Abstract
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 β-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 α-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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Abbreviations
- CHS :
-
chalcone synthase
- CLSM:
-
confocal laser-scanning microscopy
- DHF:
-
7,4′-dihydroxyflavone
- DPBA:
-
diphenylboric acid 2-aminoethyl ester
- DR5 :
-
auxin response element
- GH3::GUS :
-
auxin-responsive promoter fused to β-glucuronidase
- IAA:
-
indole-3-acetic acid
- IAAM :
-
tryptophan monooxygenase
- IGS :
-
indole-3-glycerolphosphate synthase
- MTT:
-
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
- NIT :
-
nitrilase
- NAA:
-
α-naphthaleneacetic acid
- NPA:
-
1-N-naphthylphthalamic acid
- pi:
-
post infection
- 2PLSM:
-
two-photon laser-scanning microscopy
- RT–PCR:
-
reverse transcription–polymerase chain reaction
- tt4(85):
-
transparent testa: flavonoid-deficient mutant
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Acknowledgements
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to C.I.U.; M.L. was supported by a Hessen Ph.D. fellowship. We thank Dr. Ulrike Mathesius (Genomic Interactions Group, Research School of Biological Sciences, National University, Canberra, Australia) for the kind gift of transformed white clover plants and stimulating discussions, and Dr. Rosalia Deeken (Würzburg University, Germany) for her spontaneous supply of Arabidopsis (Ler) plants.
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Dedicated to the memory of Professor Wilhelm Simonis (1909–2003)
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Schwalm, K., Aloni, R., Langhans, M. et al. Flavonoid-related regulation of auxin accumulation in Agrobacterium tumefaciens-induced plant tumors. Planta 218, 163–178 (2003). https://doi.org/10.1007/s00425-003-1104-6
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DOI: https://doi.org/10.1007/s00425-003-1104-6