Journal of Plant Growth Regulation

, Volume 6, Issue 2, pp 97–111 | Cite as

Phytohormones,Rhizobium mutants, and nodulation in legumes. VII. Identification and quantification of cytokinins in effective and ineffective pea root nodules using radioimmunoassay

  • Jane Badenoch-Jones
  • Charles W. Parker
  • D. S. Letham


Radioimmunoassays (RIA), employing antisera raised in rabbits against bovine serum albumin conjugates of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine, were used to estimate levels of these cytokinins and their corresponding bases in samples of effective (nitrogen-fixing, Fix+), ineffective (nonnitrogen-fixing, Fix) pea root nodules and uninoculated roots. Assays were done on extracts of nodule tissue, 1–2 g fresh weight, or approximately 10 g fresh weight of root tissue, and high specific activity [3H]zeatin riboside was added during preparation of the extract for use as a recovery marker. Two different purification procedures were employed, each involving several purification steps. High performance liquid chromatography (HPLC) was the final step in both procedures. Fractions from HPLC were analyzed by RIA using the appropriate antiserum. The cytokinins, zeatin, zeatin riboside, dihydrozeatin riboside, isopentenyl adenine, and isopentenyladenosine were detected and quantified in nodule tissue, and similarly, in root tissue (with the exception of zeatin, which we were unable to quantify in root tissue). Cytokinin levels in nodule tissue were higher than those in root tissue. The major cytokinins detected in nodule tissue were zeatin, followed by zeatin riboside and then dihydrozeatin riboside. The levels of zeatin and zeatin riboside estimated in nodules in the present study by RIA were of the same order of magnitude, though tending to be a little higher, than values obtained previously by bioassay. Dihydrozeatin riboside was identified with confidence for the first time in nodule tissue. There was a general decline with age in cytokinin levels in nodules, but no major qualitative change in nodule cytokinins with age. For theRhizobium strains examined, the data did not indicate a clear correlation between nodule cytokinin levels and the effectiveness of nodules in nitrogen fixation.


High Performance Liquid Chromatography Root Tissue Zeatin High Specific Activity Isopentenyl 
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  1. Akiyoshi DE, Morris RO, Hinz R, et al. (1983) Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the tDNA. Proc Natl Acad Sci USA 80:407–411Google Scholar
  2. Badenoch-Jones J, Summons RE, Djordjevic MA, Shine J, Letham DS, Rolfe BG (1982a) Mass spectrometric quantification of indole-3-acetic acid inRhizobium culture supernatants: Relation to root hair curling and nodule initiation. Appl Environ Microbiol 44:275–280Google Scholar
  3. Badenoch-Jones J, Summons RE, Entsch B, Rolfe BG, Parker CW, Letham DS (1982b) Mass spectrometric identification of indole compounds produced byRhizobium strains. Biomed Mass Spectrom 9:429–437Google Scholar
  4. Badenoch-Jones J, Rolfe BG, Letham DS (1983) Phytohormones,Rhizobium mutants, and nodulation in legumes. III. Auxin metabolism in effective and ineffective pea root nodules. Plant Physiol 43:347–352Google Scholar
  5. Badenoch-Jones J, Rolfe BG, Letham DS (1984a) Phytohormones,Rhizobium mutants, and nodulation in legumes. V. Cytokinin metabolism in effective and ineffective pea root nodules. Plant Physiol 74:239–246Google Scholar
  6. Badenoch-Jones J, Summons RE, Rolfe BG, Letham DS (1984b) Phytohormones,Rhizobium mutants, and nodulation in legumes. IV. Auxin metabolites in pea root nodules. J Plant Growth Regul 3:23–39Google Scholar
  7. Badenoch-Jones J, Rolfe BG, Letham DS (1984c) Phytohormones,Rhizobium mutants, and nodulation in legumes. VI. Metabolism of zeatin riboside applied via the tips of nodulated pea roots. J Plant Growth Regul 3:41–49Google Scholar
  8. Badenoch-Jones J, Letham DS, Parker CW, Rolfe BG (1984d) Quantitation of cytokinins in biological samples using antibodies against zeatin riboside. Plant Physiol 75:1117–1125Google Scholar
  9. Brenner ML (1981) Modern methods for plant growth substance analysis. Annu Rev Plant Physiol 32:511–538Google Scholar
  10. Brooker G, Harper JF, Terasaki WL, Moylan RD (1979) Radioimmunoassay of cyclic AMP and cyclic GMP. In: Brooker G, Greengard PG, Robison GA (eds) Advances in cyclic nucleotide research. Raven Press, New York, pp 1–31Google Scholar
  11. Bhuvaneswari TV, Turgeon G, Bauer WD (1980) Early stages in the infection of soybean (Glycine max L. Merr.) byRhizobium japnicum. I. Localization of infectible root cells. Plant Physiol 66:1027–1031Google Scholar
  12. Chen HK, Thornton HG (1940) The structure of “ineffective” nodules and its influence on nitrogen fixation. Proc R Soc Lond (Biol) 129:208–229Google Scholar
  13. Djordjevic MA, Zurkowski W, Rolfe BG (1982) Plasmids and stability of symbiotic properties ofRhizobium trifolii. J Bacteriol 151:560–568PubMedGoogle Scholar
  14. Ernst D, Schäfer W, Oesterhelt D (1983) Isolation and quantitation of isopentenyladenosine in an anise cell culture by single-ion monitoring, radioimmunoassay and bioassay. Planta 159:216–221Google Scholar
  15. Henson IE, Wheeler CT (1976) Hormones in plants bearing nitrogen-fixing root nodules: The distribution of cytokinins inVicia faba L. New Phytol 76:433–439Google Scholar
  16. Henson IE, Wheeler CT (1977) Hormones in plants bearing nitrogen-fixing root nodules: Cytokinin levels in roots and root nodules of some nonleguminous plants. Z Pflanzenphysiol 84:179–182Google Scholar
  17. Horgan R (1978) Analytical procedures for cytokinins. In: Hillman JR (ed) Isolation of plant growth substances, Society for Experimental Biology Seminar Series 4. Cambridge University Press, Cambridge, U.K., pp 97–114Google Scholar
  18. Jaiswal V, Rizvi SJH, Mukerji D, Mathur SN (1981) Cytokinins in root nodules ofPhaseolus mungo. Ann Bot 48:301–305Google Scholar
  19. Johnston AWB, Beringer JE (1976) Pea nodules containing more than oneRhizobium species. Nature 263:502–504Google Scholar
  20. Letham DS (1978) Cytokinins. In: Letham DS, Goodwin PB, Higgins TJV (eds) Phytohormones and related compounds—a comprehensive treatise, Vol I. Elsevier North-Holland, Amsterdam, pp 205–263Google Scholar
  21. MacDonald EMS, Akiyoshi DE, Morris RO (1981) Combined high performance liquid chromatography—radioimmunoassay for cytokinins. J Chromatogr 214:101–109Google Scholar
  22. Morris RO, Akiyoshi DE, MacDonald EMS, Morris JW, Regier DA, Zaerr JB (1982) Cytokinin metabolism in relation to tumor induction byAgrobacterium tumefaciens In: Wareing PF (ed) Plant growth substances. Academic Press, London, pp 175–183Google Scholar
  23. Puppo A, Rigaud J, Barthe P (1974) Sur la présence de cytokinines dans les nodules dePhaseolus vulgaris L. CR Acad Sci Paris (Ser D) 279:2029–2032Google Scholar
  24. Rodriguez-Barrueco C, Miguel C, Palni LMS (1979) Cytokinins in root nodules of the nitrogen-fixing non-legumeMyrica gale L. Z Pflanzenphysiol Bd 95S:275–278Google Scholar
  25. Short K, Torrey JG (1972) Cytokinins in seedling roots of pea. Plant Physiol 49:155–160Google Scholar
  26. Syono K, Torrey JG (1976) Identification of cytokinins of root nodules of the garden pea,Pisum sativum L. Plant Physiol 57:602–606Google Scholar
  27. Syono K, Newcomb W, Torrey JG (1976) Cytokinin production in relation to the development of pea root nodules. Can J Bot 54:2155–2162Google Scholar
  28. Vincent JM (1980) Factors controlling the legume-Rhizobium symbiosis. In: Newton WE, Orme-Johnson WH (eds) Nitrogen fixation, Vol 2. University Park Press, Baltimore, pp 103–129Google Scholar
  29. Wang T, Wood EA, Brewin NJ (1982) Growth regulators,Rhizobium and nodulation in peas. The cytokinin content of a wild-type and a Ti-plasmid-containing strain ofR. leguminosarum. Planta 155:350–355Google Scholar
  30. Weiler EW (1980) Radioimmunoassays for trans-zeatin and related cytokinins. Planta 149:155–162Google Scholar
  31. Weiler gnEW, Spanier K (1981) Phytohormones in the formation of crown gall tumors. PlantaGoogle Scholar

Copyright information

© Springer-Verlag New York Inc 1987

Authors and Affiliations

  • Jane Badenoch-Jones
    • 1
  • Charles W. Parker
    • 1
  • D. S. Letham
    • 1
  1. 1.Research School of Biological SciencesAustralian National UniversityCanberra CityAustralia

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