A non-canonical Δ9-desaturase synthesizing palmitoleic acid identified in the thraustochytrid Aurantiochytrium sp. T66

Thraustochytrids are oleaginous marine eukaryotic microbes currently used to produce the essential omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3). To improve the production of this essential fatty acid by strain engineering, it is important to deeply understand how thraustochytrids synthesize fatty acids. While DHA is synthesized by a dedicated enzyme complex, other fatty acids are probably synthesized by the fatty acid synthase, followed by desaturases and elongases. Which unsaturated fatty acids are produced differs between different thraustochytrid genera and species; for example, Aurantiochytrium sp. T66, but not Aurantiochytrium limacinum SR21, synthesizes palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7). How strain T66 can produce these fatty acids has not been known, because BLAST analyses suggest that strain T66 does not encode any Δ9-desaturase-like enzyme. However, it does encode one Δ12-desaturase-like enzyme. In this study, the latter enzyme was expressed in A. limacinum SR21, and both C16:1 n-7 and C18:1 n-7 could be detected in the transgenic cells. Our results show that this desaturase, annotated T66Des9, is a Δ9-desaturase accepting C16:0 as a substrate. Phylogenetic studies indicate that the corresponding gene probably has evolved from a Δ12-desaturase-encoding gene. This possibility has not been reported earlier and is important to consider when one tries to deduce the potential a given organism has for producing unsaturated fatty acids based on its genome sequence alone. Key points • In thraustochytrids, automatic gene annotation does not always explain the fatty acids produced. • T66Des9 is shown to synthesize palmitoleic acid (C16:1 n-7). • T66des9 has probably evolved from Δ12-desaturase-encoding genes.

GhFAD2-3 is Required for Anther Development in Gossypium hirsutum

Background In higher plants, the FAD2 gene encodes the microsomal oleate Δ12-desaturase, one of the key enzymes essential for the biosynthesis of the polyunsaturated lipids that serve many important functions in plant development and stress responses. FAD2 catalyzes the first step, in the biosynthesis of the polyunsaturated fatty acids (PUFAs) found in the cell membrane and cell wall, and it is thus of great importance to investigate the regulatory role of FAD2 in anther development.Results We reported the molecular characterization of the cotton (Gossypium hirsutum) GhFAD2 gene family and the essential role of GhFAD2-3 in cotton anther development. G. hirsutum contains four pairs of homoeologous FAD2 genes (GhFAD2-1 to GhFAD2-4). GhFAD2-3 is ubiquitously and relatively highly expressed in all analyzed tissues, particularly in anthers. Specific inhibition of GhFAD2-3 using the RNA interference approach resulted in male sterility due to impaired anther development at the stages from meiosis to maturation. The cellular phenotypic abnormality observed at the meiosis stage of the GhFAD2-3 silenced plant (fad2-3) coincides with the significant reduction of C18:2 in anthers at the same stage. Compared with that of the wild type (WT), the content of C18:1 was 41.48%, which increased by 5 fold in the fad2-3 anther at the pollen maturation stage. Moreover, the ratio of monounsaturated to polyunsaturated fatty acid was 5.43 in fad2-3 anther, which was much higher than that of the WT (only 0.39). Through compositional analysis of anthers cuticle and transcriptome data, we demonstrated it was unfavorable to the development of anther by regulating GhFAD2-3 expression level to increase the oleic acid content.Conclusions Our work demonstrated the importance of C18:2 and/or C18:3 in the development of the pollen exine and anther cuticle in cotton and provided clue for further investigation of the physiological significance of the fatty acid composition for plant growth and development.

GhFAD2-3 is Required for Anther Development in Gossypium hirsutum

Background In higher plants, the FAD2 gene encodes the microsomal oleate Δ12-desaturase, one of the key enzymes essential for the biosynthesis of the polyunsaturated lipids that serve many important functions in plant development and stress responses. FAD2 catalyzes the first step, in the biosynthesis of the polyunsaturated fatty acids (PUFAs) found in the cell membrane and cell wall, and it is thus of great importance to investigate the regulatory role of FAD2 in anther development.Results We reported the molecular characterization of the cotton (Gossypium hirsutum) GhFAD2 gene family and the essential role of GhFAD2-3 in cotton anther development. G. hirsutum contains four pairs of homoeologous FAD2 genes (GhFAD2-1 to GhFAD2-4). GhFAD2-3 is ubiquitously and relatively highly expressed in all analyzed tissues, particularly in anthers. Specific inhibition of GhFAD2-3 using the RNA interference approach resulted in male sterility due to impaired anther development at the stages from meiosis to maturation. The cellular phenotypic abnormality observed at the meiosis stage of the GhFAD2-3 silenced plant (fad2-3) coincides with the significant reduction of C18:2 in anthers at the same stage. Compared with that of the wild type (WT), the content of C18:1 was 41.48%, which increased by 5 fold in the fad2-3 anther at the pollen maturation stage. Moreover, the ratio of monounsaturated to polyunsaturated fatty acid was 5.43 in fad2-3 anther, which was much higher than that of the WT (only 0.39). Through compositional analysis of anthers cuticle and transcriptome data, we demonstrated it was unfavorable to the development of anther by regulating GhFAD2-3 expression level to increase the oleic acid content.Conclusions Our work demonstrated the importance of C18:2 and/or C18:3 in the development of the pollen exine and anther cuticle in cotton and provided clue for further investigation of the physiological significance of the fatty acid composition for plant growth and development.

GhFAD2-3 is Required for Anther Development in Gossypium hirsutum

Background In higher plants, FAD2 gene encodes the microsomal oleate Δ12-desaturase, one of the key enzymes essential for biosynthesis of polyunsaturated lipids that serve many important functions in plant development and stress responses. FAD2 catalyzes the first step, towards biosynthesis of polyunsaturated fatty acids (PUFAs) found in cell membrane and wall, and it is thus of great importance to investigate the regulatory role of FAD2 in anther development. Results We reported the molecular characterization of the cotton (Gossypium hirsutum) GhFAD2 gene family and the essential role of GhFAD2-3 in cotton anther development. G. hirsutum contains four pairs of homoeologous FAD2 genes (GhFAD2-1 to GhFAD2-4). GhFAD2-3 is ubiquitously and relatively highly expressed in all tissues analyzed, particularly in anthers. Specific inhibition of GhFAD2-3 using the RNA interference approach resulted in male sterility due to impaired anther development at the stages from meiosis to maturation. The cellular phenotypic abnormality observed at the meiosis stage of the GhFAD2-3 silenced plant (fad2-3) coincides with the significant reduction of C18:2 in anthers at the same stage. Compared with that of wild type (WT), the content of C18:1 was 41.48%, accordingly increasing by 5 fold in fad2-3 anther at pollen maturation stage. Moreover, the ratio of monounsaturated to polyunsaturated fatty acid reached to 5.43 in fad2-3 anther, which was much higher than that of the WT (only 0.39). Based on compositional analysis of anthers cuticle and transcriptome data, we demonstrated it was unfavorable to the development of anther by regulating GhFAD2-3 expression level to increase the oleic acid content. Conclusions Our work demonstrated the importance of C18:2 and/or C18:3 in development of pollen exine and anther cuticle in cotton, and provided clue for further investigation of the physiological significance of the fatty acid composition in plant growth and development.

GhFAD2-3 is Required for Anther Development in Gossypium hirsutum

Background In higher plants, FAD2 gene encodes the microsomal oleate Δ12-desaturase, one of the key enzymes essential for biosynthesis of polyunsaturated lipids that serve many important functions in plant development and stress responses. FAD2 catalyzes the first step, towards biosynthesis of polyunsaturated fatty acids (PUFAs) found in cell membrane and wall, and it is thus of great importance to investigate the regulatory role of FAD2 in anther development. Results We reported the molecular characterization of the cotton (Gossypium hirsutum) GhFAD2 gene family and the essential role of GhFAD2-3 in cotton anther development. G. hirsutum contains four pairs of homoeologous FAD2 genes (GhFAD2-1 to GhFAD2-4). GhFAD2-3 is ubiquitously and relatively highly expressed in all tissues analyzed, particularly in anthers. Specific inhibition of GhFAD2-3 using the RNA interference approach resulted in male sterility due to impaired anther development at the stages from meiosis to maturation. The cellular phenotypic abnormality observed at the meiosis stage of the GhFAD2-3 silenced plant (fad2-3) coincides with the significant reduction of C18:2 in anthers at the same stage. Compared with that of wild type (WT), the content of C18:1 was 41.48%, accordingly increasing by 5 fold in fad2-3 anther at pollen maturation stage. Moreover, the ration of monounsaturated and polyunsaturated fatty acid reached to 5.43 in fad2-3 anther, far beyond WT that of which was only 0.39. Based on compositional analysis of anthers cuticle and transcriptome data, we demonstrated it was unfavorable to the development of anther by regulating GhFAD2-3 expression level to increase the oleic acid content. Conclusions Our work demonstrated the importance of C18:2 and/or C18:3 in development of pollen exine and anther cuticle in cotton, and provided clue for further investigation of the physiological significance of the fatty acid composition in plant growth and development.

A Conserved Glycine Is Identified to be Essential for Desaturase Activity of IpFAD2s by Analyzing Natural Variants from Idesia polycarpa

High amounts of polyunsaturated fatty acids (PUFAs) in vegetable oil are not desirable for biodiesel or food oil due to their lower oxidative stability. The oil from Idesia polycarpa fruit contains 65–80% (mol%) linoleic acid (C18:2). Therefore, development of Idesia polycarpa cultivars with low PUFAs is highly desirable for Idesia polycarpa oil quality. Fatty acid desaturase 2 (FAD2) is the key enzyme converting oleic acid (C18:1) to C18:2. We isolated four FAD2 homologs from the fruit of Idesia polycarpa. Yeast transformed with IpFAD2-1, IpFAD2-2 and IpFAD2-3 can generate appreciable amounts of hexadecadienoic acid (C16:2) and C18:2, which are not present in wild-type yeast cells, revealing that the proteins encoded by these genes have Δ12 desaturase activity. Only trace amounts of C18:2 and little C16:2 were detected in yeast cells transformed with IpFAD2-4, suggesting IpFAD2-4 displays low activity. We also analyzed the activity of several FAD2 natural variants of Idesia polycarpa in yeast and found that a highly conserved Gly376 substitution caused the markedly reduced products catalyzed by IpFAD2-3. This glycine is also essential for the activity of IpFAD2-1 and IpFAD2-2, but its replacement in other plant FAD2 proteins displays different effects on the desaturase activity, suggesting its distinct roles across plant FAD2s proteins.