Yayun Xu

Aristolochia contorta Bunge has been widely used as traditional Chinese medicine materials. However, its utility faces a great challenge due to the presence of aristolochic acids (AAs), a class of benzylisoquinoline alkaloid (BIA) derivatives. The first step in BIA skeleton formation is catalysis by norcoclaurine synthase (NCS). To gain knowledge of BIA and AA biosynthesis in A. contorta, genome-wide characterizations of NCS genes were carried out. This resulted in the identification of 15 A. contorta NCSs, namely, AcNCS1–AcNCS15. The AcNCS1–AcNCS8 proteins contained one catalytic domain, whereas the AcNCS9–AcNCS15 proteins had two. Phylogenetic analysis shows that AcNCS proteins can be classified into two clades. Gene expression analysis shows that five AcNCSs, including AcNCS2, AcNCS4, AcNCS5, AcNCS14, and AcNCS15, exhibited relatively high expression in roots and flowers, where norcoclaurine accumulated. An enzyme catalytic activity assay shows that all five of the AcNCSs can catalyze norcoclaurine formation with AcNCS14 and AcNCS15, exhibiting higher catalytic efficiency. Precolumn derivatization analysis shows that the formed norcoclaurine included (S)- and (R)-norcoclaurine, with more (S)-configuration. The results provide useful information for further understanding BIA and AA biosynthesis in A. contorta and for AA elimination and bioactive compound improvement in AA-containing medicinal materials.

Aristolochia contorta Bunge is an academically and medicinally important plant species. It belongs to the magnoliids, with an uncertain phylogenetic position, and is one of the few plant species lacking a whole-genome duplication (WGD) event after the angiosperm-wide WGD. A. contorta has been an important traditional Chinese medicine material. Since it contains aristolochic acids (AAs), chemical compounds with nephrotoxity and carcinogenicity, the utilization of this plant has attracted widespread attention. Great efforts are being made to increase its bioactive compounds and reduce or completely remove toxic compounds. MicroRNAs (miRNAs) and natural antisense transcripts (NATs) are two classes of regulators potentially involved in metabolism regulation. Here, we report the identification and characterization of 223 miRNAs and 363 miRNA targets. The identified miRNAs include 51 known miRNAs belonging to 20 families and 172 novel miRNAs belonging to 107 families. A negative correlation between the expression of miRNAs and their targets was observed. In addition, we identified 441 A. contorta NATs and 560 NAT-sense transcript (ST) pairs, of which 12 NATs were targets of 13 miRNAs, forming 18 miRNA-NAT-ST modules. Various miRNAs and NATs potentially regulated secondary metabolism through the modes of miRNA-target gene–enzyme genes, NAT-STs, and NAT-miRNA-target gene–enzyme genes, suggesting the complexity of gene regulatory networks in A. contorta. The results lay a solid foundation for further manipulating the production of its bioactive and toxic compounds.

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs in plants. They play critical functions in various biological processes during plant growth and development. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant with significant medicinal, economic, and academic values. In order to elucidate the role of miRNAs in S. miltiorrhiza, six small RNA libraries from mature roots, young roots, stems, mature leaves, young leaves and flowers of S. miltiorrhiza and one degradome library from mixed tissues were constructed. A total of 184 miRNA precursors, generating 137 known and 49 novel miRNAs, were genome-widely identified. The identified miRNAs were predicted to play diversified regulatory roles in plants through regulating 891 genes. qRT-PCR and 5′ RLM-RACE assays validated the negative regulatory role of smi-miR159a in SmMYB62, SmMYB78, and SmMYB80. To elucidate the function of smi-miR159a in bioactive compound biosynthesis, smi-miR159a transgenic hairy roots were generated and analyzed. The results showed that overexpression of smi-miR159a caused a significant decrease in rosmarinic acid and salvianolic acid B contents. qRT-PCR analysis showed that the targets of smi-miR159a, including SmMYB62, SmMYB78, and SmMYB80, were significantly down-regulated, accompanied by the down-regulation of SmPAL1, SmC4H1, Sm4CL1, SmTAT1, SmTAT3, SmHPPR1, SmRAS, and SmCYP98A14 genes involved in phenolic acid biosynthesis. It suggests that smi-miR159a is a significant negative regulator of phenolic acid biosynthesis in S. miltiorrhiza.