Assessment of potential environmental risks of transgene flow in smallholder farming systems in Asia: Brassica napus as a case study in Korea.

The cultivation of genetically modified (GM) crops has raised many questions regarding their environmental risks, particularly about their ecological impact on non-target organisms, such as their closely-related relative species. Although evaluations of transgene flow from GM crops to their conventional crops has been conducted under large-scale farming system worldwide, in particular in North America and Australia, few studies have been conducted under smallholder farming systems in Asia with diverse crops in co-existence. A two-year field study was conducted to assess the potential environmental risks of gene flow from glufosinate-ammonium resistant (GR) Brassica napus to its conventional relatives, B. napus, B. juncea, and Raphanus sativus under simulated smallholder field conditions in Korea. Herbicide resistance and simple sequence repeat (SSR) markers were used to identify the hybrids. Hybridization frequency of B. napus × GR B. napus was 2.33% at a 2 m distance, which decreased to 0.007% at 75 m. For B. juncea, it was 0.076% at 2 m and decreased to 0.025% at 16 m. No gene flow was observed to R. sativus. The log-logistic model described hybridization frequency with increasing distance from GR B. napus to B. napus and B. juncea and predicted that the effective isolation distances for 0.01% gene flow from GR B. napus to B. napus and B. juncea were 122.5 and 23.7 m, respectively. Results suggest that long-distance gene flow from GR B. napus to B. napus and B. juncea is unlikely, but gene flow can potentially occur between adjacent fields where the smallholder farming systems exist.

Evaluation of maximum potential gene flow from herbicide resistant Brassica napus to its male sterile relatives under open and wind pollination conditions.

Pollen-mediated gene flow (PMGF) from genetically modified (GM) Brassica napus to its wild relatives by wind and insects is a major ecological concern in agricultural ecosystems. This study conducted is to estimate maximum potential gene flow and differentiate between wind- and bee-mediated gene flows from herbicide resistant (HR) B. napus to its closely-related male sterile (MS) relatives, B. napus, B. juncea and Raphanus sativus. Various markers, including pods formation in MS plants, herbicide resistance, and SSR markers, were used to identify the hybrids. Our results revealed the following: 1) maximum potential gene flow (a maximum % of the progeny of pollen recipient confirmed hybrid) to MS B. napus ranged from 32.48 to 0.30% and from 14.69 to 0.26% at 2-128 m from HR B. napus under open and wind pollination conditions, respectively, and to MS B. juncea ranged from 21.95 to 0.24% and from 6.16 to 0.16%, respectively; 2) estimates of honeybee-mediated gene flow decreased with increasing distance from HR B. napus and ranged from 17.78 to 0.03% at 2-128 m for MS B. napus and from 15.33 to 0.08% for MS B. juncea; 3) a small-scale donor plots would strongly favour insect over wind pollination; 4) no gene flow occurred from HR B. napus to MS R. sativus. Our approach and findings are helpful in understanding the relative contribution of wind and bees to gene flow and useful for estimating maximum potential gene flow and managing environmental risks associated with gene flow.

Functional identification of oleate 12-desaturase and ω-3 fatty acid desaturase genes from Perilla frutescens var. frutescens.

KEY MESSAGE: We described identification, expression, subcellular localization, and functions of genes that encode fatty acid desaturase enzymes in Perilla frutescens var. frutescens. Perilla (Perilla frutescens var. frutescens) seeds contain approximately 40 % of oil, of which α-linolenic acid (18:3) comprise more than 60 % in seed oil and 56 % of total fatty acids (FAs) in leaf, respectively. In perilla, endoplasmic reticulum (ER)-localized and chloroplast-localized ω-3 FA desaturase genes (PfrFAD3 and PfrFAD7, respectively) have already been reported, however, microsomal oleate 12-desaturase gene (PfrFAD2) has not yet. Here, four perilla FA desaturase genes, PfrFAD2-1, PfrFAD2-2, PfrFAD3-2 and PfrFAD7-2, were newly identified and characterized using random amplification of complementary DNA ends and sequence data from RNAseq analysis, respectively. According to the data of transcriptome and gene cloning, perilla expresses two PfrFAD2 and PfrFAD3 genes, respectively, coding for proteins that possess three histidine boxes, transmembrane domains, and an ER retrieval motif at its C-terminal, and two chloroplast-localized ω-3 FA desaturase genes, PfrFAD7-1 and PfrFAD7-2. Arabidopsis protoplasts transformed with perilla genes fused to green fluorescence protein gene demonstrated that PfrFAD2-1 and PfrFAD3-2 were localized in the ER, and PfrFAD7-1 and PfrFAD7-2 were localized in the chloroplasts. PfrFAD2 and perilla ω-3 FA desaturases were functional in budding yeast (Saccharomyces cerevisiae) indicated by the presence of 18:2 and 16:2 in yeast harboring the PfrFAD2 gene. 18:2 supplementation of yeast harboring ω-3 FA desaturase gene led to the production of 18:3. Therefore, perilla expresses two functional FAD2 and FAD3 genes, and two chloroplast-localized ω-3 FA desaturase genes, which support an evidence that P. frutescens cultivar is allotetraploid plant.

