The effect of exogenously supplied nitrogen on gene expression of GS, GOGA T and GDH in plants remains uncertain and varies with species, experimental conditions and the spe- cific member from each gene family which is examined. The expression of the three en- zymes in grapevine, especially GDH, under different nitrogen sources have received con- siderable research attention over the past decade. The results from grapevine cell suspen- sions and calluses clearly show that the nitrogen source in the culture medium strongly affects expression of GS, Fd-GOGAT and GDH at activity, protein and mRNA levels (Loulakakis and Roubelakis-Angelakis, 1996b; 1997; and our unpublished results).
Presence of ammonium or glutamine in the culture medium of grapevine cells caused a decrease in both, enzyme activity and protein of chloroplastic and cytosolic isoforms of GS, while nitrate had a positive regulatory effect. Furthermore, change in the nitrogen source resulted in differential pattern of expression of the vvgsl;l, vvgsl;2 and vvgsI;3 genes (Loulakakis and Roubelakis-Angelakis, 1996b; and our unpublished results). De- pending on the plant species, the type of tissue and the experimental conditions, either positive or negative regulatory effects of the nitrogen source on GS gene expression have been reported (Hayakawa et al., 1990; Stanford et al., 1993; Sukanya et aI., 1994). These results indicate that differential regulation of gene expression significantly affected the evolution of chloroplastic GS gene families (Li et aI., 1993). In fact, the mUltiple GS genes in plants could provide a mechanism for the differential regulation of GS synthesis in vari- ous tissues in response to different requirements for ammonium assimilation.
In the case of Fd-GOGAT, the results confirm a clear inductive effect of nitrates on Fd- GOGAT transcript level in grapevine cell cultures. In contrast, supply of anunonium, as the sole source of nitrogen, resulted in decreased Fd-GOGAT transcript levels (Loulakakis and Roubelakis-Angelakis, 1997). In other plant species, a positive response to nitrate for Fd-GOGAT transcript, polypeptide content and activity has been noticed (Hayakawa et al.,
78 K.A. LOULAKAKIS and K.A. ROUBELAKIS-ANGELAKIS
1990; Redinbaugh and Campbell, 1993), while ammonium either did not affect or sup- pressed the expression of Fd-GOGAT (Hecht et al., 1988; Hayakawa et al., 1990; Oaks, 1994). The inhibitory effect of ammonium on Fd-GOGAT could be related to a more gen- eral ammonium toxicity phenomenon, as previously reported (Hecht et al., 1988).
The total quantity of a- and ~-subunits and the isoenzymic pattern of GDH was al- tered by the exogenous nitrogen source. Protein extracts derived from calluses grown on medium containing nitrates or glutamic acid contained a slightly greater amount of ~
subunit and cathodal isoenzymes, whereas a-subunit and the more anodal isoenzymes predominated in calluses grown in the presence of either ammonium or glutamine (Lou- lakakis and Roubelakis-Angelakis, 1991; 1992). This positive effect of ammonium on the GDH enzyme system was concentration dependent. Transfer of grapevine calluses from culture medium containing only nitrates, as a nitrogen source, to culture media con- taining increasing concentrations of ammonium resulted to a significant increase in NADH-GDH activity and total GDH protein. These changes were accompanied by a decrease in the protein of the more cathodal isoenzymes and an increase in the protein of the more anodal ones (Roubelakis-Angelakis et al., 1991).
