Sucrose metabolism during Agrobacterium tumefaciens — induced tumor growth in sunflower hypocotyls

JOURNAL OF PLANT PHYSIOLOGY

J Plant Physiol. Vt11. 157. pp. 1-6 (2000) http://www.urbanfischer.de/journals/j pp

© 2000 URBAN & FISCHER Verlag

Sucrose metabolism during Agrobacterium tumefaciens induced tumor growth in sunflower hypocotyls U. Kutschera*, M. Bahrami, R. Grotha FB 19 Pflanzenphysiologie, Universitiit Kassel, Heinrich-Plett-Str. 40, 0-34109 Kassel, Germany Received November 3, 1999 . Accepted December 30, 1999

Summary

Tumor growth induced by the wild-type strain B6 of Agrobacterium tumefaciens in hypocotyls of sunflower seedlings (Hefianthus annuus L.) was investigated with respect to the catabolism of imported sucrose. Tumor development was associated with a large increase in dry mass of the infected stem region and a cessation of hypocotyl growth. In non-infected control seedlings the hypocotyls continued to elongate. 5ucrolysis in the growing tumor tissue was analyzed. The enzyme soluble (vacuolar) acid invertase (EC 3.2.1.26) was found to represent the dominant sucrose breakdown activity, whereas the specific catalytic activity of sucrose synthase (EC. 2.4.1.13) was very low. During tumor growth the level of acid invertase displayed a large increase. In the control, a decrease in the activity of this enzyme was measured. The time-course changes in the concentrations of sucrose, hexoses (glucose and fructose) and the osmolality of the tissue sap were determined during tumor development. The data indicate that the growing tumor cells import and cleave large amounts of sucrose, i.e., they represent a strong sink. As a result, in the infected sunflower seedlings, sucrose may no longer be available in sufficient amounts for the maintenance of stem elongation.

Key words: Agrobacterium tumefaciens, acid invertase, sucrose metabolism, tumor growth. Abbreviations: A.t. = Agrobacterium tumefaciens; INV = soluble (vacuolar) acid invertase; 55 = sucrose synthase.

Introduction

In the majority of green plants, the non-reducing disaccharide sucrose is the form in which fixed carbon is translocated via the phloem from the photosynthetic tissues (leaves) to the non-photosynthetic cells of the organism (stem, roots). According to current terminology, the green, light-exposed leaves represent the source, whereas the heterotrophic tissues are the sinks of the plant. 5ucrose molecules can enter the cytoplasm of a plant cell either through plasmodesmata from another cell or via membrane-associated hexose carriers after hydrolysis. Both symplastic and apoplastic pathways of sucrose import have been demonstrated to occur in various plant species (Hawker et al., 1991). Two major alternatives are available * Correspondence: e-mail: [email protected]

for the breakdown of the imported sucrose. Acid invertase (INY, E.C. 3.2.1.26) is an enzyme that exists in both an intracellular soluble (vacuolar) and in an extracellular (cell-wallassociated) form. It catalyzes an irreversible hydrolytic reaction that yields the hexoses glucose and fructose. 5ucrose synthase (55, E.C. 2.4.1.13) catalyzes a reversible reaction but preferentially converts the disaccharide into fructose and uridine diphosphate (UDP)-glucose. The relative roles ofINV and 55 in the catabolism of sucrose in various sink tissues is still a matter of ongoing debate (Wang et al., 1993). The relationships between cell elongation and sucrose catabolism in developing sunflower seedlings has been analyzed in detail in our laboratory. In the growing region of the hypocotyl both soluble INV and 55 were found to be active enzymes of sucrose breakdown. However, upon irradiation of etiolated seedlings with continuous white light the specific 0176-1617/001157/01 $ 12.00/0

