Journal of Experimental Botany, Vol. 51, No. 346, pp. 853-863,
May 2000
© 2000 Oxford University Press
Controlled induction of GUS marked clonal sectors in Arabidopsis
1 Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK
2 Laboratory of Plant Physiology and Molecular biology, University of Turku, 20520, Turku, Finland
3 Department of Biology, University of California at San Diego, La Jolla, Ca., 92093–0116 USA
Received 8 October 1999; Accepted 17 January 2000
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Abstract |
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Stably transformed Arabidopsis lines in which GUS marked cell clones are readily produced in response to heat-shock have been established and characterized. Control of GUS activation is achieved by heat-shock-induced FLP recombinase activity which ‘switches on’ expression of a GUS marker gene previously held transcriptionally silent. To obtain efficient GUS sectoring, single insert Arabidopsis lines carrying FLP recombinase under the control of a heat-shock-inducible promoter and an FLP-activatable GUS construct were generated. Analysis of GUS sectoring in lines hemizygous and homozygous for both inserts was conducted after various regimes of heat-shock were given at various developmental stages. It is shown that GUS sectoring events can be efficiently induced in most vegetative, aerial and sexual structures in Arabidopsis. Furthermore, the frequency of sectoring events, sector size and, to some extent, the tissues in which sectors are generated can be readily controlled by choice of the conditions and timing of heat-shock used.
Key words: FLP recombinase, site-specific recombination, Arabidopsis, clonal sectors, lineage analysis.
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Introduction |
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The ability unambiguously to mark cell clones in which a given gene of interest has been ‘switched off’ or ‘switched on’ provides the experimenter with the opportunity to conduct a mosaic analysis of gene function. In Drosophila, mosaic flies have been extensively exploited to dissect signal transduction pathways (Noordermeer et al., 1994
; Siegfried et al., 1994; Acharya et al., 1997; Matunis et al., 1997), to determine cell patterning events (Dang and Perrimon, 1992; Duffy et al., 1998) and to establish the cell autonomy of gene product (Doherty et al., 1997; Sturtevant et al., 1997). The principal advantage afforded by cell marking is that genotype and phenotype can be correlated with certainty.
In plants, efforts to produce marked cell clones have been principally directed towards facilitating cell lineage and/or sector boundary analysis. Of particular note in this regard are the use of periclinal chimeras (Szymkowiak and Sussex, 1996), micro-injection of single cells (Lusardi et al., 1994), induction of rare intrachromosomal recombination events (Swoboda et al., 1994), X-irradiation-induced chromosome rearrangement in plant lines heterozygous for a recessive visible marker gene (Coe et al., 1978; Poethig et al., 1986; Furner and Pumfrey, 1992, 1993) and activation of GUS expression by excision of an Activator (Ac)-based element from an Ac transcriptionally-interrupted GUS expression cassette (Bossinger and Smyth, 1996). However, none of these approaches in their reported form readily lends itself to the concomitant activation or deactivation of a given gene of interest.
The possibility of generating GUS marked cell clones in Arabidopsis has been investigated previously by exploiting site-specific recombination (Kilby et al., 1995). The basic strategy was to generate plant lines transgenic for a source of heat-shock-inducible FLP recombinase and then to cross these lines to other lines transgenic for a marker gene cassette comprised of a 35S CaMV promoter and GUS coding region, interrupted by an FLP-excisable hygromycin resistance casette which, because of its physical intervention also served, in the absence of FLP recombinase, as a GUS transcription blocking sequence. In these initial studies which used multiple insert-containing lines, it was shown that heat-shock treatment of germinating seedlings derived from such crosses produced GUS marked sectors in approximately 10% of treated plants. Sectoring events were observed in roots, cotyledons and the first true leaves, but no systematic description of sector frequency, sector size or any other parameter was undertaken. Given that heat-shock-induced FLP-mediated activation of GUS expression was the result of excision of the interlying transcription blocking sequence, heat-shocked plants were mosaic for the hygromycin gene cassette and mosaic sectors were clearly marked as such by GUS activity.
