Ectopic expression of cDNAs from larkspur (Consolida ajacis) for increased synthesis of gondoic acid (cis-11 eicosenoic acid) and its positional redistribution in seed triacylglycerol of Camelina sativa.

MAIN CONCLUSION: A ß-ketoacyl-ACP-synthase II (KAS2) like enzyme and a lysophosphatidic acid acyltransferase (LPAT2) from Consolida ajacis catalyze gondoic acid biosynthesis and incorporation into the sn-2 position of seed TAG in engineered Camelina sativa. Gondoic acid (cis-11 eicosenoic acid, 20:1∆11) is the predominant very-long-chain fatty acid (VLCFA) in camelina (Camelina sativa) seed oil accounting for 12-15% of total triacylglycerol fatty acids. To explore the feasibility of engineering increased levels of this fatty acid in camelina seed, oils from a range of plant species were analyzed to identify those producing 20-Carbon (C20) fatty acids as the only VLCFAs in their seed oil. Seeds of Consolida and Delphinium species (Ranunculaceae) were found to contain moderate levels (0.2% to 25.5%) of C20 fatty acids without accompanying longer chain fatty acids. The C20 fatty acids were abundant in both sn-2 and sn-1/3 positions of seed TAG in Consolida, but were largely absent from the sn-2 position in Delphinium seed TAG. Through generation of a developing seed transcriptome, sequences were identified and cDNAs amplified from Consolida ajacis encoding a ß-ketoacyl-ACP-synthase II like protein (CaKAS2B) that lacked a predicted chloroplast transit peptide, and two homologues of Arabidopsis thaliana lysophosphatidic acid acyltransferase 2 (CaLPAT2a and CaLPAT2b). Expression of CaKAS2B in conventional (WT) camelina and a line previously engineered for high seed oleic acid content (HO) resulted in increased seed VLCFA content. Total VLCFA levels were raised from 24 to 35% and from 7 to 23% in T3 seed from representative transformants in the WT and HO backgrounds, respectively. Gondoic acid was the predominant VLCFA in transformed HO lines with low endogenous cytoplasmic fatty acid elongation activity, suggesting limited capacity of CaKAS2B to elongate beyond C20. Expression in camelina of CaLPAT2b resulted in significantly increased C20-VLCFA esterification at the sn-2 position of seed TAG with VLCFA levels of 33.8% in this position in one transformed line compared to 0.3% at sn-2 in the corresponding control line. Only small changes in total seed VLCFA content were observed in transformed lines implying that increased VLCFA esterification capacity in camelina results in positional redistribution of VLCFAs but does not significantly enhance flux through the fatty acid elongation pathway. The full potential of CaKAS2B and CaLPAT2a for the engineering of high gondoic acid levels in camelina remains to be determined. Seed fatty acid composition of Consolida and Delphinium also provides information that may be of value in the systematics of the Ranunculaceae.

Involvement of Arabidopsis ACYL-COENZYME A DESATURASE-LIKE2 (At2g31360) in the biosynthesis of the very-long-chain monounsaturated fatty acid components of membrane lipids.

The Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A (CoA) desaturase-like (ADS) gene family contains nine genes encoding fatty acid desaturase-like proteins. The biological function of only one member of the family, fatty acid desaturase5 (AtADS3/FAD5, At3g15850), is known, and this gene encodes the plastidic palmitoyl-monogalactosyldiacylglycerol Δ7 desaturase. We cloned seven members of the gene family that are predicted not to have a chloroplast transit peptide and expressed them in the yeast Saccharomyces cerevisiae. All seven have previously undescribed desaturase activity on very-long-chain fatty acid (VLCFA) substrates and exhibit diverse regiospecificity, catalyzing introduction of double bonds relative to the methyl end of the molecule (n-x) at n-6 (AtADS4, At1g06350), n-7 (AtADS1.3, At1g06100 and AtADS4.2, At1g06360), n-9 (AtADS1, At1g06080 and AtADS2, At2g31360) or Δ9 (relative to the carboxyl end of the molecule) positions (AtADS1.2, At1g06090 and AtADS1.4, At1g06120). Through forward and reverse genetics it was shown that AtADS2 is involved in the synthesis of the 24:1(n-9) and 26:1(n-9) components (X:Y, where X is chain length and Y is number of double bonds) of seed lipids, sphingolipids, and the membrane phospholipids phosphatidylserine, and phosphatidylethanolamine. Plants deficient in AtADS2 expression showed no obvious phenotype when grown under normal growing conditions, but showed an almost complete loss of phosphatidylethanolamine(42:4), phosphatidylserine(42:4), dihydroxy-monohexosylceramide(42:2)-2, trihydroxy-monohexosylceramide(42:2)-3, and trihydroxy-glycosylinositolphosphoceramide(42:2)-3, lipid species that contain the VLCFA 24:1(n-9), and trihydroxy-glycosylinositolphosphoceramide(44:2)-3, a lipid containing 26:1(n-9). Acyl-CoA profiling of these plants revealed a major reduction in 24:1-CoA and a small reduction in 26:1-CoA. Overexpression of AtADS2 resulted in a substantial increase in the percentage of glycerolipid and sphingolipids species containing 24:1 and a dramatic increase in the percentage of very-long-chain monounsaturated fatty acids in the acyl-CoA pool. Plants deficient in AtADS1 expression had reduced levels of 26:1(n-9) in seed lipids, but no significant changes in leaf phospholipids or sphingolipids were observed. These findings indicate that the 24-carbon and 26-carbon monounsaturated VLCFAs of Arabidopsis result primarily from VLCFA desaturation, rather than by elongation of long chain monounsaturated fatty acids.

