Coixendide efficacy in combination with temozolomide in glioblastoma and transcriptome analysis of the mechanism.

The purpose of this study was to explore the role of coixendide (Coix) combine with temozolomide (TMZ) in the treatment of Glioblastoma (GBM) and explore its possible mechanism. CCK-8 was used to determine the inhibitory rate of Coix group, TMZ group and drug combination group on GBM cells, and the combination index (CI) was calculated to determine whether they had synergistic effect. Then RNA was extracted from each group, transcriptome sequencing was performed, and differentially expressed genes (DEGs) were identified. The possible mechanism was analyzed by GO enrichment analysis and KEGG enrichment analysis. The CI of Coix and TMZ indicating a synergistic effect when TMZ concentration is 0.1 mg/ml and Coix concentration is 2 mg/ml. Transcriptome sequencing analysis showed that interferon (IFN) related genes were down-regulated by Coix and up-regulated by TMZ and combined drugs, however, the up-regulation induced by combined drugs was less than that of TMZ. Besides IFN related genes, cholesterol metabolism pathway were also been regulated. Coix and TMZ have synergistic effects in the treatment of GBM at certain doses. RNA-Seq results suggested that the abnormal on genetic materials caused by DNA damage induced by TMZ treatment can be sensed by IFN related genes and activates antiviral IFN signaling, causing the activation of repairing mechanism and drug resistance. Coix inhibits IFN related genes, thereby inhibits drug resistance of TMZ. In addition, the activation of ferroptosis and the regulation of DEGs in cholesterol metabolism pathway were also contributed to the synergistic effects of Coix and TMZ.

FUSCA3-induced AINTEGUMENTA-like 6 manages seed dormancy and lipid metabolism.

FUSCA 3 (FUS3), a seed master regulator, plays critical role in seed dormancy and oil accumulation. However, its downstream regulation mechanisms remain poorly understood. Here, we explored the roles of AINTEGUMENTA-like 6 (AIL6), a seed transcription factor, in these processes. The activation of AIL6 by FUS3 was demonstrated by dual-LUC assay. Seeds of ail6 mutants showed alterations in fatty acid compositions, and both AtAIL6 (AIL6 from Arabidopsis thaliana) and BnaAIL6 (AIL6 from Brassica napus) rescued the phenotype. Over-expression (OE) of AIL6s reversed changes in seed fatty acid composition. Notably, OE lines showed low seed germination rates down to 12% compared to 100% of wild-type Col-0. Transcriptome analysis of the mutant and an OE line indicated widespread expression changes of genes involved in lipid metabolism and phytohormone pathways. In OE mature seeds, GA4 content decreased more than 15-fold, while abscisic acid and indole-3-acetic acid (IAA) contents clearly increased. Exogenous GA3 treatments did not effectively rescue the low germination rate. Nicking seed coats increased germination rates from 25% to nearly 80% while the wild-type rdr6-11 is 100% and 98% respectively, and elongation of storage time also improved seed germination. Furthermore, dormancy imposed by AIL6 was fully released in the della quintuple mutant. Together, our results indicate AIL6 acts as a manager downstream of FUS3 in seed dormancy and lipid metabolism.

Case study of a landslide continuous probability rainfall threshold analysis based on the prediction interval principle.

Bazhong City is located on stratum dominated by red-bed conditions. This type of weak geological condition with sand and mudstone interbedding is very easily affected by climatic conditions to produce rainfall-type landslides. On the basis of landslide data statistics collected in Bazhong City from 2011 to 2019, this paper uses ERA5-Land rainfall data to statistically analyze the correlation between rainfall and landslide events in Bazhong City. The landslide events in Bazhong City are greatly affected by rainfall events lasting for 10 days. Considering the influence of rainfall seepage and other processes, an effective cumulative rainfall-duration threshold curve for Bazhong City is obtained via median nonlinear fitting. Then, on the basis of the prediction interval, the rainfall threshold formula under different landslide occurrence probabilities is obtained and the critical threshold curve with a non-exceeding probability of 1% in Bazhong City is calculated and verified. Subsequently, a continuous probability distribution fitting function of landslide occurrence is established and a continuous probability distribution surface with a good fitting effect in Bazhong City is obtained. This allows a definite probability of whether future rainfall events will induce landslides to be obtained, providing an important basis for engineering disaster prevention and mitigation.

