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IN THIS ISSUE

A Genomic Analysis of Tumor Development and Source-Sink Relationships in Agrobacterium-Induced Crown Gall Disease in Arabidopsis

neckardt{at}aspb.org

Agrobacterium tumefaciens is a Gram-negative rod-shaped bacterium that is commonly found in the rhizosphere of many plants, where it survives on root exudates. It will infect a plant only through a wound site (which often occurs in nursery stock through transplanting and grafting and in vineyards through pruning). Galls typically form at the crown (the point at the soil line where the main root joins the stem) but may also develop on secondary or lateral roots and on the main stem and branches above the soil line (see figure).

View larger version (70K): [in this window] [in a new window] [as a PowerPoint slide]   Crown Gall Development in Arabidopsis. (A) An inflorescence stalk was injured at the base just above the rosette before virulent Agrobacterium C58 was inoculated at the wounded site (encircled). (B) A tumor developed 30 to 35 d later. (Figure courtesy of Rosalia Deeken.)

Agrobacterium-based vectors are the most widely used tools for plant transformation in research and commercial applications. Plant transformation vectors are created in the laboratory by removing the tumor- and disease-related oncogenes and retaining only those segments of DNA (the T-DNA border repeat sequences) responsible for T-DNA integration. T-DNA oncogenes include several encoding enzymes that synthesize the plant hormones cytokinin and auxin (indole-3-acetic acid) and novel plant metabolites known as opines (such as octopine, nopaline, and the agrocinopines). The synthesis and release of phytohormones and opines from transformed plant cells causes changes in gene expression and metabolism. Phytohormone overproduction results in the development and proliferation of tumors, and opines are small molecules that provide a carbon and energy source for the agrobacteria.

The study of the development of crown gall disease in plants is important, not only because the disease affects a wide range of dicotyledonous plants (especially those in the rose family, including fruit trees and raspberries as well as roses), but also because of the nature of the developmental changes that occur. Understanding tumorigenesis and crown gall development could provide important insights into plant hormone signaling pathways, carbon and nitrogen metabolism, and source-sink relationships.

In this issue of The Plant Cell, Deeken et al. (pages 3617–3634) provide an in-depth functional genomics analysis of the developmental changes that occur during tumorigenesis in Arabidopsis infected with virulent agrobacteria. To get a comprehensive picture of a T-DNA–induced plant tumor, the authors combined genome-wide expression analysis (using Affymetrix ATH1 arrays) with direct analysis of metabolites in tumor tissue. Tumors were induced by inoculating the nopaline-using Agrobacterium strain C58 to the base of a wounded, very young inflorescence stalk, and tumor tissue was harvested 35 d after inoculation. Wounded but uninfected tumor-free inflorescence stalk segments of the same age served as reference tissue. The micorarray data were obtained from four independent biological replicates each from tumor tissue and nontumorous reference tissue, for a total of eight independent microarray hybridizations.

The authors conducted several experiments to show convincingly that the results obtained reflect changes in plant, rather than bacterial, metabolism and that these changes are the result of plant transformation. Using in situ hybridization with antisense-RNA for the T-DNA oncogene Nopaline synthase (NOS) as probe, the authors estimated that >95% of the cells in the tumor were transformed (e.g., expressed the T-DNA–encoded NOS mRNA). This differs from early reports (completed in the 1970s and 1980s before molecular markers were available) but agrees with the more recent estimate of Rezmer et al. (1999) that 100% of tumor cells are transformed.

Next, Deeken et al. found that Glu, Arg, and Pro were markedly accumulated in tumor tissue. Tumor cells transformed by Agrobacterium C58 synthesize nopaline from Arg, and nopaline is metabolized to Glu via Pro and Orn (Dessaux et al., 1986). However, in Agrobacterium C58 bacterial cultures, amino acid levels were low or below the level of detection. Arg was found in the pelleted bacteria at a concentration approaching that of tumor tissue (0.44 µmol g^–1 pelleted bacteria compared with 0.68 µmol g^–1 of tumor tissue), but the bacterial biomass in tumor tissue is much less than that of plant cells. The authors therefore concluded that the contribution of bacterial Arg to total Arg in tumors should be negligible. Because genes encoding enzymes involved in Arg synthesis were only slightly upregulated in Arabidopsis crown galls, the authors argue that tumor Arg content most likely results from translocation by the host plant via the transpiration stream and/or phloem.

The rapid growth of crown gall tumors creates strong metabolic sinks in plant tissue, and the accompanying changes in gene expression and metabolism paint a vivid picture of this source-sink relationship. The results showed that genes involved in photosynthesis were strongly downregulated, as expected. Genes encoding enzymes of the glycolytic pathway and ethanolic fermentation were strongly upregulated in tumors, consistent with a switch to fermentative energy metabolism. This was further confirmed by an increased ethanol level and the induction of PYRUVATE DECARBOXYLASE1 and ALCOHOL DEHYDROGENASE gene transcripts. These results imply that transformation of plant cells with T-DNA of virulent Agrobacterium is accompanied by a change from autotrophic to heterotrophic metabolism, where ATP production is mainly powered by glycolysis and fermentation.