The potential of the FLP recombinase-based system for generating GUS marked cell clones in all vegetative, aerial and sexual structures in Arabidopsis is extensively characterized here. To carry out this work new, single insert, homozygous lines were generated which are conveniently maintained by self pollination. A detailed description is provided of how the generation of sectors in lines either hemizygous or doubly homozygous for both inserts can be controlled by choice of the conditions and timing of applied heat-shock, and it is shown that, by optimizing the experimental conditions, sectors can be induced in up to 100% of plants without any obvious abberant (heat-shock-induced) effect(s) on phenotype. Data presented here allow the experimenter to select the desired extent and frequency of GUS sectoring events making the system immediately available for cell lineage analysis (RA Sessions et al., unpublished results). In addition, the sector data described for plants hemizygous for both inserts is of direct relevance to the experimenter interested in generating marked cells in Arabidopsis that are mosaic for a given gene. The simplest strategy in this regard would be to heat-shock the progeny of a cross between a plant that is homozygous for a heat-shock-responsive source of FLP recombinase (e.g. HFC*, described here) and a plant homozygous for an FLP responsive gene switch (e.g. pFLP-SWITCH, Davies et al., 1999) that contains an appropriately configured gene of interest. All progeny plants from such a cross would be competent to produce marked mosaic sectors in response to heat-shock, sectors being produced within a highly defined heat-shock-inducible FLP expression background.
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Materials and methods |
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Plasmid constructs
Inducible (heat-shock) FLP source:
HSP FLP (Fig. 1A
; Kilby et al., 1995) carries the native FLP recombinase coding region placed under the control of the Gmhsp 17.6L soybean heat-shock promoter (Severin and Schöffl, 1990).
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FLP target construct:
The FLP-activatable ß-glucuronidase gene target construct pNJK14 (Fig. 1B
) was as previously described (Kilby et al., 1995).
Generation of transgenic Arabidopsis lines: transformation and selection
The FLP expression construct and the FLP target construct were mobilized into Agrobacterium tumefaciens strain C58C1rif by tri-parental mating (Ditta et al., 1980). Transformation of Arabidopsis thaliana Landsberg erecta and selection of transformants was as described (Kilby et al., 1995).
Generation of a homozygous, single-insert, heat-shock-inducible FLP recombinase expressing line
The Arabidopsis thaliana Landsberg erecta line AraHSF1 (Kilby et al., 1995) contains three HSP FLP inserts. The inserts in AraHSF1 were segregated by back-crossing AraHSF1 to wild-type Landsberg erecta, followed by one round of selfing. Two hemizygous lines carrying two different single inserts were identified by Southern analysis (Fig. 2A, tracks 3 and 4) and were designated HFC* and HFC+. Each single insert line was made homozygous by selfing. Homozygosity was confirmed by segregation analysis of kanamycin resistance (n>150).
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Generation of a homozygous, single insert, FLP-activatable ß-glucuronidase line
An FLP-activatable ß-glucuronidase line, designated TD, was identified following Agrobacterium-mediated transformation of Arabidopsis with the FLP target construct pNJK14, as described (Kilby et al., 1995). Chi-squared analysis of the segregation ratio of (hemizygous) line TD (
2=0.969; not significant at the 5% level) indicated that there was one active selectable marker insert in this transformant; this was confirmed by Southern analysis (Fig. 2B) as was the structural integrity of the corresponding T-DNA (Fig. 2C). Line TD was used as the target line for all sector generation experiments reported here. Line TD was made homozygous by appropriate rounds of selfing and kanamycin/hygromycin selection. Confirmation of homozygosity was based on the criterion that 100% of seedlings (n150>) were resistant to both kanamycin and hygromycin.