Empirical antibiotic treatment strategies for community-acquired pneumonia: a network meta-analysis.

OBJECTIVES: This network meta-analysis aimed to compare the efficacy and safety of fluoroquinolone (FQ) monotherapy, ß-lactam (BL) monotherapy and ß-lactam/macrolide (BL-M) combination therapy in hospitalized patients with community-acquired pneumonia (CAP). METHODS: Pubmed, Embase and the Cochrane Library were searched for randomized controlled trials (RCTs) comparing FQ monotherapy, BL monotherapy and BL-M combination therapy up to July 2021. The outcomes of interest included all-cause mortality, clinical success, microbiological success and drug-related adverse events. The summary relative risks (RRs) were estimated using pairwise and Bayesian network meta-analysis. RESULTS: A total of 12 RCTs involving 5009 patients were included. In pairwise meta-analysis, no significant differences were found among FQ monotherapy, BL monotherapy and BL-M dual therapy for all-cause mortality, clinical success or microbiological success. FQ monotherapy was associated with fewer adverse events compared with BL-M therapy (RR 0.80, 95% confidence interval [CI] 0.66-0.98). The network meta-analysis showed that there was no significant difference observed among FQ monotherapy, BL monotherapy and BL-M dual therapy regarding all the outcomes. CONCLUSION: FQ monotherapy, BL monotherapy and BL-M combination therapy demonstrated similar efficacy and safety for hospitalized patients with CAP in this network meta-analysis. Due to the limitations of quality and quantity of the included studies, it is difficult to make a definitive recommendation before more large-scale and high-quality RCTs are conducted.

Multiple caleosins have overlapping functions in oil accumulation and embryo development.

Caleosins are lipid droplet- and endoplasmic reticulum-associated proteins. To investigate their functions in oil accumulation, expression levels of caleosins in developing seeds of Arabidopsis thaliana were examined and four seed-expressed caleosins (CLO1, CLO2, CLO4, and CLO6) were identified. The four single mutants showed similar minor changes of fatty acid composition in seeds. Two double mutants (clo1 clo2 and clo1×clo2) demonstrated distinct changes of fatty acid composition, a 16-23% decrease of oil content, and a 10-13% decrease of seed weight. Moreover, a 40% decrease of oil content, further fatty acid changes, and misshapen membranes of smaller lipid droplets were found in seeds of quadruple CLO RNAi lines. Notably, ~40% of quadruple CLO RNAi T1 seeds failed to germinate, and deformed embryos and seedlings were also observed. Complementation experiments showed that CLO1 rescued the phenotype of clo1 clo2. Overexpression of CLO1 in seedlings and BY2 cells increased triacylglycerol content up to 73.6%. Transcriptome analysis of clo1 clo2 developing seeds showed that expression levels of some genes related to lipid, embryo development, calcium signaling, and stress responses were affected. Together, these results suggest that the major seed-expressed caleosins have overlapping functions in oil accumulation and show pleiotropic effects on embryo development.

Establishment and metabonomics analysis of nonalcoholic fatty liver disease model in golden hamster.

The aim is to establish a model of nonalcoholic fatty liver disease (NAFLD) caused by feeding with high-fat, high-fructose, and high-cholesterol diet (HFFCD) in golden hamsters, and to investigate the characteristics of the NAFLD model and metabolite changes of liver tissue. Golden hamsters were fed HFFCD or control diets for six weeks. Body weight, abdominal fat index, and liver index was assessed, serum parameters, hepatic histology, and liver metabolites were examined. The results showed that body weight, abdominal fat, and liver index of hamsters were significantly increased in the model group, the level of serum total cholesterol (TC), triglyceride (TG), and low density lipoprotein-cholesterol (LDL-C) were significantly increased in model group as well, and high density lipoprotein-cholesterol (HDL-C) was significantly decreased. In addition, lipid deposition in liver tissue formed fat vacuoles of different sizes. Metabonomics analysis of the liver showed that the metabolic pathways of sphingolipid, glycerophospholipids, and arginine biosynthesis were disordered in the NAFLD model. The modeling method is simple, short time, and uniform. It can simulate the early fatty liver caused by common dietary factors, and provides an ideal model for the study of the initial pathogenesis and therapeutic drugs for NAFLD.

