Yeast Assay Research Articles

A CCAAT-binding factor, SlNFYA10, negatively regulates ascorbate accumulation by modulating the d-mannose/l-galactose pathway in tomato

Ascorbic acid (AsA), an important antioxidant and growth regulator, and it is essential for plant development and human health. Specifically, humans have to acquire AsA from dietary sources due to their inability to synthesize it. The AsA biosynthesis pathway in plants has been elucidated, but its regulatory mechanism remains largely unknown. In this report, we biochemically identified a CCAAT-box transcription factor (SlNFYA10) that can bind to the promoter of SlGME1, which encodes GDP-Man-3’,5’-epimerase, a pivotal enzyme in the d-mannose/l-galactose pathway. Importantly, SlNFYA10 simultaneously binds to the promoter of SlGGP1, a downstream gene of SlGME1 in the d-mannose/l-galactose pathway. Binding assays in yeast and functional analyses in plants have confirmed that SlNFYA10 exerts a negative effect on the expression of both SlGME1 and SlGGP1. Transgenic tomato lines overexpressing SlNFYA10 show decreased levels of SlGME1 and SlGGP1 abundance and AsA concentration in their leaves and fruits, accompanied by enhanced sensitivity to oxidative stress. Overall, SlNFYA10 is the first CCAAT-binding factor identified to date to negatively regulate the AsA biosynthetic pathway at multiple sites and modulate plant responses to oxidative stress.

Functional characterization of an novel acyl-CoA:diacylglycerol acyltransferase 3-3 (CsDGAT3-3) gene from Camelina sativa

Diacylglycerol acyltransferases (DGAT) catalyze the final committed step of de novo biosynthesis of triacylglycerol (TAG) in plant seeds. This study was to functionally characterize DGAT3 genes in Camelina sativa, an important oil crops accumulating high levels of unsaturated fatty acids (UFAs) in seeds. Three camelina DGAT3 genes (CsDGAT3-1, CsDGAT3-2 and CsDGAT3-3) were identified, and the encoded proteins were predicted to be cytosolic-soluble proteins present as a homodimer containing the 2Fe-2S domain. They had divergent expression patterns in various tissues, suggesting that they may function in tissue-specific manner with CsDGAT3-1 in roots, CsDGAT3-2 in flowers and young seedlings, and CsDGAT3-3 in developing seeds. Functional complementation assay in yeast demonstrated that CsDGAT3-3 restored TAG synthesis. TAG content and UFAs, particularly eicosenoic acid (EA, 20:1n-9) were largely increased by adding exogenous UFAs in the yeast medium. Further heterogeneously transient expression in N. benthamiana leaves and seed-specific expression in tobacco seeds indicated that CsDGAT3-3 significantly enhanced oil and UFA accumulation with much higher level of EA. Overall, CsDGAT3-3 exhibited a strong abilty catalyzing TAG synthesis and high substrate preference for UFAs, especially for 20:1n-9. The present data provide new insights for further understanding oil biosynthesis mechanism in camelina seeds, indicating that CsDGAT3-3 may have practical applications for increasing both oil yield and quality.

Noscapine alleviates cerebral damage in ischemia-reperfusion injury in rats.

With high unmet medical needs, stroke remains an intensely focused research area. Although noscapine is a neuroprotective agent, its mechanism of action in ischemic-reperfusion (I-R) injury is yet to be ascertained. We investigated the effect of noscapine on the molecular mechanisms of cell damage using yeast, and its neuroprotection on cerebral I-R injury in rats. Yeast, both wild-type and Δtrx2 strains, was evaluated for cell growth and viability, and oxidative stress to assess the noscapine effect at 8, 10, 12, and 20μg/ml concentrations. The neuroprotective activity of noscapine (5 and 10mg/kg; po for 8days) was investigated in rats using middle cerebral artery occlusion-induced I-R injury. Infarct volume, neurological deficit, oxidative stress, myeloperoxidase activity, and histological alterations were determined in I-R rats. In vitro yeast assays exhibited significant antioxidant activity and enhanced cell tolerance against oxidative stress after noscapine treatment. Similarly, noscapine pretreatment significantly reduced infarct volume and edema in the brain. The neurological deficit was decreased and oxidative stress biomarkers, superoxide dismutase activity and glutathione levels, were significantly increased while lipid peroxidation showed significant decrease in comparison to vehicle-treated I-R rats. Myeloperoxidase activity, an indicator of inflammation, was also reduced significantly in treated rats; histological changes were attenuated with noscapine. The study demonstrates the protective effect of noscapine in yeast and I-R rats by improving cell viability and attenuating neuronal damage, respectively. This protective activity of noscapine could be attributed to potent free radical scavenging and inhibition of inflammation in cerebral ischemia-reperfusion injury.