Generation of a line homozygous for HSP FLP and pNJK14 inserts
HSP FLP and pNJK14 both carry kanamycin resistance. To generate a line containing both HSP FLP and pNJK14 as homozygous, single inserts, lines hemizygous for both inserts were first generated by crossing homozygous HFC* with homozygous TD, with subsequent selection for hygromycin-resistant F2 progeny. F3 seed from selfs of hygromycin-resistant F2 plants were germinated in the presence of hygromycin. F2 plants which gave 100% of seedlings resistant to hygromycin were thereby identified as being homozygous for the pNJK14 insert. F3 progeny of F2 TD homozygotes were tested for HSP FLP zygosity by heat-shocking F3 seedlings from each of 15 randomly selected sample lines, and quantifying the proportion of plants in each line that produced GUS sectors. A line was identified in which GUS sectoring events occurred in 100% of heat-shock-treated plants indicating that this line was doubly homozygous for both HSP FLP and pNJK14 inserts.
Heat-shock treatment of plants
Seeds and germinating seedlings maintained on DMS medium (MS medium (Murashige and Skoog, 1962) containing sucrose (1%, w/v), agar (0.8%, w/v), pH 5.7) in ‘Parafilm’-sealed plastic Petri dishes were heat-shocked after stratification (4 °C for 5 d with subsequent transfer and acclimation to growth room temperature (20 °C) prior to heat-shock) in a microbiological incubator at 42 °C, for various times (see Results). Whole plants in soil were heat-shocked in enclosed plant propagators containing a reservoir of water to maintain humidity.
Maintenance of transgenics
Transgenic plants were grown in a potting compost : sand mix (1 : 4, v : v) in a controlled temperature (23 °C) room with artificial light (Philips TLD60W/84HF) supplied on a 16 h d photoperiod.
GUS analysis
Histochemical localization of GUS activity:
Gus activity in plant tissues was localized by histochemical staining with 1 mM 5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid (X-gluc, Biosynth AG, Staad, Switzerland) as described (Jefferson, 1987). Tissues were incubated in X-gluc-containing reaction buffer at 37 °C overnight, then destained in 70% ethanol prior to photography.
Embedding and sectioning of plant material:
Plant material destined for sectioning was stained for GUS activity and fixed, as described (Peleman et al., 1989). Fixed structures were embedded in low-melting point agarose (5%, w/v) then sectioned under distilled water using a Leica VT 1000E Vibratome. Sections were floated onto microscope slides prior to photography.
Statistical analyses:
Chi-squared analysis of sector event frequency was conducted on separately pooled event data; data pools comprised sectoring events collated from all durations of heat-shock treatments given, for a given tissue type (i.e. root, cotyledon and true leaf) on a given day when heat-shock was administered. Typically, greater than 20 plants were analysed per heat-shock treatment. Similarly, ANOVA on ranks testing was done using pooled data, as described above.
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Results |
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During our earlier sector generating studies (Kilby et al., 1995
), GUS sectors were produced in approximately 10% of heat-shocked seedlings, with sectoring events being observed in roots, cotyledons and the first true leaves. These preliminary experiments used heat-shock at 37 °C in a segregating (multiple insert) population. Heat-shock experiments conducted subsequently at 42 °C on the progeny of crosses between single insert-containing lines (HFC* and TD, see below) proved to increase the frequency of GUS sectoring events relative to heat-shock treatment at 37 °C (data not shown); 42 °C was therefore adopted as this study's standard heat-shock temperature in experiments reported here. To refine the genetics of this system, single insert, homozygous HSP FLP lines (HFC* and HFC+) were generated and an FLP-activatable ß-glucuronidase (pNJK14) single insert, homozygous target line, TD (see Materials and methods). To test if the HSP FLP lines and the target line were competent to produce GUS marked sectors in response to heat-shock when appropriately combined, pairwise crosses were made between line HFC* or HFC+ and line TD. Seed from each cross was then heat-shocked for 4 h on three consecutive days immediately post-stratification and stained for GUS activity one week later. 100% (n=58) of heat-shocked seedlings from the cross between line HFC* and target line TD showed GUS sectors. No GUS sectoring was observed in any seedling derived from crosses involving line HFC+ (or from any seedling from any cross not subjected to heat-shock). In all GUS sector generating experiments described here, homozygous line HFC* was used as the heat-shock-inducible source of FLP recombinase and homozygous line TD was used as the target line.
