Access the full text.
Sign up today, get DeepDyve free for 14 days.
J. Wet, K. Woodt, D. Helinski, Marlene DELUCAt (1985)
Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.Proceedings of the National Academy of Sciences of the United States of America, 82 23
R. Palmiter, R. Behringer, C. Quaife, F. Maxwell, I. Maxwell, R. Brinster (1987)
Cell lineage ablation in transgenic mice by cell-specific expression of a toxin geneCell, 50
A. Hoekema, P. Hirsch, P. Hooykaas, R. Schilperoort (1983)
A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmidNature, 303
C. Berg, V. Willemsen, G. Hendriks, P. Weisbeek, B. Scheres (1997)
Short-range control of cell differentiation in the Arabidopsis root meristemNature, 390
C. Landel, J. Zhao, D. Bok, G. Evans (1988)
Lens-specific expression of recombinant ricin induces developmental defects in the eyes of transgenic mice.Genes & development, 2 9
J. Dawson, Z. Wilson, M. Aarts, A. Braithwaite, L. Briarty, B. Mulligan (1993)
Microspore and pollen development in six male-sterile mutants of Arabidopsis thalianaBotany, 71
A. Koltunow, J. Truettner, K. Cox, M. Wallroth, R. Goldberg (1990)
Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development.The Plant cell, 2
Dellaporta. (1983)
A plant DNA minipreparation. Version IIPlant Mol. Biol. Rep., 1
J. Sambrook, E. Fritsch, T. Maniatis (2001)
Molecular Cloning: A Laboratory Manual
M. Goldman, R. Goldberg, C. Mariani (1994)
Female sterile tobacco plants are produced by stigma‐specific cell ablation.The EMBO Journal, 13
C. Mariani, M. Beuckeleer, J. Truettner, J. Leemans, R. Goldberg (1990)
Induction of male sterility in plants by a chimaeric ribonuclease geneNature, 347
Clarke (1992)
High-frequency transformation of Arabidopsis thaliana by Agrobacterium tumefaciensPlant Mol. Biol. Rep., 10
M. Thorsness, M. Kandasamy, M. Nasrallah, J. Nasrallah (1991)
A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana.Developmental biology, 143 1
A. Spurr (1969)
A low-viscosity epoxy resin embedding medium for electron microscopy.Journal of ultrastructure research, 26 1
M. Breitman, S. Clapoff, J. Rossant, Lap-Chee Tsui, L. Glodé, I. Maxwell, A. Bernstein (1987)
Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice.Science, 238 4833
Diane Hird, D. Worrall, R. Hodge, S. Smartt, W. Paul, R. Scott (1993)
The anther-specific protein encoded by the Brassica napus and Arabidopsis thaliana A6 gene displays similarity to beta-1,3-glucanases.The Plant journal : for cell and molecular biology, 4 6
S. Kunes, H. Steller (1991)
Ablation of Drosophila photoreceptor cells by conditional expression of a toxin gene.Genes & development, 5 6
M. Czakó, Jyan-chyun Jang, J. Herr, L. Marton (1992)
Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobaccoMolecular and General Genetics MGG, 235
M. Thorsness, M. Kandasamy, M. Nasrallah, J. Nasrallah (1993)
Genetic Ablation of Floral Cells in Arabidopsis.The Plant cell, 5
Chang (1994)
Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in plantaPlant J, 5
Seok Chang, Soon-Ki Park, B. Kim, B. Kang, Dal-Ung Kim, H. Nam (1994)
Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in plantaPlant Journal, 5
Hugo Bellen, D. D'evelyn, M. Harvey, S. Elledge (1992)
Isolation of temperature-sensitive diphtheria toxins in yeast and their effects on Drosophila cells.Development, 114 3
D. Worrall, Samita Patel, K. Lindsey, D. Twell (1996)
A novel transient assay system demonstrates that DT-Atsm is a temperature-sensitive toxin in plant tissues, 113
J. Männer, W. Seidl, G. Steding (1996)
Experimental study on the significance of abnormal cardiac looping for the development of cardiovascular anomalies in neural crest-ablated chick embryosAnatomy and Embryology, 194
F. Guerineau, L. Brooks, P. Mullineaux (1991)
Effect of deletions in the cauliflower mosaic virus polyadenylation sequence on the choice of the polyadenylation sites in tobacco protoplastsMolecular and General Genetics MGG, 226
R. Scott, E. Dagless, R. Hodge, W. Paul, I. Soufleri, J. Draper (1991)
Patterns of gene expression in developing anthers of Brassica napusPlant Molecular Biology, 17
A. Feinberg, B. Vogelstein (1983)
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Analytical biochemistry, 132 1
F. Guerineau, R. Waugh (1993)
The U6 small nuclear RNA gene family of potatoPlant Molecular Biology, 22
A. Nishimura, M. Morita, Y. Nishimura, Y. Sugino (1990)
A rapid and highly efficient method for preparation of competent Escherichia coli cells.Nucleic acids research, 18 20
G. Evans (1989)
Dissecting mouse development with toxigenics.Genes & development, 3 3
W. Paul, R. Hodge, S. Smartt, J. Draper, R. Scott (1992)
