Skip to main content
PMC home page
The Plant Cell logoLink to The Plant Cell
. 1999 Oct;11(10):1981–1994. doi: 10.1105/tpc.11.10.1981

The role of opaque2 in the control of lysine-degrading activities in developing maize endosperm.

E L Kemper 1, G C Neto 1, F Papes 1, K C Moraes 1, A Leite 1, P Arruda 1
PMCID: PMC144114  PMID: 10521527

Abstract

We have isolated a cDNA clone, designated ZLKRSDH, encoding the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) from maize. The predicted polypeptide has an N-terminal LKR domain and a C-terminal SDH domain that are similar to the yeast LYS1 and LYS9 monofunctional proteins, respectively. The maize LKR/SDH protein is located in the cytoplasm of subaleurone endosperm cell layers. Transcripts and polypeptides as well as enzyme activities showed an upregulation and downregulation during endosperm development. The developmental expression of ZLKRSDH was examined in normal and opaque2 seeds. In the mutant endosperm, mRNA levels were reduced by >90%, with concomitant reductions in polypeptide levels and LKR/SDH activity. These results suggest that lysine levels in the endosperm are likely to be controlled at the transcriptional level by the Opaque2 transcription factor.

Full Text

The Full Text of this article is available as a PDF (633.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ameen M., Palmer T., Oberholzer V. G. Inhibition of bovine liver lysine-ketoglutarate reductase by urea cycle metabolites and saccharopine. Biochem Int. 1987 Apr;14(4):589–595. [PubMed] [Google Scholar]
  2. Arruda P., Sodek L., da Silva W. J. Lysine-ketoglutarate reductase activity in developing maize endosperm. Plant Physiol. 1982 Apr;69(4):988–989. doi: 10.1104/pp.69.4.988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aukerman M. J., Schmidt R. J., Burr B., Burr F. A. An arginine to lysine substitution in the bZIP domain of an opaque-2 mutant in maize abolishes specific DNA binding. Genes Dev. 1991 Feb;5(2):310–320. doi: 10.1101/gad.5.2.310. [DOI] [PubMed] [Google Scholar]
  4. Azevedo R. A., Arruda P., Turner W. L., Lea P. J. The biosynthesis and metabolism of the aspartate derived amino acids in higher plants. Phytochemistry. 1997 Oct;46(3):395–419. doi: 10.1016/s0031-9422(97)00319-1. [DOI] [PubMed] [Google Scholar]
  5. Bass H. W., Webster C., OBrian G. R., Roberts J. K., Boston R. S. A maize ribosome-inactivating protein is controlled by the transcriptional activator Opaque-2. Plant Cell. 1992 Feb;4(2):225–234. doi: 10.1105/tpc.4.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blemings K. P., Crenshaw T. D., Swick R. W., Benevenga N. J. Lysine-alpha-ketoglutarate reductase and saccharopine dehydrogenase are located only in the mitochondrial matrix in rat liver. J Nutr. 1994 Aug;124(8):1215–1221. doi: 10.1093/jn/124.8.1215. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Bright S. W., Miflin B. J., Rognes S. E. Threonine accumulation in the seeds of a barley mutant with an altered aspartate kinase. Biochem Genet. 1982 Apr;20(3-4):229–243. doi: 10.1007/BF00484421. [DOI] [PubMed] [Google Scholar]
  9. Brinch-Pedersen H., Galili G., Knudsen S., Holm P. B. Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase. Plant Mol Biol. 1996 Nov;32(4):611–620. doi: 10.1007/BF00020202. [DOI] [PubMed] [Google Scholar]
  10. Brochetto-Braga M. R., Leite A., Arruda P. Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms. Plant Physiol. 1992 Mar;98(3):1139–1147. doi: 10.1104/pp.98.3.1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cord Neto G., Yunes J. A., da Silva M. J., Vettore A. L., Arruda P., Leite A. The involvement of Opaque 2 on beta-prolamin gene regulation in maize and Coix suggests a more general role for this transcriptional activator. Plant Mol Biol. 1995 Mar;27(5):1015–1029. doi: 10.1007/BF00037028. [DOI] [PubMed] [Google Scholar]
  12. Epelbaum S., McDevitt R., Falco S. C. Lysine-ketoglutarate reductase and saccharopine dehydrogenase from Arabidopsis thaliana: nucleotide sequence and characterization. Plant Mol Biol. 1997 Dec;35(6):735–748. doi: 10.1023/a:1005808923191. [DOI] [PubMed] [Google Scholar]