Dataset for liver metabolomic profile of highland barley Monascus purpureus went extract-treated golden hamsters with nonalcoholic fatty liver disease.

Nonalcoholic Fatty Liver Disease (NAFLD) is a serious problem endangering human health in the world. The pathogenesis of this disease is often accompanied by lipid metabolism disorder and can cause liver lipid accumulation. Highland barley Monascus purpureus Went extract (HBMPWE) can inhibit the liver lipid accumulation caused by a high-fat, high-fructose, high-cholesterol diet. However, it is not clear what changes have taken place in the process of liver lipid metabolism after HBMPWE administration. To fill this knowledge gap and to support the findings published in the companion research article entitled "Highland Barley Monascus purpureus Went Extract Ameliorates High-Fat, High-Fructose, High-Cholesterol Diet Induced Nonalcoholic Fatty Liver Disease by Regulating Lipid Metabolism in Golden Hamsters" [1], we provided important information related to the liver differential metabolites and identified twenty-one differential metabolites of liver metabolism. In the model group, the levels of lactate, linoleic acid, and malic acid increased significantly. After HBMPWE treatment, the expressions of these metabolites reduced significantly. Therefore, these liver differential metabolites could be used as biological signatures reflecting the severity of NAFLD and HBMPWE treatment outcomes.

Highland barley Monascus purpureus Went extract ameliorates high-fat, high-fructose, high-cholesterol diet induced nonalcoholic fatty liver disease by regulating lipid metabolism in golden hamsters.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatocyte lipid accumulation is the main feature in the early stage of nonalcoholic fatty liver disease (NAFLD). Highland barley Monascus purpureus Went (HBMPW), a fermentation product of Hordeum vulgare Linn. var. nudum Hook. f. has traditionally been used as fermented foods in Tibet with the effect of reducing blood lipid in folk medicine. AIM OF THE STUDY: This study investigated the protective effects and molecular mechanism of highland barley Monascus purpureus Went extract (HBMPWE) on NAFLD in syrian golden hamster fed with high-fat, high-fructose, high-cholesterol diet (HFFCD). MATERIALS AND METHODS: HFFCD-induced NAFLD golden hamster model was established and treated with HBMPWE. Liver index, biochemical index, and hematoxylin and eosin (HE) staining were observed. Liver metabolomics and western blot analysis were employed. RESULTS: Our study found that HBMPWE ameliorated HFFCD induced dyslipidemia, weight gain and elevated the liver index. In addition, HBMPWE treatment significantly attenuated lipid accumulation in the liver and modulated lipid metabolism (sphingolipid, glycerophospholipid). Our data demonstrated that HBMPWE not only regulated the expression of proteins related to fatty acid synthesis and decomposition (SREBP-1/ACC/FAS/AceS1, PPARα/ACSL/CPT1/ACOX1), but also regulated the expression of proteins related to cholesterol synthesis and clearance (HMGCR, LDLR, CYP7A1). CONCLUSIONS: HBMPWE improved NAFLD through multiple pathways and multiple targets in body metabolism and could be used as a functional food to treat NAFLD and other lipid metabolic disorders.

Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses.

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N1-107 ) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant-pathogen co-evolution.

ABA-INSENSITIVE 3 with or without FUSCA3 highly up-regulates lipid droplet proteins and activates oil accumulation.

ABA-INSENSITIVE 3 (ABI3) has long been known for activation of storage protein accumulation. A role of ABI3 on oil accumulation was previously suggested based on a decrease of oil content in seeds of abi3 mutant. However, this conclusion could not exclude possibilities of indirect or pleiotropic effects, such as through mutual regulatory interactions with FUSCA3 (FUS3), an activator of oil accumulation. To identify that ABI3 functions independent of the effects of related seed transcription factors, we expressed ABI3 under the control of an inducible promoter in tobacco BY2 cells and Arabidopsis rosette leaves. Inducible expression of ABI3 activated oil accumulation in these non-seed cells, demonstrating a general role of ABI3 in regulation of oil biosynthesis. Further expressing ABI3 in rosette leaves of fus3 knockout mutant still caused up to 3-fold greater triacylglycerol accumulation, indicating ABI3 can activate lipid accumulation independently of FUS3. Transcriptome analysis revealed that LIPID DROPLET PROTEIN (LDP) genes, including OLEOSINs and CALEOSINs, were up-regulated up to 1000-fold by ABI3 in the absence of FUS3, while the expression of WRINKLED1 was doubled. Taken together, our results provide genetic evidence that ABI3 activates oil accumulation with or without FUS3, most likely through up-regulating LDPs and WRINKLED1.

A 3-ketoacyl-CoA synthase 11 (KCS11) homolog from Malania oleifera synthesizes nervonic acid in plants rich in 11Z-eicosenoic acid.

Nervonic acid (24:1) is a major component in nerve and brain tissues and it has important applications in food and pharmaceutical industries. Malania oleifera seeds contain about 40% nervonic acid. However, the mechanism of nervonic acid biosynthesis and accumulation in seeds of this endangered tree species remains unknown. In this study, developmental changes in fatty acid composition within embryos and their pericarps were investigated. Nervonic acid proportions steadily increased in developing embryos but 24:1 was not detected in pericarps at any stage. Two 3-ketoacyl-CoA synthase (KCS) homologs have been isolated from M. oleifera developing seeds by homologous cloning methods. Both KCSs are expressed in developing embryos but not detected in pericarps. Based on a phylogenetic analysis, these two KCSs were named as MoKCS4 and MoKCS11. Seed-specific expression of the MoKCS11 in Arabidopsis thaliana led to about 5% nervonic acid accumulation, while expression of the MoKCS4 did not show an obvious change in fatty acid composition. It is noteworthy that the transformation of the same MoKCS11 construct into two Brassica napus cultivars with high erucic acid did not produce the expected accumulation of nervonic acid, although expression of MoKCS11 was detected in the developing embryos of transgenic lines. In contrast, overexpression of MoKCS11 results in similar level of nervonic acid accumulation in camelina, a species which contains a similar level of 11Z-eicosenoic acid as does Arabidopsis thaliana. Taken together, the MoKCS11 may have a substrate preference for 11Z-eicosenoic acid, but not for erucic acid, in planta.

Evaluation of duplicated reference genes for quantitative real-time PCR analysis in genome unknown hexaploid oat (Avena sativa L.).

BACKGROUND: Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses. However, no suitable RGs (reference genes) for qPCR (quantitative real-time PCR) has been documented for oat yet. Single-copy gene is often selected as RG, which is challengeable or impactable in unexplored polyploids. RESULTS: In this study, eleven candidate RGs, including four duplicated genes, were selected from oat transcriptome. The stability and the optimal combination of these candidate RGs were assessed in 18 oat samples by using four statistical algorithms including the ΔCt method, geNorm, NormFinder and BestKeeper. The most stable RGs for "all samples", "shoots and roots of seedlings", "developing seeds" and "developing endosperms" were EIF4A (Eukaryotic initiation factor 4A-3), UBC21 (Ubiquitin-Conjugating Enzyme 21), EP (Expressed protein) and EIF4A respectively. Among these RGs, UBC21 was a four-copy duplicated gene. The reliability was validated by the expression patterns of four various genes normalized to the most and the least stable RGs in different sample sets. CONCLUSIONS: Results provide a proof of concept that the duplicated RG is feasible for qPCR in polyploids. To our knowledge, this study is the first systematic research on the optimal RGs for accurate qPCR normalization of gene expression in different organs and tissues of oat.

Global Profiling of Dynamic Alternative Splicing Modulation in Arabidopsis Root upon Ralstonia solanacearum Infection.

Alternative splicing (AS) is an important mechanism by which eukaryotes regulate transcription and protein diversity. The dynamic changes in AS that occur on a genome-wide scale during interactions between plant roots and pathogens remain unknown. Here, we used the interaction between Arabidopsis and Ralstonia solanacearum as a model to explore the AS changes that take place during the response of roots to infection by means of high-throughput RNA-sequencing. We showed that dynamic changes in AS occur much earlier than changes at the level of transcription during R.solanacearum infection. Comparing genes that are regulated at the transcriptional and AS levels indicated that there are few common genes between differentially spliced genes (DSGs) and differentially expressed genes (DEGs). The functional gene ontology (GO) analysis identified that the enriched GO terms for the DSGs were different from those of the DEGs. The DSGs were over-represented in GO terms associated with post-transcriptional and translational regulations, suggesting that AS may act on RNA stability and during post-translation, thus affecting the output of plant defense molecules. Meanwhile, changes in DSGs were infection stage-specific. Furthermore, the nucleotide binding domain and leucine-rich repeat proteins and receptor-like kinases, key regulators in plant immunity, were shown to undergo dynamic changes in AS in response to R. solanacearum. Taken together, AS, along with transcription, modulates plant root defense to R. solanacearum through transcriptome reprogramming.

