Soybean as a Model Crop to Study Plant Oil Genes: Mutations in FAD2 Gene Family

Plants have numerous fatty acid desaturase (FAD) enzymes regulating the unsaturation of fatty acids, which are encoded by a FAD gene family. The FAD2 genes belong to such family and play a vital role in converting monounsaturated oleic acid to polyunsaturated linoleic acid. Oleic acid has the health benefits for humans, such as reduction in cholesterol level, antioxidation property, and industrial benefits like longer shelf life. The development of genotypes with high oleic acid content in seeds has become one of the primary goals in breeding oilseed plants. The identification and characterization of the FAD2 genes in plants have been an important step to better manipulate gene expression to improve the seed oil quality. The induction of mutations in FAD2 genes to reduce FAD2 enzyme activity has been an integral approach to generate genotypes with high oleic acid. This chapter will describe the FAD2 gene family in the model organism soybean and the correction of mutations in FAD2 genes with the increase of oleic acid content. Leveraging advanced research of FAD2 gene family in soybean promotes the study of FAD2 genes in other legume species, including peanut. The future perspectives and challenges associated with mutations in FAD2 genes will be discussed.

Multiplex CRISPR/Cas9-mediated genome editing of the FAD2 gene in rice: a model genome editing system for oil palm

Background Genome editing employing the CRISPR/Cas9 system has been widely used and has become a promising tool for plant gene functional studies and crop improvement. However, most of the applied CRISPR/Cas9 systems targeting one locus using a sgRNA resulted in low genome editing efficiency. Results Here, we demonstrate the modification of the FAD2 gene in rice using a multiplex sgRNA-CRISPR/Cas9 genome editing system. To test the system’s efficiency for targeting multiple loci in rice, we designed two sgRNAs based on FAD2 gene sequence of the Oryza sativa Japonica rice. We then inserted the validated sgRNAs into a CRISPR/Cas9 basic vector to construct pYLCRISPRCas9PUbi-H:OsFAD2. The vector was then transformed into protoplast cells isolated from rice leaf tissue via PEG-mediated transfection, and rice calli using biolistic transformation. Direct DNA sequencing of PCR products revealed mutations consisting of deletions of the DNA region between the two target sgRNAs. Conclusion The results suggested that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing system may be useful for crop improvement in monocot species that are recalcitrant to genetic modification, such as oil palm.

Specialized Functions of Olive FAD2 Gene Family Members Related to Fruit Development and the Abiotic Stress Response

Three different cDNA sequences, designated OepFAD2-3, OepFAD2-4 and OepFAD2-5, encoding three microsomal oleate desaturases (FAD2) have been isolated from olive (Olea europaea cv. Picual). Sequence analysis and functional expression in yeast of the corresponding cDNAs confirm that they encode microsomal oleate desaturases. Gene expression and lipid analysis indicate that these three genes are not involved in the linoleic acid present in seed lipids, while OeFAD2-5, together with OeFAD2-2, contributes mostly to the linoleic acid present in the mesocarp and, therefore, in the olive oil. Our results have also shown that olive FAD2-3, FAD2-4 and FAD2-5 gene expression is not only spatially and temporally regulated in olive fruit, but also is cultivar-dependent, as well as regulated by water regime, temperature, light and wounding. All these data suggest specialized physiological roles for the olive FAD2 gene family members with respect to both aspects of the biosynthesis of the linoleic acid, either present in storage lipids that constitute the olive oil or being part of membrane lipids, which are involved in the response to abiotic stresses, and highlight the differences on FAD2 gene regulation between oilseeds and oil fruits.

Cloning and Functional Analysis of the FAD2 Gene Family from Desert Shrub Artemisia sphaerocephala

Background: Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members. Results: Full-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2-1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2-1, -10, and -23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2-1 and AsFAD2-10 could also produce palmitolinoleic acid. Conclusions: This research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.

Cloning and Functional Analysis of the FAD2 Gene Family from Desert Shrub Artemisia sphaerocephala

Background: Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members. Results: Full-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2-1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2-1, -10, and -23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2-1 and AsFAD2-10 could also produce palmitolinoleic acid. Conclusions: This research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.