Alicia Orea

Influence of plant age and growth conditions on nitrate assimilation in roots of Lotus japonicus plants

In roots of Lotus japonicus (Regel) Larsen cv. Gifu, the level of NADH-nitrate reductase (NR) activity and protein, as well as ferredoxin-nitrite reductase activity and nitrate accumulation, were higher in roots of young plants, and decreased in mature plants grown in seed trays. When plants were grown in larger pots, the decrease in NR activity and nitrate accumulation took place at a later stage of growth, suggesting that the cessation of nitrate assimilation in mature plants could be related to both ageing and a lower availability of space for roots. Low NR activity was detected in leaves, whereas nitrate accumulation in this tissue could reach relatively high levels. NR activity and protein, and nitrate accumulation, also decreased in leaves of mature plants, indicating that the diminution in nitrate accumulation and NR in roots from mature plants is not related to a shift of the nitrate assimilation process from roots to leaves. Measurement of the extent of total and active NR according to the phosphorylation/dephosphorylation inactivating mechanism described for spinach leaf NR, indicates that this mechanism is operative on NR from L. japonicus roots, being responsible for a great proportion of inactive NR protein. The amount of inactive NR protein in roots due to the aforementioned phosphorylation mechanism also increased in mature plants.

Nitrate and ammonium assimilatory enzymes

Protocols for extraction and assay of the key enzymes of nitrate and ammonium assimilation in Lotus japonicus are given in this chapter, together with the peculiarities observed for these assays in different organs and physiological situations of the plant.

Nitrate assimilation: Influence of nitrogen supply

We have studied the effect of nitrogen supply on growth as well as nitrate assimilation in Lotus japonicus plants. High nitrate concentrations (higher than 20 mM) were toxic for L japonicus, inhibiting growth, with the optimum nitrate concentration ranging between 4 and 8 mM. The maximum growth and levels of nitrate reductase (NR) and nitrite reductase (NiR) activity as well as nitrate accumulation was obtained in plants grown simultaneously on nitrate plus ammonium. L japonicus could also grow on ammonium as the sole nitrogen source, without a supplement of organic acids like succinate. Low external nitrate concentrations induced a higher growth of roots, while higher external nitrate concentration inhibited the growth of roots and favoured shoots growth. NR and NiR activities were always higher in roots than in shoots of L japonicus plants. An increase in external nitrate concentrations did not alter significantly the plant partitioning of nitrate assimilation among roots and shoots, being predominantly higher in the roots than in stems and leaves. The kinetics of NR activity induction by nitrate showed a different pattern in roots and in leaves, being this induction in roots independent on external nitrate concentration in the short-term. In contrast, the induction kinetics of NiR activity and nitrate accumulation showed the same pattern in roots and leaves, being dependent on the external nitrate concentration. In all cases the levels of NR and NiR activity and nitrate accumulation were higher in roots than in leaves. Significant levels of NR protein but no NR activity were detected in NH 4 + -grown plants.

TRANSGENIC PLANTS AFFECTED IN NITRATE ASSIMILATION

Plant nitrate assimilation is a process that takes place both in roots and in shoots. The partitioning of nitrate assimilation between both organs depends on the particular specie studied and environmental factors. Lotus japonicus is a temperate legume that assimilates nitrate mainly in roots. We have analysed the levels of nitrate reductase (NR) activity in roots and leaves from L japonicus transgenic plants with altered NR expression produced by means of constitutive, leaf-specific or root-specific promoters. Although NR expression was effectively modified in some of the transgenic lines, none of them showed higher levels of NR activity in leaves than in roots. Some type of mechanism may be operating in L japonicus to maintain low levels of NR enzyme activity in leaves of this plant.