The present communication reports substantial activity of gluconeogenic fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in three common heterosporous aquatic ferns (Marsilea minuta, Salvinia natans, and Azolla pinnata) and also describes a protocol for its partial purification from mature sporocarps of Marsilea minuta. The cytosolic FBPase, obtained from Marsilea minuta, Salvinia natans, and Azolla pinnata was recognized as gluconeogenic enzyme due to its drastic catabolic inactivation in presence of externally administered glucose and its insensitivity towards photosynthetic light illumination. Cytosolic gluconeogenic FBPase was partially purified from mature sporocarps of Marsilea minuta to about 22-fold over homogenate following low-speed centrifugation (11, 400 × g), 30–80% ammonium sulfate fractionation followed by subsequent chromatography using matrices like CM-Cellulose, Sephadex G-200, and Ultrogel AcA 34. The profile of partially purified FBPase in PAGE under non-denaturing condition was recorded. The enzyme activity increased linearly with respect to protein concentration to about 100 µg and with respect to time up to 75 minutes. Temperature optimum was found at 35 °C. The effect of substrate concentration and kinetic analyses for FBPase were carried out using D-fructose-1,6-bisphosphate (D-FBP, the substrate) in the range of 0.0 to 1.0 mM at an interval of 0.1 mM concentration. The K_m value for D-FBP of FBPase was 0.06129 mM and V_max was 4525 nmole P_i released (mg)^-1 protein h^-1 as determined by nonlinear regression kinetics using Prism 8 software (Graph Pad). The enzyme was functional in a constricted pH range of 7.0 to 8.0, giving maxima at pH 7.5. This cytosolic enzyme was significantly stimulated by Mg²⁺ and strongly inhibited by Hg²⁺, Cu²⁺ and Zn²⁺.

Keywords: aquatic fern, enzyme purification and characterization, FBPase, gluconeogenesis, sporocarp

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1. Adhikari J., Majumder A., Bhaduri T.J., DasGupta S., Majumder A.L. 1987. Chloroplast as a locale of L-myo-inositol-1-phosphate synthase. Plant Physiology, 85(3): 611–614. https://doi.org/10.1104/pp.85.3.611
2. Arokiyaraj S., Bharanidharan R., Agastian P., Shin H. 2018. Chemical composition, antioxidant activity and antibacterial mechanism of action from Marsilea minuta leaf hexane: methanol extract. Chemistry Central Journal, 12(1): 1–11. https://doi.org/10.1186/s13065-018-0476-4
3. Bierhorst D.W. 1971. Morphology of vascular plants. New York: MacMillan, 137 pp.
4. Biswas T., Adhikari J., Choudhuri R.B., Majumder A.L. 1981. Fructose-1,6-bisphosphatase & myo-inositol synthase: A phylogenetic search. Indian Journal of Biochemistry and Biophysics, 18(6): 442–444.
5. Bold H.C., Alexopoulos C.J., Delevoryas T. 1987. Morphology of plants and fungi. 5^th ed. Harpercollins College: Harper & Row, 912 pp.
6. Bollag D.M., Rozycki M.D., Edelstein S.J. 1996. Protein methods. New York: Wiley-Liss, 415 pp.
7. Botha F.C., Turpin D.H. 1990. Fructose 1,6-bisphosphatase in the green algae Selenastrum minutum. I. Evidence for the presence of isoenzymes. Plant Physiology, 93(4): 1460–1465. https://doi.org/10.1104/pp.93.4.1460
8. Bower F.O. 1928. The ferns, vol. 3. Cambridge: Cambridge University Press, 359 pp.
9. Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing principle of protein-dye binding. Analytical Biochemistry, 72: 248–254. https://doi.org/10.1016/0003-2697(76)90527-3
10. Chen P.S., Toribara T.Y., Warner H. 1956. Microdetermination of phosphorous. Analytical Chemistry, 28(11): 1756–1758. https://doi.org/10.1021/ac60119a033
11. Dey A., Lahiri A., Basak A., Adhikari J. 2014. Characterization of gluconeogenic fructose-1,6-bisphosphatase in the senescent thalli of an alga Cladophora bengalensis Martens. Indian Journal of Plant Physiology, 19: 8–13. https://doi.org/10.1007/s40502-013-0052-9