Acute toxicity of Potentilla anserina L. extract in mice.

Potentilla anserina L. is not only a medicinal plant, but also a traditional cuisine. Hence, an acute toxicity study was performed to confirm its safety profile. Forty Kunming mice were randomly divided into two groups: control group and P. anserina L. extract group. Using the maximum dosage method, the P. anserina L. extract group was given the maximum dose within 12 h, equivalent to 345.6 g/kg crude drug. The control group was given distilled water. After administration, toxicity symptoms of mice were observed, body weight and food intake were recorded. After 14 days, blood was collected to measure biochemical parameters, autopsy was carried out to observe the changes of organs, and the vital organs were separated, weighed, and preserved for histopathological examination. The results showed that P. anserina L. extract group had no toxic symptoms. The activity, weight, and diet of mice were normal, and no abnormality was found in organ index, renal function, liver function, anatomical observation, and histopathological examination. Therefore, the maximum oral dosage (345.6 g/kg) of P. anserina L. was good safety. This study indicated that P. anserina L. had a large safety range and the clinical application was safe.

Transcriptome Analysis Reveals Candidate Genes for Petroselinic Acid Biosynthesis in Fruits of Coriandrum sativum L.

Petroselinic acid (18:1Δ6), a monounsaturated cis Δ-6 fatty acid, has many prospective applications in functional foods and for the nutraceutical and pharmaceutical industries. Up to 80% of petroselinic acid has been found in the oil from fruits of coriander (Coriandrum sativum L.), which make it an ideal source for investigating the biosynthesis of petroselinic acid. A coriander acyl-acyl carrier protein desaturase was identified to be involved in its biosynthesis more than two decades ago, but since then little further progress in this area has been reported. In this study, the fatty acid profiles of coriander fruits at six developmental stages were analyzed. Fruit samples from three developmental stages with rapid accumulation of petroselinic acid were used for RNA sequencing using the Illumina Hiseq4000 platform. The transcriptome analysis presented 93 323 nonredundant unigenes and 8545 differentially expressed genes. Functional annotation and combined gene expression data revealed candidate genes potentially involved in petroselinic acid biosynthesis and its incorporation into triacylglycerols. Tissue-specific examination of q-PCR validation further suggested that ACPD1/3, KAS I-1, FATB-1/3, and DGAT2 may be highly involved. Bioinformatic analysis of CsFATB and CsDGAT2 identified their putative key amino acids or functional motifs. These results provide a molecular foundation for petroselinic acid biosynthesis in coriander fruit and facilitate its genetic engineering in other hosts.

A quick and efficient hydroponic potato infection method for evaluating potato resistance and Ralstonia solanacearum virulence.

BACKGROUND: Potato, the third most important crop worldwide, plays a critical role in human food security. Brown rot, one of the most destructive potato diseases caused by Ralstonia solanacearum, results in huge economic losses every year. A quick, stable, low cost and high throughout method is required to meet the demands of identification of germplasm resistance to bacterial wilt in potato breeding programs. RESULTS: Here we present a novel R. solanacearum hydroponic infection assay on potato plants grown in vitro. Through testing wilt symptom appearance and bacterial colonization in aerial part of plants, we found that the optimum conditions for in vitro potato infection were using an OD600 0.01 bacterial solution suspended with tap water for infection, broken potato roots and an open container. Infection using R. solanacearum strains with differential degree of aggressivity demonstrated that this infection system is equally efficient as soil-drench inoculation for assessment of R. solanacearum virulence on potato. A small-scale assessment of 32 potato germplasms identified three varieties highly resistant to the pathogen, which indicates this infection system is a useful method for high-throughout screening of potato germplasm for resistance. Furthermore, we demonstrate the utility of a strain carrying luminescence to easily quantify bacterial colonization and the detection of latent infections in hydroponic conditions, which can be efficiently used in potato breeding programs. CONCLUSIONS: We have established a quick and efficient in vitro potato infection system, which may facilitate breeding for new potato cultivars with high resistance to R. solanacearum.

