Functional Genome Analyses Reveal the Molecular Basis of Oil Accumulation in Developing Seeds of Castor Beans.

Castor (Ricinus communis L.) seeds produce abundant ricinoleic acid during seed maturation, which is important for plant development and human demands. Ricinoleic acid, as a unique hydroxy fatty acid (HFA), possesses a distinct bond structure that could be used as a substitute for fossil fuels. Here, we identified all homologous genes related to glycolysis, hydroxy fatty acid biosynthesis, and triacylglycerol (TAG) accumulation in castor seeds. Furthermore, we investigated their expression patterns globally during five seed development stages. We characterized a total of 66 genes involved in the glycolysis pathway, with the majority exhibiting higher expression levels during the early stage of castor bean seed development. This metabolic process provided abundant acetyl-CoA for fatty acid (FA) biosynthesis. Subsequently, we identified 82 genes involved in the processes of de novo FA biosynthesis and TAG assembly, with the majority exhibiting high expression levels during the middle or late stages. In addition, we examined the expression patterns of the transcription factors involved in carbohydrate and oil metabolism. For instance, RcMYB73 and RcERF72 exhibited high expression levels during the early stage, whereas RcWRI1, RcABI3, and RcbZIP67 showed relatively higher expression levels during the middle and late stages, indicating their crucial roles in seed development and oil accumulation. Our study suggests that the high HFA production in castor seeds is attributed to the interaction of multiple genes from sugar transportation to lipid droplet packaging. Therefore, this research comprehensively characterizes all the genes related to glycolysis, fatty acid biosynthesis, and triacylglycerol (TAG) accumulation in the castor and provides novel insight into exploring the genetic mechanisms underlying seed oil accumulation in the endosperm of castor beans.

Separation and Purification of Two Saponins from Paris polyphylla var. yunnanensis by a Macroporous Resin.

An effective method for separating and purifying critical saponins (polyphyllin II and polyphyllin VII) from a Paris polyphylla var. yunnanensis extract was developed in this study which was environmentally friendly and economical. Static adsorption kinetics, thermodynamics, and the dynamic adsorption-desorption of macroporous resins were investigated, and then the conditions of purification and separation were optimized by fitting with an adsorption thermodynamics equation and a kinetic equation. Effective NKA-9 resin from seven macroporous resins was screened out to separate and purify the two saponins. The static adsorption and dynamic adsorption were chemical and physical adsorption dual-processes on the NKA-9 resin. Under the optimum parameters, the contents of polyphyllin II and polyphyllin VII in the product were 17.3-fold and 28.6-fold those in plant extracts, respectively. The total yields of the two saponins were 93.16%. This research thus provides a theoretical foundation for the large-scale industrial production of the natural drugs polyphyllin II and polyphyllin VII.

Identification of the carotenoid cleavage dioxygenase genes and functional analysis reveal DoCCD1 is potentially involved in beta-ionone formation in Dendrobium officinale.

The carotenoids are the most widely distributed secondary metabolites in plants and can be degraded by carotenoid cleavage dioxygenase (CCD) to form apocarotenoids including an important C13 compound beta-ionone. Volatile beta-ionone can confer the violet and woody fragrance to plant essential oils, flowers, fruits, and vegetables, which therefore has been used in various industries. Dendrobium officinale is a traditional medicinal plant. However, there was limited information on the key enzymes involved in the biosynthesis of beta-ionone in D. officinale. In the present study, beta-ionone was detected in stems and leaves of D. officinale and genome-wide identification and expression profiles of CCD genes were subsequently carried out. There were nine DoCCD members in D. officinale. According to the phylogenetic relationship, DoCCD proteins were classified into six subfamilies including CCD1, CCD4, CCD7, CCD8, nine-cis-epoxycarotenoid dioxygenase (NCED) and zaxinone synthase (ZAS). DoCCD genes showed distinctive expression profiles and DoCCD1 gene was abundantly expressed in eight tissues. Induced expression of DoCCD1 gene resulted in discoloration of Escerichia coli strains that can accumulate carotenoids. Analysis of Gas Chromatography/Mass Spectrometer showed that DoCCD1 enzyme can cleave lycopene to produce 6-methyl-5-hepten-2-one and pseudoionone and also catalyze beta-carotene to form beta-ionone. Expression of DoCCD1 gene in Nicotiana benthamiana leaf resulted in production of abundant beta-ionone. Overall, the present study first provides valuable information on the CCD gene family in D. officinale, function of DoCCD1 gene as well as production of beta-ionone through genetic modification.

