Genome-wide identification and functional characterization of borneol dehydrogenases in Wurfbainia villosa.

MAIN CONCLUSION: Genome-wide screening of short-chain dehydrogenases/reductases (SDR) family reveals functional diversification of borneol dehydrogenase (BDH) in Wurfbainia villosa. Wurfbainia villosa is an important medicinal plant, the fruits of which accumulate abundant terpenoids, especially bornane-type including borneol and camphor. The borneol dehydrogenase (BDH) responsible for the conversion of borneol to camphor in W. villosa remains unknown. BDH is one member of short-chain dehydrogenases/reductases (SDR) family. Here, a total of 115 classical WvSDR genes were identified through genome-wide screening. These WvSDRs were unevenly distributed on different chromosomes. Seven candidate WvBDHs based on phylogenetic analysis and expression levels were selected for cloning. Of them, four BDHs can catalyze different configurations of borneol and other monoterpene alcohol substrates to generate the corresponding oxidized products. WvBDH1 and WvBDH2, preferred (+)-borneol to (-)-borneol, producing the predominant ( +)-camphor. WvBDH3 yielded approximate equivalent amount of (+)-camphor and (-)-camphor, in contrast, WvBDH4 generated exclusively (+)-camphor. The metabolic profiles of the seeds showed that the borneol and camphor present were in the dextrorotatory configuration. Enzyme kinetics and expression pattern in different tissues suggested WvBDH2 might be involved in the biosynthesis of camphor in W. villosa. All results will increase the understanding of functional diversity of BDHs.

The complete chloroplast genome sequence of Aconitum tschangbaischanense (Ranunculaceae).

The perennial herbal medicine species Aconitum tschangbaischanense, is endemic to Changhai Mountain, Jilin province. In this study, we attempted to uncover the complete chloroplast (cp) genome of A. tschangbaischanense based on sequencing data using the Illumina sequencing technology. As per the results: (1) the length of its complete cp genome is 155,881 bp with a typical tetrad structure; (2) the structure of its cp genome contains large single-copy and small single-copy (LSC and SSC) regions of 86,351 and 16,9444 bp, respectively, isolated by two inverted repeat regions (IRs) of 26,293 bp; (3) we annotated a total 131 genes, consisting of 86 protein-coding genes, eight rRNA genes, and 37 tRNA genes. According to the maximum-likelihood phylogenetic tree based on complete cp genomes, A. tschangbaischanense, showed close association with A. carmichaelii, which belongs to clade I. Finally, this study provides the characteristics of the cp genome of A. tschangbaischanense, and its phylogenetic position.

Chromosome-level genome assembly and functional characterization of terpene synthases provide insights into the volatile terpenoid biosynthesis of Wurfbainia villosa.

Wurfbainia villosa is a well-known medicinal and edible plant that is widely cultivated in the Lingnan region of China. Its dried fruits (called Fructus Amomi) are broadly used in traditional Chinese medicine for curing gastrointestinal diseases and are rich in volatile terpenoids. Here, we report a high-quality chromosome-level genome assembly of W. villosa with a total size of approximately 2.80 Gb, 42 588 protein-coding genes, and a very high percentage of repetitive sequences (87.23%). Genome analysis showed that W. villosa likely experienced a recent whole-genome duplication event prior to the W. villosa-Zingiber officinale divergence (approximately 11 million years ago), and a recent burst of long terminal repeat insertions afterward. The W. villosa genome enabled the identification of 17 genes involved in the terpenoid skeleton biosynthesis pathway and 66 terpene synthase (TPS) genes. We found that tandem duplication events have an important contribution to the expansion of WvTPSs, which likely drove the production of volatile terpenoids. In addition, functional characterization of 18 WvTPSs, focusing on the TPS-a and TPS-b subfamilies, showed that most of these WvTPSs are multi-product TPS and are predominantly expressed in seeds. The present study provides insights into the genome evolution and the molecular basis of the volatile terpenoids diversity in W. villosa. The genome sequence also represents valuable resources for the functional gene research and molecular breeding of W. villosa.

A Comparison of Two Monoterpenoid Synthases Reveals Molecular Mechanisms Associated With the Difference of Bioactive Monoterpenoids Between Amomum villosum and Amomum longiligulare.

The fruits of Amomum villosum and Amomum longiligulare are both used medicinally as Fructus Amomi the famous traditional Chinese medicine, however, the medicinal quality of A. villosum is better than that of A. longiligulare. Volatile terpenoids in the seeds, especially bornyl acetate and borneol, are the medicinal components of Fructus Amomi. The volatile terpenoids and transcriptome of developing seeds of A. villosum and A. longiligulare were compared in this study. The result revealed that the bornyl acetate and borneol contents were higher in A. villosum than in A. longiligulare. Additionally, six terpenoid synthase genes (AlTPS1-AlTPS6) were screened from the transcriptome of A. longiligulare, and AlTPS2 and AlTPS3 were found to share 98 and 83% identity with AvTPS2 and AvBPPS (bornyl diphosphate synthase) from A. villosum, respectively. BPPS is the key enzyme for the biosynthesis of borneol and bornyl acetate. Biochemical assays improved that AlTPS2 had an identical function to AvTPS2 as linalool synthase; however, AlTPS3 produced camphene as the major product and bornyl diphosphate (BPP) as the secondary product, whereas AvBPPS produced BPP as its major product. There was only one different amino acid between AlTPS3 (A496) and AvBPPS (G495) in their conserved motifs, and the site-directed mutation of A496G in DTE motif of AlTPS3 changed the major product from camphene to BPP. Molecular docking suggests that A496G mutation narrows the camphene-binding pocket and decreases the BPP-binding energy, thus increases the product BPP selectivity of enzyme. In addition, the expression level of AvBPPS was significantly higher than that of AlTPS3 in seeds, which was consistent with the related-metabolites contents. This study provides insight into the TPS-related molecular bases for the biosynthesis and accumulation differences of the bioactive terpenoids between A. villosum and A. longiligulare. BPPS, the key gene involved in the biosynthesis of the active compound, was identified as a target gene that could be applied for the quality-related identification and breeding of Fructus Amomi.

[Effects of lead exposure on copper and copper transporters in choroid plexus of rats].

OBJECTIVE: To investigate the effects of lead exposure on the copper concentration in the brain and serum and the expression of copper transporters in the choroid plexus among rats. METHODS: Sixty specific pathogen-free Sprague-Dawley rats were randomly divided into a control group and three lead-exposed groups, with 8 mice in each group. The lead-exposed groups were orally administrated with 500 (low-dose group)), 1 000 (middle-dose group), and 2 000 mg/L (high-dose group) lead acetate in drinking water for eight weeks. And the rats in control group were given 2 000 mg/L sodium acetate in drinking water. The content of lead and copper in the serum, hippocampus, cortex, choroid plexus, bones, and cerebrospinal fluid (CSF) was determined by inductively coupled plasma-mass spectrometry (ICP-MS). Confocal and real-time PCR methods were applied to measure the expression of copper transporters including copper transporter 1 (Ctr1), antioxidant protein 1 (ATX1), and Cu ATPase (ATP7A). RESULTS: Compared with the control group, the lead-exposed groups showed significantly higher lead concentrations in the serum, cortex, hippocampus, choroid plexus, CSF, and bones (P < 0.05) and significantly higher copper concentrations in the CSF, choroid plexus, serum, and hippocampus (P < 0.05). Confocal images showed that Ctr1 protein was expressed in the cytoplasm and cell membrane of choroid plexus in control group. However, Ctr1 migrated to CSF surface microvilli after lead exposure. Ctr1 fluorescence intensity gradually increased with increasing dose of lead, except that the middle-dose group had a higher Ctr1 fluorescence intensity than the high-dose group. In addition, the middle- and high-dose groups showed a lower ATX1 fluorescence intensity compared with the control group. Real-time PCR data indicated that the three lead-exposed groups showed significantly higher mRNA levels of Ctr1 and ATP7A compared with the control group (P < 0.05). CONCLUSION: Copper homeostasis in the choroid plexus is affected by lead exposure to induce copper homeostasis disorders in brain tissue, which may be one of the mechanisms of lead neurotoxicity.