Cell Wall Biomolecular Composition Plays a Potential Role in the Host Type II Resistance to Fusarium Head Blight in Wheat.

Fusarium head blight (FHB) is a serious disease of wheat worldwide. Cultivar resistance to FHB depends on biochemical factors that confine the pathogen spread in spikes. Breeding for cultivar resistance is considered the most practical way to manage this disease. In this study, different spectroscopy and microscopy techniques were applied to discriminate resistance in wheat genotypes against FHB. Synchrotron-based spectroscopy and imaging techniques, including focal plane array infrared and X-ray fluorescence (XRF) spectroscopy were used to understand changes in biochemical and nutrients in rachis following FHB infection. Sumai3 and Muchmore were used to represent resistant and susceptible cultivars to FHB, respectively, in this study. The histological comparison of rachis showed substantial differences in the cell wall thickness between the cultivars after infection. Synchrotron-based infrared imaging emphasized substantial difference in biochemical composition of rachis samples between the two cultivars prior to visible symptoms; in the resistant Sumai3, infrared bands representing lignin and hemicellulose were stronger and more persistent compared to the susceptible cultivar. These bands may be the candidates of biochemical markers for FHB resistance. Focal plane array infrared imaging (FPA) spectra from the rachis epidermis and vascular bundles revealed a new band (1710 cm(-1)) related to the oxidative stress on the susceptible cultivar only. XRF spectroscopy data revealed differences in nutrients composition between cultivars, and between controls and inoculated samples, with substantial increases observed for Ca, K, Mn, Fe, Zn, and Si in the resistant cultivar. These nutrients are related to cell wall stability, metabolic process, and plant defense mechanisms such as lignification pathway and callose deposition. The combination of cell wall composition and lignification plays a role in the mechanism of type II host resistance to FHB. Biochemical profiling using the synchrotron-based spectroscopy holds potential for screening wheat genotypes for FHB resistance.

Characterization of an oleate 12-desaturase from Physaria fendleri and identification of 5'UTR introns in divergent FAD2 family genes.

Mining of an EST sequence collection representing genes expressed during seed development in Physaria fendleri identified abundant sequences encoding apparent homologues of the Arabidopsis oleate 12-desaturase (AtFAD2 At3g12120). Of the 62 sequenced clones, 59 were identified as encoding the previously characterized bifunctional oleate 12-hydroxylase/desaturase (LFAH12/PfFAH12). The remaining 3 clones encoded a second FAD2 homologue. Isolation of a full length ORF and heterologous expression in yeast revealed that this sequence, designated PfFAD2, is the first full length sequence from any Physaria species that encodes an oleate 12-desaturase. PfFAD2 was expressed in both leaf and developing seed with activity on palmitate (16:1(Δ9)) and oleate (18:1(Δ9)). Sequence comparison revealed that PfFAD2 shares 93% amino acid identity with Arabidopsis FAD2 and only 84% identity with PfFAH12. By comparison of EST and genomic sequences it was revealed that the PfFAD2 gene encodes a transcript with a single intron of 1120 bp in the 5'-untranslated region (5'UTR). A short intron, 81 bp in length, was also discovered in the 5'UTR of the PfFAH12 gene, 16 bp upstream of the translation initiation codon. In silico examination of FAD2 like genes from the genome of castor (Ricinus communis) identified putative 5'UTR introns in genes encoding the castor oleate 12-desaturase (RcFAD2) and oleate 12-hydroxylase (CFAH12). By sequencing of genomic DNA the presence of single 5'UTR introns in each gene, and the size of these introns, was confirmed. These findings suggest that 5'UTR introns may be a characteristic feature of FAD2 genes and also of divergent FAD2 genes encoding fatty acid modifying enzymes, and that the selection pressure maintaining these introns is very different.

Characterization of novel triacylglycerol estolides from the seed oil of Mallotus philippensis and Trewia nudiflora.

Triacylglycerol estolides have been reported as components of the seed oil of a number of plant species and are generally associated with the presence of fatty acids containing hydroxyl groups. We have used MALDI-TOF MS to examine the intact acylglycerol species present in the seed oils of two plants that produce kamlolenic acid (18-hydroxy-Δ9cis,11trans,13trans-octadecatrienoic acid). Mallotus philippensis and Trewia nudiflora were both shown to produce seed oil rich in TAG-estolides. Analysis by MALDI-TOF MS/MS demonstrated that the TAG-estolides had a structure different to that previously proposed after enzymatic digestion of the oil. Acylglycerols containing up to 14 fatty acids were detected but fatty acid estolides were only present in a single position on the glycerol backbone, with predominantly non-hydroxyl fatty acids in the remaining two positions. Increased numbers of fatty acids per glycerol backbone were accounted for by the presence of fatty acid estolides containing a correspondingly greater number of fatty acids. For example, acylglycerols containing seven fatty acids had a fatty acid estolide of five fatty acids at one position on the glycerol backbone. Both capped and uncapped fatty acid estolides, with a free hydroxyl group, were present, with capped fatty acid estolides being more abundant in T. nudiflora and uncapped fatty acid estolides in M. philippensis.