Journal of Experimental Botany, Vol. 51, No. 353, pp. 1951-1960,
December 2000
© 2000 Oxford University Press
Review Article |
Vascularization is a general requirement for growth of plant and animal tumours
1 Institut für Botanik, Technische Universität, D-64287 Darmstadt, Germany
2 Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel
Received 8 March 2000; Accepted 12 July 2000
Abstract
Solid-tumour growth in animals as in humans depends on angiogenesis. Tumours that fail to induce the formation of new blood vessels do not enlarge beyond a few millimetres in diameter. Plant tumours induced by Agrobacterium tumefaciens can reach diameters of more than 100 mm, thus raising the question of how they are sufficiently supplied with nutrients and water. Until recently, these rapidly growing tumours were considered unorganized or partly organized masses. However, in analogy to animal and human tumours, growth of leaf and stem tumours depends on neovascularization. Plant tumour cells induce the formation of a sophisticated vascular network consisting of water-conducting vessels and assimilate-transporting sieve elements. Similar to animal and human tumours that overexpress angiogenic growth factors, plant tumours overexpress the T-DNA-encoded vascularization-promoting growth factors auxin and cytokinin upon Agrobacterium infection. High auxin levels induce ethylene emission from the tumours, which has a strong impact on tumour and host stem, as well as on root structure and function. Ethylene apparently stimulates abscisic acid synthesis in the leaves above the tumour, which reduces transpiration and thus protects the host plant from rapid wilting. Hence, for the elucidation of phytohormone-dependent vascular development in plants, such tumours are regarded as an excellent model system. The comparison of analogous requirement of neovascularization for tumour growth in plants, as in animals and humans, is discussed in terms of interdisciplinary strategies of possible prevention and therapy.
Key words: Agrobacterium tumefaciens, phytohormone-directed vascular differentiation, plant and animal/human tumours, vascularization, water and solute transport.
Introduction
Recent reports about successful antiangiogenic therapy against growth of mouse endothelial tumours by using the endogenous angiogenesis inhibitors, endostatin and angiostatin, attracted world-wide attention to the importance of tumour control by vascularization (Boehm et al., 1997
; O'Reilly et al., 1997; Marshall, 1998). In contrast, anatomical knowledge of plant tumours is still scarce, even though it has been known for over 100 years that the most studied plant tumours, crown galls, are incited by the soil bacterium Agrobacterium tumefaciens and A. vitis (Cavara, 1895; Smith and Townsend, 1907; Burr and Otten, 1999). About 50 years later, it was suggested that bacterial DNA is expressed in plant tumours (Klein, 1953). The discovery that a DNA sequence of about 20 kb (T-DNA) of a large bacterial plasmid (Ti-plasmid) is stably incorporated into the higher plant genome (Van Larebeke et al., 1974; Chilton et al., 1977) was a break-through in molecular biology and biotechnology. Molecular biological research exploded upon the discovery that disarmed agrobacteria can be used as gene vectors between pro- and eukaryotes. In the meantime most of the T-DNA-located genes have been identified. The most prominent ones are those encoding enzymes of auxin, cytokinin and opine biosynthesis (iaaM, iaaH, ipt, and nos/ocs) (Weiler and Schröder, 1987) besides phytohormone regulatory genes e, f and 5 (Körber et al., 1991; Broer et al., 1995).
Until recently such tumours were believed to contain only 10–25% transformed cells, which overproduce auxin and cytokinins, and assumed to be homogeneously distributed among the majority of non-transformed host cells (Sacristan and Melchers, 1977; Ooms et al., 1982; Van Slogteren et al., 1983). Their estimation did not take into account enucleate vascular structural peculiarities of the tumours. Recent reinvestigation based on structure and on mRNA and DNA marker analysis, resulted in an estimation of the transformation rate of up to 100% (Rezmer et al., 1999). This illustrates the importance of knowing basic tissue structures for any further evaluation of physiological, biochemical and molecular properties. The aim of this review is to highlight the sophisticated anatomy and its functionality of A.t.-induced plant tumours, which enables rapid tumour proliferation in analogy to human tumour vascularization. The detection of particular tissue structures in plant tumours, namely continuous vascular bundles, makes them a unique model system to study phytohormone-controlled vascular bundle development, including membrane pumps, channels and specific carriers of vascular bundles.
