JXB Advance Access originally published online on November 22, 2004
Journal of Experimental Botany 2005 56(412):507-513; doi:10.1093/jxb/eri028
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RESEARCH PAPER |
Reduction of cell size induced by enod40 in Arabidopsis thaliana
1University of Verona, Dipartimento Scientifico e Tecnologico, Strada le Grazie 15, Cà Vignal 1, 37134 Verona, Italy
2Wageningen University, Department of Molecular Biology, Drijenlaan 3, 6703 HA Wageningen, The Netherlands
* To whom correspondence should be addressed. Fax: +39 45 8027929. E-mail: marisa.levi{at}univr.it
Received 26 May 2004; Accepted 13 September 2004
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Abstract |
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An extensive analysis of organ and cell size was performed in three different Arabidopsis lines transformed with the early nodulin gene enod40 under control of the CaMV35S promoter. All three transgenic lines presented a significant decrease in the mean size of both epidermal internode and leaf mesophyll cells. Flow cytometric and image analysis of enod40-transfected protoplasts prepared from wild-type Arabidopsis cell suspensions showed that transient expression of the gene resulted in reduced forward light scattering (a factor correlated with particle size) and cell size. The direct administration of ENOD40 peptide to fresh protoplasts also resulted in reduced forward scattering with respect to the control and to the administration of unrelated peptides. As far as is known this is the first report documenting a biological effect of enod40 at the cellular level in non-legume plants.
Key words: Arabidopsis, cell size, enod40, flow cytometry, GFP
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Introduction |
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enod40 was identified as a gene whose expression is highly induced after infection of legume roots with Rhizobium spp. The gene is induced within 3 h after root infection in the root pericycle and later on in dividing cortical cells and in the nodule primordium (Kouchi and Hata, 1993
; Yang et al., 1993; Compaan et al., 2001). As the expression in the pericycle precedes cortical cell division, the early expression of enod40 suggests that this gene is involved in Rhizobium signal-mediated induction of cell division. Introduction of enod40 by ballistic targeting has been shown to induce cortical cell divisions in a non-autonomous-cell manner (Charon et al., 1997). Over-expression of enod40 in Medicago truncatula leads to changes in the dynamics of nodulation, but not to the formation of more nodules, indicating that expression of enod40 does not lead to cell division per se (Charon et al., 1999). enod40 expression has also been localized in non-symbiotic meristems of legume plants, including apices of lateral roots, young leaves, and stipule primordia (Papadopoulou et al., 1996; Corich et al., 1998), procambial cells (Corich et al., 1998), and embryonic tissues (Flemetakis et al., 2000).
enod40 has also been cloned from non-legumes, such as tobacco (van de Sande et al., 1996), rice (Kouchi et al., 1999), and more recently from tomato (Vleghels et al., 2003), maize (Compaan et al., 2003), Lolium perenne, and Hordeum vulgare (Knud, 2004).
The enod40 genes isolated so far display relatively low overall sequence homology. The homology among enod40 genes is restricted to two highly conserved regions (box I and box II). enod40 mRNA lacks a long open reading frame (ORF), but instead several short ORFs are present. This observation has led to the hypothesis that either peptides encoded by the short ORFs are biologically active molecules (van de Sande et al., 1996; Kouchi et al., 1999; Compaan et al., 2001; Sousa et al., 2001; Rohrig et al., 2002; Knud, 2004) or that enod40 mRNA itself possesses biological activity (Crespi et al., 1994; Sousa et al., 2001).
Within all enod40 genes identified to date, the 5' conserved sequence (box I) comprises an ORF encoding for a putative small peptide that is from 10 to 13 amino acids long depending on the species (Kouchi et al., 1999; Knud, 2004). This suggests that the peptide encoded within this ORF might be translated and that it represents the molecule that mediates the biological activity of enod40. Although the peptide has not been detected in vivo, which may be due to either a high level of intrinsic instability or the rapid masking of epitopes, the presence of an antigen related to the peptide in nodule extracts of Medicago sativa (Sousa et al., 2001) and soybean (van de Sande et al., 1996) has been detected in immunocompetition experiments, indicating that the peptide might be present in tissues that display a high expression of the gene.
