- Email: [email protected]
Future of early embryogenesis studies in Arabidopsis thaliana
C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573 © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés S0764446901013270/REV
Revue / Review
Future of early embryogenesis studies in Arabidopsis thaliana Patrick Gallois* Laboratoire génome et développement des plantes, CNRS UMR 5096, 52, avenue de Villeneuve, 66860 Perpignan cedex, France Received 23 October 2000; accepted 4 December 2000 Communicated by Christian Dumas
Abstract – Embryogenesis is a long-standing field of interest for plant scientist as recorded in the ‘notes’ of the French Science Academy. This either with fundamental or applied points of view. Since the beginning of the century techniques and questions have co-evolved, from microscope and fate map to laser ablation and cell–cell signalling. So far in plant embryogenesis, a limited use has been made of the whole range of approaches generally available to study development. This is due to technical limitations when working with plant embryos. Novel mutant screens and techniques are now at hand and are expected to unravel further the nature of cell interactions underlying embryo development. This in turn will modify the focus of our questioning. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS plant embryogenesis / cell ablations / gain-of-function screen
Résumé – Le futur de l’embryogenèse précoce chez Arabidopsis thaliana. Les notes des séances de l’Académie des sciences témoignent de l’intérêt constant porté par les scientifiques au développement de l’embryon végétal, que ce soit pour des raisons fondamentales ou appliquées. Depuis le début du siècle, les questions posées ont co-évoluées avec les outils ou les techniques disponibles : du microscope et du devenir des cellules issues du zygote, au laser et à la communication intercellulaire. Jusqu’à présent l’étude de l’embryogenèse végétale a fait un usage limité de toutes les techniques utilisées par ailleurs dans l’étude du développement. La raison est à chercher dans les limitations techniques propres aux végétaux. Néanmoins, ces barrières commencent à être levées, ce qui devrait inéluctablement faire progresser notre compréhension des interactions impliquées dans le développement de l’embryon. Ces nouveaux outils vont à leur tour faire évoluer la nature des questions posées. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS embryogenèse végétale / ablation cellulaire / mutations gain-de-fonction
. Version abrégée Le 7 août 1916, Monsieur Souèges présenta à la noble assemblée de l’Académie des sciences son travail sur l’origine des cellules du suspenseur au cours de l’embryogenèse de Capsella Bursa-pastoris [1]. Cette espèce était alors un modèle de choix pour la simplicité de son embryon et la facilité d’observation de celui-ci. Il s’agissait d’établir si la cellule basale issue de la
première division du zygote était ou non capable de division, et si ses descendantes contribuaient de quelque manière à la structure finale de l’embryon. Monsieur Souèges décrivit pour la première fois la succession des divisions initiales du zygote, ce qui établit la généalogie des cellules du suspenseur (figure 1). Pour lui, ces observations présentaient un intérêt tout particulier en embryogenèse comparative pour la classification des Brassicacées (crucifères) [2]. De nos
*Correspondence and reprints. E-mail address: [email protected] (P. Gallois).
569
P. Gallois / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573
jours, la généalogie des cellules de l’embryon est associée avec d’autres centres d’intérêt liés aux événements moléculaires qui régissent la mise en place d’un organisme multicellulaire à partir de la cellule unique qui forme le zygote. Arabidopsis thaliana se trouve être une espèce apparentée à C. Bursa-pastoris et le développement embryonnaire est très similaire entre les 2 espèces (figure 2), ce qui rend les observations de Monsieur Souèges directement transposables. A. thaliana se montre cependant un modèle alternatif nécessaire en raison de son génome plus simple et de la génétique qui lui est associée. Contrastant avec l’étude de l’embryogenèse, l’analyse du développement de la fleur fait appel à des approches beaucoup plus nombreuses : cribles pour des mutations récessives ou dominantes [14–17], expression ectopique de gènes [18, 19], cribles pour des mutants suppresseurs [20]. Ces approches multiples font donc défaut. Pourtant un crible pour des mutants gain-defonction se justifierait pleinement en raison de la redondance fonctionnelle (ou génétique) qui semble exister pour les gènes régulateurs du développement [12, 13]. Pour sa mise en place au cours de l’embryogenèse, cette stratégie requiert des systèmes
efficaces et disponibles d’activation génique [22, 23]. Ces mêmes systèmes vont permettre également l’expression de mutants dominants négatifs : une approche très fructueuse dans le domaine animal qui commence à peine à porter ses fruits dans l’étude du végétal [24].