The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 genePlant Molecular Biology, 19
C. Berg, V. Willemsen, W. Hage, P. Weisbeek, B. Scheres (1995)
Cell fate in the Arabidopsis root meristem determined by directional signallingNature, 378
F. Guerineau, A. Lucy, P. Mullineaux (1992)
Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplastsPlant Molecular Biology, 18
M. Bevan (1984)
Binary Agrobacterium vectors for plant transformation.Nucleic acids research, 12 22
Christopher Day, Bernard Galgoci, V. Irish (1995)
Genetic ablation of petal and stamen primordia to elucidate cell interactions during floral development.Development, 121 9
Dawn Worrall, Diane Hird, R. Hodge, Wyatt Paul, John Draper, Rod Scott (1992)
Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco.The Plant cell, 4
K. Moffat, J. Gould, H. Smith, C. O’Kane (1992)
Inducible cell ablation in Drosophila by cold-sensitive ricin A chain.Development, 114 3
K. Edwards, C. Johnstone, C. Thompson (1991)
A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.Nucleic acids research, 19 6
<h1>Introduction</h1> The development of a complex organism progresses through a number of cell and tissue specification and differentiation steps that are determined by a cascade of events under the control of spatially and temporally regulated gene expression. The ability to interfere with such a programme is a powerful approach for unravelling its complexity. The specific inhibition of gene expression, either through mutation or reverse genetic techniques, yields information about gene function and sometimes affects the development of the cells in which it takes place. An important adjunct to these techniques involves the specific ablation of cells and the subsequent observation of the consequences on the development of a tissue or organ ( van den Berg et al ., 1995 ). As mechanical (e.g. Manner et al ., 1996 ) and laser ablation (e.g. van den Berg et al ., 1997 ) can only be used in a limited number of situations, the production of a cytotoxin by specific cells offers a valuable alternative. This can be achieved by the expression of a gene encoding a toxin under the control of a transcriptional promoter of known specificity. Such cell ablation experiments were first done in mice (reviewed in Evans, 1989 ) by expressing the gene encoding the diphtheria toxin A chain (DTA) polypeptide in lens ( Breitman et al ., 1987 ) or pancreatic ( Palmiter et al ., 1987 ) cells. The ricin A toxin was also shown to be active when produced in mice lens cells ( Landel et al ., 1988 ). In all cases, the production of the toxin by specific cells resulted in cell type or even organ ablation in the transgenic animals. In plants, the same strategy was applied to the production of male-sterile plants, by the expression of cytotoxins in the tapetum, a specialized tissue of the anthers surrounding the sporogenous cells. The tapetum-specific promoter TA29 has been used to drive expression of a ribonuclease gene ( Mariani et al ., 1990 ) and the DTA gene which resulted in dominant male-sterility due to the specific ablation of the tapetum ( Koltunow et al ., 1990 ). Collapse of the tapetum was also observed when the A9 and A6 promoters were used to drive expression of the barnase gene in transgenic plants ( Hird et al ., 1993 ; Paul et al ., 1992 ). Similarly, when the S-locus glycoprotein gene promoter of Brassica was fused to the DTA gene and transferred into tobacco ( Thorness et al ., 1991 ) and A. thaliana ( Thorness et al ., 1993 ) it resulted in self-sterile plants due to expression of the gene in both pistil and anthers. Stigma-specific cell ablation by DTA protein induced female-sterility in tobacco plants ( Goldman et al ., 1994 ). Expression of the DTA gene fused to the pea vicilin promoter induced seed mortality in transgenic plants ( Czako et al ., 1992 ), whereas an AP3-DTA fusion resulted in the complete ablation of petals and stamen in transgenic flowers ( Day et al ., 1995 ). These cell ablation experiments all required the use of a characterized promoter coupled to a dominant-acting cytotoxin gene, an approach which has two drawbacks. Firstly, the expression of a toxin under the control of a promoter of unknown specificity may not be informative because of the early death of the organism and secondly, the dominant action of the cytotoxin reveals only the developmental impact of the first instance of gene expression. One means of avoiding such drawbacks is through the use of an inducible system of cell ablation. This would allow normal development in permissive conditions but could