Molecular cloning and characterization of an acyl-ACP thioesterase gene (AhFatB1) from allotetraploid peanut (Arachis hypogaea L.)

Acyl-acyl carrier protein (ACP) thioesterase is a nuclear encoded plastid localized enzyme which plays an essential role in chain termination during de novo fatty acid synthesis in plant. FatB genes coding for this enzyme from a variety of plant species have been isolated and characterized. However, there are few reports on such genes in peanut (Arachis hypogaea), an important edible and oilseed crop. In this study, full-length cDNA of an acyl-acyl carrier protein thioesterase (EC 3.1.2.14), designated as AhFatB1, was isolated from peanut cDNA libraries. The putative open reading frames consist of 1239 bp with five introns spliced from the corresponding genomic sequence, encoding a 413 amino acid protein, two homologous genes, AhFatB1A and AhFatB1B, with sequence difference at the 5’ non-coding regions were characterized at the nucleotide level from different cultivated peanut genotypes, and the two genes have their origin in different diploid progenitor which was evidenced by the characterization of AhFatB1 genes from Arachis duranensis and Arachis ipaensis, the putative A-genome donor and B-genome donor respectively. AhFatB1 genes are constitutively expressed in peanut tissues and the total FatB1 transcript accumulations are temporally regulated during peanut seed development.Keywords: Peanut thioesterase, palmitic acid oilsee

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

Structure and Function in Plant Ä12 Fatty Acid Desaturases and Acetylenases

This study provides insight into the structure/function relationship between desaturases and acetylenases, and indicates amino acid residues within acetylenases which influence reaction outcome. Oleate desaturases belong to a family of enzymes capable of introducing cis double bonds between C12 - C13 in oleate esters. Acetylenases are a subset of oleate desaturase enzymes which introduce a triple bond in the C12 - C13 position of linoleate. To better understand which amino acids could be responsible for differentiating the activity of acetylenases from typical desaturases, a total of 50 protein sequences were used to compare the two classes of enzymes resulting in the identification of 11 amino acid residues which are conserved within either separate family but differ between the two groups of enzymes. These identified amino acid residues were then singularly altered by site-directed mutagenesis to test their role in fatty acid modification. Specifically, the wild type acetylenase, Crep1 from Crepis alpina, and a number of point mutants have been expressed in Saccharomyces cerevisiae, followed by fatty acid analysis of the resulting cultures. Results indicate the importance of 4 amino acid residues within Crep1 (Y150, F259, H266, and V304) with regards to desaturase and acetylenase chemoselectivity, stereoselectivity, and/or substrate recognition. The F259L mutation affected the acetylenase by converting it to an atypical FAD2 capable of producing both cis and trans isomers. The V304I mutation resulted in the conversion of Crep1 into a stereoselective FAD2, where only the cis isomers of 16:2 and 18:2 were produced. The Y150F mutation led to a loss of acetylenase activity without affecting the inherent desaturase activity of Crep1. The H266Q mutation appears to affect substrate selection causing an inability to bind substrate (16:1-9c and/or 18:1-9c) in a cisoid conformation, resulting in an increased accumulation of trans product. The changes in enzyme activity detected in cultures expressing Crep1 mutants demonstrate the profound effect that exchanging as little as one amino acid can have on an enzyme properties. Enzymes retain some conservation of amino acids necessary for activity, such as those involved in metal ion binding, whereas subtle changes can affect overall enzyme function and catalysis

Identification of differentially expressed genes in seeds of two Brassica napus mutant lines with different oleic acid content

The regulation of seed oleic acid synthesis in rapeseed is largely unknown. In this study, gene expression pattern during seed development between two Brassica napus mutants was compared. Using immature seeds 27 days after pollination, differentially expressed cDNA clones were identified bysubtractive suppression hybridization (SSH). A total of 480 cDNA clones corresponding to 88 genes were found up-regulated and 18 genes down-regulated in seeds with high oleic acid content. Most ofthe differentially expressed genes are related to metabolism and regulation. The possible role of these genes in seeds was discussed. Further analysis of the function of these genes may provide novel targets for manipulation of fatty acid composition in rapeseed

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

How temperatures may affect the synthesis of fatty acids during olive fruit ripening: genes at work in the field

