Acknowledgment

Graham Coutts acknowledges a Ph.D studentship from the BBSRC.

TRANSCRIPTIONAL REGULATION OF NITROGEN ASSIMILATION BY THE PRODUCTS OF NITROGEN FIXATION

J. Bussell1, M. Ferguson1, H. Winter2, A. Mann1, P. Storer, C. Atkins1, P. Smith1

department of Botany, The University of Western Australia, Crawley, Australia

2Biologie, Universität Osnabrück, Osnabrück, Germany

De novo purine nucleotide synthesis is the major assimilation pathway for fixed N in species of the Tribes Phaseoleae, Desmodieae and Indigofereae within the Phaseoloid Group of papilionoid legumes (Atkins, Smith 2000). The amide group of glutamine provides half of the N for the purine ring while amino-N, as the amino group of aspartate and the intact glycine molecule, provides the other two. The product, IMP, is oxidized to ureides (allantoin and allantoic acid) and exported from nodules in xylem. The ureides are the major form of N for the nutrition of the host. Nine pur genes encode the 10 enzymes in the purine pathway. Regulation of pur genes by products of N2 fixation was investigated by growing inoculated cowpea plants with their root systems in 80% Ar: 20% 02. Although after 16-18 days under these conditions the plants begin to suffer N deficiency up to ca. 16 days nodule development is normal. Normal levels of nitrogenase activity are expressed and, because high rates of H2 evolution occur, the nodules utilize incoming phloem-delivered sugar and do not accumulate starch. In nodules grown in Ar:02 Vupurl (PRPP amidotransferase), 4 (FGAR amidotransferase), J (AIR synthetase), 6 (AIR carboxylase), 7 (SAICAR synthetase) and 8 (adenylosuccinate AMP lyase) are expressed at levels required to maintain basic cellular processes, while in air, expression increases dramatically as N2 fixation begins, consistent with regulation of these genes by products of fixation. Vupur2 (GAR synthetase), 3 (GAR transformylase), 9 (AICAR transformylase/IMP cyclohydrolase), IMPDH (IMP dehydrogenase) and uricase transcripts showed little change in level of expression over time. However, expression of Vupur2, 9 and IMPDH were reduced in Ar:02 compared to air while Vupur3 and uricase was the same in both treatments. These data indicate that unlike the situation in some prokaryotes the purine pathway in legume nodules is not coordinately regulated and only some of the encoding genes are transcribed in response to the flux of fixed N entering the infected cell.

Transient exposure of an established symbiosis to Ar:02 causes the activity of AIRS and uricase, but not GART, to decline rapidly. However, only AIRS protein rapidly disappeared in Ar and only the gene encoding AIRS (Vupur5) appeared to be transcriptionally regulated. Within 3 h, expression of Vupur5 was markedly reduced, returning to its initial level once plants were transferred back into air. The activity of uricase, the ultimate step in purine oxidation, was rapidly lost in Ar:02 but the protein was stable, consistent with post-translational/allosteric regulation. The prompt loss of de novo purine synthesis that occurs when N2 fixation is inhibited could be accounted for by rapid turnover of AIRS protein and by regulation of transcription and translation of Vupur5.

Taken together these data indicate that the ureide pathway is regulated closely by the flux of fixed N, that not all genes involved are equally sensitive, and that a number of different levels of regulation are involved. Current research is examining the nature of pur gene promoters with a view to identifying the transcription factors and mechanisms involved.

References

Atkins CA, Smith PMC (2000) In Triplett E (ed), Prokaryotic Nitrogen Fixation, pp 559-587,

Horizon Scientific Press, Wymondham, UK

ALFALFA NODULATION BY A PQQ-LINKED GLUCOSE DEHYDROGENASE DEFECTIVE MUTANT OF SINORHIZOBIUM MELILOTI RCR2011

C.E. Bernardelli, M.F. Luna, M.L. Galar and J.L. Boiardi

CINDEFI, Facultad de Cs. Exactas (UNLP)-CONICET, Argentina

We have previously reported extracellular oxidation of glucose, via a periplasmic PQQ-linked glucose dehydrogenase (GDH), in cultures and in bacteroids of different rhizobia. It has been previously shown that the expression of GDH provides different bacteria with energetic and competitive advantages. In this study we have investigated the possible involvement of GDH during the symbiotic association between S. meliloti and alfalfa roots. Inoculation of alfalfa with a gdh mutant of S. meliloti RCR2011 (RmH580, from T. Finan) resulted in a significant decrease in number of nodules per plant with fewer nodules above the root tip position at the time of inoculation compared with control plants inoculated with the wild-type strain. These differences were maintained irrespective of the inoculum dosage (Figures A and B).

A NODULATION

RmH580 (gdh mutant) j

10" 106 108 10s INOCULUM DOSE PER PLANT

INOCULUM DOSE PER PLANT

INOCULUM DOSE PER PLANT

In co-inoculation experiments, nodulation and infectivity increased with the ratio wild-type/mutant strain in the inoculum. Nodule occupancy by wild-type and mutant was proportional to the relative concentration of each strain in the inoculum. However, at equal concentrations of wild-type and mutant in the inoculum, significantly more nodules were formed by the wild type, particularly at low inoculum rates (Figure C). Therefore, the lack of GDH in S. meliloti seems to be associated with an impaired symbiotic phenotype indicating that expression of this metabolic route may be important during some stage(s) of nodule formation. Using an S. meliloti strain carrying a gdh::lacZ fusion (RmG212, from T. Finan) we investigated the expression of gdh at different stages of the symbiotic association. In situ expression of p-galactosidase was observed in root-colonizing rhizobia, in bacteria growing inside the infection threads, and in bacteroids at different developmental stages of alfalfa nodules (between 3 to 12 weeks after inoculation). In young nodules, the nodule core was stained but not the apical meristem. In mature nodules, staining was observed in the central tissue but not in the senescent zone nor in the apical meristem. These results show that GDH is expressed from the early root colonization and mantained thereafter all over the symbiosis.

The symbiotic defects reported here for the gdh mutant, the transcription of gdh during different symbiotic stages, and the already reported GDH activity in bacteroids from alfalfa nodules indicate a role for the extracellular oxidation of aldoses during the symbiotic association.

Supported in part by ANPCyT (PICT 97 No. 1196).

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