Heterologous Reconstitution of Omega-3 Polyunsaturated Fatty Acids in Arabidopsis.

Reconstitution of nonnative, very-long-chain polyunsaturated fatty acid (VLC-PUFA) biosynthetic pathways in Arabidopsis thaliana was undertaken. The introduction of three primary biosynthetic activities to cells requires the stable coexpression of multiple proteins within the same cell. Herein, we report that C22 VLC-PUFAs were synthesized from C18 precursors by reactions catalyzed by Δ(6)-desaturase, an ELOVL5-like enzyme involved in VLC-PUFA elongation, and Δ(5)-desaturase. Coexpression of the corresponding genes (McD6DES, AsELOVL5, and PtD5DES) under the control of the seed-specific vicilin promoter resulted in production of docosapentaenoic acid (22:5 n-3) and docosatetraenoic acid (22:4 n-6) as well as eicosapentaenoic acid (20:5 n-3) and arachidonic acid (20:4 n-6) in Arabidopsis seeds. The contributions of the transgenic enzymes and endogenous fatty acid metabolism were determined. Specifically, the reasonable synthesis of omega-3 stearidonic acid (18:4 n-3) could be a useful tool to obtain a sustainable system for the production of omega-3 fatty acids in seeds of a transgenic T3 line 63-1. The results indicated that coexpression of the three proteins was stable. Therefore, this study suggests that metabolic engineering of oilseed crops to produce VLC-PUFAs is feasible.

Coexpression of multiple genes reconstitutes two pathways of very long-chain polyunsaturated fatty acid biosynthesis in Pichia pastoris.

The introduction of novel traits to cells often requires the stable coexpression of multiple genes within the same cell. Herein, we report that C22 very long-chain polyunsaturated fatty acids (VLC-PUFAs) were synthesized from C18 precursors by reactions catalyzed by delta 6-desaturase, an ELOVL5 involved in VLC-PUFA elongation, and delta 5-desaturase. The coexpression of McD6DES, AsELOVL5, and PtD5DES encoding the corresponding enzymes, produced docosatetraenoic acid (C22:4 n-6) and docosapentaenoic acid (C22:5 n-3), as well as arachidonic acid (C20:4 n-6) and eicosapentaenoic acid (C20:5 n-3) in the methylotrophic yeast Pichia pastoris. The expression of each gene increased within 24 h, with high transcript levels after induction with 0.5 or 1 % methanol. High levels of the newly expressed VLC-PUFAs occurred after 144 h. This expression system exemplifies the recent progress and future possibilities of the metabolic engineering of VLC-PUFAs in oilseed crops.

Identification and functional characterization of polyunsaturated fatty acid elongase (McELOVL5) gene from pike eel (Muraenesox cinereus).

The cDNA coding for a polyunsaturated fatty acid elongase (McELOVL5) was isolated from the brain of the pike eel (Muraenesox cinereus) being based on available sequences in 23 types of fish. Four sequence variants were identified with different amino acid substitutions as compared with two clones of McELOVL5 gene (McELOVL5 11.7 and McELOVL5 12.4). When the two variants of McELOVL5 were expressed in Saccharomyces cerevisiae, the two recombinant yeasts elongated γ-linolenic acid (GLA, 18:3n-6) to di-homo-γ-linolenic acid (DGLA, 20:3n-6) but differed in the rate of GLA conversion to DGLA. Cells transformed with McELOVL5 12.4 also converted arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) to docosatetraenoic acid (22:4n-6) and docosapentaenoic acid (22:5n-3), respectively. However McELOVL5 11.7 lost its function for the elongation of C20 fatty acids. The four sequence variants have changed substrate specificities. Three-dimensional models of the McELOVL5 proteins are suggested.

Functional characterization of polyunsaturated fatty acid delta 6-desaturase and elongase genes from the black seabream (Acanthopagrus schlegelii).

