JXB Advance Access originally published online on February 28, 2005
Journal of Experimental Botany 2005 56(414):1263-1268; doi:10.1093/jxb/eri122
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RESEARCH PAPER |
Ectopic endoreduplication caused by sterol alteration results in serrated petals in Arabidopsis
1Department of Ion-Beam-Applied Biology, JAERI (Japan Atomic Energy Research Institute), Watanuki-machi 1233, Takasaki, Gunma 370-1292, Japan
2RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351-0198, Japan
3Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
* To whom correspondence should be addressed. Fax: +81 27 346 9480. E-mail: hase{at}taka.jaeri.go.jp
Received 9 October 2004; Accepted 14 January 2005
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
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The Arabidopsis frill1 (frl1) mutant, that has serrated petals and sepals but no other large changes in plant morphology, was studied. The frl1 had a mutation in STEROL METHYLTRANSFERASE 2 and an altered sterol composition. It was found that the frl1 mutation causes ectopic endoreduplication in petal tips that do not normally endoreduplicate. The rosette leaves of frl1 also showed an enhanced level of endoreduplication, but their morphology was hardly affected. These facts suggest that the suppression of endoreduplication is important for petal morphogenesis and the normal sterol composition is required for this suppression.
Key words: Arabidopsis thaliana, endoreduplication, frill1, petal, SMT2, sterol
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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In a screen for mutants defective in organ development, the Arabidopsis frill1 (frl1) mutant that has serrated petals and sepals was isolated and characterized (Hase et al., 2000
). In the distal region of frl1 petals, the radial cell arrangement that can be seen in the wild type was disorganized. In this region, the number of cells was reduced whereas both cell size and nuclei size dramatically increased. These results suggested that the frl1 mutation affected cell division and also caused abnormal endoreduplication in the distal part of petals.
Sterols are membrane components and have a role in regulating membrane fluidity and permeability (Hartmann, 1998). They also serve as a precursor for brassinosteroids (BRs), which have been extensively studied as growth-promoting plant steroid hormones (Bishop and Koncz, 2002). The majority of sterols in higher plants are alkylated at carbon 24. This alkylation is performed by two classes of S-adenosyl-methionine-dependent C-24 SMTs (Bouvier-Navé et al., 1998). Arabidopsis has three SMT genes (SMT1, SMT2, and SMT3). The protein encoded by SMT1 catalyses the first methylation step, and SMT2 and SMT3 work together in the second methyl-addition step. To date, several mutants defective in the early steps of the biosynthetic pathway, such as fk/hyd2, hyd1, and smt1, have been described (Diener et al., 2000; Jang et al., 2000; Schrick et al., 2000; Souter et al., 2002). They all show severe phenotypes in embryogenesis and can not be rescued by the exogenous application of BRs. The cvp1 mutant that shows defects in cotyledon vascular patterning was found to be caused by a mutation in SMT2 (Carland et al., 2002). Thus, it has been suggested that phytosterols other than BRs also have important and specific roles in plant development.
Endoreduplication, i.e. duplication of the genome without cell division, is a common phenomenon in plants. Most plant cells undergo endoreduplication during their terminal differentiation processes following the arrest of cell division (Larkins et al., 2001). In fact, most somatic tissues in Arabidopsis contain multiploid cells and the degree of endoreduplication is developmentally regulated depending on age and tissue types (Galbraith et al., 1991). Generally, endoreduplication is associated with cells that become enlarged (Melaragno et al., 1993) and it is thought to be one of the important factors that are involved in organ size control (Mizukami, 2001). However, its contribution to developmental regulation of organ shape is still poorly understood in plants.
In this report, it is shown that FRL1 is identical to SMT2. The altered sterol profile in frl1 led to ectopic endoreduplication in the distal part of petals and also enhanced endoreduplication level in rosette leaves. The results suggest that normal sterol composition is required to control endoreduplication and also suggest that the suppression of endoreduplication is important for petal morphogenesis.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Mapping and sequencing of FRL1
The frl1 mutant was generated in a Columbia (Col) ecotype using a carbon-ion beam as a mutagen. All plants were grown in pots containing Metro-Mix 350 (Scotts-Sierra Horticultural Products) in a growth room at 23 °C under 16/8 h light/dark conditions with 3000–4000 lx of white fluorescent light. frl1 was mapped using CAPS markers as described by Konieczny and Ausubel (1993)
. About 400 F2 plants with the frl1 phenotype that were obtained from a cross of frl1 and the Landsberg erecta (Ler) ecotype were used. frl1 was mapped to a 105 kb region on chromosome 1. One single base pair deletion was found after about 30% of this region had been sequenced from the frl1 mutant line.
Complementation test
A 6.3 kb BamHI–EcoRI genomic fragment containing the SMT2 coding region (1.1 kb), an upstream region of 3.7 kb, and a downstream region of 1.5 kb was excised from the insert of the BAC clone F14O10. The fragment was subcloned into the plasmid pBI101. The frl1 plants were transformed by Agrobacterium GV3101 by the infiltration method (Bechtold and Pelletier, 1998) with the 6.3 kb fragment cloned in pBI101. Transformants were selected on a 2 g l–1 gellan gum plate containing 1x MS salts, 1x B5 vitamin, 2.5% sucrose, 50 mg l–1 kanamycin, and 166 mg l–1 claforan (pH 5.7 with KOH). All ten kanamycin-resistant T1 plants showed complete restoration of the mutant phenotype.
SMT2::GUS plant
A translational fusion of a SMT2::GUS construct was made by inserting the 2.7 kb 5' region including the first 33 nucleotides of the SMT2 coding sequence upstream of the ß-glucuronidase coding sequence of the plasmid pBI101. Transformants with SMT2::GUS were made in the Columbia background. T2 plants derived from kanamycin-resistant T1 plants were submerged in 90% acetone on ice for 1 h and then in a GUS staining solution containing 2 mM X-Gluc, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, and 0.5% Triton X-100 in NaPO4-buffer (pH 7.0). Tissues was infiltrated under vacuum for 30 min and then incubated for 1–2 d at 37 °C in the dark. The stained tissues were cleared in 70% ethanol until the GUS stain became clearly visible. Two independent transgenic lines showed a similar staining pattern.
RT-PCR analysis
Total RNA was isolated from 14-d-old seedlings and the tips of petals using RNeasy Plant Mini Kit (Qiagen). The RNA was treated with DNase I according to the manufacturer's instructions. Reverse transcription was performed with the BD PowerScript Reverse Transcriptase (BD Biosciences Clontech). The sequences of the PCR primers were as follows: for SMT1 (SMT1-F, 5'-TGTTTTTGGTTATGAATCGT-3' and SMT1-R, 5'-CCGAACATCAGAAAATAAAG-3'), for SMT2 (SMT2-F, 5'-ACTGGTTCCTCTGCGTTCTC-3' and SMT2-R, 5'-TGATGGTGAGAAGTGGAAGG-3'), for SMT3 (SMT3-F, 5'-GATCTTTGTTCGTGTCCTAC-3' and SMT3-R, 5'-TTTCTCCTTCACTACCTCAA-3') and for actin (actin-F, 5'-CTACGTCTTGATCTTGCTGGTCGTG-3' and actin-R, 5'-CCACCACTGAGCACAATGTTACCG-3'). PCR was carried out in 30 µl total reaction volume with the following program: 94 °C for 5 min followed by cycles of 94 °C for 30 s, 55 °C or 60 °C for 30 s and 72 °C for 1 min. The annealing temperature was 55 °C for SMT1 and SMT3, and 60 °C for SMT2 and actin. Ten µl of PCR products, after 20, 25, 30, 35, 40, and 45 cycles, were separated on agarose gels and visualized by UV-excitation of ethidium bromide staining. The images were captured through the filter supplied with the IS8000 digital imaging system (Alpha Innotech, San Leandro, CA). Each amplification yielded a specific band at the expected length. Analysis of the band intensity was performed with the software AlphaEase FC (Alpha Innotech). Measurement was done before saturation was reached. All RT expression analysis was performed in duplicate in three independent experiments.
Analysis of endogenous sterols
Inflorescence samples (400–600 mg FW) were collected from frl1 and wild-type plants that were just starting to flower. Endogenous sterols were analysed as described by Noguchi et al. (2000).
Flow cytometry
Petal tips were collected from several fully-opened flowers. Rosette leaves were collected from the same plants. A single 4th or 5th leaf was used for each run. The collected tissues were chopped using a razor blade in about 0.5 ml nuclei extraction buffer (solution A of a High Resolution Kit for Plant DNA; Partec GmbH, Münster, Germany). After filtration through a 30 µm mesh nylon sieve, about 2.0 ml of staining solution containing DAPI (solution B of the kit) was added. Ploidy level was measured using a PAS flow cytometer (Partec). The lowest peak was assumed to be 2C nuclei (C is a haploid DNA content). For each run, more than 3000 nuclei were analysed.
