Download A Membrane-Bound NAC Transcription Factor Regulates Cell

This article is published in The Plant Cell Online, The Plant Cell Preview Section, which publishes manuscripts accepted for publication after they
have been edited and the authors have corrected proofs, but before the final, complete issue is published online. Early posting of articles reduces
normal time to publication by several weeks.
A Membrane-Bound NAC Transcription Factor Regulates
Cell Division in Arabidopsis
W
Youn-Sung Kim,a Sang-Gyu Kim,a Jung-Eun Park,a Hye-Young Park,a Mi-Hye Lim,a Nam-Hai Chua,b
and Chung-Mo Parka,1
a Department
b Laboratory
of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, Korea
of Plant Molecular Biology, The Rockefeller University, New York, New York 10021-3699
Controlled release of membrane-tethered, dormant precursors is an intriguing activation mechanism that regulates diverse
cellular functions in eukaryotes. An exquisite example is the proteolytic activation of membrane-bound transcription
factors. The proteolytic cleavage liberates active transcription factors from the membranes that can enter the nucleus and
evokes rapid transcriptional responses to incoming stimuli. Here, we show that a membrane-bound NAC (for NAM, ATAF1/2,
CUC2) transcription factor, designated NTM1 (for NAC with transmembrane motif1), is activated by proteolytic cleavage
through regulated intramembrane proteolysis and mediates cytokinin signaling during cell division in Arabidopsis thaliana.
Cell proliferation was greatly reduced in an Arabidopsis mutant with retarded growth and serrated leaves in which a
transcriptionally active NTM1 form was constitutively expressed. Accordingly, a subset of cyclin-dependent kinase (CDK)
inhibitor genes (the KIP-related proteins) was induced in this mutant with a significant reduction in histone H4 gene
expression and in CDK activity. Consistent with a role for NTM1 in cell cycling, a Ds element insertional mutant was
morphologically normal but displayed enhanced hypocotyl growth with accelerated cell division. Interestingly, cytokinins
were found to regulate NTM1 activity by controlling its stability. These results indicate that the membrane-mediated
activation of NTM1 defines a molecular mechanism by which cytokinin signaling is tightly regulated during cell cycling.
INTRODUCTION
Cell division plays a crucial role in virtually all aspects of growth
and developmental processes in plants (Meijer and Murray,
2001; De Veylder et al., 2003; Dewitte and Murray, 2003). The cell
cycle consists of alternating phase transitions of DNA replication
(S-phase) and chromosome partitioning (M-phase) separated
by gap phases (G1- and G2-phases). The phase transitions
are coordinately regulated by a battery of cell cycle regulators,
including cyclins, cyclin-dependent kinases (CDKs), and CDK
inhibitors that have been termed inhibitors of Cdc2 kinases or
KIP-related protein (KRPs) in plants (De Veylder et al., 2003).
Plant growth hormones affect cell cycle progression. The roles
of cytokinins, auxins, brassinosteroids, abscisic acid, jasmonic
acid, and ethylene have all been demonstrated (Dewitte and
Murray, 2003). Among these, the best characterized are cytokinins, which are most directly related to cell division. Cytokinins
regulate the G1/S and G2/M transitions as well as progression
through the S-phase (De Veylder et al., 2003). They induce
CYCD3-1 expression, and transgenic plants overexpressing
CYCD3-1 are cytokinin-autotrophic in leaf callus culture (RiouKhamlichi et al., 1999).
1 To
whom correspondence should be addressed. E-mail cmpark@
snu.ac.kr; fax 82-2-889-1568.
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: Chung-Mo Park
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.106.043018
Selective protein degradation is a critical component of both
growth hormone signaling and cell cycling (Deshaies, 1999;
Hellman and Estelle, 2002; Risseeuw et al., 2003; Vierstra, 2003).
Auxins direct the degradation of auxin/indole-3-acetic acid
(AUX/IAA) proteins that act as auxin signaling repressors. The
SCF complex containing the TIR1 F-box protein provokes AUX/
IAA degradation in response to auxins (Gray et al., 2001).
Interestingly, the nucleus-localized ring E3 SINAT5 regulates
NAC1, a NAC transcriptional activator that modulates auxin
signaling during lateral root formation (Xie et al., 2000). Accumulating evidence strongly suggests that the ubiquitin/proteasome
pathway also plays a key role in cytokinin signaling. An Arabidopsis thaliana mutant with an inactive RPN12 subunit of the 26S
proteasome is insensitive to cytokinins and exhibits constitutive
cytokinin responses, such as the suppression of primary root
growth and lateral root formation (Smalle et al., 2002).
Proteolytic cleavage also plays a role in the activation of
membrane-bound, dormant transcription factors. It was recently
demonstrated that a group of membrane-bound transcription
factors are activated by proteolytic cleavage (Sakai et al., 1998;
Hoppe et al., 2000, 2001; Vik and Rine, 2000). The membranebound transcription factors are activated by controlled proteolytic cleavage through either of two distinct but biochemically
related ways. In regulated intramembrane proteolysis (RIP),
active transcription factors are liberated by specific membrane-associated proteases (Vik and Rine, 2000; Hoppe et al.,
2001). In regulated ubiquitin/26S proteasome-dependent processing (RUP), they are ubiquitinated and degraded by the 26S
proteasome in a controlled manner, resulting in the release of
transcriptionally active forms. One example of RUP was recently
The Plant Cell Preview, www.aspb.org ª 2006 American Society of Plant Biologists
1 of 13
2 of 13
The Plant Cell
demonstrated during the activation of SPT23 and MGA2 transcription factors in yeast (Hoppe et al., 2000). They are expressed
as dormant, endoplasmic reticulum/nuclear membrane–associated
precursors. Upon stimulation, transcriptionally active forms are
liberated by RUP.
We demonstrate here that a membrane-bound transcription
factor, NTM1 (for NAC with transmembrane motif1), that belongs
to the plant-specific NAC transcription factor family controls cell
division in Arabidopsis. NTM1 is released from the membranes
by proteolytic cleavage. The activated NTM1 transcription factor
enters the nucleus and induces a subset of CDK inhibitor genes
(KRPs) and represses the histone H4 gene, resulting in reduced
cell division. Interestingly, cytokinins stabilize the NTM1 proteins.
Therefore, we propose that the cytokinin regulation of NTM1
stability is a molecular scheme that modulates CYCD3-mediated
cytokinin signaling during cell cycle control (Riou-Khamlichi et al.,
1999) to achieve a balanced regulation of cell division in plants.
RESULTS
ntm1-D Exhibits Reduced Growth with Serrated Leaves
To explore the genetic components and molecular schemes that
govern plant growth and development, we isolated a morphogenetic leaf mutation (designated ntm1-D) from an Arabidopsis
mutant pool that had been generated by randomly integrating
the cauliflower mosaic virus 35S enhancer into the genome of
ecotype Columbia (Col-0). ntm1-D exhibits retarded growth
(Figures 1A and 1D) with severely serrated leaves (Figure 1B).
Its phenotype is further characterized by extremely tiny petals
(Figure 1C), small plant organs, reduced apical dominance, and
reduced root growth (Figures 1A and 1E). In addition to disrupted
petal development, other floral organs are also influenced by the
ntm1-D mutation. Although sepals and stamens are morphologically normal, they are small (Figure 1C).
The most prominent feature in ntm1 is severe leaf serration
(Figure 1B). Notably, it has been reported that overexpression of
CDK inhibitor genes, such as KRP1, KRP2, KRP3, and KRP6
(Wang et al., 2000; De Veylder et al., 2001), and inactivation of
cell cycle regulators, including JAGGED (Dinneny et al., 2004;
Ohno et al., 2004), result in a similar phenotype to that seen in
ntm1-D, suggesting a correlation between the phenotype conferred by ntm1-D and disturbed cell cycling.
When ntm1-D was backcrossed to wild-type plants (Col-0),
wild-type and ntm1-D–like phenotypes were observed at a ratio
of 1:1 in the F1 progeny, indicating that the ntm1 mutation is
dominant. Furthermore, BASTA-resistant and -sensitive plants
were observed at a ratio of 1:1, and all BASTA-resistant plants
showed the ntm1-D–like phenotype, demonstrating that this
phenotype cosegregates with T-DNA insertion.
