Heterologous Expression In Yeast Research Articles

Arabidopsis ECERIFERUM2-LIKEs Are Mediators of Condensing Enzyme Function.

Condensing enzymes catalyze the committed reaction of fatty acid elongation and determine the chain length of fatty acids accepted and produced by the elongation complex. While necessary for the elongation of very-long-chain fatty acids (VLCFAs), identified plant condensing enzymes cannot efficiently produce VLCFAs longer than 28 carbons, which are precursors for the most abundant cuticular waxes of most plant species that have been surveyed. The eceriferum2 (cer2) mutant of Arabidopsis thaliana has a severe wax-deficient phenotype and specifically lacks waxes longer than 28 carbons, but the CER2 protein does not share sequence similarity with condensing enzymes. Instead, CER2 is homologous to BAHD acyltransferases. Heterologous expression in yeast previously demonstrated that CER2, and a small clade of BAHD acyltransferases with high sequence identity to CER2, can extend the chain-length specificity of the condensing enzyme CER6. This biochemical function is distinct from that of the broader BAHD acyltransferase family. The product specificity and physiological functions of individual CER2-LIKE proteins are unique. Here, we demonstrate that CER2 physically interacts with the fatty acid elongase. We cloned chimeric CER2-LIKE proteins and expressed these in yeast cells to identify the features that define the substrate specificities of CER2-LIKEs. We generated homology-based structural models to compare CER2-LIKEs and BAHD acyltransferases. In addition, based on the current phylogenetic analysis of the CER2-LIKE clade, we describe two further Arabidopsis CER2-LIKE genes, CER2-LIKE3 and CER2-LIKE4. We used yeast expression and mutant analysis to characterize these genes. Collectively, these results expand our knowledge of the functions of CER2-LIKEs, the BAHD acyltransferase family and cuticular wax metabolism.

Functional Analysis of Phosphate Transporter OsPHT4 Family Members in Rice

The transport of phosphate between cytoplasm and subcellular compartments is critical for plant metabolic regulation. We conducted bioinformatic analysis, heterologous expression in yeast, gene expression pattern and subcellular localization analysis to characterize the possible functions of OsPHT4 gene family in rice. Together with the PHT4 genes from higher plants, OsPHT4s can be classified into six distinct groups. OsPHT4;1–OsPHT4;4 are targeted to chloroplasts, and OsPHT4;6-1 and OsPHT4;6-2 are located to Golgi apparatus. OsPHT4 proteins can mediate inorganic phosphate (Pi) transport in yeast. In addition, dynamic transcriptional changes determined by qRT-PCR revealed different expression profiles of OsPHT4 genes in response to phosphate starvation, salicylic acid, abscisic acid and salt stress treatments. These results suggested that OsPHT4 proteins are involved in Pi distribution between the cytoplasm and chloroplast or Golgi apparatus and also involved in stress responses.

Functional characterization, tissue distribution and nutritional regulation of the Elovl4 gene in golden pompano, Trachinotus ovatus (Linnaeus, 1758)

The elongases of very long-chain fatty acids (Elovls) are involved in the rate-limiting of the carbon chain elongation reaction in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovls family, Elovl4, has been regarded as a critical enzyme involved in the biosynthesis pathway of polyunsaturated fatty acids (PUFAs). To explore the role of Elovl4 in PUFA synthesis in Trachinotus ovatus, the cDNA of the Elovl4b gene is cloned from T. ovatus (ToElovl4b). The ORF of ToElovl4b was 918 bp and encoded 305 amino acid (aa) protein sequences. Sequence alignment showed that the deduced amino acids contained significant structural features of the Elovl4 family, such as a histidine box motif (HXXHH), multiple transmembrane domains and an endoplasmic reticulum (ER) retention signal. Moreover, phylogenetic analysis revealed that ToElovl4b was highly conserved with that of Rachycentron canadum Elovl4b. Moreover, heterologous expression in yeast demonstrated that ToElovl4b could efficiently elongate 18:2n-6, 18:3n-6 and 20:5n-3 FAs up to 20:2n-6, 20:3n-6 and 22:5n-3, respectively. Furthermore, the tissue expression profile indicated that mRNA expression of ToElovl4b was higher in the gonads and brain than in other tissues. Additionally, nutritional regulation suggested the highest mRNA levels of ToElovl4b in liver and brain were under feeding with 1:1 FO-SO (fish oil, FO; soybean oil, SO) and 1:1 FO-CO (corn oil, CO)), respectively. These new insights were useful for understanding the molecular basis and regulation of LC-PUFA biosynthesis in fish.

Identification of triterpenes and functional characterization of oxidosqualene cyclases involved in triterpene biosynthesis in lettuce (Lactuca sativa)

Lettuce (Lactuca sativa) is a member of the family Asteraceae and is most often used for green salads. Triterpenes are the largest class of natural compounds in plants and have beneficial health effects. Here, we identified various triterpene esters (taraxasterol acetates, ψ-taraxasterol acetates, taraxerol acetates, lupeol acetates, α-amyrin acetates, β-amyrin acetates, and germanicol acetate) and free triterpenes (α-amyrin, β-amyrin, taraxerol, and taraxasterol) in both the leaves and roots of lettuce. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene. None of the OSC genes involved in triterpene biosynthesis in lettuce have been characterized. Five putative lettuce OSC genes (LsOSC1, LsOSC2, LsOSC3, LsOSC4, and LsOSC5) were selected from a transcriptome database. These five genes were functionally characterized via heterologous expression in yeast. The first two enzymes were multifunctional triterpene synthase and the last three genes were monofunctional. Transgenic yeast expressing LsOSC1 produced five triterpenes, namely, taraxasterol, Ψ-taraxasterol, α-amyrin, β-amyrin, and dammarenediol-II. Yeast expressing LsOSC2 produced baurenol and Ψ-taraxasterol. LsOSC3, LsOSC4, and LsOSC5 expression led to β-amyrin, taraxerol, and lupeol production, respectively. Transcriptional activity assessment of the five genes revealed that all the OSC genes were more actively transcribed in roots than in leaves, and LsOSC5 among the five OSC genes showed the highest expression in both the leaves and the roots. In conclusion, we identified structurally diverse free triterpenes and triterpene esters in lettuce plants and characterized five OSC genes, which are key enzymes involved in the biosynthesis of diverse triterpenes in lettuce.

Phaseolus vulgaris SUT1.1 is a high affinity sucrose-proton co-transporter.

Plant sucrose transporters are required for phloem loading, and therefore are essential for plant growth and development. In common beans (Phaseolus vulgaris) there are only two sucrose transporters functionally characterized. Through a previous RNA‐seq study, we identified a putative sucrose transporter in common bean, which we hypothesize to function in import of sucrose into plant cells. In silico analysis revealed that PvSUT1.1 is a putative sucrose‐proton co‐transporter distinct from other characterized sucrose transporters in common bean indicating that this is a previously undescribed transporter protein in beans. Further analysis revealed that PvSUT1.1 shares high protein sequence homology to the phloem loader Arabidopsis SUC2; both have 12 transmembrane domains, a typical characteristic of plant sucrose transporters. Heterologous expression in yeast further showed PvSUT1.1 to be functional and it imported sucrose into yeast cells with a Km of 0.7 mM sucrose. Import of sucrose through PvSUT1.1 is also pH‐dependent with highest uptake at pH 4.0, and activity is lost in the presence of the uncoupler carbonyl cyanide 3‐chlorophenylhydrazone. Consistent with identification of PvSUT1.1 as a Type I transporter, PvSUT1.1 also transports esculin. Finally, PvSUT1.1 showed expression in multiple tissues and the protein was localized to the plasma membrane. The results show that PvSUT1.1 is a sucrose transporter that is probably involved in the uptake of sucrose into source and sink cells. The potential role of PvSUT1.1 in leaf phloem loading of sucrose in common beans and its importance in heat tolerance of reproductive tissues are further discussed.

