Strategies on biosynthesis and production of bioactive compounds in medicinal plants.

Medicinal plants are a valuable source of essential medicines and herbal products for healthcare and disease therapy. Compared with chemical synthesis and extraction, the biosynthesis of natural products is a very promising alternative for the successful conservation of medicinal plants, and its rapid development will greatly facilitate the conservation and sustainable utilization of medicinal plants. Here, we summarize the advances in strategies and methods concerning the biosynthesis and production of natural products of medicinal plants. The strategies and methods mainly include genetic engineering, plant cell culture engineering, metabolic engineering, and synthetic biology based on multiple "OMICS" technologies, with paradigms for the biosynthesis of terpenoids and alkaloids. We also highlight the biosynthetic approaches and discuss progress in the production of some valuable natural products, exemplifying compounds such as vindoline (alkaloid), artemisinin and paclitaxel (terpenoids), to illustrate the power of biotechnology in medicinal plants.

Analysis of the Glycoside Hydrolase Family 1 from Wild Jujube Reveals Genes Involved in the Degradation of Jujuboside A.

Jujubosides are the major medicinal ingredients of Ziziphi Spinosae Semen (the seed of wild jujube). To date, a complete understanding of jujuboside's metabolic pathways has not been attained. This study has systematically identified 35 ß-glucosidase genes belonging to the glycoside hydrolase family 1 (GH1) using bioinformatic methods based on the wild jujube genome. The conserved domains and motifs of the 35 putative ß-glucosidases, along with the genome locations and exon-intron structures of 35 ß-glucosidase genes were revealed. The potential functions of the putative proteins encoded by the 35 ß-glucosidase genes are suggested based on their phylogenetic relationships with Arabidopsis homologs. Two wild jujube ß-glucosidase genes were heterologously expressed in Escherichia coli, and the recombinant proteins were able to convert jujuboside A (JuA) into jujuboside B (JuB). Since it has been previously reported that JuA catabolites, including JuB and other rare jujubosides, may play crucial roles in the jujuboside's pharmacological activity, it is suggested that these two proteins can be used to enhance the utilization potential of jujubosides. This study provides new insight into the metabolism of jujubosides in wild jujube. Furthermore, the characterization of ß-glucosidase genes is expected to facilitate investigations involving the cultivation and breeding of wild jujube.

Revealing the Roles of the JAZ Family in Defense Signaling and the Agarwood Formation Process in Aquilaria sinensis.

Jasmonate ZIM-domain family proteins (JAZs) are repressors in the signaling cascades triggered by jasmonates (JAs). It has been proposed that JAs play essential roles in the sesquiterpene induction and agarwood formation processes in Aquilaria sinensis. However, the specific roles of JAZs in A. sinensis remain elusive. This study employed various methods, including phylogenetic analysis, real-time quantitative PCR, transcriptomic sequencing, yeast two-hybrid assay, and pull-down assay, to characterize A. sinensis JAZ family members and explore their correlations with WRKY transcription factors. The bioinformatic analysis revealed twelve putative AsJAZ proteins in five groups and sixty-four putative AsWRKY transcription factors in three groups. The AsJAZ and AsWRKY genes exhibited various tissue-specific or hormone-induced expression patterns. Some AsJAZ and AsWRKY genes were highly expressed in agarwood or significantly induced by methyl jasmonate in suspension cells. Potential relationships were proposed between AsJAZ4 and several AsWRKY transcription factors. The interaction between AsJAZ4 and AsWRKY75n was confirmed by yeast two-hybrid and pull-down assays. This study characterized the JAZ family members in A. sinensis and proposed a model of the function of the AsJAZ4/WRKY75n complex. This will advance our understanding of the roles of the AsJAZ proteins and their regulatory pathways.

Fusarium oxysporum infection-induced formation of agarwood (FOIFA): A rapid and efficient method for inducing the production of high quality agarwood.

Agarwood, a non-wood product from the endangered Aquilaria and Gyrinops tress, is highly prized for its use in fragrances and medicines. The special formation process of agarwood is closely related to external injury and fungal infection. In this study, we demonstrate that infection of Aquilaria sinensis by Fusarium oxysporum, a soilborne fungus that causes vascular wilt diseases in diverse plants, induces agarwood formation. Based on these findings, an efficient method, termed F. oxysporum infection-induced formation of agarwood (FOIFA), was developed for the rapid production of quality agarwood. The agarwood formed in response to F. oxysporum infection was similar in structure and chemical composition to wild agarwood according to TLC (Thin-layer chromatography), HPLC (high performance liquid chromatography), and GC-MS (gas chromatography-mass spectrometry) analyses, except that the contents of alcohol-soluble extract, chromones, and essential oils (mainly sesquiterpenes) were higher in the formed agarwood.