A three-step thermal asymmetric interlaced PCR method was
used to map the T-DNA insertion site (Liu et al., 1995). A single
copy of the 35S enhancer was inserted into the fourth exon of an
open reading frame (At4g01540) in ntm1-D. The open reading
frame had been predicted to have five exons (Arabidopsis
Genome Initiative, 2000). However, sequencing of full-size
cDNA clones identified an additional exon with the authentic
Figure 1. The Phenotype Conferred by ntm1-D, and Mapping of the
T-DNA Insertion Site.
(A) Retarded growth, with small plant organs.
(B) Serrated leaves. Leaves were compared by scanning electron
micrography.
(C) Impaired petal development.
(D) and (E) Short stems (D) and reduced primary root growth and lateral
root formation (E). The number of lateral roots was counted for unit root
length. Measurements of 30 plants were averaged. Bars indicate SD.
The statistical significance of the measurements was determined by
Student’s t test (P < 0.01).
(F) T-DNA insertion in the ntm1-D genome. ntm1-D has a single insertion
event, as indicated by the arrow, in the fourth exon of NTM1. The hatched
box marks the exon newly identified in this work.
GT/AG boundaries between exons four and five in the original
prediction (Figure 1F).
The Phenotype Conferred by ntm1-D Is Caused by
Constitutive Expression of Active NTM1
The revised open reading frame encodes a polypeptide of 473
residues. Sequence analyses revealed that it contains a highly
Cell Cycle Regulation by NAC
Figure 2. NTM1 Structure and Transgenic Arabidopsis Plants Expressing Different NTM1 Constructs.
(A) NTM1 structure. A putative nuclear localization signal (NLS) is
recognized close to the NAC domain. A TM motif is present in the far
C-terminal region. Numbers indicate amino acid (aa) positions.
(B) Structural comparison of NTM1 with other NAC proteins. NTM1 and
NTM2 have TM motifs. Note that the DC construct in (A) has a similar size
to those of other NAC proteins.
(C) DC transcription in ntm1-D. A tubulin gene (TUB) was used as a
control for constitutive expression.
(D) Transgenic Arabidopsis plants overexpressing different NTM1 proteins.
Three different NTM1 proteins with or without the TM motif were expressed.
(E) Correlation between serrated leaves and DC expression. Four transgenic lines with different DC transcript levels were compared.
3 of 13
conserved NAC domain in the N-terminal region (Figure 2A) (Aida
et al., 1997). The NAC domain of ;130 residues has been
identified in members of the recently recognized NAC transcription factor family in plants (Figure 2B) (Takada et al., 2001; Hibara
et al., 2003). It possesses a sequence-specific DNA binding
activity. A region of 60 residues within the NAC domain contains
a novel transcription factor fold consisting of a twisted b-sheet
surrounded by a few helical elements (Duval et al., 2002).
However, the C-terminal sequences do not have any discernible
sequence similarities. The NAC proteins have been proven to be
transcription factors by in vivo assays in yeast (Duval et al., 2002)
and in planta (Xie et al., 2000), and the transcriptional activity has
been assigned to the variable C-terminal region.
Strikingly, the predicted polypeptide is distinct from other NAC
members in that it has a transmembrane (TM) motif with a
predicted a-helical conformation in the far C-terminal region
(Figures 2A and 2B). Therefore, the polypeptide was designated
NTM1 (for NAC with Transmembrane Motif1) in this work. NTM1
has a putative nuclear localization signal close to the NAC
domain (Figure 2A). An adjacent locus (At4g015550) also encodes a NAC protein (designated NTM2) with a similar structural
organization to that of NTM1 (Figure 2B). Although At4g01520
encodes another NAC-like protein of smaller size than NTM1 and
NTM2 (Figure 2B), we were not able to detect its expression by
extensive RT-PCR runs. It may be a nonfunctional gene or expressed at a subtle developmental stage.
Based on the T-DNA insertion site in NTM1, it was first
anticipated that ntm1-D would be a loss-of-function mutation.
Alternatively, the T-DNA insertion might cause the expression of
a truncated NTM1 that lacks the TM motif. It is notable that the
estimated size of the truncated NTM1 is quite similar to those of
other nuclear NAC proteins (Figure 2B). In addition, an NTM1
transcript with a smaller size was detected in ntm1-D to a level
similar to that in control plants (Figure 2C), supporting the notion
that a truncated NTM1 is expressed. Sequencing of a cDNA of
the truncated NTM1 transcript in ntm1-D showed that it covered
the NTM1 sequence up to the T-DNA insertion site followed
by the T-DNA sequence. To further address this issue, NTM1
gene sequences encoding a full-size and a couple of partial
NTM1 proteins were transformed into Arabidopsis plants. Interestingly, only the transgenic plants expressing the DC construct,
which is equivalent to the truncated NTM1, showed serrated
leaves (Figure 2D). In addition, the severity of leaf serration was
correlated with the transcript level of DC (Figure 2E). However,
those plants overexpressing the full-size or DTM constructs
showed no serrated leaves and were indistinguishable from
control plants, although they were expressed to high levels.
These results suggest that expression of the truncated NTM1 is
the molecular cause of the leaf serration observed in ntm1-D.
NAC proteins form dimers through the NAC domains (Ernst
et al., 2004). Therefore, we suspected that DC expression might
cause a dominant negative suppression of intrinsic NTM1. We
used the RNA interference method to specifically inactivate
(F) Transgenic Arabidopsis plants in which NTM1 expression is specifically blocked by RNA interference. Two transgenic plants (c and d) have
normal leaves. Con, control transgenic plant with vector alone.
4 of 13
The Plant Cell
NTM1, because no NTM1-deficient mutant was available. When
RNA interference was used to specifically inactivate intrinsic
NTM1, the majority of the 120 transgenic lines examined did not
exhibit serrated leaves, although 2 transgenic lines possessed
marginally serrated leaves (Figure 2F, lines c and d). However,
this was not related to the inactivation of intrinsic NTM1 in these
plants (Figure 2F, right panel). Furthermore, NTM1 expression
was greatly reduced in transgenic line c, which exhibited normal
leaf morphology. The normal leaf morphology of the T-DNA
insertional mutant ntm1-1 (see Figure 5 below) confirmed these
results, demonstrating that the phenotype conferred by ntm1-D
is not caused by the dominant negative suppression of intrinsic
NTM1. Therefore, it appeared that the expression of a truncated
NTM1 was responsible for the phenotype conferred by ntm1-D.
Cell Division Is Reduced in ntm1-D
Constitutive expression of the transcriptionally active nuclear
NTM1 protein (nNTM1) is directly linked to the phenotype conferred by ntm1-D (Figure 2D), which is similar to those of
transgenic plants overexpressing KRPs and of the jagged mutation with disrupted cell cycling. Therefore, we hypothesized
that NTM1 might have a role in cell cycle control and that cell
proliferation would be reduced in ntm1-D.
To examine this hypothesis, the primary roots of ntm1-D and
control plants were cut into segments of 0.5 mm in length, and the
root segments were cultured for 2 weeks on Murashige and
Skoog (MS) medium supplemented with 100 mg/L 2,4-D and
different concentrations of kinetin, ranging from 30 to 3000 mg/L
(Inoue et al., 2001). Calli were actively induced, and callus cells
rapidly proliferated in control root segments in the presence of all
combinations of growth hormone concentrations tested (Figure
3A). By contrast, callus growth was greatly reduced in the ntm1-D
root segments, especially at high concentrations of kinetin. Callus
growth was also repressed to a similar degree in the cotyledons
of ntm1-D (see Supplemental Figure 1 online). These results indicate that cell division is significantly repressed in ntm1-D and
that nNTM1 has a negative effect on cell division.
Mutant plants with reduced cell division frequently exhibit a
normal growth appearance. This can be explained by cell enlargement that compensates for reduced cell number (Donnelly
et al., 1999; Kessler and Sinha, 2004). In other cases, such as
transgenic plants overexpressing KRP1, KRP2, or KRP6, the
plants exhibit retarded growth as well as severe developmental
Figure 3. Reduced Cell Division in ntm1-D.
(A) Reduced callus growth from primary root segments. Primary root segments were cultured on a callus induction medium in the presence of different
concentrations of 2,4-D and kinetin (K).
(B) Enlarged cells. Sizes of the cells from the adaxial sides of leaves were compared by scanning electron micrography and displayed by bar graphs.