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U. Kutschera, M. Bahrami, R. Grotha

catalytic activity of INV was reduced to a large extent, whereas 55 was only slightly affected (Pfeiffer and Kutschera, 1995). In general, the rate of cell enlargement appears to be positively correlated with the activity of soluble INY, but not to that of 55. In contrast, during development of fruits such as tomatoes and potato tubers, 55 rather than INV is the dominant enzyme in metabolizing imported sucrose (5ung et aI., 1988; Wang et al., 1993). 5unflower plants were the first experimental system for the study of the growth of Agrobacterium tumefociens (A.t.)-induced hypocotyl tumors (Braun, 1941; Beiderbeck, 1977). It has been suggested that these plant tumors, which can be regarded as a mass of growing cells with a characteristic three-dimensional structure (Bopp and Leppla, 1964; Aloni et al., 1995), are strong sinks for sucrose (Pradel et al., 1999). Although the role of the wallassociated INV in A.t.-induced tumors in Ricinus-stems and leaves of Kalanchoe has been investigated (Pradel et al., 1996; 5churr et al., 1996), the question of whether the soluble INV or SS represents the dominant enzyme of intracellular sucrose metabolism during tumor growth in sunflower seedlings has not yet been analyzed. The objectives of the present work were to investigate the kinetics of INV- and 55-activities during tumor growth in juvenile sunflower plants. 5ince no recent data on the growth of A.t.-induced hypocotyl tumors are available we have included some data on the physiology of the infected seedlings and the transformed tumor tissue.

Materials and Methods Plant material Seeds (achenes) of sunflower (Helianthus annuus L. cv. Giganteus) were imbibed for 1 h in water and thereafter germinated in vermiculite that was soaked with Y2 strength Hoagland nutrient solution (Kutschera, 1990). Growth of the juvenile plants took place over a period of 16 days in open plastic trays in a controlled environment chamber at 25 ± I ·C with a 12 h : 12 h (light: dark) schedule as described by Jucknischke and Kutschera (1998). During the light period the photosrnthetic photonflux density was approximately 70 Ilmol. m- 2 • s- at plant level. To maintain a constant water potential in the vermiculite the seedling trays were watered once per week. The relative humidity within the growth chamber was about 70 %. Harvests for the determination of organ size, fresh mass, dry mass, osmotic concentrations, enzyme activities and sugar concentrations were taken at the same time each day (between 8: 15 and 9: 15). Growth measurements All measurements were carried out between days 8 and 16 after sowing. The lengths of the hypocotyls were determined with a ruler; for determination of the diameter in the sub-apical region (see Fig. 1 A) a micrometer was used. Fresh mass and dry mass of excised hypocotyl segments, 2 cm in length, were determined as described by Jucknischke and Kutschera (1998). Bacteria and inoculation The wild-type strain B6 of Agrobacterium tumefociens (octopine catabolizing) was obtained from Dr. R. Beiderbeck (Botanisches Institut, Universitat Heidelberg, Germany) and cultivated on Agarplates. One day before inoculation, bacteria were grown in a sterile