be activated by a shift to non-permissive conditions. Such systems have been developed and used in eye cell ablation experiments in Drosophila melanogaster ( Kunes et al ., 1991 ). Temperature-sensitive mutants of the DTA (DTA ts ) ( Bellen et al ., 1992 ) and cold-sensitive ricin A mutants ( Moffat et al ., 1992 ) have been isolated. Their expression in D. melanogaster cells resulted in temperature-dependent cell death. To date, the application of these systems in plants has remained untested. Another interesting application of a conditional cell ablation system is in the isolation of regulatory mutants in model plants like A. thaliana . Any mutation affecting the expression of the toxin gene would produce a suppression of toxin-induced phenotype which is easily scorable in a large population. However, any mutagenesis screen requires the production of a large quantity of seeds transgenic for the toxin gene, which would not be possible if the expression of the gene affected vital processes such as embryo development, germination or fertility. The use of a toxin with temperature-dependent activity would circumvent this problem. We demonstrate here that a mutant DTA can induce specifically conditional male sterility. This is controllable at temperatures easily obtainable in simple plant growth rooms and has been used in a screen for apomictic mutants in A. thaliana , as described in Praekelt and Scott (2001 ). <h1>Results</h1> <h2>Isolation of temperature-sensitive male-sterile plants</h2> The DTA ts gene encodes a mutant diphtheria toxin which is reportedly active at 18 °C and inactive at 25 °C ( Bellen et al ., 1992 ). To generate conditional male-sterile A. thaliana plants, we placed this gene under control of the A. thaliana tapetum-specific promoter A9 ( Paul et al ., 1992 ) to create the A9::DTA ts gene. Two separate Agrobacterium -mediated root transformation experiments were performed with the A9::DTA ts construct to assess whether the temperature regime altered the frequency of transformation. Our hypothesis was that transformation performed at 20 °C (Experiment 1) would be less efficient than at 26 °C (Experiment 2) because DTA ts is inactive at 26 °C. In both experiments, primary putative transformants (Generation-1 plants) were allowed to self-pollinate (at either 20 °C in Experiment 1 or 26 °C in Experiment 2) to produce Generation-2 seed. Plants grown from Generation-2 seed were assessed for conditional sterility by first growing the plants at 18 °C until flowering and then increasing the temperature to 26 °C. Generation-2 plants were scored as conditionally sterile if they lacked elongated pods at 18 °C but produced elongated pods at 26 °C. The number of primary transformants resulting from plant transformation at 20 °C was 43 and from transformation at 26 °C was 100. <h2>Analysis of transformants recovered from transformation Experiment 1 (20 °C)</h2> Seeds were successfully harvested from the 43 putative primary transformants (Generation-1 plants) grown at 20 °C. These were sown on soil and the resulting plants were grown at 18 °C. To assess the transformation frequency, a proportion of the Generation-2 plants were analysed for the presence of the neomycin phosphotransferase II (NPTII) gene by polymerase chain reaction (PCR) using specific primers. Of 18 lines tested, 14 produced the expected 524 base pair (bp) PCR product (not shown). Each of the PCR positive Generation-2 plants were assessed for conditional sterility as described in Experimental procedures. One of the 43 Generation-2 plants failed to produce elongated siliques at 18 °C but did produce seeds at 26 °C. This originated from primary transformant A9::DTA ts -Q. Generation-3 seeds were harvested from each of the 11 Generation-2 plants originating from transformant A9::DTA ts -Q, which will be referred to as Q1 to Q11. Generation-3 plants Q3, Q4 and Q6 were male-sterile at 18 °C but produced siliques when grown at 26 °C ( Figure 1B ), whereas untransformed control plants produced siliques at both temperatures ( Figure 1A ). All plants from the Generation-3 families Q2, Q3, Q4 and Q6 were sterile at 18 °C, whereas other families contained a mixture of conditionally sterile plants, fertile plants and plants with reduced fertility ( Table 1 ). All plants from families Q1, Q7 and Q8 were fertile at 18 °C, although Q1 plants showed a reduced fertility. To determine the cause of sterility in A9::DTA ts -Q plants, anthers from open flowers were compared from wild-type and A9::DTA ts -Q6 plants grown at 18 °C. No pollen was found on anthers from A9::DTA ts -Q6 plants ( Figure 