Antioxidant and tyrosinase inhibitory activities of traditional fermented Rosa from Dali Bai communities, Northwest Yunnan, China.

Traditional fermented Rosa (TFR) is a typical food and medical product among the Dali Bai people, and its popularity is growing. A few studies have looked into TFR's medicinal advantages, linked germplasm resources, traditional processing procedures, and functional food qualities. Our goal was to look into Rosa's traditional processing, examine the dominant strains in TFR, and prove how these strains affected antioxidant and tyrosinase inhibitory activities. We used a snowball selection strategy to pick 371 informants for a semi-structured interview, supplemented with direct observations and sample collection. A microbial strain was isolated and identified from a TFR sample collected in the field. We synthesized TFR in the lab using the traditional way. Both of 2, 2-diphenyl-1 picrylhydrazyl (DPPH) free radical scavenging and tyrosinase inhibitory properties of the fermented solution of Rosa 'Dianhong' have been tested in this study. Altogether 15 species belonging to the genus Rosa, which are utilized in herbal medicine and fermented foods. Rosa 'Dianhong' was the Bai community's principal species with considerable cultural value and consumption. Raw Rosa petals included 15 major flavonoids and phenols, which were identified as TFR's active components. TFR-1 was discovered to be the dominating microbial strain in TFR, increasing total phenolic and flavonoid content in the fermented solution of Rosa 'Dianhong' by 0.45 mg GAE/ml and 0.60 mg RE/ml, respectively, after 30 days. TFR-1 also exhibited promising activity in terms of DPPH free radical scavenging and tyrosinase inhibition. TFR showed potent antioxidant and free-radical scavenger properties and is beneficial in skincare and nutrition, according to the findings. TFR's medicinal and edible properties suggest that it could be used as a cosmetic or nutraceutical product.

Transcriptomic Analyses Shed Light on Critical Genes Associated with Bibenzyl Biosynthesis in Dendrobium officinale.

The Dendrobium plants (members of the Orchidaceae family) are used as traditional Chinese medicinal herbs. Bibenzyl, one of the active compounds in Dendrobium officinale, occurs in low amounts among different tissues. However, market demands require a higher content of thes compounds to meet the threshold for drug production. There is, therefore, an immediate need to dissect the physiological and molecular mechanisms underlying how bibenzyl compounds are biosynthesized in D. officinale tissues. In this study, the accumulation of erianin and gigantol in tissues were studied as representative compounds of bibenzyl. Exogenous application of Methyl-Jasmonate (MeJA) promotes the biosynthesis of bibenzyl compounds; therefore, transcriptomic analyses were conducted between D. officinale-treated root tissues and a control. Our results show that the root tissues contained the highest content of bibenzyl (erianin and gigantol). We identified 1342 differentially expressed genes (DEGs) with 912 up-regulated and 430 down-regulated genes in our transcriptome dataset. Most of the identified DEGs are functionally involved in the JA signaling pathway and the biosynthesis of secondary metabolites. We also identified two candidate cytochrome P450 genes and nine other enzymatic genes functionally involved in bibenzyl biosynthesis. Our study provides insights on the identification of critical genes associated with bibenzyl biosynthesis and accumulation in Dendrobium plants, paving the way for future research on dissecting the physiological and molecular mechanisms of bibenzyl synthesis in plants as well as guide genetic engineering for the improvement of Dendrobium varieties through increasing bibenzyl content for drug production and industrialization.

Research Advances in Multi-Omics on the Traditional Chinese Herb Dendrobium officinale.