Effect of duration and timing of heat-shock on the frequency of GUS sectoring events
Seed hemizygous or doubly homozygous for HFC* and TD was stratified then heat-shocked once, for either 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 h on either day 1, 2, 3, 4 or 5 post-stratification. Seedlings were stained for GUS activity 10 d after heat-shock treatment was administered and the frequency of GUS sectoring events in roots, cotyledons and first true leaves was recorded (Fig. 3A, B). The sectoring response to heat-shock of both hemizygous and doubly homozygous plant populations followed the same broad trend. Typically, there was a pronounced high frequency of sectoring events when heat-shock treatment was given 1 d after stratification, a relatively marginal response if heat-shock was applied on days 2 or 3, and a pronounced response if heat-shock was administered 4 or 5 d after stratification. Although the overall trend of sectoring events between the two populations was comparable, the frequency of sectoring events was significantly higher in the doubly homozygous population as compared to the hemizygous population. In the doubly homozygous population the boundary between sectors often became indistiguishable (i.e. sectors merged) when prolonged heat-treatment was given on days 1, 4 or 5, post-stratification; no such effect was observed in the hemizygous population when comparable heat-shock treatments were administered. Statistical analysis of pooled sector frequency data for each tissue type examined (roots, cotyledons and first true leaves) compared with, and as influenced by, the day on which heat-shock was given (Table 1) showed that sector frequency differed significantly at the 99% confidence level (P<0.01) both with tissue type and with the day on which heat-shock was applied. In contrast, the relative ratio of sectoring events between each tissue type was not influenced by the day on which heat-shock was given (Table 2).
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GUS sectoring and sector size in leaves and cotyledons
The size of GUS sectors produced in true leaves was influenced by the timing of applied heat-shock. Sector size could be broadly classified into three qualitative groups (1) small sectors (restricted to a few cells; Fig. 4L, M, N), (2) large sectors (i.e. extensive sectoring events; Fig. 4A, B, G, H, I, J, K) and (3) intermediate sectors (sector size intermediate between 1 and 2, above; Fig. 4C, F). Large sectors were frequently produced when heat-shock was administered on days 1 and 2 post-stratification. Heat-shocking on these days also produced small and intermediate sectors. These smaller-sized sectors were only evident when the frequency and/or the extent of large sectoring events was sufficiently restricted within a given tissue type or organ, to permit their observation. Large sectors were produced infrequently when heat-shock was administered later than 2–3 d post-stratification. Heat-shocking 4 and 5 d post-stratification consistently produced small and intermediate sectors which, because of the event frequency of sector production, often merged (i.e. sector boundaries overlapped) in the doubly homozygous plant population when prolonged heat-shock was administered. Cross-sections taken through half leaf blade sectors (Fig. 4E) and mid-rib sectors (Fig. 4D) confirmed the clonal nature of GUS sectoring events. It was also observed that trichomes were stained in some GUS activation events (data not shown). GUS sectoring events in cotyledons (Fig. 4O, P) were typically small to intermediate in size and ran parallel to the cotyledon margins. Cotyledon sectors were associated with lateral vein boundaries and the mid-vein, but did not appear to cross the mid-vein itself.
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GUS sectoring in root, aerial and sexual structures
Analysis of root structures showed that GUS sectors could be generated throughout the entire length of the root and that, in such cases, root hairs were also stained (Fig. 4U). GUS sectoring events restricted to the central vascularity (data not shown) and discrete lateral roots (Fig. 4S) were frequently observed. In order to extend the analysis to include a description of GUS sectoring events in aerial and sexual structures, whole plants hemizgous for HFC* and TD at the stage of bolting were heat-shocked for 4 h, then allowed to develop until floral structures at all stages of development had been produced. Alternatively, whole plants were allowed to bolt, heat-shocked for 4 h, then propagated for 3 d more. Discrete structures were then removed from plants and stained for GUS activity. GUS sectors were evident in a variety of tissues including the flank meristem of the stem apex (Fig. 4Q), cauline leaves (Fig. 4V, upper sector), siliques (data not shown), and sepals (Fig. 4V(lower sector) and Fig. 4X). GUS sectors were also observed in the stamen and carpel (data not shown) but these events were relatively rare. In one experiment, embryos in maturing siliques (3 d after pollination) were heat-shocked at 42 °C for 45 min. Corresponding inflorescences derived after germination of seed from these embryos exhibited either (1) no GUS staining, (2) complete GUS staining of the entire structure or (3) half GUS sectorization of the inflorescence, mericlinal in L1, L2 and L3 (Fig. 4R, T, W) extending down the stem (as in Fig. 4T, side view aspect of Fig. 4R). It was noted that in a parallel experiment, identical heat-shock treatment of embryos in maturing siliques given 2 d after pollination did not produce GUS sectors.
