Comprehensive evaluation, morpho-physiological and transcriptional response involving the tolerance of Semi-wild soybean (Glycine gracilis) seedlings to nitrogen starvation

Nitrogen (N) limitation significantly constrains crop growth, yield and quality. Developing crop varieties with high N deficiency tolerance represents a critical strategy for reducing N fertilizer application and promoting sustainable agriculture. Semi-wild soybean offers valuable genetic resources for the improvement of soybean varieties. Nevertheless, the mechanisms underlying N deficiency tolerance remain poorly understood. In this study, we employed a comprehensive analytical approach-including Pearson's correlation analysis, principal component analysis, subordinate function analysis, and cluster analysis-to evaluate the N starvation tolerance of 50 semi-wild soybean varieties. Shoot fresh weight, root-shoot ratio, SPAD2 value and leaf nitrate content were identified as key indicators for assessing N starvation tolerance. The variety V03 was identified as the most N starvation-tolerant. Comparative physiological analyses revealed that V03 enhances tolerance to N deficiency by optimizing root architecture and sustaining the activity of nitrogen metabolism enzymes-such as nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT)-in root and leaf tissues. Transcriptomic analysis indicated that V03 exhibits a broader transcriptional response (with more N Starvation-induced DEGs) and functional reprogramming in root tissues, showing stronger enrichment in stress-responsive processes, regulatory functions, and plasma membrane-related terms as well as environmental information processing pathways. Furthermore, V03 displayed more pronounced changes in the expression of genes related to N transport, N assimilation and transcription factor (TF) compared to the N starvation-sensitive variety V46. This study provides a robust and comprehensive methodology for evaluating N deficiency tolerance in semi-wild soybean. Our findings offer new insights into the physiological adaptions and molecular regulatory network governing N uptake and metabolism, which may support future breeding efforts aimed at enhancing NUE in leguminous crops.