown are average and SD and are representative of at
least three independent experiments. Asterisks denote Student’s t test significance compared with untreated plants: *P < 0.05 and ***P < 0.001.
Jasmonic Acid Modulates Root Stem Cell Niche3341
significantly reduced the expression levels of PLT1pro:CFP (Fig-
ures 5A and 5B) and PLT2pro:CFP (Figures 5C and 5D) in the wild
type but not in coi1-2 and myc2-2, suggesting that JA represses
PLT1 and PLT2 expression in a COI1- and MYC2-dependent
manner. Next, using qRT-PCR assays, we compared the re-
pression effect of JA on PLT expression in the wild type, coi1-2,
myc2-2, and the MYC2 overexpression line 35Spro:MYC2 (see
Supplemental Figures 2E and 2F online). In line with previous
plants did not show constitutive activation of JA responses,
Figure 4. JA-Mediated Promotion of CSC Differentiation in the Wild Type, coi1-2, and myc2-2.
(A) and (B) JA induces deregulated division of CSCs, as revealed by the CSC-specific marker J2341. Five-day-old seedlings of the J2341 marker line
were transferred to medium without (MS) or with 20 mM JA for 1 d before GFP expression in CSCs was examined. White arrows indicate the presence of
two CSC layers in JA-treated roots. Bars = 20 mm.
(C) to (H) Lugol staining showing that JA induces CSC differentiation in Col-0 ([C] and [D]) but not in coi1-2 ([E] and [F]) and myc2-2 ([G] and [H]). Five-
day-old seedlings were transferred to medium without (MS) or with 20 mM JA for 1 d before Lugol staining was performed. Nondifferentiated CSCs
(white arrows) below the QC are characterized by the absence of starch granules, whereas starch granules are visible in differentiated CSCs (red arrow).
The white dashed line in (C) indicates the well-organized columella cell layers of untreated wild-type roots. The red dashed line in (D) indicates increased
and disorganized columella cell layers of JA-treated wild-type roots. Bars = 20 mm.
(I) JA-induced CSC differentiation frequency in Col-0, coi1-2, and myc2-2. Seedlings were grown on medium without (Control) or with 20 mM JA for 6 d
before CSC differentiation frequency was determined. At least 50 seedlings were examined for each genotype for each experiment. Data shown are
average and SD and are representative of at least three independent experiments. Asterisks denote Student’s t test significance compared with
untreated plants: *P < 0.05 and ***P < 0.001.
(J) JA induces extra columella cell layers in Col-0 but not in coi1-2 and myc2-2. Seedlings were grown on medium without (Control) or with 20 mM JA for
6 d before columella cell layers were determined. At least 50 seedlings were examined for each genotype for each experiment. Data shown are average
and SD and are representative of at least three independent experiments. Asterisks denote Student’s t test significance compared with untreated plants:
*P < 0.05.
(K) Kinetics of JA-induced QC division and CSC differentiation. Col-0 seeds were germinated on medium containing 20 mM JA (JA), and QC division
frequency and CSC differentiation frequency were determined at a 2-d interval. At least 50 seedlings were examined for each biological repeat. Data
shown are average and SD and are representative of at least three independent experiments.
3342 The Plant Cell
Figure 5. JA Reduces the Expression of PLT1 and PLT2 through COI1 and MYC2.
(A) JA reduces PLT1pro:CFP expression levels in Col-0, but not in coi1-2 and myc2-2.
(B) Quantification of CFP fluorescence shown in (A).
(C) JA reduces PLT2pro:CFP expression levels in Col-0, but not in coi1-2 and myc2-2.
(D) Quantification of CFP fluorescence shown in (C).
For (A) to (D), 5-d-old seedlings were transferred to medium without (MS) or with 20 mM JA for 1 d before CFP fluorescence was monitored. Data shown
are average and SD (n = 15 to 20). Asterisks denote Student’s t test significance compared with untreated plants: ***P < 0.001. Bars = 50 mm.
Jasmonic Acid Modulates Root Stem Cell Niche3343
although enhanced inhibition of root growth was observed in
these 35Spro:MYC2 seedlings after treatment with JA (see Sup-
plemental Figure 2F online). Consistently, we showed that, in the
absence of JA, PLT1 and PLT2 expression was largely similar
among the genotypes compared (Figures 5E and 5F). JA treat-
ment markedly downregulated the transcript levels of PLT1 and
PLT2 in the wild type but not in coi1-2 and myc2-2 (Figures 5E
and 5F). Significantly, JA-induced downregulation of PLT1 and
PLT2 expression was substantially exaggerated in 35Spro:MYC2
compared with the wild type (Figures 5E and 5F), indicating an
important role of MYC2 in mediating JA-induced repression of
PLT1 and PLT2 expression. Importantly, the observation that
MYC2 represses PLT gene expression onlyin the presence of JA
suggests that this action of MYC2 may require JA perception or
other upstream signaling events.