1. Introduction
embryonic structure. At the end of his presentation Dr Souèges concluded that his observations defined the role of the basal cell in the making of embryo in C. bursapastoris. A more extensive account of his data was published later [2]. To him his study was a basis for comparative embryogenesis, the focus of the time. Additionally Dr. Souèges spent some time monitoring variations of the number and origin of cell divisions in early embryos, depending of the individual observed.
On the 7th of August 1916, in a richly decorated room of the French Science Academy Dr. R. Souèges presented his work on the development of the suspensor cells during plant embryogenesis [1]. The question was then to solve the origin of the hypophysis, the apical cell of the suspensor. Two schools of thought existed. One held that the basal cell of the first division of the zygote never divides again and that the whole embryo structure originates from the apical cell. The other proposed that the basal cell divides and that its derivatives end up forming the suspensor and the hypophysis. A model plant of choice was then Capsella Bursa-pastoris for the simple structure of its embryo and the ease of observation. Careful microscopic examination allowed Dr. Soueges to establish ‘step by step’ the successive cell divisions starting from fecondation. His observations were consigned in drawings of embryos at stages never reported before. His finding was recapitulated in figures such as figure 1, where the fate map of the suspensor cells has been indicated. It showed that during embryogenesis in C. bursa-pastoris the first zygotic division gives rise to two cells. The apical cell will contribute to most of the embryo structure, the embryoproper. The basal cell develops in a file of 7 to 9 cells called the suspensor. All but one of these cells will degenerate at the end of early embryogenesis. The surviving cell will contribute to some domains of the root apex. In conclusion, his work established that the basal cell was capable of dividing and contributed to some of the final
570
Dans notre laboratoire, nous avons montré qu’un système d’activation génique permet le développement de stratégies d’ablation cellulaire qui éliminent ou perturbent des structures, révélant ainsi des interactions insoupçonnées entre domaines cellulaires. C’est ainsi que l’expression du gène toxique barnase dans le protoderme de l’embryon altère la structure du pôle basal, à priori en modifiant l’équilibre des signaux intercellulaires (figure 3; Baroux et al., soumis). L’utilisation d’ablation cellulaire grâce au laser devrait compléter la panoplie des stratégies offertes. Il est permis d’imaginer qu’en combinant l’ablation laser avec la microscopie confocale et un système de fécondation in vitro, comme celui développé avec le maïs [26], l’étude de l’embryogenèse pourrait entrer dans une nouvelle ère qui abordera des questions que nul n’a posé jusqu’à présent.
2. Embryogenesis in Arabidopsis The same observations at the descriptive level have been made using Arabidopsis thaliana but new tools and techniques brought novel questions. The molecular mechanism underlying the shaping of the embryo is now the centre of attention. A. thaliana happens to be a relative of C. Bursa-pastoris and the development of its embryo is very similar. However A. thaliana, a diploid unlike C. bursa-pastoris which is tetraploid, is today a model of choice in development because of its genetics. The question of how a multicellular organism constructs itself starting from a single zygote cell is not new. In plants the answers lay in the study of early embryogenesis. This term covers the stages from the initial fertilised cell to the heart stage embryo (figure 2). At the end of early embryogenesis the basic structure of the plant is achieved. Molecules important for this process are being searched for, using a whole array of approaches including biochemistry, genetics, molecular biology and genetic engineering. Dr.
P. Gallois / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573
Figure 2. Schematic fate map of embryo development in A. thaliana. The apical cell will contribute to most of the embryo structure, the embryo-proper. The basal cell develops in a file of 8 cells called the suspensor and contributes to part of the root apex. Grey area corresponds to the suspensor.
Figure 1. Capsella bursa-pastoris Moench. Schematic representation of suspensor development starting from the bottom two basal cells of a 4-cell-stage embryo. In this particular case the basal cell of the pro-embryo is dividing only twice (translated from Souèges [2]).