Effect of heat-shock on plant morphology
The effect of heat-shock on plant mortality and phenotype was investigated by visual assessment of heat-shocked plants (prior to GUS staining) and by visual assessment of heat-shocked plants transferred to soil and grown to maturity. In one experiment, seeds were heat-shocked for 4 h on seven consecutive days post-stratification. Up to three consecutive heat-shocks could be given with no observed mortality of plants. No abberant phenotype was observed in heat-shocked plants grown to maturity compared to wild-type, unheat-shocked, control plants.
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Discussion |
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Here, a highly flexible, externally controllable means of marking cells is described that is immediately available for lineage or sector boundary analysis in Arabidopsis (RA Sessions et al., unpublished results). In a modified form the system is also available for the mosaic analysis of cloned genes. The basic strategy in this regard would be to use the FLP expressing line HFC* described here, to ‘switch on’ or ‘switch off’ expression of a cloned gene configured as part of an FLP responsive gene switch that concomitantly marks all cells in which an FLP-mediated gene switching event has taken place. It was noted that the use of directed site-specific recombination to undertake the mosaic analysis of gene function is an established procedure in Drosophila (Dang and Perrimon, 1992
; Noordermeer et al., 1994; Siegfried et al., 1994; Acharya et al., 1997; Doherty et al., 1997; Matunis et al., 1997; Sturtevant et al., 1997; Duffy et al., 1998) and has recently been reported in Arabidopsis where the Cre/lox site-specific recombination system from bacteriophage P1 (Austin et al., 1981) was used to conduct a mosaic analysis of AGAMOUS gene function (Sieburth et al., 1998). Importantly, the approach of this study for cell marking and that of Sieburth et al. (Sieburth et al., 1998) provides several advantages over other currently available methods for generating marked cell sectors in plants, particularly in respect of the ease with which the timing and frequency of marking events can be controlled. It was noted that the Cre recombinase-based cell marking method developed by Sieburth et al. (Sieburth et al., 1998) has yet to be characterized as extensively as the FLP recombinase-based method used here. It is anticipated, however, that by using both systems within the same genetic background it will be possible to conduct simultaneous, dual gene mosaic analysis provided, of course, that two distinct cell markers are used.
These initial observations concerning the generation of GUS marked clonal sectors in Arabidopsis were restricted to a description of GUS marking events in the first true leaves, cotyledons and roots (Kilby et al., 1995). These observations have been confirmed and this analysis extended to show the clonal nature of GUS marking events, as revealed by cross-sections through the leaf (Fig. 4D, E). It is believed that the particular ease and abundance with which GUS sectors can be generated in leaves (Fig. 4A–N) identifies this method of cell marking as being of particular value for leaf cell lineage analysis. The ability to produce GUS marked cells in both aerial and sexual structures in Arabidopsis is also highly desirable in terms of providing an experimentally flexible and informative method of cell marking. It has been shown that this system of cell marking is complete in this respect with the caveat that GUS marking events in the stamen and carpel are relatively rare; this may, of course, be indicative of the experimental conditions chosen. It is noted that although sectors could be generated in meristem, silique, stem, cauline leaf, and flowers, GUS marking of seed was never observed, presumably because such events are very rare or perhaps because of poor infiltration of GUS substrate through the seed coat during staining.