The expression of enod40 in non-symbiotic tissues and its presence in non-legume plants suggest that the gene is involved not only in symbiosis, but also that enod40 has a role in a process shared by all cell types in which the gene is expressed. Studies aimed at elucidating the role of enod40 in plant development and at finding out whether the peptide encoded within box I is biologically active would undoubtedly benefit from the availability of mutants in enod40. So far only a Mu-transposon enod40-tagged line has been described in maize (Compaan et al., 2003). However, no phenotypic growth aberration was observed, which could be explained by either the presence of a second enod40 gene in maize or by the mild phenotype. Over-expression of enod40 in tobacco leads to plants with adventitious shoots, while over-expression in Medicago interferes with nodulation (Charon et al., 1999). Apparently, plants are more responsive to over-expression of enod40 than its knock-out. To investigate further the role of enod40 in plant development, the effect of over-expression of enod40 was studied in the model plant Arabidopsis thaliana.
In this report, it is shown that enod40 over-expression affects cell size in selected tissues. This activity could be mimicked by transient over-expression of enod40 in protoplasts of A. thaliana or by administration to protoplasts of ENOD40 synthetic peptides from either soybean or tobacco.
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Materials and methods |
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Plant material
Seeds of Arabidopsis thaliana L., ecotype Wassilewskjia, were planted in a soil/agriperlite mixture, vernalized for 3 d, and then transferred to a greenhouse with a 14 h photoperiod. For root analysis, seeds were sterilized and spread on solid MS medium in Magenta vessels, vernalized, and then transferred to a growth chamber at 25 °C with a 16 h photoperiod.
Arabidopsis thaliana L. plants, ecotype Wassilewskjia, carrying CaMV35S::Gmenod40 (Van de Sande et al., 1996), were generated by vacuum infiltration (Bechtold et al., 1993). Seeds were selected on MS plates (Murashige and Skoog, 1962) containing 20 µg ml–1 methotrexate. Plants resistant to the selective antibiotic were propagated and the progeny was selected twice. Plants homozygous for the presence of the methotrexate resistance gene were further analysed for the presence of Gmenod40 DNA by Southern analysis. Expression of the transgene was confirmed by RT-PCR using Gmenod40 specific primers (Van de Sande et al., 1996). The progeny of three plants of F3, lines 1.1, 3.1, and 4.4, homogenous for the presence of the methotrexate resistance gene and CaMV35S::GmENOD40, with the higher enod40 expression, was used for further analyses together with the wild-type line.
The Arabidopsis suspension culture E157, generated from A. thaliana Columbia embryos, kindly provided by Professor Fiorella Lo Schiavo (Dipartimento di Biologia, University of Padova), was maintained in Gamborg's medium (Gamborg et al., 1968) supplemented with 2% sucrose, on a rotary shaker at 25° and subcultured every 7 d.
Organ and cell analysis
Internode length was measured with a graduated ruler. Leaves were photographed and measured as described below. Root diameter was measured on transverse sections. Fully expanded leaves and 5-mm-long segments of root tips from 40-d-old plants were fixed, dehydrated and embedded in paraplast (leaves) and in LR White resin (roots) by routine methods. Transverse leaf sections (7 µm) and transverse and longitudinal root sections (1 µm) were cut and stained with 0.1% Calcofluor (Fluorescent Brightener 28; Sigma) in water. The sections were observed with a fluorescence inverted Olympus IX70 microscope and images were acquired with a JVC KI-F58 CCD camera. Images were also collected, as described above, from fresh epidermis peelings of fully expanded first caulinar internode, and mature pollen grains.
Cell size and shape were analysed using the software ‘Image-Pro Plus’ (Media Cybernetics, LP), with manual, semi-automatic, or fully automatic measuring options. Analysis of variance of the data was performed with Microsoft Excel. At least 300 cells from at least 10 different plants were measured for each sample.
Preparation of plasmids for transient expression
For the transient expression assay, a construct (termed ‘double construct’) allowing the simultaneous expression of a reporter gene and enod40 was prepared and inserted in pGEM 3Zf(+). The construct contained a gfp (green fluorescent protein) variant reporter gene under CaMV35S promoter and an expression box X (containing CaMV35S, a small polylinker and nos terminator), in which the coding sequence of the gene of interest can be inserted. Soluble modified (sm) variants of GFP were used. The pUC118 clone of smrs (red shift)-gfp was obtained from ABRC (Arabidopsis Biological Resource Centre, Ohio State University, USA). A 442 bp fragment of Gmenod40-2 (accession number X86442) containing the ORF of the peptide cloned in pMON999 was used.
Intermediate plasmids were prepared by insertion of smrs-gfp, CaMV35S-nos, and enod40 sequences into pBluescript SK(+). Sequences excised from intermediate vectors and pGEM 3Zf(+) were used for the preparation of the following plasmids for protoplast transformation: pRSGFPX (with the expression box X empty) and pRSGFPXENOD (with the expression box X containing the enod40 sequence).
Protoplasts preparation and transformation
Ten-day-old seedlings of control and transformed plants were cut into pieces and incubated with 1% cellulase R10 and 0.25% macerozyme R10 (Yakult Pharmaceuticals) in 10 mM 2-(N-morpholino)ethane sulphonic acid (MES) buffer containing 30 mM CaCl2 and 0.5 M mannitol for 3–4 h with gentle agitation at room temperature. The suspension was then filtered through a 50 µm mesh filter, washed twice with 0.5 M mannitol and resuspended at a concentration of 200 000 protoplasts ml–1 in Gamborg's medium containing 2% sucrose, 0.5 M mannitol, 0.5 mg l–1 2,4-D (2,4-dichlorophenoxyacetic acid), and 0.25 mg l–1 6-BAP (6-benzylaminopurine). Protoplasts were analysed by flow cytometry and image analysis (see below).
For transient expression experiments, protoplasts were prepared from a suspension culture of the E-157 cell line, incubating cells (3 d post-inoculation) with enzymatic solution (2% cellulase, 1% macerozyme, and 0.5 M mannitol in 50 mM Na-citrate buffer, pH 4.8) overnight in the dark at 25 °C with gentle agitation. Thereafter, the suspension was treated as in Abel and Theologis (1998) for polyethylene glycol (PEG)-mediated transformation. Briefly, the suspension was filtered through a 50 µm mesh filter, washed in CaCl2 and mannitol and resuspended in W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM glucose, and 1.5 mM MES–KOH, pH 5.6). Plasmid DNA (20–100 µg) and an equal amount of carrier DNA were mixed, precipitated, and resuspended in 50 µl sterile water. The protoplast suspension (300 µl, corresponding to 
1.5x106 protoplasts) was added to DNA and gently mixed; 300 µl of PEG solution (400 mM mannitol, 100 mM Ca(NO3)2, and 40% v/v PEG 3350; Sigma) were added and the mixture was incubated for 30 min at room temperature. After several washes in W5 solution, protoplasts were resuspended in 3 ml of Gamborg's medium+0.5 mg l–1 2,4-D and 0.5 M mannitol, and incubated for 16–24 h in Petri dishes (60 mm diameter) in the dark at 25 °C.
For the analysis of the effect of peptide administration, cell suspensions were incubated in the enzyme mixture for 8 h and then washed three times in Gamborg's medium with 2% sucrose and 0.5 M mannitol, and cultured in the same medium.
Peptide administration
The following peptides were used: GmENOD40 (MELCWLTTIHGS) (a), NtENOD40 (MQWDEAIHGS) (b), unrelated peptide M1.2 (LLLLTVLTV) (c), and gramicidin S (d). Three Petri dishes for each treatment were prepared and analysed in each experiment. Experiments were performed at least four times with peptides (a) and (b), and twice with peptides (c) and (d).
Fluorescence microscopy
Calcofluor-stained sections were observed with an Olympus IX70 inverted microscope, equipped with a WU filter (excitation 330–385 nm, dichroic mirror 400 nm, emission >420 nm). Protoplasts were observed with the same microscope, with the filter combination NB (excitation 470–490 nm, dichroic mirror 500 nm, emission >515 nm).
Flow cytometry
Flow cytometric analysis (FCM) of protoplasts was performed with a Bryte HS (Bio-rad) cytometer, equipped with a mercury-xenon lamp. Forward-angle light scattering (FS) and large-angle light scattering (SS) include photons deflected at angles up to 2° and 15°, respectively. The following filter blocks were used: excitation block FITC 520 (excitation filter 470–490 nm; beam splitter 510 nm; emission filter >520 nm); separator block GR1 (emission filter 1, FL1, 515–565 nm; beam splitter 560 nm; emission filter 2, FL2, 590–720 nm). Fluoresbryte beads (4.5 µm diameter) were used for calibration of the instrument and as reference internal standard for light scattering detection.