OsNRAMP1 transporter contributes to cadmium and manganese uptake in rice.

Rice is a major dietary source of the toxic metal, cadmium (Cd). Previous studies reported that the rice transporter, OsNRAMP1, (Natural resistance-associated macrophage protein 1) could transport iron (Fe), Cd and arsenic (As) in heterologous yeast assays. However, the in planta function of OsNRAMP1 remains unknown. Here, we showed that OsNRAMP1 was able to transport Cd and manganese (Mn) when expressed in yeast, but did not transport Fe or As. OsNRAMP1 was mainly expressed in roots and leaves and encoded a plasma membrane-localized protein. OsNRAMP1 expression was induced by Cd treatment and Fe deficiency. Immunostaining showed that OsNRAMP1 was localized in all root cells, except the central vasculature, and in leaf mesophyll cells. The knockout of OsNRAMP1 resulted in significant decreases in root uptake of Cd and Mn and their accumulation in rice shoots and grains, and increased sensitivity to Mn deficiency. The knockout of OsNRAMP1 had smaller effects on Cd and Mn uptake than knockout of OsNRAMP5, while knockout of both genes resulted in large decreases in the uptake of the two metals. Taken together, OsNRAMP1 contributes significantly to the uptake of Mn and Cd in rice, and the functions of OsNRAMP1 and OsNRAMP5 are similar but not redundant.

Two CYP71AJ enzymes function as psoralen synthase and angelicin synthase in the biosynthesis of furanocoumarins in Peucedanum praeruptorum Dunn.

Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.

Features of fungal colonization of newborns from mothers with vulvovaginitis

The aim of this study was to determine the frequency and etiological structure of fungal colonization of newborns from mothers with vaginal infections taking into account the methods of delivery, gestational age and body weight of the child. Material and methods. 80 women clinically suspicious of fungal vulvovaginitis were examined at 16—22 weeks of gestation. Samples from the loci of probable fungal colonization in their children (81 newborns, one twins) were collected on 2—3 and 10—5 days of life. Samples were plated on Sabouraud agar and Czapek agar, followed by species identification by API Candida — api 20 C AUX yeast assay. Results. Positive results were found in 23 (28.8%) mothers (Candida spp.) And 44 (54.3%) newborns (Candida spp., Aspergillus flavus, Penicillium expansum Link, Mucor racemosus). Isolates of 7.4% of full-term infants were completely identical to the maternal microflora (Candida spp.). The remaining 19 (23.5%) premature infants were found to have molds of fungi, which indicates the nosocomial nature of the infection. A statistically significant relationship between the type of delivery and fungal colonization in newborns was found in the early neonatal period (2—3 days of life).

Functional characterisation of fatty acyl desaturase, Fads2, and elongase, Elovl5, in the Boddart's goggle-eyed goby Boleophthalmus boddarti (Gobiidae) suggests an incapacity for long-chain polyunsaturated fatty acid biosynthesis.