  13. Falco S. C., Guida T., Locke M., Mauvais J., Sanders C., Ward R. T., Webber P. Transgenic canola and soybean seeds with increased lysine. Biotechnology (N Y) 1995 Jun;13(6):577–582. doi: 10.1038/nbt0695-577. [DOI] [PubMed] [Google Scholar]
  14. Frankard V., Ghislain M., Jacobs M. Two Feedback-Insensitive Enzymes of the Aspartate Pathway in Nicotiana sylvestris. Plant Physiol. 1992 Aug;99(4):1285–1293. doi: 10.1104/pp.99.4.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fujioka M., Takata Y., Ogawa H., Okamoto M. The inactivation of saccharopine dehydrogenase (L-lysine-forming) by diethyl pyrocarbonate. J Biol Chem. 1980 Feb 10;255(3):937–942. [PubMed] [Google Scholar]
  16. Fujioka M., Takata Y. Role of arginine residue in saccharopine dehydrogenase (L-lysine forming) from baker's yeast. Biochemistry. 1981 Feb 3;20(3):468–472. doi: 10.1021/bi00506a004. [DOI] [PubMed] [Google Scholar]
  17. Gallie D. R., Feder J. N., Schimke R. T., Walbot V. Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression. Mol Gen Genet. 1991 Aug;228(1-2):258–264. doi: 10.1007/BF00282474. [DOI] [PubMed] [Google Scholar]
  18. Gallusci P., Varotto S., Matsuoko M., Maddaloni M., Thompson R. D. Regulation of cytosolic pyruvate, orthophosphate dikinase expression in developing maize endosperm. Plant Mol Biol. 1996 Apr;31(1):45–55. doi: 10.1007/BF00020605. [DOI] [PubMed] [Google Scholar]
  19. Gaziola S. A., Teixeira C. M., Lugli J., Sodek L., Azevedo R. A. The enzymology of lysine catabolism in rice seeds--isolation, characterization, and regulatory properties of a lysine 2-oxoglutarate reductase/saccharopine dehydrogenase bifunctional polypeptide. Eur J Biochem. 1997 Jul 1;247(1):364–371. doi: 10.1111/j.1432-1033.1997.00364.x. [DOI] [PubMed] [Google Scholar]
  20. Giroux M. J., Boyer C., Feix G., Hannah L. C. Coordinated Transcriptional Regulation of Storage Product Genes in the Maize Endosperm. Plant Physiol. 1994 Oct;106(2):713–722. doi: 10.1104/pp.106.2.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goncalves-Butruille M., Szajner P., Torigoi E., Leite A., Arruda P. Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize. Plant Physiol. 1996 Mar;110(3):765–771. doi: 10.1104/pp.110.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Habben J. E., Kirleis A. W., Larkins B. A. The origin of lysine-containing proteins in opaque-2 maize endosperm. Plant Mol Biol. 1993 Nov;23(4):825–838. doi: 10.1007/BF00021537. [DOI] [PubMed] [Google Scholar]
  23. Hinnebusch A. G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev. 1988 Jun;52(2):248–273. doi: 10.1128/mr.52.2.248-273.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jacobson A., Peltz S. W. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem. 1996;65:693–739. doi: 10.1146/annurev.bi.65.070196.003401. [DOI] [PubMed] [Google Scholar]
  25. Karchi H., Shaul O., Galili G. Lysine synthesis and catabolism are coordinately regulated during tobacco seed development. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2577–2581. doi: 10.1073/pnas.91.7.2577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kemper E. L., Cord-Neto G., Capella A. N., Gonçalves-Butruile M., Azevedo R. A., Arruda P. Structure and regulation of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase in maize. Eur J Biochem. 1998 May 1;253(3):720–729. doi: 10.1046/j.1432-1327.1998.2530720.x. [DOI] [PubMed] [Google Scholar]
  27. Larkins B. A., Hurkman W. J. Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiol. 1978 Aug;62(2):256–263. doi: 10.1104/pp.62.2.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lohmer S., Maddaloni M., Motto M., Di Fonzo N., Hartings H., Salamini F., Thompson R. D. The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene. EMBO J. 1991 Mar;10(3):617–624. doi: 10.1002/j.1460-2075.1991.tb07989.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lohmer S., Maddaloni M., Motto M., Salamini F., Thompson R. D. Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence. Plant Cell. 1993 Jan;5(1):65–73. doi: 10.1105/tpc.5.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. MERTZ E. T., BATES L. S., NELSON O. E. MUTANT GENE THAT CHANGES PROTEIN COMPOSITION AND INCREASES LYSINE CONTENT OF MAIZE ENDOSPERM. Science. 1964 Jul 17;145(3629):279–280. doi: 10.1126/science.145.3629.279. [DOI] [PubMed] [Google Scholar]