Monoparametric histograms and biparametric cytograms of FS, SS, FL1, and FL2 were acquired in listmode with log amplification. For each acquisition at least 1200 cells were measured. For each sample, three different acquisitions were collected, with highly reproducible results. The experiments were performed at least three times with similar results. The figures show one representative experiment.
For the identification of transformed protoplasts, a threshold was fixed on the basis of the autofluorescence of the untransformed control. In the transformed sample, protoplasts with green fluorescence higher than the threshold were considered transformed. For the analysis of the effect of enod40 expression, FS values of smRS-GFP- and enod40-expressing protoplasts were compared with those of a control expressing rsGFP only.
In peptide administration experiments, protoplasts were stained with 10 µg ml–1 fluorescein diacetate (FDA) for 5 min at room temperature prior to FCM analysis. A threshold was fixed on the basis of the autofluorescence of the unstained control; protoplasts with green fluorescence higher than the threshold were considered viable and analysed for their FS values.
Flow cytometry data were converted in FCS (flow cytometry standard) format and analysed with WinMDI software (http://facs.scripps.edu/software.html).
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Results |
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Flow cytometric analysis of protoplasts from plants over-expressing enod40 revealed a reduction in cell size
Three enod40-transformed Arabidopsis lines (1.1, 3.1, and 4.4) were chosen for their high enod40 expression. Initially, protoplasts were isolated from 10-d-old whole plantlets and from mature leaves of wild-type (wt) and 1.1, 3.1, 4.4 enod40-over-expressing lines, and analysed by flow cytometry. Differences in forward light scattering (FS) were found between the protoplasts isolated from transformed lines and wt protoplasts. The mean FS values of protoplasts from transformed lines (1.1, 1260; 3.1, 1265; and 4.4, 1275 arbitrary units) were significantly lower (P <0.01) than those from the wt (1337). Image analysis of the same protoplasts showed that the area of protoplasts isolated from the transgenic lines was significantly smaller (P <0.01) than that of protoplasts isolated from wt plants.
Arabidopsis plants over-expressing enod40 showed reduced cell size in some tissues
The overall appearance of transgenic plants was very similar to that of wt, with the same number of organs, phyllotaxis, and branching in vegetative and reproductive shoots. As preliminary flow cytometry experiments suggested that transgenic plants should have some cells that are reduced in size, an extensive analysis of the size of different organs and cells was performed. No significant differences were found between organ sizes in all three transgenic lines compared with wt. With regard to individual cells, in all three transgenic lines the size of epidermal cells of the first internode and of mesophyll cells was significantly lower than in the wild type; that of pollen grains and of endodermal cells was lower in two transgenic lines (Table 1).
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Protoplasts transiently expressing enod40 showed reduced expansion
As a second approach, the effect of transient expression of enod40 in Arabidopsis protoplasts was examined by preparing a ‘double construct’ that allowed the detection of the simultaneous expression of a reporter gene and another gene of interest. sm(soluble modified; Davis et al., 1998
)rs(red shifted)-gfp was used as the reporter gene. Protoplasts were transiently transformed with pRSGFPXENOD, in which enod40 was inserted in the expression box ‘X’ of pRSGFPX. RT-PCR performed on protoplasts 24 h after transformation showed the simultaneous expression of the two genes, whereas in control protoplasts transfected with pRSGFPX (with an empty expression box), only smRSGFP mRNA was detected (not shown). smrs-gfp was chosen as the reporter gene instead of smgfp due to its higher excitation with blue light, which also permitted light scattering analysis.