Souèges would have been probably delighted to see how this changed the nature of the questions asked about the suspensor. At his time the suspensor had been confined to a structural role in plant embryogenesis. Two structural roles had been proposed. One is being a physical support and anchorage device for the developing embryo in the seed. The second one is to carry out an embryo feeding role in which the suspensor cells would transport nutrients from the mother plant to the embryo as well as being themselves metabolically active for protein, auxin and gibberellin synthesis [3]. More recently mutants have been isolated that are affected in suspensor development (reviewed in [4]) and are divided in 2 classes: susp and twin. The model based on the observed phenotypes of these two classes is that the embryo-proper produces signals responsible for (i) maintaining suspensor cell identity and (ii) inducing suspensor cell death. Additionally in wild type, embryos form their axis with their basal pole towards suspensor cells. In the twin mutants, suspensor cells can form secondary embryos with an axis of polarity in the same or reverse orientation to that of the primary embryo [5]. One possibility is that secondary embryos can form the apical–basal axis at random in either orientation because of the presence of suspensor cells on two opposite sides. This would imply the existence of a second signalling pathway from suspensor to embryo-proper. Cloning of genes corresponding to a susp and a twin mutant has not been informative since the sequences encode housekeeping proteins indirectly involved in abnormal embryo development [6, 7]. The nature of the signalling molecules produced either by the embryoproper or the suspensor remains totally unknown although the plant hormone auxin is a candidate [reviewed in 8].
Revisiting the suspensor function will require molecular markers of its identity, and identification of putative signal molecules. This very focussed example epitomises what mutants have brought to the field of plant embryogenesis and what they unexpectedly failed to do. Mutants are instrumental to study the processes of organised cell divisions and orientated cell expansion [9] and interesting data are gathered by crossing signalling mutants and mutants presenting altered pattern formation [10, 11]. However many mutants with altered early embryogenesis resulted in the cloning of genes not directly involved in the decision making. Noticeably only loss-of-function screens have been carried out so far in embryogenesis. This makes mutant screens for embryogenesis hostage of a possible lethality induced by the mutation of key genes and hostage of genetic/functional redundancy that may mask the role of particular regulatory genes. For the latter, known examples are the two CUC genes that have to be both mutated to give the phenotype of fused cotyledons [12] or the three SEPALLATA genes that give a phenotype in flower once all mutated [13]. This redundancy calls for gain-of-function screen. Unlike what has been done in embryogenesis, it is striking to note that in the study of flower development several mutant approaches have already been used: screen for recessive or dominant mutations [14–17], targeted misexpression of genes [e.g. 18, 19], suppressor screen [20]. In particular gain-of-function screens are already being carried out for some aspects of plant development and recent publications have demonstrated the power of the approach [16]. That is not as easy to carry out during embryogenesis. This because dominant mutations affecting dramatically the structure of the embryo will be lost since the original mutated individual is unlikely to survive and give a progeny. To remove this stumbling block, technological development is necessary, allowing conditional misexpression of genes. A striking example of the potential of such an approach in embryogenesis is the misexpression of the gene LEC-1 resulting in embryonic structures developing on leaves albeit at low frequency
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P. Gallois / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573
[21]. Activation systems that could be used to misexpress gene in embryo have been developed [22, 23] and hope fully novel screen will be soon available that will give us access to new genes implicated in embryo development. The basic idea is that the potentially deleterious gene is not expressed in the mother plant until its promoter is activated in the progeny by the experimentator either by crossing with an activating parent line or using specific chemical compound. A promoter, that can be activated, is to be inserted at random in the genome of A. thaliana creating a collection of plant containing silent insertions. Upon activation in the embryo of the next generation some of the insertions will lead to misexpression of a nearby coding sequence, either in time or space. This will create dominant mutation that can be screened for. On a similar note, dominant negative forms of regulatory genes will be expressed in embryo in the future. Currently one limiting factor is that their effect might lead to counterselection of the transgene in parental lines. In that case no transgenic line can be recovered and analysed. The study of Hemerly et al. [24] exemplifies this powerful but under-used approach by introducing in plants a ‘dead’ form of a cell cycle regulatory gene (CDC2a). The mutated protein interferes with the endogenous wild type protein thus reducing the frequency of cell division in embryo development. This affected the apico-basal pattern but cell differentiation was not affected. Generalising this type of approach in embryogenesis would be made easy by the use of a two-component activation system as described above. Another approach that has not been yet applied to embryos is cell ablation [reviewed in 25]. A laser beam can be used to destroy temporarily specific cells in the embryo. Modification of subsequent development would give indications on possible signals emitted by the destroyed cell and on the determination/totipotency of adjacent cells. The technical hurdle here is to keep the embryo alive in such a way that development would continue after dissecting out the seed. Alternatively genetic ablation can be carried out where a toxin is expressed under a specific promoter so to remove a given embryonic structure by killing the corresponding cells. Using constructs in which the toxin gene is linked to a tissue specific promoter is very limiting since promoters have to be tissue and embryo specific so that a whole transgenic plant can be regenerated and the deleterious effect of the transgene observed in the progeny only. A conditional expression system allowing the toxin to accumulate in the progeny of two healthy parents is therefore a very valuable tool. New experiments in our laboratory have indicated that informative phenotypes can be obtained using the Barnase gene as a toxin and a two-component activation system. And this even with promoters that carry on to be expressed during vegetative development. Using for example the Ltp2promoter, that drives expression in the embryo protoder-
572
Figure 3. Effect of Barnase expression under the LTP2 promoter. A two-component activation system has been used to express Barnase in the protodermis cells of the embryo. A. wild type embryo; B. Barnase expressing embryo with an enlarged basal pole (arrow).
