Nitrogen Isotope Fractionation Associated with Nitrate Reductase

Nitrogen isotope fractionation by Pearl Millet (Pennisetwun anwerianum L. and P. moNlssimum L.) grown on nitrate was associated with nitrate reductase activity. Fractionation was evidenced at the step of nitrate reduction when the substrate-to-enzyme ratio was high (possibly saturating for the active sites of the nitrate reductase enzyme), for instance in young seedlngs having a low nitrate reductase activity or in seedlings grown on high nitrate concentration. When the substrate concentration was low (and, hence, the active sites of the enzyme were possibly not saturated), the isotopic discrimination could only be associated with the uptake of nitrate into the cell. In that case, isotopic fractionation was null. It is concluded that the uptake of nitrate does not discriminate among nitrogen isotopes. In order to use the variation in the natural abundance of '5N as the basis for estimating N2-fixation, it is necessary to know whether there is a significant alteration of the isotopic content (isotopic fractionation) as atmospheric N2 moves from its origin into plant tissue. Information about possible fractionation of soil-derived N, usually NO3-N, is less crucial, since the 15N abundance of the total N ofnonfixing plants, the most useful control when estimates ofN2-fixation are attempted, provides an estimate ofthe integrated effect of any isotopic fractionation associated with uptake and incorporation (3, 5). Mariotti et al. (5) reported that nitrogen isotope fractionation during nitrate absorption by plants of 42 different species (38 nonlegume and four noninoculated legume) after a long period of growth (more than 2 months) was very low or even null. Nitrogen isotope abundance did not change in the plant as compared to the nutrient. The mean isotope enrichment factor (defined below) was -0.27 ± 0.63 %o (SD). Experiments were run with nutrient solutions containing nitrate at 8 meq/l. The frequency of nutrient renewal was appropriate to maintain an infinite nitrate reservoir. A report by Kohl and Shearer (3) was not in agreement with this result. With three plants-rye grass, marigold, and nonnodulating soybeans-they observed mean enrichment factors of -4 to -5 %o. In two plants, rye grass and marigold, the isotope enrichment factors clearly decreased when plants aged from 23 to 65 d (the enrichment factor for rye grass varied from -6.7 %o at 23 d to 'Support for this research was provided by Centre National de la Recherche Scientifique (E. R. A. 604, L. A. 40). -1.9 %o at 65 d). Nonnodulating soybeans discriminated against '5NO3 about 3 %o throughout the growing season. Miyake and Wada (8) and Sweeney et al. (10) reported that the nitrogen enrichment between nitrate and phytoplankton was null. The purpose of the experiment presented in this study was to investigate the source of the discrepant results described above by studying the effect of time during the growing season and external N03 concentration on isotopic fractionation associated with N03 uptake. The results given in this paper suggest that the apparent discrepancy of the previous reports are not surprising. It will be shown in this paper that the observed isotope effect for NO3 uptake varies systematically in response to environmental conditions which have an impact on the relative rates of the relevant reactions. MATERIALS AND METHODS Isotopic Terminology (6). The nitrogen isotope composition is expressed in 8'5N value with atmospheric nitrogen as a standard: ('5N %o [ 15N/4N sample 11 x 1000 L15N/'4N standard J The isotope fractionation factor, iproduct/substrate (ap/,8), is referred to as: ap= Rpi/R8 where Rpi is the isotopic ratio (15N/14N) of the increment of product which appears in an infinitely short time at time t, and R, the isotope ratio of the substrate at the same time. The isotope enrichment factor is: (p/ff= (ap/81) X 1,000 When the quantity ofsubstrate is infinite compared to the quantity of product (6), the isotopic ratio of the product Rp is constant. apl. RpIR. and Ep/s = . X 1,000 B, + 1,000 Sp is the isotopic composition of the product; S. is the isotopic composition of the substrate. In natural conditions, S. is very smal compared to 1,000, therefore: