Screening of differentially expressed microRNAs and target genes in two potato varieties under nitrogen stress

Abstract

Background A reasonable supply of nitrogen (N) fertilizer is essential for obtaining high-quality, high-level, and stable potato yields, and an improvement in the N utilization efficiency can effectively reduce N fertilizer use. It is important to use accurate, straightforward, and efficient transgenic breeding techniques for the identification of genes that can improve nitrogen use efficiency, thus enabling us to achieve the ultimate goal of breeding N-efficient potato varieties. In recent years, some of the mechanisms of miRNAs have been elucidated via the analysis of the correlation between the expression levels of potato miRNA target genes and regulated genes under conditions of stress, but the role of miRNAs in the inhibition/expression of key genes regulating N metabolism under N stress is still unclear. Our study aimed to identify the role played by specific enzymes and miRNAs in the responses of plants to N stress. Results The roots and leaves of the N-efficient potato variety, Yanshu4 ("Y"), and N-inefficient potato variety, Atlantic ("D"), were collected at the seedling and budding stages after they were exposed to different N fertilizer treatments. The miRNAs expressed differentially under the two types of N stress and their corresponding target genes were first predicted using miRNA and degradome analysis. Then, quantitative polymerase chain reaction (qRT-PCR) was performed to verify the expression of differential miRNAs that were closely related to N metabolism. Finally, the shearing relationship between stu-miR396-5p and its target gene StNiR was determined by analyzing luciferase activity levels. The results showed that NiR activity increased significantly with an increase in the applied N levels from the seedling stage to the budding stage, and NiR responded significantly to different N treatments. miRNA sequencing enabled us to predict 48 families with conserved miRNAs that were mainly involved in N metabolism, carbon metabolism, and amino acid biosynthesis. The differences in the expression of the following miRNAs were identified via screening (high expression levels and P < 0.05): stu-miR396-5p, stu-miR408b-3p_R-1, stu-miR3627-3p, stu-miR482a-3p, stu-miR8036-3p, stu-miR482a-5p, stu-miR827-5p, stu-miR156a_L-1, stu-miR827-3p, stu-miR172b-5p, stu-miR6022-p3_7, stu-miR398a-5p, and stu-miR166c-5p_L-3. Degradome analysis showed that most miRNAs had many-to-many relationships with target genes. The main target genes involved in N metabolism were NiR, NiR1, NRT2.5, and NRT2.7. qRT-PCR analysis showed that there were significant differences in the expression levels of stu-miR396-5p, stu-miR8036-3p, and stu-miR482a-3p in the leaves and roots of the Yanshu4 and Atlantic varieties at the seedling and budding stages under conditions that involved no N and excessive N application; the expression of these miRNAs was induced in response to N stress. The correlation between the differential expression of stu-miR396-5p and its corresponding target gene NiR was further verified by determining the luciferase activity level and was found to be strongly negative. Conclusion The activity of NiR was significantly positively correlated with N application from the seedling to the budding stage. Differential miRNAs and target genes showed a many-to-many relationship with each other. The expression of stu-miR396-5p, stu-miR482a-3p, and stu-miR8036-3p in the roots and leaves of the Yanshu4 and Atlantic varieties at the seedling and budding stages was notably different under two types of N stress. Under two types of N stress, stu-miR396-5p was down-regulated in Yanshu4 in the seedling-stage and shoot-stage roots, and up-regulated in seedling-stage roots and shoot-stage leaves; stu-miR482a-3p was up-regulated in the seedling and shoot stages. The expression of stu-miR8036-3p was up-regulated in the leaves and roots at the seedling and budding stages, and down-regulated in roots under both types of N stress. The gene expressing the key enzyme involved in N metabolism, StNiR, and the stu-miR396-5p luciferase assay reporter gene had a strong regulatory relationship with each other. This study provides candidate miRNAs related to nitrogen metabolism and highlights that differential miRNAs play a key role in nitrogen stress in potato, providing insights for future research on miRNAs and their target genes in nitrogen metabolic pathways and breeding nitrogen-efficient potatoes.

Root Reduction Caused Directly or Indirectly by High Application of Nitrogen Fertilizer Was the Main Cause of the Decline in Biomass and Nitrogen Accumulation in Citrus Seedlings

“…Nitrate reductase (NR) is one of the key enzymes in plant N assimilation and root structural remodeling [17], which is responsible for the reduction of NO 3 − to NO 2 − in plant cells [18]. Nitrite reductase is the second enzyme involved in NO 3 − reduction process, which to some extent directly reflects the nutritional status and N assimilation level of plants [19]. Glutamine synthetase and glutamate synthetase are the two main enzymes of NH 4 + assimilation, with glutamine synthetase catalyzing the first step of N assimilation in plant cells [20].…”

Genome-wide identification of nitrate-responsive microRNAs by small RNA sequencing in the rice restorer cultivar Nanhui 511

“…Furthermore, several miRNAs have been identified and characterized in plants, including miR167 and miR174 involved in signaling during root development (Meng et al, 2011;Liang et al, 2015). More importantly,miR156,miR159,miR160,miR166,miR169,miR171,miR172,miR319,miR396,miR397,miR398,miR399, and miR408, which are involved in plant growth and development, have been shown to regulate N absorption and assimilation (Xu et al, 2011;Liang et al, 2012;Nguyen et al, 2015;Tiwari et al, 2020;Lin et al, 2022;Lu et al, 2022). Previous studies have summarized the miRNAs involved in plant sensory functions, nutrient uptake, long-distance root transport, and physiological functions related to nutrients (Chiou, 2007;Islam et al, 2022b).…”