1D ) but cross-pollination with wild-type pollen ( Figure 1C ) resulted in elongated pods (not shown), therefore the plants were female fertile. When the same A9::DTA ts -Q6 plants were transferred to 26 °C, pollen was present on the anthers within 5 days (data not shown). This data suggested that the cause of the observed sterility in line A9::DTA ts -Q was temperature-sensitive male-sterility. <h2>Analysis of the number of transformants from Experiment 2 (26 °C)</h2> Of the 100 putative primaries generated in Experiment 2, only three plants showed conditional male sterility in Generation-2 progenies. This suggested that performing the transformation at 26 °C produced no significant improvement in the frequency of conditional sterile plants as compared to 20 °C. <h2>Molecular characterization of conditional male-sterile line A9::DTA ts -Q</h2> To determine the number of transgene loci in A9::DTA ts -Q, Generation-3 seeds from segregants Q1 to Q11 were sown on a germination medium containing kanamycin to give the segregation data of kanamycin sensitive plants to resistant plants ( Table 1 ). A plant carrying a single locus when selfed would be expected to give a 3 : 1 ratio of resistant to sensitive plants. A plant carrying two transgenes at separate loci would be expected to give a 15 : 1 ratio of resistant to sensitive plants. A9::DTA ts -Q7 showed a 3 : 1 segregation which indicated the presence of the NPTII gene at a single locus. The ratio of resistant to sensitive plants for A9::DTA ts -Q5, A9::DTA ts -Q10 and A9::DTA ts -Q11 was between these figures (8 : 1, 8.3 : 1 and 5 : 1, respectively) which is consistent with the integration of the transgene at two or more linked loci. Families A9::DTA ts -Q3, Q4 and Q6 appear to contain transgene integrations at more than two independent loci as there are resistant plants but no sensitive plants. Families A9::DTA ts -Q3, Q4 and Q6 account for all of the homogeneously sterile (at 18 °C) families, which suggested a link between the number of transgene loci and the degree of sterility. The A9::DTA ts -Q8 family appeared not to have inherited the NPTII transgene. To further investigate the link between transgene copy number and sterility, we quantified the number of DTA ts gene loci by Southern analysis. DNA was digested with Hin dIII (which cleaves upstream of the A9 promoter but not between the DTA ts gene and the transformed DNA (T-DNA) left border) so that each T-DNA insertion event in the plant genome resulted in the presence of one band of specific molecular weight on the Southern blot which would be different from any other insertion. When probed with the DTA ts coding sequence, it appeared that all but one family contained several copies of the transgene ( Figure 2A ). No signal was produced by wild-type or A9::DTA ts -Q8 DNA, which confirmed the NPTII segregation data. Results from other plants were inconsistent with the segregation data. For example, the Q7 family showed a 3 : 1 segregation for kanamycin resistance ( Table 1 ) which suggested a single gene insertion, but Southern analysis showed the presence of several DTA ts gene insertions ( Figure 2A ) which might indicate that there are tandem gene insertions. When RNA dot blots were probed with the DTA ts coding sequence, it became apparent that the level of expression was very variable between families ( Figure 2B ). The highest levels of RNA were found in plants from families Q2, Q3, Q4 and Q6. This supported a correlation between the gene copy number, as seen by Southern analysis ( Figure 2A ), and the strength of the sterility phenotype observed in the populations ( Table 1 ). This was consistent with the hypothesis that the expression of the DTA ts gene was the cause of the temperature-dependent male-sterility observed in A9::DTA ts -Q plants. However, the gene copy number in Q5 is inconsistent with this, because although the mRNA levels are high as in Q3, Q4 and Q6, the temperature dependent sterility is 30% for Q5 compared to 100% for the Q3, Q4 and Q6, which indicates that the Q5 mRNAs are not all translated. <h2>Cytological analysis of anthers from pA9::DTA ts transformant</h2> To assess the physiological effect of the DTA ts gene upon pollen development, flower buds were viewed by light and electron microscopy. Although no pollen grains were visible at the time of dehiscence on the surface of the A9::DTA ts -Q anthers grown at 18 °C ( Figure 1D ), some grains were observed using light microscopy when the anthers were crushed. Pollen grains from wild-type plants ( Figure 3A ) were different from those from sterile plants