FCM analysis of protoplasts was performed at the end of the transformation procedure and 24 h later. As shown in Table 2, the mean FS values increased in this period and those attained by protoplasts expressing smrs-gfp and enod40 were significantly lower than those of control protoplasts (expressing smrs-gfp alone). The shoulder in the distribution of FS values of enod40-transfected protoplasts (Fig. 1) indicates that enod40 expression results in a higher proportion of protoplasts with low FS, which should have a smaller size. Accordingly, image analysis made on 350 protoplasts for each sample showed that the area of enod40-transformed protoplasts was significantly lower than that of controls (3566 versus 4282 µm2; P <0.005). This was due to a higher content in small protoplasts, as shown in Fig. 2. These differences were evident after 24 h of protoplast culture, when cell division is not yet restored in transfected protoplasts.
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Protoplasts treated with ENOD40 peptide showed reduced FS
To verify whether the effect of over-expression of enod40 could be due to the peptide, protoplasts were directly treated with ENOD40 peptide. Flow cytometric analysis of FS was performed on fresh protoplasts (T0) and after 17 h of culture. To identify viable protoplasts, the suspensions were stained for 5 min with FDA prior to FCM analysis.
Initial experiments with concentrations of the peptide between 10–8 and 10–5 M showed that the maximal effect on FS values was found at a concentration of 10–6 M. Protoplasts were then treated with ENOD40 peptide and two unrelated peptides at this concentration. As shown in Table 3, the mean FS value attained by NtENOD40-treated protoplasts was significantly lower than that attained by untreated protoplasts and protoplasts treated with unrelated peptides. The effect of GmENOD40 peptide was very similar to that of the tobacco peptide (data not shown).
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Discussion |
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In the present report, three different systems were used to verify a possible effect of enod40 in the non-legume model plant Arabidopsis thaliana: (i) three lines of Arabidopsis plants transformed with enod40 under control of the CaMV35S promoter, obtained from independent transformation events; (ii) Arabidopsis protoplasts transiently expressing the enod40 gene; (iii) Arabidopsis protoplasts treated with ENOD40 peptide. The results obtained in all these systems are highly concordant and demonstrate that enod40 induces a limitation of cell size, both in intact plants and in protoplasts derived from suspension cell culture.
In earlier flow cytometric studies, when isolated from whole plantlets or mature leaves, protoplasts from all three transgenic lines showed mean forward scattering levels that were significantly lower than those of protoplasts isolated from wt plants. This was due to an increase in protoplasts with lower FS values. Since FS is a parameter correlated with particle size (Kerker, 1983), these results suggested that a certain number of protoplasts from transformed plants (and hence cells from which these protoplasts had been isolated) had smaller sizes than wt. Direct size measurement of protoplasts with a microscope-based image analysis program confirmed this conclusion. FS can thus be considered a good marker of protoplast size in the present system. Nonetheless, the location of the cells with reduced size could not be identified after protoplast isolation.
Therefore, an extensive analysis of cell size in different cells and tissues (freshly prepared or sectioned) was performed. It was found that epidermal cells of the first internode and mesophyll cells of fully expanded leaves in all three transgenic lines were significantly smaller than those in the wt.
It cannot be excluded that the reduction in cell size induced by enod40 could be the result of an attempt to develop the proper intrinsic organ size as a consequence of increased cell division. Plants are generally believed to control organ size by monitoring the size of the entire organ rather than by monitoring the number and size of individual cells (Day and Lawrence, 2000). Therefore a variation in cell number could result in normal-sized organs with altered cell volume. Many authors reported an increase in cell volume compensating a decrease in cell number (Hemerly et al., 1995; Mizukami and Fischer, 2000; Wang et al., 2000). On the other hand, compensation of an increased division rate or division extent with a corresponding decrease in cell volume has never been reported.
A discrimination between the effect on cell division or cell expansion is difficult to achieve in whole plants. Nonetheless, freshly prepared protoplasts cultured in suspension expand before cell division starts again, and could represent a good system with which to determine an independent effect on cell expansion.
Actually, as shown both from FS values and image analysis (Figs 1, 2), 24 h after transfection enod40-expressing protoplasts were smaller than the control ones. Therefore, the expression of enod40 inhibited cell expansion, since within 24 h no cell division had occurred in transfected protoplasts.
The protoplast system also allowed the effect of exogenous administration of the ENOD40 peptide, which cannot be tested in planta, to be studied. Fresh protoplasts treated with tobacco and soybean ENOD40 peptide attained lower FS values with respect to both the untreated control and protoplasts treated with two unrelated peptides.
