Methylglyoxal destroys <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> crown gall tumours in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> without any adverse effect on the host plant

Methylglyoxal (MG) is a highly reactive α-oxoaldehyde, demonstrating anticancer effect on plant neoplastic tumours. In in vivo studies it was observed that MG destroyed crown gall tumours in Nicotiana tabacum produced by Agrobacterium tumefaciens, without any adverse effect on the host. The efficacy of MG in comparison to other anticancer drugs viz. cisplatin and ellagic acid in the treatment of crown gall was investigated. A slight degeneration of galls was noted in plants treated with cisplatin and ellagic acid but the plants died subsequently. With MG however, crown galls were completely cured and the plants completed their usual life cycle by flowering and producing seeds. MG inhibited the respiration of crown gall calluses suggesting that energy depletion resulted in tumour destruction.     

Interspecific differences in differentiation and dedifferentiation patterns in the genusNicotiana

Differentiation on hormoneless media, habituation ability and crown gall induction inNicotiana tissue cultures have been used as physiological parameters of evolutionary differentiation between species. Some of them on hormone free media differentiated whole plantlets, others produced only shoots or roots or showed undifferentiated growth (habituation), some eventually died. Moreover, the same genotypes showed a differential behaviour as far as tumor formation byAgrobacterium tumefaciens was concerned. Particularly, the competence for crown gall transformation inNicotiana species seems negatively correlated with differentiation capacity and may be ascribed to differences in the plants capacity to synthesize growth regulators. The correlation between the results obtained and the phylogenetic position of the genotypes tested is finally discussed.     

The Never ripe Mutant Provides Evidence That Tumor-Induced Ethylene Controls the Morphogenesis of Agrobacterium tumefaciens-Induced Crown Galls on Tomato Stems

We confirm the hypothesis that Agrobacterium tumefaciens-induced galls produce ethylene that controls vessel differentiation in the host stem of tomato (Lycopersicon esculentum Mill.). Using an ethylene-insensitive mutant, Never ripe (Nr), and its isogenic wild-type parent we show that infection by A. tumefaciens results in high rates of ethylene evolution from the developing crown galls. Ethylene evolution from isolated internodes carrying galls was up to 50-fold greater than from isolated internodes of control plants when measured 21 and 28 d after infection. Tumor-induced ethylene substantially decreased vessel diameter in the host tissues beside the tumor in wild-type stems but had a very limited effect in the Nr stems. Ethylene promoted the typical unorganized callus shape of the gall, which maximized the tumor surface in wild-type stems, whereas the galls on the Nr stems had a smooth surface. The combination of decreased vessel diameter in the host and increased tumor surface ensured water-supply priority to the growing gall over the host shoot. These results indicate that in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown-gall morphogenesis.     

Polycentric vascular rays in Suaeda monoica and the control of ray initiation and spacing

Summary The early-formed xylem of Suaeda monoica Forssk. ex J. F. Gmel (Chenopodiaceae) is temporarily rayless. Vascular rays differentiate during later stages of its xylem ontogeny. The rays in Suaeda are heterogeneous, and some of them are aggregated. The mature xylem of this species is characterized by two unique types of vascular rays: (1) rays with several inside initiation centres of small cells formed by local frequent cell divisions in the cambium, and (2) huge xylem rays with radial phloem strands that are connected to the axial phloem. The spacing of the xylem rays is not even, and possible mechanisms controlling ray spacing are discussed. Our observations indicate that rays do not have an inhibitory zone around them in which ray initiation is prevented. The initiation of radial patterns of small cells which appear like inside rays within a large vascular ray suggests that initiation and spacing of rays is controlled by radial signal flows in relationship with axial signal fluxes.     