YEB medium (5 g beef extract, 1g yeast extract, 5 g peptone, 5 g sucrose/L). For the induction of virulence, the bacteria were treated with acetosyringon (4-Acetyl-2,6-dimethoxyphenol, 100llmollL) for 12 h. For inoculation, a syringe was used. The bacteria (5 ILL of suspension, 1,8· 10 9 colony forming units/mL, optical density = 1,0) were injected into the sub-apical region of the hypocotyl 3 em below the onset of the cotyledons (Fig. I A). For all experiments of this study 6-day-old seedlings of average size were selected. In the contro\, 5 ILL of nutrient broth (without bacteria) was injected as described above. Enzyme assays Sections, 2 em in length, were excised from the hypocotyls (Fig. 1A) and collected on ice. The tissue (1 g of fresh mass per sample) was homogenized using a mortar and pestle, which contained 5 mL of extraction buffer (5 mmollL EDTA, 100 mmollL L-cysteine, 5 % [w/vl glycine, 100 mmollL Tris/HC\' pH 7,6) and 0.1 g of quarz sand. Extraction of soluble enzymes was carried out as described by Pfeiffer and Kutschera (1995). The protein concentration in the extract was determined by the method of Bradford (1976). For determination of the activities of soluble INV and 55 0.2 mL of the same extract (eluate) was used. The activities of both enzymes were measured in two-step spectrophotometric assays (Pfeiffer and Kutschera, 1995). Oxidation or reduction of NAD(P)+/NAD(P)H+H+ was measured at 340 nm. All enzyme activities were corrected against controls (eluate that was kept for 5 min in a boiling water bath). The specific catalytic activities of the two enzymes were calculated and expressed in the 51-unit nkatal/mg protein. All enzymes and substrates were purchased from Boehringer, Mannheim, Germany. Determination of osmotic concentrations Hypocotyl sections, 2 cm in length, were excised from the seedlings as indicated in Fig. I A. The organ fragments (0.5 g of tissue) were cut into small pieces, frozen and stored at -20·C. The samples were thawed, homogenized and centrifuged. The osmotic concentrations of the supernatants (tissue saps) were determined cryoscopically as described by Kutschera (1991 a, b). Determination ofsucrose and hexoses

The concentration of sucrose was determined by the method of Jones et al. (1977). Freshly harvested hypocotyl segments (1 g) were homogenized in 2.5 mL of NaOH (20 mmollL). The extracts were heated for 30 min, placed on ice and centrifuged for 10 min at 10.000 gn. Aliquots of 300 ILL were used for measurement of sucrose concentration (Jones et aI., 1977). For determination of glucose and fructose tissue saps were prepared as described by Kutschera (1991 a, b). The concentrations of glucose and fructose were determined spectrophotometrically using a diagnostic kit (Boehringer, Mannheim, Germany). Statistics All data points shown in Figs. 2-6 are the means of 4-8 independent measurements. The estimated standard errors of the means are only shown if they exceed the size of the symbols.

Results

Growth measurements The morphological changes in the sub-apical region of the hypocotyl of seedlings that were inoculated with the wild-

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Sucrose metabolism and tumor growth type strain B6 of Agrobacterium tumefaciens (A.t.) are depicted in Fig. 1 B. At day 8 after sowing (2 d after infection) a hole can be detected in the hypocotyl, which was caused by the tip of the needle. Growing tumor tissue was observed by day 10 after sowing; thereafter, a tuber-shaped hypocotyl tumor developed and replaced the epidermal cell layer in the infected plants. Fig. 2A shows that the hypocotyls of the control seedlings elongated at a rate of approximately 2.5 mml day over the time interval studied here. This demonstrates that the physical damage caused by the injection (nutrient broth - Bacteria) at day 6 after sowing did not cause a disturbance of growth. The infected stems ceased to elongate by day 12 after sowing. In the sub-apical region of infected hypocotyls a steady increase in stem diameter was measured (tumor growth); only minor changes were observed in the control (Fig.2B). The fresh masses of excised 2 em-segments that contained the growing hypocotyl tumor were significantly larger than that of the uninfected control (Fig. 3A). It is obvious that the fresh mass increase measured in the uninfected hypocotyls was in part attributable to the thickening of the stem (see Fig. 2 B). The time-course changes in dry mass of the hypocotyl segments are shown in Fig. 3 B. In the control this parameter remained largely constant. At day 6 after infection (day 12 after sowing) a large increase in dry mass occurred. A comparison with Fig. 2 A reveals that this dry matter accumulation of the 2 em-segment caused by the growing tumor cells is correlated with the cessation of hypocotyl elongation. In addition, we have measured the accumulation of fresh- and dry mass of entire seedlings. No significant differences between infected and control plants were observed (data not shown). In summary, our growth measurements demonstrate that the A.t.-induced tumor had a large inhibitory effect on stem growth in the juvenile sunflower plant.