Polyunsaturated fatty acid metabolism in three fish species with different trophic level

Reducing the dependency of fishfeed for marine ingredients and species diversification are both considered crucial factors for the sustainable development of aquaculture. The substitution of fish oil (FO) by vegetable oils (VO) in aquafeeds is an economically feasible solution. However, such substitution may compromise the fish flesh content of essential n-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA) and, therefore, its nutritional value for human consumption. Likewise, there is a wide range of strategies to select new target species for sector diversification, among which, the capacity to biosynthesize n-3 LC-PUFA from their C18 precursors abundant in VO might be considered as a fair preliminary strategy. Therefore, the aim of the present study was to analyze the metabolic fate of [1-14C] labeled 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3 in isolated hepatocytes and enterocytes from wild individuals of three fish species with different trophic level: the marine herbivorous salema (Sarpa salpa), the strict carnivorous sand sole (Pegusa lascaris) and the omnivorous thicklip grey mullet (Chelon labrosus). These species were selected for their phylogenetic proximity to consolidated farmed species such as gilthead seabream (Sparus aurata), senegalese sole (Solea senegalensis), and golden grey mullet (Liza aurata), respectively. The study also assessed the molecular cloning, functional characterization and tissue distribution of the fatty acyl elongase (Elovl) gene, elovl5, involved in the biosynthetic metabolism of n-3 LC-PUFA. The three species were able to biosynthesize docosahexaenoic acid (22:6n-3). S. salpa seems to have similar biosynthetic capacity than S. aurata, with a fatty acyl desaturase 2 (Fads2), with Δ6, Δ8 and Δ5 activities. P. lascaris showed a wider Fads2 activity repertory than S. senegalensis, including Δ4 and residual Δ6/Δ5 activities. In C. labrosus, both Δ8 and Δ5 activities but not the Δ6 described for L. aurata were detected in the incubated cells. Elongation from C18 and C20 precursors to C20 and C22 products occurred in hepatocytes and enterocytes as well as in the functional characterization of Elovl5 by heterologous expression in yeast. Elovl5 showed a species specific expression pattern, with the highest rates observed in the liver, gut and brain in S. salpa and P. lascaris, and in the brain for C. labrosus. In summary, the LC-PUFA biosynthesis capacity from S. salpa, P. lascaris and C. labrosus greatly resembled that of their phylogenetic closer species. The three studied species could be further explored as candidates for the aquaculture diversification from their potential ability to biosynthesize LC-PUFA.

Comparsion of Activities of Fatty Acyl Desaturases and Elongases Among Six Teleosts With Different Feeding and Ecological Habits

Fatty acyl desaturases 2 (Fads2) and elongases of very long chain fatty acid 5 (Elovl5) are two key enzymes involved in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA), and their activities determine the LC-PUFA biosynthetic ability of teleost. In order to investigate the relation of enzymic activities with fish’s feeding habits and ecological habits, the activities of Fads2 and Elovl5 were compared among six teleosts, namely, freshwater carnivorous mandarin fish (Siniperca chuatsi), freshwater herbivorous grass carp (Ctenopharyngodon idellus), marine carnivorous orange-spotted grouper (Epinephelus coioides), marine herbivorous rabbitfish (Siganus canaliculatus), anadromous Atlantic salmon (Salmo salar) and catadromous Japanese eel (Anguilla japonica). Among them, the enzymatic features of Fads2 and Elovl5 from the last five fish species have been characterized, while those of S. chuatsi were unknown. And thus, the coding sequences (CDS) of S. chuatsi fads2 and elovl5 (elovl5a and elovl5b) were isolated and their function were further characterized by heterologous expression in yeast. The results showed that S. chuatsi Fads2 has a monofunctional Δ6 desaturase, and Elovl5a has a higher activity towards C18-C20 PUFAs than that in Elovl5b, which showed noteworthy activity towards C22 PUFAs. The comparison of enzymatic activities among the six teleosts showed that the ∆6 Fad and Elovl5 activities varied markedly among fish species, especially, the activity of Δ6 Fad in C. idellus, S. canaliculatus, and A. japonica was significantly higher than that in S. chuatsi, S. salar and E. coioides. For C18 PUFA substrates, A. japonica Elovl5 has higher elongation than that in other tested fish, and exhibits higher activity towards the C20 PUFAs. The results suggest that the ∆6 Fad activity is influenced by both feeding habits and ecological habits, while the Elovl5 activity was more affected by the feeding habits. These data enrich our knowledge on LC-PUFA biosynthesis diversity of fatty acid desaturation and elongations in teleosts, and provide guidance for the choice of dietary PUFA precursors for farmed fish.