Structure and organization of plant tumours
In contrast to sieve elements, structures of lignified vessels are easy to identify and have been shown in tumours of various plant species such as Datura stramonium, Helianthus annuus, Kalanchoë daigremontiana, Rubus loganobaccus, R. procerus or Vitis vinifera (McKeen, 1954; Bopp and Leppla, 1964; Kupila-Ahvenniemi and Therman, 1968; Tarbah and Goodman, 1988; Meyer, 1987; Sachs, 1991). Due to the three-dimensional organization and tree-like architecture of the bundles throughout the tumours, in thin tissue sections tracheary elements appear as if they were isolated idioblasts. This interpretation was supported by the finding that isolated single green mesophyll cells can differentiate into single tracheary elements when treated with a certain combination of cytokinins and auxin under vigorous shaking of the cultures (Fukuda, 1996; McCann, 1997). However, until now only cells isolated from Zinnia could be induced to form tracheary elements. For tumours it was assumed that those idioblasts finally connect to the main host bundles (Kupila-Ahvenniemi and Therman, 1968; Beiderbeck, 1977).
The question, how cell masses of up to 100 mm in diameter (Fig. 1B), supposed to be unorganized (Gordon, 1982; Tarbah and Goodman, 1988; Weiler and Schröder, 1987; Sachs, 1991; Schell et al., 1994) are rapidly and sufficiently supplied with inorganic and organic nutrients and water, led to a reinvestigation of the tumour structure (Malsy et al., 1992; Aloni et al., 1995). To obtain a more complete picture of the assumed three-dimensional bundle architecture, tumour tissue sections of about 3 mm thickness were cleared with lactic acid and stained with lacmoid, a method which had been successful in simultaneously revealing the three-dimensional structure of both vessels, by staining their lignified walls dark blue, and sieve elements by staining the callose of the sieve plates sky blue (Aloni and Sachs, 1973). It became apparent that at the onset of tumour growth, 2–3 d after infection, vessels and sieve elements differentiated within the growing tumour tissue (Fig. 1A, D). Kalanchoë leaf tumours display a net of single vessel and sieve element strands (Malsy et al., 1992) while stem tumours develop concentric bundles with an inner xylem surrounded by phloem (Aloni et al., 1995). Those bundles extend close to the tumour surface indicating a production of auxin and cytokinins by the surface cell layer. The xylem and phloem bundles are interconnected by a dense net of phloem anastomoses (Fig. 2D; Aloni et al., 1995), as they are also common in healthy stems (Aloni and Sachs, 1973; Aloni and Peterson, 1990; Aloni and Barnett, 1996).
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Similar to plant tumours, tissue cultures are still often regarded as consisting of homogeneous parenchyma cells. The formation of nodules of vascular tissue has been described as consisting of tracheas and islands of sieve elements in callus tissue grown in vitro (Wetmore and Rier, 1963
). A possible interconnection of nodules and sieve elements across the nodules from one part of a callus to another could not be excluded (Abbott et al., 1977). Phloem differentiation was attributed to a crucial role of sucrose (Wetmore and Rier, 1963). This interpretation was revised by authors who could prove the fundamental importance of auxin in sieve element and vessel differentiation in tissue cultures (Bornman et al., 1977; Aloni, 1980). A low auxin concentration induced sieve elements, while high auxin levels induced xylem (Aloni, 1980), similar to the normal pattern of phloem and xylem development in the apical plant parts (Esau, 1945). Techniques were developed for using plant tissue cultures to study phloem development in vitro (Sjölund, 1997). Hence, homogeneous-looking callus tissue cultures also consist of different cell types and must have different, tissue-specific membrane transporters.
Interestingly, fibres are nearly absent from tumour tissue (Aloni et al., 1995), probably due to low gibberellin levels, a limiting factor for fibre differentiation (Aloni, 1979). This may cause the sometimes crumbly structure of herbaceous plant tumours like those in Arabidopsis (Fig. 2A
).