that appeared to be abnormally shaped ( Figure 3B ) and were sometimes aggregated in one mass inside the anther ( Figure 3C ). Thin transverse sections taken across flower buds from untransformed control-plants and A9::DTA ts -Q plants at the tetrad stage of development were observed by light microscopy ( Figure 4A,C ). Dramatic degeneration of the tapetal cells is associated with expression of the barnase gene ( Paul et al ., 1992 ), but DTA ts did not appear to produce such acute effects. However, the tapetal cells in male-sterile A9::DTA ts -Q plants ( Figure 4C ) were less intensely stained than tapetal cells from the control plants ( Figure 4A ), which indicated a change in cell composition. There was also a reduction in the number of vacuoles in A9::DTA ts -Q plants ( Figure 4C , arrows) compared with the control plants ( Figure 4A , arrows). Electron micrographs showed that there was a reduced ribosome density between the cells of the control ( Figure 4B ) and those expressing the DTA ts gene ( Figure 4D ) which could be the cause of the less intense staining seen in Figure 4C . In addition, dense material was visible between the pollen grains, by light and electron microscopy, which could be callose ( Figure 4E,F , arrows). Callose is normally digested by a mixture of enzymes (callase) secreted by the tapetal cells shortly after the completion of male cytokinesis. These results suggest that there is a reduction in the ability of the tapetal cells to either synthesize or secrete callase enzyme and this could indicate a general cell dysfunction. <h1>Discussion</h1> This is the first report of an engineered temperature-dependent phenotype in transgenic A. thaliana . The gene encoding the temperature-sensitive protein was expressed under the control of the A9 tapetum promoter and caused discrete alterations in tapetum development leading to pollen malformation and aggregation. As a result, transgenic plants were male-sterile at 18 °C but were fully male-fertile at 26 °C whilst being fully female-fertile at both temperatures. The activity of the A9 promoter was previously shown to be subject to tight temporal and spatial regulation. Studies of temporal expression with A9::Gus and A9::RNase in tobacco showed expression in the tapetum before sporogenous cell meiosis, increased dramatically reaching a plateau and then fell sharply and ceased in anthers with premitotic microspores ( Paul et al ., 1992 ). Transgenic A9 expression patterns closely match the temporal accumulation of B. napus A9 transcript determined by Scott et al . (1991 ). The A9 spatial expression was found to be tapetum-specific ( Paul et al ., 1992 ; Scott et al ., 1991 ) and was confirmed when the A9 promoter was used to drive the expression of the firefly luciferase reporter gene in A. thaliana . No luciferase was detected in pollen grains (A.-M. Sorensen, unpublished results) indicating that the promoter was inactive in male gametophytic cells. This suggests that the phenotypic alterations visible in the A9::DTA ts -Q pollen grains also resulted from tapetal dysfunction and not from the production of the toxin by the pollen grains. Since it has been established that the DTA acts in a cell autonomous manner when expressed in transgenic cells ( Evans, 1989 ), passive diffusion of the toxin from the tapetal cells towards the sporogenous cells can also be ruled out. Therefore, our evidence strongly suggests that temperature-sensitive male sterility resulted from the expression and action of the DTA ts within the tapetal cells and not via non-cell autonomous effects on adjacent cell types. Our results therefore indicate that the DTA ts gene is a potentially useful tool for addressing cell function in plants. Our data is consistent with previous reports that indicated that the DTA ts toxin was of very low activity compared to the wild-type DTA ( Bellen et al ., 1992 ; De Wet et al ., 1985 ). This low activity might account for the low frequency of completely male-sterile transgenic plants in those lines that contained the A9-DTA ts gene. This contrasts with the outcome of experiments where expression of the barnase gene or the wild-type DTA toxin gene under control of the tapetum-specific promoter TA29 resulted in a high number of male sterile plants and a complete collapse of the tapetum ( Koltunow et al ., 1990 ; Mariani et al ., 1990 ). This was also the case when the A9 promoter was used to drive the expression of the barnase gene ( Paul et al ., 1992 ). In the A9::DTA ts -Q plants, expression of the DTA ts resulted in discrete alterations in tapetal cells. The lighter