Functional and Predictive Structural Characterization of WRINKLED2, A Unique Oil Biosynthesis Regulator in Avocado

WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors regulates fatty acid biosynthesis and triacylglycerol (TAG) accumulation in plants. Among the four known Arabidopsis WRI1 paralogs, only WRI2 was unable to complement and restore fatty acid content in wri1-1 mutant seeds. Avocado (Persea americana) mesocarp, which accumulates 60-70% dry weight oil content, showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3, during fruit development. While the role of WRI1 as a master regulator of oil biosynthesis is well-established, the function of WRI1 paralogs is poorly understood. Comprehensive and comparative in silico analyses of WRI1 paralogs from avocado (a basal angiosperm) with higher angiosperms Arabidopsis (dicot), maize (monocot) revealed distinct features. Predictive structural analyses of the WRI orthologs from these three species revealed the presence of AP2 domains and other highly conserved features, such as intrinsically disordered regions associated with predicted PEST motifs and phosphorylation sites. Additionally, avocado WRI proteins also contained distinct features that were absent in the nonfunctional Arabidopsis ortholog AtWRI2. Through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. We predict that the unique features and activities of ancestral PaWRI2 were likely lost in orthologous genes such as AtWRI2 during evolution and speciation, leading to at least partial loss of function in some higher eudicots. This study provides us with new targets to enhance oil biosynthesis in plants

Mining the bitter melon (momordica charantia l.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

Abstract Background Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids. Results Deep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis. Conclusions 454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in developing bitter melon seeds. The transcriptomic data presented provide a resource for the study of novel fatty acid metabolism and for the biotechnological production of conjugated fatty acids and possibly other novel fatty acids in established oilseed crops.</p

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions

Background: Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine. Results: Plant LPCATs were expressed in yeast and biochemically characterized. Conclusion: LPCATs can edit acyl composition of phosphatidylcholine through their combined forward and reverse reactions. Significance: Plant LPCATs play a role in editing both sn-positions of PC and remove ricinoleic acid with high selectivity from this lipid

Expression of tung tree diacylglycerol acyltransferase 1 in E. coli

<p>Abstract</p> <p>Background</p> <p>Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Database search has identified at least 59 DGAT1 sequences from 48 organisms, but the expression of any DGAT1 as a full-length protein in <it>E. coli </it>had not been reported because DGAT1s are integral membrane proteins and difficult to express and purify. The objective of this study was to establish a procedure for expressing full-length DGAT1 in <it>E. coli</it>.</p> <p>Results</p> <p>An expression plasmid containing the open reading frame for tung tree (<it>Vernicia fordii</it>) DGAT1 fused to maltose binding protein and poly-histidine affinity tags was constructed and expressed in <it>E. coli </it>BL21(DE3). Immunoblotting showed that the recombinant DGAT1 (rDGAT1) was expressed, but mostly targeted to the membranes and insoluble fractions. Extensive degradation also occurred. Nonetheless, the fusion protein was partially purified from the soluble fraction by Ni-NTA and amylose resin affinity chromatography. Multiple proteins co-purified with DGAT1 fusion protein. These fractions appeared yellow in color and contained fatty acids. The rDGAT1 was solubilized from the insoluble fraction by seven detergents and urea, with SDS and Triton X-100 being the most effective detergents. The solubilized rDGAT1 was partially purified by Ni-NTA affinity chromatography. PreScission protease digestion confirmed the identity of rDGAT1 and showed extensive precipitation following Ni-NTA affinity purification.</p> <p>Conclusions</p> <p>This study reports the first procedure for expressing full-length DGAT1 from any species using a bacterial expression system. The results suggest that recombinant DGAT1 is degraded extensively from the carboxyl terminus and associated with other proteins, lipids, and membranes.</p

Mining the bitter melon (\u3ci\u3emomordica charantia\u3c/i\u3e l.) seed transcriptome by 454 analysis of nonnormalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

Background: Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids. Results: Deep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis. Conclusions: 454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in developing bitter melon seeds. The transcriptomic data presented provide a resource for the study of novel fatty acid metabolism and for the biotechnological production of conjugated fatty acids and possibly other novel fatty acids in established oilseed crops

Arabidopsis Contains a Large Superfamily of Acyl-Activating Enzymes. Phylogenetic and Biochemical Analysis Reveals a New Class of Acyl-Coenzyme A Synthetases

Acyl-activating enzymes are a diverse group of proteins that catalyze the activation of many different carboxylic acids, primarily through the formation of a thioester bond. This group of enzymes is found in all living organisms and includes the acyl-coenzyme A synthetases, 4-coumarate:coenzyme A ligases, luciferases, and non-ribosomal peptide synthetases. The members of this superfamily share little overall sequence identity, but do contain a 12-amino acid motif common to all enzymes that activate their acid substrates using ATP via an enzyme-bound adenylate intermediate. Arabidopsis possesses an acyl-activating enzyme superfamily containing 63 different genes. In addition to the genes that had been characterized previously, 14 new cDNA clones were isolated as part of this work. The protein sequences were compared phylogenetically and grouped into seven distinct categories. At least four of these categories are plant specific. The tissue-specific expression profiles of some of the genes of unknown function were analyzed and shown to be complex, with a high degree of overlap. Most of the plant-specific genes represent uncharacterized aspects of carboxylic acid metabolism. One such group contains members whose enzymes activate short- and medium-chain fatty acids. Altogether, the results presented here describe the largest acyl-activating enzyme family present in any organism thus far studied at the genomic level and clearly indicate that carboxylic acid activation metabolism in plants is much more complex than previously thought

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA

Synthesis of <SUP>31</SUP>P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [&#947;-<SUP>32</SUP>P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture ofoxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate

The N termini of Brassica and tung omega-3 fatty acid desaturases mediate proteasome-dependent protein degradation in plant cells

The regulation of fatty acid desaturase activity in plants is important for determining the polyunsaturated fatty acid content of cellular membranes, which is often rapidly adjusted in plant cells in response to temperature change. Recent studies have demonstrated that the endoplasmic reticulum (ER)-localized omega-3 fatty acid desaturases (Fad3s) are regulated extensively at the post-transcriptional level by both temperature-dependent changes in translational efficiency, as well as modulation of protein half-life. While the N-terminal sequences of Fad3 proteins were shown to contain information that mediates their rapid, proteasome-dependent protein turnover in both plant and yeast cells, it is currently unknown whether these sequences alone are sufficient to direct protein degradation. In this report, we fused the N-terminal sequences of two different Fad3 proteins to an ER-localized fluorescent protein reporter, consisting of the green fluorescent protein and the ER integral membrane protein cytochrome b5, and then measured (via microscopy) the degradation of the resulting fusion proteins in plant suspension-cultured cells relative to a second, co-expressed fluorescent reporter protein. Overall, the results demonstrate that the N-termini of both Fad3 proteins are sufficient for conferring rapid, proteasome-dependent degradation to an ER-bound marker protein