Plant Molecular Biology 46: 205–213, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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An upstream region of the Arabidopsis thaliana CDKA;1 (CDC2aAt) gene directs transcription during trichome development Yoshiro Imajuku, Yohei Ohashi, Takashi Aoyama∗ , Koji Goto and Atsuhiro Oka Laboratory of Molecular Biology, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan (∗ author for correspondence; e-mail:
[email protected]) Received 7 August 2000; accepted in revised form 13 February 2001
Key words: CDC2aAt, CDK, cell morphogenesis, GUS fusion, promoter, trichome
Abstract The cell cycle of eukaryotes is tightly regulated through the activity of cyclin-dependent kinases. The Arabidopsis thaliana CDKA;1 (CDC2aAt) gene is thought to encode such a protein kinase, since it is actively transcribed in proliferating tissues and can complement defects in the Schizosaccharomyces pombe cdc2 gene. We analyzed the functional structure of the CDKA;1 promoter, using fusion genes between various upstream regions of CDKA;1 and the Escherichia coli β-glucuronidase (GUS) gene. A 595 bp DNA fragment upstream from the transcription start site conferred GUS activity on developing trichomes, but not on proliferating tissues. On the other hand, another upstream fragment extending to the 50 non-coding transcribed region gave GUS activity to both proliferating tissues and developing trichomes. Against the gl2 mutant background, GUS activity directed by the 595 bp fragment was detected in single-stalk cells, but not in giant cells without obvious polar extension growth. These results revealed that the 595 bp fragment lacks cis element(s) essential for proliferating-cell-specific promoter activity, but can direct transcription in a specific period during trichome development, which does not include cell division. This suggests that CDKA;1 functions during cell morphogenesis as well as cell proliferation. Abbreviations: CDK, cyclin-dependent kinase; GUS, β-glucuronidase; PCR, polymerase chain reaction
Introduction Proliferation of eukaryotic cells proceeds according to a common cell cycle program. The cell cycle is regulated at two checkpoints at least (i.e., the G1 to S-phase transition and entry into mitosis) through a particular class of protein kinase activity (for reviews see Pines, 1993; Nigg, 1995). Since these kinases require an associating protein, cyclin, for their activity, they are called cyclin-dependent kinases (CDKs). The cdc2 gene product (p34cdc2) of the fission yeast Schizosaccharomyces pombe was the first CDK whose involvement in cell cycle regulation was demonstrated at a molecular level (for reviews, see Forsburg and Nurse, 1991; Reed, 1992). Subsequently, p34cdc2 kinase has been found to be highly conserved in all eukaryotes analyzed, including higher plants (for reviews, see Mironov
et al., 1999; Joubes et al., 2000). In Arabidopsis, five CDK-related kinase genes, CDKA;1 (CDC2aAt), CDKB1;1 (CDC2bAt), CDKC;1 (CDC2cAt), CDKC;2 (CDC2dAt), and CDKD;1 (CAK2At) have been identified (Hirayama et al., 1991; Ferreira et al., 1991; Imajuku et al., 1992; Burssens et al., 1998; Joubes et al., 2000). These kinases are classified into four subclasses by their amino acid sequences in the cyclin-binding domain (Joubes et al., 2000). Yeast complementation analysis has demonstrated that CDKA;1, but not CDKB1;1, encodes a functional homologue of p34cdc2 (Hirayama et al., 1991; Ferreira et al., 1991; Imajuku et al., 1992). Expression analysis has revealed that CDKA;1 transcription occurs throughout the cell cycle in proliferating cells, whereas CDKB1;1 is preferentially transcribed during S and G2 phases (Hermerly et al., 1993; Segers et al., 1996). Although CDKA;1 tran-
206 scription seems to be constant throughout the cell cycle, it is tightly regulated throughout plant development. Accumulation of CDKA;1 mRNA is observed in cells that are proliferating or competent for proliferation (Martinez et al., 1992; Hermerly et al., 1993). Histochemical analysis using a fusion gene between a CDKA;1 upstream region and the Escherichia coli β-glucuronidase (GUS) gene has revealed that CDKA;1 transcription is induced upon auxin or cytokinin treatment or wounding, all of which lead to cell proliferation (Hermerly et al., 1993). These facts suggest that CDKA;1 expression is closely linked to cell proliferation or to competence of cell proliferation. In addition to the normal cell cycle, plant cells undergo various developmental processes without cell division, such as polar extension growth and endoreduplication (for reviews, see Hulskamp et al., 1998; Traas et al., 1998). In Arabidopsis, the development of the trichome and root hair cells includes polar extension growth of a single cell (Dolan et al., 1993; Hulskamp et al., 1994). Endoreduplication is known to occur in Arabidopsis epidermal cells including trichomes (Melaragno et al., 1993; Gendreau et al., 1997). By analogy with animal cell biology, these processes are also thought to be regulated by CDKs (Nikolic et al., 1996; Sigrist et al., 1997; MacAuley et al., 1998). So far, however, very little is known about the involvement of CDKs in these processes in plants. To shed light on the involvement of CDKA;1 in the regulation of cell proliferation and other developmental processes, we analyzed the functional structure of its promoter. As a first step, we determined its transcription start site, and then dissected the promoter to localize the cis element for transcription in proliferating cells using a series of promoter-GUS fusion genes. Our results indicated that a region downstream from the transcription start site is essential for the proliferating-cell-specific transcription of CDKA;1. On the other hand, a 595 bp fragment upstream from the transcription start site can direct transcription in developing trichomes, suggesting that CDKA;1 functions in cell morphogenesis as well as cell proliferation.