Results of experiments that involved quantifying GUS cell marking events in vegetative structures of Arabidopsis (Fig. 3A, B) serve to highlight the flexibility of the FLP recombinase-based system with regard to the degree of control that the experimenter has over the frequency of marking events. Data obtained using lines hemizygous and doubly homozygous for HFC* and TD indicate that heat-shock-induced GUS marking event frequency is a function of (a) the choice of duration of heat-shock, (b) the choice of day on which heat-shock is administered, (c) the zygosity of the inserts in the plant lines used, and (d) the developmental time window (e.g. germination versus bolting stage) when heat-shock was applied. Statistical analysis of the event frequency of sectoring events in roots, cotyledons and first true leaves (Table 1) confirmed that sector event frequency is a function of which day post-stratification heat-shock was administered and further established that the frequency of sectoring is different in each of the different tissue types examined with the highest event frequency being observed in cotyledons. The response of each tissue type to heat-shock in terms of the relative ratio of sectoring events observed, appears to be independent of the day when heat-shock is given (Table 2).
The relatively high sector event frequency observed in cotyledons as compared to the sector event frequency observed in roots and true leaves is intriguing. It is suggested that this phenomenon is simply a reflection of the relative number of cells in each tissue type. Each embryonic cotyledon is comprised of approximately 5200 cells (Bowman, 1994). In contrast, the embryonic shoot meristem is comprised of approximately 110 cells (Irish and Sussex, 1992; Schnittger et al., 1996) and the embryonic root (pro) meristem (i.e. shared epidermal/lateral root cap initials, shared cortical/endodermal initials, columella initials and the quiescent centre) contains 40 cells (Dolan et al., 1993). Assuming that cells in the root and shoot meristems and cells in each of the cotyledons are all as equally likely to undergo a sectoring event, then purely on the basis of relative cell number alone, sector event frequency would be expected to be higher in the cotyledons than in embryonic root or shoot meristems. An alternative hypothesis to explain the relatively high sector event frequency in cotyledons, is that the anatomy of the mature seed may influence how each tissue type ‘experiences’ the application of heat. Heat shielding by one tissue type overlaying another could influence the activity of heat-shock-induced FLP recombinase in a spatially determined, pseudo tissue-specific manner.
The observation that sectors can be produced readily and repeatedly in the cotyledons of germinating seeds supports the view that the increase in size of cotyledons during germination (distinct from the formation of cotyledons in embryogenesis, where cell divisions are restricted to the first 144 h after fertilization (Bowman, 1994)) involves both cell expansion and cell division (Hou et al., 1993; Fridlender et al., 1996). The alternative view that the increase in the size of cotyledons during germination does not involve cell division, merely cell expansion (Tsukaya et al., 1994) is not consistent with these observations. In this latter case, cotyledon sectors (which were typically small to intermediate in size and similar in appearance and distribution to those described by Fridlender et al., 1996) would not have been observed, except in the unlikely event that they arose from multiple adjacent independent single cell sectoring events. Our interpretation remains, that the generation of cotyledon GUS marked sectors provides strong evidence that cell division does occur in the cotyledons of germinating Arabidopsis embryos. Furthermore, the close association of cotyledon sectors with vascular tissue suggests a post-germinative role for cell division in organizing late events required to establish or ‘fine tune’ correct cotyledon vascular patternation.
As already noted, the profile of sectoring events in both hemizygous and homozygous populations was characterized by a pronounced high frequency of sectoring when heat-shock treatment was given 1 d after stratification, with a relatively marginal response if heat-shock was applied on days 2 or 3, and another pronounced response if heat-shock was administered 4 or 5 d after stratification. A possible explanation for this response profile is that on day 1 post-stratification seeds destined for heat-shock had been moved from 4 °C to 20 °C (until acclimated to 20 °C; a process taking approximately 1–2 h), then heat-shocked at 42 °C. This rapid temperature gradient (not experienced by seeds that were heat-shocked after day 1 post-stratification since they were incubated at 20 °C for a minimum of 24 h) may have had the effect of ‘priming’ the heat-shock promoter used to drive FLP recombinase activity in advance of application of the principal heat-shock treatment. The consequence of promoter priming may have been to amplify the heat-shock sectoring response (further discussed below). After prolonged incubation at 20 °C, promoter priming may no longer be relevant, leading to a reduction in the response to heat-shock observed on days 2 and 3 post-stratification. The upturn of the heat-shock response on days 4 and 5 post-stratification is consistent with the view that as each seedling emerged from its seed coat, it received a greater relative level of direct exposure to heat and, therefore, to the activity of FLP recombinase. It is also possible that the frequency of cell division at this time was relatively high, making a marking event more likely.
