Acknowledgements

This work was supported by the EC project FYSAME (ERBIC18CT960081) and by AUPELF and INRA postdoctoral fellowships for M. Soussi.

USE OF PEA (PISUMSATIVUM L.) MUTANTS IMPAIRED IN ROOT FORMATION TO STUDY THE ROLE OF AUXIN IN NODULE DEVELOPMENT

Z.B. Pavlova1, N.V. Ischenko1, V.A. Voroshilova2, V.E. Tsyganov2, A.Y. Borisov2,

I.A. Tikhonovich2, L.A. Lutova1

1 Dcpt of Genetics, St.-Petersburg State University, Universitetskaya nab. 7/9, 199034, St-Petersburg, Russia

2Lab. of Genetics of Plant-Microbe Interactions, All-Russia Research Institute for Agricultural Microbiology, Podbelsky h. 3, 196608, St.-Petersburg, Pushkin-8, Russia

Studying symbiotic traits of plant hormonal mutants has been proposed as an approach to investigate the functions of phytohormones in the legume-Rhizobium symbiosis. It is well known that root development and especially the response of roots to environmental stimuli are regulated mainly by the plant hormone auxin. Thus, mutants of leguminous plants impaired in root development are the very convenient tools for studying the role of auxin in the symbiotic root nodule formation.

Two allelic mutant lines JI819 and JI1743 (age) characterized by root agravitropic response (Blixt, 1970) and recently obtained mutant line SGEcrt (crt) characterized by curly roots (Tsyganov et al. 2000) were used in the present research. These mutants were compared with parental lines in in vitro culture for exogenous auxin sensitivity. As a result the increased sensitivity to auxins was revealed for all analyzed mutants. Also mutant line SGEcrt has been characterized by two-fold increased content of free IAA (Tsyganov et al. 2000). Thus the significant role of auxin in accomplishment of mutant phenotypes for all characterized mutants has been demonstrated.

The comparative analysis of nodulation ability and nodule histological differentiation of mutants and parental lines was performed. All mutants had significantly decreased (1.8-2.5 times) number of nodules in comparison with parental genotypes after inoculation with effective R. leguminosarum bv. viciae strain CIAM 1026 (Safronova, Novikova 1996). At the same time the nodules of line SGEcrt formed after inoculation by strain R. I. bv. viciae VF39 with constitutive expression of reporter gene gusA did not differ from initial line SGE by their histological differentiation.

Thus, auxin influences nodulation ability but it is unlikely to be involved in control of nodule histological differentiation.

References

Blixt S (1970) Pisum Newslett. 2, 11-12

Safronova, Novikova (1996) J. Microbiol. Methods 24, 231-237

Acknowledgements

The seeds of age lines were kindly provided by Dr M. Ambrose (JI Centre, UK) and strain R. I. bv. viciae VF39 with constitutive expression of reporter gene gusA by Prof. U.B. Prifier (Oekologie des Bodens, RWTH-Aahen, Germany). This work was financially supported by the Netherlands Organization for Scientific Research (NWO) grant 0147-007.017 and Russian Ministry of Education grant E00-6.0-248.

ROLE OF A CATALASE-PEROXIDASE IN RHIZOBIUM LEGUMINOSARUM

K.M. Gray, J.-Y. Chen, A J. Crockford, A.M. Carey, H.D. Williams

Dept of Bio. Sciences, Imperial College of Science, Tech. and Medicine, London, UK

1. Introduction

Catalase levels in Rhizobium leguminosarum biovar phaseoli strain 4292 are cell-density regulated and are controlled by the accumulation of extracellular molecules (Crockford et al. 1995). Catalase activity reaches its peak in mid-exponential phase before declining on approach to stationary phase. This control is different to enteric bacteria where catalase activity reaches its peak in stationary phase. We were interested in investigating the role of catalases in R. leguminosarum.

2. Results

Southern blotting indicated that R. leguminosarum has potential homologs to the E. coli katG and katE genes, which encode a catalase-peroxidase and a monofunctional catalase respectively. A 4.7kb insert, containing a potential R. leguminosarum katG homolog, was cloned and sequenced. Sequencing identified three open reading frames (ORFs), including a divergently arranged unit consisting of an oxyR homolog gene (0.9 kb) and a katG homolog gene (2.2 kb) plus a partial panB homolog (0.8 kb). The R. leguminosarum KatG showed strong homology to Escherichia coli KatG (60.3% similiarity) but strongest homology to catalase-peroxidase from Streptomyces reticuli (66.8% similarity). The ORF adjacent to the katG gene is most similar to the oxidative stress sensing transcription factor OxyR.

A katG::Q mutant was constructed. Two potential mutants were identified as they showed weak bubbling when colonies were flooded with 3% H2O2 compared to the wild type, which is indicative of loss of catalase activity. These two mutants have an 80% decrease in catalase activity compared to the wild type in exponential phase and activity stained gels indicated that they were defective in the production of a catalase-peroxidase (KatG).

Using a katG mutant we investigated the role of KatG in protecting R. leguminosarum against LbOj- We showed that KatG protects against H2O2 in exponential phase but not stationary phase cultures. KatG also appears to have a critical role in the H2O2 induced adaptive response as, unlike the wild type, pre-treatment of the katG mutant with a sub-lethal dose of H2O2 does not result in resistance to subsequent lethal doses. Interestingly KatG does not have a role in symbiosis as the mutant nodulated and promoted plant growth as efficiently as the wild type.

To investigate the regulation of katG in R. leguminosarum we constructed a plasmid-borne katG-lacZ transcriptional gene-fusion. During growth katG expression was shown to parallel the pattern of catalase activity, with maximal expression in mid-exponential phase and minimal levels throughout stationary phase. This suggests that KatG is regulated, at least in part, at the level of transcription.

3. Conclusions

We have cloned and sequenced a catalase-peroxidase, KatG, in R. leguminosarum. KatG has a role in oxidative stress protection, particularly in exponential phase. However the catalase-peroxidase does not have a role in symbiosis. Preliminary regulatory studies, using a katG-lacZ gene-fusion, suggest that the catalase-peroxidase is controlled at the level of transcription.

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