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. 1990 Nov;2(11):1107–1119. doi: 10.1105/tpc.2.11.1107

cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

A Sturm 1, M J Chrispeels 1
PMCID: PMC159958  PMID: 2152110

Abstract

We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

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Selected References

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  1. Bennett M. D., Smith J. B. Nuclear dna amounts in angiosperms. Philos Trans R Soc Lond B Biol Sci. 1976 May 27;274(933):227–274. doi: 10.1098/rstb.1976.0044. [DOI] [PubMed] [Google Scholar]
  2. Cooper R. A., Greenshields R. N. The partial purification and some properties of two sucrases of Phaseolus vulgaris. Biochem J. 1964 Aug;92(2):357–364. doi: 10.1042/bj0920357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Corbin D. R., Sauer N., Lamb C. J. Differential regulation of a hydroxyproline-rich glycoprotein gene family in wounded and infected plants. Mol Cell Biol. 1987 Dec;7(12):4337–4344. doi: 10.1128/mcb.7.12.4337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. EDELMAN J., HALL M. A. ENZYME FORMATION IN HIGHER-PLANT TISSUES. DEVELOPMENT OF INVERTASE AND ASCORBATE-OXIDASE ACTIVITIES IN MATURE STORAGE TISSUE OF HELIANTHUS TUBEROSUS L. Biochem J. 1965 May;95:403–410. doi: 10.1042/bj0950403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Edge A. S., Faltynek C. R., Hof L., Reichert L. E., Jr, Weber P. Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Anal Biochem. 1981 Nov 15;118(1):131–137. doi: 10.1016/0003-2697(81)90168-8. [DOI] [PubMed] [Google Scholar]
  6. Edman P., Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. doi: 10.1007/978-3-662-25813-2_14. [DOI] [PubMed] [Google Scholar]
  7. Giaquinta R. T., Lin W., Sadler N. L., Franceschi V. R. Pathway of Phloem unloading of sucrose in corn roots. Plant Physiol. 1983 Jun;72(2):362–367. doi: 10.1104/pp.72.2.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hasilik A., Tanner W. Carbohydrate moiety of carboxypeptidase Y and perturbation of its biosynthesis. Eur J Biochem. 1978 Nov 15;91(2):567–575. doi: 10.1111/j.1432-1033.1978.tb12710.x. [DOI] [PubMed] [Google Scholar]
  9. Joshi C. P. An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res. 1987 Aug 25;15(16):6643–6653. doi: 10.1093/nar/15.16.6643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Karuppiah N., Vadlamudi B., Kaufman P. B. Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue. Plant Physiol. 1989;91:993–998. doi: 10.1104/pp.91.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Krishnan H. B., Blanchette J. T., Okita T. W. Wheat invertases : characterization of cell wall-bound and soluble forms. Plant Physiol. 1985 Jun;78(2):241–245. doi: 10.1104/pp.78.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kunst F., Pascal M., Lefesant J. A., Walle J., Dedonder R. Purification and some properties of an endocellular sucrase from a constitutive mutant of Bacillus subtilis Marburg 168. Eur J Biochem. 1974 Mar 1;42(2):611–620. doi: 10.1111/j.1432-1033.1974.tb03376.x. [DOI] [PubMed] [Google Scholar]
  13. Laurière C., Laurière M., Sturm A., Faye L., Chrispeels M. J. Characterization of beta-fructosidase, an extracellular glycoprotein of carrot cells. Biochimie. 1988 Nov;70(11):1483–1491. doi: 10.1016/0300-9084(88)90285-4. [DOI] [PubMed] [Google Scholar]
  14. Laurière M., Laurière C., Chrispeels M. J., Johnson K. D., Sturm A. Characterization of a xylose-specific antiserum that reacts with the complex asparagine-linked glycans of extracellular and vacuolar glycoproteins. Plant Physiol. 1989 Jul;90(3):1182–1188. doi: 10.1104/pp.90.3.1182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lemoine R., Daie J., Wyse R. Evidence for the presence of a sucrose carrier in immature sugar beet tap roots. Plant Physiol. 1988 Feb;86(2):575–580. doi: 10.1104/pp.86.2.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lois R., Dietrich A., Hahlbrock K., Schulz W. A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light responsive cis-acting elements. EMBO J. 1989 Jun;8(6):1641–1648. doi: 10.1002/j.1460-2075.1989.tb03554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lütcke H. A., Chow K. C., Mickel F. S., Moss K. A., Kern H. F., Scheele G. A. Selection of AUG initiation codons differs in plants and animals. EMBO J. 1987 Jan;6(1):43–48. doi: 10.1002/j.1460-2075.1987.tb04716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Martin I., Débarbouillé M., Ferrari E., Klier A., Rapoport G. Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase. Mol Gen Genet. 1987 Jun;208(1-2):177–184. doi: 10.1007/BF00330439. [DOI] [PubMed] [Google Scholar]
  19. Matsushita K., Uritani I. Change in invertase activity of sweet potato in response to wounding and purification and properties of its invertases. Plant Physiol. 1974 Jul;54(1):60–66. doi: 10.1104/pp.54.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Reddy V. A., Johnson R. S., Biemann K., Williams R. S., Ziegler F. D., Trimble R. B., Maley F. Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons. J Biol Chem. 1988 May 25;263(15):6978–6985. [PubMed] [Google Scholar]
  22. Ripp K. G., Viitanen P. V., Hitz W. D., Franceschi V. R. Identification of membrane protein associated with sucrose transport into cells of developing soybean cotyledons. Plant Physiol. 1988 Dec;88(4):1435–1445. doi: 10.1104/pp.88.4.1435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sarokin L., Carlson M. Upstream region required for regulated expression of the glucose-repressible SUC2 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Dec;4(12):2750–2757. doi: 10.1128/mcb.4.12.2750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schmalstig J. G., Geiger D. R. Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast. Plant Physiol. 1985 Sep;79(1):237–241. doi: 10.1104/pp.79.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shannon L. M., Uritani I., Imaseki H. De novo synthesis of peroxidase isozymes in sweet potato slices. Plant Physiol. 1971 Apr;47(4):493–498. doi: 10.1104/pp.47.4.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Straus J. Invertase in Cell Walls of Plant Tissue Cultures. Plant Physiol. 1962 May;37(3):342–348. doi: 10.1104/pp.37.3.342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sturm A., Chrispeels M. J. The high mannose oligosaccharide of phytohemagglutinin is attached to asparagine 12 and the modified oligosaccharide to asparagine 60. Plant Physiol. 1986 May;81(1):320–322. doi: 10.1104/pp.81.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sturm A., Johnson K. D., Szumilo T., Elbein A. D., Chrispeels M. J. Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides. Plant Physiol. 1987 Nov;85(3):741–745. doi: 10.1104/pp.85.3.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sung Z. R. Turbidimetric measurement of plant cell culture growth. Plant Physiol. 1976 Mar;57(3):460–462. doi: 10.1104/pp.57.3.460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]
  31. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]

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