Isozyme gene expression in potato tumors incited by Agrobacterium

Summary Two plant tumors (crown galls and hairy roots) were experimentally provoked on potato cv. Désirée by oncogenic strains of Agrobacterium tumefaciens and A. rhizogenes. A marked shift in the expression of some organ-specific genes occurred in crown galls derived from the central zone of tubers: two novel isozyme genes (Est-B and Pox-E) were expressed, two others (Est-C and Pox-F) were suppressed and the remaining ones were maintained in the original state. When the starting tissue was the stem segment, a smaller shift occurred, namely the activation of Adh-A and the suppression of Pox-F. In all cases, the isozyme profiles characterizing all crown galls, whatever their origin, were identical. Under normal aeration conditions, Adh-A was not expressed in either tumoral or non-tumoral roots. However, under the relative anaerobic conditions of in vitro cultures, a difference existed between both types of roots: Adh-A was expressed in normal but not in tumoral roots. This means that hairy roots can tolerate higher levels of anaerobiosis without giving rise to an anaerobic response. For the remaining isozymes, no alteration occurred in either organized (hairy root) or unorganized (crown gall) tumors, as compared to the corresponding non-tumoral tissues (normal root and callus, respectively).     

Detection of nopaline dehydrogenase and lysopine dehydrogenase activities in crown gall tumors of different plant species

The LpDH and NpDH activities in crown gall tumors incited byAgrobacterium tumefaciens strain C58 were followed in 7 plant species and 100 tumors were assayed in each experimental variant. The NpDH activity was found in 790 out of 800 crown galls observed. The LpDH activity was tested, after induction of tumors withA. tumefaciens strain B6-806 and 37400, in three experimental variants. The LpDH activity was found in 290 out of 300 crown galls. In the small fraction of the LpDH and NpDH negative tumors, the activity was possibly actually present, but it was below the limits of the sensitivity of the detection technique used.     

The development of phloem anastomoses between vascular bundles and their role in xylem regeneration after wounding in Cucurbita and Dahlia

The differentiation of phloem anastomoses linking the longitudinal vascular bundles has been studied in stem internodes of Cucurbita maxima Duchesne, C. pepo L. and Dahlia pinnata Cav. These anastomoses comprise naturally occurring regenerative sieve tubes which redifferentiate from interfascicular parenchyma cells in the young internodes. In all three species, severing a vascular bundle in a young internode resulted in regeneration of xylem to form a curved by-pass immediately around the wound. The numerous phloem anastomoses in these young internodes were not involved in this process, the regenerated vessels originating from interfascicular parenchyma alone. Conversely, in mature internodes of Dahlia, the regenerated vessels originated from initials of the interfascicular cambia, and their phloem anastomoses did not influence the pattern of xylogenesis. On the other hand, in old internodes of Cucurbita, in which an interfascicular cambium was not yet developed, the parenchyma cells between the bundles had lost the ability to redifferentiate into vessel elements, and instead, regenerated vessels were produced in the phloem anastomoses. Thus, the wounded region of the vascular bundle was not bypassed via the shortest, curved pathway, but by more circuitous routes further away from the wound. Some of the regenerated vessels produced in the phloem anastomoses were extremely wide, and presumably efficient conductors of water. It is proposed that the dense network of phloem anastomoses developed during evolution as a mechanism of adaptation to possible damage in mature internodes by providing flexible alternative pathways for efficient xylem regeneration in plants with limited or no interfascicular cambium.This paper is dedicated to the memory of the late Isaac Blachmann (deceased 19 November 1995), father-in-law of the senior author, for encouragement and advice throughout the yearsThis research was supported by an International Scientific Exchange Award to R.A. from the Israel Academy of Sciences and The Royal Society.     

Occurrence of Agrobacterium tumefaciens Biovar 3 on Grapevine in Brazil

Occurrence of crown gall on grape was observed in a vineyard in Minas Gerais State, Brazil. Isolations of the pathogen yielded a bacterium which incited galls on grapevine, tomato and bryophillum. The bacterium was confirmed as Agrobacterium tumefaciens and its characterization showed it to be biovar 3. This is the first report on the occurrence of the biovar 3 of A. tumefaciens on grapevine in Brazil.     

