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First published online January 11, 2006; 10.1104/pp.105.072173 Plant Physiology 140:693-703 (2006) © 2006 American Society of Plant Biologists Mitosis-Specific Promoter of the Alfalfa Cyclin-Dependent Kinase Gene (Medsa;CDKB2;1) Is Activated by Wounding and Ethylene in a Non-Cell Division-Dependent Manner1,[W]Institute of Plant Biology, Biological Research Center, Szeged H–6701, Hungary (M.K.Z., A.P.-S., Z.N., S.B., D.D., A.F.); Laboratory of Tumorpathology and Molecular Diagnostics, Institute for Biotechnology, Bay Zoltan Foundation for Applied Research, Szeged H–6726, Hungary (E.S.); 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University of Medicine, Budapest H–1085, Hungary (T.K.); and Szilak Laboratories, Bioinformatics and Molecule-Design, Szeged H–6723, Hungary (L.S.)
Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.
In recent years, the general understanding of the cell proliferation and cell cycle control has increased considerably (Inzé, 2005  ). The phosphorylation cascade that moves the cell cycle on is understood quite well, but the regulation of cell cycle-dependent gene expression is still to be elucidated. In the eukaryotic cell cycle, there are two active phases, DNA synthesis (S-phase) and segregation of chromosomes (M-phase), preceded by the regulatory gap phases G1 and G2, respectively. G1/S and G2/M transitions are controlled by cyclin-dependent Ser/Thr kinases (CDKs), which are key regulators of the cell cycle. CDK activities are controlled by various mechanisms, including phosphorylation, dephosphorylation, and binding of regulatory cyclin subunits. Usually, the expression pattern of CDKs is constitutive throughout the cell cycle. Meanwhile, the periodic expression of cyclins provides the characteristic cell cycle phase-specific timing of CDK activities (Morgan, 1997). However, plants contain a unique class of CDKs (B-type CDKs [CDKBs]) with periodic gene expression and elevated protein accumulation at G2/M-phases (Fobert et al., 1996; Segers et al., 1996; Magyar et al., 1997; Umeda et al., 1999; Sorrell et al., 2001; Menges and Murray, 2002). The alfalfa (Medicago sativa) Cdc2MsD and Cdc2MsF (according to recent nomenclature, Medsa;CDKB1;1 and Medsa;CDKB2;1, respectively; Joubès et al., 2000) belong in this plant-specific CDK class (Magyar et al., 1997).
The transcriptional regulation of cell cycle phase-specific genes may involve several mechanisms based on multiple regulatory elements within the promoter regions of the genes. In the case of the mitotic B-type cyclin genes of Catharanthus roseus (Catro;CycB1;1/CYM promoter; Ito et al., 1998
The regulation of G2-to-M-phase transition has special significance, considering plant development, because it is linked to the synthesis of the new cell wall and the possibility of endoreduplication. Endoreduplication involves repetitive chromosomal DNA replications without intervening mitosis or cytokinesis, leading to increased ploidy (Larkins et al., 2001
Up to now, just a couple of cell cycle-dependent CDK promoters have been analyzed, to our knowledge, including the promoters of the genes coding for Arath;CDKA;1 (Hemerly et al., 1993
An open wound caused by mechanical injury is a potential infection site for pathogens; thus, expression of defense genes at the wound site is necessary for the plants to build a barrier against opportunistic microorganisms. The inducible defense genes are regulated by several signal pathways involving jasmonic acid, salicylic acid, and ethylene (Ryals et al., 1996 In this work, experimental evidence is shown that the promoter-driven reporter genes and the endogenous Medsa;CDKB2;1 kinase gene are expressed in highly proliferating regions, but are also activated in consequence of wounding and ethephon in a non-cell cycle-dependent manner.
