Nitrate is the primary form of nitrogen uptake in plants, mainly transported by nitrate transporters (NRTs), including NPF (NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY), NRT2 and NRT3. In this study, we identified a total of 78 NPF, seven NRT2, and two NRT3 genes in maize. Phylogenetic analysis divided the NPF family into eight subgroups (NPF1-NPF8), consistent with the results in Arabidopsis thaliana and rice. The NRT2 family appears to have evolved more conservatively than the NPF family, as NRT2 genes contain fewer introns. The promoters of all NRTs are rich in cis-acting elements responding to biotic and abiotic stresses. The expression of NRTs varies in different tissues and developmental stages, with some NRTs only expressed in specific tissues or developmental stages. RNA-seq analysis using Xu178 revealed differential expression of NRTs in response to nitrogen starvation and nitrate resupply. Moreover, the expression patterns of six key NRTs genes (NPF6.6, NPF6.8, NRT2.1, NRT2.5 and NRT3.1A/B) varied in response to alterations in nitrogen levels across distinct maize inbred lines with different nitrogen uptake rates. This work enhances our understanding of the structure and expression of NRTs genes, and their roles in nitrate response, paving the way for improving maize nitrogen efficiency through molecular breeding.

Bio-solubilization of lignite is a promising technology to transform coal into humic acids (HAs) which are broadly used in agriculture. In this work, HAs were extracted from lignite using the cell-free filtrate (CFF) of Penicillium ortum MJ51. The extraction method was optimized using response surface methodology (RSM) based on the interactive effects of nitric acid concentrations, coal loading ratio, extraction temperature and time as input factors, and the absorbance of HAs at 450 nm wavelength as the output response. Under optimized conditions (lignite pretreated with 4.7 N HNO₃, coal loading ratio of 4.9%, temperature of 77.3 °C and time of 8.6 hours), the absorbance at 450 nm peaked at 70.28, and the concentration and extraction yield of HAs were 31.3 g L⁻¹ and 63.9%, respectively, which were dramatically higher than those observed for traditional biological methods (0.7 g L⁻¹ and 14.1%, respectively). The qualities of HAs produced under optimized conditions were evaluated and compared with those extracted by the conventional chemical method. The optimized process resulted in better HA quality indices, including lower molecular mass; higher nitrogen; less aromatic carbon; more aliphatic and carboxylic carbon; and higher bioactivity for promoting plant growth. Moreover, the anti-flocculation ability was improved, thereby supporting its applicability in agriculture. Extraction of HAs from lignite using the CFF of P. ortum MJ51 provides a novel technological approach for the efficient conversion of lignite to bio-active HAs.