Proliferating sieve elements present in bud phloem anastomoses connect sieve tubes of axillary bud traces to stelar vascular bundles in the aquatic monocotyledonPotamogeton natans L. (Potamogetonaceae)

Summary The stem ofPotamogeton natans is characterized by a central stelar vascular system with reduced xylem and abundant phloem. Wide sieve tubes composed of short sieve-tube members joined by simple sieve plates and associated with companion cells establish an effective conduit for assimilates. At each node the phloem forms a network of parallel sieve elements connecting the stem phloem to leaf and bud traces. InP. natans an axillary bud rarely develops into a side branch, its procambial vascular bundles are each connected to the nodal complex via separate anastomoses. Their most unusual components are the anastomosai sieve elements (ANSE), characterized by thin cell walls pitted all over by tiny callose-lined pores resembling plasmodesmata, which can be detected as bright areas by fluorescence microscopy after staining with aniline blue. Several layers of ANSE make up the centre of an anastomosis and link to both the nodal and bud stelar sieve tubes via mediating (MSE) and connecting sieve elements (CSE). The ultrastructural differentiation of ANSE, MSE, and CSE corresponds to that of normal sieve elements, i.e., in the mature stage they are enucleate, evacuolate, and have lost most of their cytoplasm. Their plastids are of form-P2c, containing many cuneate protein crystals, typical of monocotyledonous sieve elements. Quantitative aspects of the pore areas are discussed in relation to the functional significance of bud anastomoses.Abbreviations ANSE anastomosai sieve elements - CSE connecting sieve elements - FM fluorescence microscopy - LM light microscopy - MSE mediating sieve elements - TEM transmission electron microscopy Dedicated to Professor Dr. Rainer Kollmann on the occasion of his retirement     

Modelling the development and arrangement of the primary vascular structure in plants

Background and Aims

The process of vascular development in plants results in the formation of a specific array of bundles that run throughout the plant in a characteristic spatial arrangement. Although much is known about the genes involved in the specification of procambium, phloem and xylem, the dynamic processes and interactions that define the development of the radial arrangement of such tissues remain elusive.

Methods

This study presents a spatially explicit reaction–diffusion model defining a set of logical and functional rules to simulate the differentiation of procambium, phloem and xylem and their spatial patterns, starting from a homogeneous group of undifferentiated cells.

Key Results

Simulation results showed that the model is capable of reproducing most vascular patterns observed in plants, from primitive and simple structures made up of a single strand of vascular bundles (protostele), to more complex and evolved structures, with separated vascular bundles arranged in an ordered pattern within the plant section (e.g. eustele).

Conclusions

The results presented demonstrate, as a proof of concept, that a common genetic–molecular machinery can be the basis of different spatial patterns of plant vascular development. Moreover, the model has the potential to become a useful tool to test different hypotheses of genetic and molecular interactions involved in the specification of vascular tissues.     

Symplastic continuity in Agrobacterium tumefaciens-induced tumours

The Agrobacterium tumefaciens-induced plant tumour is regarded as a strong sink, containing a well-developed vascular system that guarantees an efficient supply of water and nutrients from the host plant into the tumour. The phloem transport and unloading of the fluorescent dye carboxyfluorescein (CF) was studied to examine the potential pathways for unloading of a low-molecular-mass solute, and was compared with the symplastic movement of potato virus X expressing a green fluorescent protein-coat protein fusion (PVX.GFP-CP). The distribution of both CF and PVX.GFP-CP in the host plant, Nicotiana benthamiana, demonstrated a clear symplastic pathway between the phloem of the host stem and the cells of the tumour, and also a considerable capacity for subsequent cell-to-cell transport between tumour cells. This same pattern of CF transport was also demonstrated independently for the host species Cucurbita maxima and Ricinus communis. In addition to entering the tumour, CF and PVX both moved through the vascular rays of the host stem towards the stele. The results confirm that host and tumour tissues in the Agrobacterium gall are in direct symplastic continuity and emphasize an important symplastic pathway for radial solute transport in stems.Key words: Agrobacterium tumefaciens, carboxyfluorescein, GFP, symplastic phloem unloading, plant tumour, vascular rays      