Cloning of a 360-bp Length Upstream Region of Medsa;CDKB2;1
Cloning of genomic DNA fragments from tetraploid alfalfa (cv Regen S) upstream of the Medsa;CDKB2;1 coding sequence was carried out with the method of PCR-based genome walking (Siebert et al., 1995
In Silico Analysis of the Upstream Region of Medsa;CDKB2;1
The cis-elements of the Medsa;CDKB2;1 promoter region were analyzed by two different programs available online: the PLACE database (http://www.dna.affrc.go.jp/htdocs/PLACE/; Higo et al., 1999
Based on the G2/M-specific expression of the Medsa;CDKB2;1 (Magyar et al., 1997 Comparison of the cloned upstream regions of Medsa;CDKB2;1 and the orthologous M. truncatula CDKB2;1 genes confirmed that most, probably all, of the cis-elements necessary to regulate the genes in a cell cycle-dependent manner are present on the relatively short fpr15 fragment (360 bp). The longer M. truncatula genomic sequence does not include other known essential cis-elements in addition to those present in the fpr15 fragment. The in silico promoter analysis revealed several other potential regulatory elements in the fpr15 sequence that are unrelated to cell cycle regulation. Among these, light-responsive elements seem to dominate the promoter region with more than 10 different transcription factor-binding sites for light (data not shown). In addition, fpr15 contains wound-related elements, like ethylene-responsive element (ERE, –110ATTTGAAA), TCA-box (wound-responsive element, –24/–19/–13/+36TCATTT/C), WRKY-binding site (W-box, –181/–121/+16GATC/T), and five WUN motifs (AATTT). Interestingly, a gibberellin-responsive element (P-box, +8CCTTTTC) was found in the 5'-untranslated region.
The structures of three CDKB promoters were analyzed (Table I). The distances of the elements were calculated from the ATG start codon. The absolute positions of some cis-elements in the promoters of CDKB2;1 genes of alfalfa and Arabidopsis are almost identical, so the arrangement of the E2Fb, WUN, ERE, MSA, TATA, and TCA motifs seems to be fixed. The location of the putative hormonal response elements like gibberellin (P-box) can vary within the entire promoter sequence, but they are definitely part of it. The promoters of the orthologous CDKB2;1 genes share the same basic structure, whereas the Arath;CDKB1;1 gene promoter possesses similar elements found in a different order. In addition, a putative abscisic acid-responsive element (ABRE) is present in the Arabidopsis CDKB promoters, but it was not recognized in alfalfa or M. truncatula.
Histochemical Analysis of the Reporter Gene Activity Driven by the Medsa;CDKB2;1 Promoter Transgenic approach was chosen for the in planta characterization of the fpr15 promoter activity. Therefore, transgenic alfalfa and Arabidopsis plants were generated with the plasmid constructs using the fpr15 promoter fragment linked to the GUS (fpr15:GUS) or luc (fpr15:luc) reporter genes. Several independent transgenic lines were tested by PCR and selected for the analysis of the reporter activities. Transgenic fpr15:luc calli with high cell division activity exhibited a strong signal after addition of luciferin substrate (Fig. 2A). Different organs of fpr15:GUS-transformed alfalfa plants were stained for GUS activity to monitor promoter function (Fig. 2, B–H). GUS expression was preferentially localized to meristematic regions, including the shoot apex (Fig. 2B). In young leaves (Fig. 2C), strong GUS expression was characteristic for the junction between the leaf and petiole, and moderate staining was detected in the region of developing vascular tissues as well. No expression was detected in mature leaves. Figure 2D shows the fpr15-regulated GUS expression at different stages of lateral root development. The staining was strongest in the central cylinder (stele) of the main root, as well as in the pericycle-derived lateral root meristem. In ultrathin cross sections of the root, GUS expression was visible only in the stele and in emerging lateral root meristem but not in the epidermal and cortical layers (Fig. 2E). The fpr15 activity was also detected in flowers. Weak blue staining was characteristic for flower buds (Fig. 2F) but not for open flowers. Anthers showed a patchy distribution of fpr15 activity during microsporogenesis (Fig. 2G). GUS activity was also detected in seed primordia (Fig. 2H).
