Genetic Improvement of Oilseed Crops Using Modern Biotechnology

In 2009, big challenges facing the agricultural sector in the twenty-first century were presented to the world. Human population growth, increased life expectancy, loss of biodiversity, climate change and accelerated land degradation are the main factors contributing to rethink agriculture system production. In that scenery, modern biotechnology has set a stage for the advancement of agricultural practices and it is clearly an important ally to apply a broad array of technologies and innovative systems where they are most needed, such as enhancing crop productivity, increasing yields, and ultimately ensuring food security. One of the biggest challenges is related to technify production systems, but with no doubt, developing genetic improvement toward getting an efficient and sustainable agriculture, generating new seed qualities (new traits), such as, among others, to upset fatty acids content in oilseed crops have been growing up significantly due to industry interest. In this study, a review about the main advances in genetic improvement of some oilseed crops, starting with omics to understand metabolic routes and to find out key genes in seed oil production, and also, getting in use of modern biotechnology to alter the production of fatty acids, and to face biotic challenges in oilseed crops is presented

Genome-wide profiling of soybean WRINKLED1 transcription factor binding sites provides insight into seed storage lipid biosynthesis.

Understanding the regulatory mechanisms controlling storage lipid accumulation will inform strategies to enhance seed oil quality and quantity in crop plants. The WRINKLED1 transcription factor (WRI1 TF) is a central regulator of lipid biosynthesis. We characterized the genome-wide binding profile of soybean (Gm)WRI1 and show that the TF directly regulates genes encoding numerous enzymes and proteins in the fatty acid and triacylglycerol biosynthetic pathways. GmWRI1 binds primarily to regions downstream of target gene transcription start sites. We showed that GmWRI1-bound regions are enriched for the canonical WRI1 DNA binding element, the ACTIVATOR of Spomin::LUC1/WRI1 (AW) Box (CNTNGNNNNNNNCG), and another DNA motif, the CNC Box (CNCCNCC). Functional assays showed that both DNA elements mediate transcriptional activation by GmWRI1. We also show that GmWRI1 works in concert with other TFs to establish a regulatory state that promotes fatty acid and triacylglycerol biosynthesis. In particular, comparison of genes targeted directly by GmWRI1 and by GmLEC1, a central regulator of the maturation phase of seed development, reveals that the two TFs act in a positive feedback subcircuit to control fatty acid and triacylglycerol biosynthesis. Together, our results provide unique insights into the genetic circuitry in which GmWRI1 participates to regulate storage lipid accumulation during seed development

Identification and characterization of genes involved in metabolism of n5 monoene precursors to n5 anacardic acids in the trichomes of Pelargonium x hortorum.

Unusual monoenoic fatty acids (UMFA’s) and specialized metabolites called anacardic acids (AnAc) are produced in glandular trichomes of Pelargonium ´ hortorum (geranium). The UMFA’s, 16:1∆11 and 18:1∆13 are precursors for the synthesis of unsaturated AnAc 22:1n5and 24:1n5 that contribute to pest resistance in geraniums. UMFAs and their derived AnAc metabolites not only provide a useful biological marker that differentiates the biosynthetic pathway for unusual mononenes from the common fatty acids (i.e. stearic, palmitic, oleic, linoleic and linolenic) but also have industrial, medical and agricultural applications. Fatty acid biosynthesis enzymes like acyl carrier proteins (ACPs); thioesterases (TEs) and β-ketoacyl-ACP synthases (KASs) are required for common fatty acid as well as the UMFA biosynthesis. Based on this, it is hypothesized that the specific isoforms of the fatty acid biosynthesis enzymes are highly expressed in trichomes and are involved specifically in metabolic channeling of UMFAs to anacardic acid synthesis within trichomes of geranium. This hypothesis is based on the knowledge that there is a novel Δ9 myristoyl-ACP desaturase (MAD) that directs acyl-ACP into UMFA biosynthesis and the products of MAD are correlated with the dominant congeners of AnAc (22:1n5 and 24:1n5). Transcription of MAD as well as production of 16:1∆11 and 18:1∆13 and AnAc 22:1n5 and AnAc 24:1n5 has been found to be highly trichome specific. This dissertation reports the identification of the complete nucleotide and protein sequences of genes for 2 ACPs, 3 FAT-As, 3 FAT-Bs, 4 KAS Is, 1 KAS II and 1 KAS III from a geranium EST database. Quantitative real-time PCR (qRT-PCR) was used to analyze tissue-specific expression patterns of the target genes, which indicated that ACP 1, ACP 2, KAS I-a/b, KAS Ic, FAT-A1, and FAT-A2 are highly expressed in trichomes. To further this research, a de novo RNA and micro-RNA transcriptome was generated from trichomes and bald pedicle of geranium, which helped in identification of several genetic components involved in UMFA synthesis. Bioinformatics analysis of RNA-transcriptome along with qRT-PCR and biochemical assays (HPLC and GC) were used to correlate the effect of temperature (18°C, 23°C and 28°C) on gene expression (ACPs, KASs, FAT-As) and changes in production of 16:1Δ11 and 18:1Δ13 UMFAs and 22:1n5and 24:1n5 AnAc. Results of this work show that expression of ACP 1, ACP 2, KAS I-c, KAS I-a/b were correlated with changes in UMFAs and AnAc production with temperature, thus indicating their potential role in UMFA metabolism. We also determined that 23°C is an optimal temperature for production of UMFAs and AnAcs as compared to 18°C and 28°C. To determine and verify the function of ACP 1 and ACP 2, we co-expressed these genes in conjunction with a Δ9 myristoyl-ACP (MAD) desaturase in both E. coli and tobacco. E. coli assay results show that expression of ACP 2 with MAD increased the production of UMFAs significantly, thus validating the novel role of ACP 2 in UMFA production. This work, in addition to the generation of a de novo transcriptome, provides a platform for further defining UMFA metabolism within trichomes of geranium

