Response Of Legume Roots To Hog Gastric Mucin Blood Group Ah Substance A Carbohydrate Ligand For Legume Root

Section Mol. Cell. Biol., U. Calif., Davis, CA, USA

Previous studies from this laboratory established that Db-LNP, a lectin nucleotide phosphohydrolase present in the roots of the legume, Dolichos biflorus, is a Nod factor-binding protein present on the surface of root hair and epidermal cells in the nodulation zone of roots and that this protein most probably plays a role in the initiation of the rhizobium-legume symbiosis. This LNP is isolated from roots by affinity chromatography on hog gastric mucin blood group A+H substance (A+H BGS) and binds well to this carbohydrate ligand. Treatment of roots with A+H BGS was found to mimic the effect of rhizobial symbionts in causing a redistribution of LNP to the tips of the root hairs. We now show that treatment of D. biflorus roots with low concentrations of this ligand results in root hair deformations that include swelling, branching and curling as well as in the formation of pseudonodules. The ability of this carbohydrate ligand of LNP to mimic the effect of rhizobia on the roots provides further evidence that LNP is involved in the signaling events that initiate the plant response to rhizobia.

SINORHIZOBIUM SP. STRAIN NGR234 LIPOPOLYSACCHARIDES: STRUCTURAL FEATURES AND COMPARISON OF LPS FROM UNINDUCED AND APIGENIN-INDUCED CELLS

S.K. Gudlavalleti, S.B. Stevens, B.R. Reuhs, L.S. Forsberg

Complex Carbohydrate Research Center, U Georgia, Athens, GA, USA

Sinorhizobium sp. NGR234 is a wide host range endosymbiont with a prominent agricultural role; for this reason it is a subject of study in several laboratories, and the nucleotide sequence of the symbiosis plasmid has recently been determined (Freiberg et al. 1997). However, there is little detailed structural information on the cell surface macromolecules produced by these bacteria, or the structural alterations that occur in these molecules during differentiation and bacteroid development. We describe an initial structural investigation of the cell surface lipopolysaccharides (LPS), including a detailed structural analysis of the lipid A moiety, and an investigation into the effects of apigenin on structural alterations in the total LPS. Complementary structural information on this LPS will prove to be useful for the ongoing genetic study of this organism.

The NGR234 strain is a member of the Rf205 serogroup (group B) (Bhat et al. 1994), as defined by the core region carbohydrate epitopes of the rough LPS (R-LPS, lacking O-antigen). Unlike Rhizobium etli, strain NGR234 produces very little smooth LPS (S-LPS, containing O-antigen), when grown in culture. The major biosynthetic product is the R-LPS, and the core region of this LPS yields approximately 20 different oligosaccharide components upon mild acid hydrolysis. This contrasts with R. etli LPS which essentially contains two major oligosaccharide components in the core region. We have found that exposure of NGR234 cells to apigenin results in a marked alteration in the chromatographic properties and carbohydrate composition of the R-LPS fraction. The R-LPS fraction from apigenin-induced cultures shows high levels of rhamnose and altered fatty acids in addition to the normal residues. These results extend those of an earlier study (Forsberg, Carlson 1998) and provide further evidence that changes in LPS structure can be induced by plant flavonoids.

The lipid A portion of the NGR234 R-LPS has structural features in common with both the enterobacterial lipid As and the lipid As of Rhizobium etli and R. leguminosarum (Jabbouri S et al. 1996; Reuhs BL et al. 1998). In contrast to R. etli, the NGR234 lipid A consists of a bisphosphorylated GlcNAc disaccharide backbone, similar to those of many enteric bacteria. However, the NGR234 lipid A displays a fatty acylation profile similar to that of R. etli and R. leguminosarum, characterized by extensive amide-linked fatty acyl heterogeneity, the absence of ester-linked acyloxyacyl residues, and the occurrence of very long chain fatty acids (29-hydroxy-30-carbon acids), attached as acyloxyacyl residues to amide-linked 3-OH-18-carbon fatty acid.