The ‘gall constriction hypothesis’
Growing crown galls dramatically affect the structure of the developing host tissues at the tumour developing site. The contact zone between the tumour and host stem is considerably different from healthy stems. It is characterized by numerous narrow vessels adjacent to the tumour, which might limit water flow to the shoot organs above the gall (Aloni et al., 1995). However, vessel diameters below the tumour are normal and, therefore, the gall itself is well supplied with water. This structural evidence led to suggest the ‘gall constriction hypothesis’ (Aloni et al., 1995) for explaining the possible hydraulic constrictions in the gall junction, necessary for efficient competition for water and nutrient supply between the developing gall and the host shoot. The hypothesis proposes that growing galls retard the development of their host shoot by a signal that reduces vessel expansion and thus limits the diameter of the vessels in the host. It was, therefore, suggested that this controlling signal is the hormone ethylene which is known to reduce vessel width in plants substantially (Yamamoto et al., 1987). The ‘gall constriction hypothesis’ has been proposed in analogy to Zimmermann's ‘segmentation hypothesis’ applying to branch junctions of trees (Zimmermann, 1983; Aloni et al., 1997). It was experimentally confirmed (Aloni et al., 1998; Wächter, 1999; Wächter et al., 1999) by showing that tumour-induced ethylene is a limiting and controlling factor of crown gall morphogenesis (Fig. 1C). This signal leads to reduced vessel diameters in the host stem–gall interface and enlargement of the tumour surface, thus giving priority in water supply to the growing gall over the host shoot. The physiological changes in water transport and the relation between transpiration of host shoot and the growing gall are discussed below.
Role of phytohormones
The expression of the oncogenes iaaM and iaaH for auxin synthesis, via a pathway from tryptophan over indoleacetamide (IAM), is well documented in bacteria. It was found in Pseudomonas syringae subsp. savastanoi, which causes galls in olive and oleander (Yamada et al., 1985), in Agrobacterium tumefaciens (Thomashaw et al., 1986), and in Erwinia herbicola pv. gypsophilae, which induces galls in Gypsophila (Manulis et al., 1998). Auxin accumulation within the tumours is abnormally high with a 500-fold increase over that found in control tissues (Kado, 1984). Similarly, the cytokinin synthesis pathway via isopentenyltransferase (ipt) has only been found in microorganisms until now. In synergism with auxin it leads, for example, to fasciation-like galls, as incited by Rhodococcus fascians (Thimann and Sachs, 1966; Vereecke et al., 2000). Cytokinins accumulate up to a 1600 times higher level in tumours (Kado, 1984). Auxins are known to be causally involved in the regulation of vascular bundle development (Sachs, 1981; Aloni and Zimmermann, 1983; Aloni, 1995; Klee and Lanahan 1995; Aloni et al., 2000). The detection of such high auxin concentrations in crown galls raises the question, how these levels are maintained, whether the auxin oxidation is slowed down, or the basipetal export is inhibited. A positive feed-back system is known for Rhizobia-induced legume nodules and nematode-induced root galls, where flavonoids regulate auxin accumulation by auxin-induced flavonoid synthesis. Flavonoids prevent oxidation and basipetal transport of auxin, thus enhancing local nodule growth (Mathesius et al., 1998; Hutangura et al., 1999). Such a mechanism is also conceivable for crown galls, where 7,4' dihydroxyflavone and formononetin were found to accumulate in tissue areas of GUS-labelled chalcone synthase selectively in crown galls of transgenic Trifolium repens (Schwalm and Ullrich, 2000). The development of circular vascular bundles in crown galls (Aloni et al., 1995, 1998) is supposed to originate from inhibition of basipetal flow of auxin, which then will be diverted to lateral or even apical directions and thus induce own transport systems, here abnormally in a circular manner. This assumption is based on anatomical studies of injured plants and on theoretical modelling (Sachs and Cohen, 1982; Aloni et al., 1997).
The conspicuously increased xylem below stem tumours (Aloni et al., 1995; Schurr et al., 1996) indicates an additional mode of inhibition of basipetal auxin transport by ethylene, emitted from the tumours. A relation between ethylene perception and auxin transport was found upon cloning of Arabidopsis EIR1 (ethylene-insensitive root 1), indicating that EIR1 codes for a protein, a target for regulation of auxin transport by ethylene (Luschnig et al., 1998). In spite of this ethylene effect, the retardation of overall root development and suppression of lateral root formation in tumourized plants (Mistrik et al., 2000) may be due to the transient 4-fold oversupply of auxin to the sensitive Ricinus roots (D Veselov, unpublished results), as had also been suggested for iaaM and iaaH transgenic hybrid aspen (Tuominen et al., 1995). A possible role of tumour ethylene in root development is not yet proven.