  31. Markovitz P. J., Chuang D. T., Cox R. P. Familial hyperlysinemias. Purification and characterization of the bifunctional aminoadipic semialdehyde synthase with lysine-ketoglutarate reductase and saccharopine dehydrogenase activities. J Biol Chem. 1984 Oct 10;259(19):11643–11646. [PubMed] [Google Scholar]
  32. Markovitz P. J., Chuang D. T. The bifunctional aminoadipic semialdehyde synthase in lysine degradation. Separation of reductase and dehydrogenase domains by limited proteolysis and column chromatography. J Biol Chem. 1987 Jul 5;262(19):9353–9358. [PubMed] [Google Scholar]
  33. Mauri I., Maddaloni M., Lohmer S., Motto M., Salamini F., Thompson R., Martegani E. Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast. Mol Gen Genet. 1993 Nov;241(3-4):319–326. doi: 10.1007/BF00284684. [DOI] [PubMed] [Google Scholar]
  34. Müller M., Knudsen S. The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box. Plant J. 1993 Aug;4(2):343–355. doi: 10.1046/j.1365-313x.1993.04020343.x. [DOI] [PubMed] [Google Scholar]
  35. Ogawa H., Fujioka M. The reaction of pyridoxal 5'-phosphate with an essential lysine residue of saccharopine dehydrogenase (L-lysine-forming). J Biol Chem. 1980 Aug 10;255(15):7420–7425. [PubMed] [Google Scholar]
  36. Ogawa H., Okamoto M., Fujioka M. Chemical modification of the active site sulfhydryl group of saccharopine dehydrogenase (L-lysine-forming). J Biol Chem. 1979 Aug 10;254(15):7030–7035. [PubMed] [Google Scholar]
  37. Rao V. V., Pan X., Chang Y. F. Developmental changes of L-lysine-ketoglutarate reductase in rat brain and liver. Comp Biochem Physiol B. 1992 Sep;103(1):221–224. doi: 10.1016/0305-0491(92)90435-t. [DOI] [PubMed] [Google Scholar]
  38. Schmidt R. J., Ketudat M., Aukerman M. J., Hoschek G. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes. Plant Cell. 1992 Jun;4(6):689–700. doi: 10.1105/tpc.4.6.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schuler G. D., Altschul S. F., Lipman D. J. A workbench for multiple alignment construction and analysis. Proteins. 1991;9(3):180–190. doi: 10.1002/prot.340090304. [DOI] [PubMed] [Google Scholar]
  40. Shaul O., Galili G. Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase. Plant Mol Biol. 1993 Nov;23(4):759–768. doi: 10.1007/BF00021531. [DOI] [PubMed] [Google Scholar]
  41. Singletary G. W., Doehlert D. C., Wilson C. M., Muhitch M. J., Below F. E. Response of enzymes and storage proteins of maize endosperm to nitrogen supply. Plant Physiol. 1990 Nov;94(3):858–864. doi: 10.1104/pp.94.3.858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sodek L., Wilson C. M. Incorporation of leucine-14C and lysine-14C into protein in the developing endosperm of normal and opaque-2 corn. Arch Biochem Biophys. 1970 Sep;140(1):29–38. doi: 10.1016/0003-9861(70)90006-8. [DOI] [PubMed] [Google Scholar]
  43. Sullivan M. L., Green P. J. Post-transcriptional regulation of nuclear-encoded genes in higher plants: the roles of mRNA stability and translation. Plant Mol Biol. 1993 Dec;23(6):1091–1104. doi: 10.1007/BF00042344. [DOI] [PubMed] [Google Scholar]
  44. Tang G., Miron D., Zhu-Shimoni J. X., Galili G. Regulation of lysine catabolism through lysine-ketoglutarate reductase and saccharopine dehydrogenase in Arabidopsis. Plant Cell. 1997 Aug;9(8):1305–1316. doi: 10.1105/tpc.9.8.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tolbert N. E. Isolation of subcellular organelles of metabolism on isopycnic sucrose gradients. Methods Enzymol. 1974;31:734–746. doi: 10.1016/0076-6879(74)31077-4. [DOI] [PubMed] [Google Scholar]
  46. Varagona M. J., Schmidt R. J., Raikhel N. V. Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants. Plant Cell. 1991 Feb;3(2):105–113. doi: 10.1105/tpc.3.2.105. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES