Studies on the Characteristics of Nitrate Reductase (NR) Activity in Some Germinating Tropical Seeds

Author: Udoh, Agnes Michael

Supervisor: Orebanjo T. O.

Substrate induction of nitrate reductase (NR) was monitored in seeds and seedlings of Oryza sativa (cv.Faro II old (1982) and new (1987) and Arachis hypogaen. Selected analyses were done on samples taken from Abemoschus esculentus as well as Colocynthus citrullus. An appreciable level of endogenous enzyme was obtained in un-imbibed and seeds of A.hupogaea as shown by results obtained from zero hour of incubation with the substrate. NR activity was higher in the embryos than in the corresponding cotyledons after 24 hours of imbibition. However, induction of the enzyme became more pronounced in the cotyledons than in the embryos from the 3rd day of germination and this trend was maintained till the 7th day of germination. In 0. Sativa (cv. paro II) NR was more easily induced in the less viable seeds than the viable ones at the dry and early imbibed stages. After 3 days of germination, the endosperms demonstrated higher nitrate reduction and kept it unchanged for more than 2 weeks. Lag phases 5 to 6 hours were demonstrated in unimbibed rice seeds while induction was delayed for between 2 to 3 hours in the controls of intact seedlings and cotyledons of mostly 6- and 7-days seedlings of A. hypogaen and A. esculentus. Excised embryos of A. hypogaen maintained a 3-hour lag phase. Pre-treatment with NO3- ions generally enhanced more NR induction as shown in the in vivo results. In the in vivo analyses, the availability of NO3- also reduced NR activity because of nitrite reductase effect. The presence of nitrate seemed to favour nitrite reductase (Nir) activity in A. hypogaea. The effect of ammonia- the feedback inhibitor of NR was both stimulatory and inhibitory in 0. Sativa as well as A. hypogaea depending on the age of the seed. Also the involvement of blosynthesis of NR in young viable seeds was demonstrated. The nucleic acid synthesis inhibitor-6-methyl purine inhibited nitrate reduction in young viable rice seeds while it had little or no effect on the older seeds. On the other hand, the proteinase inhibitor – phenyl methyl sulphonyl flouride (PMSF) inhibited enzymatic activity in young viable seeds while it stimulated NR activity in older seeds. A proteinase was therefore implicated for the activation of NR activity in older rice seeds with reduced viability. NR in the seeds sampled showed low affinity to low molecular weight salts as there was remarkable decrease in activity after sephadex G25 filtration. The removal of these salts favoured nitrate reduces activity. This report demonstrates the dual nature of the mechanism involved in nitrate reduction in the seeds/seedlings. The mechanism involves- (1) The activation of the latent enzyme in older non-viable rice and groundnut seeds, and (2) the induction of the enzyme in all the younger viable seeds. The importance of the endosperms and cotyledons not only as storage organs but also organs for the biosynthesis of NR was also shown. It was also demonstrated that the embryo tissues lost their functional mechanisms before the endosperms and cotyledons. From the results obtained in this study, two forms of NR seem to be present in dormant seeds: the constitutive and the inducible NR. Non-viable seeds lack inducible NR in their embryos. This finding can be used to determine seed viability by testing for inducible NR in excised embryos.