, Volume 48, Issue 1–3, pp 173–181 | Cite as

Thin-layer chromatographic analysis of lumichrome, riboflavin and indole acetic acid in cell-free culture filtrate ofPsoralea nodule bacteria grown at different pH, salinity and temperature regimes



Using thin-layer chromatography, 16 bacterial isolates from root nodules of 8 differentPsoralea species were quantitatively assessed for their exudation of the metabolites lumichrome, riboflavin and IAA in response to pH, salinity and temperature. Our data showed that the bacterial strains tested differed in their levels of secretion of the three metabolites. For example, strain AS2 produced significantly greater amounts of lumichrome at both pH 5.1 and 8.1, while strains RT1 and PI produced more lumichrome per cell at only pH 8.1. Strains API and RP2 also produced more riboflavin at pH 5.1 than at pH 8.1; conversely strain RTl secreted more riboflavin at pH 8.1 than at pH 5.1. TwoP. repens strains (RP1 and RP2) isolated from very saline environments close to the Indian Ocean produced significant levels of lumichrome and riboflavin at both low and high salinity treatments. However, strains ACI and LI (fromP. aculeata andP. laxa) even produced greater amounts of lumichrome and riboflavin at higher salinity (i.e. 34.2 mM NaCl) and probably originated from naturally saline soils. In this study, high acidity and high temperature induced the synthesis and release of high levels of IAA by bacterial cells. In contrast, there was greater strain secretion of lumichrome at lower temperature (10°C) than at high temperature (30°C). The variations in the secretion of lumichrome, riboflavin and IAA by bacterial strains exposed to different pH, salinity and temperature regimes suggest that genes encoding these metabolites are regulated differently by the imposed environmental factors. The data from this study also suggest that natural changes of pH, salinity and/or temperature in plant rhizospheres could potentially elevate the concentrations of lumichrome, riboflavin and IAA in soils. An accumulation of these molecules in the rhizosphere would have consequences for ecosystem functioning as both lumichrome and riboflavin have been reported to act as developmental signals that affect species in all three plant, animal, and microbial kingdoms.