Multi-genomic analysis of the cation diffusion facilitator transporters from algae.

Metal transport processes are relatively poorly understood in algae in comparison to higher plants and other eukaryotes. A screen of genomes from 33 taxonomically diverse algal species was conducted to identify members of the Cation Diffusion Facilitator (CDF) family of metal ion transporter. All algal genomes contained at least one CDF gene with four species having >10 CDF genes (median of 5 genes per genome), further confirming that this is a ubiquitous gene family. Phylogenetic analysis suggested a CDF gene organisation of five groups, which includes Zn-CDF, Fe/Zn-CDF and Mn-CDF groups, consistent with previous phylogenetic analyses, and two functionally undefined groups. One of these undefined groups was algal specific although excluded chlorophyte and rhodophyte sequences. The majority of sequences (22 out of 26 sequences) from this group had a putative ion binding site motif within transmembrane domain 2 and 5 that was distinct from other CDF proteins, such that alanine or serine replaced the conserved histidine residue. The phylogenetic grouping was supported by sequence cluster analysis. Yeast heterologous expression of CDF proteins from Chlamydomonas reinhardtii indicated Zn2+ and Co2+ transport function by CrMTP1, and Mn2+ transport function by CrMTP2, CrMTP3 and CrMTP4, which validated the phylogenetic prediction. However, the Mn-CDF protein CrMTP3 was also able to provide zinc and cobalt tolerance to the Zn- and Co-sensitive zrc1 cot1 yeast strain. There is wide diversity of CDF transporters within the algae lineage, and some of these genes may be attractive targets for future applications of metal content engineering in plants or microorganisms.

A cycloartenol synthase from the steroidal saponin biosynthesis pathway of Paris polyphylla

Steroidal saponins named polyphyllin are the major active components of Paris polyphylla. Cycloartenol synthase (CAS) is a key enzyme that catalyzes the formation of the sterol scaffold. In this study, we cloned a putative CAS gene from Paris polyphylla. Heterologous expression in yeast indicated that PpCAS can convert 2,3-oxidosqualene into cycloartenol. qRT-PCR analysis showed that the expression of PpCAS was highest in leaves and lowest in roots. To our best knowledge, this is the first report of the functional characterization of cycloartenol synthase from Paris polyphylla, which lays the foundation for further analysis of the biosynthesis pathway of polyphyllins.

Palmitvaccenic Acid (Δ11-cis-hexadecenoic acid) Is Synthesized by an OLE1-like Desaturase in the Arbuscular Mycorrhiza Fungus Rhizophagus irregularis

Arbuscular mycorrhiza (AM) fungi deliver mineral nutrients to the plant host in exchange for reduced carbon in the form of sugars and lipids. Colonization with AM fungi upregulates a specific host lipid synthesis pathway resulting in the production of fatty acids. Predominantly palmitic acid (16:0) and the unusual palmitvaccenic acid (16:1Δ11cis) accumulate in the fungus Rhizophagus irregularis. Here, we present the isolation and characterization of RiOLE1-LIKE, the desaturase involved in palmitvaccenic acid synthesis, by heterologous expression in yeast and plants. Results are in line with the scenario in which RiOLE1-LIKE encodes an acyl-CoA desaturase with substrate specificity for C15-C18 acyl groups, in particular C16. Phylogenetic analysis of RiOLE1-LIKE-related sequences revealed that this gene is conserved in AM fungi from the Glomales and Diversisporales but is absent from nonsymbiotic Mortierellaceae and Mucoromycotina fungi, suggesting that 16:1Δ11cis provides a specific function during AM colonization.

