Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase, and its spatial and temporal changes during leaf development.
The grapevine (Vitis vinifera L.) partial fragment of cDNA clone pGOGAT1 [Loulakakis and Roubelakis-Angelakis (1997) Physiol Plant 101:220-228], encoding the ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), was overexpressed in Escherichia coli cells. A hybrid between the Fd-GOGAT fragment and maltose-binding protein was purified and used to raise a polyclonal antibody in a rabbit. The prepared antibody appeared to be specific towards Fd-GOGAT; it recognized a protein band of approximately 160 kDa on nitrocellulose blots after SDS-PAGE of total proteins from leaves, internodes, roots and calluses, and precipitated most of the enzyme activity present in grapevine protein extracts. The quantity of Fd-GOGAT protein was substantially higher in leaves than in other grapevine tissues tested, coincident with a similar distribution of the enzyme specific activity. Intracellular localization studies revealed that both the enzyme activity and the 160-kDa immunoreactive protein were associated with the chloroplastic fraction. Furthermore, the accumulation of Fd-GOGAT, glutamine synthetase (GS) and glutamate dehydrogenase (GDH), at the activity and protein levels, was monitored during leaf development of field-grown plants, from the stage of the newly expanding leaf to the senescing old leaf. Both the specific activity and quantity of the 160-kDa polypeptide of Fd-GOGAT were higher in the mature, full sized leaves and substantially lower in young and senescing leaves. GS specific activity and immunoreactive protein followed the same trend as Fd-GOGAT, while GDH showed opposite developmental patterns of accumulation. The biological significance of the presence of Fd-GOGAT in the various grapevine tissues and its physiological role during early development and natural senescence of the leaves are discussed.
Loulakakis
Konstantinos A
KA
Department of Horticulture, Technological Educational Institute of Crete, 71 500 Heraklion, Crete, Greece.
Primikirios
Nikolas I
NI
Nikolantonakis
Michael A
MA
Roubelakis-Angelakis
Kalliopi A
KA
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002374:Catalase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D015151:Immunoblotting; D010788:Photosynthesis / Q000502:physiology; D018515:Plant Leaves / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism; D027843:Vitis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D011993:Recombinant Fusion Proteins; D002374:Catalase; D013385:Succinate Dehydrogenase; D000594:Amino Acid Oxidoreductases; D005969:Glutamate Dehydrogenase; C042233:glutamate synthase (ferredoxin)