The statistical significance of the measurements was determined by Student’s t test (P < 0.01). The error shown is SD (n ¼ 85). Bars ¼ 50 mm.
(C) Reduced leaf sizes. Twenty measurements of the sizes of fifth leaves were averaged. The error shown is SD.
(D) Increased KRP expression. The transcript levels of KRPs were examined by RT-PCR–based DNA gel blot analysis using the leaves of 2-week-old
plants.
(E) Reduced H4 gene expression. H4 expression was analyzed by RNA gel blot analysis.
(F) Reduced CDK activities. CDK activities were compared using histone H1 as a general phosphorylation substrate.
Cell Cycle Regulation by NAC
defects. It has been proven that although the phenotype is
caused mainly by reduced cell number, it is at least partially
compensated for by increased cell expansion (De Veylder et al.,
2001). Based on the compensation system, cell enlargement is
widely used as an indicator of reduced cell division in Arabidopsis. Scanning electron micrographic comparison of leaf cells
showed that cells from the adaxial side of rosette leaves in
ntm1-D were enlarged by ;40% compared with those of control
plants (Figure 3B). Cell enlargement was more prominent on the
adaxial side than on the abaxial side, which may be related to
the morphogenesis of leaf margins (Wang et al., 2000). Because
the leaf size of ntm1-D is ;60% smaller than that of control
plants (Figure 3C), total cell number in ntm1-D leaves is estimated to be reduced by ;80%.
The morphological similarity between KRP-overexpressing
plants and ntm1-D suggests that KRPs may be upregulated
in ntm1-D. As expected, a subset of KRPs was significantly
upregulated in ntm1-D. The transcript levels of KRP2, KRP3,
KRP6, and KRP7 significantly increased, whereas those of
KRP1, KRP4, and KRP5 were unaffected (Figure 3D), indicating
that NTM1 functions, at least in part, by inducing KRPs. Another
criterion frequently used to measure cell division rate is the
histone H4 gene, which is highly expressed in actively dividing
cells (Riou-Khamlichi et al., 1999). Total RNA samples were
extracted from leaf tissues, and the H4 transcript levels were
analyzed by RNA gel blot analysis. It was drastically reduced in
ntm1-D (Figure 3E), further supporting the idea of reduced cell
division in ntm1-D. That cell division was reduced in ntm1-D was
further supported by a 50% decrease in CDK activity (Figure 3F)
in ntm1-D, as judged by phosphorylation of the histone H1 as a
general substrate.
To further confirm the correlation between the phenotype
conferred by ntm1-D and reduced cell division, a kinematic
examination of leaf growth in ntm1-D was performed. The total
cell number per leaf was estimated (Figure 4C) using average
total leaf size (Figure 4B) and epidermal cell size (Figure 4A) on
the adaxial side of the first leaves. The number of adaxial
epidermal cells in mature leaves rapidly increased in control
plants until day 7, whereas the rate was much slower in ntm1-D
(Figure 4C). By day 10, the leaf cell number in ntm1-D was 25% of
that in control plants. Comparison of average cell division rates,
calculated as the slope of total cell number per leaf using threepoint differentiation formulas (De Veylder et al., 2001), showed
that the cell division rate was much slower in ntm1-D until day 8
(Figure 4D). Altogether, these observations clearly demonstrate
that cell division was significantly reduced in ntm1-D. We also
obtained similar results from 35S::DC transgenic plants. Cell
number per leaf and leaf growth kinematics were reduced
significantly, and KRP expression was upregulated in the transgenic plants (see Supplemental Figure 2 online).
Consistent with a role for NTM1 in cell division, a knockout
mutant, ntm1-1, displayed elongated hypocotyls (Figures 2A and
5A). However, the size of ntm1-1 hypocotyl cells was essentially
identical to that of control cells (Figure 5B). Furthermore, expression of the G1/S cell cycle regulators KRP2 and KRP7 was
significantly reduced in ntm1-1 (Figure 5C), providing further
evidence that NTM1 regulates cell division. The expression of
CYCD3;1 was also reduced in ntm1-1, indicating the possibility
5 of 13
Figure 4. Kinematic Analysis of Leaf Growth in ntm1-D.
Kinematic analysis of leaf growth was performed on plants grown on MS
medium. Error bars indicate SD. The statistical significance of the
measurements was determined by Student’s t test (P < 0.01). DAG,
days after germination.
(A) Cell size measurements. Cell sizes were determined from three
independent measurements, each consisting of at least 100 adaxial
epidermal cells, and averaged (P < 0.01).
(B) Leaf area measurements.
(C) Cell number per leaf.
(D) Estimated cell division rates.
6 of 13
The Plant Cell
Figure 5. Characterization of ntm1-1.
(A) Growth phenotype. Thirty hypocotyls were measured from 5-d-old seedlings grown in the light and averaged. The error shown is SD.
(B) Hypocotyl cells. Midhypocotyl regions were compared from the seedlings shown in (A). Scanning electron micrographic images of 11 hypocotyls of
2-week-old seedlings were used to count epidermal cells and averaged. The statistical significance of the measurements was determined by Student’s
t test (P < 0.01). The error shown is SD. Bars ¼ 100 mm.
(C) Expression of KRPs. For detection of KRP expression in ntm1-1, plants were sprayed with an N6-benzyladenine solution (BA; 500 mM), and total
RNAs were extracted from the leaf tissues. MO indicates mock treatment. A tubulin gene (TUB) was used as a control for constitutive expression.
(D) Relative levels of CYCD3;1 expression. The relative levels of CYCD3;1 expression in (C) were quantitated. The levels were compared with those in
the mock-treated (MO) Col-0.
of negative feedback regulation between cell cycle regulators,
especially KRPs and CYCs (Figure 5D). Although the reduction
was not prominent, it was reproducible in repeated experiments.
NTM1 Is Associated with Intracellular Membranes
NTM1 has a potential TM motif (Figure 2A). Therefore, we
hypothesized that NTM1 might be expressed as a membraneassociated, dormant form but released from the membranes and
localized in the nucleus, where it might trigger a molecular event
that is linked to the phenotype conferred by ntm1-D.
To examine this hypothesis, a myc-NTM1 fusion polypeptide
was transiently expressed in Nicotiana benthamiana leaf cells.
Cell fractionation and protein gel blot assays showed that total
cellular extract contained two major NTM1-specific bands (Figure 6A). The upper band was predicted to be the full-length,
membrane-associated NTM1 protein (mNTM1) and was detected
exclusively in the microsomal fraction. The estimated molecular
size of the lower band was close to that of DC and other nuclear
NACs, suggesting that it was a processed nNTM1. The nNTM1
polypeptide was not detected in the soluble fraction, suggesting
that it might be unstable, as has been reported for the soluble
forms of other membrane-associated transcription factors
(Brown et al., 2000; Hoppe et al., 2001). However, only a fullsize NTM1 transcript was detected (Figure 6B), excluding the
possibility of alternative mRNA splicing for the generation of two
NTM1 forms. The association of mNTM1 with the intracellular
membranes was further confirmed by extensive cell fractionation
analysis. It was always detected only in the microsomal fractions
and removed from the microsomal fractions by detergent treatment (Figure 6C; see Supplemental Figure 3 online). Therefore, it
was evident that mNTM1 is associated with the intracellular
membranes, from which a soluble form (nNTM1) is processed.
To determine whether nNTM1 is processed from mNTM1, the
transgenic plants were treated with cycloheximide. Levels of
mNTM1 decreased significantly after cycloheximide treatment,
certainly as a result of decreased protein synthesis and continued mNTM1 processing. Whereas the level of nNTM1 was
relatively high in mock-treated plants even after 18 h of incubation, it increased slightly after 3 h of incubation with cycloheximide and decreased slowly after longer incubation (Figure 6D),
indicating that nNTM1 was processed from mNTM1.
To confirm the membrane association of mNTM1 and the
nuclear localization of nNTM1, a series of GFP-NTM1 fusion
polypeptides were transiently expressed in onion (Allium cepa)
epidermal cells. It was found that mNTM1 was densely localized
to the nuclear membrane and/or the endoplasmic reticulum
membrane around the nucleus (Figure 6E). To a lesser degree,
nNTM1 signal was detected within the nucleus, possibly as a
result of intrinsic NTM1 processing. In comparison, DC and DTM
were detected exclusively within the nucleus. Although both DC
and DTM were localized in the nucleus, only the DC transgenic
plants had serrated leaves (Figure 2D), suggesting that the
nuclear localization is not sufficient for NTM1 function. An
inhibitory activity obviously resides in the C-terminal region that
is present in DTM but missing in DC.