References

Bhat UR, Forsberg LS, Carlson RW (1994) J. Biol. Chem. 269, 14402-14410

Freiberg, CR et al. (1997) Nature 387, 394-401

Forsberg LS, Carlson RW (1998) J. Biol. Chem. 273, 2141-2151

Jabbouri S et al. (1996) In Stacey G, Mullin B, Gresshoff PM (eds) Biology of Plant Microbe

Interactions, pp. 319-324, ISMPMI, St. Paul, USA Reuhs BL et al. (1998) Appl. Environ. Microbiol. 64, 4930-4938

Acknowledgement

Supported by NRI/USDA CSREES Grant 99-35305-8143 (to LSF) and by DOE Grant DE-FG09-93ER20097 (to the CCRC).

GROWTH AND SYMBIOTIC RESPONSE OF COWPEA (VIGNA UNGUCULATA) TO ELEVATED LEVELS OF UV-B RADIATION

'Botany Dept, UCT, P/B Rondebosch 7701, Cape Town

2Ecology and Conservation, Claremont 7735, South Africa

Depletion of the stratospheric ozone layer has led to increased levels of solar ultraviolet-B radiation (UV-B, 290-315 nm) with uncertain consequences on crop plants. Among other effects, UV-B radiation is reported to damage the photosynthetic machinery (Teramura and Sullivan 1994) and induce accumulation of flavonoids and anthocyanins in leaves of plant species as defense mechanisms (Jansen et al. 1998). These compounds are however known to serve as plant signals to symbiotic bacteria in the Rhizobiaceae (Hungria et al. 1991; Phillips 1992). We therefore hypothesized that a rise in UV-B radiation will (1) increase nodulation and N2 fixation in legumes if the increase in flavonoid concentration in parent plants and in seeds includes node gene inducers, or (2) reduce nodulation and N2 fixation if the damaged photosynthetic machinery results in reduced synthesis and release of root exudate compounds into the rhizosphere. The aim of this study was to assess the symbiotic performance of cowpea plants exposed to UV-B radiation.

Cowpea (Vigna unguculata) plants were grown outdoors in potted sand under ambient and two levels of elevated UV-B at Kistenbosch Botanical Institute, Cape Town. Elevation in UV-B above ambient conditions was achieved by artificial supplementation with filtered UV fluorescent sun lamps. Weighted dose in the ambient UV-B treatment was 6.70 kJ m"2 d"1 (average amounts for the experimental time covering January to March in Cape Town). The two levels of elevated UV-B were 8.87 kJ m"2 d"1 (UV-B1) and 10.90 kJ rn2 d"1 (UV-B2). The elevated UV-B amounts simulated the levels that would be received in the southern latitudes of the tropical region. The plants were harvested at 65 DAP and assessed for nodulation, biomass accumulation, and concentration of metabolites.

Elevated UVB1 significantly reduced nodule numbers, nodule mass and total biomass, but increased the concentration of ureides in xylem. Flavonoid concentration in tissue was not altered by UV-B1. By contrast, the higher level of elevated UV-B (UV-B2) markedly increased nodule mass, tissue concentration of flavonoids, and %N in leaves and stems. The reduction in symbiotic parameters with UV-B1 was possibly due to the unaltered tissue flavonoid concentrations, in contrast to UV-B2 where increased nodulation and tissue nitrogen concentration was accompanied by increased plant flavonoids levels. These accumulated flavonoids could protect plant cells by absorbing UV-B radiation, but whether UV-B induced flavonoids are involved in signaling rhizobia for nodule formation is still being investigated.

References

Hungria M et al. (1991) Plant Physiol. 97, 751-758 Jansen MAK et al. (1998) Trends in Plant Sci. 3, 131-135 Phillips DA (1992) Recent Adv. Phytochem. 26, 201-231 Teramura AH, Sullivan JH (1994) Photosyn. Res. 39, 463-473

Acknowledgements

The authors thank AAU and DAAD for a fellowship awarded to SBMC, NBI for financial support to CM, the NRF and URC for research grant to FDD.

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