Functional Characterization of the Effects of CsDGAT1 and CsDGAT2 on Fatty Acid Composition in Camelina sativa.

Triacylglycerols (TAGs) are the storage oils of plant seeds, and these lipids provide energy for seed germination and valuable oils for human consumption. Three diacylglycerol acyltransferases (DGAT1, DGAT2, and DGAT3) and phospholipid:diacylglycerol acyltransferases participate in the biosynthesis of TAGs. DGAT1 and DGAT2 participate in the biosynthesis of TAGs through the endoplasmic reticulum (ER) pathway. In this study, we functionally characterized CsDGAT1 and CsDGAT2 from camelina (Camelina sativa). Green fluorescent protein-fused CsDGAT1 and CsDGAT2 localized to the ER when transiently expressed in Nicotiana benthamiana leaves. To generate Csdgat1 and Csdgat2 mutants using the CRISPR/Cas9 system, camelina was transformed with a binary vector carrying Cas9 and the respective guide RNAs targeting CsDGAT1s and CsDGAT2s via the Agrobacterium-mediated floral dip method. The EDD1 lines had missense and nonsense mutations in the CsDGAT1 homoeologs, suggesting that they retained some CsDGAT1 function, and their seeds showed decreased eicosaenoic acid (C20:1) contents and increased C18:3 contents compared to the wild type (WT). The EDD2 lines had a complete knockout of all CsDGAT2 homoeologs and a slightly decreased C18:3 content compared to the WT. In conclusion, CsDGAT1 and CsDGAT2 have a small influence on the seed oil content and have an acyl preference for C20:1 and C18:3, respectively. This finding can be applied to develop oilseed plants containing high omega-3 fatty acids or high oleic acid.

Plant-made pharmaceuticals: exploring studies for the production of recombinant protein in plants and assessing challenges ahead.

The production of pharmaceutical compounds in plants is attracting increasing attention, as plant-based systems can be less expensive, safer, and more scalable than mammalian, yeast, bacterial, and insect cell expression systems. Here, we review the history and current status of plant-made pharmaceuticals. Producing pharmaceuticals in plants requires pairing the appropriate plant species with suitable transformation technology. Pharmaceuticals have been produced in tobacco, cereals, legumes, fruits, and vegetables via nuclear transformation, chloroplast transformation, transient expression, and transformation of suspension cell cultures. Despite this wide range of species and methods used, most such efforts have involved the nuclear transformation of tobacco. Tobacco readily generates large amounts of biomass, easily accepts foreign genes, and is amenable to stable gene expression via nuclear transformation. Although vaccines, antibodies, and therapeutic proteins have been produced in plants, such pharmaceuticals are not readily utilized by humans due to differences in glycosylation, and few such compounds have been approved due to a lack of clinical data. In addition, achieving an adequate immune response using plant-made pharmaceuticals can be difficult due to low rates of production compared to other expression systems. Various technologies have recently been developed to help overcome these limitations; however, plant systems are expected to increasingly become widely used expression systems for recombinant protein production.

Increasing Monounsaturated Fatty Acid Contents in Hexaploid Camelina sativa Seed Oil by FAD2 Gene Knockout Using CRISPR-Cas9.

Seed oils are used as edible oils and increasingly also for industrial applications. Although high-oleic seed oil is preferred for industrial use, most seed oil is high in polyunsaturated fatty acids (PUFAs) and low in monounsaturated fatty acids (MUFAs) such as oleic acid. Oil from Camelina, an emerging oilseed crop with a high seed oil content and resistance to environmental stress, contains 60% PUFAs and 30% MUFAs. Hexaploid Camelina carries three homoeologs of FAD2, encoding fatty acid desaturase 2 (FAD2), which is responsible for the synthesis of linoleic acid from oleic acid. In this study, to increase the MUFA contents of Camelina seed oil, we generated CsFAD2 knockout plants via CRISPR-Cas9-mediated gene editing using the pRedU6fad2EcCas9 vector containing DsRed as a selection marker, the U6 promoter to drive a single guide RNA (sgRNA) covering the common region of the three CsFAD2 homoeologs, and an egg-cell-specific promoter to drive Cas9 expression. We analyzed CsFAD2 homoeolog-specific sequences by PCR using genomic DNA from transformed Camelina leaves. Knockout of all three pairs of FAD2 homoeologs led to a stunted bushy phenotype, but greatly enhanced MUFA levels (by 80%) in seeds. However, transformants with two pairs of CsFAD2 homoeologs knocked out but the other pair wild-type heterozygous showed normal growth and a seed MUFAs production increased up to 60%. These results provide a basis for the metabolic engineering of genes that affect growth in polyploid crops through genome editing.

Assessment of Metabolic Profiles in Florets of Carthamus Species Using Ultra-Performance Liquid Chromatography-Mass Spectrometry.