The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA), a process to convert C18 polyunsaturated fatty acids into eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or arachidonic acid (ARA), requires the concerted activities of two enzymes, the fatty acyl desaturase (Fads) and elongase (Elovl). This study highlights the cloning, functional characterisation and tissue expression pattern of a Fads and an Elovl from the Boddart's goggle-eyed goby (Boleophthalmus boddarti), a mudskipper species widely distributed in the Indo-Pacific region. Phylogenetic analysis revealed that the cloned fads and elovl are clustered with other teleost orthologs, respectively. The investigation of the genome of several mudskipper species, namely Boleophthalmus pectinirostris, Periophthalmus schlosseri and Periophthalmus magnuspinnatus, revealed a single Fads2 and two elongases, Elovl5 and Elovl4 for each respective species. A heterologous yeast assay indicated that the B. boddarti Fads2 possessed low desaturation activity on C18 PUFA and no desaturation on C20 and C22 PUFA substrates. In comparison, the Elovl5 showed a wide range of substrate specificity, with a capacity to elongate C18, C20 and C22 PUFA substrates. An amino acid residue that affects the capacity to elongate C22:5n-3 was identified in the B. boddarti Elovl5. Both genes are highly expressed in brain tissue. Among all tissues, DHA is highly concentrated in neuron-rich tissues, whereas EPA is highly deposited in gills. Taken together, the results showed that due to the inability to perform desaturation steps, B. boddarti is unable to biosynthesise LC-PUFA, relying on dietary intake to acquire these nutrients.

Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants

Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1/HpDTC1 gene and the "dehydrogenase momilactone A synthase" gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (Oryza and Echinochloa) and C. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.

Tricresyl phosphate isomers exert estrogenic effects via G protein-coupled estrogen receptor-mediated pathways

Tricresyl phosphates (TCPs), as representative aromatic organophosphate flame retardants (OPFRs), have received much attention due to their potential neurotoxicity and endocrine-disrupting effects. However, the role of estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) in their estrogen disrupting effects remains poorly understood. Therefore, in this study, three TCP isomers, tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) and tri-p-cresyl phosphate (TpCP), were examined for their activities on ERα by using two-hybrid yeast assay, and action on GPER by using Boyden chamber assay, cAMP production assay, calcium mobilization assay and molecular docking analysis. The results showed that three TCP isomers were found to act as ERα antagonists. Conversely, they had agonistic activity on GPER to promote GPER-mediated cell migration of MCF7 cells and SKBR3 cells. Both ToCP and TpCP activated GPER-mediated cAMP production and calcium mobilization, whereas TmCP had different mode of action, it only triggered GPER-mediated calcium mobilization, as evidenced by using the specific GPER inhibitor (G15) and GPER overexpressing experiments. Molecular docking further revealed that the way of interaction of TmCP and TpCP with GPER was different from that of ToCP with GPER, and higher activity of ToCP in activating GPER-mediated pathways might be associated with the alkyl substitution at the ortho position of the aromatic ring. Our results, for the first time, found a new target, GPER, for TCPs exerting their estrogen-disrupting effects, and demonstrated complex estrogen-disrupting effects of three TCP isomers involved their opposite activities toward ERα and GPER.

Characterization of cancer-related somatic mutations in the adenosine A2B receptor

In cancer, G protein-coupled receptors (GPCRs) are involved in tumor progression and metastasis. In this study we particularly examined one GPCR, the adenosine A2B receptor. This receptor is activated by high concentrations of its endogenous ligand adenosine, which suppresses the immune response to fight tumor progression. A series of adenosine A2B receptor mutations were retrieved from the Cancer Genome Atlas harboring data from patient samples with different cancer types. The main goal of this work was to investigate the pharmacology of these mutant receptors using a ‘single-GPCR-one-G protein’ yeast assay technology. Concentration-growth curves were obtained with the full agonist NECA for the wild-type receptor and 15 mutants. Compared to wild-type receptor, the constitutive activity levels in mutant receptors F141L4.61, Y202C5.58 and L310P8.63 were high, while the potency and efficacy of NECA and BAY 60–6583 on Y202C5.58 was lower. A 33- and 26-fold higher constitutive activity on F141L4.61 and L310P8.63 was reduced to wild-type levels in response to the inverse agonist ZM241385. These constitutively active mutants may thus be tumor promoting. Mutant receptors F259S6.60 and Y113F34.53 showed a more than one log-unit decrease in potency. A complete loss of activation was observed in mutant receptors C29R1.54, W130C4.50 and P249L6.50. All mutations were characterized at the structural level, generating hypotheses of their roles on modulating the receptor conformational equilibrium. Taken together, this study is the first to investigate the nature of adenosine A2B receptor cancer mutations and may thus provide insights in mutant receptor function in cancer.