Next, using transgenic plants containing the promoters of
PLT1 and PLT2 combined with PLT-YFP (for yellow fluorescent
protein) protein fusions (Galinha et al., 2007), we found that JA
5G and 5H) and PLT2pro:PLT2-YFP (Figures 5I and 5J) in the wild
type but not in coi1-2 and myc2-2. Together, our results support
the hypothesis that JA represses the transcriptional expression
of PLT1 and PLT2 through the COI1- and MYC2-mediated
jasmonate signaling pathway.
Given that JA promotes auxin biosynthesis (Dombrecht et al.,
2007; Sun et al., 2009) and auxin itself regulates PLT expression
(Aida et al., 2004), we designed experiments to test whether the
above-described effect of JA on PLT expression is achieved
of PLT1 and PLT2 in the wild type and the auxin biosynthesis
mutants asa1-1 (Sun et al., 2009) and yuc1D (previously known
as yucca; Zhao et al., 2001). Our recent work demonstrates that
the asa1-1 mutant contains a mutation in the Anthranilate Syn-
thase a1 (ASA1/WEI2) gene and therefore is defective in JA-
induced auxin biosynthesis (Sun et al., 2009). The dominant
yuc1D mutant contains elevated free IAA levels, resulting from
the overexpression of a rate-limiting auxin biosynthesis gene
YUCCA1 (YUC1) (Zhao et al., 2001; Zhao, 2010). As revealed by
our qRT-PCR assays, in wild-type roots, a slight reduction of
PLT1 and PLT2 transcripts was observed 2 h after JA treatment
and, a marked reduction of these transcripts was observed at 6
online). Importantly, JA-induced reduction levels and kinetics of
PLT1 and PLT2 expression were largely similar among asa1-1,
yuc1D, and the wild type (see Supplemental Figures 6A and 6B
online), indicating that the effect of JA on PLT expression is not
achieved through the ASA1- and YUC1-dependent auxin bio-
synthesis. Consistently, the asa1-1 and yuc1D mutations did not
affect JA-induced inhibition of root growth (see Supplemental
Figure 6C online) and JA-induced reduction of root meristem cell
number (see Supplemental Figure 6D online). Together, these
data support the notion that the effect of JA on PLT expression is
independent of the auxin pathway.
MYC2 Directly Regulates the Expression of PLT1 and PLT2
The basic helix-loop-helix–type transcription factor MYC2 acts
as a master regulator of JA-mediated gene transcription and
regulates versatile aspects of JA responses, including defense
gene expression and root growth inhibition (Dombrecht et al.,
2007; Memelink, 2009). Our finding that JA represses the tran-
scriptional expression of PLT1 and PLT2 in a MYC2-dependent
manner suggests that MYC2 may associate with promoters of
PLT1 and PLT2. It is well known that MYC2 preferably binds to
the G-box–related hexamer 59-CACATG-39 of its target genes
(Dombrecht et al., 2007). Our sequence analysis revealed one
upstream of the translational start codon; Figure 6A) of the PLT1
promoter. Sequence analysis also identified two 59-CACATG-39
motifs in the P2 region (located 2940 to 2945 and 21098 to
21103 bp upstream of the translational start codon; Figure 6A)
and one 59-CACATG-39 motif close tothe P3region(located+288
of PLT2. To test the in vivo interaction between MYC2 and the
chromatin regions of PLT1 and PLT2, we performed chromatin
immunoprecipitation (ChIP) assays using the 35Spro:MYC2
transgenic plants (containing the 35Spro:MYC2-4Myc construct)
and anti-Myc antibodies (Roche). As shown in Figure 6B, chro-
matin immunoprecipitated with anti-Myc antibodies was pro-
foundly enriched in the P1 region of the PLT1 promoter and in
the P3 region of the PLT2 promoter. These results indicate a
specific association of MYC2 with the promoters of PLT1 and
A DNA electrophoretic mobility shift assay (EMSA) was con-
ducted to confirm that MYC2 directly binds these G-box–related
motifs presented in the upstream regions of PLT1 and PLT2.
Figure 5. (continued).
(E) and (F) qRT-PCR assay showing that JA downregulates the transcription of PLT1 (E) and PLT2 (F) in a COI1- and MYC2-dependent manner. Five-
day-old seedlings germinated on MS medium were transferred to medium without (Control) or with 20 mM JA for 1 d, and 2-mm root tips were harvested
for RNA extraction and qRT-PCR analysis. The transcript levels of PLT1 and PLT2 were normalized to the ACTIN2 expression. PLT1 and PLT2
transcription levels of Col-0 without JA treatment were arbitrarily set to 1. Data presented are mean values of four biological repeats with SD. Samples
with different letters are significantly different: P < 0.01.
(G) JA reduces PLT1pro:PLT1-YFP expression levels in Col-0, but not in coi1-2 and myc2-2.