A major concern for olive cultivation in many extra-Mediterranean regions is the adaptation of recently introduced cultivars to environmental conditions different from those prevailing in the original area, such as the Mediterranean basin. Some of these cultivars can easily adapt their physiological and biochemical parameters in new agro-environments, whereas others show unbalanced values of oleic acid content. The objective of this study was to evaluate the effects of the thermal regime during oil synthesis on the expression of fatty acid desaturase genes and on the unsaturated fatty acid contents at the field level. Two cultivars (Arbequina and Coratina) were included in the analysis over a wide latitudinal gradient in Argentina. The results suggest that the thermal regime exerts a regulatory effect at the transcriptional level on both OeSAD2 and OeFAD2-2 genes and that this regulation is cultivar-dependent. It was also observed that the accumulated thermal time affects gene expression and the contents of oleic and linoleic acids in cv. Arbequina more than in Coratina. The fatty acid composition of cv. Arbequina is more influenced by the temperature regime than Coratina, suggesting its greater plasticity. Overall, findings from this study may drive future strategies for olive spreading towards areas with different or extreme thermal regimes serve as guidance for the evaluation olive varietal patrimony.EEA San JuanFil: Contreras Valentín, Ana Cibeles. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Juan; Argentina.Fil: Contreras Valentín, Ana Cibeles. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pierantozzi, Pierluigi. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Juan; Argentina.Fil: Pierantozzi, Pierluigi. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Maestri, Damian. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Maestri, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tivani, Martín. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Juan; Argentina.Fil: Tivani, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Searles, Peter. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja; ArgentinaFil: Brizuela, Magdalena. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja; ArgentinaFil: Fernández, Fabricio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Catamarca; Argentina.Fil: Toro, Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Cerro Azul; Argentina.Fil: Puertas, Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Junin; Argentina.Fil: Trentacoste, Eduardo R. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Junin; Argentina.Fil: Kiessling, Juan. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Alto Valle. Agencia de Extensión Rural Centenario; Argentina.Fil: Mariotti, Roberto. National Research Council. Institute of Biosciences and Bioresources; ItaliaFil: Baldoni, Luciana. National Research Council. Institute of Biosciences and Bioresources; ItaliaFil: Mousavi, Soraya. National Research Council. Institute of Biosciences and Bioresources; ItaliaFil: Fernández, Paula. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Fernández, Paula. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; ArgentinaFil: Fernández, Paula. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Moschen, Sebastián. Instituto Nacional de Tecnología Agropecuaria (INTA).Estación Experimental Agropecuaria Famaillá; Argentina.Fil: Moschen, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres, Myriam Mariela.Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Juan; Argentina.Fil: Torres, Myriam Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Modification of the fatty acid composition in Arabidopsis and maize seeds using a stearoyl-acyl carrier protein desaturase-1 (ZmSAD1) gene

Composition of fatty acids in the transgenic ZmSAD1 Arabidopsis mature seeds (DOCX 17 kb

Cloning of a novel stearoyl-acyl desaturase gene from white ash (Fraxinus americana) and evolution analysis with those from other plants

Using reverse transcription polymerase chain reaction (RT- PCR Based on evolution analysis, it was clear that the genes from the same family were approximately clustered into a group, but all genes from woody plants were not clustered into a separate group. In woody plants, it was indicated that all sequences clustered into two major groups and the FaSAD from white ash was closely related to the SAD gene from Macfadyena unguis-cati

Evidence-based gene models for structural and functional annotations of the oil palm genome

The advent of rapid and inexpensive DNA sequencing has led to an explosion of data waiting to be transformed into knowledge about genome organization and function. Gene prediction is customarily the starting point for genome analysis. This paper presents a bioinformatics study of the oil palm genome, including comparative genomics analysis, database and tools development, and mining of biological data for genes of interest. We have annotated 26,059 oil palm genes integrated from two independent gene-prediction pipelines, Fgenesh++ and Seqping. This integrated annotation constitutes a significant improvement in comparison to the preliminary annotation published in 2013. We conducted a comprehensive analysis of intronless, resistance and fatty acid biosynthesis genes, and demonstrated that the high quality of the current genome annotation. 3,658 intronless genes were identified in the oil palm genome, an important resource for evolutionary study. Further analysis of the oil palm genes revealed 210 candidate resistance genes involved in pathogen defense. Fatty acids have diverse applications ranging from food to industrial feedstocks, and we identified 42 key genes involved in fatty acid biosynthesis in oil palm. These results provide an important resource for studies of plant genomes and a theoretical foundation for marker-assisted breeding of oil palm and related crops