Fatty acid delta 6-desaturase (D6DES) and elongases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs) including arachidonic acid (ARA) and eicosapentaenoic acid (EPA) from microorganisms to higher animals. To identify the genes encoding D6DES and elongases for PUFAs, we isolated each cDNA with a high similarity to the D6DES and ELOVL5-like elongases of mammals and fishes via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii. A recombinant vector expressing AsD6DES was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity toward n-6 and n-3 fatty acids in the PUFA biosynthesis. The heterologously expressed AsD6DES produced γ-linolenic acid (GLA, C18:3 n-6) and stearidonic acid (STA, C18:4 n-3) at conversion rates of 26.3-35.6% from exogenous linoleic acid (LA, C18:2 n-6) and α-linolenic acid (ALA, C18:3 n-3) substrates, respectively. When AsELOVL5 was expressed in yeast, it conferred an ability to elongate GLA to di-homo-γ-linolenic acid (DGLA, C20:3 n-6). In addition, AsELOVL5 showed an ability to convert ARA (C20:4 n-6) and EPA (C20:5 n-3) to dodecylthioacetic acid (DTA, C22:4 n-6) and docosapentaenoic acid (DPA, C22:5 n-3), respectively. In these results, the AsD6DES encodes a delta 6-fatty acid desaturase and the AsELOVL5 encoding a long-chain fatty acid elongase shows activity to enlongate C18Δ6/C20Δ5, but not C22.

Ectopic overexpression of castor bean LEAFY COTYLEDON2 (LEC2) in Arabidopsis triggers the expression of genes that encode regulators of seed maturation and oil body proteins in vegetative tissues.

The LEAFY COTYLEDON2 (LEC2) gene plays critically important regulatory roles during both early and late embryonic development. Here, we report the identification of the LEC2 gene from the castor bean plant (Ricinus communis), and characterize the effects of its overexpression on gene regulation and lipid metabolism in transgenic Arabidopsis plants. LEC2 exists as a single-copy gene in castor bean, is expressed predominantly in embryos, and encodes a protein with a conserved B3 domain, but different N- and C-terminal domains to those found in LEC2 from Arabidopsis. Ectopic overexpression of LEC2 from castor bean under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis plants induces the accumulation of transcripts that encodes five major transcription factors (the LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON1-LIKE (L1L), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) transcripts for seed maturation, and WRINKELED1 (WRI1) transcripts for fatty acid biosynthesis), as well as OLEOSIN transcripts for the formation of oil bodies in vegetative tissues. Transgenic Arabidopsis plants that express the LEC2 gene from castor bean show a range of dose-dependent morphological phenotypes and effects on the expression of LEC2-regulated genes during seedling establishment and vegetative growth. Expression of castor bean LEC2 in Arabidopsis increased the expression of fatty acid elongase 1 (FAE1) and induced the accumulation of triacylglycerols, especially those containing the seed-specific fatty acid, eicosenoic acid (20:1(Δ11)), in vegetative tissues.

Isolation and functional characterization of a delta 6-desaturase gene from the pike eel (Muraenesox cinereus).

Stearidonic acid (STA; 18:4n-3) and γ-linolenic acid (GLA; 18:3n-6) are significant intermediates in the biosynthetic pathway for the very-long-chain polyunsaturated fatty acids of eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6), respectively. To develop a sustainable system for the production of dietary polyunsaturated fatty acids, we focused on the action of the enzyme delta 6-desaturase (D6DES) on the essential acids, linoleic acid (LA; 18:2n-6) and α-linolenic acid (ALA; 18:3n-3). A 1,335-bp full-length cDNA encoding D6DES (McD6DES) was cloned from Muraenesox cinereus using degenerate PCR and RACE-PCR methods. To investigate the enzymatic activity of McD6DES in the production of n-6 and n-3 fatty acids, a recombinant plasmid expressing McD6DES (pYES-McD6DES) was transformed into and expressed in Saccharomyces cerevisiae. The exogenously expressed McD6DES produced GLA and STA at conversion rates of 14.2% and 45.9%, respectively, from the exogenous LA and ALA substrates. These results indicate that McD6DES is essentially a delta 6-desaturase involved in very-long-chain polyunsaturated fatty acid synthesis.

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea.

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.

Molecular cloning and functional analysis of two FAD2 genes from American grape (Vitis labrusca L.).

The synthesis of polyunsaturated fatty acids (PUFAs), the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase 2 (FAD2; EC 1.3.1.35), also called microsomal oleate Δ12-desaturase. Since the FAD2 gene was first identified in Arabidopsis thaliana, FAD2 research has gained wide interest as the essential enzyme for synthesizing PUFA. Grapes are one of the most frequently cultivated fruits in the world, with most commercial growers cultivating Vitis vinifera and V. labrusca. Grapeseed oil contains a high proportion, 60-70% of linoleic acid (18:2). We cloned two putative FAD2 genes from V. labrusca cv. Campbell Early based on V. vinifera genome sequences. Deduced amino acid sequences of two putative genes showed that VlFAD2s show high similarity to Arabidopsis FAD2 and commonly contain six transmembrane domain, three histidine boxes and endoplasmic reticulum (ER) retrieval motif representing the characteristics of fatty acid desaturase. Phylogenetic analyses of various plant FAD2s showed that VlFAD2-1 and VlFAD2-2 are separately grouped with constitutive and seed-type FAD2s, respectively. Southern blot showed that one or two bands are found in each lane. Because Campbell Early is a hybrid cultivar, FAD2-1 and FAD2-2 genes may exist as one copy in V. labrusca. Expression analysis in different tissues indicated that VlFAD2-1 is a constitutive gene but VlFAD2-2 is a seed-type gene. Complementation experiments of fad2-1 mutant Arabidopsis with VlFAD2-1 or VlFAD2-2 demonstrated that VlFAD2-1 and VlFAD2-2 can restore low PUFA proportion of fad2 to normal PUFA proportion.