Materials and methods Plant material The Columbia ecotype was used as the wild-type Arabidopsis thaliana. The trichome mutants ttg, gl1, gl2, and gl3 (ecotype Lansberg erecta; Koornneef et al., 1982) were obtained from the Arabidopsis Seed Stock Center at Miyagi University of Education (Japan). The GUS-fusion genes were transformed into Agrobacterium tumefaciens strain LBA4404. Transgenic Arabidopsis plants were produced by the root transformation method of Valvekens et al., (1988) or the vacuum infiltration method of Bechtold et al., (1993). Plants for histochemical analysis were grown on agar medium at 22 ◦ C with a 16 h photoperiod. Primer-extension analysis and S1-nuclease mapping Total RNA from mature plants was isolated as previously described (Hirayama et al., 1991). Primerextension analysis and S1-nuclease mapping were carried out as outlined in Sambrook et al., (1989). For primer-extension analysis, 64 µg of total RNA and 0.2 pmol of primer DNA 32 P-labeled at the 50 end were incubated in 30 µl of hybridization buffer (80% formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM PIPES pH 6.4) at 30 ◦ C for 12 h. After ethanol precipitation, cDNA was synthesized at 37 ◦ C for 2 h in 50 µl of reaction mixture (50 mM Tris-HCl pH 8.2, 50 mM KCl, 10 mM MgCl2 , 1 mM dithiothreitol, 2 mM each of the four dNTPs) containing 200 units of Superscript reverse transcriptase (Gibco-BRL). For S1-nuclease mapping, single-stranded probe was made by elongating the labeled primer used in the primer-extension analysis as previously described (Aoyama et al., 1989). Total RNA (5 µg) and probe DNA (0.01 pmol) were heated in 10 µl of the hybridization buffer at 75 ◦ C for 10 min, cooled to 45 ◦ C over more than 2 h, and kept at 45 ◦ C for 12 h. Then 90 µl of S1-nuclease buffer (0.1 M NaCl, 4 mM ZnCl2 , 40 mM sodium acetate pH 4.5) containing 200 units of S1 nuclease (Takara Shuzo) was added and the sample was incubated at 20 ◦ C for 20 min. Each sample was electrophoresed on an 8% polyacrylamide-urea gel together with a sequence ladder made with the same primer. Construction of GUS-fusion genes GUS-fusion genes were constructed by inserting upstream regions of the CDKA;1 gene into the multi-
207 cloning site of the vector plasmid pBI101 (Clontech). The upstream DNA fragment in the fusion gene designated as P(–591/+4)::GUS was cut from a cloned Arabidopsis genomic fragment by digestion with BclI and HindIII. For P(−1299/+677)::GUS, P(+4/+677)::GUS, and P(−591/+677)::GUS, the upstream DNA fragments were cut from a cloned Arabidopsis genomic fragment by the polymerase chain reaction (PCR) using appropriate primers so that they could be fused to the GUS gene in frame. Histochemical GUS assay GUS histochemical staining was carried out using the basic procedure described by Jefferson et al., (1987). Arabidopsis organs (hand-cut into small pieces) or whole seedlings were submerged in cold 90% acetone for 1 h at −20 ◦ C. After several washes in 100 mM sodium phosphate buffer pH 7.4, the tissues were incubated 6 h at 37 ◦ C in a solution containing 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-βD-glucuronide (X-Gluc), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 100 mM sodium phosphate buffer pH 7.4. The reaction was stopped by several washes in 100 mM sodium phosphate buffer pH 7.4 and plant pigments were removed from the tissues with 70% ethanol.