The genus Carthamus is a diverse group of plants belonging to the family Compositae. Florets of Carthamus species exhibit various colors, including white, yellow, orange, and red, which are related to their metabolite compositions. We aimed to investigate the metabolites accumulated in florets of three wild (C. lanatus, C. palaestinus, and C. turkestanicus) and one cultivated (C. tinctorius) species of safflower at three developmental stages. Metabolites were extracted from freeze-dried florets using 70% methanol; qualification and quantification were carried out using liquid chromatography quadrupole time-of-flight mass spectrometry in positive and negative ion modes followed by extraction of the peaks. Fifty-six metabolites, including phenylpropanoids, chalcones, isoflavonoids, flavanones, flavonols, flavones, and other primary metabolites, were identified for the first time in safflower wild species. The orange florets contained high abundances of safflomin A, anhydrosafflor yellow B, and baimaside, whereas white/cream and light-yellow pigmented florets had high abundances of 1,5-dicaffeoylquinic acid, luteolin 7-O-glucuronide, and apigenin 7-O-ß-D-glucuronide. The principal component analysis clearly distinguished the samples based on their pigment types, indicating that color is a dominant factor dictating the identity and amount of the metabolites. Pearson correlation data based on levels of metabolites showed that orange and yellow florets were significantly correlated to each other. White and cream pigmented species were also highly correlated. Comparison between three developmental stages of safflower wild species based on their metabolite profile showed inconsistent. The findings of this study broaden the current knowledge of safflower metabolism. The wide diversity of metabolites in safflower materials also helps in efforts to improve crop quality and agronomic traits.

Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants.

Meloidogyne incognita is a devastating plant parasitic nematode that causes root knot disease in a wide range of plants. In the present study, we investigated host-induced RNA interference (RNAi) gene silencing of chitin biosynthesis pathway genes (chitin synthase, glucose-6-phosphate isomerase, and trehalase) in transgenic tobacco plants. To develop an RNAi vector, ubiquitin (UBQ1) promoter was directly cloned, and to generate an RNAi construct, expression of three genes was suppressed using the GATEWAY system. Further, transgenic Nicotiana benthamiana lines expressing dsRNA for chitin synthase (CS), glucose-6-phosphate isomerase (GPI), and trehalase 1 (TH1) were generated. Quantitative PCR analysis confirmed endogenous mRNA expression of root knot nematode (RKN) and revealed that all three genes were more highly expressed in the female stage than in eggs and in the parasitic stage. In vivo, transformed roots were challenged with M. incognita. The number of eggs and root knots were significantly decreased by 60-90% in RNAi transgenic lines. As evident, root galls obtained from transgenic RNAi lines exhibited 0.01- to 0.70-fold downregulation of transcript levels of targeted genes compared with galls isolated from control plants. Furthermore, phenotypic characteristics such as female size and width were also marginally altered, while effect of egg mass per egg number in RNAi transgenic lines was reduced. These results indicate the relevance and significance of targeting chitin biosynthesis genes during the nematode lifespan. Overall, our results suggest that further developments in RNAi efficiency in commercially valued crops can be applied to employ RNAi against other plant parasitic nematodes.

A Metabolic Perspective and Opportunities in Pharmacologically Important Safflower.

Safflower (Carthamus tinctorius L.) has long been grown as a crop due to its commercial utility as oil, animal feed, and pharmacologically significant secondary metabolites. The integration of omics approaches, including genomics, transcriptomics, metabolomics, and proteomics datasets, has provided more comprehensive knowledge of the chemical composition of crop plants for multiple applications. Knowledge of a metabolome of plant is crucial to optimize the evolution of crop traits, improve crop yields and quality, and ensure nutritional and health factors that provide the opportunity to produce functional food or feedstuffs. Safflower contains numerous chemical components that possess many pharmacological activities including central nervous, cardiac, vascular, anticoagulant, reproductive, gastrointestinal, antioxidant, hypolipidemic, and metabolic activities, providing many other human health benefits. In addition to classical metabolite studies, this review focuses on several metabolite-based working techniques and updates to provide a summary of the current medical applications of safflower.

Perception of severe osteoporosis amongst medical doctors in South Korea: Awareness, impact, and treatment.

OBJECTIVES: Little is currently known about the issues surrounding management and treatment of severe osteoporosis in South Korea. Our objective was to assess doctors' views on the perception, diagnosis, and treatment of severe osteoporosis. METHODS: Face-to-face interviews were conducted (16 February-13 March 2015) with 100 doctors (specialists in orthopedic surgery, endocrinology, neurosurgery, family medicine, or rheumatology) who treated ≥5 severe osteoporosis (T-score ≤ -2.5, plus fracture) patients per month. Respondent demographic characteristics, their perception of severe osteoporosis, its impact and treatment, and their views on current practice and unmet needs were assessed. RESULTS: Of 416 doctors approached, 100 completed the survey (24% response rate). Most doctors (90%) specialized in orthopedic surgery, endocrinology, or neurosurgery. When diagnosing severe osteoporosis, most doctors (79%) considered both bone mineral density and fracture. Almost all doctors (≥91%) ranked disease impact and seriousness highly, but much fewer (≤25%) doctors thought society agreed. Most doctors (89%) had concerns with current treatments, switching treatments because of the efficacy and safety of bisphosphonates (>89%), the efficacy of selective estrogen receptor modulators (>71%), and the high cost of parathyroid hormone (>73%). Parathyroid hormone was ranked highest for efficacy and was preferentially prescribed to severe osteoporosis patients (mean 32.2% of prescriptions) compared with osteoporosis patients overall (3.7%). "Limitations with reimbursement" was the most commonly cited (76%) unmet need. CONCLUSIONS: There are concerns with the safety, efficacy, and affordability of current treatments for severe osteoporosis in South Korea, as well as a perceived lack of disease awareness amongst patients and doctors.