Rice Heavy Metal P-type ATPase OsHMA6 Is Likely a Copper Efflux Protein

P1B-type heavy metal ATPases (HMAs) are transmembrane metal-transporting proteins that play a key role in metal homeostasis. We here reported the characterization of rice OsHMA6, a member of the P1B-type ATPase family. Phylogenetic tree analysis showed that OsHMA6 belonged to the Cu/Ag subgroup of the HMA family and had a close evolutionary relationship with OsHMA9. Amino acid sequence alignment showed 82.78% consistency between OsHMA6 and OsHMA9. OsHMA6 expressed in all organs at different growth stages, including spikelet, and abundant in leaf blades, however, OsHMA9 most strongly expressed in roots, but very low in spikelet. Excessive Cu2+ can up-regulate the expression of OsHMA6 and OsHMA9 in rice seedlings. The heterologous expression in yeast showed that OsHMA6 can significantly rescue the growth of yeast strain CM52 when supplied with 3 or 6mmol/L Cu2+. Compared with the empty vector pYES2, the Cu concentration in OsHMA6-pYES2 decreased by 23.4% and 30.3% under 3 or 6mmol/L Cu2+, respectively. Subcellular localization revealed that OsHMA6 was located in the plasma membrane. These results suggested that OsHMA6, similar to OsHMA9, is likely a copper efflux protein located in the plasma membrane.

Characterization of an alkylresorcinol synthase that forms phenolics accumulating in the cuticular wax on various organs of rye (Secale cereale).

Alkylresorcinols are bioactive compounds produced in diverse plant species, with chemical structures combining an aliphatic hydrocarbon chain and an aromatic ring with characteristic hydroxyl substituents. Here, we aimed to isolate and characterize the enzyme that forms the alkylresorcinols accumulating in the cuticular wax on the surface of all above-ground organs of rye. Based on sequence homology with other type-III polyketide synthases, a candidate alkylresorcinol synthase was cloned. Yeast heterologous expression showed that the enzyme, ScARS, is highly specific for the formation of the aromatic resorcinol ring structure, through aldol condensation analogous to stilbene synthases. The enzyme accepts long-chain and very-long-chain acyl-CoA starter substrates, preferring saturated over unsaturated chains. It typically carries out three rounds of condensation with malonyl-CoA prior to cyclization, with only very minor activity for a fourth round of malonyl-CoA condensation and cyclization to 5-(2'-oxo)-alkylresorcinols or 5-(2'-hydroxy)-alkylresorcinols. Like other enzymes involved in cuticle formation, ScARS is localized to the endoplasmic reticulum. ScARS expression patterns were found correlated with alkylresorcinol accumulation during leaf development and across different rye organs. Overall, our results thus suggest that ScARS synthesizes the cuticular alkylresorcinols found on diverse rye organ surfaces.

Lipid metabolism in Tinca tinca and its n-3 LC-PUFA biosynthesis capacity

Carps, barbels and other cyprinids are the major contributors to freshwater aquaculture at global scale. Nevertheless, freshwater fish aquaculture needs to diversify their production in order to offer consumers new species. Tench (Tinca tinca) is a freshwater species with great interest for the diversification of continental aquaculture. However, up to date, no commercial formulated diet exists for this species in order to optimize their nutritional requirements and the quality of its final product. Using multiple methodological approaches, the aim of this study was to evaluate the long chain polyunsaturated fatty acid (LC-PUFA) metabolism of T. tinca. Firstly, the molecular cloning and functional characterisation by heterologous expression in yeast of a desaturase (Fads2) and two elongases (Elovl2 and Elovl5) involved in LC-PUFA biosynthesis, and the analysis of gene expression among tissues were performed. Secondly, in order to confirm the LC-PUFA biosynthesis capacity of isolated hepatocytes and enterocytes, cells were incubated with [1-14C] labelled linoleic acid (18:2n-6, LA), linolenic acid (18:3n-3, ALA) and eicosapentaenoic acid (20:5n-3, EPA). In yeast, Fads2 showed a Δ6/Δ5 bifunctional activity. Elovl2 was more active over C20 and C22 substrates, whereas Elovl5 was over C18 and C20. Liver displayed the highest expression for the three target genes (fads2, elovl2 and elovl5). Incubated cells also showed Fads2 bifunctional activity as well as elongation products in concordance with yeast heterologous expression results. Importantly, our results demonstrated that tench is able to biosynthesise docosahexaenoic acid (DHA) from 18:3n-3 in both hepatocytes and enterocytes, a capacity that seems to explain in part the surprisingly high levels of DHA found in the fish flesh compared to its dietary supply. Tench is a promising freshwater species with a potential capacity to endogenously increase its flesh DHA contents, reducing the impact that the usage of fish oils from forage fisheries may have on the aquaculture industry.