mis, to activate the Barnase gene, induced a specific defect during embryogenesis that resulted into an enlarged basal pole (figure 3) (Baroux et al., submitted). Since mutations altering specifically the epidermis don’t exist, this type of experiment is filling a gap in the possible experimental designs used by plant developmental biologist. We expect that genetics and later laser ablation will provide invaluable information on cell–cell communication between specific tissues during the generation of the plant embryo. Additional tools would be in vitro fertilisation techniques followed by zygote development such as those designed in maize [26]. Confocal techniques are as well being developed and one could dream of days to come when A. thaliana embryos would survive and take shape under laser scrutiny during the 5 days of early embryogenesis.
3. Conclusion Our current understanding of the regulation of plant embryogenesis at molecular level is still in its infancy. However the picture is evolving rapidly and the trend is likely to accelerate as many of the approaches described here are underway. Sadly applying global transcript analysis and proteomics to A. thaliana embryos seems out of reach at present since it would require a miniaturisation of the technique that is not yet available. In the mean time other model species might fill the gap. Only eighty years after his seminar at the Academy of Sciences, Dr. Soueges would hardly recognise the content and focus of today’s textbook dealing with plant development. It is easy to predict that the same feeling will hit the current generation of plant scientist well before the middle of the 21rst Century. That is what keeps plant embryogenesis exciting.
P. Gallois / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573
References
[15] Kobayashi Y., Kaya H., Goto K., Iwabuchi M., Araki T., A pair of related genes with antagonistic roles in mediating flowering signals, Science 286 (1999) 1960–1962.
[1] Souèges R., Les premiers cloisonnements de l’œuf et l’origine de l’hypophyse chez le Capsella Bursa-Pastoris Moench, C. R. Acad. Sci. Paris CLXIII (1916) 158–160. [2] Souèges R., Les premières divisions de l’œuf et les differenciations du suspenseur chez le Capsella Bursa-Pastoris Moench, Ann. Sci. Bot. I (1919) 1–28. [3] Brady T., Walthall E.D., The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein content, Dev. Biol 107 (1985) 531–536. [4] Yeung E.C., Meinke D., Embryogenesis in Angiosperms: Development of the suspensor, Plant Cell 10 (1993) 1371–1381. [5] Vernon D.M., Meinke D.W., Embryonic transformation of the suspensor in twin, a polyembryonic mutant in Arabidopsis, Dev. Biol 165 (1994) 566–573. [6] Schwartz B.W., Yeung E.C., Meinke D.W., Disruption of morphogenesis and transformation of the suspensor in abnormal suspensor mutants of Arabidopsis, Development 120 (1994) 3235–3245. [7] Zhang J.Z., Somerville C.R., Suspensor-derived polyembryony caused by altered expression of valyl-tRNA synthetase in the twin2 mutant of Arabidopsis, Proc. Natl. Sci. Acad. USA 9 (1997) 7349–7355. [8] Souter M., Lindsey K., Polarity and signalling in plant embryogenesis, J. Exp. Bot. 347 (2000) 971–983. [9] Mayer U., Jürgens G., Pattern formation in plant embryogenesis: a reassessment, Sem. Cell Dev. Biol. 9 (1998) 187–193. [10] Vroemen C.W., de Vries S.C., Quatrano R., Signalling in plant embryos during the establishment of the polar axis, Sem. Cell Dev. Biol. 10 (1999) 157–164. [11] Hamann T., Mayer U., Jürgens G., The auxin-insensitive bodenlos mutation affects primary root formation and apical basal patterning in the Arabidopsis embryo, Development 126 (1999) 1387–1395. [12] Aida M., Ishida T., Fukaki H., Fujisawa H., Tasaka M.,Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant, Plant Cell. 9 (1997) 841–857. [13] Pelaz S., Ditta G.S., Baumann E., Wisman E., Yanofsky M.F., B and C floral organ identity functions require SEPALLATA MADS-box genes, Nature 11 (2000) 200–203. [14] Koornneef M., Hanhart C.J., van der Veen J.H., A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana, Mol. Gen. Genet. 229 (1991) 57–66.