Global Perspective of Drought Impacts on Ozone Pollution Episodes.

Ozone (O3) pollution threatens global public health and damages ecosystem productivity. Droughts modulate surface O3 through meteorological processes and vegetation feedbacks. Unraveling these influences is difficult with traditional chemical transport models. Here, using an atmospheric chemistry-vegetation coupled model in combination with a suite of existing measurements, we investigate the drought impacts on global surface O3 and explore the main driving processes. Relative to the mean state, accelerated photochemical rates dominate the surface O3 enhancement during droughts except for eastern U.S. and western Europe, where reduced stomatal uptakes make comparable contributions. During 1990-2012, the simulated frequency of O3 pollution episodes in western Europe decreases greatly with a negative trend of -5.5 ± 6.6 days per decade following the reductions in anthropogenic emissions if meteorology is fixed. However, such decreased trend is weakened to -2.1 ± 3.8 days per decade, which is closer to the observed trend of -2.9 ± 1.1 days per decade when year-to-year meteorology is applied because increased droughts alone offset 43% of the effects from air pollution control. Our results highlight that more stringent controls of O3 precursors are necessary to mitigate the higher risks of O3 pollution episodes by more droughts in a warming world.

Identifying the dominant climate-driven uncertainties in modeling gross primary productivity.

Accurate simulation of gross primary productivity (GPP) is essential for estimating the global carbon budget. However, GPP modeling is subject to various sources of uncertainties, among which the impacts of biases in climate forcing data have not been well quantified. Here, using a well-validated vegetation model, we compare site-level simulations using either ground-based meteorology or assimilated reanalyses to identify climate-driven uncertainties in the predicted GPP at 91 FLUXNET sites. Simulations yield the lowest root mean square errors (RMSE) in GPP relative to observations when all site-level meteorology and CO2 concentrations are used. Sensitivity tests conducted with Modern-Era Retrospective Analysis (MERRA) reanalyses increase GPP RMSE by 30%. Replacement of site-level CO2 with global annual average values provides limited contributions to these changes. In contrast, GPP uncertainties increase almost linearly with the biases in meteorology. Among all factors, photosynthetically active radiation (PAR), especially diffuse PAR, plays dominant roles in modulating GPP uncertainties. Simulations using all MERRA forcings but with site-level diffuse PAR help reduce over 50% of the climate-driven biases in GPP. Our study reveals that biases in meteorological forcings, especially the variabilities at diurnal to seasonal time scales, can induce significant uncertainties in the simulated GPP at FLUXET sites. We suggest cautions in simulating global GPP using climate reanalyses for dynamic global vegetation models and urgent improvements in climatic variability in reanalyses data, especially for diffuse radiation.

Integrated metabolomic and transcriptomic profiling reveals the tissue-specific flavonoid compositions and their biosynthesis pathways in Ziziphora bungeana.

BACKGROUND: Ziziphora bungeana Juz. is a folk medicine from the Xinjiang Uygur Autonomous Region. The herb or the aerial parts of it have been used to medicinally treat cardiovascular diseases. Flavonoids are the main pharmacologically active ingredients in Z. bungeana. Identification of the tissue-specific distribution of flavonoids in Z. bungeana is crucial for effective and sustainable medicinal use of the plant. Furthermore, understanding of the biosynthesis pathways of these flavonoids in Z. bungeana is of great biological significance. METHODS: The flavonoids from different tissues of Z. bungeana were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The full-length transcriptome of Z. bungeana was determined using a strategy based on a combination of Illumina and PacBio sequencing techniques. The functions of differentially expressed unigenes were predicted using bioinformatics methods and further investigated by real-time quantitative PCR and phylogenetic relationship analysis. RESULTS: Among the 12 major flavonoid components identified from Z. bungeana extracts, linarin was the most abundant component. Nine flavonoids were identified as characteristic components of specific tissues. Transcriptome profiling and bioinformatic analysis revealed that 18 genes were putatively involved in flavonoid biosynthesis. The gene expression and phylogenetic analysis results indicated that ZbPALs, Zb4CL3, ZbCHS1, and ZbCHI1 may be involved in the biosynthesis of the main flavonoid intermediate. ZbFNSII, ZbANS, and ZbFLS may be involved in the biosynthesis of flavones, anthocyanins, and flavonols, respectively. A map of the biosynthesis pathways of the 12 major flavonoids in Z. bungeana is proposed. CONCLUSIONS: The chemical constituent analysis revealed the compositions of 9 characteristic flavonoids in different tissues of Z. bungeana. Linarin can be hydrolysed into acacetin to exert a pharmaceutical role. Apigenin-7-O-rutinoside is hypothesised to be the precursor of linarin in Z. bungeana. There was greater content of linarin in the aerial parts of the plant than in the whole herb, which provides a theoretical basis for using the aerial parts of Z. bungeana for medicine. These results provide a valuable reference for further research on the flavonoid biosynthesis pathways of Z. bungeana and will be significant for the effective utilisation and ecological protection of Z. bungeana.

[Transcriptome analysis reveals candidate genes involved in flavonoid biosynthesis in Ziziphora bungeana].

Ziziphora bungeana is a kind of medicinal plants belongs to Labiatae,and it also a kind of geoherbs in Xinjiang. The main active ingredient linarin has a higher content in inflorescence than in other parts. In this study,high-throughput sequencing technology was used to reveal the transcriptome of the inflorescence of Z. bungeana,77 366 unigenes were acquired,of which 56 375 unigenes were annotated based on search of the database and classification. Through the analysis of metabolic pathways,sixty unigenes were probably encoding some enzymes involved in the flavonoid biosynthesis pathways. The contents of linarin in different parts were determined and the key genes were verified by qRT-PCR. The discovery provides the research basis for further analysis of the enzyme genes involved in the biosynthesis of the major flavonoid components in Z. bungeana.

Functional Analysis of Polyprenyl Diphosphate Synthase Genes Involved in Plastoquinone and Ubiquinone Biosynthesis in Salvia miltiorrhiza.

Polyprenyl diphosphate synthase (PPS) plays important roles in the biosynthesis of functionally important plastoquinone (PQ) and ubiquinone (UQ). However, only few plant PPS genes have been functionally characterized. Through genome-wide analysis, two PPS genes, termed SmPPS1 and SmPPS2, were identified from Salvia miltiorrhiza, an economically significant Traditional Chinese Medicine material and an emerging model medicinal plant. SmPPS1 and SmPPS2 belonged to different phylogenetic subgroups of plant trans-long-chain prenyltransferases and exhibited differential tissue expression and light-induced expression patterns. Computational prediction and transient expression assays showed that SmPPS1 was localized in the chloroplasts, whereas SmPPS2 was mainly localized in the mitochondria. SmPPS2, but not SmPPS1, could functionally complement the coq1 mutation in yeast cells and catalyzed the production of UQ-9 and UQ-10. Consistently, both UQ-9 and UQ-10 were detected in S. miltiorrhiza plants. Overexpression of SmPPS2 caused significant UQ accumulation in S. miltiorrhiza transgenics, whereas down-regulation resulted in decreased UQ content. Differently, SmPPS1 overexpression significantly elevated PQ-9 content in S. miltiorrhiza. Transgenic lines showing a down-regulation of SmPPS1 expression exhibited decreased PQ-9 level, abnormal chloroplast and trichome development, and varied leaf bleaching phenotypes. These results suggest that SmPPS1 is involved in PQ-9 biosynthesis, whereas SmPPS2 is involved in UQ-9 and UQ-10 biosynthesis.

Identification of genes involved in metabolism and signalling of abscisic acid and gibberellins during Epimedium pseudowushanense B.L.Guo seed morphophysiological dormancy.

KEY MESSAGE: Key genes involved in metabolism and signalling of abscisic acid and gibberellins during Epimedium pseudowushanense B.L.Guo seed morphophysiological dormancy release were identified using phytochemistry, transcriptomics, and bioinformatic methods. The molecular mechanism of seed morphophysiological dormancy of Epimedium pseudowushanense B.L.Guo. remains largely unknown. The endogenous abscisic acid (ABA) and gibberellin (GA) content of E. pseudowushanense seeds at three developmental stages were quantitatively determined. The results showed the levels of ABA in E. pseudowushanense seeds decreased during seed embryo growth and development, while levels of GA3 increased during seed embryo growth, and levels of GA4 increased during seed dormancy release and seed sprouting. A high-throughput sequencing method was used to determine the E. pseudowushanense seed transcriptome. The transcriptome data were assembled as 178,613 unigenes and the numbers of differentially expressed unigenes between the seed development stages were compared. Computer analysis of reference pathways revealed that 12 candidate genes were likely to be involved in metabolism and signalling of ABA and GAs. The expression patterns of these genes were revealed by real-time quantitative PCR. Phylogenetic relationships among the deduced E. pseudowushanense proteins and their homologous proteins in other plant species were analysed. The results indicated that EpNCED1, EpNCED2, EpCYP707A1, and EpCYP707A2 are likely to be involved in ABA biosynthesis and catabolism. EpSnRK2 is likely implicated in ABA signalling during seed dormancy. EpGA3ox is likely to be involved in GA biosynthesis. EpDELLA1 and EpDELLA2 are likely implicated in GA signalling. This study is the first to provide the E. pseudowushanense seed transcriptome and the key genes involved in metabolism and signalling of ABA and GAs, and it is valuable for studies on the mechanism of seed morphophysiological dormancy.

A Portable Farmland Information Collection System with Multiple Sensors.

Precision agriculture is the trend of modern agriculture, and it is also one of the important ways to realize the sustainable development of agriculture. In order to meet the production requirements of precision agriculture-efficient use of agricultural resources, and improving the crop yields and quality-some necessary field information in crop growth environment needs to be collected and monitored. In this paper, a farmland information collection system is developed, which includes a portable farmland information collection device based on STM32 (a 32-bit comprehensive range of microcontrollers based on ARM Crotex-M3), a remote server and a mobile phone APP. The device realizes the function of portable and mobile collecting of multiple parameters farmland information, such as chlorophyll content of crop leaves, air temperature, air humidity, and light intensity. UM220-III (Unicore Communication Inc., Beijing, China) is used to realize the positioning based on BDS/GPS (BeiDou Navigation Satellite System, BDS/Global Positioning System, GPS) dual-mode navigation and positioning system, and the CDMA (Code Division Multiple Access, CDMA) wireless communication module is adopted to realize the real-time remote transmission. The portable multi-function farmland information collection system is real-time, accurate, and easy to use to collect farmland information and multiple information parameters of crops.

Leaf Chlorophyll Content Estimation of Winter Wheat Based on Visible and Near-Infrared Sensors.

The leaf chlorophyll content is one of the most important factors for the growth of winter wheat. Visual and near-infrared sensors are a quick and non-destructive testing technology for the estimation of crop leaf chlorophyll content. In this paper, a new approach is developed for leaf chlorophyll content estimation of winter wheat based on visible and near-infrared sensors. First, the sliding window smoothing (SWS) was integrated with the multiplicative scatter correction (MSC) or the standard normal variable transformation (SNV) to preprocess the reflectance spectra images of wheat leaves. Then, a model for the relationship between the leaf relative chlorophyll content and the reflectance spectra was developed using the partial least squares (PLS) and the back propagation neural network. A total of 300 samples from areas surrounding Yangling, China, were used for the experimental studies. The samples of visible and near-infrared spectroscopy at the wavelength of 450,900 nm were preprocessed using SWS, MSC and SNV. The experimental results indicate that the preprocessing using SWS and SNV and then modeling using PLS can achieve the most accurate estimation, with the correlation coefficient at 0.8492 and the root mean square error at 1.7216. Thus, the proposed approach can be widely used for winter wheat chlorophyll content analysis.

The phenylalanine ammonia-lyase gene family in Salvia miltiorrhiza: genome-wide characterization, molecular cloning and expression analysis.

Salvia miltiorrhiza Bunge is a well-known material of traditional Chinese medicine. Hydrophilic phenolic acids, such as rosmarinic acid and salvianolic acid B, are a group of pharmaceutically important compounds in S. miltiorrhiza. The biosynthesis of rosmarinic acid requires the coordination of the phenylpropanoid pathway and the tyrosine-derived pathway. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. Systematic analysis of the SmPAL gene family has not been carried out. We report here the identification of three SmPALs through searching the recently obtained working draft of the S. miltiorrhiza genome and full-length cDNA cloning. Bioinformatic and phylogenetic analyses showed that SmPAL1 and SmPAL3 clustered in a sub-clade of dicot PALs, whereas SmPAL2 fell into the other one. Some important cis-elements were conserved in three SmPAL promoters, whereas the others were not. SmPAL1 and SmPAL3 were highly expressed in roots and leaves of S. miltiorrhiza, but SmPAL2 were predominately expressed in stems and flowers. It indicates that SmPAL1 and SmPAL3 function redundantly in rosmarinic acid biosynthesis. All SmPALs were induced in roots treated with PEG and MeJA, but the time and degree of responses were different, suggesting the complexity of SmPAL-associated metabolic network in S. miltiorrhiza. This is the first comprehensive study dedicated to SmPAL gene family characterization. The results provide a basis for elucidating the role of SmPAL genes in the biosynthesis of bioactive compounds.

High-throughput sequencing and characterization of the small RNA transcriptome reveal features of novel and conserved microRNAs in Panax ginseng.

microRNAs (miRNAs) play vital regulatory roles in many organisms through direct cleavage of transcripts, translational repression, or chromatin modification. Identification of miRNAs has been carried out in various plant species. However, no information is available for miRNAs from Panax ginseng, an economically significant medicinal plant species. Using the next generation high-throughput sequencing technology, we obtained 13,326,328 small RNA reads from the roots, stems, leaves and flowers of P. ginseng. Analysis of these small RNAs revealed the existence of a large, diverse and highly complicated small RNA population in P. ginseng. We identified 73 conserved miRNAs, which could be grouped into 33 families, and 28 non-conserved ones belonging to 9 families. Characterization of P. ginseng miRNA precursors revealed many features, such as production of two miRNAs from distinct regions of a precursor, clusters of two precursors in a transcript, and generation of miRNAs from both sense and antisense transcripts. It suggests the complexity of miRNA production in P. ginseng. Using a computational approach, we predicted for the conserved and non-conserved miRNA families 99 and 31 target genes, respectively, of which eight were experimentally validated. Among all predicted targets, only about 20% are conserved among various plant species, whereas the others appear to be non-conserved, indicating the diversity of miRNA functions. Consistently, many miRNAs exhibited tissue-specific expression patterns. Moreover, we identified five dehydration- and ten heat-responsive miRNAs and found the existence of a crosstalk among some of the stress-responsive miRNAs. Our results provide the first clue to the elucidation of miRNA functions in P. ginseng.

Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza.

Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering.

Comprehensive transcriptome analysis reveals novel genes involved in cardiac glycoside biosynthesis and mlncRNAs associated with secondary metabolism and stress response in Digitalis purpurea.

BACKGROUND: Digitalis purpurea is an important ornamental and medicinal plant. There is considerable interest in exploring its transcriptome. RESULTS: Through high-throughput 454 sequencing and subsequent assembly, we obtained 23532 genes, of which 15626 encode conserved proteins. We determined 140 unigenes to be candidates involved in cardiac glycoside biosynthesis. It could be grouped into 30 families, of which 29 were identified for the first time in D. purpurea. We identified 2660 mRNA-like npcRNA (mlncRNA) candidates, an emerging class of regulators, using a computational mlncRNA identification pipeline and 13 microRNA-producing unigenes based on sequence conservation and hairpin structure-forming capability. Twenty five protein-coding unigenes were predicted to be targets of these microRNAs. Among the mlncRNA candidates, only 320 could be grouped into 140 families with at least two members in a family. The majority of D. purpurea mlncRNAs were species-specific and many of them showed tissue-specific expression and responded to cold and dehydration stresses. We identified 417 protein-coding genes with regions significantly homologous or complementary to 375 mlncRNAs. It includes five genes involved in secondary metabolism. A positive correlation was found in gene expression between protein-coding genes and the homologous mlncRNAs in response to cold and dehydration stresses, while the correlation was negative when protein-coding genes and mlncRNAs were complementary to each other. CONCLUSIONS: Through comprehensive transcriptome analysis, we not only identified 29 novel gene families potentially involved in the biosynthesis of cardiac glycosides but also characterized a large number of mlncRNAs. Our results suggest the importance of mlncRNAs in secondary metabolism and stress response in D. purpurea.

Molecular analysis of rice plants harboring a multi-functional T-DNA tagging system.

About 25,000 rice T-DNA insertional mutant lines were generated using the vector pCAS04 which has both promoter-trapping and activation-tagging function. Southern blot analysis revealed that about 40% of these mutants were single copy integration and the average T-DNA insertion number was 2.28. By extensive phenotyping in the field, quite a number of agronomically important mutants were obtained. Histochemical GUS assay with 4,310 primary mutants revealed that the GUS-staining frequency was higher than that of the previous reports in various tissues and especially high in flowers. The T-DNA flanking sequences of some mutants were isolated and the T-DNA insertion sites were mapped to the rice genome. The flanking sequence analysis demonstrated the different integration pattern of the right border and left border into rice genome. Compared with Arabidopsis and poplar, it is much varied in the T-DNA border junctions in rice.