Localization of wound-tumor virus antigen in plant tumors by the use of fluorescent antibodies.
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A cooperative reinvestigation of the possibility that filtrates of crowngall bacterial cultures can induce autonomous tumors in plants has been conducted. The results indicate that the neoplasms formed after treatment do not fulfill the criteria for crown gall since they are not autonomous.
Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.
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The development of Agrobacterium tumefaciens-induced plant tumors primarily depends on the excessive production of auxin and cytokinin by enzymes encoded on T-DNA genes integrated into the plant genome. The aim of the present study was to investigate the involvement of additional phytohormone signals in the vascularization required for rapid tumor proliferation. In stem tumors of Ricinus communis L., free auxin and zeatin riboside concentrations increased within 2 weeks to 15-fold the concentrations in control stem tissue. Auxin and cytokinin immunolocalization revealed the highest concentrations within and around tumor vascular bundles with concentration gradients. The time-course of changes in free auxin concentration in roots was inversely correlated with that in the tumors. The high ethylene emission induced by increased auxin- and cytokinin correlated with a 36-fold accumulation of abscisic acid in tumors. Ethylene emitted from tumors and exogenously applied ethylene caused an increase in abscisic acid concentrations also in the host leaves, with a diminution in leaf water vapor conductance. Jasmonic acid concentration reached a maximum already within the first week of bacterial infection. A wound effect could be excluded. The results demonstrate the concerted interaction of a cascade of transiently induced, non-T-DNA-encoded phytohormones jasmonic acid, ethylene and abscisic acid with T-DNA-encoded auxin and zeatin riboside plus trans-zeatin, all of which are required for successful plant tumor vascularization and development together with inhibition of host plant growth.
A simple method for inserting foreign genes into the T-region of Agrobacterium Ti-plasmids is described. A modified cosmid (pHC 79) was introduced into a predetermined site of the T-region of pTi C58. An Agrobacterium strain harboring this modified Ti-plasmid was used as an acceptor strain into which genes, cloned in pBR322, can be introduced by mobilization from Escherichia coli. pBR322-derived plasmids cannot replicate in Agrobacterium, but can be maintained by integration into the T-region of the modified Ti-plasmids by homologous recombination. This method was used to introduce the genes for ovalbumin and alpha-actin from chicken into tobacco tumors. Southern blotting and re-isolation of the inserted genes by reverse cloning showed that the animal DNA was transferred and integrated into the plant genome without rearrangements. The alpha-actin gene is not transcribed in plant tumors, whereas transcription of the ovalbumin gene was observed, however the initiation point of transcription was different from the one used in the chicken oviduct. The RNA transcribed from the chicken ovalbumin gene is polyadenylated and ranges in size between 2 and 7 kb.
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Agrobacterium tumefaciens-induced tumors of dicotyledonous plants consist of well-defined vascular bundle-like structures originating from transformed cells. The current view that 25% of the tumor cells are transformed has been re-investigated by using beta-glucuronidase (gus)-gene-containing wild-type bacteria (A281 p35S gus-int). Regularly growing stem and leaf tumors showed irregular GUS-staining patterns in the different plant species, Ricinus communis L., Cucurbita maxima L., Vicia faba L. and Kalanchoe daigremontiana Hamet et Perrier. Variable staining and inconsistency between staining and tumor growth suggested an inhibition of gus expression. By polymerase chain reaction (PCR) and reverse transcriptase-PCR analyses it became evident that gus is also integrated into the DNA of unstainable tumor parts but not expressed. These results and area calculations of tissues unable to contain the bacterial transferred-DNA with gus provide strong evidence that in A. tumefaciens-induced tumors most cells, or even all, are transformed, i.e. ca. 100%.
Vegetable tumoral tissue are more radiosensitive than the homologous normal tissue. Tumoral tissue are more stimulated by yeast extract and yeast s-ARN than normal tissue. Yeast extracts and s-RNA extracts from yeast also restore more the growth of tumorous tissue than normal tissue, which were subjected to gamma radiation from 60Co. A mixture of mononucleotides obtained from fractionated s-RNA has the same stimulatory effect. It is suggested that the active principle from yeast and s-RNA is a cytokinin like substance, which have been detected in yeast extract and in soluble ribonucleic acid from yeast.
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