Transcriptome Analysis Revealed the Dynamic Oil Accumulation in \u3ci\u3eSymplocos paniculata\u3c/i\u3e Fruit

Background Symplocos paniculata, asiatic sweetleaf or sapphire berry, is a widespread shrub or small tree from Symplocaceae with high oil content and excellent fatty acid composition in fruit. It has been used as feedstocks for biodiesel and cooking oil production in China. Little transcriptome information is available on the regulatory molecular mechanism of oil accumulation at different fruit development stages. Results The transcriptome at four different stages of fruit development (10, 80,140, and 170 days after flowering) of S. paniculata were analyzed. Approximately 28 million high quality clean reads were generated. These reads were trimmed and assembled into 182,904 non-redundant putative transcripts with a mean length of 592.91 bp and N50 length of 785 bp, respectively. Based on the functional annotation through Basic Local Alignment Search Tool (BLAST) with public protein database, the key enzymes involved in lipid metabolism were identified, and a schematic diagram of the pathway and temporal expression patterns of lipid metabolism was established. About 13,939 differentially expressed unigenes (DEGs) were screened out using differentially expressed sequencing (DESeq) method. The transcriptional regulatory patterns of the identified enzymes were highly related to the dynamic oil accumulation along with the fruit development of S. paniculata. In addition, quantitative real-time PCR (qRT-PCR) of six vital genes was significantly correlated with DESeq data. Conclusions The transcriptome sequences obtained and deposited in NCBI would enrich the public database and provide an unprecedented resource for the discovery of the genes associated with lipid metabolism pathway in S. paniculata. Results in this study will lay the foundation for exploring transcriptional regulatory profiles, elucidating molecular regulatory mechanisms, and accelerating genetic engineering process to improve the yield and quality of seed oil of S. paniculata. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3275-0) contains supplementary material, which is available to authorized users

Transcriptome analysis revealed the dynamic oil accumulation in Symplocos paniculata fruit

BACKGROUND: Symplocos paniculata, asiatic sweetleaf or sapphire berry, is a widespread shrub or small tree from Symplocaceae with high oil content and excellent fatty acid composition in fruit. It has been used as feedstocks for biodiesel and cooking oil production in China. Little transcriptome information is available on the regulatory molecular mechanism of oil accumulation at different fruit development stages. RESULTS: The transcriptome at four different stages of fruit development (10, 80,140, and 170 days after flowering) of S. paniculata were analyzed. Approximately 28 million high quality clean reads were generated. These reads were trimmed and assembled into 182,904 non-redundant putative transcripts with a mean length of 592.91 bp and N50 length of 785 bp, respectively. Based on the functional annotation through Basic Local Alignment Search Tool (BLAST) with public protein database, the key enzymes involved in lipid metabolism were identified, and a schematic diagram of the pathway and temporal expression patterns of lipid metabolism was established. About 13,939 differentially expressed unigenes (DEGs) were screened out using differentially expressed sequencing (DESeq) method. The transcriptional regulatory patterns of the identified enzymes were highly related to the dynamic oil accumulation along with the fruit development of S. paniculata. In addition, quantitative real-time PCR (qRT-PCR) of six vital genes was significantly correlated with DESeq data. CONCLUSIONS: The transcriptome sequences obtained and deposited in NCBI would enrich the public database and provide an unprecedented resource for the discovery of the genes associated with lipid metabolism pathway in S. paniculata. Results in this study will lay the foundation for exploring transcriptional regulatory profiles, elucidating molecular regulatory mechanisms, and accelerating genetic engineering process to improve the yield and quality of seed oil of S. paniculata. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-3275-0) contains supplementary material, which is available to authorized users