In so far as the suitability of use of a given method for cell marking is concerned, it is essential that the method used is non-lethal, and does not perturb the phenotype of the plant. In experiments reported here, using optimal sector generating conditions (Fig. 3A, B) no lethality of plants was observed. This, however, does not exclude the possibility that more subtle changes in phenotype might have occurred as a result of heat-shock. This suggestion was not examined in detail, but plants that were heat-shocked and grown to maturity, developed normally. Interestingly, in a study exploiting heat-shock treatment of transgenic tobacco to activate expression of an introduced isopentenyl transferase gene from a heat-shock responsive promoter, Medford et al. (Medford et al., 1989) showed that in heat-shocked wild-type control plants, the only discernible phenotypic effect of weekly heat-shock treatment (2 h at 45 °C) was a limited degree of ovule abortion and heterostylic development in a small percentage of cases. With regard to increasing the confidence of the experimenter that heat-shock treatment has the least possible effect (if any) on phenotype, it is noted that heat-shock pretreatment of restricted duration prior to application of the principal heat-shock might serve to protect heat-shocked plants by up-regulating heat-shock-induced proteins in advance. Such a strategy is commonly employed during heat-shock-induced, FLP-mediated cell marking in Drosophila, where it is used not only to protect heat-shocked flies from the physiological insult of elevated temperature, but also to up-regulate the induction of recombinase activity (Brand et al., 1994).
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Acknowledgments |
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This work was supported by BBSRC grant G04197.
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Notes |
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4 To whom correspondence should be addressed in Finland. Fax: +358 2 333 8075. E-mail:Nigel.Kilby{at}utu.fi
5 Present address: Plant Centre, LIBA, Irene Manton Building, University of Leeds, Leeds LS2 9JT, UK.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Acharya JK, Jalink K, Hardy RW, Hartenstein V, Zuker CS.1997. InsP3 receptor is essential for growth and differentiation but not for vision in Drosophila.Neuron 18, 881–887.[Web of Science][Medline]
Austin S, Ziese M, Sternberg N.1981. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 25, 729–736.[Web of Science][Medline]
Bossinger G, Smyth, DR.1996. Initiation patterns of flower and floral organ development in Arabidopsis thaliana. Development 122, 1093–1102.[Abstract]
Bowman J.1994. Arabidopsis: an atlas of morphology and development. Heidelberg: Springer-Verlag.
Brand AH, Manoukian AS, Perrimon N.1994. Ectopic expression in Drosophila. In: Wilson L, Matsudaira P, eds. Methods in cell biology. Drosophila melanogaster, practical uses in cell and molecular biology. California: Academic Press, 635–654.
Coe EH, Neuffer MG.1978. Embryo cells and their destinies in the corn plant. In: Subtelny S, Sussex IM, eds. The clonal basis of development. New York: Academic Press, 113–129.
Dang TD, Perrimon N.1992. Use of a yeast site-specific recombinase to generate embryonic mosaics in Drosophila. Developmental Genetics 13, 367–375.[Web of Science][Medline]
Davies GJ, Kilby NJ, Riou-Khamlichi C, Murray JAH.1999. Somatic and germinal inheritance of an FLP-mediated deletion in transgenic tobacco. Journal of Experimental Botany 50, 1447–1456.
Ditta G, Stanfield S, Corbin D, Helinski DR.1980. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium mellilotti. Proceedings of the National Academy of Sciences, USA 77, 7347–7351.
