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Tetrastigma hemsleyanum Diels et Gilg (Sanyeqing, SYQ) is a perennial climbing liana and an endemic plant to southern China. Its tuberous roots (TRs) are used in traditional Chinese medicine for treating some diseases such as high fever, pneumonia, asthma, hepatitis, and cancers. However, the mechanisms underlying the development of TR and the content of flavonoids and phenylpropanoids (FPs) are not well-understood. In this study, we performed a transcriptomic analysis of 12 fully developed TR (FD-TR) samples harvested in four seasons [spring (Sp), summer (Su), autumn (Au), and winter (Wi)] using the RNA-Sequencing (RNA-Seq). We obtained a total of 78.54 Gb raw data and 65,578 unigenes. Then, the unigenes were annotated by using six databases such as non-redundant protein database (NR), Pfam, eggNOG, SWISSProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene ontology (GO). The transcriptomic profiling showed closer relationships between the samples obtained in Su and Au than those obtained in Sp and Wi based on the results of both total unigenes and differentially expressed genes (DEGs). Three pathways, including the biosynthesis of FPs, metabolism of starch and sucrose, and signaling of phytohormones, were highly enriched, suggesting a gene-level seasonal variation. Based on the numbers of DEGs, brassinosteroid (BR) signal transduction factors appeared to play a key role in modulating the development of TRs while most of the auxin signaling genes were mainly activated in Wi and Sp FD-TRs. Most genes in the biosynthesis and biodegradation of starch and biodegradation of cellulose were activated in Wi FD-TRs. As determined by the high performance liquid chromatography (HPLC) and aluminum nitrate colorimetric method, the contents of total flavonoids and most detected FP components increased from Sp to Au but decreased in Wi. Enhanced expression levels of some genes in the biosynthetic pathways of FPs were detected in Su and Au samples, which corroborated well with metabolite content. Our findings provide the first transcriptomic and biochemical data on a seasonal variation in the composition of medically important metabolites in SYQ FD-TRs.

Gray mold of tomato is caused by the pathogen Botrytis cinerea. MicroRNAs play a crucial role in the biotic and abiotic stress responses of plants and regulate their targets by gene silencing. miR394 is an ancient and conserved miRNA in plants, and it participates in the regulation of plant development and stress responses. In our previous study, miR394 was found to respond to B. cinerea infection in tomato, but the roles and regulatory mechanisms of miR394 in B. cinerea-infected tomato remain unclear. miR394 was down-regulated in tomato in response to B. cinerea infection, showing an expression pattern opposite to the previous finding that miR394 was up-regulated in tomato cv. Jinpeng 1 infected by B. cinerea. We obtained transgenic Arabidopsis overexpressing miR394, which resulted in low expression levels of its target LEAF CURLING RESPONSIVENESS (LCR). Leaf lesion size and trypan blue staining showed that miR394 overexpression led to increased sensitivity of transgenic Arabidopsis to B. cinerea compared to wild type. We also detected changes in the expression levels of stress-related miRNAs, including miR159, miR156, miR168, and miR172. In the transgenic plants, it indicated potential cross talk between these miRNAs and miR394, except for miR159. miR394 also enhanced the expression of ARGONAUTE 1 (AGO1), DSRNA-BINDING PROTEIN 4 (DRB4) and the RNA-binding protein gene DAWDLE (DDL), which are involved in the pathways of miRNA biosynthesis and regulation, suggesting that miR394 overexpression has a feedback effect on these genes. Our data indicate that overexpression of miR394 in Arabidopsis increased the susceptibility of plants to B. cinerea by affecting the expression of its target gene LCR along with a number of key genes involved in plant miRNA metabolism (AGO1). Thus, miR394 is a negative regulator of Arabidopsis resistance to B. cinerea infection by targeting LCR.