Furthermore, the latter experiments allowed the conclusion that the effect of enod40 on cell size can be ascribed, at least in part, to the activity of the peptide, although RNA activity cannot be ruled out. Recently, Campalans et al. (2004) showed that enod40 RNA mediates cytoplasmic relocalization of a nuclear protein in Medicago truncatula.
As enod40 was effective both when the peptide was administered exogenously and when the gene was over-expressed in the cytoplasm, it can be surmised that either receptors or enod40-responsive machinery exist both on the plasmalemma and inside the cell or, alternatively, that the peptide is active on the cell surface and its RNA acts in the cytoplasm.
Recently, Hardin et al. (2003) found that the ENOD40 peptide antagonizes the in vitro phosphorylation of serine 170 of maize sucrose synthase SUS1, which promotes SUS1 proteolysis. enod40 could therefore preserve the level of SUS1, whose products are utilized in the synthesis of structural polymers, such as cell wall polysaccharides, which are in turn involved in cell expansion. Sugar sensing and signalling are involved in the control of plant growth and development (Rolland et al., 2002). Sucrose itself is considered to be a signal molecule for plants, controlling the expression of genes such as patatin, phloem-specific rol-C, and the leucine zipper gene ATB2 (Smeekens and Rook, 1997). The mechanisms behind sucrose-sensing are still largely unknown, although there is evidence that plant cells, rather than monitoring the sucrose concentration directly, may sense changes in the ratio between sucrose and glucose, which is affected by sucrose synthase activity (Sturm and Tang, 1999; Smeekens and Rook, 1997).
Hanson et al. (2001) found that the over-expression of the transcription factor HDZhdip in Arabidopsis thaliana resulted in sugar-dependent alteration of the development of leaves and cotyledons, which presented larger cells, showing a possible direct effect of a sugar-signalling pathway on cell size control.
All this evidence suggests that enod40, by stabilizing sucrose synthase, could trigger a sugar-signalling pathway that modulates cell expansion. enod40 might also affect cell expansion by directly modulating the activity of other genes or enzymes. It would be interesting to verify whether the expression of expansin genes is affected in transformed cells or in protoplasts treated with the peptide. It is also possible that the development of turgor pressure could be affected.
An additional effect of enod40 on cell division cannot be ruled out by the present data. An increase in cell division, with normal-sized organs having a higher number of smaller cells, could explain why no clear differences in organ size between all the three transgenic lines and the wt were found despite the differences in cell size.
The effect of enod40 on cell size was found only in selected types of cells. This finding is probably due to tissues responding differently. Moreover, with regard to protoplasts, only a fraction of transfected protoplasts attained a reduced size. Since only protoplasts expressing the reporter gene were taken into consideration for size and FS analysis, and as these protoplasts should also express enod40, it can be surmised that different populations of protoplasts with differing responsiveness to enod40 expression were present. Studies of the binding of fluorescent ENOD40 peptide to protoplasts could clarify this point.
Along these lines, there is recent evidence that protoplasts isolated from cell suspension cultures are actually a mixture of different populations. In protoplasts from carrot suspension cultures, by cell sorting, it was possible to separate two subpopulations that differ in their cytoplasm: vacuole ratio and morphogenetic potential, which have been interpreted as being derived from ‘meristematic’ and ‘differentiated’ cells (Guzzo et al., 2002). Preliminary experiments in Arabidopsis suspension cultures also showed that two subpopulations exist that respond differently to 2,4-D (F Guzzo et al., unpublished results).
As far as is known, the reduction of cell expansion shown in the present paper is the first report of a biological effect of enod40 at the cellular level for a non-legume plant. The phenotype induced by enod40 reported in this work is somewhat subtle, but it was clearly demonstrated using different, independent approaches. Using reverse genetics, Compaan et al. (2003) were unable to show any phenotype in enod40-mutated lines. The ability to detect subtle phenotypes depends strongly on the techniques used for evaluation. In the present case, flow cytometric experiments, which allowed the measurement of thousands of cells for each sample, gave highly reproducible and reliable results allowing for the detection of even small differences.
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Acknowledgements |
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The authors are grateful to Dr Renato Longhi (ICRM-CNR, Milano, Italy) for providing M1.2 peptide. This research was supported by University of Verona.
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References |
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