Figure 1. Primer-extension analysis and S1-nuclease mapping of the CDKA;1 transcript. The products of the reverse transcriptase (lane P in a) and S1-nuclease (lane S in b) reactions were electrophoresed together with reference sequence ladders (lanes A, G, C, and T). All the labeled products have the same 50 end, so the position of the signals can be inferred directly. The sequences of both strands in the relevant region are shown on the right sides and the signal positions are indicated by arrows.
Scanning electron microscopy GUS-stained young leaves were dehydrated, subjected to critical point drying, mounted on stubs, and coated with gold. The preparations were then examined by standard methods.
Results
Figure 2. Structures of the GUS-fusion genes constructed for dissection of the CDKA;1 promoter. The CDKA;1 upstream regions fused to the GUS-coding sequence are illustrated. Open and filled bars represent coding and non-coding regions of CDKA;1, respectively. Shaded bars represent the GUS-coding sequence. A vertical arrow indicates the position of the CDKA;1 transcription start site. Numbers above the bars indicate the position corresponding to the distance (bp) upstream (−) or downstream (+) from the transcription start site (+1).
Assignment of the transcription start site of CDKA;1 As a first step of the promoter analysis, we determined the transcription start site of CDKA;1. Total RNA was prepared from seedlings of A. thaliana and subjected to primer-extension analysis and S1-nuclease mapping. An intense signal band 677 bp upstream from the CDKA;1 initiation codon and additional weak bands were observed in the primer-extension analysis (Figure 1a). In S1-nuclease mapping, signal bands were detected only around the position corresponding to the intense signal band observed above (Figure 1b). Since two or three consecutive bands are generally
seen around the actual transcription start site in S1nuclease mapping, we concluded that the transcription of CDKA;1 starts at the A residue 677 bp upstream from the initiation codon. The non-coding transcribed region of CDKA;1 is essential for proliferating-cell-specific promoter activity Activity of the CDKA;1 promoter in proliferating cells has been demonstrated in both histochemical analysis with a translational GUS-fusion
208 gene (Hemerly et al., 1993) and in situ hybridization analysis (Martinez et al., 1992). In order to dissect the functional structure of the CDKA;1 promoter, we constructed four GUS-fusion genes, P(−1299/+677)::GUS, P(−591/+677)::GUS, P(+4/+677)::GUS, and P(−591/+4)::GUS, in which different upstream regions of CDKA;1 were followed by the GUS-coding sequence (illustrated in Figure 2), and introduced them into transgenic Arabidopsis plants. More than 10 independent lines of 5-day old transgenic seedlings for each construct were histochemically examined for GUS activity. GUSstaining patterns typically observed for each construct are shown in Figure 3. Strong GUS activity was observed in apical shoot and root meristems of all the seedlings carrying P(−1299/+677)::GUS (Figure 3a) and P(−591/+677)::GUS (Figure 3b–d) as reported before (Martinez et al., 1992; Hermerly et al., 1993). In addition, cotyledons and root vascular systems also showed GUS activity. Although the intensity tended to be stronger in P(−1299/+677)::GUS plants than in P(−591/+677)::GUS plants, it varied also within the transgenic lines for each construct. On the other hand, P(+4/+677)::GUS (data not shown) and P(−591/+4)::GUS (Figure 3e and f) did not confer GUS activity on seedlings in any line. These results indicate that the non-coding transcribed region contains the cis element(s) essential for the promoter activity in proliferating cells in seedlings, and that the region from −591 to the initiation codon (+677) contains enough cis element(s) for the proliferatingcell-specific promoter activity. The 595 bp fragment mainly upstream from the CDKA;1 transcription start site has promoter activity in developing trichomes As shown above, transgenic seedlings carrying P(−591/+4)::GUS did not show any GUS activity. In young plants, the fusion gene conferred no GUS activity on the shoot apical meristem. Instead, strong GUS activity was observed in immature trichomes for 10 out of 12 transgenic lines (Figure 4b and c). The activity was limited to developing immature trichomes in young leaves and scarcely any was detected in mature trichomes (Figure 4b and c). This staining pattern in trichomes was also observed in all analyzed transgenic lines carrying P(−591/+677)::GUS (Figure 4a) and P(−1299/+677)::GUS (data not shown), indicating that the authentic CDKA;1 promoter has
