Rehmannia glutinosa L., a perennial plant of Scrophulariaceae, is one of the most commonly used herbs in traditional Chinese medicine (TCM) that have been widely cultivated in China. However, to date, the biosynthetic pathway of its two quality-control components, catalpol and acteoside, are only partially elucidated and the mechanism for their tissue-specific accumulation remains unknown. To facilitate the basic understanding of the key genes and transcriptional regulators involved in the biosynthesis of catalpol and acteoside, transcriptome sequencing of radial striation (RS) and non-radial striation (nRS) from four R. glutinosa cultivars was performed. A total of 715,158,202 (~107.27 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 150,405 transcripts. Functional annotation with multiple public databases identified 155 and 223 unigenes involved in catalpol and acteoside biosynthesis, together with 325 UGTs, and important transcription factor (TF) families. Comparative analysis of the transcriptomes identified 362 unigenes, found to be differentially expressed in all RS vs. nRS comparisons, with 143 upregulated unigenes, including those encoding enzymes of the catalpol and acteoside biosynthetic pathway, such as geranyl diphosphate synthase (RgGPPS), geraniol 8-hydroxylase (RgG10H), and phenylalanine ammonia-lyase (RgPAL). Other differentially expressed unigenes predicted to be related to catalpol and acteoside biosynthesis fall into UDP-dependent glycosyltransferases (UGTs), as well as transcription factors. In addition, 16 differentially expressed genes were selectively confirmed by real-time PCR. In conclusion, a large unigene dataset of R. glutinosa generated in the current study will serve as a resource for the identification of potential candidate genes for investigation of the tuberous root development and biosynthesis of active components.

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WRKYs play important roles in plant metabolism, but their regulation mechanism in Rehmannia glutinosa remains elusive. In this study, 37 putative WRKY transcription factors (TFs) with complete WRKY domain from R. glutinosa transcriptome sequence data were identified. Based on their conserved domains and zinc finger motif, the R. glutinosa WRKY TFs were divided into five groups. Structural feature analysis shows that the 37 RgWRKY proteins contain WRKYGQK/GKK domains and a C2H2/C2HC-type zinc finger structure. To identify the function of RgWRKY members involved in acteoside biosynthesis, transcriptional profiles of 37 RgWRKYs in hairy roots under salicylic acid (SA), methyl jasmonate (MeJA), and hydrogen peroxide (H2O2) treatments were systematically established using RNA-seq analysis. Based on the correlationship between the expression levels of RgWRKY genes and acteoside content, RgWRKY7, RgWRKY23, RgWRKY34, RgWRKY35, and RgWRKY37 were suggested to be involved in acteoside biosynthesis in R. glutinosa, and RgWRKY37 was selected for gene functional research. Overexpression of RgWRKY37 increased the content of acteoside and total phenylethanoid glycosides (PhGs) in hairy roots and enhanced the transcript abundance of seven enzyme genes involved in the acteoside biosynthesis pathway. These results strongly suggest the involvement of the WRKY transcription factor in the regulation of acteoside biosynthesis.

The soybean (Glycine max) node number on the main stem (NNMS) is closely related to seed yield of soybean. The aim of this study was to identify important loci affecting soybean NNMS using meta-analysis based on a reference physical map. Twenty-nine NNMS-related QTLs were mapped across 8 years with a recombination inbred line (RIL) population. Fifty-four QTLs related to NNMS of soybean identified from the database and our research were collected and each QTL was projected onto the soybean physical map. Through meta-analysis, 11 consensus soybean NNMS QTLs were obtained and located on LG D1b, LG C2, LG B1, LG F, LG L, and LG I. The map distance was from 0.02 to 4.22 Mb, the variation of original QTLs was from 2 to 4, and the mean R 2 values ranged from 6.90% to 27.40%. Furthermore, 488 candidate genes were located in these consensus QTLs, 6 of which had a relationship with NNMS. These results may help lay a foundation for fine mapping of QTLs/genes related to NNMS that can be used to help breed high-yield soybean cultivars.

Opioid analgesic tolerance, a root cause of opioid overdose and misuse, can develop through an associative learning. Despite intensive research, the locus and central pathway subserving the associative opioid analgesic tolerance (AOAT) remains unclear. Using a combination of chemo/optogenetic manipulation with calcium imaging and slice physiology, here we identify neuronal ensembles in a hierarchically organized pathway essential for AOAT. The association of morphine-induced analgesia with an environmental condition drives glutamatergic signaling from ventral hippocampus (vHPC) to dorsomedial prefrontal cortex (dmPFC) cholecystokininergic (CCKergic) neurons. Excitation of CCKergic neurons, which project and release CCK to basolateral amygdala (BLA) glutamatergic neurons, relays AOAT signal through inhibition of BLA μ-opioid receptor function, thereby leading to further loss of morphine analgesic efficacy. This work provides evidence for a circuit across different brain regions distinct for opioid analgesic tolerance. The components of this pathway are potential targets to treat opioid overdose and abuse.