ИСТИНА

Nutrients are needed for plant growth and development. Expression level alterations of microRNAs (miRNAs) in plants were shown under different nutrient concentrations in soil or nutrient solutions. We sequenced three flax (Linum usitatissimum L.) small RNA libraries and obtained 7.2M (inorganic phosphate – Pi - deficiency), 11.6M (normal nutrition), and 7.6M (excessive fertilizer) raw reads. We identified 76 conserved miRNA homologues which belong to 20 distinct miRNA families. Pi deficiency and excess fertilizer responsive miRNAs were identified in flax for the first time. The most significant down-regulation was shown for miR395 (fold change = –2.3, p-value = 4.4*10 6) and miR169 (fold change = –1.9, p-value = 3.4*10 4) in flax grown in redundancy of macro- and micronutrients. Under Pi deficiency up-regulation of miR398 (2.0-fold change; p-value 1.5*10 11), miR399 (2.2-fold change; p-value 0.02) and miR408 (2.3-fold change; p-value 6.6*10 7) was observed. We identified 156 potential novel flax miRNAs with predicted hairpins secondary structures. Predicted target genes for new miRNAs are involved in a wide range of processes occurring in the plant, including cell differentiation, immune responses, phosphate homeostasis regulation, plant growth and development. We identified potential novel flax miRNA lus-miR-N1 which was down-regulated under Pi deficiency. Quantitative real-time PCR (qPCR) on the extended sampling was performed to validate the results obtained from the highthroughput sequencing for miR169, miR395 and lus-miR-N1. However, qPCR confirmed differential expression only for miR395 and lus-miR-N1, but not for miR169. Target genes of miR395 are involved in sulfate uptake and assimilation. We speculate that alterations of the expression level of miR395 could be associated not only with excess sulfur application but also with redundancy of other macro- and micronutrients especially phosphorus and nitrogen which are necessary for protein synthesis. Target gene of lus-miR-N1 encodes ubiquitin-activating enzyme E1. This enzyme catalyzes the first step in the ubiquitination reaction, and in this way this miRNA could regulate phosphate starvation response in flax. Revealed alterations of flax miRNA expression under excess fertilizer and Pi deficiency clarify molecular mechanisms of nutrition regulation in plants and provide a wide area for further investigations.