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Plant Cell. Nov 2006; 18(11): 3132–3144.
PMCID: PMC1693948

A Membrane-Bound NAC Transcription Factor Regulates Cell Division in Arabidopsis[W]

Abstract

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 (Riou-Khamlichi et al., 1999).

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 membrane-bound 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 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 and1D)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 and1E).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).

Figure 1.
The Phenotype Conferred by ntm1-D, and Mapping of the T-DNA Insertion Site.

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 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 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 β-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.

Figure 2.
NTM1 Structure and Transgenic Arabidopsis Plants Expressing Different NTM1 Constructs.

Strikingly, the predicted polypeptide is distinct from other NAC members in that it has a transmembrane (TM) motif with a predicted α-helical conformation in the far C-terminal region (Figures 2A and and2B).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 ΔC 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 ΔC (Figure 2E). However, those plants overexpressing the full-size or ΔTM 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 ΔC expression might cause a dominant negative suppression of intrinsic NTM1. We used the RNA interference method to specifically inactivate 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.

Figure 5.
Characterization of ntm1-1.

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 μg/L 2,4-D and different concentrations of kinetin, ranging from 30 to 3000 μg/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.

Figure 3.
Reduced Cell Division in ntm1-D.

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 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 three-point 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::ΔC 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).

Figure 4.
Kinematic Analysis of Leaf Growth in ntm1-D.

Consistent with a role for NTM1 in cell division, a knockout mutant, ntm1-1, displayed elongated hypocotyls (Figures 2A and and5A).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 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 membrane-associated, 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 ΔC 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 full-size 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.

Figure 6.
NTM1 Release from the Intracellular Membranes by Proteolytic Cleavage.

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, ΔC and ΔTM were detected exclusively within the nucleus. Although both ΔC and ΔTM were localized in the nucleus, only the ΔC 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 ΔTM but missing in ΔC.

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 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 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 μM (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.

Figure 7.
Cytokinin Regulation of NTM1 Function.

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::ΔC 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 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 ΔC construct was fused in-frame to the GAL4 DNA binding domain in the yeast expression vector pGBKT7. The GAL4-ΔC fusion was expressed in a yeast strain containing a reporter gene (LacZ), and α-galactosidase activity was measured. The yeast cells expressing ΔC clearly showed LacZ activation (Figure 8A). Furthermore, whereas the C-terminal region of ΔC strongly activated the LacZ reporter gene, the NAC domain alone did not activate it. These results clearly demonstrate that NTM1 is a transcriptional activator.

Figure 8.
A Schematic Working Model for the NTM1 Transcription Factor.

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 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 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 ΔC and ΔTM polypeptides are localized in the nucleus. However, only the 35S::ΔC transgenic plants, and not the 35S::ΔTM 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 ΔTM 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 CYCD3-mediated 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.

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 24°C with RH of 60% under long-day conditions (16 h of light and 8 h of dark) with white light illumination (120 μmol·m−2·s−1). 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 μg 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 65°C for 10 min, resolved on a 1.0% denaturing agarose gel, and transferred to a Hybond-N+ nylon membrane (Amersham-Pharmacia). The membrane was hybridized with gene-specific 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 μg of total RNA in a 20-μL 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 94°C for 1 min, at 55°C for 30 s, and at 72°C for 3 min. One additional cycle at 72°C 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-N+ nylon membrane (Amersham-Pharmacia). The membrane was hybridized with gene-specific probes labeled with [32P]dCTP using the Megaprime DNA labeling system (Amersham-Pharmacia).

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 (23°C, 60% RH, and 120 μmol·m−2·s−1). Starting from 5 d after germination, 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 5′ 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 23°C, 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 μM IAA or 5 to 100 μM 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 μg of protein extract was added to p13Suc1-conjugated agarose beads (Upstate). The phosphorylation reaction was initiated by adding 1 μg/μL histone H1, 25 μM ATP, and 0.1 μCi/μL [32P]γATP 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

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.

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::ΔC 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.

Supplementary Material

[Supplemental Data]

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 R08-2004-000-10066-0 to Y.-S.K.), and the Korea Institute of Science and Technology Evaluation and Planning (M1-0219-00-0003).

Notes

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 (rk.ca.uns@krapmc).

[W]Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.106.043018

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