(H) Quantification of YFP fluorescence shown in (G). Data shown are average and SD (n = 15 to 20). Asterisks denote Student’s t test significance
compared with untreated plants: ***P < 0.001.
(I) JA reduces PLT2pro:PLT2-YFP expression levels in Col-0, but not in coi1-2 and myc2-2.
(J) Quantification of YFP fluorescence shown in (I). Data shown are average and SD (n = 15 to 20). Asterisks denote Student’s t test significance
compared with untreated plants: ***P < 0.001.
For (G) to (J), 5-d-old seedlings were transferred to medium without (MS) or with 20 mM JA for 1 d before YFP fluorescence was monitored. Bars = 50 mm.
3344The Plant Cell
Full-length MYC2 protein was expressed as a maltose binding
protein (MBP) fusion protein in Escherichia coli and affinity
purified. As shown in Figure 6C, the MYC2-MBP fusion proteins
were able to bind DNA probes containing the 59-CACATG-39
motif located in the P1 region of the PLT1 promoter and in the P3
region of the PLT2 gene. Furthermore, additional unlabeled DNA
probes competed for binding in a dose-dependent manner
(Figure 6C). Parallel experiments indicated that MYC2-MBP
failed to bind DNA probes containing the mutant form of the
59-CACATG-39 motif (Figure 6C). Together, these results reveal
that MYC2 regulates PLT1 and PLT2 expression through direct
association with their promoters.
Next, using the well-established transient expression assay of
Nicotiana benthamiana leaves, we verified the repression effect of
fused with the firefly luciferase gene (LUC). When the PLT1pro:LUC
LUC activity could be detected (Figures 7A and 7B), indicating that
intensity (Figure 7), suggesting that the manipulation process of the
assay could have simulated the JA signaling of the infiltrated N.
2009; Sheard et al., 2010), onto the N. benthamiana leaf area
coexpressing PLT1pro:LUC and 35Spro:MYC2, led to a more dra-
matic reduction effect of 35Spro:MYC2 on PLT1pro:LUC expression
(Figure 7). In a parallel experiment, PLT1-mpro:LUC, in which the
G-box of the PLT1 promoter was deleted and fused with LUC, was
coexpressed with 35Spro:MYC2 in N. benthamiana leaves. As
shown in Figure 7, in the absence or presence of COR, the
repression effect of 35Spro:MYC2 on PLT1-mpro:LUC expression
was largely attenuated. The observation that the PLT1-mpro:LUC
be explainedbythe presence ofthe59-GAGTA-39motifinthePLT1
promoter (see Supplemental Figure 7 online), which has been
shown to be responsive to JA (Boter et al., 2004). Together, our
transient assays of N. benthamiana leaves confirmed that MYC2
represses PLT1 expression in vivo.
The above-described results suggest that MYC2-mediated
modulation of root meristem activity and stem cell niche mainte-
nance.Todeterminethe geneticrelationshipbetweenJAand PLT
proteins, we assessed the effect of JA on root meristems of the
8A and 8B, JA reduced the root meristem cell number by 33% in
the wildtype but onlyby 19.7% in plt1-4 plt2-2, indicating thatthe
Figure 6. MYC2 Associates with PLT1 and PLT2 Promoters.
(A) Schematic diagram of potential MYC2 binding sites (white and black triangles), DNA fragments (P1, P2, and P3) used for ChIP, and probes used for
EMSA. The sequence 2 kb upstream of the start site and part of the coding sequence for PLT1 and PLT2 are shown. The translational start site (ATG) is
shown at position +1.
(B) Enrichment of the indicated DNA fragments (P1 and P3) following ChIP using anti-Myc antibodies. Chromatin of transgenic plants expressing 35Spro:
MYC2-4Myc was immunoprecipitated with anti-Myc antibodies, and the presence of the indicated DNA in the immune complex was determined by RT-
PCR. The Actin2 promoter fragment was used as a negative control. The experiment was repeated three times with similar results.
(C) EMSA showing that the MYC2-MBP fusion protein binds to the DNA probes of PLT1 and PLT2 in vitro. Biotin-labeled probes were incubated with
MYC2-MBP protein, and the free and bound DNAs (arrows) were separated in an acrylamide gel. As indicated, unlabeled probes were used as
competitors. Mu, mutated probe in which the 59-CACATG-39 motif was deleted.
Jasmonic Acid Modulates Root Stem Cell Niche 3345
double mutations significantly attenuated the reduction effect of
JA on the root meristem cell number. To test whether over-
expression of PLT genes could somehow rescue the reduction
effect of JA on root meristem cell number, we used trans-
genic plants containing the inducible construct 35Spro:PLT2-GR
(Galinha et al., 2007). Figures 8C and 8D indicated that, without
dexamethasone (DEX) induction (i.e., without PLT2 overexpres-
sion), JA reduced the root meristem cell number of 35Spro:PLT2-
GR plants by 31.4%. In the presence of DEX induction; however,
JA reduced the root meristem cell number of the PLT2 over-
expressor by 20% (Figures 8C and 8D). The finding that the
JA-induced reduction of root meristem cell number is reduced in
plt1-4 plt2-2 and PLT2 overexpression plants supports the sce-
nario that PLT proteins act genetically downstream of JA in
regulating root meristem maintenance. The fact that plt1-4 plt2-2
double mutants and PLT2 overexpression plants are still respon-
sivetoJAcould beexplainedbyatleast two possibilities: first, the
existence of PLT family members other than PLT1 and PLT2 (i.e.,
PLT3 and BABY BOOM) (Galinha et al., 2007); second, in addition
to the PLT family of transcription factors, JA could employ other
mechanisms to regulate root meristem maintenance.
Postembryonic root growth of higher plants exhibits remarkable
plasticity to adapt to the ever-changing environmental condi-
the postembryonic activity of cells within the root meristem that
is coordinated by several phytohormones, including jasmonate
(Benkova ´ and Heja ´tko, 2009; Petra ´sek and Friml, 2009; Wolters
and Ju ¨rgens, 2009). Here, we address the cellular and molecular
mechanisms underlying JA-induced inhibition of primary root
growth of Arabidopsis plants. We show that, at the cellular level,
JA reduces cell number and cell elongation of the DZ and EZ,
inhibits cell division activity of the root meristem, and alters the
cellular organization of the stem cell niche. We also show that, at
the molecular level, MYC2 directly represses the expression of
PLT1 and PLT2 upon JA treatment, suggesting that MYC2-
mediated repression of PLT expression is important for JA-
induced modulation of root meristem activity and stem cell niche
maintenance. This study demonstrates a mode of jasmonate-
auxin crosstalk, in which a major transcription factor of the JA
known to actin the auxin pathway. Ourresults extend the view of
how plants can translate stress cues into growth response and
JA Modulates Maintenance of the Root Stem Cell Niche
Although jasmonate-induced root growth inhibition has been em-
ployed as the most prominent genetic assay to identify jasmonate-
related mutants in Arabidopsis (Wasternack, 2007; Browse,
2009), the cellular and molecular basis of jasmonate action on
root growth is hitherto not clear. We show here that JA reduces
Figure 7. MYC2 Represses PLT1 Expression, as Revealed by Transient Assays of N. benthamiana leaves.
(A) Transient expression assays showing that MYC2 represses the expression of PLT1. Representative images of N. benthamiana leaves 72 h after
infiltration are shown. The bottom panel indicates the infiltrated constructs and treatments.
(B) Quantitative analysis of luminescence intensity in (A). Five independent determinations were assessed. Error bars represent SD. Asterisks denote
Student’s t test significance compared with control plants: **P < 0.01 and ***P < 0.001.
(C) qRT-PCR analysis of MYC2 expression in the infiltrated leaf areas shown in (A). Total RNAs were extracted from leaves of N. benthamiana
coinfiltrated with the constructs. Five independent determinations were assessed. Error bars represent SD.
3346 The Plant Cell
both cell number and cell length of the root, indicating that JA-
induced inhibition of primary root growth is a complex process
that involves multiple cellular processes in distinct root tissues.
reduced meristem size and decreased expression levels of the
CYCB1;1pro:GUS reporter gene and a group of cell cycle–related
genes. The reduced cell proliferation rate in the JA-treated root
meristem could contribute to the observed reduction of cell
number in the EZ. Our observations reveal that the mechanism
by which JA inhibits primary root growth is distinct from that of
talk with JA (Lorenzo et al., 2003). Several groups showed that
ethylene does not affect cell cycle activity of the root meristem
but strongly reduces cell elongation in the EZ (Ru ˚zicka et al.,
2007; Stepanova et al., 2007; Swarup et al., 2007).
Consistent with a reduced root meristem activity by JA, we
niche. For example, JA triggers the division of QC cells and
JA treatment, the occurrence of QC division is generally earlier
thanthatof CSC differentiation. Inthe contextthat properexpres-
sion of WOX5 in the QC is required to maintain CSCs as undiffer-
entiated (Sarkar et al., 2007), we examined the possible effect of
JA on WOX5 expression and found that JA treatment has little, if
any, effect on WOX5 expression. Given that ethylene has been
shown to be able to induce QC division (Ortega-Martı ´nez et al.,
2007), it is important to determine whether this hormone and JA
Two lines of evidence favor that JA and ethylene modulate the
distinct mechanisms. First, whereas the supernumerary QC cells
produced by ethylene-induced QC division still display their QC
function to maintain the stem cell identity of CSCs (Ortega-
Martı ´nez et al., 2007), JA-induced supernumerary QC cells par-
tially lose their ability to maintain the stem cell identity of CSCs.
Second, blocking of the ethylene pathway by genetic or chemical
manipulation does not affect JA-induced QC division.
Interestingly, our results reveal that, depending on the tissue
context, JA exerts opposite effects on cell division. For example,
JA represses cell division in the proximal root meristem, as
evidenced by reduced meristem cell number, reduced expres-
sion of the mitotic cell cycle marker CYCB1;1pro:GUS, and
reduced expression levels of several cell cycle–related genes in
JA-treated roots. These results are consistent with previous
Figure 8. JA-induced Root Meristem Cell Number in plt1-4 plt2-2 and PLT2-overexpression Plants.
(A) JA-induced reduction of meristem size in wild-type (Wassilewskija [WS]) and plt1-4 plt2-2 plants. Seedlings were grown on medium without (MS) or
with 20 mM JA for 4 d before meristem cell number was determined. The MZ is marked with a red line. Bars = 50 mm.
(B) Quantification of meristem cell number in (A). Data shown are average and SD (n = 20). Samples with different letters are significantly different: P <
0.05. These experiments were repeated at least three times with similar results.
(C) JA-induced reduction of root meristem size in transgenic plants expressing 35Spro:PLT2-GR. Bars = 50 mm.
(D) Statistics of the meristem cell number shown in (C). Data shown are average and SD (n = 15 to 20). Samples with different letters are significantly
different: P < 0.01. These experiments were repeated at least three times with similar results.
For (C) and (D), 4-d-old seedlings germinated on MS medium were transferred to control medium (MS) or medium containing 2 mM DEX for another 2 d
before root meristem cell number was determined. Four-day-old seedlings germinated on medium with 20 mM JA were transferred to medium with 20
mM JA + 2 mM DEX (DEX + JA) or 20 mM JA only (JA) for another 2 d before root meristem cell number was determined.
Jasmonic Acid Modulates Root Stem Cell Niche 3347
observations showing that, as a growth regulator, JA generally
inhibits cell division and therefore represses growth (Dathe et al.,
1981; Swiatek et al., 2004; Pauwels et al., 2008; Zhang and
Turner, 2008). On the other hand, JA promotes cell division in the
QC, as evidenced by the presence of supernumerary QC cells in
JA-treated roots, QC-specific marker gene expression, and EdU
staining assays. It will be interesting in future studies to unravel
the molecular mechanisms underlying these distinct aspects of
JA action. In the context that JA plays an important role in
transmitting environmental cues into developmental responses,
our finding that JA impacts QC division presents an attractive
scenario on how plants adapt their growth to environments by
modulating stem cell division and activity.
MYC2-Mediated Repression of PLT Expression Integrates
Jasmonate and Auxin Pathways in Regulating Maintenance
of the Root Stem Cell Niche
It is noteworthy that the above-described effects of JA on the
root meristem and stem cell niche require the function of MYC2,
a major transcription factor thatpositively ornegatively regulates
different aspects of jasmonate responses, including defense
gene expression and root growth inhibition. Therefore, our in-
vestigation hints at the involvement of MYC2-mediated tran-
scriptional regulation in JA-induced root growth inhibition.
PLT genes encode proteins that act as dose-dependent regu-
lators for root development (Aida et al., 2004; Galinha et al., 2007).
For example, high levels of PLT2 maintain the stem cells, and
intermediate levels facilitate transit amplifying cell divisions that
induced reduction of root meristem activity and disturbance of
stem cell niche maintenance, we found that JA downregulates the
Inthe contextthatJA affects auxin biosynthesis (Dombrecht et al.,
2007; Sun et al., 2009) and that auxin itself regulates the transcrip-
tional expression of PLT genes (Aida et al., 2004), we provided
several lines of evidence that the effect of JA on PLT expression
of the effect of JA on auxin biosynthesis. First, our previous work
revealed that JA promotes auxin biosynthesis through transcrip-
tional activation of the auxin biosynthetic gene ASA1/WEI2 (Sun
etal., 2009; Stepanovaet al., 2005). We show here thatthe asa1-1
mutant, which harbors a loss-of-function mutation of ASA1/WEI2
normal response to JA in terms of root growth inhibition and
meristem cell number reduction. Second, the asa1-1 (Stepanova
et al., 2005; Sun et al., 2009) and yuc1D (Zhao et al., 2001)
mutations, which are defective in auxin biosynthesis, do not affect
JA-induced repression levels and kinetics of PLT1 and PLT2
expression. Third, the action mode of JA on PLT expression is
and PLT2 expression (Aida et al., 2004), we show here that JA
negatively regulates their expression. These, together with other
PLT1 and PLT2 expression is independent of the auxin pathway.
