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Essay 22.1

Tumor-Induced Ethylene Controls Crown Gall Morphogenesis

Roni Aloni, Plant Sciences, Tel Aviv University, Israel; Cornelia I. Ullrich, Botany, Darmstadt University of Technology, Germany

April, 2005

Agrobacterium tumefaciens-induced galls produce very high ethylene concentrations. The tumor-induced ethylene is a limiting and controlling factor in gall development. It reduces the diameter of vessels in the host stem adjacent to the tumor and enlarges the gall surface through which high transpiration occurs, thus giving priority in water supply to the growing tumor over the host shoot.

Infection of sensitive plants by Agrobacterium tumefaciens induces crown galls. Tumor growth is initiated by the integration and expression of the T-DNA of the bacterial Ti plasmid within the plant nuclear DNA. The T-DNA encodes enzymes catalyzing the synthesis of high levels of auxin, cytokinin and opines (Zambryski et al. 1989). Such tumors are characterized by high percentage of transformed cells, which may reach up to 100% transformation (Rezmer et al. 1999).

For many years, plant tumors induced by A. tumefaciens were considered unorganized, or only partly organized masses (Sachs 1991). However, an analysis of the three-dimensional pattern of phloem and xylem in Agrobacterium-induced crown galls unveiled a sophisticated vascular network of continuous vascular bundles extending from the host plant up to the tumor margin (Aloni et al. 1995). The development of these bundles indicates release of free auxin by the A. tumefaciens-transformed plant cells up to the surface of the fast-growing tumor.

Agrobacterium vitis-induced crown galls cause poor xylem development in grapevine, which impairs water flow into the young parts of the shoot above the gall (Agrios 1988). Furthermore, crown galls do not regenerate the disrupted epidermis and cuticle to protect against water evaporation (Aloni et al. 1995; Schurr et al. 1996), and the enlarged and unorganized callus shape of the tumor surface increases transpiration substantially at the tumor surface, about 15 times higher compared with host leaves, and 7.5 times higher compared with leaves of noninfected castor bean plants (Schurr et al. 1996; Wächter et al. 2003). In the centripetal direction the crown gall causes the development of pathologic xylem within the host stem characterized by narrow vessels, giant vascular rays and by the absence of fibers (Aloni et al. 1995). These anatomical features have triggered the propounding of the gall-constriction hypothesis (Aloni et al. 1995) to explain the mechanism that gives priority in water supply to the growing gall over the host shoot. This hypothesis proposes that a growing gall retards the development of its host shoot by decreasing vessel diameter in the shoot tissues close to the tumor, which substantially reduces water supply to the upper parts of the shoot. It was further postulated that the controlling signal that induces the narrow vessels in the host is the phytohormone ethylene (Aloni et al. 1995), which is known to reduce the diameter of vessels (Yamamoto et al. 1987). It was also suggested that the high auxin levels induced by the T-DNA-encoded oncogenes stimulate this ethylene production (Aloni et al. 1995). These ideas were experimentally confirmed by showing that tumor-induced ethylene is a limiting and controlling factor of crown gall morphogenesis; very high ethylene levels are produced continuously by growing crown galls during several weeks (Figure 1); up to 140 times more ethylene than in wounded, but not infected control stems, reaching a maximum at five weeks after infection (Aloni et al. 1998; Wächter et al. 1999). The vigorous ethylene synthesis in galls is enhanced by high levels of auxin and cytokinin (Wächter et al. 1999, 2003). Furthermore, this ethylene emission induces the synthesis of considerable concentrations of abscisic acid in the tumor and host leaves; as a consequence, transpiration in the leaves slows down to 10% of that of uninfected plants (Veselov et al. 2003).

Figure 1   Time course of ethylene emission from growing crown galls on stems of castor bean (Ricinus communis) plants, reaching a peak on the fifth week after infection. (Click image to enlarge.)

In wild-type tomato stems, tumor-induced ethylene substantially decreases vessel diameter in the host tissues adjacent to the tumor, but has a very limited effect in the ethylene-insensitive mutant, the Never ripe (Nr) (Aloni et al. 1998). Ethylene promotes the typical unorganized callus shape of the gall, which greatly enlarges the surface (through which high transpiration occurs) of the tumor in the wild-type stems (Figure 2A), while the galls on the Nr shoots have a smooth surface (Figure 2B). The combination of decreased vessel diameter (limiting water flow to the shoot above the gall) in the host stem, and increased tumor surface (promoting water flow into the tumor) ensures priority in water supply to the growing gall (Figure 2C) over the host shoot, whereas an uninterrupted water supply to the upper parts of the Nr shoot through its wide vessels (Aloni et al. 1998) reduces tumor growth, or even inhibits gall development (Figure 2D). Therefore, it is not surprising that inhibitors of ethylene synthesis suppress tumor growth. These results demonstrate that, in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown gall morphogenesis. Thus, the T-DNA-encoded oncogenes, namely iaaH, iaaM and ipt, trigger a cascade of the following phytohormones: auxin, cytokinin, ethylene, abcisic acid, and jasmonic acid, which together with gene 6b expression-dependent flavonoid accumulation promote crown gall growth (Veselov et al. 2003; Schwalm et al. 2003; Galis et al. 2004).