activity in developing trichomes. In addition to developing trichomes, P(−591/+4)::GUS conferred GUS activity to the edges of young leaves (Figure 4b). P(+4/+677)::GUS conferred no GUS activity to young plants (data not shown). The promoter activity of the 595 bp fragment was detected in developing single-stalk cells on gl2 mutant leaves Trichome development involves several cell morphological events such as cell enlargement, endoreduplication, cell outgrowth, polar extension, and branching (Hulskamp et al., 1994). To determine which process the promoter activity detected above is associated with, we crossed a transgenic line carrying P(−591/+4)::GUS with mutants having defects in trichome development, including ttg, gl1, gl2, and gl3 (Koornneef, 1981; Koornneef et al., 1982). We established F3 plants homozygous with both each mutant gene and the transgene, and analyzed their GUS activity in young leaves. A similar staining pattern to that under the wild-type background was observed in F3 gl3 mutant plants (Figure 5d), where trichome cells are less branched and less endoreduplicated (Hulskamp et al., 1994). Against the ttg and gl1 mutant backgrounds (Figure 5a and b), where no initiation of trichome development occurs (Hulskamp et al., 1994), no GUS activity was detected except in the edges of young leaves. Against the gl2 mutant background, the transgene conferred GUS activity on developing single-stalk cells, as well as the edges of young leaves (Figure 5c). In gl2 mutant plants, giant cells and single-stalk cells are formed as mutant trichomes (Hulskamp et al., 1994). At an early stage of leaf development, when development of single-stalk cells is not clear, only those mutant trichome cells expanding perpendicularly to the leaf surface in comparison with other mutant trichome cells showed GUS activity (indicated by arrows in Figure 5e and f). On the other hand, no GUS activity was detected in the middle part of the leaf surface (Figure 5e), where many giant cells exist (Figure 5f). Single-stalk cells on fully expanded leaves showed no GUS activity (data not shown). These results suggest that the promoter activity is limited to trichome cells under polar extension growth.
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Figure 3. Histochemical analysis of CDKA;1 promoter activity in seedlings. Transgenic Arabidopsis 5 days after germination carrying P(−1299/+677)::GUS (a), P(−591/+677)::GUS (b–d), or P(−591/+4)::GUS (e and f) were examined histochemically. Close-up pictures of apical and root meristems are shown for P(−591/+677)::GUS (c and d, respectively) and P(−591/+4)::GUS (f). The bars in a, b, and e stand for 1 mm, and the bars in c and d for 0.2 mm.
Figure 4. Histochemical analysis of CDKA;1 promoter activity in young plants. GUS activity in an apical part of 14-day old transgenic Arabidopsis carrying P(−591/+677)::GUS (a) and P(−591/+4)::GUS (b), and in a young leaf of the P(−591/+4)::GUS plant (c) are shown. Leaf edges showing GUS activity are indicated by arrows in b. Bars: 0.2 mm.
Discussion Transcription start site of CDKA;1 We determined the transcription start site of CDKA;1 by two methods, viz. primer-extension analysis and S1-nuclease mapping. Both results are consistent and indicate that the transcript starts at the A residue 677 bp upstream from the initiation codon. The length
of the CDKA;1 mRNA deduced from the start site is consistent with that determined in northern analysis (Hirayama et al., 1991). In addition, the most upstream sequence of CDKA;1 cDNA that has been cloned so far (Ferreira et al., 1991) extends to a position very close to the start site determined here. These facts validate our assignment of the transcription start site. Consequently, it was revealed that the promoter
210
Figure 5. Histochemical analysis of developing-trichome-specific promoter activity against trichome mutant backgrounds. GUS activity in an apical part of 14-day old P(−591/+4)::GUS plants with ttg (a), gl1 (b), gl2 (c), and gl3 (d) mutant backgrounds is shown. The same young leaf of the P(−591/+4)::GUS plant with the gl2 mutant background was analyzed both histochemically (e) and by scanning electron microscopy (f). A mutant trichome cell expanding perpendicularly to the leaf surface in comparison with other mutant trichome cells is indicated by arrows in e and f. Bars: 0.2 mm.
contains no typical TATA-box sequences within the upstream region near the start site. cis element(s) for proliferating-cell-specific promoter activity of CDKA;1 The CDKA;1 promoter was dissected for cis-element analysis. The fragments extending from −1299 and
−591 to the initiation codon (+677) showed promoter activity in proliferating cells of seedlings, while the fragment containing only the upstream region from +4 did not. From these results, we concluded that the region from −591 to the initiation codon is sufficient for promoter activity in proliferating cells, and that the non-coding transcribed region (+4 to +677)
211 contains cis element(s) essential for the proliferatingcell-specific activity of the CDKA;1 promoter. In the non-coding transcribed region, there are sequences the same as or similar to the cis elements required for the proliferating-cell-specific promoter activity of the Arabidopsis histone H4 gene (Chaubet et al., 1996). The hexamer sequence (CCGTCG) and sequences similar to the nonamer (AGATCGACG) and octamer (CGCGGATC) exist at +67, +100, and +244, respectively. These sequences might also act as cis elements for proliferating-cell-specific expression in the CDKA;1 gene. The non-coding transcribed region, however, did not show any promoter activity by itself. This suggests that the region does not contain minor transcription start sites.