NTM1 Is Activated by Proteolytic Cleavage
A few examples of membrane-associated transcription factors
have been functionally characterized in prokaryotes, yeast, and
animals. Membrane-associated transcription factors characterized to date are released either by RUP or by RIP (Vik and Rine,
2000; Hoppe et al., 2001). One example of RUP is the activation
of SPT23/MGA2 transcription factors in yeast (Hoppe et al.,
2000), whereas the RIP process has been best characterized in
Cell Cycle Regulation by NAC
7 of 13
Figure 6. NTM1 Release from the Intracellular Membranes by Proteolytic Cleavage.
The aerial parts of 2-week-old plants were used to examine cycloheximide and MG132 effects on NTM1 processing and stability. The arrowheads and
arrows indicate putative membrane-associated (mNTM1) and nuclear (nNTM1) forms, respectively.
(A) Cell fractionation assays. Total extract (T) of N. benthamiana leaf cells expressing a myc-NTM1 fusion was fractionated into soluble (S) and
microsomal (M) fractions, and the myc-NTM1 proteins were detected using an anti-myc antibody. Control leaves (Con) injected with the vector alone
were analyzed for comparison.
(B) NTM1 transcript profile in the transgenic plants overexpressing myc-NTM1. Twenty-five micrograms of total RNA sample was analyzed by RNA gel
blot hybridization and exposed for up to 48 h.
(C) Membrane association of NTM1. Aliquots of each fraction were mixed with an equal volume of the SDS buffer and subjected to protein gel blot
analysis (top panel) using an anti-myc antibody. The Commassie blue–stained membrane is displayed as a loading control (bottom panel). T, total
extract; S, soluble fraction; M, membrane fraction; B, buffer-extracted fraction; SD, SDS-extracted fraction; F, final membrane fraction (see
Supplemental Figure 3 online).
(D) Cycloheximide (CHX) effect on the NTM1 processing. MO, mock treatment.
(E) Subcellular localization of NTM1 proteins. The inset shows a magnified view of the nuclear region to illustrate dense localization of the GFP-NTM1
fusion on the nuclear/endoplasmic reticulum membranes around the nucleus.
(F) MG132 effect on NTM1 stability. Asterisk marks a putative intermediate form.
(G) NTM1 cleavage by calpain protease. A calpain inhibitor (ALLN) was included in the assays. K, kinetin.
the activation of Sterol-Regulatory Element Binding Protein
(Sakai et al., 1998; Hoppe et al., 2001).
To examine more fully the mechanism of NTM1 activation, a
26S proteasome–specific inhibitor (Lee and Goldberg, 1998),
MG132, was first used. Treatment of transgenic Arabidopsis
plants expressing a myc-NTM1 fusion with MG132 resulted in a
significant increase of both mNTM1 and nNTM1 levels in the total
cellular extract (Figure 6F). One additional band, which was
smaller than mNTM1, was also increased (Figure 6F, asterisk). It
is thus likely that the 26S proteasome is not involved in the
release of NTM1 from the membrane but instead regulates the
stability of both mNTM1 and nNTM1 proteins.
The next step was to determine the mechanism through which
NTM1 was released from the membranes. Calpains are known to
play essential roles in cell proliferation, differentiation, and apoptosis in eukaryotes. Plant species studied to date have been
shown to possess a single calpain gene that encodes a membrane-associated, Defective Kernel1 (DEK1)–type protease
(Margis and Margis-Pinheiro, 2003). It was also recently demonstrated that the tobacco (Nicotiana tabacum) calpain (Nt
DEK1) regulates cell proliferation and differentiation and is critical
for normal organ development (Ahn et al., 2004). Particularly
interesting are transgenic Arabidopsis plants that overexpress
the Arabidopsis DEK1 gene with a phenotype of serrated leaves
and floral defects (Lid et al., 2005), suggesting that calpain might
be involved in the NTM1 processing. To examine the role of
calpains in NTM1 processing, transgenic Arabidopsis plants
expressing a myc-NTM1 fusion were treated with a calpain
inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal (ALLN). mNTM1
level increased, whereas nNTM1 level almost completely disappeared after ALLN treatment (Figure 6G). This finding indicated that NTM1 was proteolytically released from the
8 of 13
The Plant Cell
intracellular membranes by calpain or its functional homologue.
However, because ALLN is not effective exclusively on calpains,
other proteases may also be involved in NTM1 processing.
To further examine these observations, a membrane-associated NTM1-like protein (At2g27300), which has an identical
structural organization to NTM1 in having a NAC domain in the
N-terminal region and a TM domain in the far C-terminal region,
was similarly analyzed. The processing of the NAC protein was
unaffected by kinetin as well as by ALLN (see Supplemental
Figure 4 online), indicating that the effects of kinetin and ALLN
are specific to NTM1.
Cytokinins Regulate NTM1 Stability
One of the major challenges with regard to membrane-bound
transcription factors is the identification of the input signals that
affect the release and degradation of transcriptionally active
forms and regulate their functions. For example, SPT23 processing by RUP is completely repressed by unsaturated fatty
acids, suggesting that the membrane-associated precursor of
SPT23 may be a sensor for membrane fluidity (Hoppe et al.,
2000).
Therefore, the next issue to be addressed is how NTM1
function is regulated. We observed that cell division was markedly reduced in ntm1-D plants that exhibited dwarfed growth and
serrated leaves, suggesting that signaling from growth hormones, particularly cytokinins and/or auxins, might be disturbed
(Meijer and Murray, 2001; Dewitte and Murray, 2003). To examine this possibility, transgenic Arabidopsis plants overexpressing
a myc-NTM1 fusion were treated with kinetin or IAA, and profiles
of the NTM1 polypeptides were examined. Interestingly, it was
found that kinetin, but not IAA, significantly affected NTM1
stability. When the transgenic Arabidopsis plants were treated
with kinetin, both mNTM1 and nNTM1 levels increased significantly soon after kinetin treatment, and the high levels were
maintained during treatment (Figure 7A). Furthermore, the kinetin
effect on NTM1 stability was proportional to the increasing
concentrations of kinetin used, and the stabilizing effect was
evident at a concentration as low as 5 mM (Figure 7B). However,
NTM1 transcription was unaffected by kinetin treatment (Figure
7C), indicating that the kinetin effect on NTM1 activity occurs not
at the transcriptional level but at the protein level.
As KRPs are induced by nNTM1 (Figure 3D) and kinetin
stabilizes the NTM1 proteins (Figure 7A), it was hypothesized
that KRPs would also be induced by kinetin. As expected, KRP7
expression in leaves was greatly induced by kinetin treatment
(Figure 7D). However, KRP7 induction by kinetin was abolished in
the ntm1-1 knockout mutant, confirming the role of cytokinins in
NTM1-mediated KRP expression and suggesting that NTM1
may mediate cytokinin signaling during cell division. When root
segments were cultured on MS medium supplemented with
zeatin and IAA, root greening and thickening were greatly reduced in ntm1-D (see Supplemental Figure 5 online) as well as in
the 35S::DC transgenic plants, probably reflecting the high level
of nNTM1, which inhibits cell division. ntm1-1 was also less
sensitive to cytokinins. Inhibition of growth and leaf development
by cytokinins was obviously reduced in ntm1-1 by the lack of
nNTM1 (Figure 7E; see Supplemental Figure 6 online). These
Figure 7. Cytokinin Regulation of NTM1 Function.
The mNTM1 (arrowheads) and nNTM1 (arrows) polypeptides are indicated. Parts of Commassie blue–stained gels are displayed as loading
controls.
(A) Kinetics of kinetin effects on NTM1 stability. Transgenic Arabidopsis
plants overexpressing a myc-NTM1 fusion construct were treated with
100 mM kinetin (K). MO refers to mock treatment.
(B) Treatment with different concentrations of kinetin. Transgenic plants
were treated with increasing concentrations of kinetin for 14 h.