[16] Weigel D., Ahn J.H., Blazquez M.A., Borevitz J.O., Christensen S.K., Fankhauser C., Ferrandiz C., Kardailsky I., Malancharuvil E.J., Neff M.M., Nguyen J.T., Sato S., Wang Z.Y., Xia Y., Dixon R.A., Harrison M.J., Lamb C.J., Yanofsky M.F., Chory J., Activation tagging in Arabidopsis, Plant Physiol. 122 (2000) 1003–1013. [17] Kardailsky I., Shukla V.K., Ahn J.H., Dagenais N., Christensen S.K., Nguyen J.T., Chory J., Harrison M.J., Weigel D., Activation tagging of the floral inducer FT, Science 286 (1999) 1962–1965. [18] Goodrich J., Puangsomlee P., Martin M., Long D., Meyerowitz E.M., Coupland G., A Polycomb-group gene regulates homeotic gene expression in Arabidopsis, Nature 386 (1997) 44–51. [19] Parcy F., Nilsson O., Busch M.A., Lee I., Weigel D., A genetic framework for floral patterning, Nature 395 (1998) 561–566. [20] Onouchi H., Igeno M.I., Perilleux C., Graves K., Coupland G., Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes, Plant Cell 12 (2000) 885–900. [21] Lotan T., Ohto M., Yee K.M., West M.A., Lo R., Kwong R.W., Yamagishi K., Fischer R.L., Goldberg R.B., Harada J.J., Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells, Cell 26 (1998) 1195–1205. [22] McNellis T.W., Mudgett M.B., Li K., Aoyama T., Horvath D., Chua N.H., Staskawicz B.J., Glucocorticoid-inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death, Plant J. 14 (1998) 247–257. [23] Moore I., Galweiler L., Grosskopf D., Schell J., Palme K., A transcription activation system for regulated gene expression in transgenic plants, Proc. Natl. Acad. Sci. USA 95 (1998) 376–381. [24] Hemerly A.S., Ferreira P.C., Van Montagu M., Engler G., Inze D., Cell division events are essential for embryo patterning and morphogenesis: studies on dominant-negative cdc2aAt mutants of Arabidopsis, Plant J. 23 (2000) 123–130. [25] Berger F., Cell ablation studies in plant development, Cell. Mol. Biol. 44 (1998) 711–719. [26] Antoine A.F., Faure J., Cordeiro S., Dumas C., Rougier M., Feijo J.A., A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants, Proc. Natl. Acad. Sci. USA 97 (2000) 10643–10648.
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Revue / Review
Future of early embryogenesis studies in Arabidopsis thaliana Patrick Gallois* Laboratoire génome et développement des plantes, CNRS UMR 5096, 52, avenue de Villeneuve, 66860 Perpignan cedex, France Received 23 October 2000; accepted 4 December 2000 Communicated by Christian Dumas
Abstract – Embryogenesis is a long-standing field of interest for plant scientist as recorded in the ‘notes’ of the French Science Academy. This either with fundamental or applied points of view. Since the beginning of the century techniques and questions have co-evolved, from microscope and fate map to laser ablation and cell–cell signalling. So far in plant embryogenesis, a limited use has been made of the whole range of approaches generally available to study development. This is due to technical limitations when working with plant embryos. Novel mutant screens and techniques are now at hand and are expected to unravel further the nature of cell interactions underlying embryo development. This in turn will modify the focus of our questioning. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS plant embryogenesis / cell ablations / gain-of-function screen
Résumé – Le futur de l’embryogenèse précoce chez Arabidopsis thaliana. Les notes des séances de l’Académie des sciences témoignent de l’intérêt constant porté par les scientifiques au développement de l’embryon végétal, que ce soit pour des raisons fondamentales ou appliquées. Depuis le début du siècle, les questions posées ont co-évoluées avec les outils ou les techniques disponibles : du microscope et du devenir des cellules issues du zygote, au laser et à la communication intercellulaire. Jusqu’à présent l’étude de l’embryogenèse végétale a fait un usage limité de toutes les techniques utilisées par ailleurs dans l’étude du développement. La raison est à chercher dans les limitations techniques propres aux végétaux. Néanmoins, ces barrières commencent à être levées, ce qui devrait inéluctablement faire progresser notre compréhension des interactions impliquées dans le développement de l’embryon. Ces nouveaux outils vont à leur tour faire évoluer la nature des questions posées. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS embryogenèse végétale / ablation cellulaire / mutations gain-de-fonction
. Version abrégée Le 7 août 1916, Monsieur Souèges présenta à la noble assemblée de l’Académie des sciences son travail sur l’origine des cellules du suspenseur au cours de l’embryogenèse de Capsella Bursa-pastoris [1]. Cette espèce était alors un modèle de choix pour la simplicité de son embryon et la facilité d’observation de celui-ci. Il s’agissait d’établir si la cellule basale issue de la
première division du zygote était ou non capable de division, et si ses descendantes contribuaient de quelque manière à la structure finale de l’embryon. Monsieur Souèges décrivit pour la première fois la succession des divisions initiales du zygote, ce qui établit la généalogie des cellules du suspenseur (figure 1). Pour lui, ces observations présentaient un intérêt tout particulier en embryogenèse comparative pour la classification des Brassicacées (crucifères) [2]. De nos
*Correspondence and reprints. E-mail address: [email protected] (P. Gallois).
