Analysis of changes in blue native/SDS-PAGE profiles of nitrate reductase and glutamine synthetase in maize (Zea mays, L.) roots after nitrate induction

Nitrate is the main form of nitrogen absorbed by plants for proper growth and development. It is the mineral nutrient absorbed by plants in the highest amount, promoting the efficient transport and assimilation of nutrients under changing status of the soil. Collectively, Nitrate Reductase and Glutamine Synthetase are two key enzymes that are crucial for nitrogen use efficiency in plants. The Nitrate Reductase enzyme converts nitrate to nitrite, an essential step in nitrogen assimilation, and is regulated by post-translational modifications such as phosphorylation and interaction with 14-3-3 proteins. The Glutamine Synthetase enzymes, existing in cytosolic (GS1) and chloroplastic (GS2) forms, convert ammonia to glutamine, playing a vital role in both nitrogen assimilation and recycling. 2D-BN/SDS-PAGE (Two-Dimensional Blue Native/Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a sophisticate biochemical technique to analyze the intricate details of protein complexes, combining the strengths of both native and denaturing electrophoresis methods. It combines the advantages of two different electrophoresis methods to provide detailed information about the size, composition, and native state of protein complexes. BN-PAGE excels in maintaining and resolving native protein complexes across a broad MW range (20 and 1200 kDa), while SDS-PAGE provides high-resolution separation of denatured proteins within a more defined MW range (5 kDa to 150 kDa). This study aimed to observe the changes in BN/SDS-PAGE profiles of NR and GS in maize (Zea mays, L. var. Lavaredo) roots after nitrate induction. Nine-day-old seedlings, grown hydroponically in absence of N, were exposed to 5 mM of nitrate for 30 hours. Although the supplemented nitrate did not result in notable changes in the plant's morphological parameters, significant increase in leaf chlorophyll, root protein, and nitrate contents were observed. Moreover, the 2D-BN/SDS-PAGE analysis, coupled with immunodetection with antibodies against Nitrate Reductase or GS1 and GS2 polypeptides, allowed to detected interesting differences in the root profile after nitrate induction. To interpret and validate these results, the mass spectrometry analysis is in progress.