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

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.

Enhancing bioactive compounds accumulation in red beet (Beta Vulgaris L.) plants by managing N nutrition. The identification of the 'critical' zone as a cultivation technique.

The increasing interest in natural health-promoting compounds, which are mostly plant secondary metabolites, inspired attempts to stimulate mechanisms strengthening their bioaccumulation in crop plants via abiotic stress while maintaining the yield potential. This study investigates the long-term effects of limiting nitrogen (N) supply on the concentration of total phenolics, free radical activity of natural antioxidants, betacyanin content, biomass production, net photosynthetic rate, total chlorophyll content, and plant water relations in red beetroot plants (Beta vulgaris L.) grown hydroponically. Depending on fertilization, the range of N supply for evaporative demand comprises two contrasted nutrient zones, in which N is limiting (zone-1) or non-limiting (zone-2). Based on the carbon-nutrient-balance hypothesis, at the transition from 1st-zone to 2nd-zone, there is a narrow transition zone in which the plant nutrient status is considered 'critical'. Herein, to determine the 'critical' zone, a modified Michaelis-Menten (M-M) model was used using a piecewise linear regression on two indexes: net photosynthetic rates and free radical-scavenging capacity of phenolic antioxidants. The model showed that the 'critical' transition points of net photosynthetic rate and phenolic free radical content are located in a narrow zone ranging between 196.70 ± 8.75 and 271.54 ± 75.50 ppm NO3-, while the cropping season appears to affect slightly the range of 'critical' (transition) zone. Thus, supplying N to red beetroot plants to levels ranging within this 'critical' zone may be an efficient, profitable and sustainable way to increase the accumulation of health-promoting plant bioactive compounds (total phenolic compounds with radical activity and betacyanins) in hydroponically cultivated reed beetroot plants.