Optimization of carbon and nitrogen utilization for enhanced photosynthesis, biomass and yield by coexpression of <i>phosphoenolpyruvate carboxylase</i> , <i>aspartate aminotransferase</i> and <i>glutamine synthetase</i> in <i>Brassica juncea</i>

Coexpression of genes involved in carbon (C) and nitrogen (N) metabolism offers a promising avenue for improving crop yield. This study investigated the impact of coexpressing phosphoenolpyruvate carboxylase [ZmPEPC (P)], aspartate aminotransferase [GmAspAT (A)] and glutamine synthetase [NtGS (G)] in Brassica juncea to enhance plant yield through improved C and N utilization. Plants coexpressing three genes (BjPAG) showed a significant increase in plant growth, biomass, chlorophyll and carotenoid content, while exhibiting decreased anthocyanin and hydrogen peroxide production. These improvements were observed under both ambient (~400 ppm; ACO₂) and low (~200 ppm; LCO₂) atmospheric CO₂ conditions. Chlorophyll fluorescence study revealed that BjPAG plants had higher photosynthetic efficiency, particularly increased maximum quantum yield of PSII and performance index. This facilitated increased CO₂ assimilation, total soluble sugar and starch accumulation, along with altered stomatal traits under both ACO₂ and LCO₂ conditions. Additionally, BjPAG plants accumulated less ammonium than control plants under both CO₂ levels, indicating effective functioning of the overexpressed genes. Improved performance (enhanced photosynthetic rates, biomass and seed yield) of BjPAG plants under various N environments (6, 3 and 0.3 mm N) also supported improved N utilization. NMR analysis suggested that PAG-coexpression resulted in a higher flux of assimilated CO₂ and NH₃ towards sugars, amino acids, organic acids, polyphenols and glucosinolates. In conclusion, this study highlights the effectiveness of coexpressing ZmPEPC, GmAspAT and NtGS in B. juncea, leading to efficient C and N utilization, ultimately resulting in enhanced photosynthesis, plant biomass and seed yields, and as a promising strategy for increasing the yield of food crops.