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Background Mulberry cultivars are well known for their flavonoid content. However, flavonoid content and the key genes involved in flavonoid biosynthesis in mulberry cultivars Zhong Shen 1 Hao (ZS) and Lv Shenzi (LSZ) are not well documented. This study aimed to analyze the flavonoid content and the key genes involved in flavonoid biosynthesis in ZS and LSZ cultivars. In this study, biochemical, transcriptomic, and functional analyses via virus-induced gene silencing (VIGS) were performed to analyze flavonoid content and key genes involved in flavonoid biosynthesis in ZS and LSZ. Results The content of flavonoids in LSZ is higher than in ZS, increasing by 38.1% compared to ZS. Transcriptome analysis identified 1938 differentially expressed genes (DEGs), with 1124 downregulated and 814 upregulated, and 14 DEGs were identified as key genes significantly enriched in flavonoid biosynthesis. Transient silencing of MaCYP73A16 notably decreased not only the expression of downstream genes, including MaCYP73A16 , MaLAR , MaCHS , MaCYP93B1 , and MaCYP98A2 , but also reduced flavonoid content, with peak silencing efficiency observed on the ninth day, underscoring the impact of silencing MaCYP73A16 on other genes involved in the flavonoid biosynthesis pathway. Conclusion This study provides a fundamental overview of flavonoid biosynthesis in LSZ and ZS and the key genes involved, which will help facilitate a comprehensive analysis of this pathway and provide significant insight into the large-scale biosynthesis of medicinal flavonoids, thereby improving flavonoid content in these cultivars.

Background 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 (MgSO 4 ) 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. Results 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. Conclusion These findings enhance our understanding of plant miRNA function under nutrient stress and provide a foundation for improving mulberry Mg tolerance.