Sclerenchyma cell thickening through enhanced lignification induced by OsMYB30 prevents fungal penetration of rice leaves.

Broad-spectrum resistance is highly preferred in crop breeding programmes. Previously, we have reported the identification of the broad-spectrum resistance-Digu 1 (bsr-d1) allele from rice Digu. The bsr-d1 allele prevents activation of Bsr-d1 expression by Magnaporthe oryzae infection and degradation of H2 O2 by peroxidases, leading to resistance to M.oryzae. However, it remains unknown whether defence pathways other than H2 O2 burst and peroxidases contribute to the bsr-d1-mediated immunity. Blast resistance was determined in rice leaves by spray and punch inoculations. Target genes of OsMYB30 were identified by one-hybrid assays in yeast and electrophoretic mobility shift assay. Lignin content was measured by phloroglucinol-HCl staining, and acetyl bromide and thioacidolysis methods. Here, we report the involvement of the OsMYB30 gene in bsr-d1-mediated blast resistance. Expression of OsMYB30 was induced during M.oryzae infection or when Bsr-d1 was knocked out or downregulated, as occurs in bsr-d1 plants upon infection. We further found that OsMYB30 bound to and activated the promoters of 4-coumarate:coenzyme A ligase genes (Os4CL3 and Os4CL5) resulting in accumulation of lignin subunits G and S. This action led to obvious thickening of sclerenchyma cells near the epidermis, inhibiting M.oryzae penetration at the early stage of infection. Our study revealed novel components required for bsr-d1-mediated resistance and penetration-dependent immunity, and advanced our understanding of broad-spectrum disease resistance.

Investigating (anti)estrogenic activities within South African wastewater and receiving surface waters: Implication for reliable monitoring

Natural and synthetic steroid hormones and many persistent organic pollutants are of concern for their endocrine-disrupting activities observed in receiving surface waters. Apart from the demonstrated presence of estrogen- and estrogen-mimicking compounds in surface waters, antagonistic (anti-estrogenic) responses originating from wastewater effluent have been reported but are less known. Estrogenicity and anti-estrogenicity were assessed using recombinant yeast estrogen receptor binding assays (YES/YAES) at ten South African wastewater treatment works (WWTWs) and receiving rivers in two separate sampling campaigns during the summer- and winter periods in the area. Four WWTWs were then further investigated to show daily variation in estrogenic endocrine-disrupting activities during the treatment process. Although estrogenicity was notably reduced at most of the WWTWs, some treated effluent and river water samples were shown to be above effect-based trigger values posing an endocrine-disrupting risk for aquatic life and potential health risks for humans. Furthermore, estrogenicity recorded in samples collected upstream from some WWTW discharge points also exceeded some calculated risk trigger values, which highlights the impact of alternative pollution sources contributing towards endocrine disrupting contaminants (EDCs) in the environment. The YAES further showed variable anti-estrogenic activities in treated wastewater. The current study highlights a variety of factors that may affect bioassay outcomes and conclusions drawn from the results for risk decision-making. For example, mismatches were found between estrogenic and anti-estrogenic activity, which suggests a potential masking effect in WWTW effluents and highlights the complexity of environmental samples containing chemical mixtures having variable endocrine-disrupting modes of action. Although the recombinant yeast assay is not without its limitations to show endocrine-disrupting modulation in test water systems, it serves as a cost-effective tier-1 scoping assay for further risk characterisation and intervention.

GARS-related disease in infantile spinal muscular atrophy: Implications for diagnosis and treatment.

