Nitrogen Utilization in Lemna

Abstract

ABSTRACITCultures of Lemna gibba L. G3 were maintained at a constant, Nlimited growth rate by adding nitrate daily in amounts calculated to sustain a rate of culture N increment of 0.20 day-'. Nitrate added to the culture was consumed within 8 to 10 hours and the partitioning to reduction and accumulation during this phase corresponded to, on the average, 75 and 25% of net uptake, respectively. The calculated rate of nitrate reduction was stimulated by onset of net uptake without delay and decreased when net uptake ceased. NADH-nitrate reductase (NR) activity measured in vitro without inclusion of antiproteolytic agents more than doubled during the first hour after nitrate addition and then gradually fell to its original level over the rest of the 24 hour interval. In the presence of the proteinase inhibitor leupeptin during extraction, however, NR activity was in general much higher and without any apparent cycles. The relative stabilizing effect of leupeptin was greatest on NADH-NR and reduced flavin adenine mononucleotide-NR activities whereas the effect was less on NADH-cytochrome c reductase activity (diaphorase) and reduced methylviologen-NR activity. The constant nitrate reductase activity measured in the presence of proteinase inhibitors is assumed to reflect the physiological situation. It thus appeares that short-term changes in nitrate assimilation by N-limited Lemma is related to the flux of nitrate to the reducing site and not to changes in nitrate reductase activity.Assimilation ofNO3-in higher plants is dependent on a variety of environmental factors, the availability of NO3-being one of the most obvious (2, 6). The flux rather than the tissue content of NO3-was shown to be decisive for the NRA2 in maize (22).However, although NRA fluctuates in response to the flux or availability of NO3-in barley (8) and pea (19), the actual N03-reduction (estimated from the tissue N03-balance) is better correlated to net NO3-uptake than to the NRA in vitro (8,19 complicated by the labile nature of NR in most in vitro preparations and several precautions must be taken to stabilize the enzyme (5, 16, 21). Proteinases more or less specific for NR have been isolated from both roots (28, 31) and shoots ( 11) of higher plants although their importance in regulating the amount or activity of NR is not as yet thoroughly understood. If extracted and assayed in an appropriate way, however, the in vitro NR assay indicates the potential NRA in the tissue (29) which might be essential for the understanding of the regulation of NR.The aim of the present work was to study the relationship between NO3-fluxes and NO3-reduction, and to compare actual NO3-reduction with NRA assayed in vitro, taking into consideration especially the stability ofthe enzyme towards proteolysis. These parameters were investigated in N-limited Lemna gibba growing at a stable suboptimal growth rate controlled by exponentially increasing the daily doses of NO3-, while keeping other nutrients at a sufficient level (13,14,19). In plants maintained in this ...

Nitrate reductase activity quantitatively predicts the rate of nitrate incorporation under steady state light limitation: A revised assay and characterization of the enzyme in three species of marine phytoplankton

“…The fact that protease inhibitors had no effect in the present study does not necessarily refute this idea because the effectiveness of the tested protease inhibitors varies with the species, and proteases resistant to the particular inhibitors used in this study may have been present. For example, lcupeptin is effective against NRspecific proteases found in Lemna (Ingemarsson 1987), while PMSF is not. The effectiveness of BSA has also been noted by Ingemarsson ( 1987), but in these cases casein was also effective; this was not true in the present study (data not shown).…”

“…For example, lcupeptin is effective against NRspecific proteases found in Lemna (Ingemarsson 1987), while PMSF is not. The effectiveness of BSA has also been noted by Ingemarsson ( 1987), but in these cases casein was also effective; this was not true in the present study (data not shown). BSA may act by providing a protein in higher concentration that the proteases can degrade in preference to NR, but there may be other effects.…”

Ensaio in vitro da enzima nitrato redutase e efeito da disponibilidade de nitrato e fosfato em variantes pigmentares de Hypnea musciformis (Wulfen) J. V. Lamour. (Gigartinales, Rhodophyta)

“…ii) realização do ensaio sob condições ótimas de atividade, que são espécies-específi cas (Lobban & Harrison 1994) e requerem condições apropriadas como pH, temperatura e concentração de substrato (Berges 1997, Granbom et al 2004, Chow et al 2007. A adição de albumina sérica bovina (BSA) no tampão de extração pode ajudar a preservar a proteína e a atividade da NR, uma vez que este composto atua como substrato preferencial para as proteases endógenas e liga-se aos compostos fenólicos (Ingemarsson 1987.…”

Rapid incorporation of 13N03 by NH4- limited phytoplankton

“…The activity of the enzyme is usually determined by colorometric assay of nitrite formation in cell-free extracts (Morris & Syrett 1965, Eppley et al 1969; the sensitivity of this assay is low unless incubations are relatively long (tens of minutes). NR activity is labile in vitro, and susceptible to proteolysis unless precautions are taken (Ingemarsson 1987). Consequently, low levels of enzyme activity are difficult to detect and preserve.…”