Microscopy
Three-day-old roots grown on an agar plate were fixed in 4% paraformaldehyde, dehydrated through an ethanol series and embedded in Technovit 7100 resin (Heraeus Kulzer GmbH & Co. KG, Wehrheim, Germany). To observe the size of the nuclei, petals and sepals cleared in 70% ethanol were dyed in 1 µg ml–1 DAPI solution.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The frl1 mutant was isolated as a morphological mutant having serrated petals and sepals (Fig. 1A). The gross morphology and size of frl1 plants are similar to those of the wild type. The fertility was 40% of that of the wild type based on the number of fertile ovules in the 3rd to 5th flowers (n=30 siliques). The frl1 locus was closely linked to the CAT3 marker (29.91 cM) on chromosome 1. Finally, frl1 was mapped to the 105 kb region that spans three BAC clones: T20H2, F14O10, and F5M15. Sequencing of this region revealed a single base deletion in the F14O10.7 gene that encodes SMT2. The frl1 gene has a single base deletion in the middle of the coding region (Fig. 2). This results in the aberrant translation of 44 amino acids before the new stop codon at the 218th amino acid. Three motifs conserved in SMT proteins are lost as a result of the mutation. The phenotype of frl1 mutant was completely restored by the transformation with genomic SMT2 fragment (Fig. 1B), indicating that the frl1 phenotype is caused by the single mutation of the SMT2 gene.
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The cvp1 mutants that were originally isolated as a mutant with a discontinuous cotyledon venation pattern have been reported to have a mutation in SMT2 (Carland et al., 2002
), i.e. frl1 is allelic to cvp1. Vascular strands in cvp1 cotyledons are discontinuous and thicker than those in wild-type cotyledons. RNA gel blotting analyses showed that SMT2 is expressed in all developing tissues. It was also mentioned that the cvp1 mutant has a serrated petal margin, however, the floral phenotype has not been investigated in detail.
To investigate the expression level of SMT2 in petals, semi-quantitative RT-PCR was carried out. The transcript level of SMT2 was significantly reduced not only in petal tips but also all over the frl1 seedlings (Fig. 3A), although the mechanism is unknown. This reduction did not affect the transcription of two other SMT genes, SMT1 and SMT3 (Fig. 3A). A reduction in the level of the SMT2 transcript was also observed in four cvp1 alleles, even though they were caused by a nucleotide substitution (Carland et al., 2002). Promoter–GUS analysis of SMT2 showed that the GUS activity in petals was relatively low compared with the levels in the other floral organs (Fig. 3B). The GUS activity was particularly strong in newly-formed lateral roots (Fig. 3C), however, longitudinal sections of root tips showed normal morphology (Fig. 3D). These results show that the organ specificity of the frl1 phenotype can not be explained by the expression pattern of the SMT2 gene.
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Endogenous sterol levels were measured in inflorescences of frl1 and wild type (Table 1). In the frl1 mutant, the amounts of C24-ethyl sterols, such as sitosterol, were between 20% and 38% of the amounts in the wild type, whereas the amount of C24-methyl sterols, such as campesterol, were more than four times higher than the amounts in the wild type. Similar changes in sterol profiles were observed in the stem and rosette leaves of frl1 plants (data not shown). This alteration of sterol profile is in good agreement with the loss of SMT2 function and is similar to the sterol profile reported in the cvp1 mutant (Carland et al., 2002
). Any attempts to rescue the mutant by applying synthetic intermediates (2 µl of 10–5 M 22
-hydroxy-sitosterol or 10–7 M brassinolide applied daily to the inflorescences for 1 week) were ineffective, although the brassinolide treatment induced pedicel elongation in both frl1 and the wild type (data not shown).
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It was found that the nuclei in the distal part of frl1 petals are larger and of various sizes (Fig. 4A) in contrast to nuclei of uniform size in the equivalent part of wild-type petals (Fig. 4B). A similar difference was observed at the borders of the sepals (Fig. 4C, D). Therefore, unusual endoreduplication has been suggested to be the cause of the marginal serration (Hase et al., 2000
). To investigate in detail, flow cytometric analysis was carried out in petal tips and rosette leaves. Almost all of the nuclei were at the 2C level in the wild-type petals, indicating no evidence of endoreduplication in petal tips (Fig. 5A), whereas the 4C peak was obvious in the frl1 petals (Fig. 5B). In rosette leaves, both the wild type and frl1 showed polyploidy up to 32C (Fig. 5C), but frl1 had a higher percentage of 16C and 32C nuclei (Fig. 5D). These results suggest that the altered sterol profile in the frl1 mutant leads to ectopic endoreduplication in petal tips and also enhances the endoreduplication level in rosette leaves. A question to be addressed is how the mutation in SMT2 results in serrated petals and sepals. Because the sterol alteration is seen all over the frl1 plant, petals and sepals are thought to be the most sensitive organs for the altered sterol profile found in the frl1 mutant. The rosette leaves of frl1 do not show visible morphological changes. This suggests that the rosette leaves have a plasticity that can adaptively regulate morphogenesis in response to the enhanced endoreduplication level. However, the petals of frl1 can not, probably because the petal tip does not naturally endoreduplicate and it has a regular cell arrangement. Suppression of endoreduplication is thought to be important for petal morphogenesis.