12. Eames A.J.1936. Morphology of vascular plants. New York: McGraw-Hill, 433 pp.
13. Eckstein A., Zięba P., Gabryś H. 2012. Sugar and light effects on the condition of the photosynthetic apparatus of Arabidopsis thaliana cultured in vitro. Journal of Plant Growth Regulation, 31(1): 90–101. https://doi.org/10.1007/s00344-011-9222-z
14. Gifford E.M., Foster A.S. 1987. Morphology and evolution of vascular plants. 3rd ed. New York: W.H. Freeman and Company, pp. 306–313.
15. Jardon R., Gancedo C., Flores C.L. 2008. The gluconeogenic enzyme fructose-1,6-bisphosphatase is dispensable for growth of the yeast Yarrowia lipolytica in gluconeogenic substrates. Eukaryotic Cell, 7(10): 1742–1749. https://doi.org/10.1128/EC.00169-08
16. Johnson D.M. 1985. New records for longevity of Marsilea sporocarps. American Fern Journal, 75: 30–31. https://doi.org/10.2307/1546579
17. Kobr M.J., Beevers H. 1971. Gluconeogenesis in the castor bean endosperm. I. Changes in glycolytic intermediates. Plant Physiology, 47(1): 48–52. https://doi.org/10.1104/pp.47.1.48
18. Kramer K.U. 1990. Marsileaceae. In: Kramer K.U., Green P.S. (eds). Pteridophytes and Gymnosperms. The Families and Genera of Vascular Plants, vol. 1. Berlin; Heidelberg: Springer, pp. 180–183. https://doi.org/10.1007/978-3-662-02604-5_32
19. Nagalingum N.S., Schneider H., Pryer K.M. 2006. Comparative morphology of reproductive structures in heterosporous water ferns and a re-evaluation of the sporocarp. International Journal of Plant Sciences, 167(4): 805–815. https://doi.org/10.1086/503848
20. Raubeson L.A., Stein D.B., Conant D.S. 1994. Insights into fern evolution from mapping chloroplast genomes. American Journal of Botany, 81(6): 123.
21. Rothwell G.W., Stockey R.A. 1994. The role of Hydropteris pinnata gen. et sp. nov. in reconstructing the cladistics of heterosporous ferns. American Journal of Botany, 81(4): 479–492.
22. Say R.A., Fuchs G. 2010. Fructose 1,6-bisphosphate aldolase/phosphatase may be an ancestral gluconeogenic enzyme. Nature, 464 (7291): 1077–1081. https://doi.org/10.1038/nature08884
23. Soltis D.E., Soltis P.S. 1986. Active enzymes from megaspores of Marsilea and Regnellidium. American Fern Journal, 76(1): 17–20. https://doi.org/10.2307/1547395
24. Sood A., Uniyal P.L., Prasanna R., Ahluwalia A.S. 2012. Phytoremediation potential of aquatic macrophyte, Azolla. Ambio, 41(2): 122–137. https://doi.org/10.1007/s13280-011-0159-z
25. Stitt M., Heldt H.W. 1985. Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate. Planta, 164(2): 179–188. https://doi.org/10.1007/BF00396080
26. Tryon R.M., Tryon A.F. 1982. Ferns and allied plants, with special reference to tropical America. New York: Springer, 857 pp. https://doi.org/10.1007/978-1-4613-8162-4
27. Yu S. 1989. Partial purification of fructose 1,6-bisphosphatase from the marine macro alga Bryopsis corticulans (Chlorophyceae). Journal of Phycology, 25: 608–612.
28. Zimmermann G., Kelly G.J., Latzko E. 1978. Purification and properties of spinach leaf cytoplasmic fructose-1,6-bisphosphatase. Journal of Biological Chemistry, 253(17): 5952–5956.