Dendrobium officinale Kimura et Migo is an important epiphytic plant, belonging to the Orchidaceae family. There are various bioactive components in D. officinale plants, mainly including polysaccharides, alkaloids, and phenolic compounds. These compounds have been demonstrated to possess multiple functions, such as anti-oxidation, immune regulation, and anti-cancer. Due to serious shortages of wild resources, deterioration of cultivated germplasm and the unstable quality of D. officinale, the study has been focused on the biosynthetic pathway and regulation mechanisms of bioactive compounds. In recent years, with rapid developments in detection technologies and analysis tools, omics research including genomics, transcriptomics, proteomics and metabolomics have all been widely applied in various medicinal plants, including D. officinale. Many important advances have been achieved in D. officinale research, such as chromosome-level reference genome assembly and the identification of key genes involved in the biosynthesis of active components. In this review, we summarize the latest research advances in D. officinale based on multiple omics studies. At the same time, we discuss limitations of the current research. Finally, we put forward prospective topics in need of further study on D. officinale.

Blue genome: chromosome-scale genome reveals the evolutionary and molecular basis of indigo biosynthesis in Strobilanthes cusia.

Natural plant dyes have been developed and used across many traditional societies worldwide. The blue pigment indigo has seen widespread usage across South America, Egypt, Europe, India and China for thousands of years, mainly extracted from indigo-rich plants. The utilization and genetic engineering of indigo in industries and ethnobotanical studies on the effects of cultural selection on plant domestication are limited due to lack of relevant genetic and genomic information of dye plants. Strobilanthes cusia (Acanthaceae) is a typical indigo-rich plant important to diverse ethnic cultures in many regions of Asia. Here we present a chromosome-scale genome for S. cusia with a genome size of approximately 865 Mb. About 79% of the sequences were identified as repetitive sequences and 32 148 protein-coding genes were annotated. Metabolic analysis showed that the main indigoid pigments (indican, indigo and indirubin) were mainly synthesized in the leaves and stems of S. cusia. Transcriptomic analysis revealed that the expression level of genes encoding metabolic enzymes such as monooxygenase, uridine diphosphate-glycosyltransferase and ß-glucosidase were significantly changed in leaves and stems compared with root tissues, implying their participation in indigo biosynthesis. We found that several gene families involved in indigo biosynthesis had undergone an expansion in number, with functional differentiation likely facilitating indigo biosynthesis in S. cusia. This study provides insight into the physiological and molecular bases of indigo biosynthesis, as well as providing genomic data that provide the basis for further study of S. cusia cultivation by Asia's traditional textile producers.

Genomic Characterization and Expression Analysis of Basic Helix-Loop-Helix (bHLH) Family Genes in Traditional Chinese Herb Dendrobium officinale.

Dendrobium officinale Kimura et Migo is of great importance as a traditional Chinese herb due to its abundant metabolites. The family of basic helix-loop-helix (bHLH) transcription factors widely exists in plants and plays an essential role in plant growth and development, secondary metabolism as well as responses to environmental changes. However, there is limited information on bHLH genes in D. officinale. In the present study, a total of 98 putative DobHLH genes were identified at the genomic level, which could be classified into 18 clades. Gene structures and conserved motifs in DobHLH genes showed high conservation during their evolution. The conserved amino acids and DNA bindings of DobHLH proteins were predicted, both of which are pivotal for their function. Furthermore, gene expression from eight tissues showed that some DobHLH genes were ubiquitously expressed while other DobHLH genes were expressed in the specific tissues. Expressional changes of DobHLH genes under MeJA and ABA treatments were detected by qRT-PCR. The protein-protein interactions between DobHLHs were predicted and several interactions were confirmed by yeast two hybrid. Therefore, our results here contribute to the understanding of bHLH genes in D. officinale and lay a foundation for the further functional study of its biological processes.

Microbial transformation of diosgenin to diosgenone by Wickerhamomyces anomalus JQ-1 obtained from Naxi traditional Jiu Qu.

