Production of Indole-3-Acetic Acid by Rhizobium phaseoli 8002 in Relation to Nodulation of Phaseolus vulgaris L

Smith, Janice A (1992) Production of Indole-3-Acetic Acid by Rhizobium phaseoli 8002 in Relation to Nodulation of Phaseolus vulgaris L. PhD thesis, University of Glasgow.

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The research reported in this thesis was concerned with the production of indolic compounds, particularly indole-3-acetic acid (IAA) by the nitrogen-fixing bacterium Rhizobium phaseoli strain 8002 and its leguminous host, Phaseolus vulgaris. These organisms are involved in a highly complex symbiosis in which bacterial IAA production has been postulated as playing an important physiological role, either in the early stages of infection or later during the development and maintenance of the mature nodule. Experiments were designed in particular to determine whether (a) differences in pathways of IAA metabolism between bacterium and host plant might be used as 'markers' for IAA production by the bacterium in the symbiotic state and (b) whether there is a correlation between IAA production by Rhizobium and the ability to nodulate the leguminous host plant. The first part of this project examined production of indoles by Rhizobium phaseoli in culture in tryptophan-supplemented and minus media. The spectrum of indoles detected was identical in both (+) and (-) media, with indole acetic acid (IAA), indole lactic acid (ILA), indole ethanol (IEt), indole aldehyde (IAId) and indole methanol (IM) being identified by reversed-phase and normal phase HPLC. MS analysis confirmed the presence of IAA, ILA, IEt and IAId. The tryptophan-supplemented cultures however, showed a large increase in accumulation, particularly of IAA, IEt and ILA. Labelled metabolic feeds of [3H]-tryptophan, the precursor of IAA, to Rhizobium in culture, resulted in the production of labelled IAA, ILA, IEt, IAId and IM, while feeding with [14C]-IAA, led to production of labelled IEt, IM and IAId. Preliminary results investigating synergism between IAA and gibberellic acid (GA3), both of which are synthesised by the bacterium, indicated an increase in production at low GA3 concentrations but this effect was not reproducible in subsequent experiments. The second part of the research focused on the feeding of radiolabelled IAA and tryptophan to detached root and nodule segments of Phaseolus vulgaris in order to compare the spectrum of indolic compounds produced with the bacterium. The addition of radiolabelled tryptophan gave rise to labelled IAA, ILA, IEt, IAId and IM in nodule tissue, while in roots no indolic compounds were detected. The addition of [14C]-IAA resulted in the production of labelled IM and IAId in both root and nodule tissue. The spectrum of metabolic products in roots and nodules was therefore identical to those produced by the bacterium in vitro and hence it was impossible to discriminate between bacterial and host IAA production in the nodule. From the identification of indoles secreted by R. phaseoli and the analysis of metabolism of radiolabelled tryptophan and IAA, it was postulated that the pathway of IAA synthesis from tryptophan is via IPyA and IAAId, and additionally that IAA is catabolised by sequential decarboxylative oxidation, involving loss of the acidic side chain, to IAId and IM. The final part of the project involved the isolation of R. phaseoli mutants with altered levels of IAA accumulation using transposon mutagenesis. A number of colonies with increased or decreased IAA accumulation in the medium, significantly different from the wild-type, were isolated. Hybridisation showed that transposon DNA was present only in the mutant DNA and that transposition, not plasmid recombination, was responsible for the mutations observed. Differences between the lowest and the highest IAA-producers and the wild-type were of the order of 4.3-fold higher and I00-fold lower per litre of culture medium. Analysis with time of the mutants with the highest and lowest IAA accumulation revealed that the differences were significant at all stages of culture growth. These strains were inoculated onto Phaseolus vulgaris but no significant differences were found for nitrogen fixation and dry weight between plants inoculated with wild-type and mutant bacteria. The wild-type however, was apparently more infective than the mutant strains. Re-isolation of the cells from the nodules revealed that only a residual resistance to neomycin remained in the population and additionally, there were no significant differences between re-isolated wild-type and mutant (neomycin resistant and sensitive mixture) bacteria in IAA accumulation. It appeared therefore that the Tn5 had been lost from the mutant cells through multiplication in the rhizosphere or inside the root, where there was obviously no antibiotic selective pressure. This may have resulted in the loss of the transposon and the reversion of the mutant to the wild-type. The foundations have been laid for further work on Rhizobium mutants with altered IAA levels. It is clear however that a mutation stable from generation to generation, created through either chemical mutagenesis or a transposon with improved stability, will be required for future mutant studies in planta.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Chris Wheeler
Keywords: Microbiology, Plant sciences
Date of Award: 1992
Depositing User: Enlighten Team
Unique ID: glathesis:1992-75015
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 13 Nov 2019 15:58
Last Modified: 13 Nov 2019 15:58

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