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Fig. 1: Morphology of a 12-day-old sunflower seedling that was infected with the wild-type strain B6 of Agrobacterium tumefaciens at day 6 after sowing (A). The 2 em-regions that were cut from the hypocotyls and used for analysis are indicated (bars). Time course of tumor growth in an infected stem between days 8 and 16 after sowing (B).

The changes in the levels of soluble acid invertase (INV) and sucrose synthase (55) were followed throughout the entire time period of tumor growth. At each time point infected samples and uninfected controls were collected and analysed within 3 h after harvest. In both types of plants the specific catalytic activities of INV were much larger than those of 55: The enzyme levels were in the range 5-14 nkatallmg protein (INV) and 0.7-1.8 nkatallmg protein (55), respectively (Fig. 4A, B). These data demonstrate that INV and not 55 is the dominant enzyme of sucrose cleavage during A.t.-induced tumor growth. In the controls the level of INV steadily declined, whereas in the infected stems a large increase in the

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specific activity of the enzyme occurred. The level of INY in the infected hypocotyl reached a maximum at days 12-14 after sowing and thereafter declined. Although the specific activity of SS was much lower than that of INY a significant A.t.-induced rise in the level of this enzyme was apparent (Fig. 4 B). These data demonstrate that tumor growth is accompanied by substantial increases in the levels of both enzymes of sucrose catabolism.

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Time-course changes in the concentrations ofsucrose and hexoses In the control, sucrose concentrations were low throughout the period of hypocotyl elongation (Fig. 5 A). At day 4 after infection (day 10 after sowing) a large increase in sucrose level was measured that reached a maximum at day 12 after onset of seedling development. Sucrose levels declined on the

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subsequent days but remained significantly larger than in the uninfected control. Fig. 5 B shows the changes in the osmotic concentrations of tissue saps obtained from infected and uninfected hypocotyl segments. Tumor growth was associated with a large increase in tissue sap osmolality, indicating that the growing cells accumulate significant amounts of various osmotica within their vacuoles. The changes in the concentration of glucose are shown in Fig. 6. In the infected stems a transient increase in the level of glucose occurred, whereas in the control a steady decline was measured. The tissue concentration of fructose was low and remained approximately constant over the time period studied here. However, in the infected hypocotyls a significant rise in fructose concentration was detected. The levels of both hexoses reached a maximum at day 12, which coincides with the increase of sucrose concentration (Fig. 5 A), and thereafter declined.

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Sucrose metabolism and tumor growth Discussion

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Several studies have documented a close aSSOCIation between cell elongation and the activity of INV (Pfeiffer and Kutschera, 1995; Rabe and Kutschera, 1998). Recent data indicate that this positive correlation may only be established in the case of the soluble (vacuolar) form of the enzyme. In sunflower seedlings, the specific catalytic activity of the wall-associated (ionically bound) INV is not correlated with the growth rate of the cells (Rabe and Kutschera, 1999). We have therefore restricted our experimental analysis to the soluble (vacuolar) form of this enzyme. For comparison, the specific catalytic activity of the second enzyme for sucrose breakdown (55) was also measured.

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Fig. 5: Time-course changes in the concentration of sucrose (A) and the osmotic concentration of the tissue sap (B) during Agrobacterium tumtfociens-induced tumor growth. Hypocotyl segments, 2 cm in length, were cut from the stems and analysed. Closed symbols: infected, open symbols: uninfected seedlings.