Growth requirements ofArabidopsis thaliana crown galls

Crown galls induced onArabidopsis thaliana plants by octopine or nopaline strains ofAgrobacterium tumefaciens were grownin vitro on different media. Dark growth of all tumor tissues was strictly hormone-dependent. In contrast, hormonal autonomy was observed in the light where crown gall calli readily differentiated into teratomas and (sometimes fertile) plants. Differentiating tissues always grew more vigorously than subtended calli. The growth of transformed calli was stimulated by vitamins and partly inhibited by growth regulators in concentrations used for the maintenance of untransformed calli. Crown gall calli, teratomas and sometimes regenerated plants were shown to express lysopine or nopaline dehydrogenase activities.     

The Galls on Forsythia intermedia Zab.

The galls on Forsythia intermedia Zab. consist of much-branched,root-like structures embedded in corky material which appearsto be sloughed-off from them. These gall ‘roots’have 7 to 14 vascular bundles compared with the 5 of normalroots and contain much more IAA. When galls were incubated in damp chambers the gall ‘roots’extended their growth. These extensions had a root-cap and root-hairsbut also had more vascular bundles than normal and they didnot respond geotropically like normal roots. Two fungi, Gibberella baccata (Wallr.) Sacc. (conid, stat. Fusarlumlateritium Nees.) and Phomopsis dominici Trav. were associatedwith die-back of gall ‘roots’ and two bacteria resemblingCorynebacterium fascians (Til.) Dowson and Agrobacterium tumefaciens(Sm. and Towns.) Conn respectively were isolated from galls.All produced IAA in culture media but their role in the etiologyof the gall, if any, remains in doubt.     

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.     

Biosynthesis and bioactivity of trichosanthin in cultured crown gall tissues of Trichosanthes kirilowii Maximowicz

Trichosanthin (TCS) from Trichosanthes kirilowii Maximowicz (T. kirilowii) can be used to treat choriocarcinoma. In this work, we established a novel system to produce TCS in crown gall tissues of T. kirilowii infected by Agrobacterium tumefaciens C58 (A. tumefaciens). In the crown gall tissues, a nopaline synthase (NOS) gene of A. tumefaciens was identified by polymerase chain reaction (PCR), and nopaline accumulation was confirmed by a high-voltage filter paper electrophoresis. Furthermore, we optimized conditions to culture the crown gall tissues able to grow fast and produce TCS in an auxin-free medium, and found that a fungal elicitor of Armillaria mellea was capable of stimulation of TCS secretion into the medium. Moreover, we identified that the TCS purified from the crown gall tissues could induce gastric cancer cell death. These data underscore the usefulness of our system as an inexpensive and virtually unlimited source of TCS.     

Quantitative differences detected in the histology of galls induced by the same aphid species in different varieties of the same host

• Plant galls are abnormal growths caused by an inducer that determines their morphology and anatomy. We qualitatively and quantitatively compared the histological anatomy of five aphid species (Paracletus cimiciformis, Forda marginata, Forda formicaria, Baizongia pistaciae and Geoica wertheimae) that induce galls in Pistacia terebinthus shrubs growing in Israel. We also quantitatively compared these galls to those that the aphids create on the same host in Spain.
• Histological study was conducted following methods described previously by the authors.
• Quantitative differences among the galls were found in five of 12 common anatomical traits: gall thickness, stomatal number in the epidermis‐air, size of vascular bundles, distance of phloem ducts from the lumen and number of intraphloematic schizogenous ducts. Other structures were particular to one or some species: number of cracks in the epidermis–lumen, a sclereid layer, trichomes and microcrystal inclusions. Fisher's tests of combined probabilities showed that the galls induced in Israel were statistically different from those in Spain. In particular, the number of intraphloematic schizogenous ducts was higher in the galls induced in P. terebinthus in Israel. Such differences were also found in other traits related to defence of the gall inhabitant.
• In conclusion, while the gall shape and size are determined mainly by the cecidogenic insect, it seems that the host plant also plays an important role in determining the number/size of quantitative traits, in this case mainly protective structures.

