Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

Characterization of the predominant cytosolic enzyme from green and ripe pericarp

Abstract

The activity of NADP+-specific isocitrate dehydrogenase (NADP+-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP+-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP+-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP+-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K m values of the enzymes from green and ripe pericarp for NADP+, isocitrate and Mg2+ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP+-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C6 organic acids and in glutamate accumulation in ripe tissues.

This is a preview of subscription content, log in to check access.

Abbreviations

DEAE:

diethylaminoethyl

GADPH:

NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase

GOGAT:

glutamate synthase

GS:

glutamine synthetase

Mr :

relative molecular mass

NAD+-IDH:

NAD+-specific isocitrate dehydrogenase

NADP+ -IDH:

NADP+-specific isocitrate dehydrogenase

PEPCase:

phosphoenolpyruvate carboxylase

References

  1. Arnozis, P.A., Nelemans, J.A., Findenegg, G.R. (1988) Phophoenolpyruvate carboxylase activity in plants grown with either NO 3 su− or NH 4 + as inorganic nitrogen source. J. Plant Physiol. 132, 23–27

  2. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248–254

  3. Brady, C.J. (1987) Fruit ripening. Annu. Rev. Plant. Physiol. 38, 155–178

  4. Brouquisse, R., Nishimura, M., Gaillard, J., Douce, A. (1987) Characterization of a cytosolic aconitase in higher plant cells. Plant Physiol. 84, 1402–1430

  5. Chen, R.D., Gadal, P. (1990a) Do the mitochondria provide the 2-oxoglutarate needed for glutamate synthesis in higher plant chloroplasts? Plant Physiol. Biochem. 28, 141–145

  6. Chen, R.D., Gadal, P. (1990b) Structure, functions and regulation of NAD+ and NADP+ dependent isocitrate dehydrogenases in higher plants and in other organisms. Plant Physiol. Biochem 28, 411–427

  7. Chen, R.D., Bismuth, E., Champigny, M.L., Gadal, P. (1989 a) Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenase are distinct isoenzymes. Planta 178, 157–163

  8. Chen, R.D., Le Marechal, P., Vidal, J., Jacquot, J.P., Gadal, P. (1988) Purification and comparative properties of the cytosolic isocitrate dehydrogenases (NADP) from pea (Pisum sativum) roots and green leaves. Eur. J. Biochem. 175, 565–572

  9. Gallardo, F. (1992) Evolución de las enzimas de la asimilación de amonio durante la diferenciación de plastidios de Lycopersicon esculentum L. Ph. D. thesis, Universidad de Málaga

  10. Gallardo, F., Cánovas, F.M. (1992) A macromolecular inhibitor of glutamine synthetase activity in tomato root extracts. Phytochemistry 31, 2267–2271

  11. Gallardo, F., Gálvez, S., Quesada, M.A., Cánovas, F.M., Nuñez de Castro, I. (1988) Glutamine synthetase activity during the ripening of tomato. Plant Physiol. Biochem. 26, 747–752

  12. Gallardo, F., Cantón, F.R., Garcia-Gutiérrez, A., Cánovas, F.M. (1993) Changes in photorespiratory enzymes and glutamate synthases in ripening tomatoes. Plant Physiol. Biochem. 31, 189–196

  13. Gálvez, S., Bismuth, E., Sarda, C., Gadal, P. (1994) Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from mixotrophic tobacco cells. Comparison with the cytosolic isoenzyme. Plant Physiol. 105, 593–600

  14. Goodenough, P.W., Prosser, I.M., Young, K. (1985) NADP-linked malic enzyme and malate metabolism in ageing tomato fruit. Phytochem. 24, 1157–1162

  15. Grierson, D., Maunders, M.J., Slater, A., Ray, J., Bird, C.R., Schuch, W., Holdsworth, M.J., Tucker, G.A., Knapp, J.E. (1986) Gene expression during tomato ripening. Phil. Trans. R. Soc. Lond. B. 314, 399–410

  16. Grierson, D., Slater, A., Maunders, M., Crookes, P., Tucker, G.A., Schuch, W., Edwards, K. (1985) In: Ethylene and plant development, pp. 147-161, Roberts, J.A., Tucker, G.A., eds. Butterworth, London

  17. Gupta, V.K., Singh, R. (1988) Partial purification and characterization of NADP+-isocitrate dehydrogenase from immature pod walls of chickpea (Cicer arietinum L.). Plant Physiol. 87, 741–744

  18. Hanning, I., Heldt, H.W. (1993) On the function of mitochondrial metabolism during photosynthesis in spinach (Spinacia oleracea L.) leaves. Plant Physiol. 103, 1147–1154

  19. Jeffery, D., Goodenough, P.W., Weitzman, P.D.J. (1986) Enzyme activities in mitochondria isolated from ripening tomato fruit. Plant 168, 390–394

  20. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685

  21. Laval-Martin, D., Farineau, J., Diamond, J. (1977) Light versus dark carbon metabolism in cherry tomato fruits. Plant Physiol. 60, 872–876

  22. Leshem, Y.Y., Havely, A.H., Frenkel, C., eds. (1986) Processes and control of plant senescence. Elsevier Publishers B.V., Amsterdam

  23. Miflin, B.J., Wallsgrove, R.M., Lea, P.J. (1981) Glutamine metabolism in higher plants. Curr. Top. Cell. Regul. 20, 1–43

  24. Morrisey, J.H. (1981) Silver stain for proteins in polyacrylamide gels, A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117, 307–310

  25. Ni, W., Robertson, E.F., Reeves, H.C. (1987) Purification and characterization of cytosolic NADP specific isocitrate dehydrogenase from Pisum sativum. Plant Physiol. 83, 785–788

  26. Piechulla, B., Glick, R.E., Bahl, H., Melis, A., Gruissem, W. (1987) Changes in photosynthetic capacity and photosynthetic protein pattern during tomato fruit ripening. Plant Physiol. 84, 911–917

  27. Randall, D.D., Givan, C.V. (1981) Subcellular location of NADP-isocitrate dehydrogenase in Pisum sativum leaves. Plant Physiol. 68, 70–73

  28. Rasmusson, A.G., Möller, I.M. (1990) NADP-Utilizing enzymes in the matrix of plant mitochondria. Plant Physiol. 94, 1012–1018

  29. Stevens, M.A. (1972) Citrate and malate concentrations in tomato fruits: genetic control and maturational effects. J. Am. Soc. Hort. Sci. 97, 655–685

  30. Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76, 4350–4354

  31. Willmer, C.M., Johnston, W.R. (1976) Carbon dioxide assimilation in some aerial plant organs and tissues. Planta 130, 33–37

Download references

Author information

Correspondence to Fernando Gallardo.

Additional information

We thank to M. Hodges (Université Paris Sud) for the revision and criticism of the manuscript. This work was partially supported by a Grant from Direction General de Investigatión Científica y Técnica (PB 92-0423) to F.M.C. F.G. was supported by a fellowship from the Programa General de Formatión de Personal Investigador, Junta de Andalucia, and by a short-term fellowship from Ministerio de Educacion y Ciencia. S.G. is recipient of a fellowship from the Programa de Formatión de Personal Investigador en el Extranjero; Ministerio de Educación y Ciencia, Spain.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gallardo, F., Gálvez, S., Gadal, P. et al. Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening. Planta 196, 148–154 (1995). https://doi.org/10.1007/BF00193228

Download citation

Key words

  • Carbon-nitrogen interaction
  • Fruit ripening
  • Lycopersicon
  • NADP+-isocitrate dehydrogenase