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In mammalian cells, cholesterol is a major sterol that is essential to drive cell cycle progression. Martínez-Botas et al. (1999)
found that human cell lines cultured in a cholesterol-deficient medium were arrested specifically at the G2 phase. The addition of cholesterol or an activator of CDK1, UCN-01, caused the cell cycle to resume (Suárez et al., 2002). The molecular mechanism of the endocycle is poorly understood in plants, but it has been suggested that important changes in the cell cycle machinery, such as inactivation of mitotic CDK/cyclin complexes, are required for the transition into the endocycle from the mitotic cycle (Traas et al., 1998; Joubés and Chevalier, 2000). In Arabidopsis, it has been reported that overexpression of CDK inhibitor (CKI) leads to serrated leaves and petals (De Veylder et al., 2001; Jasinski et al., 2001). The reduction of cell number and the increase in cell size was observed in the leaves of the transformant that overexpresses one of the Arabidopsis CKIs, named KRP2 (De Veylder et al., 2001). In contrast to the frl1 mutant, the cell enlargement observed in the transformant was not accompanied by enhanced endoreduplication. Another transformant that overexpresses a tobacco-derived CDK inhibitor, NtKISa, had serrated leaves and petals (Jasinski et al., 2001). The marginal serration of petals of the transformant differs from that of the frl1 mutant because the cells in the distal part of the petal in the transformant had an elongated shape. It is probable that the reduction of CDK activity inhibited the differentiation into round-shape cells that are normally found in the distal part of the petal. In addition, strong expression of NtKISa inhibited endoreduplication in rosette leaves. Taken together, the marginal serration of leaves and petals of the CKI overexpressors is thought to be caused by the suppression not only of the mitotic cell cycle but also of cell differentiation and endoreduplication. Although the mechanism by which the ectopic endoreduplication occurred in frl1 petals is still unclear, the results suggest that the normal sterol composition is necessary to control the switch to endoreduplication and also support the hypothesis that some other factors that suppress endoreduplication are affected by the altered sterol composition of the frl1 mutant. The frl1 mutant provides a useful tissue to investigate the molecular mechanism of endoreduplication.
The fact that the frl1 phenotype is mostly observed in petals and sepals raises the possibility of changing petal shape (without making other large changes in plant morphology) by changing SMT2 activity. SMTs have been isolated from Arabidopsis, tobacco, rice, and soybean, and are thought to be conserved in most plant species (Schaeffer et al., 2000). Therefore, the possibility of using SMTs to change petal shape may be applicable to many plant species. Schaeffer et al. (2001) examined transgenic Arabidopsis plants showing co-suppression of SMT2 and they concluded that SMT2 regulates the ratio of campesterol to sitosterol and this ratio modulates the plant growth. Carland et al. (2002) also mentioned that the ratio of campesterol to sitosterol is proportional to the severity of the phenotype and only the strongest allele of cvp1 showed retarded growth. The ratios of the co-suppressed lines were 1.1 and 2.2, in contrast to 0.19 of the control plants. These values are comparable to the values of frl1 (1.7) and the wild type (0.16). However, the co-suppressed line had more severe phenotypes, such as bushy appearance, reduced height, increased branching, and very low fertility. Since the co-suppressed lines were generated on the C24 ecotype, the difference in phenotype between frl1 and the co-suppressed lines may be due to the difference in their backgrounds. During the gene-mapping experiments, it was noticed that the F2 mutant plants derived from frl1 (Col background) x Ler often had more severe phenotypes than did the original frl1 plants (Fig. 1C, D). frl1 was crossed with five kinds of Arabidopsis ecotypes and it appeared that the mutant phenotype was most enhanced in the cross with Ler or C24. By contrast, F2 plants derived from the cross with Ws seemed to be less severe. Because a close linkage could not be found between the enhanced phenotype and any DNA markers, several factors, possibly including sterol profile, appear to be related to the phenotype induced by the reduction of SMT2 activity.
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Acknowledgements |
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We thank Y Oono, A Sakamoto, N Shikazono, S Takahashi, and all the other members of A Tanaka's laboratory for their assistance and helpful comments; the ABRC for providing BAC clones used in the cloning of FRL1. We also acknowledge S Takatsuto for 22
-hydroxy-sitosterol and deuterated sterols; and M Kobayashi for his technical assistance. |
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