     

Induction of crown galls by Agrobacterium tumefaciens (strain C58) reverses assimilate translocation and accumulation in Kalanchoë daigremontiana

The mechanism of hormone-enhanced solute accumulation was investigated in crown galls of Kalanchoë diagremontiana, induced by Agrobacterium tumefaciens (C58). Electrophysiological, cytological staining, and ¹⁴CO₂-tracer techniques were used. Intracellular auxin and zeatinriboside concentration increased in tumours by three and five times, respectively, compared with the mesophyll. In contrast, the electrical membrane potential difference, in particular the energy-dependent component across the plasma membrane of tumours was at least 60 mV smaller than that of mesophyll cells. The detection of functional sieve tubes, metabolically active companion cells and also developed xylem vessels correlated well with evidence for reversed, long-distance, ¹⁴C-labelled assimilate flow from CAM–performing mesophyll to sugar and potassium-accumulating tumours. The change in hormone production, encoded by the iaaM, iaaH and ipt T-DNA genes, apparently induced functioning vascular bundles and established a strong sink. From a comparison with tobacco suspension cells (SR1 and SR1-C58), the changes in the amino acid pattern in the mesophyll/tumour complex can be attributed to the enzymatic activities incited by the nos encoding gene. Differences in sugar and inorganic ion content were not apparent in the transformed suspension cells, indicating that the increase in extracellular concentration by enhanced phloem unloading may be the crucial factor for the pathological solute accumulation in the tumours.     

A cytochemical study of acid phosphatases and esterases during the induction of crown gall in the tomatoLycopersicon esculentum

Synopsis A cytochemical study has been made of acid -glycerophosphatase, acid naphthol-AS-BI-phosphatase and naphthol esterase activities during the induction of crown gall inLycopersicon esculentum byAgrobacterium tumefaciens. There was an increased activity of these enzymes with time after infection, being more marked for the phosphatases than the esterases. It is suggested that the esterase activities may play a part in cell wall development of the maturing gall.     

Phytohormones in the formation of crown gall tumors

Crown gall tumors were initiated in a variety of plant species by infection with Agrobacterium tumefaciens strain B6 and the concomitant changes in the tissue levels of phytohormones, mainly indole-3-acetic acid (IAA) and cytokinins, were analyzed. A comparison was made of these hormones with those produced by virulent and avirulent strains of the bacterium in liquid culture and with those of bacteria-free crown gall callus cultures. Specific radioimmunoassays were employed for hormone determinations. An assay for the quantitation of femto-mol amounts of isopentenyladenosine and related cytokinins was newly developed and is described in detail. The results can be summarized as follows: Virulence in strain B 6 is associated with the ability to release trans-zeatin and increased amounts of IAA into the surrounding environment. In many, but not all plants analyzed, the development of crown gall tumors is also associated with a sharp rise in the levels of trans-zeatin-type zytokinins and IAA (e.g., Euphorbia lathyris, Catharanthus roseus). Crown gall calli growing on hormone-free media varied greatly in their cytokinin levels. In a culture of Nicotiana tabacum, both trans-zeatin and isopentenyladenine or related cytokinins were not detected. Thus, tumor growth cannot be explained on the basis of elevated levels of IAA and/or cytokinins alone.Abbreviations ABA abscisic acid - GC-MS gas chromatography-mass spectroscopy - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - RIA radioimmunoassay - TLC thin layer chromatography Part 19 in the series Use of immunoassay in plant science