Multivariate Analysis of Root Architecture, Morpho-Physiological, and Biochemical Traits Reveals Higher Nitrogen Use Efficiency Heterosis in Maize Hybrids During Early Vegetative Growth

Maize (<i>Zea mays</i> L.) is a globally significant crop with high economic and nutritional importance. Its productivity, however, relies heavily on nitrogen (N) inputs, often resulting in low nitrogen use efficiency (NUE). Enhancing NUE necessitates a comprehensive understanding of the biochemical and physiological mechanisms driving N uptake and utilization. The study evaluated the NUE heterosis of 7 inbred lines and their 12 hybrids under low and high N conditions during early vegetative growth. Significant genotypic variations across traits were analyzed using analysis of variance, principal component analysis, correlation, regression, and structural equation modeling. The key contributors to genetic variation included shoot dry weight, N accumulation, and NUE. Hybrids demonstrated enhanced root architecture, superior enzymatic activities of nitrate reductase (NR) and glutamine synthetase (GS), and improved morphological traits, photosynthetic efficiency, and N accumulation, resulting in greater biomass production, N accumulation, and NUE compared to inbred lines. Among hybrids, Zheng58 × PH4CV exhibited the highest NUE, driven by efficient N uptake, robust enzymatic activity, and substantial N accumulation. Nitrogen uptake efficiency (NUpE) correlated strongly with root traits such as activity (r = 0.80 ***), length (r = 0.73 ***), surface area (r = 0.67 ***), GS activity (r = 0.84 ***), and dry weight (r = 0.92). Similarly, nitrogen utilization efficiency (NutE) was positively correlated with shoot NR activity (r = 0.90 ***), shoot GS activity (r = 0.56 ***), leaf area (r = 0.73 ***), shoot dry weight (r = 0.82 ***), and shoot N accumulation (r = 0.55 ***), particularly under high N conditions. Based on key traits such as shoot dry weight, N accumulation, and NUE, hybrids Zheng58 × PH4CV, 444 × PH4CV, 444 × MO17, and B73 × MO17 emerged as N-efficient genotypes, confirmed by contrasting root systems, enhanced N metabolism, and superior NUE. These findings reveal the pivotal roles of root architecture and N metabolism in optimizing NUE, emphasizing the biochemical and physiological traits crucial for developing highly N-efficient maize hybrids.