MicroRNA-mediated responses to magnesium-induced stress coordinate target genes involved in porphyrin and biosynthesis of secondary metabolites metabolisms in mulberry (Morus alba L.)

MicroRNAs (miRNAs) play essential regulatory roles in magnesium (Mg) imbalance by targeting key defense genes for cleavage or translational repression. miRNA targets in response to Mg deficiency and toxicity remain uncharacterized in mulberry (Morus alba) in Mg-induced stress. We, for the first time, investigated Mg-stress-responsive miRNAs and their targets in mulberry leaves using transcriptome-wide sequencing to identify miRNAs putatively involved in Mg-stress tolerance. In this study, Mg (MgSO4) treatment applied in six concentration ranging from 0 mM/L (T0) as Mg deficiency, 1 mM/L and 2 mM/L (T1 and T2) as low Mg, 3 mM/L as the sufficiency or control (CK), and 6 mM/L and 9 mM/L (T6 and T9) as Mg excess for 20 days under controlled environment. Our analysis of differentially expressed miRNAs (DE-miRNAs) revealed that 254 out of the 893 miRNAs expressed across all samples showed differential expression, representing 28.44%. The study further identified 64 known miRNAs and 190 novel miRNAs within this set of differentially expressed miRNAs. These DE-miRNAs targeted a total of 39 Mg-induced genes and were predominantly involved in porphyrin, polyphenol oxidase, and phenylalanine ammonia-lyase metabolisms. For instance, miR477-x with ─25.1 minimum folding free energy and a minimum free energy ratio of 67.11% targeted four PAL genes, whereas novel miRNA (novel-m0001-5p) targeted eight genes in the polyphenol oxidase gene family involved in tyrosine metabolism. Additionally, four genes, including red chlorophyll catabolite reductase and uroporphyrinogen-III synthase, were targeted by miR529-x under Mg-induced treatments. These findings enhance our understanding of plant miRNA function under nutrient stress and provide a foundation for improving mulberry Mg tolerance.