To compare the accumulation of the endogenous CDKB2;1 protein and of fpr15-controlled luc reporter in roots, ultrathin cross sections were generated from young roots of fpr15:luc transgenic alfalfa distantly from the root tip in the maturation zone. Single-labeling immunohistochemistry was performed on consecutive sections with antibodies against Medsa;CDKB2;1 and luc, respectively. In both cases, immunopositivity showed similar patterns inside the stele (Fig. 2, I and J). The isolated Medsa;CDKB2;1 promoter fragment was also tested in a plant species other than Leguminosae. For this reason, the construct fpr15:GUS was introduced into Arabidopsis plants. Similarly to alfalfa, actively proliferating regions of different organs (shoot apical meristem, expanding leaves, and root apical meristem) of these transformed plants showed GUS signals (Supplemental Fig. 1).
The histochemical analysis showed that the cloned promoter could provide cell division-dependent expression for the reporter genes, but the correct timing of the promoter activity (i.e. the cycle specificity) remained to be confirmed. The regulation of gene expression was characterized by comparisons between the expression pattern of the endogenous CDKB2;1 and fpr15-driven reporter genes in synchronized cell suspension cultures generated from the proper transgenic plants. Figure 3 shows the results obtained by the synchronization of the cell suspension culture expressing the fpr15:luc construct. Cell cycle progression was synchronized by the S-phase inhibitor hydroxyurea (HU). After releasing the block, samples were taken at 3-h intervals for flow-cytometric analysis and mitotic index determination (Fig. 3A), as well as for RNA isolation. The transcript levels of the genes of interest were compared by northern hybridization-blot assay (Fig. 3B). Transcript levels of Medsa;CDKA;1, a cell cycle-related kinase with constitutive expression during the cell cycle (Hirt et al., 1993
Activation of Medsa;CDKB2;1 by Wounding and Hormones
The wound response of the CDKB2;1 promoter, assumed on the basis of the in silico promoter analysis (see above), was tested on leaf explants. Mature leaves from alfalfa plants grown in greenhouse were detached, wounded by cuttings, and then immediately placed onto solid medium. Similarly, the hormonal response of the CDKB2;1 gene was analyzed by exposing nonwounded leaves to jasmonic acid, salicylic acid, or ethephon (an ethylene precursor), and 2,4-dichlorophenoxyacetic acid (2,4-D), known to induce cell proliferation (Hemerly et al., 1993
The inducible defense genes can be regulated mostly via jasmonic acid-, salicylic acid-, and ethylene-related signal pathways (Reymond and Farmer, 1998 ; Wang et al., 2002). Therefore, we tested these possible activating hormones, if they could turn the CDKB2;1 promoter on in detached leaves. The jasmonic acid had no effect at all, and the salicylic acid could hardly activate the promoter (data not shown); however, the effect of ethephon, which was assumed by the in silico analysis, was more pronounced (Fig. 4C). Luc activity was measured and quantified in detached leaves treated with ethephon (Fig. 4C). The emitted light was significant from the first day and became stronger by time. The level of the endogenous CDKB2;1 protein was determined by western blot in ethephon-treated leaf explants from nontransgenic greenhouse plants (Fig. 4C). The CDKB2;1 protein was detectable 1 d after the treatment and showed a similar accumulation pattern as in the case of wounded leaves. These experiments were performed in parallel with 2,4-D-treated leaf explants (Fig. 4D). The GUS activity staining of the leaf explants showed that the CDKB2;1 promoter is activated after 3 d (Fig. 4D). Western-blot assay on 2,4-D-treated nontransgenic leaves confirmed this result; the CDKB2;1 kinase appeared on the third day, but its level became highly elevated from the fourth day (Fig. 4D). For the analysis of the effects of wounding and hormone treatments on activation of cell cycle, nuclei were isolated for flow cytometry. Figure 4E shows that there was no change in the DNA content of the wounded or ethephon-treated leaf cells during the examined period similarly to the negative control (not wounded leaves cultivated on hormone-free medium). However, in the 2,4-D-treated leaf explants, increased frequency of S- and G2-phase cells indicated that cell cycle was activated after 3 d of treatment. After 10 d, the 2,4-D-treated leaf explants indeed started to produce calli (Fig. 2A). Taken together, these data suggested that the expression of CDKB2;1 can be controlled, not only in relation to the cell cycle but also independently of it, by wounding. To further confirm this phenomenon, wounded