(C) Kinetin effects on NTM1 expression. Wild-type plants were treated
with 100 mM kinetin for up to 6 h. NT, no treatment.
(D) Kinetin effects on KRP expression. Twelve-day-old plants were
treated with 100 mM kinetin for 6 h. Twenty-five micrograms of total RNA
samples extracted from the leaves was loaded onto each lane.
(E) Growth response of ntm1-1 to kinetin. ntm1-1 plants were germinated
and cultured on MS medium supplemented with 1 mM kinetin for 3 weeks.
Thirty plants per plant group were measured and averaged. Bars denote
SD. The statistical significance of the measurements was determined by
Student’s t test (P < 0.01). Bars ¼ 0.5 mm. Ler, Landsberg erecta.
Cell Cycle Regulation by NAC
9 of 13
observations strongly support the notion that NTM1 mediates
cytokinin signals.
NTM1 Mediates Cytokinin Signaling Specifically during
Cell Division
Cytokinins regulate diverse aspects of plant growth and developmental processes, such as seed germination, stem and root
growth, senescence, and cell division (Mok and Mok, 2001;
Smalle et al., 2002). Numerous cytokinin-responsive genes have
been identified through molecular genetic approaches (Hwang and
Sheen, 2001; Heyl and Schmülling, 2003). The two-component
signaling scheme, including ARR response regulators, is apparently a critical component of cytokinin signaling. Type-A ARR
genes, such as ARR4 and ARR5, are rapidly induced by cytokinin
treatment (D’Agostino et al., 2000). Therefore, it is evident that
cytokinin signals are modulated through a complex network of
multiple cascades.
To examine whether cytokinin responses, other than that in cell
cycle control, are influenced by NTM1, cytokinin effects on ntm1-D
root growth were analyzed. Root growth is inhibited by high
concentrations of cytokinins and auxins. Measurements of relative
root length showed that ntm1-D root growth was inhibited by both
N6-benzyladenine (see Supplemental Figure 7A online) and IAA
(see Supplemental Figure 7B online) to a similar degree as in
control plants, indicating that cytokinin and auxin signaling pathways in root growth regulation are not influenced by the ntm1
mutation.
The cytokinin effect on ARR5 induction was also unaffected in
ntm1-D (see Supplemental Figure 8 online). ARR5 was rapidly
induced by cytokinin treatment in ntm1-D by a similar kinetics as
in control plants. Therefore, it is obvious that not all of the
cytokinin signaling pathways are regulated by NTM1. The primary role of NTM1 appears to be specific to the cytokinin
signaling in cell cycle control.
NTM1 Is a Transcriptional Activator
All of our observations indicate that nNTM1 is released from the
membranes and localized in the nucleus, where it may function
as a transcriptional activator like other known NAC transcription
factors.
To examine this, the DC construct was fused in-frame to the
GAL4 DNA binding domain in the yeast expression vector
pGBKT7. The GAL4-DC fusion was expressed in a yeast strain
containing a reporter gene (LacZ), and a-galactosidase activity
was measured. The yeast cells expressing DC clearly showed
LacZ activation (Figure 8A). Furthermore, whereas the C-terminal
region of DC strongly activated the LacZ reporter gene, the NAC
domain alone did not activate it. These results clearly demonstrate that NTM1 is a transcriptional activator.
Altogether, our data support the notion that NTM1 mediates
cytokinin signaling specifically during cell division. The NTM1
transcription factor is stabilized by cytokinins and negatively
regulates cell division by inducing KRP genes (Figure 8B). The
NTM1-mediated cytokinin signaling pathway is likely to be
required for the fine-tuning of the CYCD3-mediated cytokinin
signaling that promotes cell division, as described for the dual
Figure 8. A Schematic Working Model for the NTM1 Transcription
Factor.
(A) Analysis of transcriptional activities. P, positive control (full-size
GAL4); N, negative control (DNA binding domain alone); DC-N and DC-C,
N-terminal and C-terminal regions of DC, respectively. Four independent
measurements were averaged (P < 0.01). The error shown is SD.
(B) Proposed mechanism for NTM1 function in cell cycle control. The
nuclear form (nNTM1) is liberated from the nuclear/endoplasmic reticulum (ER) membranes through regulated intramembrane proteolysis (RIP).
Cytokinins stabilize the NTM1 proteins, possibly by blocking the 26S
proteasome activity. In this scheme, the CYCD3-mediated cytokinin
signaling is likely to be balanced with the negative regulatory effect
exerted by NTM1, similar to that described for c-MYC function in cell
division in animals (O’Donnell et al., 2005).
roles of c-MYC in the regulation of cell division in animals
(O’Donnell et al., 2005).
DISCUSSION
NTM1 Is a Membrane-Bound NAC Transcription Factor
There are up to 105 NAC transcription factors predicted in the
Arabidopsis genome (Duval et al., 2002; Ooka et al., 2003). A
group of NAC transcription factors have been genetically characterized in various growth and developmental processes, including floral development (Sablowski and Meyerowitz, 1998),
apical meristem formation (Hibara et al., 2003), stress responses
(Tran et al., 2004; He et al., 2005), and growth hormone signaling
(Xie et al., 2000; Fujita et al., 2004).
NTM1 is distinct from other NAC members studied in that it is
an integral membrane protein. Interestingly, there are up to 14
additional NAC proteins, including NTM1 and NTM2, that possess strong TM motifs (Schwacke et al., 2003). One of these was
recently characterized. An Arabidopsis transcription factor, At
10 of 13
The Plant Cell
bZIP60, is predicted to be membrane-associated and to regulate
endoplasmic reticulum stress responses (Iwata and Koizumi,
2005). Like NTM1, membrane release is essential for At bZIP60
function.
The membrane-bound NAC proteins certainly belong to a
unique set of NAC transcription factors whose transcriptional
activities are regulated by the liberation step from the membranes. A recent report provides additional insight into how the
membrane-bound NAC proteins are liberated. The RING-H2
domain is a specific type of zinc finger that is present in many
multiple-subunit E3 ligases, such as the SCF complex (Deshaies,
1999). This domain interacts with ANAC, an abscisic acid–
responsive NAC, via the NAC domain (Greve et al., 2003). Both
RIP and RUP pathways may be involved in the activation of the
membrane-bound NAC transcription factors.
A calpain inhibitor, ALLN, inhibits NTM1 processing, suggesting that NTM1 is released from the membranes by calpain
protease. Arabidopsis has a single calpain gene (DEK1).
Notably, N. benthamiana DEK1 has been shown to regulate
cell proliferation (Ahn et al., 2004). Particularly, transgenic
Arabidopsis plants overproducing Arabidopsis DEK1 exhibit
serrated leaves and floral defects (Lid et al., 2005), similar to
those observed in ntm1-D, supporting the above notion. However, ALLN is not highly specific to calpain protease. Therefore, it
is possible that other proteases, such as S1P, S2P, and presenilins, may also contribute to NTM1 processing (Hoppe et al.,
2001). It will be interesting to examine the patterns of NTM1
processing in mutant plants that lack or overexpress functional
S1P and S2P.
Our data support the idea that NTM1 function is further
modulated through the regulation of protein stability. NTM1
protein levels (both mNTM1 and nNTM1) were greatly increased
in 35S::myc-NTM1 transgenic plants after treatment with
MG132, a 26S proteasome–specific inhibitor (Figure 6F). This
finding suggests that NTM1 is ubiquitinated and that the ubiquitinated NTM1 is degraded by the 26S proteasome. We observed that the NTM1 proteins are stabilized by cytokinins.
Therefore, we postulated that cytokinins may regulate NTM1
stability either by inhibiting NTM1 ubiquitination or by blocking
26S proteasome activity. The role of the 26S proteasome activity
has been demonstrated in cytokinin signaling. RPN12a, a component of the Arabidopsis 26S proteasome, functions as a
positive regulator in cytokinin signaling (Smalle et al., 2002).
Therefore, we expected that NTM1 stability might be regulated
by an E3 ligase complex that is inhibited by cytokinins. Together,
our data suggest that NTM1 activity is regulated by a dual
mechanism: liberation from the membranes and regulation of its
stability.
Both the DC and DTM polypeptides are localized in the
nucleus. However, only the 35S::DC transgenic plants, and
not the 35S::DTM transgenic plants, exhibited reduced growth
and leaf serration, suggesting that removal of the TM domain
should be precise for NTM1 activity. By contrast, overexpression of a DTM construct of At bZIP60 could activate target
genes, showing that it is transcriptionally active (Iwata and
Koizumi, 2005). It seems that molecular events underlying the
processing of membrane-bound transcription factors would be
diverse.
NTM1 Defines a Novel Cytokinin Signaling Pathway
in Cell Division
Our results indicate that NTM1 negatively regulates cell division
by inducing KRPs. In addition, cytokinins stabilize both the
membrane-associated and nuclear forms of NTM1 (mNTM1 and
nNTM1). Consistent with this, cytokinins activate a subset of
KRP genes (Figure 7D). Similar results have also been obtained
by other research groups. It has been reported that expression of
KRP2, KRP3, and KRP7 is induced by auxins and cytokinins and
that the growth hormone effects on KRP induction disappear in
the proporz1 (prz1) mutant, suggesting that the PRZ1 transcriptional adaptor mediates growth hormone signals in KRP expression (Sieberer et al., 2003). This regulatory scheme is similar to
the role of abscisic acid in cell cycle control. Abscisic acid
represses cell cycling by inducing KRP1 (Wang et al., 1998).
However, this is opposite the known role of cytokinins in cell
cycle control. The only molecular event verified to date concerning the effects of cytokinins on cell division is the induction of
CYCD3-1, which is subsequently incorporated into the typical
retinoblastoma pathway during cell cycling and accelerates cell
division (Riou-Khamlichi et al., 1999). The previous and present
observations suggest that cytokinins simultaneously activate cell
cycle activators and inhibitors. How can these two contrasting
mechanisms be compromised for cytokinin function in cell cycle
control?
We propose that the promotion of cell division by CYCD3mediated cytokinin signaling would be countered by the
NTM1-mediated induction of CDK inhibitors (KRPs) to maintain
the cell division rate to an optimal level under a given growth
condition. This view explains the previous and present observations, in which KRP genes are induced by cytokinin treatment (Sieberer et al., 2003; this work). Interestingly, a similar
mechanism was recently proposed for the regulation of cell
division in animal cells. c-MYC regulates cell division by activating E2F1. It also activates two microRNAs, miR-17-5p and
miR-20a, that negatively regulate E2F1 (O’Donnell et al.,
2005). Similar to the roles of cytokinins in our proposed
working scheme, c-MYC simultaneously activates cell cycle
activators (E2F1) and inhibitors (miR-17-5p and miR-20a), providing a self-sustained mechanism for the tight control of cell
division.
In physiological terms, the proposed regulatory mechanisms
governing cytokinin signaling in cell cycle control are quite similar
to the negative feedback controls that are prevalent in growth
hormone signaling in plants. Auxins stimulate the degradation of
AUX/IAA proteins to release auxin response factors, thereby
activating downstream genes (Dharmasiri and Estelle, 2004;
Woodward and Bartel, 2005). The AUX/IAA proteins are negative
regulators of auxin signaling. However, the AUX/IAA genes are
also induced by auxins. Another example is the GH3 enzymes,
which inactivate active IAA by catalyzing its adenylation or
conjugation with amino acids (Staswick et al., 2005). The GH3
genes are also auxin-inducible. Simultaneous activation of both
positive and negative signaling components by auxins would be
necessary for the fine-tuning of auxin action and auxin homeostasis. A similar mechanism is also envisioned for cytokinin
function in cell cycle control.
Cell Cycle Regulation by NAC
METHODS
Plant Materials and Growth Conditions
All Arabidopsis thaliana lines used were in the Col-0 background. Plants
were grown in a controlled culture room at 23 to 248C with RH of 60%
under long-day conditions (16 h of light and 8 h of dark) with white light
illumination (120 mmolm2s1). Nicotiana benthamiana was also grown
under the same growth conditions.
Isolation of ntm1-D and ntm1-1
Ecotype Col-0 was transformed with an activation-tagging vector,
pSKI015 (Weigel et al., 2000). Collected seeds were sown in soil, and a
Finale solution (AgrEvo), which contains 5.78% BASTA, was diluted 1000
times and sprayed twice per week. Among the herbicide-resistant transformants, a morphogenetic mutant (ntm1-D) that showed retarded
growth with serrated leaves was chosen for analysis.
The single insertion event of T-DNA in ntm1-D was verified by genomic
DNA gel blot analysis using the 35S enhancer sequence as a probe. The
flanking sequences of the insertion site were determined by the thermal
asymmetric interlaced PCR method (Liu et al., 1995).
The knockout mutant ntm1-1 was isolated from a pool of Ds gene trap
insertion lines (NASC N180483; Nottingham Arabidopsis Stock Centre,
University of Nottingham). It contained a single copy of the insertion in the
first exon of NTM1. The flanking sequence of the insertion site encodes
the NAC domain (Figure 2A). The absence of the NTM1 transcript in
ntm1-1 was verified by RT-PCR.
RNA Gel Blot Hybridization and Comparative RT-PCR
Total RNA samples were extracted from plant materials using the RNeasy
Plant Total RNA Isolation kit (Qiagen). Routinely, 20 mg of each RNA
sample was denatured in denaturation buffer (20 mM MOPS, 8 mM
sodium acetate, and 1 mM EDTA) supplemented with 50% (v/v) formamide and 2.2 M formaldehyde at 658C for 10 min, resolved on a 1.0%
denaturing agarose gel, and transferred to a Hybond-Nþ nylon membrane
(Amersham-Pharmacia). The membrane was hybridized with genespecific probes labeled with [32P]dCTP using the Megaprime DNA labeling kit (Amersham-Pharmacia).
Before semiquantitative RT-PCR, total RNA samples were pretreated
with RNase-free DNaseI to eliminate any contaminating genomic DNA.
The first-strand cDNA was synthesized from 1 to 2 mg of total RNA in a
20-mL reaction volume using SuperScript II reverse transcriptase (Invitrogen). RT-PCR runs were performed for 15 to 40 cycles, depending on
the linear range of PCR amplification for each gene. Each PCR cycle
included incubations at 948C for 1 min, at 558C for 30 s, and at 728C for
3 min. One additional cycle at 728C for 7 min was run after the last cycle to
allow trimming of incomplete polymerizations. Whenever possible, positive and negative control genes were included in the reactions to ensure
the feasibility of the assay conditions. The PCR primers used in this work
are summarized in Supplemental Table 1 online.
For RT-PCR–based DNA gel blot hybridization, PCR products were
electrophoresed on a 1% agarose gel and transferred to a Hybond-Nþ
nylon membrane (Amersham-Pharmacia). The membrane was hybridized
with gene-specific probes labeled with [32P]dCTP using the Megaprime
DNA labeling system (Amersham-Pharmacia).
11 of 13
nation, six healthy plants from each plant group were harvested at the
indicated time intervals. The harvested plants were incubated in methanol
overnight to get rid of chlorophyll and subsequently placed in lactic acid
for light microscopy (BX21; Olympus). The adaxial side of the first leaf
on individual plants was photographed for cell size measurements. At
least 100 cells from each leaf were measured and averaged, with leaf
growth and cell division rates calculated as described previously
(De Veylder et al., 2001). Alternatively, the leaves were fixed in glutaraldehyde, postfixed in osmium tetroxide, and subjected to critical
point drying and scanning electron micrography. Three nonmarginal
parts of each leaf were photographed, and 40 to 50 cells were randomly
chosen for cell size measurements. The surface area of each cell was
determined by the Labwork image-acquisition and analysis program
(Media Cybernetics).
Subcellular Localization of NTM1
The GFP-coding sequence was fused in-frame to the 59 ends of the NTM1
gene constructs, and the gene fusions were subcloned into the pBA002
vector (Kost et al., 1998) for transient expression in onion (Allium cepa)
epidermal cells. After incubation for 24 h at 238C, the cells were subject to
bright-field and fluorescence microscopy.
NTM1 Processing
The myc-NTM1 transgenic Arabidopsis plants were incubated in liquid
MS medium supplemented with 100 mM IAA or 5 to 100 mM kinetin for 1 to
18 h in white light. The plant materials were then ground in liquid nitrogen,
and the cellular extracts were suspended in SDS-PAGE sample loading
buffer.