Toluidine Blue staining of the cells expressing the DTA ts gene could be attributed to a lower ribosome density in the cytoplasm, as suggested by electron micrographs ( Figure 4D ). Such a phenotype was also found in D. melanogaster cells expressing the DTA ts gene ( Bellen et al ., 1992 ). A more dramatic phenotype was visible in the A9::DTA ts pollen grains which appeared to aggregate to a greater or lesser extent in the anthers ( Figure 3C,B , respectively). This was confirmed by the observation of electron-dense material between the pollen grains ( Figure 4E , see arrow). A very similar type of agglutination was described in the A. thaliana ms1 mutant ( Dawson et al ., 1993 ). A hypothesis for the origin of this material is the incomplete dissolution of the callose wall by the tapetal ॆ-1,3-glucanase as a result of tapetal dysfunction. It has been shown that the timing of the production of this catalytic activity is critical for fertility ( Worrall et al ., 1992 ). This aggregation might explain why pollen grains could not be seen on the surface of the A9::DTA ts anthers after dehiscence ( Figure 1D ). Therefore the DTA ts protein appears to have a chronic rather than an acute effect on anther development and/or function. Due to the postulated low activity of the temperature sensitive version of the toxin, a high level of expression is probably required for sterility. This was illustrated by several transgenic lines including A9::DTA ts -Q7 ( Figure 2 ), which contained several copies of the T-DNA but was nevertheless male fertile ( Table 1 ). The poor recovery of transgenic lines capable of complete male-sterility at 18 °C may rest with the technique used to produce the transgenic plants. The Generation-2 seeds were harvested from the primary transformants grown in vitro . Since regeneration and seed set are impeded at 26 °C (the non-permissive temperature) the primary transformants were grown at 20 °C. It is possible that some transformants failed to produce seeds because of the activity of the toxin. Thus, the selection could have been biased against the recovery of transgenic lines, giving high expression of the DTA ts gene when hemizygous in Generation-1 and biased towards lines expressing the gene sufficiently highly to cause sterility when homozygous in Generation-2. For future applications, the problem of recovering Generation-2 plants at 26 °C could be easily addressed by using one of the A. thaliana transformation methods that avoid in vitro regeneration ( Chang et al ., 1994 ). A9::DTA ts -Q has been used in a mutagenesis screen for the isolation of parthenogenetic mutants which involves pollinating large numbers of M1 plants with wild-type pollen; controllable male sterility greatly facilitates this process. These mutants can be switched to permissive temperatures to allow self-pollination and use as males in out-crosses. This mutagenesis screen is further discussed in Praekelt and Scott (2001 ). In conclusion, it has been shown that a DTA ts gene induces a temperature-dependent loss of cell function in transgenic A. thaliana . Since sterility is controllable by applying permissive and non-permissive temperatures, the system is useful in a range of experiments. The transgenic line A9::DTA ts -Q could have applications which include the isolation of regulatory mutants following mutagenesis, the analysis of gene expression patterns or cell fate, or screening of parthenogenetic and apomictic mutants capable of producing seed embryos in the absence of pollination. <h1>Experimental procedures</h1> <h2>Plant material and reagents</h2> Restriction endonucleases were purchased from Gibco-BRL and Promega, T4 ligase from Gibco-BRL and Taq polymerase (with buffer) from Promega. Antibiotics, media and growth regulators for plant tissue culture were obtained from Sigma. Compounds for cytology were purchased from Agar Scientific Ltd. <h2>Construction of pA9::DTA ts</h2> As a first step to constructing pA9::DTA ts , the 850 bp DTA ts gene was isolated from pLAT59-DTM ( Worrall et al ., 1996 ), using Nco I and Eco RI and cloned into the Nco I and Sma I sites of pAF0, creating pAF78. pAF0 was a pUC-derived plasmid carrying the firefly luciferase gene ( De Wet et al ., 1985 ) inserted between 1 k bp of A9 promoter sequence ( Paul et al ., 1992 ) and the 750 bp CaMV polyadenylation sequence of pJIT60 ( Guerineau et al ., 1992 ). The Nco I- Sma I treatment of pAF0 removed the luciferase gene, which was replaced by the DTA ts gene in pAF78. To construct the binary vector (pA9::DTA ts ) the A9 promoter–DTA ts –polyadenylation sequence (2.6 k bp) was