Nitrogen effects on the induction of ferredoxin-dependent glutamate synthase and its mRNA in maize leaves under the light

Changes in the levels of ferredoxin (Fd)-dependent glutamate synthase (EC 1.4.7.1) and its mRNA were investigated in leaves of maize (Zea mays L. cv DEA), In etiolated leaves, detached from nitrogen-starved dark-grown seedlings, Fd-glutamate synthase was present at a low level. The enzyme protein and the activity were induced from 3- to 5-fold during the 35 h after transfer of etiolated leaves to a medium containing either 10 mM KNO3, 10 mM NH4Cl or 10 mM NH4NO3 under the continuous photosynthetic photon flux density of 300 mu mol quanta/m(2) per s. A slight increase in the activity occurred in the leaves grown in a nitrogen-free medium under continuous light. The increase in the enzyme activity was paralleled by the incorporation of L-[S-35]methionine in the medium into the Fd-glutamate synthase polypeptide under the same conditions. The production of the enzyme protein and the uptake of labeled amino acid into the enzyme protein were blocked by adding 71 mu M cycloheximide to the medium supplemented with KNO3, NH4Cl or NH4NO3. The addition of the different nitrogenous compounds under continuous darkness did not significantly alter the Fd-glutamate synthase activity and L-[S-35]methionine incorporation into the enzyme protein. A partial cDNA of 2479 base pairs long encoding maize Fd-glutamate synthase was cloned and characterized. Using this cDNA as hybridization probe, Fd-glutamate synthase mRNA was observed to be present at a low level in nitrogen-starved etiolated leaves, The mRNA increased about 5-fold when etiolated leaves were incubated under continuous light in a medium containing either 10 mM KNO3, 10 mM NH4Cl or LD mM NH4NO3. The level of mRNA was also slightly enhanced in the leaves incubated in a nitrogen-free medium under the light. The exogenous nitrogen compounds did not increase the level of the mRNA under continuous darkness. The presence of 71 mu M cycloheximide in either of the media did not significantly change the level of the transcript during the initial 6 h. The induction of mRNA in the presence of exogenous nitrogen under the light was consistent with a protein synthesis-independent process in maize leaves.