Comparison between the development of plant and animal tumors has shown an analogous requirement for neovascularization in both, suggesting possible strategies for prevention (inhibition of tumor-induced ethylene, or propagation of ethylene-insensitive crops) and therapy (Ullrich and Aloni 2000; Kempf et al. 2002).

Figure 2   Comparison of A. tumefaciens-induced crown galls on wild-type tomato (A and C) and the Never ripe (ethylene insensitive) mutant (B and D) stems. (A and B) Front view of 3-week-old tumor developed on: (A) a wild-type plant showing the typical unorganized callus shape of a young crown gall and the epinastic response of the leaves (a typical ethylene effect) above and below the tumor, and (B) the Nr mutant characterized by the smooth surface and leaves in a normal orientation. Note that the lower half of the gall is protected by epidermis. (C and D) Side view of a 2-month-old crown gall on: (C) a wild-type stem with numerous adventitious roots (white spots), known to be promoted by ethylene, appearing both above and below the tumor, and (D) the Nr mutant showing a degenerated fibrous hard gall and stem almost free of adventitious roots.

References

Agrios, G. N. (1988) Bacterial Galls. In Plant Pathology, G. N. Agrios ed., Academic Press, New York, pp. 483-488.

Aloni, R., Pradel, K. S., and Ullrich, C. I. (1995) The three–dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stem of Ricinus communis L. Planta 196: 597-605.

Aloni, R., Wolf, A., Feigenbaum, P., Avni, A., and Klee, H. J. (1998) The Never ripe mutant provides evidence that tumor-induced ethylene controls the morphogenesis of Agrobacterium tumefaciens-induced crown galls on tomato stems. Plant Physiol. 117: 841-847.

Gális, I., Kakiuchi, Y, Simek, P. and Wabiko H. (2004) Agrobacterium tumefaciens AK-6b gene modulates phenolic compound metabolism in tobacco. Phytochemistry 65: 169-179.

Kempf, V.A.J., Hitziger, N., Riess, T., and Autenrieth, I. B. (2002) Do plant and human pathogens have a common pathogenicity strategy? Trends Microbiol. 10: 269—275.

Rezmer, C., Schlichting, R., Wächter, R., and Ullrich, C. I. (1999) Identification and localization of transformed cells in Agrobacterium tumefaciens-induced plant tumors. Planta 209: 399-405.

Sachs, T. (1991) Callus and tumor development. In, Pattern Formation in Plant Tissues, by T. Sachs. Cambridge University Press, Cambridge, pp. 38-55.

Schurr, U., Schuberth, B., Aloni, R., Pradel, K. S., Schmundt, D., Jähne, B., and Ullrich, C. I. (1996) Structural and functional evidence for xylem-mediated water transport and high transpiration in Agrobacterium tumefaciens-induced tumors of Ricinus communis. Bot. Acta. 109: 405-411.

Ullrich, C. I., and Aloni, R. (2000) Vascularization is a general requirement for growth of plant and animal tumours. J. Exp. Bot. 51: 1951-1960.

Veselov, D., Langhans, M., Hartung, W., Aloni, R., Feussner, I., Götz, C.,Veselova, S., Schlomski, S., Dickler C., Bächmann, K., and Ullrich, C. I. (2003) Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid, auxin, cytokinin, ethylene, and abscisic acid. Planta 216: 512-522.

Wächter, R., Fischer, K., Gäbler, R., Kühnemann, F., Urban, W., Bögemann, G. M., Voesenek, L. A. C. J., Blom, C. W. P. M., and Ullrich, C. I. (1999) Ethylene production and ACC-accumulation in Agrobacterium tumefaciens-induced plant tumours and their impact on tumour and host stem structure and function. Plant Cell Environ. 22: 1263-1273.

Wächter, R., Langhans, M., Aloni, R. Götz, S., Weilmünster, A., Koops, A., Temguia, L., Mistrik, I., Pavlovkin, J., Rascher, U., Schwalm, K., Koch, K. E., Ullrich, C. I. (2003) Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens. Plant Physiol. 133: 1024—1037.

Yamamoto, F., Angeles, G., and Kozlowski, T. T. (1987) Effect of ethrel on stem anatomy of Ulmus americana seedlings. IAWA Bull. N.S. 8: 3-9.

Zambryski, P., Tempé, J., and Schell, J. (1989) Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56: 193-201.