Diosgenone [(20S,22R,25R)-spirost-4-en-3-one, C27H40O3] has been considered as a potential therapeutic alternative remedy for malaria. An efficient and economical approach of microbial transformation with diosgenin to diosgenone by the yeast strain Wickerhamomyces anomalus JQ-1 from Naxi traditional Jiu Qu was developed in this study. Chromatographic analysis confirmed that 85% of 0.1 mM diosgenin was transformed to diosgenone within 72 h. This research demonstrates that diosgenin could be converted to diosgenone through two-step pathway including 3ß-hydroxyl oxidation and double bond isomerization rather than through one-step pathway, which prompted a further inference that the oxidation activity in W. anomalus JQ-1 has the same function with the Oppenauer-type oxidation which can convert diosgenin into diosgenone. Gaining specific functional strains from traditional fermented products will be a potential direction and ethnobotanical researches could provide helps with discovery and utilization of microbial resources.

Genomic Characterization and Expressional Profiles of Autophagy-Related Genes (ATGs) in Oilseed Crop Castor Bean (Ricinus communis L.).

Cellular autophagy is a widely-occurring conserved process for turning over damaged organelles or recycling cytoplasmic contents in cells. Although autophagy-related genes (ATGs) have been broadly identified from many plants, little is known about the potential function of autophagy in mediating plant growth and development, particularly in recycling cytoplasmic contents during seed development and germination. Castor bean (Ricinus communis) is one of the most important inedible oilseed crops. Its mature seed has a persistent and large endosperm with a hard and lignified seed coat, and is considered a model system for studying seed biology. Here, a total of 34 RcATG genes were identified in the castor bean genome and their sequence structures were characterized. The expressional profiles of these RcATGs were examined using RNA-seq and real-time PCR in a variety of tissues. In particular, we found that most RcATGs were significantly up-regulated in the later stage of seed coat development, tightly associated with the lignification of cell wall tissues. During seed germination, the expression patterns of most RcATGs were associated with the decomposition of storage oils. Furthermore, we observed by electron microscopy that the lipid droplets were directly swallowed by the vacuoles, suggesting that autophagy directly participates in mediating the decomposition of lipid droplets via the microlipophagy pathway in germinating castor bean seeds. This study provides novel insights into understanding the potential function of autophagy in mediating seed development and germination.

Association between referral source and duration of untreated psychosis in pathways to care among first episode psychosis patients in Northern Malawi.

AIMS: To examine the association between referral source and duration of untreated psychosis (DUP) and explore determinants of referral source; when adjusting for pathways to care, positive and negative symptoms, diagnosis and socio-demographic characteristics. METHODS: A total of 140 subjects with first episode psychosis (FEP) were enrolled from a pilot early intervention service for psychosis in Northern Malawi between June 2009 and September 2012. Logistic regression analyses were used to quantify the associations between variables of interest. RESULTS: Age ranged between 18 and 65 at assessment, with median, 33. Median DUP was 12.5 months. First contact did not independently determine DUP. Long DUP (>6 months) was associated with referral from community based volunteer (CBV) or traditional healer (TH), a unit increase in severity of negative symptoms and having schizophrenia, which was also associated with referral from CBV or TH. Additionally, being unemployed was associated with referral from CBV or TH. However, a unit increase in the number of times religious advice (RA) was sought, GP was contacted and severity of positive symptoms was associated with referral by GP. CONCLUSIONS: Mental health awareness is justified for this population and collaboration with THs in identifying and treating patients with psychosis may help reduce treatment delays. Access to mental health services ought to improve, particularly for the unemployed group. Future studies should consider adjusting for referral source when ascertaining first contact source as a predictor of DUP.

Indigenous knowledge of dye-yielding plants among Bai communities in Dali, Northwest Yunnan, China.

BACKGROUND: Bai people in the Dali Prefecture of Northwest Yunnan, China, have a long history of using plant extracts to dye their traditional costumes and maintain this culture for posterity. However, the development of modern technology, while vastly improving the dyeing efficiency, is also replacing indigenous knowledge which threatens the indigenous practice, causing the latter disappearing gradually. This study sought to examine the indigenous knowledge of plants used for textile dyeing in Bai communities, so as to provide a foundation for their sustainable development. METHODS: We conducted a semi-structured interview among 344 informants (above age 36) selected through a snowball sampling method. Free lists and participant observation were used as supplementary methods for the interviews. Three quantitative indicators (informant consensus factor [ICF], use frequency, and cultural importance index [CI]) were used to evaluate the indigenous knowledge of the dye-yielding plants. RESULTS: Twenty-three species belonging to 19 plant taxonomic families were used for dye by Bai communities. We summarized them into four life forms, eight used parts, five colors, three processing methods, and four dyeing methods. Among them, Strobilanthes cusia (Nees) O. Kuntze was the most traditional dyeing plant and has an important cultural value. Location, age, and gender were found to have a significant effect on indigenous knowledge, and the dyeing knowledge was dynamic and influenced by social factors. CONCLUSIONS: Diverse plant resources and rich indigenous knowledge of textile dyeing persist at settlements of Bai communities in Dali Prefecture. However, high labor costs and thinning market of traditional products that use plant dye cause repulsion toward traditional practice. To that, a good income in other profession attracts indigenous people to shift from their tradition of making plant-based dye and associated cultural systems at risk of extinction. More research for market development for products that use plant-based dye is necessary for the conservation of this valuable knowledge and biodiversity protection in Bai communities.

Transcriptome analysis reveals crucial genes involved in the biosynthesis of nervonic acid in woody Malania oleifera oilseeds.

BACKGROUND: Malania oleifera Chun et Lee (Olacaceae), an evergreen broad-leaved woody tree native to southwest China, is an important oilseed tree. Its seed oil has a high level of nervonic acid (cis-tetracos-15-enoic acid, over 60%), which is essential for human health. M. oleifera seed oil is a promising source of nervonic acid, but little is known about the physiological and molecular mechanisms underlying its biosynthesis. RESULTS: In this study, we recorded oil accumulation at four stages of seed development. Using a high-throughput RNA-sequencing technique, we obtained 55,843 unigenes, of which 29,176 unigenes were functionally annotated. By comparison, 22,833 unigenes had a two-fold or greater expression at the fast oil accumulation stage than at the initial stage. Of these, 198 unigenes were identified as being functionally involved in diverse lipid metabolism processes (including de novo fatty acid synthesis, carbon chain elongation and modification, and triacylglycerol assembly). Key genes (encoding KCS, KCR, HCD and ECR), putatively responsible for nervonic acid biosynthesis, were isolated and their expression profiles during seed development were confirmed by quantitative real-time PCR analysis. Also, we isolated regulatory factors (such as WRI1, ABI3 and FUS3) that are putatively involved in the regulation of oil biosynthesis and seed development. CONCLUSION: Our results provide novel data on the physiological and molecular mechanisms of nervonic acid biosynthesis and oil accumulation in M. oleifera seeds, and will also serve as a starting point for biotechnological genetic engineering for the production of nervonic acid resources.

Development of an efficient chromatin immunoprecipitation method to investigate protein-DNA interaction in oleaginous castor bean seeds.

Chromatin immunoprecipitation (ChIP) is usually a reliable technique to find the binding sites of a transcription factor. In the current study, we developed a suitable ChIP method using developing castor bean seeds. A castor bean seed with large and persistent endosperm contains high amounts of storage lipids (ca. 50-60%) and is often considered as a model material to studying seed biology. In oleaginous seeds, due to the rich oils which could seriously affect immunoprecipitation and DNA isolation, it is often difficult to carry out a successful ChIP experiment. Thus, the development of an efficient ChIP method for oleaginous seeds is required. In this study, we modified different steps, including tissue preparation for cross-linking, chromatin washing, sonication and immunoprecipitation of other existing methods. As exemplified by the targeted gene identification of a master regulator WRI1, which regulates fatty acid biosynthesis, we found that the improved ChIP method worked well. We analyzed percentage input and fold changes of the ChIPed DNA. We also made successful ChIP-seq libraries using this method. This method provides a technical support not only for use on castor bean seeds; it might be used equally to analyze protein-DNA interaction in vivo in other oleaginous seeds.

High light exposure on seed coat increases lipid accumulation in seeds of castor bean (Ricinus communis L.), a nongreen oilseed crop.

Little was known on how sunlight affects the seed metabolism in nongreen seeds. Castor bean (Ricinus communis L.) is a typical nongreen oilseed crop and its seed oil is an important feedstock in industry. In this study, photosynthetic activity of seed coat tissues of castor bean in natural conditions was evaluated in comparison to shaded conditions. Our results indicate that exposure to high light enhances photosynthetic activity in seed coats and consequently increases oil accumulation. Consistent results were also reached using cultured seeds. High-throughput RNA-Seq analyses further revealed that genes involved in photosynthesis and carbon conversion in both the Calvin-Benson cycle and malate transport were differentially expressed between seeds cultured under light and dark conditions, implying several venues potentially contributing to light-enhanced lipid accumulation such as increased reducing power and CO2 refixation which underlie the overall lipid biosynthesis. This study demonstrated the effects of light exposure on oil accumulation in nongreen oilseeds and greatly expands our understanding of the physiological roles that light may play during seed development in nongreen oilseeds. Essentially, our studies suggest that potential exists to enhance castor oil yield through increasing exposure of the inflorescences to sunlight either by genetically changing the plant architecture (smart canopy) or its growing environment.

An ethnobotanical study of the less known wild edible figs (genus Ficus) native to Xishuangbanna, Southwest China.

BACKGROUND: The genus Ficus, collectively known as figs, is a key component of tropical forests and is well known for its ethnobotanical importance. In recent decades an increasing number of studies have shown the indigenous knowledge about wild edible Ficus species and their culinary or medicinal value. However, rather little is known about the role of these species in rural livelihoods, because of both species and cultural diversity. METHODS: In this study we 1) collected the species and ethnic names of wild edible Ficus exploited by four cultural groups in Xishuangbanna, Southwest China, and 2) recorded the collection activities and modes of consumption through semi-structured interviews, 3) investigated the resource management by a statistical survey of their field distribution and cultivation, and 4) compared and estimated the usage intensities by the grading method. RESULTS: The young leaves, leaf buds and young or ripe syconia of 13 Ficus species or varieties are traditionally consumed. All the species had fixed and usually food-related ethnic names. All four cultural groups are experienced in the collection and use of edible Ficus species as vegetables, fruits or beverages, with the surplus sold for cash income. Different cultural groups use the Ficus species at different intensities because of differences in availability, forest dependency and cultural factors. Both the mountain and basin villagers make an effort to realize sustainable collection and meet their own and market needs by resource management in situ or cultivation. CONCLUSIONS: In comparison with reports from other parts of the world, ethnic groups in Xishuangbanna exploited more edible Ficus species for young leaves or leaf buds. Most of the edible species undergo a gradient of management intensities following a gradient of manipulation from simple field gathering to ex situ cultivation. This study contributes to our understanding of the origins and diffusion of the knowledge of perception, application and managing a group of particular plant species, and how the local culture, economic and geographical factors influence the process.

Expression of genes controlling unsaturated fatty acids biosynthesis and oil deposition in developing seeds of Sacha inchi (Plukenetia volubilis L.).

Sacha inchi (Plukenetia volubilis L., Euphorbiaceae) seed oil is rich in α-linolenic acid, a kind of n-3 fatty acids with many health benefits. To discover the mechanism underlying α-linolenic acid accumulation in sacha inchi seeds, preliminary research on sacha inchi seed development was carried out from one week after fertilization until maturity, focusing on phenology, oil content, and lipid profiles. The results suggested that the development of sacha inchi seeds from pollination to mature seed could be divided into three periods. In addition, investigations on the effect of temperature on sacha inchi seeds showed that total oil content decreased in the cool season, while unsaturated fatty acid and linolenic acid concentrations increased. In parallel, expression profiles of 17 unsaturated fatty acid related genes were characterized during seed development and the relationships between gene expression and lipid/unsaturated fatty acid accumulation were discussed.