Furthermore, in vivo labelling of de novo synthesized proteins in grapevine calluses with 35S-methionine revealed a considerable increase in the synthesis of a-subunits in the presence of ammonium while there was not detectable synthesis of ~-subunits. Thus, the increase in the activity, a-subunit content and in the intensity of staining of the more anodic isoenzymes is caused by the de novo synthesis of the a-subunit of GDH (Lou- lakakis and Roubelakis-Angelakis, 1992). Two-dimensional analysis of the de novo syn- thesized isoenzymes of GDH indicated a low rate of synthesis of all isoenzymes in the presence of nitrates and confirmed the synthesis of the more anodic isoenzymes in the presence of ammonium. These results support that in nitrate-grown calluses the combination of the two de novo synthesized subunits results in the appearance of radioactivity in all isoenzymes. In contrast, in ammonium-grown calluses the radioactive more anodic isoenzymes are produced by the assembly of de novo synthesized a- subunits and preexisting ~-subunits (Loulakakis and Roubelakis-Angelakis, 1992). These results could support that ammonium does not cause activation of GDH, as it was proposed (Srivastava and Singh, 1987), but that it induces expression of subunit a, and therefore the regulation of GDH is exerted by differential expression of the respective genes. In fact, grapevine cell suspensions cultured in the presence of ammonium expressed significantly higher levels of the wgdhl mRNA. In addition, similarly to calluses, cell suspensions cultured in ammonium containing media showed significantly higher specific activity of GDH accompanied by an accumulation of the a-subunit of the enzyme and predominance of the more anodal isoenzymes (our unpublished results, Fig.
3.l3). The increased accumulation of GDH in the ammonium-grown cells may suggest a role of this enzyme in the direction of nitrogen assimilation, at least as a detoxification reaction (Primikirios and Roubelakis-Angelakis, 1999; Paschal ides et aI., this book).
Based on GDH activities determined in crude plant extracts several authors have re- ported a considerable variation of the NADH-GDHlNAD-GDH activity ratio, depending on
NITROGEN ASSIMILATlON IN GRAPEVINE 79 Time, days
o 2 3 4
A
B
Figure 3.13. Isoenzymic patems and subunit composition ofGDH from grapevine cell suspension.
Cells were cultured in the presence of ammonium for 4 days. A, 5% native electrophoresis gel stained for GDH activity by using the tetrazolium system method. B, protein blot analysis after 7.5% SDS gel electrophoresis. NADH-GDH polypeptides were probed with anti-GDH serum (our unpublished results).
the developmental stages or the environmental conditions (Loyola-Vargas and de Jimenez 1984, Cammaerts and Jacobs, 1985; Leon et ai., 1990; Watanabe et ai., 1992) and have proposed a variable anabolic and catabolic function of GDH and its isoen- zymes in plant metabolism. Furthermore the two activities of GDH were found to re- spond differently to the supply of different nitrogenous salts and amino acids in maize root and shoot tissues (Singh and Srivastava, 1982; 1983), and were proposed to be due to changes in the physicochemical nature of the enzyme. However, these results have been recently reconsidered; it was shown that malate in the crude extracts resulted in erroneous overestimation of the NAD-GDH activity through the malate dehydrogenase reaction (Fricke and Pahlich, 1992; Loulakakis and Roubelakis-Angelakis, 1996a). Thus, in dialyzed protein extracts of grapevine the ratio of the two in vitro GDH activities (aminating-/deaminating-activity) remained relatively constant although the isoenzyme profile could vary, and was dependent on the nitrogen source (Loulakakis and Roube- lakis-Angelakis, 1996a).
Furthermore, the aminating and deaminating activities of each of the seven grapevine GDH isoenzymes were identified by staining the two activities, following separation of the isoenzymes on polyacrylamide gels (Loulakakis and Roubelakis-Angelakis, 1996a).
80 K.A. LOULAKAKIS and KA ROUBELAKIS-ANGELAKIS
Transfer of calluses from a nitrate- to an ammonium-containing culture medium resulted in similar alterations of the isoenzyme profile using both, the aminating and the deami- nating staining methods. Also, leaf isoenzymes showed similar ratios of in vitro activi- ties and affinity for their substrates (Loulakakis and Roubelakis-Angelakis, J996a).
Therefore, it was concluded that each of the seven GOB isoenzymes have similar in vi- tro anabolic and catabolic activities. Further study on the expression of GOB, GS and GOGAT in both cell cultures and whole grapevine plants, under varying trophic and environmental conditions, would further elucidate their relative contribution in ammonia assimilation.