Abstract
Multiple isoforms of β-fructofuranosidase (invertase, EC 3.2.1.26) were identified in mature green leaves of the cruciferous plant Arabidopsis thaliana (L.) Heynh. There were four major and one minor isoforms of soluble acid invertase and an additional activity which could be released from the cell wall by buffers of high ionic strength. This study reports the separation and characterisation of three soluble isoforms following ammonium sulphate and polyethylene glycol 6000 precipitations, Concanavalin A, MonoQ ion exchange, Superose 12 sizeexclusion chromatography and chromatofocusing. These isoforms, designated INV1, INV2 and INV3, had isoelectric points of 4.75, 4.70 and 4.65 and a K m for sucrose of 5, 12 and 5 mM, respectively. Each had a pH optimum of 5.5, exhibited optimal activity at 45 °C and used sucrose as the preferred substrate. All fractions containing these isoforms contained a 52-kDa polypeptide which was specifically detected by immunoblotting with an antibody raised against deglycosylated wheat invertase. The N-terminal amino-acid sequence of this polypeptide was homologous to acid invertases isolated from other plant species. The possible origin of isoforms of soluble acid invertase is discussed.
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Abbreviations
- PEG:
-
polyethylene glycol
- pI:
-
isoelectric point
- PMSF:
-
phenylmethylsulphonyl fluoride
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410
Arnold WN (1965) β-fructofuranosidase from grape berries. Biochim Biophys Acta 110: 134–147
Benhamou N, Grenier J, Chrispeels MJ (1991) Accumulation of beta-fructosidase in the cell walls of tomato roots following infection by a fungal wilt pathogen. Plant Physiol 97: 739–750
Bracho GE, Whitaker JR (1990) Purification and partial characterization of potato (Solanum tuberosum) invertase and its endogenous proteinaceous inhibitor. Plant Physiol 92: 386–394
Chen JQ, Black CC (1992) Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls. Arch Biochem Biophys 295: 61–69
Dickinson CD, Altabella T, Chrispeels MJ (1991) Slow-growth phenotype of transgenic tomato expressing apoplastic invertase. Plant Physiol 95: 420–425
Elliott KJ, Butler WO, Dickinson CD, Konno Y, Vedvick TS, Fitzmaurice L, Mirkov TE (1993) Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening. Plant Mol Biol 21: 515–524
Fahrendorf T, Beck E (1990) Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: A comparison. Planta 180: 237–244
Faye L (1981) A new enzymatic staining method for the detection of radish beta-fructosidase in gel electrophoresis. Anal Biochem 112: 90–95
Faye L, Mouatassim B, Ghorbel A (1986) Cell wall and cytoplasmic isozymes of radish (Raphanus sativus cultivar Longue rave saumonee) beta-fructosidase have different N-linked oligosaccharides. Plant Physiol 80: 27–33
Foyer CH (1987) The basis for source-sink interaction in leaves. Plant Physiol Biochemistry 25: 649–657
Huber SC (1989) Biochemical mechanism for regulation of sucrose accumulation in leaves during photosynthesis. Plant Physiol 91: 656–662
Karuppiah N, Vadlamudi B, Kaufman PB (1989) Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue. Plant Physiol 91: 993–998
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Matsushita K, Uritani I (1974) Change in invertase activity of sweet potato in response to wounding and purification and properties of its invertases. Plant Physiol 54: 60–66
Mercier RW, Gogarten JP (1995) A second cell wall acid invertase gene in Arabidopsis thaliana. Plant Physiol 107: 659–660
Obenland DM, Simmen U, Boller T, Wiemken A (1993) Purification and characterization of three soluble invertases from barley (Hordeum vulgare L.) leaves. Plant Physiol 101: 1331–1339
Ruffner HP, Hürlimann M, Skrivan R (1995) Soluble invertase from grape berries: purification, deglycosylation and antibody specificity. Plant Physiol Biochem 33, in press
Scholes JD, Lee PJ, Horton P, Lewis DH (1994) Invertase: Understanding changes in the photosynthetic and carbohydrate metabolism of barley leaves infected with powdery mildew. New Phytol 126: 213–222
Schwebel-Dugue N, El-Mtili N, Krivitzky M, Jean-Jacques I, Williams JHH, Thomas M, Kreis M, Lecharny A (1994) Arabidopsis gene and cDNA encoding cell-wall invertase. Plant Physiol 104: 809–810
Sonnewald U, Brauer M, vonSchaewen A, Stitt M, Willmitzer L (1991) Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: A powerful tool for studying sucrose metabolism and sink/source interactions. Plant J 1: 95–106
Spencer CM, Cai K, Martin R, Gaffney SH, Goulding PN, Magnolato D, Lilley TH Haslam E (1988) Polyphenol complexation — some thoughts and observations. Phytochemistry 27: 2397–2409
Stitt M, vonSchaewen A, Willmitzer U (1991) “Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes. Planta 183: 40–50
Stommel JR, Simon PW (1990) Multiple forms of invertase from Daucus carota cell cultures. Phytochemistry 29: 2087–2090
Storr T, Hall JL (1992) The effect of infection by Erysiphe pisi DC on acid and alkaline invertase activities and aspects of starch biochemistry in leaves in Pisum sativum L. New Phytol 121: 535–543
Sturm A, Chrispeels MJ (1990) cDNA cloning of carrot extracellular β-fructosidase and its expression in response to wounding and bacterial infection. Plant Cell 2: 1107–1119
Sung HY, Huang WC (1994) Purification and characterization of cell-wall-bound invertase from rice (Oryza sativa) grains. Biotechnol Applied Biochem 19: 75–83
Unger C, Hardegger M, Lienhard S, Sturm A (1994) cDNA cloning of carrot (Daucus carota) soluble acid β-fructofuranosidases and comparison with the cell wall isoenzyme. Plant Physiol 104: 1351–1357
vonSchaewen A, Stitt M, Schmidt R, Sonnewald U, Willmitzer L (1990) Expression of a yeast-derived invertase in the cell-wall of tobacco and Arabidopsis plants leads to an accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants. EMBO J 9: 3033–3044
Walker RP, Pollock CJ (1993) The purification and characterisation of soluble acid invertase from coleoptiles of wheat (Triticum aestivum L. cv. Avalon). J Exp Bot 44: 1029–1037
Wu LL, Song I, Kim D, Kaufman PB (1993) Molecular basis of the increase in invertase activity elicited by gravistimulation of oatshoot pulvini. J Plant Physiol 142: 179–183
Yelle, S, Chetelat RT, Dorais M, Deverna JW, Bennett AB (1991) Sink metabolism in tomato fruit: IV. Genetic and biochemical analysis of sucrose accumulation. Plant Physiol 95: 1026–1035
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We wish to acknowledge the support of the British/Swiss Joint Research Programme and the Sheffield University Research Support Fund. X.T. was in receipt of an Overseas Research Scholarship and a University of Sheffield Research Scholarship. We wish to thank Dr A. Moir for his help in N-terminal amino-acid sequencing.
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Tang, X., Ruffner, HP., Scholes, J.D. et al. Purification and characterisation of soluble invertases from leaves of Arabidopsis thaliana . Planta 198, 17–23 (1996). https://doi.org/10.1007/BF00197581
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DOI: https://doi.org/10.1007/BF00197581