For analysis in N. benthamiana leaf cells, Agrobacterium tumefaciens
cells containing the vector construct with the myc-NTM1 gene fusion
were injected directly into the leaves. After incubation for 4 d, the cellular
extracts were subjected to protein gel blot analysis as described above.
CDK Activity
p13Suc1-associated Cdc2-like kinase activity was assayed as described
(Wang et al., 1998). Cellular extracts were prepared from aerial plant
parts, and 150 mg of protein extract was added to p13Suc1-conjugated
agarose beads (Upstate). The phosphorylation reaction was initiated by
adding 1 mg/mL histone H1, 25 mM ATP, and 0.1 mCi/mL [32P]gATP and
stopped by adding SDS-PAGE sample loading buffer.
Cytokinin Responsiveness
Roots of 14-d-old plants were used for cytokinin responsiveness analyses. Primary root segments (0.5 mm in length) or cotyledons were
cultured for 2 weeks on MS medium plates supplemented with 2,4-D and
kinetin.
For root growth inhibition assays, seedlings were grown on MS medium
supplemented with various concentrations of 2,4-D or kinetin for 7 d, and
primary root lengths were measured and averaged using 25 seedlings for
each measurement. Relative root lengths, in which each root’s growth is
expressed relative to the mean root length of untreated seedlings, were
compared.
Transcriptional Activity Measurement
Kinematic Analysis of Leaf Growth
Kinematic analysis was performed essentially as described previously
(De Veylder et al., 2001). Wild-type and ntm1-D plants were germinated
and grown on MS medium plates under long-day growth conditions
(238C, 60% RH, and 120 mmolm2s1). Starting from 5 d after germi-
Transcriptional activity assays were performed essentially as described
previously (Duval et al., 2002). The pGBKT7 vector and the yeast strain
AH109 were used (Clontech). The NTM1 gene sequences were fused to
the GAL4 DNA binding domain of pGBKT7, and the vector constructs
were transformed into the AH109 cells.
12 of 13
The Plant Cell
Scanning Electron Micrography and Cell Size Measurements
The fifth rosette leaves of 20-d-old plants were used. Leaves were fixed in
glutaraldehyde and subsequently in osmium tetroxide. They were then
subjected to critical point drying and scanning electron micrography.
Three nonmarginal parts of each leaf were photographed, and 40 to 50
cells were randomly chosen for cell size measurements. The surface area
of each cell was determined by the Labwork image-acquisition and
analysis program (Media Cybernetics).
Accession Numbers
Arabidopsis Genome Initiative locus identifiers for the major genes
mentioned in this article are as follows: NTM1, At4g01540; NTM2,
At4g01550; KRP1, At2g23430; KRP2, At3g50630; KRP3, At5g48820;
KRP4, At2g32710; KRP5, At3g24810; KRP6, At3g19150; KRP7,
At1g49620; CycB1;2, At1g77390; and CycD3;1, At4g34160.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Callus Induction from Cotyledons.
Supplemental Figure 2. Characterization of 35S::DC Transgenic
Plants.
Supplemental Figure 3. Cell Fractionation Procedure.
Supplemental Figure 4. Effects of ALLN and Kinetin on NTL8
Cleavage.
Supplemental Figure 5. Root Greening of ntm1-D.
Supplemental Figure 6. Cytokinin Responses of ntm1-1.
Supplemental Figure 7. Effects of N6-Benzyladenine and IAA on
Root Growth.
Supplemental Figure 8. N6-Benzyladenine Effects on ARR5 Expression.
Supplemental Table 1. Primer Information for the Arabidopsis Genes
Used in This Work.
ACKNOWLEDGMENTS
We thank D. Weigel for kindly providing the pSKI015 vector and J. Kim
for his discussion of important scientific questions. This work was
supported by the Brain Korea 21, Biogreen 21 (20050301034456), and
National Research Laboratory Programs, by a grant from the Plant
Signaling Network Research Center, and by grants from the Korean
Science and Engineering Foundation (R02-2003-000-10001-0), the
Korea Research Foundation (2005-070-C00129 to C.-M.P. and R082004-000-10066-0 to Y.-S.K.), and the Korea Institute of Science and
Technology Evaluation and Planning (M1-0219-00-0003).
Received April 3, 2006; revised September 1, 2006; accepted October 23,
2006; published November 10, 2006.
REFERENCES
Ahn, J., Kim, M., Lim, J.H., Kim, K.-T., and Pai, H.-S. (2004).
Phytocalpain controls the proliferation and differentiation fates of
cells in plant organ development. Plant J. 38, 969–981.
Aida, M., Ishida, T., Fukaki, H., Fujisawa, H., and Tasaka, M. (1997).
Genes involved in organ separation in Arabidopsis: An analysis of the
cup-shaped cotyledon mutant. Plant Cell 9, 841–857.
Arabidopsis Genome Initiative (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.
Brown, M.S., Ye, J., Rawson, R.B., and Goldstein, J.L. (2000).
Regulated intramembrane proteolysis: A control mechanism conserved from bacteria to humans. Cell 100, 391–398.
D’Agostino, I.B., Deruère, J., and Kieber, J.J. (2000). Characterization
of the response of the Arabidopsis response regulator gene family to
cytokinin. Plant Physiol. 124, 1706–1717.
Deshaies, R.J. (1999). SCF and cullin/ring H2-based ubiquitin ligases.
Annu. Rev. Cell Dev. Biol. 15, 435–467.
De Veylder, L., Beeckman, T., Beemster, G.T., Krols, L., Terras, F.,
Landrieu, I., van der Schueren, E., Maes, S., Naudts, M., and Inzé,
D. (2001). Functional analysis of cyclin-dependent kinase inhibitors of
Arabidopsis. Plant Cell 13, 1653–1668.
De Veylder, L., Joubès, J., and Inzé, D. (2003). Plant cell cycle
transitions. Curr. Opin. Plant Biol. 6, 536–543.
Dewitte, W., and Murray, J.A.H. (2003). The plant cell cycle. Annu. Rev.
Plant Biol. 54, 235–264.
Dharmasiri, N., and Estelle, M. (2004). Auxin signaling and regulated
protein degradation. Trends Plant Sci. 9, 302–308.
Dinneny, J.R., Yadegari, R., Fischer, R.L., Yanofsky, M.F., and
Weigel, D. (2004). The role of JAGGED in shaping lateral organs.
Development 131, 1101–1110.
Donnelly, P.M., Bonetta, D., Tsukaya, H., Dengler, R.E., and Dengler,
N.G. (1999). Cell cycling and cell enlargement in developing leaves of
Arabidopsis. Dev. Biol. 215, 407–419.
Duval, M., Hsieh, T.F., Kim, S.Y., and Thomas, T.L. (2002). Molecular
characterization of AtNAM: A member of the Arabidopsis NAC domain
superfamily. Plant Mol. Biol. 50, 237–248.
Ernst, H.A., Olsen, A.N., Skriver, K., Larsen, S., and Lo Leggio, L.
(2004). Structure of the conserved domain of ANAC, a member of the
NAC family of transcription factors. EMBO Rep. 5, 297–303.
Fujita, M., Fujita, Y., Maruyama, K., Seki, M., Hiratsu, K., OhmeTakagi, M., Tran, L.S., Yamaguchi-Shinozaki, K., and Shinozaki,
K. (2004). A dehydration-induced NAC protein, RD26, is involved in a
novel ABA-dependent stress-signaling pathway. Plant J. 39, 863–876.
Gray, W.M., Kepinski, S., Rouse, D., Leyser, O., and Estelle, M.
(2001). Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA
proteins. Nature 414, 271–276.
Greve, K., La Cour, T., Jensen, M.K., Poulsen, F.M., and Skriver, K.
(2003). Interactions between plant RING-H2 and plant-specific NAC
(NAM/ATF1/2/CUC2) proteins: RING-H2 molecular specificity and
cellular localization. Biochem. J. 371, 97–108.
He, X.J., Mu, R.L., Cao, W.H., Zhang, Z.G., Zhang, J.S., and Chen,
S.Y. (2005). AtNAC2, a transcription factor downstream of ethylene
and auxin signaling pathways, is involved in salt stress response and
lateral root development. Plant J. 44, 903–916.
Hellman, H., and Estelle, M. (2002). Plant development: Regulation by
protein degradation. Science 297, 793–797.
Heyl, A., and Schmülling, T. (2003). Cytokinin signal perception and
transduction. Curr. Opin. Plant Biol. 6, 480–488.
Hibara, K., Takada, S., and Tasaka, M. (2003). CUC1 gene activates
the expression of SAM-related genes to induce adventitious shoot
formation. Plant J. 36, 687–696.
Hoppe, T., Matuschewski, K., Rape, M., Schlenker, S., Ulrich, H.D.,
and Jentsch, S. (2000). Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.
Cell 102, 577–586.
Hoppe, T., Rape, M., and Jentsch, S. (2001). Membrane-bound
transcription factors: Regulated release by RIP or RUP. Curr. Opin.
Cell Biol. 13, 344–348.
Cell Cycle Regulation by NAC
Hwang, I., and Sheen, J. (2001). Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413, 383–389.
Inoue, T., Higuchi, M., Hashimoto, Y., Seki, M., Kobayashi, M., Kato,
T., Tabata, S., Shinozaki, K., and Kakimoto, T. (2001). Identification
of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409,
1060–1063.
Iwata, Y., and Koizumi, N. (2005). An Arabidopsis transcription factor,
AtbZIP60, regulates the endoplasmic reticulum stress response in a
manner unique to plants. Proc. Natl. Acad. Sci. USA 102, 5280–5285.
Kessler, S., and Sinha, N. (2004). Shaping up: The genetic control of
leaf shape. Curr. Opin. Plant Biol. 7, 65–72.
Kost, B., Spielhofer, P., and Chua, N.-H. (1998). A GFP-mouse talin
fusion protein labels plant actin filaments in vivo and visualizes the
actin cytoskeleton in growing pollen tubes. Plant J. 16, 393–401.
Lee, D.H., and Goldberg, A.L. (1998). Proteasome inhibitors: Valuable
new tools for cell biologists. Trends Cell Biol. 8, 397–403.
Lid, S.E., et al. (2005). Mutation in the Arabidopsis thaliana DEK1
calpain gene perturbs endosperm and embryo development while
over-expression affects organ development globally. Planta 221,
339–351.
Liu, Y.G., Mitsukawa, N., Oosumi, T., and Whittier, R.F. (1995).
Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert
junctions by thermal asymmetric interlaced PCR. Plant J. 8, 457–463.
Margis, R., and Margis-Pinheiro, M. (2003). Phytocalpains: Orthologous calcium-dependent cysteine proteinases. Trends Plant Sci. 8,
58–62.
Meijer, M., and Murray, J.A.H. (2001). Cell cycle controls and the
development of plant form. Curr. Opin. Plant Biol. 4, 44–49.
Mok, D.W., and Mok, M.C. (2001). Cytokinin metabolism and action.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 89–118.
O’Donnell, K.A., Wentzel, E.A., Zeller, K.I., Dang, C.V., and Mendell,
J.T. (2005). c-Myc-regulated microRNAs modulate E2F1 expression.
Nature 435, 839–843.
Ohno, C.K., Reddy, G.V., Heisler, M.G., and Meyerowitz, E.M. (2004).
The Arabidopsis JAGGED gene encodes a zinc finger protein that
promotes leaf tissue development. Development 131, 1111–1122.
Ooka, H., et al. (2003). Comprehensive analysis of NAC family genes in
Oryza sativa and Arabidopsis thaliana. DNA Res. 10, 239–247.
Riou-Khamlichi, C., Huntley, R., and Murray, J.A.H. (1999). Cytokinin
activation of Arabidopsis cell division through a D-type cyclin. Science
283, 1541–1544.
Risseeuw, E.P., Daskalchuk, T.E., Banks, T.W., Liu, E., Cotelesage,
J., Hellmann, H., Estelle, M., Somers, D.E., and Crosby, W.L.
(2003). Protein interaction analysis of SCF ubiquitin E3 ligase subunits
from Arabidopsis. Plant J. 34, 753–767.
Sablowski, R.W., and Meyerowitz, E.M. (1998). A homolog of NO
APICAL MERISTEM is an immediate target of the floral homeotic
genes APETALA3/PISTILLATA. Cell 92, 93–103.
13 of 13
Sakai, J., Rawson, R.B., Espenshade, P.J., Cheng, D., Seegmiller,
A.C., Goldstein, J.L., and Brown, M.S. (1998). Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and
controls lipid composition of animal cells. Mol. Cell 2, 505–514.
Schwacke, R., Schneider, A., van der Graaff, E., Fischer, K., Catoni,
E., Desimone, M., Frommer, W.B., Flügge, U.I., and Kunze, R.
(2003). ARAMEMNON, a novel database for Arabidopsis integral
membrane proteins. Plant Physiol. 131, 16–26.
Sieberer, T., Hauser, M.T., Seifert, G.J., and Luschnig, C. (2003).
PROPORZ1, a putative Arabidopsis transcriptional adaptor protein,
mediates auxin and cytokinin signals in the control of cell proliferation.
Curr. Biol. 13, 837–842.
Smalle, J., Kurepa, J., Yang, P., Babiychuk, E., Kushnir, S., Durski,
A., and Vierstra, R.D. (2002). Cytokinin growth responses in Arabidopsis involve the 26S proteasome subunit RPN12. Plant Cell 14,
17–32.
Staswick, P.E., Serban, B., Rowe, M., Tiryaki, I., Maldonado, M.T.,
Maldonado, M.C., and Suza, W. (2005). Characterization of an
Arabidopsis enzyme family that conjugates amino acids to indole-3acetic acid. Plant Cell 17, 616–627.
Takada, S., Hibara, K., Ishida, T., and Tasaka, M. (2001). The CUPSHAPED COTYLEDON gene of Arabidopsis regulates shoot apical
meristem formation. Development 128, 1127–1135.
Tran, L.S.P., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y.,
Maruyama, K., Fujita, M., Seki, M., Shinozaki, K., and YamaguchiShinozaki, K. (2004). Isolation and functional analysis of Arabidopsis
stress-inducible NAC transcription factors that bind to a droughtresponsive cis-element in the early responsive to dehydration stress
1 promoter. Plant Cell 16, 2481–2498.
Vierstra, R.D. (2003). The ubiquitin/26S proteasome pathway, the
complex last chapter in the life of many plant proteins. Trends Plant
Sci. 8, 135–142.
Vik, Å., and Rine, J. (2000). Membrane biology: Membrane-regulated
transcription. Curr. Biol. 10, R869–R871.
Wang, H., Fowke, L.C., and Crosby, W.L. (1998). ICK1, a cyclindependent protein kinase inhibitor from Arabidopsis thaliana interacts
with both Cdc2a and CycD3, and its expression is induced by
abscisic acid. Plant J. 15, 501–510.
Wang, H., Zhou, Y., Gilmer, S., Whitwill, S., and Fowke, L.C. (2000).
Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects
cell division, plant growth, and morphology. Plant J. 24, 613–623.
Weigel, D., et al. (2000). Activation tagging in Arabidopsis. Plant
Physiol. 122, 1003–1013.
Woodward, A.W., and Bartel, B. (2005). Auxin: Regulation, action, and
interaction. Ann. Bot. 95, 707–735.
Xie, Q., Frugis, G., Colgan, D., and Chua, N.-H. (2000). Arabidopsis
NAC1 transduces auxin signal downstream of TIR1 to promote lateral
root development. Genes Dev. 14, 3024–3036.
A Membrane-Bound NAC Transcription Factor Regulates Cell Division in Arabidopsis
Youn-Sung Kim, Sang-Gyu Kim, Jung-Eun Park, Hye-Young Park, Mi-Hye Lim, Nam-Hai Chua and
Chung-Mo Park
Plant Cell; originally published online November 10, 2006;
DOI 10.1105/tpc.106.043018
This information is current as of June 17, 2017
Supplemental Data
/content/suppl/2006/11/07/tpc.106.043018.DC1.html
Permissions
https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X
eTOCs
Sign up for eTOCs at:
http://www.plantcell.org/cgi/alerts/ctmain
CiteTrack Alerts
Sign up for CiteTrack Alerts at:
http://www.plantcell.org/cgi/alerts/ctmain
Subscription Information
Subscription Information for The Plant Cell and Plant Physiology is available at:
http://www.aspb.org/publications/subscriptions.cfm
© American Society of Plant Biologists
ADVANCING THE SCIENCE OF PLANT BIOLOGY