Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase, and its spatial and temporal changes during leaf development

Abstract

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.

Spatial and Temporal Distribution of Polyamine Levels and Polyamine Anabolism in Different Organs/Tissues of the Tobacco Plant. Correlations with Age, Cell Division/Expansion, and Differentiation

“…A steady-state equilibrium may also be established between the rates of Put biosynthesis and its degradation (K. Paschalidis and K. Roubelakis-Angelakis, unpublished data), as both of them are responsible for the regulation of endogenous concentrations of Put in cells. Our previous results suggest that under conditions of abiotic stress a similar equilibrium would continuously recycle NH 3 , therefore minimizing its toxicity (Loulakakis et al, 2002).…”

Arabidopsis Roots and Shoots Show Distinct Temporal Adaptation Patterns toward Nitrogen Starvation

“…In tobacco and grape (Vitis vinifera), there is evidence for a role of GDH in recycling ammonium in companion cells (Dubois et al, 2003;Tercé-Laforgue et al, 2004;Fontaine et al, 2006). Indeed, intracellular ammonia, due to exogenous ammonium (Restivo, 2004;Tercé-Laforgue et al, 2004), senescence-induced high proteolytic activities (Masclaux et al, 2000;Loulakakis et al, 2002), or abiotic stress (Lutts et al, 1999;Hoai et al, 2003), has resulted in increased aminating GDH activity in vitro. Under N starvation conditions, ammonia is produced by several catabolic processes, and GDH activity has also been shown to increase by 200% in plants grown under limited N (Tschoep et al, 2009).…”

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

“…The exact physiological roles of GDH in plant carbon and nitrogen metabolism and remobilization in plants remain largely speculative (reviewed in Miflin and Habash, 2002;Stitt et al, 2002;Dubois et al, 2003). Intracellular hyperammonia due to either exogenous ammonium (Loulakakis and RoubelakisAngelakis, 1992;Restivo, 2004;Tercé -Laforgue et al, 2004a), senescence-induced high proteolytic activities (Masclaux et al, 2000;Loulakakis et al, 2002;Tercé -Laforgue et al, 2004b), or abiotic stress (Lutts et al, 1999;Hoai et al, 2003) results in increased aminating GDH activity in vitro. Early in the season, in planta, fully developed and physiologically active leaves in the basal part of the shoot exhibit high GOGAT expression, protein accumulation, and activity, whereas GDH concentration and activity are low.…”

“…Early in the season, in planta, fully developed and physiologically active leaves in the basal part of the shoot exhibit high GOGAT expression, protein accumulation, and activity, whereas GDH concentration and activity are low. Later during development, GS and GOGAT decrease in the senescing leaves, accompanied by expression of gdh-NAD;A1, GDH protein accumulation, and increased in vitro aminating activity in tissues from senescing leaves (Masclaux et al, 2000;Loulakakis et al, 2002;Tercé -Laforgue et al, 2004a, 2004b. In salt-tolerant rice (Oryza sativa) cultivars, aminating GDH activity increases with increasing salt stress, whereas it decreases in the salt-sensitive ones (Kumar et al, 2000), and in pea (Pisum sativum) plants, an ammonium-tolerant plant, the aminating GDH activity in roots is high (Lasa et al, 2002).…”