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. 1994 Feb;6(2):201–213. doi: 10.1105/tpc.6.2.201

ENOD12, an early nodulin gene, is not required for nodule formation and efficient nitrogen fixation in alfalfa.

G Csanádi 1, J Szécsi 1, P Kaló 1, P Kiss 1, G Endre 1, A Kondorosi 1, E Kondorosi 1, G B Kiss 1
PMCID: PMC160427  PMID: 8148645

Abstract

To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medicago sativa subspecies were tested for the presence or absence of ENOD12 alleles. In M. s. ssp coerulea w2 (Mcw2), two ENOD12 genes were detected, whereas in M. s. ssp quasifalcata k93 (Mqk93) only one gene was present. In both plants, the ENOD12 genes were expressed in nodules induced by Rhizobium meliloti. The nucleotide sequence of the ENOD12 genes showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M. s. ssp sativa. ENOD12 from Mqk93 was similar to the corresponding gene found in M. truncatula. From the aligned ENOD12 sequences, an evolutionary tree was constructed. Genetic analysis of the progenies of a cross between Mqk93 and Mcw2 showed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only lacking the ENOD12 gene appeared. Surprisingly, the ENOD12-deficient plants were similar to their wild-type parents in viability, nodule development, nodule structure, and nitrogen fixation efficiency. Therefore, we concluded that in Medicago the ENOD12 gene is not required for symbiotic nitrogen fixation. Furthermore, we proposed that the heterozygous nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the construction of plant mutants deficient in these genes.