Our finding that JA represses PLT1 and PLT2 expression in a
MYC2-dependent manner raises the interesting possibility that
MYC2 may directly regulate PLT expression. Indeed, our EMSA
and ChIP assays indicate that MYC2 associates with the pro-
moters of both PLT1 and PLT2. Using the well-established
transient assay of N. benthamiana leaves, we show that MYC2
indeed represses PLT1pro:LUC expression and, importantly, that
the G-box-related motif CACATG in the promoter of PLT1 is
of JA on root meristem cell number was substantially reduced in
the plt1-4 plt2-2 double mutant and transgenic plants over-
expressing PLT2. Collectively, these results support the notion
that the PLT1 and PLT2 transcription factors act downstream of
MYC2 in the JA-mediated regulation of stem cell niche mainte-
nance and root meristem activity. Considering the established
role of the PLT transcription factors in mediating developmental
responseto auxininthe rootmeristem(Aidaetal.,2004;Dinneny
and Benfey, 2008), we propose that MYC2-induced repression
of PLT1 and PLT2 expression integrates JA action into the auxin
signaling pathway in regulating postembryonic root growth. It
has been shown that auxin upregulates PLT1 and PLT2 tran-
scripts and positively regulates stem cell niche maintenance and
meristem activity (Aida et al., 2004). In contrast with auxin, this
study reveals that JA downregulates PLT1 and PLT2 expression
and negatively regulates stem cell niche maintenance and mer-
istem activity. PLT1 and PLT2 therefore represent a key junction
for crosstalk between the JA and auxin signaling pathways in the
regulation of root stem cell niche maintenance.
Plant Materials and Growth Conditions
Arabidopsis thaliana ecotypes Columbia-0(Col-0), C24,and Wassilewskija
were used. Some of the plant materials used in this study were previously
described: J2341 and QC25 (Sabatini et al., 1999), PLT1pro:PLT1-YFP,
PLT2pro:PLT2-YFP, PLT1pro:CFP, and PLT2pro:CFP (Galinha et al., 2007),
GUS (Colo ´n-Carmona et al., 1999), WOX5pro:GFP (Blilou et al., 2005),
myc2-2 (Boter et al., 2004), plt1-4 plt2-2 (Aida et al., 2004), and 35Spro:
PLT2-GR (Galinha et al., 2007). jaz10 (CS872819, SAIL_92_D08) was
identified from the SIGnAL T-DNA collection (Alonso et al., 2003b).
MYC2pro:GUS and COI1pro:GUS plants were obtained from transfor-
35Spro:MYC2 plants were obtained from transformation of Col-0 with the
Arabidopsis seeds were surface sterilized for 15 min in 10% bleach,
washed four times with sterile water, and plated on half-strength
Murashige and Skoog (MS) medium. Plants were stratified at 48C for 2
light/8-h-dark photoperiod (light intensity 120 mmol m22s21). Nicotiana
benthamiana was grown under a 16-h-light (288C)/8-h-dark (228C) pho-
toperiod. All experiments were repeated at least three times.
JA (Sigma-Aldrich) was dissolved with ethanol and prepared as a 200
mM stock solution. The 1-amino-1-cyclopropane carboxylic acid (Sigma-
Aldrich)wasdissolved withwaterand preparedasa 10mMstocksolution.
DNA Constructs and Plant Transformation
The COI1 promoter region was PCR amplified with the primers COI1pro-
F, 59-CCGCTGCAGGAGTCAACACCAAGTACAATGAC-39, and COI1pro-
R, 59-CACCTCTTGATATCAGGATCCTCCATCGGATCG-39. The PCR
product was cloned into the PstI-BamHI sites of the binary vector
3348 The Plant Cell
pCAMBIA1391-Z (CAMBIA) to generate the COI1pro:GUS construct. The
MYC2 promoter region was PCR amplified with the primers MYC2pro-F,
59-CACCGACCCTTCCTAAAAAGTAAG-39, and MYC2pro-R, 59-CATTC-
CATAAACCGGTGACCGGT-39. The PCR product was cloned using a
pENTR Directional TOPO cloning kit (Invitrogen) and then recombined
with the plant binary vector pGWB3 (Nakagawa et al., 2007) to generate
the MYC2pro:GUS construct. MYC2 cDNA was PCR amplified from the
reverse transcription product with primers 59-CACCATGACTGAT-
TACCGGCTACA-39 and 59-ACCGATTTTTGAAATCAAACTTGC-39. The
PCR product was cloned using the pENTR Directional TOPO cloning kit
and then recombined with the binary vector pGWB17 (35S promoter,
C-4Myc) to generate the 35Spro:MYC2-4Myc construct.