569
P. Gallois / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573
jours, la généalogie des cellules de l’embryon est associée avec d’autres centres d’intérêt liés aux événements moléculaires qui régissent la mise en place d’un organisme multicellulaire à partir de la cellule unique qui forme le zygote. Arabidopsis thaliana se trouve être une espèce apparentée à C. Bursa-pastoris et le développement embryonnaire est très similaire entre les 2 espèces (figure 2), ce qui rend les observations de Monsieur Souèges directement transposables. A. thaliana se montre cependant un modèle alternatif nécessaire en raison de son génome plus simple et de la génétique qui lui est associée. Contrastant avec l’étude de l’embryogenèse, l’analyse du développement de la fleur fait appel à des approches beaucoup plus nombreuses : cribles pour des mutations récessives ou dominantes [14–17], expression ectopique de gènes [18, 19], cribles pour des mutants suppresseurs [20]. Ces approches multiples font donc défaut. Pourtant un crible pour des mutants gain-defonction se justifierait pleinement en raison de la redondance fonctionnelle (ou génétique) qui semble exister pour les gènes régulateurs du développement [12, 13]. Pour sa mise en place au cours de l’embryogenèse, cette stratégie requiert des systèmes
efficaces et disponibles d’activation génique [22, 23]. Ces mêmes systèmes vont permettre également l’expression de mutants dominants négatifs : une approche très fructueuse dans le domaine animal qui commence à peine à porter ses fruits dans l’étude du végétal [24].
1. Introduction
embryonic structure. At the end of his presentation Dr Souèges concluded that his observations defined the role of the basal cell in the making of embryo in C. bursapastoris. A more extensive account of his data was published later [2]. To him his study was a basis for comparative embryogenesis, the focus of the time. Additionally Dr. Souèges spent some time monitoring variations of the number and origin of cell divisions in early embryos, depending of the individual observed.
On the 7th of August 1916, in a richly decorated room of the French Science Academy Dr. R. Souèges presented his work on the development of the suspensor cells during plant embryogenesis [1]. The question was then to solve the origin of the hypophysis, the apical cell of the suspensor. Two schools of thought existed. One held that the basal cell of the first division of the zygote never divides again and that the whole embryo structure originates from the apical cell. The other proposed that the basal cell divides and that its derivatives end up forming the suspensor and the hypophysis. A model plant of choice was then Capsella Bursa-pastoris for the simple structure of its embryo and the ease of observation. Careful microscopic examination allowed Dr. Soueges to establish ‘step by step’ the successive cell divisions starting from fecondation. His observations were consigned in drawings of embryos at stages never reported before. His finding was recapitulated in figures such as figure 1, where the fate map of the suspensor cells has been indicated. It showed that during embryogenesis in C. bursa-pastoris the first zygotic division gives rise to two cells. The apical cell will contribute to most of the embryo structure, the embryoproper. The basal cell develops in a file of 7 to 9 cells called the suspensor. All but one of these cells will degenerate at the end of early embryogenesis. The surviving cell will contribute to some domains of the root apex. In conclusion, his work established that the basal cell was capable of dividing and contributed to some of the final
570
Dans notre laboratoire, nous avons montré qu’un système d’activation génique permet le développement de stratégies d’ablation cellulaire qui éliminent ou perturbent des structures, révélant ainsi des interactions insoupçonnées entre domaines cellulaires. C’est ainsi que l’expression du gène toxique barnase dans le protoderme de l’embryon altère la structure du pôle basal, à priori en modifiant l’équilibre des signaux intercellulaires (figure 3; Baroux et al., soumis). L’utilisation d’ablation cellulaire grâce au laser devrait compléter la panoplie des stratégies offertes. Il est permis d’imaginer qu’en combinant l’ablation laser avec la microscopie confocale et un système de fécondation in vitro, comme celui développé avec le maïs [26], l’étude de l’embryogenèse pourrait entrer dans une nouvelle ère qui abordera des questions que nul n’a posé jusqu’à présent.