The majority of patients with spinal muscular atrophy (SMA) identified to date harbor a biallelic exonic deletion of SMN1. However, there have been reports of SMA-like disorders that are independent of SMN1, including those due to pathogenic variants in the glycyl-tRNA synthetase gene (GARS1). We report three unrelated patients with de novo variants in GARS1 that are associated with infantile-onset SMA (iSMA). Patients were ascertained during inpatient hospital evaluations for complications of neuropathy. Evaluations were completed as indicated for clinical care and management and informed consent for publication was obtained. One newly identified, disease-associated GARS1 variant, identified in two out of three patients, was analyzed by functional studies in yeast complementation assays. Genomic analyses by exome and/or gene panel and SMN1 copy number analysis of three patients identified two previously undescribed de novo missense variants in GARS1 and excluded SMN1 as the causative gene. Functional studies in yeast revealed that one of the de novo GARS1 variants results in a loss-of-function effect, consistent with other pathogenic GARS1 alleles. In sum, the patients' clinical presentation, assessments of previously identified GARS1 variants and functional assays in yeast suggest that the GARS1 variants described here cause iSMA. GARS1 variants have been previously associated with Charcot-Marie-Tooth disease (CMT2D) and distal SMA type V (dSMAV). Our findings expand the allelic heterogeneity of GARS-associated disease and support that severe early-onset SMA can be caused by variants in this gene. Distinguishing the SMA phenotype caused by SMN1 variants from that due to pathogenic variants in other genes such as GARS1 significantly alters approaches to treatment.

Antioxidant and Glucose Lowering Effects of Hydroethanolic Extract of Baillonella toxisperma Pulp

Hyperglycemia and oxidative stress play an important role in the pathogenesis of diabetes. Their management is a key point in the prevention and treatment of this disease which is a potential cause of mortality in the world. We evaluated the antioxidant and glucose lowering effects of hydroethanolic extract of Baillonella toxisperma pulp. The total polyphenol and flavonoid contents were determined and the antioxidant activity was evaluated by 3 mechanisms: scavenging by DPPH•, ABTS• and NO• radicals; reducing property by MoO42+ and Fe3+ reduction; metal chelation by Cu2+ and Fe2+. Glucose adsorption capacity was evaluated followed by the capacity to promote insulino-sensitivity through glucose uptake by yeast and muscle cell assays. The hydroethanolic extract of B. toxisperma pulp possessed high total polyphenols and flavonoids; 459.55 µg Equivalent Gallic Acid/mg and 252.15 µg of Equivalent Catechin /mg respectively. This showed their ability to scavenge DPPH•, ABTS• and NO• radicals with SC50 of 3.49, 3.24 and 4.28 mg/ml respectively. The extract also reduced MoO42+ and Fe3+ and chelated Cu2+ through inhibiting their capacity to induce hemolysis with the IC50 of 3.49 mg/ml. The extract showed a high glucose binding capacity with a glucose binding percentage rise of 60 %. It increased yeast cell absorption of glucose with the increasing percentage varying from 42.97 to 56.62 %. In the muscle cells, after 30 min of administration of the extract, we also noted an increased glucose absorption with the percentage glucose reducing to 22 %. We demonstrated that hydroethanolic extract of B. toxisperma pulp possess antiradical, reducing, metal chelating, glucose binding and insulino-sensitivity promoting properties. These mechanisms imply that B. toxisperma pulp is both a good antioxidant and an antihyperglycemiant, thus a potential agent in the management of diabetes and its complications.

The transcription factor VaNAC17 from grapevine (Vitis amurensis) enhances drought tolerance by modulating jasmonic acid biosynthesis in transgenic Arabidopsis.