Doherty D, Jan LY, Jan YN.1997. The Drosophila neurogenic gene big brain, which encodes a membrane-associated protein, acts cell autonomously and can act synergistically with Notch and Delta. Development 124, 3881–3893.[Abstract]
Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, Scheres-B.1993. Cellular organisation of the Arabidopsis thaliana root. Development 119, 71–84.[Abstract]
Duffy JB, Harrison DA, Perrimon N.1998. Identifying loci required for follicular patterning using directed mosaics. Development 125, 2263–2271.[Abstract]
Furner IJ, Pumfrey JE.1992. Cell fate in the shoot apical meristem of Arabidopsis thaliana. Development 115, 755–764.[Abstract]
Furner IJ, Pumfrey JE.1993. Cell fate in the inflorescence meristem and floral buttress of Arabidopsis thaliana. The Plant Journal 4, 917–931.[Web of Science]
Fridlender M, Lev-Yadun S, Baburek I, Angelis K, Levy AA.1996. Cell divisions in cotyledons after germination: localization, time-course and utilization for a mutagenesis assay. Planta 199, 307–313.
Hou Y, von Arnim AG, Deng X-W.1993. A new class of Arabidopsis constitutive photomorphogenic genes involved in regulating cotyledon development. The Plant Cell 5, 329–339.[Abstract]
Irish VF, Sussex IM.1992. A fate map of the Arabidopsis embryonic shoot apical meristem. Development 115, 745–753.[Abstract]
Jefferson RA.1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reporter 5, 387–405.
Kilby NJ, Davies JD, Snaith MR, Murray JAH.1995. FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis. The Plant Journal 8, 637–652.[Web of Science][Medline]
Lusardi MC, Neuhaus-Url G, Potrykus I, Neuhaus G.1994. An approach towards genetically engineered cell fate mapping in maize using the Lc gene as a visible marker: transactivation capacity of Lc vectors in differentiated maize cells and microinjection of Lc vectors into somatic embryos and shoot apical meristems. The Plant Journal 5, 571–582.[Web of Science][Medline]
Matunis E, Tran J, Gonczy P, Caldwell K, DiNardo S.1997. punt and schnurri regulate a somatically derived signal that restricts proliferation of committed progenitors in the germline. Development 124, 4383–4391.[Abstract]
Medford JI, Horgan R, El-Sawi Z, Klee HJ.1989. Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. The Plant Cell 1, 403–413.
Murashige T, Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.
Noordermeer J, Klingensmith J, Perrimon N, Nusse R.1994. Dishevelled and armadillo act in the Wingless signalling pathway in Drosophila. Nature 367, 80–83.[Medline]
Peleman J, Boerjan W, Engler G, Seurinck J, Botterman J, Alliotte T, van Montague M, Inzé D.1989. Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase. The Plant Cell 1, 81–93.
Poethig RS, Coe EH, Johri MM.1986. Cell lineage patterns in maize embryogenesis: a clonal analysis. Developmental Biology 117, 392–404.[Web of Science]
Schnittger A, Grini PE, Folkers U, Hulskamp M.1996. Epidermal fate map of the Arabidopsis shoot meristem. Developmental Biology 175, 248–255.[Web of Science][Medline]
Severin K, Schöffl F.1990. Heat-inducible hygromycin resistance in transgenic tobacco. Plant Molecular Biology 15, 827–833.[Web of Science][Medline]
Sieburth LE, Drews GN, Meyerowitz EM.1998. Non-autonomy of AGAMOUS function in flower development: use of a Cre/loxP method for mosaic analysis in Arabidopsis. Development 125, 4303–4312.[Abstract]
Siegfried E, Wilder EL, Perrimon N.1994. Components of wingless signalling in Drosophila. Nature 367, 76–80.[Medline]
Sturtevant MA, Biehs B, Marin E, Bier E.1997. The spalt gene links the A/P compartment boundary to a linear adult structure in the Drosophila wing. Development 124, 21–32.[Abstract]
Swoboda P, Gal S, Hohn B, Puchta H.1994. Intrachromosomal homologous recombination in whole plants. EMBO Journal 13, 484–489.[Web of Science][Medline]
Szymkowiak EJ, Sussex IM.1996. What plant chimeras can tell us about plant development. Annual Review of Plant Physiology and Plant Molecular Biology 47, 351–376.[Web of Science]
Tsukaya H, Tsuge T, Uchimiya H.1994. The cotyledon—a superior system for studies of leaf development. Planta 195, 309–312.[Web of Science]
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