Supplementary file 1_Quinoa CqNLP9: a possible regulator of nitrogen metabolism under low nitrogen stress.docx

IntroductionNitrogen metabolism constitutes a fundamental physiological process governing plant growth and development. Nin-like protein (NLP) transcription factors act as central regulators in nitrate signaling, coordinating nitrogen uptake and assimilation. Quinoa (Chenopodium quinoa), known for its adaptability to nutrient-poor soils, serves as an excellent model for dissecting nitrogen-acquisition mechanisms. This study aims to explore the role of CqNLP9 in quinoa under low-nitrogen (LN) stress and its potential regulatory mechanisms.
MethodsThe expression level of CqNLP9 in quinoa was examined by qRT-PCR after 30 days of LN treatment. Gain- and loss-of-function approaches were employed by overexpressing CqNLP9 in Arabidopsis and silencing its homologous gene NbNLP9 in tobacco. Phenotypic changes and related physiological and biochemical parameters were analyzed under LN conditions. Additionally, yeast two-hybrid and luciferase complementation imaging assays were performed to identify proteins interacting with CqNLP9.
ResultsFollowing 30 days of LN treatment, CqNLP9 expression increased 24-fold in quinoa compared with plants grown in complete nutrient solution. Under LN conditions, CqNLP9-overexpressing Arabidopsis lines exhibited greater biomass, chlorophyll content, soluble protein, and total nitrogen compared with wild-type (Col-0). Activities of key nitrogen metabolism enzymes —nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase —were elevated, along with enhanced peroxidase, superoxide dismutase, and catalase activities. Malondialdehyde content decreased, indicating reduced oxidative damage. Conversely, silencing of the homologous gene NbNLP9 in tobacco resulted in opposite phenotypic changes. Furthermore, yeast twohybrid and luciferase complementation imaging assays revealed a physical interaction between CqNLP9 and CqMOB1B, a core component of the Hippo signaling pathway.
DiscussionThese findings suggest that CqNLP9 might contribute to improved low-nitrogen tolerance through modulation of nitrogen metabolism enzyme activities, enhancement of ROS scavenging capacity, and maintenance of chlorophyll and soluble protein balance, which in turn could facilitate nitrogen uptake and assimilation. The interaction between CqNLP9 and CqMOB1B offers new perspectives on the crosstalk between nutrient signaling and developmental regulatory pathways in plants.