The above constructs were then transformed into Agrobacterium
tumefaciens strain GV3101 (pMP90), which was used for transformation
Phenotypic Analysis, Statistics, and Microscopy
Seedlings were photographed and root length was measured (Image J;
National Institutes of Health; http://rsb.info.nih.gov/ij). Root meristem size
and QC division were analyzed on seedlings mounted in HCG solution
(chloroacetaldehyde:water:glycerol = 8:3:1). Microscopy was performed
on a Leica Microsystems DM5000B microscope and DFC490 charge-
number between the QC and the first elongating cell in the cortex cell file
(Casamitjana-Martı ´nez et al., 2003; Dello Ioio et al., 2007). The statistical
significance was evaluated by Student’s t test analysis. For multiple
comparisons, an analysis of variance followed by Fisher’s LSD mean
separation test (SPSS) was performed on the data. Samples with different
letters are significantly different at P < 0.01 or P < 0.05. Data presented are
mean values of at least three biological repeats with SD. Images were
processed with Adobe Photoshop CS and Spot Flex software.
GFP, YFP, CFP, and propidium iodide fluorescence was imaged under
a Leica confocal laser scanning microscope (Leica Microsystems). For
imaging GFP, YFP, CFP, and propidium iodide, the 488-, 514-, 458-, and
543-nm lines of the laser were used for excitation, and emission was
detected at 510, 530, 480, and 620 nm, respectively. Fluorescence was
quantified with the LAS AF Lite program on confocal sections acquired
with the same microscope settings (Ru ˚zicka et al., 2007; Zhou et al.,
2010). Approximately 10 seedlings/images were examined, and at least
three independent experiments were performed, giving the same statis-
tically significant results.
Whole seedlings or different tissues were cleared in HCG solution for
several minutes before microscopy analysis. For Lugol staining of roots,
tissues were incubated in Lugol solution (Sigma-Aldrich) for 3 to 5 min,
was performed as described previously (Jefferson et al., 1987).
An EdU incorporation assay was performed as previously described
MS containing 10 mM EdU (Click-iT EdU Alexa Fluor 647 imaging kit;
Invitrogen). After growth, seedlings were fixed in 3.7% formaldehyde in
PBS, pH 7.4, for 15 min. Samples were then washed in 3% BSA in PBS,
EdU to the Alexa fluor substrate occurred in the dark in the Click-iT
reaction mixture, prepared according to the manufacturer’s instructions,
and observations were made using confocal microscopy.
Whole-Mount in Situ Hybridization
Whole-mount in situ hybridization was performed according to the reported
protocols (Weigeland Glazebrook, 2002;Heja ´tko et al., 2006). Antisense and
using T7 and SP6 RNA polymerases, respectively. The following primers
were used to amplify the DNA template for the probe synthesis: WOX5
Gene Expression Analysis
For qRT-PCR analysis, seedling roots were harvested and frozen in liquid
nitrogen for RNA extraction. RNA was extracted with the RNeasy kit
(Qiagen). Poly(dT) cDNA was prepared from 10 mg of total RNA with
Superscript III reverse transcriptase (Invitrogen) and quantified with a
cycler apparatus (Bio-Rad) with the Real Master Mix kit (SYBR Green;
Tiangen) according to the manufacturer’s instructions. PCR was per-
formed in 96-well optical reaction plates heated for 5 min at 958C to
activate hot start Taq DNA polymerase, followed by 40 cycles of dena-
turation for 30 s at 958C, annealing for 30 s at 598C, and extension for 20 s
at 688C. Expression levels of target genes were normalized to those of
ACTIN2 or ACTIN7. The statistical significance was evaluated by Stu-
dent’s t test. For multiple comparisons, an analysis of variance followed
by Fisher’s LSD mean separation test (SPSS) was performed on the data.
Samples with different letters are significantly different at P < 0.01 or P <
0.05. Primers used to quantify gene expression levels are listed in
Supplemental Table 1 online.
One gram of 8-d-old 35Spro:MYC2 (35Spro:MYC2-4Myc) seedlings was
used for ChIP experiments. ChIP was performed as previously described
(Gendrel et al., 2005). The enrichment of DNA fragments was determined
by semiquantitative PCR using the following primer pairs: ACT2proF,
59-CGTTTCGCTTTCCTTAGTGTTAGCT-39; ACT2proR, 59-CACAACG-
CATGCTAAACAGATCTAG-39; PLT1pro(P1)F, 59-GCCCCCTTATTGAAT-
TGGCTCTT-39; PLT1pro(P1)R, 59-GCATACTTGATCCAGTATATGCA-39;
PLT2pro(P2)F, 59-CACATGCAATGAACTCGGGGATC-39; PLT2pro(P2)R,
59-CCCTCTGATCTCTACATACTAAC-39; PLT2pro(P3)F, 59-CAGTCCCT-
CCTTGATGAGATCAT-39; and PLT2pro(P3)R, 59-CATTTCCCTTTTCTTG-
To construct a plasmid for the expression of recombinant MYC2 protein
in Escheichia coli, the full-length cDNA fragment was amplified by
PCR using primers 59-GCTAGCATGACTGATTACCGGCTACAAC-39
and 59-CTGCAGGACCGATTTTTGAAATCAAACTTGC-39 and cloned into
the pMAL-c2 vector via XbaI and PstI restriction sites. Oligonucleotide
probes were synthesized and labeled with biotin at the 39 end (Invitrogen).
EMSA was performed using a LightShift Chemiluminescent EMSA kit
(Thermo Scientific). Briefly, biotin-labeled probes were incubated in 13
binding buffer, 2.5% glycerol, 50 mM KCl, 5 mM MgCl2, and 10 mM EDTA
with or without proteins at room temperature for 20 min. For nonlabeled
probe competition, nonlabeled probes were added to the reactions. The
probe sequences were as follows: PLT1 probe sequence, 59-GAAT-
TTCGGGGATCTACAATAA-39; PLT2 probe sequence, 59-AAGACATG-
ATTTTCTTG-39; PLT1 probe without the CACATG motif (Mu), 59-GAA-
GGATCTACAATAA-39; PLT2 probe without the CACATG motif (Mu),
Transinhibition of PLT1 Promoter Activity by MYC2 in
N. benthamiana Leaves
The transient expression assays were performed in N. benthamiana
leaves as previously described (Matsui et al., 2008; Shang et al., 2010; Qi
Jasmonic Acid Modulates Root Stem Cell Niche 3349
et al., 2011). The PLT1 promoter was amplified with the primer pairs
59-CACCCCTAGCACAATACAATGCAAAGGAC-39 and 59-CTACCACTT-
TGGTATGATCAATATACC-39 and cloned into pENTR using the pENTR
mutations, site-directed mutagenesis was used to delete the CACATG
motif in the P1 region of the PLT1 promoter (Figure 6A) using the TaKaRa
MutanBEST kit. Then, both PLT1 promoter versions were fused with the
binary vector pGWB35 (Nakagawa et al., 2007) to generate the reporter
constructs PLT1pro:LUC and PLT1-mpro:LUC. The MYC2 effector con-
struct was the above-described 35Spro:MYC2-4Myc (35Spro:MYC2).
Agrobacterium-mediated infiltration of N. benthamiana leaves was
performed as described (Shang et al., 2010; Qi et al., 2011). Infiltrated
plants were incubated at 228C for 72 h before CCD imaging. For COR
treatment, the infiltration buffer containing 5 mM COR (+COR) (or solvent
buffer as control) was infiltrated into the N. benthamiana leaf area
coexpressing the indicated constructs 10 h before CCD imaging.
We used a low-light cooled CCD imaging apparatus (NightOWL II
LB983 with indigo software) to capture the LUC image and to count
luminescence intensity. The leaves were sprayed with 100 mM luciferin
and were placed in darkness for 3 min before luminescence detection.
The experiments were repeated at least five times with similar results.
Sequence data from this article can be found in the Arabidopsis Genome
Initiative under the following accession numbers: At3g18780 (ACTIN2),
At5g09810 (ACTIN7), At4g37490 (CYCB1;1), At3g48750 (CDC2A),
At2g23430 (KRP1), At1g07370 (PCNA1), At3g20840 (PLT1), At1g51190
(PLT2), At3g11260 (WOX5), At1g32640 (MYC2), and At2g39940 (COI1).
The following materials are available in the online version of this article.
Supplemental Figure 1. JA Reduces Both Cell Number and Cell
Length of the DZ and EZ.
Supplemental Figure 2. COI1 and MYC2 Are Involved in JA-Induced
Inhibition of Root Growth.
Supplemental Figure 3. JA-Mediated Promotion of QC Division in
the Ethylene Signaling Mutants ein2-5 and ein3-1 eil1-1.
Supplemental Figure 4. The JA Overproduction Mutant cev1 Is
Defective in Maintenance of the Root Stem Cell Niche.
Supplemental Figure 5. JA-Induced Modulation of Root Meristem
Size and Stem Cell Niche Maintenance in the JA-Hypersensitive
Supplemental Figure 6. JA-Induced Repression of PLT Expression in
the Auxin Biosynthesis Mutants asa1-1 and yuc1D.
Supplemental Figure 7. Presence of the JA-Responsive 59-GAGTA-39
Motif in Promoter Regions of PLT1 and PLT2.
Supplemental Table 1. DNA Primers Used for qRT-PCR Assays in
We thank Ben Scheres and Daoxin Xie for kindly providing seeds used in
this study. This work was supported by grants from The Ministry of
Science and Technology of China (2011CB915400 and 2007CB948200)
and The National Natural Science Foundation of China (31030006,
90717007, and 31070251).
C.L. designed theresearch, analyzed thedata, andwrote thearticle. Q.C.
and J.S. designed and performed the research and analyzed the data.
Q.Z., W.Z., L.Q., L.X., B.W., R.C., H.J., J.Q., and X.L. performed the
research and analyzed the data. K.P. analyzed the data.
2011; published September 27, 2011.
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