Increase in the number of unlignified rays and differentiation of smaller vessels is also known to occur in flooded or wounded trees (Lev-Yadun and Aloni, 1995). In addition, such symptoms could be artificially induced by the application of ethylene as ethrel. The similar symptoms observed in the crown gall/host stem interface may have the same cause, namely ethylene production by the tumours. Indeed, stem tumours of tomato plants (Aloni et al., 1998) and of Ricinus (Wächter et al., 1999) were shown to produce up to 140 times more ethylene than wounded, but not infected control stems, with a maximum at 5 weeks after infection. In control experiments ethrel application to non-infected stems of both plant species caused similar symptoms. The importance of ethylene for vascularization and thus for tumour development has been unequivocally demonstrated by inhibited tumour induction in the Never ripe (Nr) tomato mutant (Fig. 1C), which is almost insensitive to ethylene (Aloni et al., 1998), and otherwise by treating developing tumours with aminoethoxyvinylglycine (AVG), the inhibitor of ethylene synthesis, by blocking the aminocyclopropanecarboxylate (ACC) synthase (Wächter, 1999). In both cases tumour growth was suppressed. ACC accumulation precedes ethylene production in crown galls with a maximum at 2 weeks after infection (Wächter et al., 1999). Synthesis of ethylene precursors is then enhanced by the high auxin concentration in tumours and by oxygen deficiency within the compact and metabolically active tissue, since both factors are known to stimulate ACC-synthase.
The crown galls offer a wide field for the study of the role of phytohormone signalling and regulation related to tissue differentiation. The earliest prominent signal in Ricinus tumours was jasmonic acid with an accumulation maximum 1 week after infection (I Feussner, unpublished results). The role of jasmonic acid is generally assumed to be stress- and plant development-related signal transduction (Creelman and Mullet, 1995; O'Donnell et al., 1996). Auxin and cytokinin levels are highest 2 weeks after infection (C Götz and D Veselov, unpublished results). The subsequent maximum ethylene production is accompanied by maximum abscisic acid (ABA) synthesis in tumours (I Feussner, unpublished results). ABA is probably exported into shoot and root, but it is not yet clear if it is involved in the inhibition of root growth and nitrate uptake in tumourized plants (Mistrik et al., 2000). Stomata closure in leaves of tumour-bearing plants inversely correlates with their distance from the tumour. The ABA concentration in such leaves is five times that in leaves of control plants or in leaves inserted closer to the plant apex (W Hartung, unpublished results). The fact that the ACC content of the stem above and below tumours is not significantly different from that of the control stems (Mistrik et al., 2000) suggests that ethylene emission from the tumour itself induces ABA synthesis in the neighbouring leaves, thus preventing extreme water stress of the host plant. Ethylene is known to inhibit foliar gas exchange and to stimulate ABA synthesis (Mayak and Halevy, 1972; Gunderson and Taylor, 1988).
Assimilate translocation and water transport
Phloem unloading and assimilate accumulation
Tumours are generally regarded as strong metabolite sinks for their host plants (Beiderbeck, 1977). In comparison to non-infected tissue, solute accumulation amounts to: 14x for sucrose, 26x for glucose, 18x for fructose, 40x for total amino acids, 5x for cations, including 2x for Fe2+, and 8x for anions (Marx and Ullrich-Eberius, 1988; Brown et al., 1990; Malsy et al., 1992; Pradel et al., 1996; Mistrik et al., 2000). The finding of consistently increased activity (8-fold) of acid cell wall invertase (CWI) in tumours on leaves of Kalanchoë and stems of tobacco inferred that they acquire the sugars by the sequence of apoplastic unloading of sucrose from the phloem complex, hydrolysis to hexoses and absorption into the tumour parenchyma cells with hexose carriers (Weil and Rausch, 1990). Transport studies with fluorescent dyes in sieve elements, such as carboxyfluorescein (CF) and injected Lucifer Yellow, and by infecting leaves of Nicotiana benthamiana with potato virus X, which expresses a green fluorescent protein-coat protein fusion (PVX.GFP-CP), revealed without doubt that assimilates are transported symplastically from the sieve elements into the parenchyma cells all over the tumour, where all cells are tightly connected by functioning plasmodesmata (Pradel et al., 1996, 1999). The question remains to be answered, which function the concomitant high CWI-activity, up to 18 times higher (Pradel et al., 1996), may have that is different from that in phloem unloading. Recent results suggest a correlation of CWI activity with abscisic acid-regulated water stress induction. In the tumour periphery, a high cell transformation rate by wild-type T-DNA, as visualized by using ß-glucuronidase gene-containing wild-type agrobacteria (A281p35gus int) (Fig. 2B), indicates a particularly high level of auxin and cytokinins, which leads to bundle differentiation. In the outermost cell layers, where bundles are not yet visible, the CWI activity was the highest of the whole tumour. It was 50 times that of the control stem and of the inner tumour bundles (I Mistrik, J Pavlovkin, A Weilmünster, C Ullrich, unpublished results). These results are not consistent with the cytokinin responsiveness of CWI (CIN1)-mRNA induction in Chenopodium rubrum (Ehness and Roitsch, 1997).