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Selected References

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  1. Allison L. A., Kiss G. B., Bauer P., Poiret M., Pierre M., Savouré A., Kondorosi E., Kondorosi A. Identification of two alfalfa early nodulin genes with homology to members of the pea Enod12 gene family. Plant Mol Biol. 1993 Jan;21(2):375–380. doi: 10.1007/BF00019952. [DOI] [PubMed] [Google Scholar]
  2. Bergmann H., Preddie E., Verma D. P. Nodulin-35: a subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules. EMBO J. 1983;2(12):2333–2339. doi: 10.1002/j.1460-2075.1983.tb01743.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bánfalvi Z., Sakanyan V., Koncz C., Kiss A., Dusha I., Kondorosi A. Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti. Mol Gen Genet. 1981;184(2):318–325. doi: 10.1007/BF00272925. [DOI] [PubMed] [Google Scholar]
  4. Caetano-Anollés G., Gresshoff P. M. Plant genetic control of nodulation. Annu Rev Microbiol. 1991;45:345–382. doi: 10.1146/annurev.mi.45.100191.002021. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Dunn K., Dickstein R., Feinbaum R., Burnett B. K., Peterman T. K., Thoidis G., Goodman H. M., Ausubel F. M. Developmental regulation of nodule-specific genes in alfalfa root nodules. Mol Plant Microbe Interact. 1988 Feb;1(2):66–74. doi: 10.1094/mpmi-1-066. [DOI] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Franssen H. J., Nap J. P., Gloudemans T., Stiekema W., Van Dam H., Govers F., Louwerse J., Van Kammen A., Bisseling T. Characterization of cDNA for nodulin-75 of soybean: A gene product involved in early stages of root nodule development. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4495–4499. doi: 10.1073/pnas.84.13.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kapros T., Bögre L., Németh K., Bakó L., Györgyey J., Wu S. C., Dudits D. Differential Expression of Histone H3 Gene Variants during Cell Cycle and Somatic Embryogenesis in Alfalfa. Plant Physiol. 1992 Feb;98(2):621–625. doi: 10.1104/pp.98.2.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980 Dec;16(2):111–120. doi: 10.1007/BF01731581. [DOI] [PubMed] [Google Scholar]
  11. Kiss G. B., Csanádi G., Kálmán K., Kaló P., Okrész L. Construction of a basic genetic map for alfalfa using RFLP, RAPD, isozyme and morphological markers. Mol Gen Genet. 1993 Apr;238(1-2):129–137. doi: 10.1007/BF00279539. [DOI] [PubMed] [Google Scholar]
  12. Kiss G. B., Vincze E., Végh Z., Tóth G., Soós J. Identification and cDNA cloning of a new nodule-specific gene, Nms-25 (nodulin-25) of Medicago sativa. Plant Mol Biol. 1990 Apr;14(4):467–475. doi: 10.1007/BF00027493. [DOI] [PubMed] [Google Scholar]
  13. Kiss G. B., Végh Z., Vincze E. Nucleotide sequence of a cDNA clone encoding leghemoglobin III (LbIII) from Medicago sativa. Nucleic Acids Res. 1987 Apr 24;15(8):3620–3620. doi: 10.1093/nar/15.8.3620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kouchi H., Hata S. Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Mol Gen Genet. 1993 Apr;238(1-2):106–119. doi: 10.1007/BF00279537. [DOI] [PubMed] [Google Scholar]
  15. Legocki R. P., Verma D. P. Identification of "nodule-specific" host proteins (nodoulins) involved in the development of rhizobium-legume symbiosis. Cell. 1980 May;20(1):153–163. doi: 10.1016/0092-8674(80)90243-3. [DOI] [PubMed] [Google Scholar]
  16. Lerouge P., Roche P., Faucher C., Maillet F., Truchet G., Promé J. C., Dénarié J. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature. 1990 Apr 19;344(6268):781–784. doi: 10.1038/344781a0. [DOI] [PubMed] [Google Scholar]
  17. Long S. R. Rhizobium-legume nodulation: life together in the underground. Cell. 1989 Jan 27;56(2):203–214. doi: 10.1016/0092-8674(89)90893-3. [DOI] [PubMed] [Google Scholar]
  18. Pichon M., Journet E. P., Dedieu A., de Billy F., Truchet G., Barker D. G. Rhizobium meliloti elicits transient expression of the early nodulin gene ENOD12 in the differentiating root epidermis of transgenic alfalfa. Plant Cell. 1992 Oct;4(10):1199–1211. doi: 10.1105/tpc.4.10.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  20. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  21. Scheres B., McKhann H. I., Zalensky A., Löbler M., Bisseling T., Hirsch A. M. The PsENOD12 Gene Is Expressed at Two Different Sites in Afghanistan Pea Pseudonodules Induced by Auxin Transport Inhibitors. Plant Physiol. 1992 Dec;100(4):1649–1655. doi: 10.1104/pp.100.4.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Scheres B., Van De Wiel C., Zalensky A., Horvath B., Spaink H., Van Eck H., Zwartkruis F., Wolters A. M., Gloudemans T., Van Kammen A. The ENOD12 gene product is involved in the infection process during the pea-Rhizobium interaction. Cell. 1990 Jan 26;60(2):281–294. doi: 10.1016/0092-8674(90)90743-x. [DOI] [PubMed] [Google Scholar]
  23. Spaink H. P., Sheeley D. M., van Brussel A. A., Glushka J., York W. S., Tak T., Geiger O., Kennedy E. P., Reinhold V. N., Lugtenberg B. J. A novel highly unsaturated fatty acid moiety of lipo-oligosaccharide signals determines host specificity of Rhizobium. Nature. 1991 Nov 14;354(6349):125–130. doi: 10.1038/354125a0. [DOI] [PubMed] [Google Scholar]
  24. Stanley S. L., Jr, Jackson T. F., Reed S. L., Calderon J., Kunz-Jenkins C., Gathiram V., Li E. Serodiagnosis of invasive amebiasis using a recombinant Entamoeba histolytica protein. JAMA. 1991 Oct 9;266(14):1984–1986. [PubMed] [Google Scholar]
  25. Thummler F., Verma D. P. Nodulin-100 of soybean is the subunit of sucrose synthase regulated by the availability of free heme in nodules. J Biol Chem. 1987 Oct 25;262(30):14730–14736. [PubMed] [Google Scholar]
  26. Tindall K. R., Kunkel T. A. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry. 1988 Aug 9;27(16):6008–6013. doi: 10.1021/bi00416a027. [DOI] [PubMed] [Google Scholar]
  27. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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