Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1996 Sep;112(1):89–97. doi: 10.1104/pp.112.1.89

Evidence that a 77-kilodalton protein from the starch of pea embryos is an isoform of starch synthase that is both soluble and granule bound.

A Edwards 1, J Marshall 1, K Denyer 1, C Sidebottom 1, R G Visser 1, C Martin 1, A M Smith 1
PMCID: PMC157927  PMID: 8819321

Abstract

In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein. A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is a different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes. Consistent with this, expression in E. coli of an N-terminally truncated version of SSII gives rise to starch synthase activity.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

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

  1. Baba T., Nishihara M., Mizuno K., Kawasaki T., Shimada H., Kobayashi E., Ohnishi S., Tanaka K., Arai Y. Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds. Plant Physiol. 1993 Oct;103(2):565–573. doi: 10.1104/pp.103.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bevan M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 1984 Nov 26;12(22):8711–8721. doi: 10.1093/nar/12.22.8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhattacharyya M. K., Smith A. M., Ellis T. H., Hedley C., Martin C. The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Cell. 1990 Jan 12;60(1):115–122. doi: 10.1016/0092-8674(90)90721-p. [DOI] [PubMed] [Google Scholar]
  4. Blackwell J. R., Horgan R. A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett. 1991 Dec 16;295(1-3):10–12. doi: 10.1016/0014-5793(91)81372-f. [DOI] [PubMed] [Google Scholar]
  5. Dry I., Smith A., Edwards A., Bhattacharyya M., Dunn P., Martin C. Characterization of cDNAs encoding two isoforms of granule-bound starch synthase which show differential expression in developing storage organs of pea and potato. Plant J. 1992 Mar;2(2):193–202. [PubMed] [Google Scholar]
  6. Edwards A., Marshall J., Sidebottom C., Visser R. G., Smith A. M., Martin C. Biochemical and molecular characterization of a novel starch synthase from potato tubers. Plant J. 1995 Aug;8(2):283–294. doi: 10.1046/j.1365-313x.1995.08020283.x. [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. Furukawa K., Tagaya M., Inouye M., Preiss J., Fukui T. Identification of lysine 15 at the active site in Escherichia coli glycogen synthase. Conservation of Lys-X-Gly-Gly sequence in the bacterial and mammalian enzymes. J Biol Chem. 1990 Feb 5;265(4):2086–2090. [PubMed] [Google Scholar]
  9. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Marston F. A. The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J. 1986 Nov 15;240(1):1–12. doi: 10.1042/bj2400001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Martin C., Carpenter R., Sommer H., Saedler H., Coen E. S. Molecular analysis of instability in flower pigmentation of Antirrhinum majus, following isolation of the pallida locus by transposon tagging. EMBO J. 1985 Jul;4(7):1625–1630. doi: 10.1002/j.1460-2075.1985.tb03829.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sivak M. N., Wagner M., Preiss J. Biochemical Evidence for the Role of the Waxy Protein from Pea (Pisum sativum L.) as a Granule-Bound Starch Synthase. Plant Physiol. 1993 Dec;103(4):1355–1359. doi: 10.1104/pp.103.4.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  16. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES