Project description:microRNAs (miRNAs) play important roles in responses to abiotic stresses, including nutrition stress, by regulating target gene expression. Phosphate (Pi) is often lacking in natural and agro-climatic environments, and plants have developed strategies to cope with low Pi (LP) availability. However, the miRNA-mediated regulation of these adaptive responses and their underlying coordinating signals are still poorly understood in forestry trees such as Betula luminifera. Four small RNA (sRNA) libraries, a mixed degradome cDNA library, and four transcriptomic libraries of B. luminifera roots and shoots treated under LP and normal conditions (CK) were constructed and sequenced using next-generation deep sequencing. sRNA sequencing analyses indicated that 66 and 60 miRNAs were differentially expressed in roots and shoots, respectively, under LP conditions. A total of 109 and 112 miRNA–target pairs were further validated in the roots and shoots, respectively, using degradome sequencing, including several novel target genes that were cleaved by isomiRNAs with lengths of 18 or 19 nucleotides, which were only differentially expressed in roots. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differential miRNA targets indicated that the “ascorbate and aldarate metabolism” pathway responded to LP, and “circadian rhythm – plant” was specifically enriched in shoots. A comprehensive B. luminifera transcriptome derived from its roots and shoots was constructed, and a total of 76,899 unigenes were generated. A comparison of transcriptome identified 8,095 and 5,584 differentially expressed genes in roots and shoots, respectively, under LP conditions. Integrated analysis uncovered 14 and 16 miRNA–target pairs that showed negatively correlated expression profiles in roots and shoots, respectively. Moreover, a putative model of miRNA–target interaction involved in plant responses to LP stress is proposed. These results suggest that comprehensive analyses of sRNAs, degradome, and transcriptome provide a useful platform for investigating LP stress in B. luminifera, and may provide new insights into the genetic engineering of high use efficiency of Pi in forestry trees.

Project description:microRNAs (miRNAs) play vital roles in plants regulating a panoply of biological processes, such as development, hormone signaling, and the stress response, by inhibiting target genes at the post-transcriptional level. However, the roles of miRNAs in Betula luminifera remain elusive. To mine for B. luminifera miRNAs and targets, we used a deep sequencing approach to analyze the sRNAs and degradome sequencing of mixed samples, including roots, stems, and leaves. A total of 114 known miRNAs or miRNA*s from 44 families, and 24 novel miRNAs and 17 miRNA*s plus 15 plausible miRNA candidates were identified, of which 36 known miRNAs, 29 miRNA*s, and all novel miRNAs had precursor sequences. Additionally, 49 targets for 19 known miRNA families and seven miRNA*s, and seven targets for novel miRNAs were identified using a high-throughput degradome-sequencing approach. The conserved miRNA targets were mainly transcription factors, whereas the miRNA* targets were mainly protein-coding genes, with preferential propensity to functional enzymes. A Gene Ontology analysis showed that the predicted targets were classified into 62 biological processes, 20 cellular components, and 28 molecular functions, respectively. We found two different targets for 396a* and miR396c*, and the target changed when the miR156d precursor cleavage site was shifted toward the 5′-end by two nucleotides, indicating the diverse regulatory roles of MIRNA genes. Furthermore, three targets identified by degradome sequencing were validated further through 5′ rapid amplification of cDNA ends. The expression patterns of the randomly selected miRNAs varied among different tissues. miR164 expression was induced under nitrogen starvation, with tissue-specific expression patterns, and was negatively correlated with the NAC1 target gene in roots and leaves, but not in stems. This study is a transcriptome-based analysis of miRNAs and the degradome in a B. luminifera pre-model tree and provides useful information to explore the functions of miRNAs and their target genes in B. luminifera.

Dataset Information

Betula luminifera strain:1V25-2

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