of promoter activity. Considering all these facts, the promoter is most likely to be activated during polar extension growth in trichome development, which occurs in stages 2 to 4. However, it cannot be ruled out that a low level of promoter activity, which was not detected in our histochemical analysis, exists in developing trichomes at stage 1 and in mature trichomes. In addition to developing trichomes, P(−591/+4)::GUS conferred GUS activity on the edges of young leaves. This region is composed of elongated cells, whose developmental process might be different from that of other leaf epidermal cells.
Activity of the CDKA;1 promoter in developing trichomes
The finding that the CDKA;1 promoter is activated during a particular period in trichome development strongly suggests that CDKA;1 operates some cell morphological process(es) in trichome development. Since promoter activity could be detected in stage 2 or later, CDKA;1 does not seem to be involved in endoreduplication, which occurs in stage 1. It has been reported that the expression of CDKA;1 is present only in mitotically dividing cells, but not in endoreduplicating tissues on the vegetative shoot apices (Jacqmard et al., 1999). Endoreduplication processes in Arabidopsis might operate independently from the function of CDKA;1. CDKA;1 might regulate branching and polar extension growth of trichomes, since the CDKA;1 promoter becomes active before or during these processes. Recently, both microtubules and actin microfilaments have been found to play important roles in these processes (Oppenheimer et al., 1997; Mathur et al., 1999; Szymanski et al., 1999; Mathur and Chua, 2000). The Xenopus cdc2 kinase has been shown to regulate microtubule dynamics in cell-free extracts from eggs (Verde et al., 1990). Other lines of evidence have indicated that p34cdc2 phosphorylates microtubule-associated proteins in yeast and animal systems (Nabeshima et al., 1995; Blangy et al., 1995). In plants, CDC2 of Medicago sativa has been observed to co-localize with microtubular structures, including the pre-prophase band, the spindle, and the phragmoplast (Stals et al., 1997). These findings suggest that CDKA;1 is involved in trichome morphogenesis through the regulation of microtubule formation. As another possibility, CDKA;1 may regulate the formation of actin microfilaments, since a mammalian CDK, CDK5, has been found to play a critical role
An unexpected result was obtained from histochemical analysis of young transgenic plants carrying P(−591/+4)::GUS. Strong GUS activity was observed specifically in developing trichomes, while no GUS activity was detected in the shoot apical meristem. The GUS activity in developing trichomes was also observed in transgenic plants carrying P(−1299/+677)::GUS or P(−591/+677)::GUS. These results indicate that the authentic CDKA;1 promoter is activated during trichome development and that the 595 bp upstream region contains enough cis element(s) to direct this activity. The development of Arabidopsis trichomes has been divided into six stages based on morphological criteria (Szymanski et al., 1998). These stages include radial expansion of a committed cell and endoreduplication (stage 1), perpendicular growth to the epidermal plane (stage 2), branch formation (stage 3), expansion via diffuse growth (stage 4), pointed-tip formation (stage 5), and maturation of the surface (stage 6). The developing-trichome-specific GUS activity was most clearly observed in small branched trichomes and not in mature trichomes under the wild-type background. Under the gl2 mutant background, GUS activity was detected in developing single-stalk cells, but not in giant cells. Giant cells are thought to have undergone endoreduplication four times without proceeding to polar extension growth (Hulskamp et al., 1994). These facts suggest that promoter activity is not associated with endoreduplication. Since no branching occurs in gl2 mutant trichomes, it can be said that the branching process is not essential for the induction
Involvement of CDKA;1 protein function in trichome development
212 in neurite outgrowth during neuronal cell differentiation through the regulation of actin microfilament formation (Nikolic et al., 1996, 1998). Conclusion CDKA;1 is thought to be a major CDK that plays a critical role in cell proliferation in Arabidopsis. In this report, we analyzed the functional structure of the CDKA;1 promoter and found evidence for the expression of CDKA;1 during trichome development, which does not include cell division. This suggests that CDKA;1 functions in cell morphogenesis as well as in cell proliferation.
Acknowledgements We thank Mr Masayoshi Ohara, Dr Masaki Tsuji and Dr Shinzo Kohjiya for technical support with the scanning electron microscopic work. This work was supported by grants to T.A. from BRAIN, Japan and from the Japanese Ministry of Education, Science and Culture (10182215) and a grant to A.O. from the Japanese Ministry of Science and Technology.
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