The response of nitrogen assimilation to changes in light availability was compared in three Australian rainforest species from contrasting ecological niches. Solanum aviculare, a pioneer species, and the shade tolerant Alocasia macrorrhiza and Alpinia coerulea were grown in a glasshouse under three light intensities, 60, 12 and 2.5% incident sunlight. The activities of nitrogen assimilatory enzymes as well as plant biomass, and leaf amino acid composition were contrasted in plants grown at the three light levels. Chlorophyll fluorescence, including measurements of electron transport rates and Fv/Fm, were also compared in plants grown at the three light levels. Leaf nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase activities in all three species and mRNA levels of nitrate reductase, nitrite reductase and chloroplastic glutamine synthetase in Solarium leaves were measured over the course of a diurnal cycle and after transfers of plants between low and high light.
Activity of leaf nitrate reductase in Solarium, Alocasia and Alpinia, assayed with MgCl2, responded rapidly and reversibly to light-dark transitions indicating the potential for rapid post-translational regulation of nitrate reductase by protein phosphorylation. Nitrate reductase activity (+Mg2+) is a measure of active nitrate reductase and nitrate reductase activity (-Mg2+) a measure of total nitrate reductase activity. Thirty minutes after transfer into the dark, nitrate reductase activity (+Mg2+) was reduced by 55 to 76% of initial activity in the three species.
Comparisons of growth and nitrogen assimilation in plants grown at the three light intensities indicated Solanum had the greatest potential for the uptake and assimilation of nitrate, followed by Alocasia then Alpinia. Activities of nitrogen assimilating enzymes, soluble protein contents and free amino acid concentrations decreased with decreasing light, in parallel with a decreasing requirement for nitrogen as a result of slower growth rates. Asparagine accumulated in leaves of Solanum and Alpinia grown at 2.5% incident sunlight. Of the primary nitrogen assimilatory enzymes, leaf nifrate reductase activity was the most responsive to decreases in growth irradiance in all three species. When grown at low light Solanum had negligible nitrate reductase activity (-Mg2+), however Alocasia and Alpinia had activities of 20 and 40 nmol gfw min-1 respectively.
The mRNA levels and activities of the nitrogen assimilatory enzymes in Solanum and the enzyme activities in Alocasia and Alpinia were measured at regular intervals throughout the diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase mRNA accumulated in Solanum during the night and decreased during the photoperiod. However, high levels of nitrate and nitrite reductase mRNA were also measured in Solanum leaves at midday. Nitrate reductase activity (-Mg2+) in Solanum leaves increased five-fold after the beginning of the light period to maximum levels, 100-110 nmol gfw-1 min-1 at midday then declined in the afternoon. In comparison, there were only small increases in nitrate reductase activity during the photoperiod in Alocasia and Alpinia leaves. Relatively high nitrate reductase activities (-Mg2+), 65 nmol gfw-1 min-1 were maintained during the night period in Alocasia. Slower increases of nitrate reductase activities (+Mg2+) compared to total nitrate reductase activity (-Mg2+) in all three species indicate that the increase in nitrate reduction after the onset of the light period is delayed by post-translational modification of nitrate reductase. Changes in nitrite reductase, glutamine synthetase and glutamate synthase activity during the diurnal cycle were minimal in all three species. The lack of correlation of mRNA levels with enzyme activities measured in Solanum leaves may indicate the operation of post-translational regulation.
Nitrate reduction rapidly declined after transfer of all three species from high to low light. Leaf nitrate reductase activities (+Mg2+, -Mg2+) were reduced within 24 hours. Significant reductions in glutamine synthetase activity were measured in all three species after one week at low light.
Nitrate reductase in Solanum leaves responded most rapidly to increasing light availability. Nitrate reductase activity (-Mg2+) increased from 10 to 40 nmol gfw-1min-1 after three hours and to 110 nmol gfw-1min-1 after one week at high light. Nitrate reductase activity (+Mg2+) in Solanum increased from 0 to 34 nmol gfw-1min-1 after three hours and remained at that level for the following two weeks. Nitrate reductase activities (-Mg2+, +Mg2+) in Alocasia increased from 15 to 37 nmol gfw-1min-1 after 24 hours in high light and maximum activities of approximately 75 nmol gfw-1min-1 were reached after two weeks. Pre-existing leaves of Alpinia had little potential for increased nitrate reduction after transfer to high light. In contrast to Solanum and Alocasia, Alpinia showed no recovery from photoinhibition as indicated by leaf Fv/Fm ratios less than 0.72. Alpinia transferred from 2.5 to 60% incident sunlight did not develop new leaves.
Large fluctuations of nitrate reduction over the diurnal period, the ability to store nitrate and rapidly increased nitrate reduction in response to increased light availability are consistent with the rapid growth rates of Solanum and its prevalence in higher light environments. In contrast, the relatively high nitrate reductase activities in low light and the maintenance of high nitrate reductase activity over the diurnal cycle in Alocasia leaves and to a lesser extent Alpinia may offer an advantage in understorey environments with fluctuating light intensities.