or 2,4-D-treated leaves of fpr15:luc transgenic plants were placed onto HU-containing or control media for 3 d. HU, an inhibitor of S-phase progression, was expected to block only the 2,4-D-induced, cell cycle-dependent, but not the wound-induced, cell cycle-independent, expression of the fpr15-driven reporter. Figure 4F shows the luciferin-treated leaves after the 3-d treatment. The luc gene was switched on in wounded as well as in 2,4-D-treated leaves incubated in the absence of HU. The presence of HU, however, selectively blocked the luc expression in 2,4-D-treated leaves but did not affect the wound-induced expression of the reporter, which indicated that it is indeed independent of cell division. Other hormones, such as kinetin, abscisic acid, and gibberellin, did not show any significant influence on the activity of CDKB2;1 promoter in leaf explants (data not shown). Because of the lack of any indication of cell proliferation in the wounded and ethephon-treated leaves, we could conclude that there must be an independent regulation of the Medsa;CDKB2;1 gene for wounding and cell division, and the mitosis-inhibitor ethylene could be a putative mediator of the wound response.
In the eukaryotic cell cycle, the active phases (DNA synthesis [S-phase] and chromosome segregation [M-phase]) are separated by two regulatory gap phases, G1 and G2. The cell cycle phase boundaries, controlled by a family of CDKs, serve as key checkpoints in the progression of the cell cycle (Stals and Inzé, 2001 ; Vandepoele et al., 2002; De Veylder et al., 2003; Dewitte and Murray, 2003; Inzé, 2005). It has been shown that in plants, as in other eukaryotes, the transition from G1- to S-phase, which is triggered by A-type CDKs (Mironov et al., 1999; Boniotti and Gutiérrez, 2001; Nakagami et al., 2002), is mediated by the activated E2F/DP transcription factors (Harbour and Dean, 2000; Müller et al., 2001). The regulation of the mitotic entry (from G2 to M) is unique in plants, as it is governed by a specific class of CDKs (designated as plant CDKBs) with cell cycle-dependent expression (Fobert et al., 1996; Segers et al., 1996; Magyar et al., 1997; Umeda et al., 1999; Mészáros et al., 2000; Sorrell et al., 2001; Menges and Murray, 2002). Understanding the regulation of G2/M transition is exceptionally important in plants because at this point cell cycle progression can diverge toward mitosis or endoreduplication. To study the transcriptional regulation of plant genes coding for CDKBs, a 360-bp promoter fragment (fpr15) of alfalfa CDKB2;1 was cloned and linked to GUS (fpr15:GUS) or luc (fpr15:luc) reporter genes and characterized in both transgenic alfalfa and Arabidopsis plants to unravel the propensity of the promoter.
Transgenic plants and cultured cell suspensions carrying the reporter gene constructs produced similar expression patterns of the fpr15-driven reporters to the endogenous Medsa;CDKB2;1 kinase. The GUS/luc activity was restricted to the actively dividing regions of intact plants (root and shoot meristems, petiole-to-leaf junctions, etc.) where the accumulation of the CDKB2;1 kinase can be expected (Fig. 2; Supplemental Fig. 1). The observed in planta Medsa;CDKB2;1 expression pattern is characteristic for the proliferating region, as shown in the case of the in situ hybridization of the CDKB1;1 (Fobert et al., 1996 We demonstrated with synchronized cell cultures that the 360-bp-long promoter is sufficient to restrict gene expression activity to the G2/M cell cycle phases. It was found that the transcript levels of the reporter gene and the endogenous Medsa;CDKB2;1 changed simultaneously with a maximum level during the G2/M-phases, verified by flow-cytometric analysis and by determination of the mitotic index. This observation was confirmed by reverse transcription-PCR experiments, as well, on independent transgenic lines (data not shown). It can be concluded therefore that the cloned 360-bp-long upstream region of the Medsa;CDKB2;1 gene has promoter activity that is characteristic for the proliferating cells with G2/M cell cycle phase specificity. Immunohistochemical approach was chosen to show that the fpr15-driven reporter activity was localized into the same area as the Medsa;CDKB2;1 protein in roots. Consecutive cross sections made in the maturation zone of young roots were reacted with antibodies against Medsa;CDKB2;1 and luc, respectively, revealing very similar immunolocalization patterns in a small group of cells (Fig. 2, I and J). Further investigations are needed to determine the biological significance of this specific expression pattern. Taken together, the expression pattern of fpr15-driven reporter genes was in good correlation with that of the endogenous Medsa;CDKB2;1 gene.