2. Embryogenesis in Arabidopsis The same observations at the descriptive level have been made using Arabidopsis thaliana but new tools and techniques brought novel questions. The molecular mechanism underlying the shaping of the embryo is now the centre of attention. A. thaliana happens to be a relative of C. Bursa-pastoris and the development of its embryo is very similar. However A. thaliana, a diploid unlike C. bursa-pastoris which is tetraploid, is today a model of choice in development because of its genetics. The question of how a multicellular organism constructs itself starting from a single zygote cell is not new. In plants the answers lay in the study of early embryogenesis. This term covers the stages from the initial fertilised cell to the heart stage embryo (figure 2). At the end of early embryogenesis the basic structure of the plant is achieved. Molecules important for this process are being searched for, using a whole array of approaches including biochemistry, genetics, molecular biology and genetic engineering. Dr.
P. Gallois / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573
Figure 2. Schematic fate map of embryo development in A. thaliana. The apical cell will contribute to most of the embryo structure, the embryo-proper. The basal cell develops in a file of 8 cells called the suspensor and contributes to part of the root apex. Grey area corresponds to the suspensor.
Figure 1. Capsella bursa-pastoris Moench. Schematic representation of suspensor development starting from the bottom two basal cells of a 4-cell-stage embryo. In this particular case the basal cell of the pro-embryo is dividing only twice (translated from Souèges [2]).
Souèges would have been probably delighted to see how this changed the nature of the questions asked about the suspensor. At his time the suspensor had been confined to a structural role in plant embryogenesis. Two structural roles had been proposed. One is being a physical support and anchorage device for the developing embryo in the seed. The second one is to carry out an embryo feeding role in which the suspensor cells would transport nutrients from the mother plant to the embryo as well as being themselves metabolically active for protein, auxin and gibberellin synthesis [3]. More recently mutants have been isolated that are affected in suspensor development (reviewed in [4]) and are divided in 2 classes: susp and twin. The model based on the observed phenotypes of these two classes is that the embryo-proper produces signals responsible for (i) maintaining suspensor cell identity and (ii) inducing suspensor cell death. Additionally in wild type, embryos form their axis with their basal pole towards suspensor cells. In the twin mutants, suspensor cells can form secondary embryos with an axis of polarity in the same or reverse orientation to that of the primary embryo [5]. One possibility is that secondary embryos can form the apical–basal axis at random in either orientation because of the presence of suspensor cells on two opposite sides. This would imply the existence of a second signalling pathway from suspensor to embryo-proper. Cloning of genes corresponding to a susp and a twin mutant has not been informative since the sequences encode housekeeping proteins indirectly involved in abnormal embryo development [6, 7]. The nature of the signalling molecules produced either by the embryoproper or the suspensor remains totally unknown although the plant hormone auxin is a candidate [reviewed in 8].