Expression of VaNAC17 improved drought tolerance in transgenic Arabidopsis by upregulating stress-responsive genes, modulating JA biosynthesis, and enhancing ROS scavenging. Water deficit severely affects the growth and development of plants such as grapevine (Vitis spp.). Members of the NAC (NAM, ATAF1/2, and CUC2) transcription factor (TF) family participate in drought-stress-induced signal transduction in plants, but little is known about the roles of NAC genes in drought tolerance in grapevine. Here, we explored the role of VaNAC17 in Vitis amurensis, a cold-hardy, drought-tolerant species of grapevine. VaNAC17 was strongly induced in grapevine by drought, exogenous abscisic acid (ABA), and methyl jasmonate (MeJA). A transient expression assay in yeast indicated that VaNAC17 functions as a transcriptional activator. Notably, heterologous expression of VaNAC17 in Arabidopsis thaliana enhanced drought tolerance. VaNAC17-expressing Arabidopsis plants showed decreased reactive oxygen species (ROS) accumulation compared to wild-type plants under drought conditions. RNA-seq analysis indicated that VaNAC17 expression increased the transcription of downstream stress-responsive genes after 5days of drought treatment, especially genes involved in jasmonic acid (JA) biosynthesis (such as LOX3, AOC1 and OPR3) and signaling (such as MYC2, JAZ1, VSP1 and CORI3) pathways. Endogenous JA levels increased in VaNAC17-OE plants under drought stress. Taken together, these results indicate that VaNAC17 plays a positive role in drought tolerance by modulating endogenous JA biosynthesis and ROS scavenging.

The biochemistry of headgroup exchange during triacylglycerol synthesis in canola.

Many pathways of primary metabolism are substantially conserved within and across plant families. However, significant differences in organization and fluxes through a reaction network may occur, even between plants in closely related genera. Assessing and understanding these differences is key to appreciating metabolic diversity, and to attempts to engineer plant metabolism for higher crop yields and desired product profiles. To better understand lipid metabolism and seed oil synthesis in canola (Brassica napus), we have characterized four canola homologues of the Arabidopsis (Arabidopsis thaliana) ROD1 gene. AtROD1 encodes phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), the enzyme that catalyzes a major flux of polyunsaturated fatty acids (PUFAs) in oil synthesis. Assays in yeast indicated that only two of the canola genes, BnROD1.A3 and BnROD1.C3, encode active isozymes of PDCT, and these genes are strongly expressed during the period of seed oil synthesis. Loss of expression of BnROD1.A3 and BnROD1.C3 in a double mutant, or by RNA interference, reduced the PUFA content of the oil to 26.6% compared with 32.5% in the wild type. These results indicate that ROD1 isozymes in canola are responsible for less than 20% of the PUFAs that accumulate in the seed oil compared with 40% in Arabidopsis. Our results demonstrate the care needed when translating results from a model species to crop plants.

The Lipid Flippases ALA4 and ALA5 Play Critical Roles in Cell Expansion and Plant Growth.

Aminophospholipid ATPases (ALAs) are lipid flippases involved in transporting specific lipids across membrane bilayers. Arabidopsis (Arabidopsis thaliana) contains 12 ALAs in five phylogenetic clusters, including four in cluster 3 (ALA4-ALA7). ALA4/5 and ALA6/7, are expressed primarily in vegetative tissues and pollen, respectively. Previously, a double knockout of ALA6/7 was shown to result in pollen fertility defects. Here we show that a double knockout of ALA4/5 results in dwarfism, characterized by reduced growth in rosettes (6.5-fold), roots (4.3-fold), bolts (4.5-fold), and hypocotyls (2-fold). Reduced cell size was observed for multiple vegetative cell types, suggesting a role for ALA4/5 in cellular expansion. Members of the third ALA cluster are at least partially interchangeable, as transgenes expressing ALA6 in vegetative tissues partially rescued ala4/5 mutant phenotypes, and expression of ALA4 transgenes in pollen fully rescued ala6/7 mutant fertility defects. ALA4-GFP displayed plasma membrane and endomembrane localization patterns when imaged in both guard cells and pollen. Lipid profiling revealed ala4/5 rosettes had perturbations in glycerolipid and sphingolipid content. Assays in yeast revealed that ALA5 can flip a variety of glycerolipids and the sphingolipid sphingomyelin across membranes. These results support a model whereby the flippase activity of ALA4 and ALA5 impacts the homeostasis of both glycerolipids and sphingolipids and is important for cellular expansion during vegetative growth.