Isolation and functional characterization of polyunsaturated fatty acid elongase (AsELOVL5) gene from black seabream (Acanthopagrus schlegelii).

To identify the genes encoding fatty acid elongases for the biosynthesis of polyunsaturated fatty acids (PUFAs), we isolated a cDNA via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii with a high similarity to the ELOVL5-like elongases of mammals and fishes. This gene is termed AsELOVL5 and encodes a 294 amino acid protein. When AsELOVL5 was expressed in Saccharomyces cerevisiae, it conferred an ability to elongate γ-linolenic acid (18:3 n-6) to di-homo-γ-linolenic acid (20:3 n-6). In addition, the transformed cells converted arachidonic acid (20:4 n-6) and eicosapentaenpic acid (20:5 n-3) to docosatetraenoic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-3), respectively. These results indicate that the AsELOVL5 gene encodes a long-chain fatty acid elongase capable of elongating C(18)Δ6/C(20)Δ5 but not C(22) PUFA substrates.

Exploiting leaf starch synthesis as a transient sink to elevate photosynthesis, plant productivity and yields.

Improvements in plant productivity (biomass) and yield have centered on increasing the efficiency of leaf CO(2) fixation and utilization of products by non-photosynthetic sink organs. We had previously demonstrated a correlation between photosynthetic capacity, plant growth, and the extent of leaf starch synthesis utilizing starch-deficient mutants. This finding suggested that leaf starch is used as a transient photosynthetic sink to recycle inorganic phosphate and, in turn, maximize photosynthesis. To test this hypothesis, Arabidopsis thaliana and rice (Oryza sativa L.) lines were generated with enhanced capacity to make leaf starch with minimal impact on carbon partitioning to sucrose. The Arabidopsis engineered plants exhibited enhanced photosynthetic capacity; this translated into increased growth and biomass. These enhanced phenotypes were displayed by similarly engineered rice lines. Manipulation of leaf starch is a viable alternative strategy to increase photosynthesis and, in turn, the growth and yields of crop and bioenergy plants.

Functional characterization of a delta 6-desaturase gene from the black seabream (Acanthopagrus schlegeli).

Delta 6-fatty acid desaturase (D6DES) is used in the synthesis of polyunsaturated fatty acids (PUFAs) from microorganisms to higher animals, including arachidonic acid (ARA) and eicosapentaenoic acid (EPA). A 1,338 bp full-length cDNA encoding D6DES was cloned from Acanthopagrus schlegeli (AsD6DES) through degenerate- and RACE-PCR methods. A recombinant vector expressing AsD6DES (pYES-AsD6DES) was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity of AsD6DES towards the production of n-6 and n-3 fatty acids. The exogenously expressed AsD6DES produced γ-linolenic acid (18:3 n-6) and stearidonic acid (18:4n-3) at 26 and 36% from exogenous linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), respectively, indicating that it is essentially a delta 6-fatty acid desaturase.

Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa).

The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches.

Genes up-regulated during red coloration in UV-B irradiated lettuce leaves.

Molecular analysis of gene expression differences between green and red lettuce leaves was performed using the SSH method. BlastX comparisons of subtractive expressed sequence tags (ESTs) indicated that 7.6% of clones encoded enzymes involved in secondary metabolism. Such clones had a particularly high abundance of flavonoid-metabolism proteins (6.5%). Following SSH, 566 clones were rescreened for differential gene expression using dot-blot hybridization. Of these, 53 were found to overexpressed during red coloration. The up-regulated expression of six genes was confirmed by Northern blot analyses. The expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and dihydroflavonol 4-reductase (DFR) genes showed a positive correlation with anthocyanin accumulation in UV-B-irradiated lettuce leaves; flavonoid 3',5'-hydroxylase (F3',5'H) and anthocyanidin synthase (ANS) were expressed continuously in both samples. These results indicated that the genes CHS, F3H, and DFR coincided with increases in anthocyanin accumulation during the red coloration of lettuce leaves. This study show a relationship between red coloration and the expression of up-regulated genes in lettuce. The subtractive cDNA library and EST database described in this study represent a valuable resource for further research for secondary metabolism in the vegetable crops.