Genome wide identification and functional characterization of two LC-PUFA biosynthesis elongase (elovl8) genes in rabbitfish (Siganus canaliculatus)

Elongases of very long-chain fatty acids (Elovls) catalyze the rate-limiting step of the elongation pathway that results in net two‑carbon elongation of pre-existing fatty acyl chains. As a set of crucial enzymes involved in the long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, Elovls of fish have been investigated extensively in recent years. In the present study, we first identified two novel fish-specific elovl genes (named as elovl8a and elovl8b) from the herbivorous marine teleost rabbitfish (Siganus canaliculatus) by genomic survey and molecular cloning methods. Subsequently, their functional characteristics, tissue distribution patterns and transcriptional changes in response to different nutritional states were investigated. Full-length coding sequences of the elovl8a and elovl8b genes were 804 and 792 bp, encoding 267 and 263 amino acids, respectively. Multiple alignment, genomic synteny and phylogenetic analyses further suggested that elovl8 genes were unique to teleosts. Functional characterization by heterologous expression in yeast showed that Elovl8b could elongate C18 (18:2n-6, 18:3n-3 and 18:4n-3) and C20 (20:4n-6 and 20:5n-3) polyunsaturated fatty acids (PUFAs) to longer-chain polyunsaturated fatty acids (LC-PUFAs) whereas Elovl8a lacked this ability. In vitro, the expression of elovl8b but not elovl8a in rabbitfish hepatocytes was significantly up-regulated by incubation with 18:2n-6, 18:3n-3, 20:4n-6 and 20:5n-3, respectively. In vivo, compared with fish oil, dietary vegetable oil enriched in C18 PUFA enhanced the expression of elovl8b in rabbitfish brain, liver, intestine and gill tissues. These findings suggest that elovl8b but not elovl8a is a novel active member of the Elovl protein family involved in the LC-PUFAs biosynthesis pathway in rabbitfish, and provide novel insight into the mechanisms of LC-PUFAs biosynthesis in teleost.

CsHT11 encodes a pollen-specific hexose transporter and is induced under high level sucrose in pollen tubes of cucumber (Cucumis sativus)

Pollen tubes require a high amount of sugars to sustain high growth rate. Sucrose, the main transport form of sugars, can serve as energy supply and as signaling molecules for pollen tube growth. We report the functional characterization of CsHT11, which is a sugar transporter protein/hexose transport protein (STP/HT). CsHT11 shares high homology with the characterized cucumber hexose transporter CsHT1, a pollen-specific gene. Analysis of CsHT11 mRNA and CsHT11 promoter-reporter gene studies revealed CsHT11 specifically expressed in pollen and pollen tubes. Subcellular localization indicated that CsHT11 is a plasma membrane transporter. Heterologous expression in yeast suggests that CsHT11 is an energy-dependent hexose/H+ symporter, with a wide variety of substrate specificity, including glucose, fructose, galactose, and mannose. In vitro pollen germination of different sugars shows that the expression of CsHT11 is significantly increased with higher sucrose content, but not with higher glucose or fructose content, thereby implying that CsHT11 may be involved in sucrose signal transduction. Thus, CsHT11 might have an essential effect on pollen development and pollen tube growth in cucumber.

Identification and functional characterization of squalene epoxidases and oxidosqualene cyclases from Tripterygium wilfordii.

We cloned two squalene epoxidases and five oxidosqualene cyclases, and identified their function using CRISPR/Cas9 tool and yeast heterologous expression. Triterpenes are the main active ingredients of Tripterygium wilfordii Hook.f., a traditional Chinese medicinal plant with many encouraging preclinical applications. However, the biosynthetic pathways of triterpenes in this plant are poorly understood. Here, we report on the isolation and identification of two squalene epoxidases (SQE6 and SQE7) and five oxidosqualene cyclases (OSC4-8) from T. wilfordii. Yeast complementation assays showed that TwSQE6 and TwSQE7 can functionally complement an erg1 yeast mutant that was constructed using the CRISPR/Cas9 system. The putative OSC genes were functionally characterized by heterologous expression in yeast. GC/MS analysis of the fermentation products of the transgenic yeast showed that both TwOSC4 and TwOSC6 are cycloartenol synthases, while TwOSC8 is a β-amyrin synthase. The discovery of these genes expands our knowledge of key enzymes in triterpenoid biosynthesis, and provides additional target genes for increasing the production of triterpenes in T. wilfordii tissue cultures by disrupting competing pathways, or in chassis cells by reconstituting the triterpenoid biosynthetic pathway.

