It is demonstrated that the Smi-miR164a module acts as a pivotal regulator, positively influencing phenolic acid biosynthesis while negatively regulating tanshinone production in S. miltiorrhiza.
Abstract
Introduction MicroRNAs (miRNAs) are key post-transcriptional regulators of plant secondary metabolism. Their primary mechanism involves silencing target genes through mRNA cleavage or translational inhibition, which is a major focus of current research in this field. However, the specific regulatory roles of individual miRNAs in coordinating different secondary metabolic pathways in medicinal plants remain largely uncharacterized. Methods This study investigated the roles of Smi-miR164a in Salvia miltiorrhiza. We generated Smi-miR164a-overexpressing (OE-miR164a) transgenic lines and performed comprehensive metabolic profiling and gene expression analysis. Results Overexpression of Smi-miR164a resulted in significant accumulation of phenolic acids, with rosmarinic acid (RA) and salvianolic acid B (SalB) levels increased by up to 2.8-fold compared to wild-type (WT). Conversely, it markedly reduced the accumulation of tanshinones, decreasing tanshinone I (T-I) and tanshinone IIA (T-IIA) to 25-68% of WT levels. Transcriptional analysis showed that expression changes in key biosynthetic genes were tightly correlated with the metabolic alterations. Genes involved in the tanshinone pathway (e.g., HMGR1, DXS2) were downregulated, whereas those in the salvianolic acid pathway (e.g., PAL1, C4H) were upregulated, consistent with the reciprocal accumulation of their corresponding metabolites. Conclusion These findings demonstrate that the Smi-miR164a module acts as a pivotal regulator, positively influencing phenolic acid biosynthesis while negatively regulating tanshinone production in S. miltiorrhiza. This gene presents a promising target for molecular breeding aimed at enhancing the yield of specific bioactive compounds.
Although drought stress typically suppresses plant growth and reduces crop yields, it paradoxically enhances the accumulation of bioactive compounds in the medicinal plant Salvia miltiorrhiza, such as salvianolic acids and tanshinones. However, the regulatory mechanisms behind this phenomenon remain poorly understood. In this study, an NAC transcription factor (TF), SmNAC36, has been identified as a pivotal regulator of bioactive compound biosynthesis and drought response in S. miltiorrhiza. Overexpression of SmNAC36 enhances the accumulation of tanshinones and salvianolic acids, and drought resistance. Conversely, knockout of SmNAC36 attenuated drought tolerance and markedly decreased bioactive compound content. Further mechanistic studies revealed that SmNAC36 directly binds to the promoters of SmPAL and Sm4CL in the salvianolic acids pathway, and to the promoter of SmDXS in the tanshinones biosynthesis pathway, thereby activating key enzymes that promote the accumulation of lignin as well as bioactive compounds. Moreover, SmNAC36 interacts with SmDREB1C, an AP2/ERF family TF that regulates drought tolerance. This interaction activates the expression of enzymes, such as superoxide dismutase and peroxidase, which are responsible for mitigating oxidative stress, thereby improving drought stress tolerance. Our results elucidate a dual-function mechanism whereby SmNAC36 integrates drought stress adaptation with bioactive compound biosynthesis, providing a potential metabolic engineering target for improving the quality and stress resilience of S. miltiorrhiza.
Zheng Zhou, Cuicui Han, Yun Wang et al.· Plant Communications· 0 citations
Results provide a genetic entry point for metabolic engineering and breeding efforts aimed at modulating grain phenolic profiles and indicate that SbmiR397-5p negatively regulates the expression of SbLAC14 in relation to CT biosynthesis, identifying it as a potential target for manipulating CT metabolism in sorghum.
Yannan Shi, Yongchao Guo, Jinping Wang et al.· Plants· 0 citations
Apple rust, caused by the fungal pathogen Gymnosporangium yamadae, leads to substantial yield losses and significant economic damage. In the rust-resistant cultivar Malus ‘Profusion’, rust infection triggers anthocyanin synthesis at infection sites as a defense mechanism to restrict fungal proliferation. Although small noncoding RNAs (miRNAs) play important roles in regulating anthocyanin biosynthesis, their specific functions under rust stress remain poorly characterized. In this study, small RNA sequencing revealed that miR166a is a key rust-responsive regulator. Its direct targeting and negative regulation of MpATHB8 were confirmed through luciferase assays, GUS staining, and gene expression analyses. Functional validation via transient and stable transformation in Malus demonstrated that suppressing miR166a expression using short tandem target mimics or overexpressing MpATHB8 promoted anthocyanin accumulation and enhanced resistance to rust. In contrast, overexpressing miR166a or silencing MpATHB8 suppressed anthocyanin synthesis and increased susceptibility to the pathogen. Further evidence indicates that the MpATHB8 protein activates anthocyanin biosynthesis by binding to and inducing the promoter of MpMYB10b. These findings reveal a miR166a-MpATHB8-MpMYB10b regulatory module that enhances rust resistance through anthocyanin metabolism in M. ‘Profusion’. Our findings provide novel insights into the miRNA-mediated regulation of anthocyanin metabolism and facilitate the breeding of rust-resistant and anthocyanin-enriched Malus cultivars.
Soil salinization restricts agricultural production. Identifying key regulatory genes and molecular mechanisms underlying saline-alkali stress is crucial for developing tolerant crops. In this study, the grapevine cultivars ‘Hotan Red’ (HTR) and ‘Yongyou No. 1’ (YY), which differ in saline-alkali tolerance, were used for physiological, microRNA, and transcriptomic analyses. HTR accumulated more osmotic regulatory substances and maintained relatively intact chloroplast structures, exhibiting stress resistance superior to YY. Fourier transform infrared (FTIR) analysis revealed that saline-alkali treatment did not alter the basic composition of functional groups in the leaf cell wall, but affected their relative contents. 26 miRNAs notably responsive to saline-alkali stress were identified. In HTR, vvi-miR395a expression was significantly downregulated under the saline-alkali stress, whereas the repression of vvi-miR395a in YY was less pronounced. RLM-5′RACE and GUS histochemical staining indicated that vvi-miR395a directly targets VvExpA10. Overexpression of vvi-miR395a suppressed VvExpA10 expression and reduced pectin and cellulose contents in grape calli and roots, but increased malondialdehyde contents. Simultaneous overexpression of VvExpA10-OE and STTM395a in transgenic calli led to significant enhancement of saline-alkali tolerance. These findings indicate that suppression of vvi-miR395a impaired its targeted cleavage of VvExpA10, thereby enhancing the levels of cell wall components and significantly improving grapevine tolerance to saline-alkali stress. This study validated the regulatory effect of the vvi-miR395a-VvExpA10 module in grapevine response to saline-alkali stress, offering potential targets used for the genetic improvement of grape with enhanced tolerance to saline-alkali stress.