Root architecture plays a fundamental role in crop yield, which is sensitive to nitrogen fertilizer. Although it is well studied that nitrogen fertilizer significantly promotes peanut (Arachis hypogaea L.) growth and yield, less information was available on how its root development responds to nitrogen deficiency. In this study, the growth and development of roots were inhibited, as indicated by the significantly decreased root dry weight and length and the lateral root number, especially under 10 days of nitrogen deficiency treatment. The activities and the expression of the genes related to nitrogen assimilation enzymes including nitrate reductase, glutamine synthetase, glutamate dehydrogenase, and glutamine oxoglutarate aminotransferase and the genes encoding the nitrate transporters were significantly decreased under 10 days of nitrogen deficiency treatment, which may lead to a decrease in nitrate content, as indicated by the significantly decreased nitrogen balance index. Transcriptome sequencing revealed a total of 293 (119 up- and 174 downregulated) and 2271 (1165 up- and 1106 downregulated) differentially expressed genes (DEGs) identified after five and ten days of nitrogen deficiency treatments, respectively. Bioinformatic analysis showed that these DEGs were mainly involved in nitrate transportation and assimilation, phytohormone signal transduction, and the lignin biosynthesis pathway. Furthermore, a putative schematic diagram of nitrogen deficiency inhibiting root growth was established, which gives us a better understanding of nitrogen metabolism in peanut roots and a theoretical basis for improving nitrogen use efficiency.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a critical protein for photosynthesis. With small subunits, RBCSs catalyze the fixation of CO₂ onto ribulose-1,5-bisphosphate (RuBP). However, the gene characteristics of peanut RBCSs, and the relationship between their expression and peanut growth under nitrogen (N)-deficiency conditions are not yet known. This study targeted the impact of sufficient N (5 mmol/L NO₃⁻) and deficient N (0.1 mmol/L NO₃⁻) on peanut seedlings’ growth, physiology, and molecular aspects. Herein, eight RBCS protein encoding genes were identified in the peanut genome, and their characteristics were described. The leaf areas of Yu Hang Hua 1 (YH1) and An Hua 3 (AH3) were reduced significantly after 10 days while “Pu Ke Hua 18” (PK18) and “Luo Hua 4087” (LH4087) had higher N-deficiency adaptations in the initial N-deficiency stage. The Rubisco carboxylation activities of YH1 and PK18 were significantly increased after 5 days of N-deficiency stress while they decreased under 15 days of N-deficiency conditions. Furthermore, the qRT-PCR results showed that the expressions of AhRBCS 1/5 and AhRBCS2/6 had negative relationships with the morpho-physiological traits of peanut under low-N stress. This study provides the biological information of peanut RBCSs and lays a theoretical foundation for N utilization in crop plants.