extracted from pAF78 using Hin dIII and Xho I, and inserted into the pBIN19 ( Bevan, 1984 ) site created by digestion with Hin dIII and Sal I. In pA9::DTA ts the A9 promoter was such that the transcription of the DTA ts gene was towards the T-DNA left border. <h2>Bacterial and plant transformation experiments</h2> E. coli competent cells (strain JM101) were prepared and transformed as described by Nishimura et al . (1990 ): a 50 mL culture inoculated with 0.5 mL of overnight culture was grown in medium A (LB supplemented with 10 m m MgSO 4 .7H 2 O and 0.2% (w/v) glucose) to mid-logarithmic phase. The cells were kept on ice for 10 min, then pelleted at 1500 g for 10 min at 4 °C. The cells were resuspended gently in 0.5 mL of medium A pre-cooled on ice, then 2.5 mL of storage solution B (36% (v/v) glycerin, 12% (w/v) PEG (M W 7500), 12 m m MgSO 4 .7H 2 O added to LB broth (pH 7.0) and sterilized by filtration) was added, and mixed without vortexing. The competent cells were divided in aliquots of 0.1 mL each and stored at −80 °C. For transformation, the frozen cells were thawed on ice, mixed immediately with 5 µL (100 pg) of plasmid, and incubated at 4 °C for 30 min. The cells were then subjected to a heat pulse at 42 °C for 60 s, chilled on ice for 1 min, diluted 10-fold into pre-warmed L broth, and incubated at 37 °C for 1 h. Samples (10 µL and 50 µL) were plated on agar plates containing 50 µg/mL antibiotic (sodium salt of ampicillin for pAF0 and pAF78). A. tumefaciens strain LBA4404 ( Hoekema et al ., 1983 ) was transformed in an electroporation cuvette (Bio-Rad) across which 2.5 kV was discharged using an electroporator power unit (Bio-Rad, capacity 25 µF, resistance 200 ष). A. thaliana (ecotype C24) root transformation experiments were conducted as described by Clarke et al . (1992 ) either at 20 °C (Transformation Experiment 1) or 26 °C (Transformation Experiment 2). Generation-2 seeds were harvested from the in vitro grown primary transformants and sown on soil. Plants were grown under continuous light at 18 °C or 26 °C. For the segregation analysis, Generation-3 seeds were sterilized (10% v/v bleach, 10 min) and placed on germination medium ( Clarke et al ., 1992 ) containing kanamycin at 35 µg/mL and grown in a growth chamber prior to segregation analysis. A separate batch of seed was grown in soil in a growth chamber at 18 °C to assess the ratio of sterile to fertile plants. <h2>Nucleic acid preparation and analysis</h2> Plant DNA mini-preparations for analysis by PCR were done as described by Edwards et al . (1991 ). Centrifugation was performed at 16 060 g in a bench-top centrifuge unless otherwise stated. Leaf material was collected using the lid of a sterile Eppendorf tube to pinch out a disc of material into the tube. The tissue was macerated in the tube with a disposable grinder (Bel-Art Products) without buffer for 15 s. A 400 µL aliquot of extraction buffer (200 m m Tris(Tris[hydroxymethyl]aminomethane)-HCl pH 7.5, 250 m m NaCl, 25 m m EDTA (ethylenediaminetetraacetic acid), 0.5% (w/v) sodium dodecyl sulphate (SDS) was added and the tube vortexed for 5 s and centrifuged for 1 min. 300 µL of the supernatant was transferred to a fresh tube and precipitated with 300 µL isopropanol and left at room temperature for 2 min. Following centrifugation for 5 min, the pellet was air dried and dissolved in 100 µL TE buffer (10 m m Tris-HCl pH 8.0, 1 m m EDTA). PCR was carried out as described previously ( Guerineau and Waugh, 1993 ) in a Perkin-Elmer thermo-cycler 480. The final volume of 50 µL buffer contained 0.2 m m dNTPs, 1 ng of each primer, 10–300 ng plant DNA and 2.5 units of Taq polymerase. Thirty-five cycles were performed at 96 °C for 40 s, 60 °C for 1 min, 72 °C for 1 min, with extension at 72 °C for 6 min in the last cycle. The primers used were 5′-CTTCGTACCACGGGACTAAACC-3′ and 5′-CCTGACACGATTTCCTGCACAG-3′, which amplified a 524-bp region. For Southern analysis, DNA was extracted from at least eight Generation-3 plants from each of the 11 A9::DTA ts -Q families, and from C24 wild-type plants as a control. DNA was extracted using a protocol derived from Dellaporta et al . (1983 ): 100 mg of leaf tissue were ground in 300 µL buffer (100 m m Tris-HCl pH 8.0, 50 m m EDTA, 500 m m NaCl, 0.5% (w/v) diethyldithiocarbamate), 40 µL 10% (w/v) SDS was added and then incubated at 65 °C for 10 min 100 µL 5 m KC 2 H 3 O 2 was added and then incubated on ice for 30 min. Following centrifugation for 10 min, 400 µL of the supernatant was added to 240 µL isopropanol, cooled at −20 °C for 30 min then centrifuged for 10 min. The pellet was re-suspended in 200 µL of TE buffer and centrifuged for 5 min to eliminate the insoluble material. 