Water transport and transpiration
Growing tumours do not regenerate epidermal layers and cuticles (Aloni et al., 1995; Schurr et al., 1996). Crown galls grown on wild-type stems develop a substantially enlarged surface area due to the unorganized callus shape of the gall, which is maximized by tumour-induced ethylene, while in the ethylene-insensitive tomato mutant (Nr) the tumours have smooth appearance with minimum surface area (Aloni et al., 1998). Hence the transpiration rate of tumours increases up to 10-fold that of the non-infected Ricinus stem. Under dark conditions the transpiration rate of the 3-week-old Ricinus tumours is 28 mmol m-2 s-1, that of leaves of control plants is about 12 mmol m-2 s-1 and that of leaves of tumourized plants is only 6 mmol m-2 s-1 (Schurr et al., 1996; Wächter, 1999). These physiological changes are due to alterations in water transport structures of both the increased unprotected gall surface and the reduction in diameter of tumour adjacent vessels in wild-type plants, as discussed above. Therefore, under moderate water stress, the older leaves above the tumour turn yellow and senesce in wild-type tomato, whereas the leaves of the ethylene-insensitive mutants (Nr) remain green and healthy (Aloni et al., 1998).
The lateral nutrient transport between xylem and phloem across the cambial zone is still underestimated in its importance (Van Bel, 1990). This pathway in the rays from the main host vessels into the phloem next to the tumour appears to be activated upon tumour growth. Predominantly in the tangential walls, primary pit fields of high density are conspicuously labelled by aniline blue staining of callose (Wächter et al., 1999). Callose is not necessarily regarded as an indicator of plugging plasmodesmata (Reichelt et al., 1999). In contrast, plant specific myosin VIII seems to incite callose deposition by high intra- and intercellular movement activity specifically in plasmodesmata. This would confirm high symplastic transport activity within the pathologically altered, unlignified and multiseriate rays between host stem and tumour. Indeed, CF and PVX-virus rapidly moved through these pathological vascular rays from the host stem into the tumour (Pradel et al., 1999), thus confirming the suggested importance of vascular rays as symplastic pathways for radial transport.
Comparison of plant tumours with animal/human tumours
Comparison of plant tumours with animal tumours on nude mice reveals striking similarities and analogies. Solid-tumour growth in animals and humans depends on angiogenesis, the formation of new capillaries from pre-existing vasculature by migration and proliferation of endothelial cells. Conversely, vasculogenesis, the development of new vessels by the assembly of angioblast islands, does not seem to be involved in tumourigenesis. Tumours that fail to induce the formation of new blood vessels do not enlarge beyond a few millimetres in diameter (Folkman et al., 1989; Folkman, 1990, 1995; Folkman and Shing, 1992). Actually, animal and human tumours overexpress angiogenic growth factors, of which TNF-
(tumour necrosis factor), FGF (fibroblast growth factor) and VEGF (vascular endothelial growth factor) are considered to be major mediators of angiogenesis in many different tumour types (Risau, 1990). Human squamous carcinoma cells induce a dense network of blood vessels that supplies the tumour stroma with nutrients, water and oxygen (Fig. 1E). In analogy, plant tumours develop a sophisticated and continuous system of vascular bundles. On minor leaf veins of Kalanchoë blossfeldiana tumour development is slow, resulting in small tumours (Fig. 1B, arrowhead). On major leaf veins tumour development is rapid (Fig. 1B, small arrow, tumour about 20 mm in diameter) and it is most rapid on stem bundles yielding the largest tumours (Fig. 1B, large arrow, up to 100 mm in diameter). This positive correlation between the size of the supplying bundle and the growth of the tumour demonstrates the important role of vascularization and nutrient supply in tumour development. Tumour cells proliferate only in vascularized regions (Fig. 1D), whereas in non-vascularized areas they necrotize as they do in animal/human tumours (Fig. 1D, arrow, and F).