Revisiting the suspensor function will require molecular markers of its identity, and identification of putative signal molecules. This very focussed example epitomises what mutants have brought to the field of plant embryogenesis and what they unexpectedly failed to do. Mutants are instrumental to study the processes of organised cell divisions and orientated cell expansion [9] and interesting data are gathered by crossing signalling mutants and mutants presenting altered pattern formation [10, 11]. However many mutants with altered early embryogenesis resulted in the cloning of genes not directly involved in the decision making. Noticeably only loss-of-function screens have been carried out so far in embryogenesis. This makes mutant screens for embryogenesis hostage of a possible lethality induced by the mutation of key genes and hostage of genetic/functional redundancy that may mask the role of particular regulatory genes. For the latter, known examples are the two CUC genes that have to be both mutated to give the phenotype of fused cotyledons [12] or the three SEPALLATA genes that give a phenotype in flower once all mutated [13]. This redundancy calls for gain-of-function screen. Unlike what has been done in embryogenesis, it is striking to note that in the study of flower development several mutant approaches have already been used: screen for recessive or dominant mutations [14–17], targeted misexpression of genes [e.g. 18, 19], suppressor screen [20]. In particular gain-of-function screens are already being carried out for some aspects of plant development and recent publications have demonstrated the power of the approach [16]. That is not as easy to carry out during embryogenesis. This because dominant mutations affecting dramatically the structure of the embryo will be lost since the original mutated individual is unlikely to survive and give a progeny. To remove this stumbling block, technological development is necessary, allowing conditional misexpression of genes. A striking example of the potential of such an approach in embryogenesis is the misexpression of the gene LEC-1 resulting in embryonic structures developing on leaves albeit at low frequency
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P. Gallois / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573
[21]. Activation systems that could be used to misexpress gene in embryo have been developed [22, 23] and hope fully novel screen will be soon available that will give us access to new genes implicated in embryo development. The basic idea is that the potentially deleterious gene is not expressed in the mother plant until its promoter is activated in the progeny by the experimentator either by crossing with an activating parent line or using specific chemical compound. A promoter, that can be activated, is to be inserted at random in the genome of A. thaliana creating a collection of plant containing silent insertions. Upon activation in the embryo of the next generation some of the insertions will lead to misexpression of a nearby coding sequence, either in time or space. This will create dominant mutation that can be screened for. On a similar note, dominant negative forms of regulatory genes will be expressed in embryo in the future. Currently one limiting factor is that their effect might lead to counterselection of the transgene in parental lines. In that case no transgenic line can be recovered and analysed. The study of Hemerly et al. [24] exemplifies this powerful but under-used approach by introducing in plants a ‘dead’ form of a cell cycle regulatory gene (CDC2a). The mutated protein interferes with the endogenous wild type protein thus reducing the frequency of cell division in embryo development. This affected the apico-basal pattern but cell differentiation was not affected. Generalising this type of approach in embryogenesis would be made easy by the use of a two-component activation system as described above. Another approach that has not been yet applied to embryos is cell ablation [reviewed in 25]. A laser beam can be used to destroy temporarily specific cells in the embryo. Modification of subsequent development would give indications on possible signals emitted by the destroyed cell and on the determination/totipotency of adjacent cells. The technical hurdle here is to keep the embryo alive in such a way that development would continue after dissecting out the seed. Alternatively genetic ablation can be carried out where a toxin is expressed under a specific promoter so to remove a given embryonic structure by killing the corresponding cells. Using constructs in which the toxin gene is linked to a tissue specific promoter is very limiting since promoters have to be tissue and embryo specific so that a whole transgenic plant can be regenerated and the deleterious effect of the transgene observed in the progeny only. A conditional expression system allowing the toxin to accumulate in the progeny of two healthy parents is therefore a very valuable tool. New experiments in our laboratory have indicated that informative phenotypes can be obtained using the Barnase gene as a toxin and a two-component activation system. And this even with promoters that carry on to be expressed during vegetative development. Using for example the Ltp2promoter, that drives expression in the embryo protoder-
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Figure 3. Effect of Barnase expression under the LTP2 promoter. A two-component activation system has been used to express Barnase in the protodermis cells of the embryo. A. wild type embryo; B. Barnase expressing embryo with an enlarged basal pole (arrow).
mis, to activate the Barnase gene, induced a specific defect during embryogenesis that resulted into an enlarged basal pole (figure 3) (Baroux et al., submitted). Since mutations altering specifically the epidermis don’t exist, this type of experiment is filling a gap in the possible experimental designs used by plant developmental biologist. We expect that genetics and later laser ablation will provide invaluable information on cell–cell communication between specific tissues during the generation of the plant embryo. Additional tools would be in vitro fertilisation techniques followed by zygote development such as those designed in maize [26]. Confocal techniques are as well being developed and one could dream of days to come when A. thaliana embryos would survive and take shape under laser scrutiny during the 5 days of early embryogenesis.
3. Conclusion Our current understanding of the regulation of plant embryogenesis at molecular level is still in its infancy. However the picture is evolving rapidly and the trend is likely to accelerate as many of the approaches described here are underway. Sadly applying global transcript analysis and proteomics to A. thaliana embryos seems out of reach at present since it would require a miniaturisation of the technique that is not yet available. In the mean time other model species might fill the gap. Only eighty years after his seminar at the Academy of Sciences, Dr. Soueges would hardly recognise the content and focus of today’s textbook dealing with plant development. It is easy to predict that the same feeling will hit the current generation of plant scientist well before the middle of the 21rst Century. That is what keeps plant embryogenesis exciting.
P. Gallois / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 569–573
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