The regulation of G2/M-phase-specific genes may be altered in animal and plant cells. In plants, cell cycle-specific transcription factors of the Myb family were identified to enhance the promoter activity of these types of genes (Tréhin et al., 1999
The in silico analysis also revealed a putative E2Fb-binding site in the fpr15 sequence. More and more evidence supports the involvement of E2F transcription factors in the G2/M-phase progression in addition to their basic role in the regulation of G1/S-phase-specific genes (Ishida et al., 2001
In the cell cycle-specific regulation of human cdc2 and cyclin B1 genes, Myb, E2F, CCAAT-, and cell cycle homology region-binding factors together coordinate the proper timing of expression (Zhu et al., 2004
According to the response speed of the tissues for mechanical injury, the wound answer can be classified to short and long term. The short-term answer starts by minutes after wounding, and the long-term response has effect after 12 h or even later as a consequence of mechanical injury (Reymond et al., 2000 Although the fpr15 activity was induced by the mechanical injury, proliferating cells could not be detected in the wounded leaves by flow cytometry. This means that the wounding could induce the activity of the promoter via a cell proliferation independent pathway. This observation was confirmed by the fact that HU treatment on wounded leaves did not inhibit the isolated promoter activity. In contrast, the accumulation of the CDKB2;1 kinase in the presence of 2,4-D was likely a consequence of the entry to the cell cycle; it started only at the time when the cells already entered into the cell division cycle, which happened later (from the third day) in comparison to the wound response (from the first day), and the promoter activity could be blocked with HU (Fig. 4F).
It was indeed shown that the cell cycle-related A-type Arabidopsis CDK kinase expressed constitutively during the cycle could also be induced by wounding without cell division. The authors concluded that the expression of this gene by wounding was a consequence of an increased competence for cell proliferation (Hemerly et al., 1993
The wound response can be activated by various pathways involving jasmonic acid, salicylic acid, and ethylene (Creelman and Mullet, 1997
The fact that the ethephon has positive effect for the regulation of the CDKB2;1 kinase is intriguing. The ethylene, which is a very promiscuous hormone (Zhong and Burns, 2003
The accumulation of Medsa;CDKB2;1 kinase due to G2/M progression or by ethephon treatment considered as a G2/M-phase inhibitor emphasizes the multifunctional role of this kinase. To reach the full kinase activity, the CDK kinases need their cyclin partners. Indeed, the ethylene treatment could also stimulate mitotic cyclin expressions in rice (Oryza sativa) stem cuttings (Lorbiecke and Sauter, 1999 Although our knowledge is continuously increasing on the regulation of the core cell cycle machinery, we hardly know molecular mechanisms that link the cell cycle to external and developmental signals. If cell cycle-related proteins are involved in processes unrelated to cell division, as indicated by the presented observations, there are additional levels of complexity to be unraveled by further experimentation.
Cloning of the Promoter of Medsa;CDKB2;1 Genomic DNA fragments, upstream of CDKB2;1, were isolated from tetraploid alfalfa (Medicago sativa L. cv Regen S) with the GenomWalker kit (BD Biosciences, CLONTECH). The fpr15 fragment was cloned into pCambia3301 as a replacement of the cauliflower mosaic virus 35S promoter region (fpr15pC3301 or fpr15:GUS). In the fpr15pC3301 construct, the GUS reporter was replaced with the luc gene, resulting in fpr15pC3305 (fpr15:luc) plasmid. The resulting plasmids were introduced into Agrobacterium tumefaciens (LBA4404).
Agrobacterium-mediated transformation of alfalfa leaf discs was performed according to Barbulova et al. (2002b)
A. tumefaciens-transformed Arabidopsis (Arabidopsis thaliana, Columbia-0) lines were generated by in planta transformation using vacuum infiltration (Bechtold and Pelletier, 1998
GUS staining was carried out by the method described by Jefferson et al. (1987)
For immunohistochemistry, glycol methacrylate sections (5 µm) were subjected to wet heat-induced antigen retrieval in citrate buffer (pH 2.5; Krenacs et al., 1999
The activity of the luc enzyme was detected after addition of 1 or 2 mM luciferin substrate (Biosynth). Light excitation was detected with a CCD camera system (Visilux Imager, Visitron Systems GmbH). Bioluminescence images were processed using the Metaview 4.5r6 software (Universal Imaging Corporation).
Two-week-old callus tissue from transformed alfalfa plants was used for the generation of cell suspensions according to Bögre et al. (1988)
Synchronization of the cell division in exponentially growing cell suspension cultures by 10 mM HU (Sigma) was done according to Ayaydin et al. (2000)
Total RNA was isolated using TRIzol reagent (GIBCO) according to the manufacturer's instructions. Twenty micrograms of total RNA were loaded into denaturing agarose gels for northern hybridization that was carried out according to standard procedures (Sambrook et al., 1989
Mature leaves from greenhouse plants were detached, sterilized, wounded randomly by cutting with a blade, and cultivated on solid B5II medium with or without 2,4-D (1 mg/L or 4,5 µM) or ethephon (10 mg/L or 0.7 µM) hormones (stated, where applied). Samples were taken on 0.5, 1, 3, and 4 d after the treatment. For western blotting, samples from nontransgenic alfalfa plants were used and processed according to Magyar et al. (1997) Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: DQ136188, promoter region of Medsa;CDKB2;1 (fpr15); X97317, Medsa;CDKB2;1; M58365, Medsa;CDKA;1; AC144481 bacterial artificial chromosome clone containing the 5' promoter region of Medicago truncatula CDKB2;1; D10851 (At3g54180), Arath;CDKB1;1; and AJ297936 (At1g76540), Arath;CDKB2;1.
We are grateful for the useful scientific advice and help of Ariana Perhald, Krisztina  tvös, and Manuela Jurka. We also thank Katalin Török and Róza Nagy for excellent technical assistance. Received September 30, 2005; returned for revision November 16, 2005; accepted December 11, 2005.
1 This work was supported by the Hungarian National Research Foundation (Országos Tudományos Kutatás: Alap, grant nos. T037910 and T042672); the Center of Excellence UNESCO, Richter Geodon Centenáriumi and Domus Hungarica Scientiarium et Artium, Hungarian Academy of Sciences and Ministry of Education (scholarships to M.K.Z.); and the János Bólyai research fellowship (to A.F.). 
2 These authors contributed equally to the paper. 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.plantphysiol.org) is: László Szilák (laszlo.szilak{at}freemail.hu).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.072173. * Corresponding author; e-mail laszlo.szilak{at}freemail.hu; fax 36–62–433434.
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ABSTRACT
RESULTS
-glucuronidase [GUS] and luciferase [luc]) regulated by a cloned 360-bp promoter fragment of the Medsa;CDKB2;1 gene was perfectly matched with the expression profile of the endogenous gene in synchronously dividing cultured cells. In silico analysis suggested some putative cell cycle-dependent regulatory elements, including MSA and E2Fb. In addition to the cell cycle-related elements, some others involved in the response for wounding and ethylene have been recognized in the promoter region. 