Lumichrome ironmental stress Psoralea species signal molecules 


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  1. Bacher, A., Eberhardt, S., Fischer, M., Kis, K., and Richter, G. 2000. Biosynthesis of vitamin b12 (riboflavin).Annual Review of Nutrition 25: 153–167.CrossRefGoogle Scholar
  2. Barazani, O. and Friedman, J. 1999. Is IAA the major root growth factor secreted from plant-growth mediating bacteria?Journal of Chemical Ecology 25: 911–922.CrossRefGoogle Scholar
  3. Becard, G., Doubs, D.D., and Pfeffer, P.E. 1992. Extensivein vitro hyphal growthof vesicular arbuscular mycorrhizalfungi in the presence of CO2 and flavonols.Applied and Environmental Microbiology 58: 821–825.PubMedGoogle Scholar
  4. Boone, C.M., Olsthoom, M.M.A., Dakora, F.D., Spaink, H.P., and Thomas-Oates, J.E. 1999. Structural characterisation of lipochitin oligosaccharides isolated fromBradyrhizobium aspalati, microsymbionts of commercially important South African legumes.Carbohydrate Research 317: 155–163.CrossRefPubMedGoogle Scholar
  5. Bowen, G.D. and Kennedy, M.M. 1959. Effect of high soil temperature onRhizobium spp.Journal of Agricultural Sciences 16: 177–179.Google Scholar
  6. Bresler, S.E., Perumov, D.A., Shevchenko, T.N., Glazunov, E.A., and Chemik, T.P. 1975. Operon of riboflavin synthesis inBacillus subtilis. IX. Preparation and properties of lumiflavinor lumichrome-resistant mutants.Genetika 11: 129–138.PubMedGoogle Scholar
  7. Capenter, C.C. 1943. Riboflavin-vitamin B2 in soil.Science 98: 109–110.CrossRefGoogle Scholar
  8. Dakora, F.D. 2004. Effects of symbiotic legumes and rhizobia on plant and microbial biodiversity in natural and agricultural ecosystems.Annals ofArid Zone 43: 377–390.Google Scholar
  9. Dakora, F.D., and Phillips, D.A. 2002. Root exudates as mediators of mineral acquisition in low-nutrient soil.Plant and Soil 245: 35–47.CrossRefGoogle Scholar
  10. Danso, S.K.A. 1977. The ecology ofRhizobium and recent advances in the study of the ecology ofRhizobium. In:Biological Nitrogen Fixation in Farming Systems of the Tropics. Ayanaba, A. and Dart, P.J., eds. Wiley, Chichester, pp. 115–125.Google Scholar
  11. de Jong, A.J., Heidstra, R., Spaink, H.P., Hartog, M.Y., Hendriks, T., Lo Schavia, F., Terzi, M., Besseling, T., van Kammen, A., and de Vries, S. 1993. A plant somatic embryo mutant is rescued by rhizobial lipo-oligosaccharides.Plant Cell 5: 615–620.CrossRefPubMedGoogle Scholar
  12. Dong, H. and Beer, S.V. 2000. Riboflavin induces disease resistance in plants by activating a novel signal transduction pathway.Phytopathology 90: 801–811.CrossRefPubMedGoogle Scholar
  13. Ghosh, S. and Basu, P.S. 2006. Production and metabolism of indole acetic acid in roots and root nodules ofPhaseolus mungo.Microbiological Research 161: 362–366.CrossRefPubMedGoogle Scholar
  14. Gordon, S.A. and Weber, R.P. 1951. Colorimetric estimation of indole acetic acid.Plant Physiology 26: 192–195.CrossRefPubMedGoogle Scholar
  15. Graham, P.H. and Vance, C.P. 2000. Nitrogen fixation in perspective: an overview of research and extension needs.Field Crops Research 65: 93–106.CrossRefGoogle Scholar
  16. Kanu, S.A., Matiru, Y.N., and Dakora, F.D. 2007. Strain and species differences in rhizobial secretion of lumichrome and riboflavin, measured using thin-layer chromatography.Symbiosis 43: 37–43.Google Scholar
  17. Khan, W., Prithiviraj, B., and Smith, D.L. 2008. Nod factor [Nod Bj V (C18:1, MeFuc)] and lumichrome enhance photosynthesis and growth of com and soybean.Journal of Plant Physiology doi:10.1016/j.jpiph.2007.11.00.Google Scholar
  18. Lowe, R.H. and Evans, H.J. 1962.Carbon dioxide requirement for growth of legumenodule bacteria.Soil Science 94: 351–356.CrossRefGoogle Scholar
  19. Matiru, V.N., Jaffer, M.A., and Dakora, F.D. 2005. Rhizobial infection of African landracesof sorghum (Sorghum bicolor L.) and finger millet (Eleucine coracana L.) promotes plant growth and alters tissue nutrient concentration under axenic conditions.Symbiosis 40: 7–15.Google Scholar
  20. Matiru, V.N. and Dakora, F.D. 2005a. The rhizosphere rhizobial signal molecule lumichrome alters seedling development in both legumes and cereals.New Phytologist 166: 439–444.CrossRefPubMedGoogle Scholar
  21. Matiru, V.N. and Dakora, F.D. 2005b. Xylem transport and shoot accumulation of lumichrome, a newly recognized rhizobial signal, alters root respiration, stomatal conductance, leaf transpiration and photosynthetic rates in legumes and cereals.New Phytologist 165: 847–855.CrossRefPubMedGoogle Scholar
  22. Moron, B., Soria-Diaz, M.E., Ault, J., Verroios, G., Sadaf, N., Rodriguez-Navarro, D.N., Gil-Serrano, A., Thomas-Oates, J., Megias, M., and Sousa, C. 2005. Low pH changes the profile of nodulation factors Produced by CIAT899.Chemistry and Biology 12: 1029–1040.CrossRefPubMedGoogle Scholar