190 µL of the supernatant was added to 12 µL 5 m NaCl and 410 µL ethanol, cooled on ice for 5 min and centrifuged for 5 min. The pellet was washed with 70% (v/v) ethanol and dissolved in 100 µL TE buffer and purified as follows: one volume (100 µL) of a solution containing 50 m m Tris-HCl pH 8.0, 10 m m EDTA, 2% (w/v) cetyldimethylammonium bromide (CTAB), 2 m NaCl were added to the DNA solution. The mixture was incubated at 65 °C for 10 min. 200 µL of 50 m m Tris-HCl pH 8.0, 10 m m EDTA, 1% (w/v) CTAB were added to the previous DNA/CTAB solution and the mixture was left at room temperature for 10 min, before being centrifuged in a microfuge for 2 min. The CTAB-DNA pellet was re-dissolved in 400 µL of TE buffer containing 1 m NaCl. A chloroform/isoamyl alcohol (24 : 1, v/v) extraction was performed and to the upper phase obtained after centrifugation two volumes of ethanol were added for 3 min. The DNA was cooled on ice for 5 min, and the precipitant was collected by centrifugation for 5 min, and dissolved in 100 µL of TE buffer. 5 µg DNA was digested with Hin dIII and electrophoresed through a 0.8% (v/v) agarose gel. For Southern analysis the gel was blotted onto a Hybond-N blotting membrane (Amersham) as described by Maniatis et al . (1982 ). Pre-hybridization was carried out for 3 h at 65 °C in 5 × SSC (1 × SSC is 15 m m sodium citrate, 150 m m NaCl), 25 m m sodium phosphate buffer pH 6.5, 0.1% (w/v) SDS, 5 m m EDTA, 5 × Denhardt's solution (1 × Denhardt's solution is 0.02% (v/v) Ficoll, 0.02% (v/v) PVP, 0.02% (v/v) (BSA), 100 µg/mL denatured calf thymus DNA). The DNA probe was labelled with [ 32 P] dCTP using the random oligonucleotide priming method ( Feinberg and Volgelstein, 1983 ) to a specific activity higher than 10 9 c.p.m./µg. Hybridization was carried out overnight at 65 °C in the pre-hybridization solution. The membrane was washed in 2 × SSC, 0.1% (w/v) SDS for 30 min at room temperature, then in 2 × SSC, 0.1% (w/v) SDS for 30 min at 65 °C, then twice in 0.1 × SSC, 0.1% (w/v) SDS, for 30 min at 65 °C. The membrane was exposed to pre-flashed X-ray film for 3 days. RNA was extracted from flower buds taken from at least eight Generation-3 plants from each of the 11 A9::DTA ts -Q families. RNA was prepared ( Guerineau et al ., 1991 ) by grinding leaf samples in TLES buffer (50 m m Tris-HCl pH 9.0, 150 m m LiCl, 5 m m EDTA, 5% (w/v); SDS). The lysate was twice treated with phenol/chloroform (1 : 1 v/v) followed by centrifugation for 1 min. The supernatant was removed and total nucleic acids were precipitated with two volumes of ethanol. After 5 min centrifugation the pellet was dissolved in 50 µL of sterile distilled water. One volume of 4 m LiCl was added and RNA was precipitated for 30 min on ice. After centrifugation for 5 min, the pellet was washed with 70% (v/v) ethanol, air dried and re-dissolved in 20 µL of sterile distilled water. 20 µg of each RNA sample was dotted onto Hybond-N membrane as recommended by the manufacturer. <h2>Light and electron microscopy</h2> For observation by light microscopy, flower buds were fixed in glutaraldehyde, dehydrated in an ethanol series and embedded in araldite. Sections of 0.2–0.5 µm were stained with 0.5% (w/v) Toluidine Blue. Electron microscopy samples were fixed using glutaraldehyde and osmium tetroxide 2% (w/v), dehydrated in ethanol and embedded in Spurr resin ( Spurr, 1969 ). Sections were stained with uranyl acetate 2% (w/v) in 50% (v/v) ethanol and 80 m m lead citrate in 120 m m sodium citrate (pH 12 with NaOH). Observations were carried out on a JEOL (Tokyo, Japan) 100CX transmission electron microscope operating at 80 kV.
Plant Biotechnology Journal – Wiley
Published: Jan 1, 2003
Keywords: anther; conditional; diphtheria toxin; male-sterility; pollen; tapetum
You can share this free article with as many people as you like with the url below! We hope you enjoy this feature!
Read and print from thousands of top scholarly journals.
Already have an account? Log in
Bookmark this article. You can see your Bookmarks on your DeepDyve Library.
To save an article, log in first, or sign up for a DeepDyve account if you don’t already have one.
Copy and paste the desired citation format or use the link below to download a file formatted for EndNote
Access the full text.
Sign up today, get DeepDyve free for 14 days.
All DeepDyve websites use cookies to improve your online experience. They were placed on your computer when you launched this website. You can change your cookie settings through your browser.