Characterization of the glutamate dehydrogenase isoenzyme system in germinating soybean

• Published in: Plant science (Limerick) Vol. 135; no. 2; pp. 137 - 148
• Main Authors: Turano, Frank J., Dashner, Ralph, Upadhyaya, Abha, Caldwell, Charles R., Bauchan, Gary
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 10-07-1998; Elsevier Science
• Subjects: Agronomy. Soil science and plant productions; Biological and medical sciences; cortex; dark; Economic plant physiology; Embryo development. Germination; enzyme activity; epidermis; Fundamental and applied biological sciences. Psychology; Germination and dormancy; glutamate dehydrogenase; glutamic acid; Glycine max; Growth and development; hypocotyls; Immunoblot; immunohistochemistry; isozymes; NAD(H)-GDH; NADP(H)-GDH; phloem; Plant physiology and development; root hairs; root tips; seed germination; Soybean; Tissue print; Immunoblot; β-ME, β-mercaptoethanol; GDH, glutamate dehydrogenase; PMSF, phenylmethylsulfonyl fluoride; NADP(H)-GDH; NAD(H)-GDH, NAD(H)-dependent GDH; NADP(H)-GDH, NADP(H)-dependent GDH; NAD(H)-GDH; Soybean; PPFD, photosynthetic photon flux density; PVP, polyvinylpyrrolidone; Tissue print; Glutamate dehydrogenase; Enzyme; Isozyme; Germination; Tissue specificity; Glycine max; NAD; Grain legume; Leguminosae; Enzymatic activity; Dicotyledones; Seed; Angiospermae; Spermatophyta; Oxidoreductases; NADP
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/S0168-9452(98)00082-X

Protein extracts from different soybean (
Glycine max L. Merr. cv. Williams) organs contained four glutamate dehydrogenase (GDH) isoenzymes (EC 1.4.1.2–1.4.1.4) when native polyacrylamide gels were stained for NAD-dependent activity. The four isoenzymes were designated GDH0, GDH1, GDH2 and GDH3. A combination of NAD- or NADP-specific GDH stains and immunoblot analysis of native polyacrylamide gels was used to characterize the four GDH isoenzymes. Isoenzymes GDH0, GDH1 and GDH2 demonstrated both NAD- and NADP-dependent activity, while GDH3 only had NAD-dependent activity. Based on the intensity of the different stains, GDH1 and GDH2 had greater activity with NAD than with NADP, while GDH0 had less activity with NAD than with NADP. Both GDH2 and GDH3 cross-reacted with rabbit serum raised against grape leaf NAD(H)-GDH, but the reaction was more intense with GDH3 than GDH2. Tissue print analyses were used to demonstrate the tissue-specific accumulation of GDH3 activity throughout the axis during germination in the dark. Negative controls (GDH stain solution lacking glutamate) and immunodetection with antiserum to grape NAD(H)-GDH validated the specificity of the NAD-GDH tissue print analyses. NAD-GDH activity was most abundant in the phloem of the hypocotyl hook with diffuse activity throughout the cortex. In addition, NAD-GDH activity was apparent in the epidermis of the hypocotyl hook. In the region of the hypocotyl below the hook and above the root hairs, enzyme activity was evident throughout the cortex and epidermis and absent in the vascular tissues. In the root, NAD-GDH activity was localized exclusively in the epidermal layer, particularly in the region of the functional root hairs. All other tissue types in this region of the root, including the xylem, phloem and cortex, were devoid of GDH activity. In emerging lateral roots, NAD-GDH activity was localized in the root tip and epidermis. Since only GDH3 was detected in these tissues, the data suggest that GDH3 may play a unique role in the mobilization of carbon or nitrogen in germinating seedlings.