WRINKLED1 homologs highly and functionally express in oil-rich endosperms of oat and castor.

Oat (Avena sativa) and castor (Ricinus communis) accumulate a large amount of lipids in their endosperms, however the molecular mechanism remains unknown. In this study, differences in oil regulators between oat and wheat (Triticum aestivum) as well as common features between oat and castor were tested by analyzing their transcriptomes with further q-PCR analysis. Results indicated that WRINKLED1 (WRI1) homologs and their target genes highly expressed in the endosperms of oat and castor, but not in the starchy endosperms of wheat. Expression pattern of WRI1s was in agreement with that of oil accumulation. Three AsWRI1s (AsWRI1a, AsWRI1b and AsWRI1c) and one RcWRI1 were identified in the endosperms of oat and castor, respectively. AsWRI1c lacks VYL motif, which is different from the other three WRI1s. Expressions of these four WRI1s all complemented the phenotypes of Arabidopsis wri1-1 mutant. Overexpression of these WRI1s in Arabidopsis and tobacco BY2 cells increased oil contents of seeds and total fatty acids of the cells, respectively. Moreover, this overexpression also resulted in up-regulations of WRI1 target genes, such as PKp-ß1. Taken together, our results suggest that high and functional expression of WRI1 play a key role in the oil-rich endosperms and the VYL motif is dispensable for WRI1 function.

Deep Sequencing Reveals Early Reprogramming of Arabidopsis Root Transcriptomes Upon Ralstonia solanacearum Infection.

Bacterial wilt caused by the bacterial pathogen Ralstonia solanacearum is one of the most devastating crop diseases worldwide. The molecular mechanisms controlling the early stage of R. solanacearum colonization in the root remain unknown. Aiming to better understand the mechanism of the establishment of R. solanacearum infection in root, we established four stages in the early interaction of the pathogen with Arabidopsis roots and determined the transcriptional profiles of these stages of infection. A total 2,698 genes were identified as differentially expressed genes during the initial 96 h after infection, with the majority of changes in gene expression occurring after pathogen-triggered root-hair development observed. Further analysis of differentially expressed genes indicated sequential activation of multiple hormone signaling cascades, including abscisic acid (ABA), auxin, jasmonic acid, and ethylene. Simultaneous impairment of ABA receptor genes promoted plant wilting symptoms after R. solanacearum infection but did not affect primary root growth inhibition or root-hair and lateral root formation caused by R. solanacearum. This indicated that ABA signaling positively regulates root defense to R. solanacearum. Moreover, transcriptional changes of genes involved in primary root, lateral root, and root-hair formation exhibited high temporal dynamics upon infection. Taken together, our results suggest that successful infection of R. solanacearum on roots is a highly programmed process involving in hormone crosstalk.

Strong co-suppression impedes an increase in polyunsaturated fatty acids in seeds overexpressing FAD2.

Fatty acid desaturase2 (FAD2) catalyses the conversion of oleic acid to linoleic acid and is the main determinant of the levels of essential poly-unsaturated fatty acids (PUFAs) in seed oils. The very limited number of successful examples of overexpression of FAD2 over the last two decades and a shortage of reports on co-suppression make it uncertain whether FAD2 can increase PUFAs effectively across a broad range of oil crops. In this study, strong co-suppression was observed in about 80% of over 100 transgenic lines when FAD2 was overexpressed in three oilseed crops, namely flax (Linum usitatissimum), carinata (Brassica carinata), and camelina (Camelina sativa), as well as in the model plant Arabidopsis. Further analyses of Arabidopsis transgenic lines revealed both endogenous and transgenic FAD2 gene-silencing. Thus, the commonality and potency of FAD2 co-suppression seemingly imposes an obstacle to engineering oilseed PUFA enhancement by direct FAD2 overexpression. AtFAD2, driven by the 35S promoter, also caused co-suppression in Arabidopsis roots. The FAD2 co-suppression was unstable and PUFA phenotypes of T4 lines were similar to the wild-type, further indicating that high PUFA content cannot be achieved by screening advanced generations. However, we demonstrate that the obstacle of FAD2 co-suppression can be overcome in the Arabidopsis rdr6 mutant, which is impaired in post-transcriptional gene-silencing, and that lines with high PUFA content are stable through four generations.