TOBACCO MUTANTS WITH A DECREASED NUMBER OF FUNCTIONAL NIA GENES COMPENSATE BY MODIFYING THE DIURNAL REGULATION OF TRANSCRIPTION, POSTTRANSLATIONAL MODIFICATION AND TURNOVER OF NITRATE REDUCTASE

Although nitrate reductase (NR, EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40-45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacc o (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two inste ad of four functional ilia genes compensate. (i) The nia transcript wa s higher in the leaves of the mutants, However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3-4 h light in WT leaves, and then de creased by 50-60% during the second part of the photoperiod and the fi rst part of the night. Leaves of mutants contained 40-60% less NR prot ein and NRA after 3-4 h illumination, but NR did not decrease during t he photoperiod. At the end of the photoperiod the WT and the mutants c ontained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1-3 h darkness, Calyculi n A prevented this reversal, When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants co ntained 50% more activity than the WT during the night. Conversion of [N-15]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in t he dark. (v) Increased stability of NR in the light and reversal of da rk-inactivation correlated with decreased levels of glutamine in the l eaves. When glutamine was supplied to detached leaves it accelerated t he breakdown of NR, and led to inactivation of NR,even in the light. ( vi) Diurnal changes were also investigated in roots. In the WT, the am ount of nia transcript rose to a maximum after 4 h illumination and th en gradually decreased. The amplitude of the changes in transcript amo unt was smaller in roots than in leaves, and there were no diurnal cha nges in NRA. In mutants, nicr transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower dur ing the day but rose during the night to an activity almost as high as in the WT. The rate of [N-15]-nitrate assimilation in the roots of th e mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants w ith very low NRA, and in nitrate-deficient WT plants. Both sets of pla nts had similar low growth rates. Nitrate reductase did not show a diu rnal rhythm in leaves or roots of Nia30(145), the leaves contained ver y low glutamine, and NR did not inactivate in the dark. Nitrate-defici ent WT plants were watered each day with 0.2 mM nitrate. After waterin g, there was a small peak of nia transcript, NR protein and NRA and, s lightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not ina ctivate. In the roots, there was a very marked increase of nitrate, ni a transcript and NRA 2-3 h after the daily watering with 0.2 mM nitrat e. (viii) It is concluded that WT plants have excess capacity for nitr ate assimilation. They only utilise this potential capacity for a shor t time each day, and then downregulate nitrate assimilation in respons e, depending on the conditions, to accumulation of the products of nit rate assimilation or exhaustion of external nitrate, Genotypes with a lower capacity for nitrate assimilation compensate by increasing expre ssion of NR and weakening the feedback regulation, to allow assimilati on to continue for a longer period each day.