The main sources of carbon and nitrogen supplied to tumor cells appear to be glucose and amino acids supplied by the host plant. This was substantiated by elevated transcription of cell wall invertase, sucrose synthase, and amino acid transporters accompanying high levels of glucose and amino acids in tumor tissue. Consistent with previous work (e.g., Mistrik et al., 2000), nitrate levels were very low and nitrate reductase activity almost unmeasurable, confirming the notion that elevated amino acid levels are not the result of higher nitrate assimilation. The authors concluded that the nitrogen supply for amino acid and protein biosynthesis in tumors is most likely derived from Gln and Glu, which are translocated by the host plant through the vascular system and imported into tumor cells by amino acid transporters.

All of these changes in gene expression and metabolism likely result from the increased production of cytokinin and auxin hormones in tumor tissue, resulting from expression of genes encoded by the T-DNA. A comparison with the transcriptome of plant cells treated with the auxin indole-3-acetic acid and the cytokinin zeatin for 3 h indicated that the expression of a number of genes related to phytohormone signaling or metabolism that are expressed in tumor tissue might be regulated by auxin and cytokinin. However, hormonal signaling and metabolism were not analyzed in detail in this study, and this is clearly an area that is ripe for further investigation.

Ditt et al. (2006) recently completed a genome-wide microarray study of Arabidopsis cell cultures inoculated with virulent Agrobacterium, with sampling conducted up to 48 h after inoculation. The degree of overlap between the results of this work and that of Deeken et al. was minimal. However, Deeken et al. point out that these differences likely reflect a difference in starting material (cell cultures versus tumor tissue) and timing (24 to 48 h versus 35 d after inoculation). Another study by Veena et al. (2003), in this case using suppressive subtractive hybridization and DNA macroarrays, showed that Agrobacterium infection leads to early induction of plant defense-related genes, but these genes are then repressed during later stages of Agrobacterium-mediated plant transformation. Veena et al. (2003) used several different virulent but nononcogenic Agrobacterium strains, specifically to identify plant genes differentially expressed during Agrobacterium-mediated transformation and to identify factors involved in regulating the transformation process. By contrast, Deeken et al. set out to investigate the process of tumor development. Future work might include samples of tumor tissue collected at earlier time points to reflect the early stages of tumorigenesis and to investigate further the role of hormones in tumor development.

The work of Deeken et al. mainly details the large changes in gene expression and metabolite content that accompany the switch to anaerobic and heterotrophic metabolism characteristic of developing tumors. In many respects, the results confirm what is expected or has been shown previously, but the amount of high-quality data presented (and made publicly available) allow for a comprehensive picture of the development of Agrobacterium-induced tumorigenesis in Arabidopsis and provide a rich resource for further study.

	Footnotes

 
www.plantcell.org/cgi/doi/10.1105/tpc.107.050294

	REFERENCES

TOP REFERENCES

 
Deeken, R., Engelmann, J.C., Efetova, M., Czirjak, T., Müller, T., Kaiser, W.M., Tietz, O., Krischke, M., Mueller, M.J., Palme, K., Dandekar, T., and Hedrich, R. (2006). An integrated view of gene expression and solute profiles of Arabidopsis tumors: A genome-wide approach. Plant Cell 18, 3617–3634.[Abstract/Free Full Text]

Dessaux, Y., Petit, A., Tempe, J., Demarez, M., Legrain, C., and Wiame, J.M. (1986). Arginine catabolism in Agrobacterium strains-Role of the Ti-plasmid. J. Bacteriol. 166, 44–50.[Abstract/Free Full Text]

Ditt, R.F., Kerr, K.F., de Figueiredo, P., Delrow, J., Comai, L., and Nester, E.W. (2006). The Arabidopsis thaliana transcriptome in response to Agrobacterium tumefaciens. Mol. Plant Microbe Interact. 19, 665–681.[Web of Science][Medline]

Gelvin, S.B. (2003). Agrobacterium-mediated plant transformation: The biology behind the "gene-jockeying" tool. Microbiol. Mol. Biol. Rev. 67, 16–37.[Abstract/Free Full Text]

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Nester, E.W., Gordon, M.P., Amasino, R.M., and Yanofsky, M.F. (1984). Crown gall: A molecular and physiological analysis. Annu. Rev. Plant Physiol. 35, 387–413.[CrossRef][Web of Science]

Rezmer, C., Schlichting, R., Wachter, R., and Ullrich, C.I. (1999). Identification and localization of transformed cells in Agrobacterium tumefaciens-induced plant tumors. Planta 209, 399–405.[CrossRef][Web of Science][Medline]

Veena, Jiang, H., Doerge, R.W., and Gelvin, S.B. (2003). Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression. Plant J. 35, 219–236.[CrossRef][Web of Science][Medline]

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An Integrated View of Gene Expression and Solute Profiles of Arabidopsis Tumors: A Genome-Wide Approach

Rosalia Deeken, Julia C. Engelmann, Marina Efetova, Tina Czirjak, Tobias Müller, Werner M. Kaiser, Olaf Tietz, Markus Krischke, Martin J. Mueller, Klaus Palme, Thomas Dandekar, and Rainer Hedrich
Plant Cell 2006 18: 3617-3634. [Abstract] [Full Text]  

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