Occurrence and AhR activity of brominated parabens in the Kitakami River, North Japan

Parabens are used as preservatives in pharmaceuticals and personal care products (PPCPs). Parabens react with aqueous chlorine, which is used in disinfection processes, leading to the formation of halogenated parabens. In the presence of Br−, parabens and HOBr (formed via oxidation of Br−) can react to form brominated parabens. Brominated parabens may result in pollution of river water through effluent discharge from sewage treatment plants. The present study involved measuring brominated paraben concentrations in the Kitakami River, northern Japan, which flows through urban and agricultural areas. Aryl hydrocarbon receptor (AhR) agonist activity was also assessed using a yeast (YCM3) reporter gene and HepG2 ethoxyresorufin O-deethylase (EROD) assays. Dibrominated methylparaben (Br2MP), ethylparaben (Br2EP), propylparaben (Br2PP), butylparaben (Br2BP), and benzylparaben (Br2BnP), and monobrominated benzylparaben (Br1BnP) were detected in 25–100% of river samples during the sampling period from 2017 to 2018 at median concentrations of 8.1–28 ng/L; the highest concentrations were measured during the low flow season (November) in urban areas (P < 0.01). In the yeast assay, 12 compounds exhibited AhR activity (activity relative to β-naphthoflavone; 4.4 × 10−4−7.1 × 10−1). All monobrominated parabens exhibited higher activity than their parent parabens, however, further bromination reduced or eliminated their activity. In the EROD assay, five compounds caused significant induction of CYP1A-dependent activity at 100 μM (P < 0.05). Monobrominated i-butylparaben (Br1iBP) and s-butylparaben (Br1sBP), Br1BnP, and Br2BP exhibited activity in both yeast and EROD assays. We found novel aspects of brominated parabens originating from PPCPs.

Bi-allelic Variants in TKFC Encoding Triokinase/FMN Cyclase Are Associated with Cataracts and Multisystem Disease

We report an inborn error of metabolism caused by TKFC deficiency in two unrelated families. Rapid trio genome sequencing in family 1 and exome sequencing in family 2 excluded known genetic etiologies, and further variant analysis identified rare homozygous variants in TKFC. TKFC encodes a bifunctional enzyme involved in fructose metabolism through its glyceraldehyde kinase activity and in the generation of riboflavin cyclic 4',5'-phosphate (cyclic FMN) through an FMN lyase domain. The TKFC homozygous variants reported here are located within the FMN lyase domain. Functional assays in yeast support the deleterious effect of these variants on protein function. Shared phenotypes between affected individuals with TKFC deficiency include cataracts and developmental delay, associated with cerebellar hypoplasia in one case. Further complications observed in two affected individuals included liver dysfunction and microcytic anemia, while one had fatal cardiomyopathy with lactic acidosis following a febrile illness. We postulate that deficiency of TKFC causes disruption of endogenous fructose metabolism leading to generation of by-products that can cause cataract. In line with this, an affected individual had mildly elevated urinary galactitol, which has been linked to cataract development in the galactosemias. Further, in light of a previously reported role of TKFC in regulating innate antiviral immunity through suppression of MDA5, we speculate that deficiency of TKFC leads to impaired innate immunity in response to viral illness, which may explain the fatal illness observed in the most severely affected individual.

Improvement of reporter gene assay for highly sensitive dioxin detection using protoplastic yeast with inactivation of CWP and PDR genes.

A yeast reporter gene assay system with improved performance for dioxin detection was established. Since yeast reporter gene assays are relatively simple, easy to handle, and inexpensive, they have been used for various assessments of environmental contaminants. We previously constructed a yeast assay strain expressing the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) carrying the lacZ reporter gene, for detection of dioxins. In the present study, genes encoding cell wall mannoproteins and ATP-binding cassette transporters in the yeast assay strains were deleted in order to increase the substance influx and prevent its efflux. We also established an assay procedure for protoplasts of these yeasts. These modifications improved the detection limit 40-fold and reduced the duration of the assay by 40%. By combining the yeast protoplast and a rapid sample preparation technique using disposal multilayer solid-phase extraction columns to remove unintended aryl hydrocarbons, this yeast reporter gene assay system detected the ligand activities of dioxins and related compounds in 1g of forest soil containing dioxins at a concentration 10 times lower than the Japanese environmental standard for dioxins in soil.