Candidate genes-association study to identify loci related to oleic acid in Brassica napus using SNP markers and their heterologous expression in yeast

Brassica napus (rapeseed) serves as a main source of edible oil, and the oil's quality is mainly determined by the relative proportions of fatty acids. A high oleic acid concentration in B. napus oil increases its shelf life and oxidative stability. Therefore, attaining a high oleic acid concentration is necessary to enhance the nutritional quality of rapeseed oil. Here, an association study of candidate genes was conducted using a population of 324 genetically diverse rapeseed accessions, and several loci related to oleic acid content were identified. Furthermore, these loci were functionally characterized in Saccharomyces cerevisiae to assess their functions, and the promising candidate loci were validated using single nucleotide polymorphic markers in an independent inbred population. The results increased our understanding of fatty acid metabolism in B. napus. Moreover, these findings may assist in marker-based breeding efforts to improve the fatty acid composition and quality of B. napus oil.

Mycorrhizal symbiosis induces divergent patterns of transport and partitioning of Cd and Zn in Populus trichocarpa

We investigated how arbuscular mycorrhizal symbiosis can alter trace element uptake, distribution and toxicity in plants by examining some of the molecular mechanisms behind Populus trichocarpa tolerance to Cd and Zn, and the effects of AMF in metal homeostasis. Plants were grown under Cd and Zn contamination, with and without Rhizophagus irregularis inoculation. We determined organ metal concentrations, the expression of genes involved in trace element homeostasis, and the function of metallothionein PtMT2b by heterologous expression in yeast. P. trichocarpa was highly tolerant to both elements, with AMF increasing Zn accumulation. AMF altered the partitioning of Cd, but maintained the same patterns for Zn, indicating that despite being geochemically similar and carried mostly by the same transporters, the nutrient metal (Zn) is handled differently from the non-essential metal (Cd). High Zn and Cd down-regulated PtHMA4 (roots), and up-regulated PtZIP1 (leaves), suggesting their involvement in transporting both metals in poplar. PtMT2b was highly up-regulated in mycorrhizal roots and enhanced Cd tolerance in transformed yeast. R. irregularis reduced Cd transfer to poplar shoots, but did not affect Zn partitioning. The gene expression patterns observed offer a glimpse into the mechanisms behind trace element uptake/translocation dynamic in poplars, influenced by AMF symbiosis.

The catadromous teleost Anguilla japonica has a complete enzymatic repertoire for the biosynthesis of docosahexaenoic acid from α-linolenic acid: Cloning and functional characterization of an Elovl2 elongase.

The Japanese eel Anguilla japonica is a catadromous fish species with considerable farming scale. Previous studies showed that dietary α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) satisfied essential fatty acid requirements in eel, which suggested that Japanese eel should have a complete pathway for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA). However, existing knowledge was insufficient to explain the molecular basis of LC-PUFA biosynthetic capacity in eel. In order to further characterize this pathway in eel, a full-length cDNA of a putative fatty acyl elongase was isolated, with the ORF encoding a protein with 294 amino acids. The putative elongase displayed high homology to Elovl2 of other teleosts. Functional characterization by heterologous expression in yeast showed the protein product of the cDNA had high activity towards C20 and C22 PUFA substrates and low activity towards C18 PUFA substrates, characteristic of Elovl2 elongases. Tissue distribution of the elovl2 mRNA showed highest expression in brain and eyes, which was different from freshwater and anadromous species. This may reflect an important role for this enzyme in the in situ endogenous biosynthesis of docosahexaenoic acid (DHA) in neural tissues in eel. This is the first report of an Elovl2 in a catadromous teleost and demonstrates that Japanese eel has a complete enzyme repertoire required for the endogenous biosynthesis of DHA via the Sprecher pathway. These data have increased our knowledge of the diversity of LC-PUFA biosynthesis in vertebrates, and provided further insight into the regulatory mechanisms of LC-PUFA biosynthesis in teleost fish.

ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas.

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.