A precondition of T-DNA transfer from the Ti plasmid of A. tumefaciens into the plant genome is wounding of host tissue. Similarly in animals, tumour development can be promoted by wounding. In plants, wounding stimulates cell division, during which the T-DNA incorporates into the plant's DNA and thus enables integration of the prokaryotic gene. Furthermore, in both animals and plants, gradients of growth factors are established, in plants by basipetal auxin transport, inducing and controlling vascular differentiation. Strangely enough, in human squamous cell carcinoma a 10-fold accumulation of auxin was also found, with a still unknown function (Shimojo et al., 1997). Increased permeability of blood vessels in animal tumours (Senger et al., 1983) corresponds to a considerable decrease in electrical membrane potential difference in plant tumour cells in comparison to healthy plant tissue (Marx and Ullrich–Eberius, 1988).
Of course, structure and quality of vascular tissue is different in animals and plants; in animals the interior space of vessels is non-cellular, in plants it is at least originally a cellular system. In human and animal, neovascularization of tumours increases the probability of metastatic spread which is unknown in plant tumours (Doonan and Hunt, 1996; Gaspar, 1998). Whereas connective tissue stroma is prominent in many animal and human tumours (Dvorak, 1986), plant tumours are devoid of fibres due to lack of gibberellin synthesis (Aloni et al., 1995) and to the high levels of tumour-induced ethylene known to inhibit fibre differentiation (Yamamoto et al., 1987).
Perspectives
The analogy between plant and human tumour growth is fascinating, in both cases neoplastic growth strictly depends on vascularization. In humans some tumour therapy aims at preventing vascularization by suppressing the function of angiogenesis factors (Ingber et al., 1990), in plants curing or protecting crop plants from tumourigenesis requires completely different strategies. In grapevine, apple or pear trees, prevention by using A. tumefaciens- or A. vitis-resistant varieties or by raising bacteria-free cuttings is highly superior to individual therapy by antibiotics, which are very costly and legally restricted in use for plant protection. Moreover, once the tumour is formed treatment of the transformed cells by antibiotics cannot change their genome. A screening for ethylene sensitivity may be useful. Precious individual trees in parks or orchards may be treated with ethylene synthesis inhibitors.
Recently, development of human Morbus Hodgkin has been suggested to result from a similar sequence of infection and DNA transfer as A. tumefaciens-induced plant tumours. Thus, comparison of plant and animal tumour development may provide insights into general principles of cancer pathogenesis and eventually open new strategies for tumour therapy or prevention (Sauter, 1995). A novel, oxidative stress-induced protein Oxy5 from Arabidopsis thaliana, which protected human tumour cells almost completely from TNF-induced apoptosis has been cloned and characterized (Jänicke et al., 1998). This protection, transferred across evolutionary boundaries, was accompanied by increased MnSOD (manganous superoxide dismutase) and decreased O-2 levels.
Since in the Columbia strain of Arabidopsis thaliana highly transformed and vascularized tumours could easily be induced, and attained sizes of up to 10 mm (Fig. 2A–C), studies of vascular development can be greatly enhanced by using mutants and transformants either overexpressing or deficient in expression of the phytohormones. One of the main questions is still open, whether or not in intact plant tissues a spontaneous differentiation is possible, as recently reported for aggressive intraocular cutaneous melanomas, where by spontaneous genetic reversion vascular channels without epithel develop to a pluripotent embryo-like genotype, independent of tumour angiogenesis and termed vasculogenic mimicry (Maniotis et al., 1999). Though such spontaneous vascular differentiation also appears to proceed in callus cultures derived from plant pith tissue explants (Bornman et al., 1977), only detection of inducing and/or repressing factors on the transcriptional level may reveal if such pith explants had already been induced when they were still in the intact plant tissue. It has been shown that, in Arabidopsis embryos, vascularization is induced by neighbouring cells which code for inducing, but not yet identified factors (Hardtke and Berleth, 1998). For the elucidation of vascular development, such Agrobacterium tumefaciens-induced tumours seem to be an excellent model system because of their reduced but functional pattern of differentiation, which is based on overproduction, in sequence, of five important phytohormones, jasmonate, auxin, cytokinins, ethylene, and abscisic acid.
Acknowledgments
We thank Dr Mihaela Skobe and Professor Norbert E Fusenig (DKFZ Heidelberg, Germany) for valuable suggestions and for kindly providing photographs of mouse epithelial tumours, Professor George Redei (University of Columbia MO, USA) for original seeds of the Columbia strain of Arabidopsis and Katja Schwalm and Kai Tragesser (TU-Darmstadt, Germany) for photographs of Arabidopsis tumour sections. This work was supported by DFG-SFB 199 to CIU.
Notes
3 To whom correspondence should be addressed. Fax: +49 6151 164 630. uleb{at}bio.tu\|[hyphen]\|darmstadt.de 
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