NITROGEN-METABOLISM IN CULTURED COTYLEDON EXPLANTS OF PINUS-RADIATA DURING DE-NOVO ORGANOGENESIS

Nitrogen metabolism was investigated under shoot-forming (SF) and non- shoot-forming (NSF) conditions in cultured cotyledon explants of Pinus radiata by following the incorporation of [C-14]-1,2-acetate into var ious metabolites. Early in culture, the lipid fraction contained the m ost C-14; however, this percentage decreased in favor of increased lab el in the amphoteric fraction. Label in the amphoteric fraction of SF cultures decreased by day 21 but plateaued in NSF cultures at this tim e. Radioactive labeling of the principle nitrogen metabolites, glutama te acid glutamine, which made up the majority of the amphoteric fracti on, paralleled labeling patterns in the amphoteric fraction. Percentag e label in glutamate remained at similar levels throughout the 21-day culture period for both SF and NSF cultures. Specific activity of glut amate (kBq mg(-1)) was significantly greater during promeristemoid for mation in SF compared to that in NSF tissues. Glutamine labeling incre ased during shoot bud initiation in SF cultures, but dropped to lower levels during shoot bud development. In contrast, in NSF cultures, the re was a continual and substantial increase in glutamine labeling thro ughout the 21-day culture period. These trends were similar when the s pecific activities of glutamine were determined, as there was a contin ual decrease from culture initiation to the end of shoot bud different iation in SF cultures. In NSF cultures, in contrast, specific activity of glutamine increased substantially from day 5 to 21 relative to tha t in SF cultures. The nitrogen assimilation enzymes glutamate synthase and glutamine synthetase increased in activity from day 0 to 21 for b oth SF and NSF tissues. Enzyme activities for glutamate dehydrogenase were similar in both treatments to day 10 in culture but subsequently diverged, with activities in NSF cultures being substantially greater than those of SF cultures by day 21. Taken together, labeling and enzy me data indicate that nitrogen metabolism is enhanced during culture, especially in SF tissues at the time of promeristemoid formation, and in non-organ-forming tissue senescence-like metabolism was exhibited l ater in culture.