Our results indicate that, during the early phase of tumor development, INV and not 55 appears to be the enzyme responsible for the catabolism of imported sucrose. Moreover, the level of INV displays a large rise over the first 4 days of tumor development (Fig. 4). The positive correlation between INV-activity and induction of tumor growth indicates that this enzyme of sucrose catabolism may playa causal role in the chain of events that leads to the tumorous growth of the hypocotyl cells. Sung et al. (1988) postulated that in plant organs that grow by cell elongation (stems, roots) INV may be the dominant enzyme of sucrose catabolism, whereas in fruits and storage organs such as potatoes the alternative enzyme (55) predominates. Subsequent experiments with growing potato tubers, developing tomato fruits and sunflower seedlings corroborated this hypothesis (e.g. Wang et aI., 1993; Pfeiffer and Kutschera, 1995; Rabe and Kutschera, 1998). Based on the morphological changes of the A.t.-infected hypocotyl region (Fig. 1) one would predict that the growing plant tumor behaves more like a tuber than an elongating axial organ. In addition, the results of Weiler and Spanier (1981) demonstrate that tumor growth in stems of Helianthus is accompanied by a large increase in the level of extractable cytokinins. In contrast, the concentration of auxin (indole-3-acetic acid) remained constant. Our data show that this prediction is not correct. In accordance with Sung et al. (1988) we have to conclude that, owing to the large INV- and the very low 55-activities, the growing tumor displays the biochemical characteristics of an organ that grows by cell elongation. A detailed structural analysis of the different cells of the growing tumor in the Helianthus hypocotyl is necessary in order to further explore this three-dimensional (isotropic) growth process.

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u. Kutschera, M. Bahrami, R. Grotha

Pradel et al. (1996) analyzed several aspects of A.t.-induced stem tumor development in mature plants of Ricinus communis. In the tumor tissue the activities of both types of invertases (cell-wall-associated and vacuolar) were found to be much higher than in the stem regions above and below (noninfected control). However, the activity of the second enzyme of sucrose catabolism (55) was not determined. Schurr et al. (1996) reported that, in the experimental system described above, tumor growth caused a disruption of the epidermis of the infected stem. As a result, no cuticle was present for the inhibition of water loss via transpiration of the ball-shaped tumor. Based on water- and gas exchange measurements these authors concluded that the tumor must be regarded as a strong water sink on the host plant. Our experiments with developing sunflower seedlings that were infected in the middle of the growing hypocotyls yielded a similar result. Between days 14 and 16 after sowing the growing cells of the cortex pierced the epidermis, resulting in a mass of tumor tissue that was not protected by a cuticle (unpublished observations). Hence, transpiration via the tumor cells may be significant. The concomitant inhibition of hypocotyl elongation (Fig. 2 A) may in part be due to the fact that the growing (sub-apical) cells of the stem are no longer supplied with a sufficient amount of water. A second cause of the tumor-induced inhibition of hypocotyl elongation is certainly due to the fact that the growing tumor cells represent an additional metabolic sink tissue along the axis of the developing sunflower plant. Our data show that the osmotic concentration of the tissue sap obtained from the 2-cm-region that contains the developing tumor was higher than in the control. This accumulation of osmotic solutes during tumor growth (Fig. 5 B) is in part due to the uptake of sucrose and glucose by the tumor cells (Figs. 5A, 6). However, sugar accumulation was transient, reaching maxima by day 12 after sowing, whereas solute accumulation rose until day 14 and thereafter remained constant. We suggest that other osmotica that accumulate, such as potassium ions, are responsible for the rise in osmotic concentration of the tumor cells. Pradel et al. (1996) have shown that A.t.induced tumors on Ricinus stems contain large amounts of K+ -ions. Corresponding data for sunflower are not available. The question why the levels of sucrose and glucose decline between days 12 and 16 after sowing is open. We suggest that these sugars are used for biosynthesis of cell walls and other constituents of the growing tumor. This assumption is supported by the fact that during the period of sugar loss the dry mass of the tumor displayed a large increase (Fig. 3 B). The imported sucrose and the resulting hexoses may have provided the substrate for this mass of newly synthesized plant tissue. Classical biochemical studies on human and animal tumors revealed that these rapidly growing cells display a high rate of glycolysis under aerobic conditions (Dang and Semenza, 1999). The question of whether or not plant tumors show a similar change in cell metabolism has long been under discussion (Beiderbeck, 1977). This problem is currently under investigation. Acknowledgements

We thank Mrs. B. Teubert for technical assistance. This work was supported by the Fonds der Chemischen Industrie.

References

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