Increasing oil content in Brassica oilseed species

Brassica oilseed species are the third most important in the world, providing approximately 15 % of the total vegetable oils. Three species (Brassica rapa, B. juncea, B. napus) dominate with B. napus being the most common in Canada, China and Europe. Originally, B. napus was a crop producing seed with high erucic acid content, which still persists today, to some extent, and is used for industrial purposes. In contrast, cultivars which produce seed used for food and feed are low erucic acid cultivars which also have reduced glucosinolate content. Because of the limit to agricultural land, recent efforts have been made to increase productivity of oil crops, including Brassica oilseed species. In this article, we have detailed research in this regard. We have covered modern genetic, genomic and metabolic control analysis approaches to identifying potential targets for the manipulation of seed oil content. Details of work on the use of quantitative trait loci, genome-wide association and comparative functional genomics to highlight factors influencing seed oil accumulation are given and functional proteins which can affect this process are discussed. In summary, a wide variety of inputs are proving useful for the improvement of Brassica oilseed species, as major sources of global vegetable oil

A molecular genetic approach to reducing the saturated fatty acid content of canola oil

xxi, 155 leaves : ill. ; 29 cm.Brassica napus is known to contain an endogenous and soluble stearoyl-acyl carrier protein (^918:0-ACP) desaturase, but does not express a palmitic (16:0)-ACP desaturase. Levels of 16:0 are low in canola oil and are associated with enhanced cholesterol biosynthesis in humans. In an attempt to further reduce the saturated fatty acid (SFA) content of canola oil, B. Napus L. cv Westar was transformed with a cDNA encoding a ^916:0-ACP desaturase from cat's claw (Doxantha unguis-cati L.). Arabidopsis thaliana was also transformed with this cDNA. Transformation of both oilseeds resulted in increased production of palmitoleic acid (^16:1) and many other effects of fatty acid composition. Overall, the SFA content did not decrease in either oilseed and investigation to why this effect occurred was examined using transgenic B. napus. Molecular genetic testing on second generation B. napus also determined the plants contained the cDNA of interest and were transcribing the cDNA

Comparison of quantitative trait loci (QTLs) associated with yield components in two commercial Dura × Pisifera breeding crosses

The high yielding tenera is the commercial oil palm planting material of choice in Southeast Asia. Notwithstanding this, there is continuous effort to further improve the yield and one way to do this is by addressing the yield components (YCs ). Using 4,451 SNP and over 600 SSR markers , this study revealed quantitative trait loci (QTL) associated with YCs in two breeding populations, a Deli dura x Yangambi pisifera (P2) and a Deli dura x AVROS pisifera (KULIM DxP). Thirteen and 29 QTLs were identified in P2 and KULIM DxP, respectively . They were compared to other YC-linked QTLs reported previously for different genetic backgrounds by mapping the QTL-linked markers to the oil palm genome . The comparison revealedfour common chromosomes containing QTLs influencing various YCs . The results reveal the possible presence of closely linked loci or pleiotropic genes influencing YCs in oil palm. Exploiting the genome data has also facilitated the discovery of candidate genes within or near the QTL regions including those related to glycosylation, fatty acid and oil biosynthesis, and development of flower, seed and fruit

Over-expression and characterisation of Brassica napus and Escherichia coli 3-oxoacyl-[acyl carrier protein] reductase

A full length cDNA clone of Brassica napus 3-oxoacyl-ACP reductase (β-ketoacyl-ACP reductase; E.C. 1.1.1.100; βKR) and the Escherichia coli gene for the same enzyme, have been over-expressed in E. coli. Both the Brassica napus seed and Escherichia coli βKR proteins have been purified by a rapid two-step, single chromatography matrix method. Glutaraldehyde cross-linking studies show the plant βKR is expressed as a tetramer and the E. coli enzyme is expressed as a dimer. The secondary structure of the two proteins was predicted via analysis of circular dichroism spectra, which also show dilution dependent unfolding of a-helical structure in the plant enzyme, a possible explanation for the dilution inactivation of βKRs. Ultrafiltration substrate binding studies and a bireactant initial velocity study show that Brassica napus βKR employs a fixed order ternary complex mechanism with NADPH binding to the enzyme first. One-dimensional western blot analysis indicates two isoforms of βKR (28 kDa and 31 kDa) in crude B. napus seed extracts. Further analysis using two- dimensional western blots demonstrates the presence of four major isoforms. Comparison with 2D blots from B. campestris suggests that one of the major isoforms has originated from that source. The crystal structure of the E. coli βKR enzyme is also discussed