  23. Muofhe, M.L. and Dakora, F.D. 1998.Bradyrhizobium species isolated from indigenous legumes of the Western Cape exhibit high tolerance of low pH. In:Biological Nitrogen Fixation for the 21st Century. Elmerich, C., Kondorosi, A. and Newton, W.E., eds, Kluwer, Dordrecht, p. 519.Google Scholar
  24. Munévar, F. and Wollum, A.G. 1981. Effects of high root temperature and rhizobium strain on nodulation, nitrogen fixation, and growth of soybean.Soil Science Society of American Journal 45: 1113–1120.CrossRefGoogle Scholar
  25. Payakapong, W., Tittabutr, P., Teaumroong, N., Boonkerd, N., Singleton, P.W., and Borthakur, D. 2006. Identification of two clusters of genes involved in salt tolerance inSinorhizobium sp. strain BL3.Symbiosis 41: 47–53.Google Scholar
  26. Phillips, D.A, Tama, C.F., King, M.D., Bhuvaneswari, T.V., and Teuber, L.R. 2004. Microbial products trigger amino acid exudation from plant roots.Plant Physiology 136: 2887–2894.CrossRefPubMedGoogle Scholar
  27. Phillips, D.A., Ferris, H., Cook, D.R, and Strong, D.R. 2003. Molecular control points in rhizosphere food webs.Ecology 84: 816–826.CrossRefGoogle Scholar
  28. Phillips, D.A, Joseph, C.M., Yang, G-P., Martinez-Romero, E., Sanborn, J.R., and Volpin, H. 1999. Identification of lumichrome as aSinorhizobium enhancer of alfalfa root respiration and shoot growth.Proceedings of the Academy of Sciences USA 96: 12275–12280.CrossRefGoogle Scholar
  29. Piha, M.J. and Munns, D.N. 1987. Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature.Plant and Soil 98: 183–324.CrossRefGoogle Scholar
  30. Rajamani, S., Phillips, D.A., Bauer, W.D., Robinson, J.B., Farrow, III J.M., Pesci, E.C., Teplitski, M., Gao, M., and Sayre, R.T. 2008. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor.Molecular Plant-Microbe interactions 21: 1184–1192.CrossRefPubMedGoogle Scholar
  31. Rodelas, B., Salmeron, V., Martinez-Toledo, M.B., and GonzalezLopez, J. 1993. Production of vitamins byAzospirillum brasilense in chemically-defined media.Plant and Soil 153: 97–101.CrossRefGoogle Scholar
  32. Sierra, S., Rodelas, B., Martinez-Toledo, M.V., Pozo, C., and Gonzalez-Lope, J. 1999. Production of B-group vitamins by two rhizobium strains in chemically-defined media.Journal of Applied Microbiology 86: 851–858.CrossRefGoogle Scholar
  33. Streit, W.R., Joseph, C.M., and Phillips, D.A. 1996. Biotin and other water-soluble vitamins are key growth factors for alfalfa root colonization byRhizobium meliloti 1021.Molecular PlantMicrobe interactions 5: 330–338.Google Scholar
  34. Tsukamoto, S., Kato H., Hirota, H., and Fusetani, N. 1999. Lumichrome: A larval metamorphosis-inducing substance in the ascidianHalocynthia roretzi.European Journal of Biochemistry 264: 785–769.CrossRefPubMedGoogle Scholar
  35. Ueno, K. and Natori, S. 1984. Possible involvement of lumichrome in the binding of storage protein to its receptor inSarcophaga peregrine.Journal of Biological Chemistry 259: 12107–12111.PubMedGoogle Scholar
  36. Verma, D.P.S., Hu, C.A, and Zhang, M. 1992. Root nodule development: origin, function and regulation of nodular genes.Physiologia Plantarum 85: 253–265.CrossRefGoogle Scholar
  37. Vessey, J.K. 2003. Plant growth-promoting rhizobacteria as biofertilizers.Plant and Soil 255: 571–586.CrossRefGoogle Scholar
  38. Vincent, J.M. 1970.A Manual for the Practical Study of Root-Nodule Bacteria. IBP Handbook No. 15. Blackwell, Oxford.Google Scholar
  39. Xiao, S., Dai, L., Liu, F., Wang, Z., Peng, W., and Xie, D. 2004. COSI: An Arabidopsis coronatine insensitive I suppressor essential for regulation on jasmonate-mediated plant defense and senescence.Plant Cell 16: 1132–1142.CrossRefPubMedGoogle Scholar
  40. Yang, G., Bhuvaneswari, T. V., Joseph, C.M, King, M.D., and Phillips, D.A. 2002. Roles forRhizobium in theSinorhizobium alfalfa association.Molecular Plant-Microbe interactions 15: 456–462.CrossRefPubMedGoogle Scholar
  41. Zahran, H.H. 1999.Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate.Microbiology and Molecular Biology Reviews 63: 968–989.PubMedGoogle Scholar
  42. Zhang, F. and Smith, D.L. 2001. Interorganismal signalling in suboptimum environments; the legume-rhizobium, symbiosis.Advances in Agronomy 76: 125–161.CrossRefGoogle Scholar
  43. Zilberstein, D., Agmon, V., Schuldiner, S., and Padan, E. 1984.Escherichia coli intracellular pH, membrane potential, and cell growth.Journal of Bacteriology 158: 246–252.PubMedGoogle Scholar

Copyright information

© Springer 2009

Authors and Affiliations

  1. 1.Department of Crop ScienceTshwane University of TechnologyPretoriaSouth Africa
  2. 2.Chemistry DepartmentTshwane University of TechnologyPretoriaSouth Africa

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