N plays a crucial role in many metabolic and biochemical processes in plants, directly and effectively affecting the formation of chlorophyll and assimilation through photosynthesis[23]. The N metabolism in millet is closely related to the growth, development, and nutrient quality. Based on the low N-tolerance of millet, the mining of genes closely related to the target traits in the N metabolism pathway formed the basis for obtaining high-yield and high-quality millet. Transcriptomic sequencing can effectively mine the genes essential for controlling a certain trait in plants and obtain all information about the expression levels of the associated genes[24]. This study used the millet genome as a reference and combined it with previous research results to conduct a comparative analysis of the genes related to low N-tolerance in millet and explore their regulatory mechanism. N metabolism, absorption, transport, and amino acid metabolism pathways related to low N-tolerance traits with more differentially annotated gene sequences selected as the basis to identify genes critical to regulating these traits.
N metabolism is an essential pathway for converting inorganic N into organic N and the biosynthesis of amino acids. After external N enters the plant, the synthesis of nitrogenous compounds is catalyzed by a series of enzymes, such as NR, to provide the nutrients required for plant growth and development[25]. The transcriptomic data obtained in this study intuitively reflected the molecular information in millet leaves under LN to understand the related genes and the regulatory pathways involved in millet growth under LN. Compared with NN, 428 genes were up-regulated, and 466 were down-regulated at LN. GO enrichment indicated that more DEGs were enriched in the process of amino acid synthesis and metabolism at LN, and it could be inferred that low N-tolerance in millet was related to transaminase activity. KEGG analysis showed that most of the DEGs were concentrated in MP; genes related to N metabolism, amino acid (glutamic acid, aspartate, tryptophan, serine, and lysine) biosynthesis, nitrate transporters, and TFs were screened.
Under LN, the upregulated genes were mainly related to amino acid synthetase, nitrate transporter, and AAP, suggesting that an active amino acid metabolic pathway may majorly lead to low N-tolerance in millet. The glutamine synthase (GS)/glutamate synthase (GOGAT) cycle, as the focal point of the entire N metabolism, is the main pathway of crop N metabolism[26]. The GS/GOGAT circulating enzyme system has been detected in the roots, cotyledon, leaves, and other organs of various plants. Through studying mutant plants lacking GOGAT, such as pea (Pisum sativum) and Arabidopsis thaliana, it was found that when grown in air, the glutamic acid accumulated rapidly in the leaves, while amino acid synthesis reduced. A double barley mutant (GS and Fd-GOGAT deficient) grown at high concentrations of CO2 was exposed to air to inhibit growth, decrease CO2 utilization, and increase NH3 concentration in the leaves[27]. Therefore, improving the efficiency of the GS/GOGAT cycle is an effective way to improve N use efficiency and enhance tolerance to low N-induced stress. In this study, the gene encoding GS was upregulated under LN, demonstrating that it plays a vital role in N metabolism and utilization in millet. NR is a rate-limiting enzyme in the conversion of NO3− to NH4+ (ammonia assimilation) [28, 29]; regulating N metabolism, nitrate assimilation, and other vital metabolic pathways[30]; and has a marked impact on the yield and quality of plants[31]. In this study, the gene encoding NR was upregulated, indicating that an increase in its activity significantly promoted the efficiency of ammonia N assimilation in millet under low N stress, thus accelerating the absorption and utilization of N by millet and improving the tolerance of millet to low N-induced stress.
Transcription factors play a crucial role in the growth, development, and morphogenesis of plants. Among these factors, the C2C2-Dof transcription factor has shown a close association with nitrogen metabolism, as revealed from the analysis of transcriptome data. The Dof transcription factors are known to participate in various biological processes such as photoresponse, photoperiod regulation, flowering, seed development, germination, plant hormone signaling, defense response, and nitrogen metabolism[32]. Notably, ZmDof1, identified in both Arabidopsis and rice, has been found to enhance nitrogen assimilation and promote plant growth, particularly under conditions of nitrogen deficiency[33]. Additionally, apart from the transcription factors specifically linked to nitrogen metabolism, the discovery of ERF transcription factors, closely related to seed development[34], has provided further insights into the genetic landscape of millet growth and development. These findings serve as valuable clues and foundations for the investigation of the nitrogen metabolism pathway in millet.
This study performed transcriptomic sequencing and functional annotation of DEGs in recombinant glutamic inbred lines under NN and LN. Through GO functional and KEGG pathway enrichment analyses, 15 genes closely related to amino acid and N metabolism and Dof TFs were screened and verified by RT-qPCR. The mechanism of their regulation during the tolerance of millet to LN was analyzed. However, further verification using overexpression and gene knockout is needed to determine the functions of the candidate genes. This study laid a foundation for the mining, cloning, and further verification of the functionally essential genes determining low N-tolerance in millet and also provided a theoretical basis for the in-depth analysis of the underlying molecular mechanism and variety improvement.