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In vivo isotopic tracing indicates Physaria fendleri incorporates industrially valuable unusual fatty acids into seed storage oil through a dynamic triacylglycerol remodeling process., Oilseed plants accumulate triacylglycerol (TAG) up to 80% of seed weight with the TAG fatty acid composition determining its nutritional value or use in the biofuel or chemical industries. Two major pathways for production of diacylglycerol (DAG), the immediate precursor to TAG, have been identified in plants: de novo DAG synthesis and conversion of the membrane lipid phosphatidylcholine (PC) to DAG, with each pathway producing distinct TAG compositions. However, neither pathway fits with previous biochemical and transcriptomic results from developing Physaria fendleri seeds for accumulation of TAG containing >60% lesquerolic acid (an unusual 20 carbon hydroxylated fatty acid), which accumulates at only the sn-1 and sn-3 positions of TAG. Isotopic tracing of developing P. fendleri seed lipid metabolism identified that PC-derived DAG is utilized to initially produce TAG with only one lesquerolic acid. Subsequently a nonhydroxylated fatty acid is removed from TAG (transiently reproducing DAG) and a second lesquerolic acid is incorporated. Thus, a dynamic TAG remodeling process involving anabolic and catabolic reactions controls the final TAG fatty acid composition. Reinterpretation of P. fendleri transcriptomic data identified potential genes involved in TAG remodeling that could provide a new approach for oilseed engineering by altering oil fatty acid composition after initial TAG synthesis; and the comparison of current results to that of related Brassicaceae species in the literature suggests the possibility of TAG remodeling involved in incorporation of very long-chain fatty acids into the TAG sn-1 position in various plants.

9 Páginas.-- 8 Figuras, Castor beans accumulate large amounts of triacylglycerols (TAGs) in the seed endosperm. This oil contains hydroxylated ricinoleic levels close to 90%, which is unique among oil seeds. The capacity to accumulate such high levels of such an unusual fatty acids is due to its specific accumulation and channeling. Here, the ability of the castor biosynthetic machinery to accumulate unusual fatty acids in the form of TAGs was investigated, focusing on ricinoleic acid and the structurally analogous lesquerolic and coriolic fatty acids. The metabolism of different radioactive precursors in active membrane fractions from castor bean's were studied, and the rates and accumulation of these fatty acids provided evidence of the different mechanisms involved in the accumulation of hydroxylated fatty acids in this species. In particular, these studies highlighted the potential of castor to accumulate unusual fatty acids other than ricinoleic acid, showing that castor endosperm can efficiently accumulate lesquerolic acid., This work was funded by General Secretariat for Universities, Research and Technology of the Andalusian Knowledge System, within the scope of the Andalusian Plan for Research, Development and Innovation (PAIDI, 2020) with the expedient number P20_00072.

Lesquerella (Physaria fendleri) contains a major unusual hydroxy fatty acid, lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid, C20:1-OH), at 55–60% of seed oil which has industrial value. The remaining seed oil comprises mainly common fatty acids including α-linolenic acid (octadec-cis-9,12,15-enoic acid, C18:3) at 10.7–15.8%. C18:3 is produced from linoleic acid (octadec-cis-9,12-enoic acid, C18:2) by FATTY ACID DESATURASE3. Previous seed transcriptome analysis uncovers two fatty acid desaturase 3 (FAD3) transcripts, PfFAD3-1 and PfFAD3-2. To determine the activity of PfFAD3-1 and PfFAD3-2, PfFAD3-1 and PfFAD3-2 were introduced into an Arabidopsis FAD3-deficient mutant (fad3-2) which has reduced C18:3 from 20.0% in wild-type to 1.6% in fad3-2. Among 20 T2 transgenic lines expression PfFAD3-1, C18:3 increased variably from 2.5 to 29.9% demonstrating that PfFAD3-1 acted as a functional FAD3. Among 32 T2 transgenic lines expressing PfFAD3-2, C18:3 content ranged from 1.0 to 3.6%, showing that PfFAD3-2 failed to recover the loss of C18:3 in fad3-2. Sequence comparison among known FAD3s revealed putative variation in PfFAD3-2 which might cause the absence of PfFAD3-2. In addition, lesquerella accumulates a minor hydroxy fatty acid, densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid, C18:2 OH) at about 1%. C18:2 OH has been shown to be produced by a FAD3 in Arabidopsis (AtFAD3) using ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid, 18:1-OH) as substrate. To test if either of PfFAD3s is able to convert C18:1-OH to C18:2-OH, PfFAD3-1 or PfFAD3-2 was transferred into a CL37 Arabidopsis which already expresses a castor (Ricinus communis) fatty acid hrdroxylase FAH12 gene (RcFAH12) and consequently accumulates C18:1-OH and C18:2-OH at 13.7% and 3.4%, respectively. Among 43 transgenic CL37 lines expressing PfFAD3-1, C18:2-OH level varied from 0.2 to 7.2%, and four of these lines exceeded to the background level of 3.4% in CL37. Whereas among 23 transgenic CL37 lines expressing PfFAD3-2, C18:2-OH level ranged from 0.4 to 3.4%, none exceeding 3.4%. The results consist with our notion that PfFAD3-1, not PfFAD3-2, exerts FAD3 function which includes converting C18:1-OH to C18:2-OH. Factors limiting PfFAD3s function in CL37 are discussed.

Seeds of castor (Ricinus communis) are enriched in oil with high levels of the industrially valuable fatty acid ricinoleic acid (18:1OH), but production of this plant is limited because of the cooccurrence of the ricin toxin in its seeds. Lesquerella (Physaria fendleri) is being developed as an alternative industrial oilseed because its seeds accumulate lesquerolic acid (20:1OH), an elongated form of 18:1OH in seed oil which lacks toxins. Synthesis of 20:1OH is through elongation of 18:1OH by a lesquerella elongase, PfKCS18. Oleic acid (18:1) is the substrate for 18:1OH synthesis, but it is also used by fatty acid desaturase 2 (FAD2) and FAD3 to sequentially produce linoleic and linolenic acids. To develop lesquerella that produces 18:1OH-rich seed oils such as castor, RNA interference sequences targeting KCS18, FAD2 and FAD3 were introduced to lesquerella to suppress the elongation and desaturation steps. Seeds from transgenic lines had increased 18:1OH to 1.1–26.6% compared with that of 0.4–0.6% in wild-type (WT) seeds. Multiple lines had reduced 18:1OH levels in the T2 generation, including a top line with 18:1OH reduced from 26.7% to 19%. Transgenic lines also accumulated more 18:1 than that of WT, indicating that 18:1 is not efficiently used for 18:1OH synthesis and accumulation. Factors limiting 18:1OH accumulation and new targets for further increasing 18:1OH production are discussed. Our results provide insights into complex mechanisms of oil biosynthesis in lesquerella and show the biotechnological potential to tailor lesquerella seeds to produce castor-like industrial oil functionality.

Hydroxy fatty acids (HFAs) are valuable industrial raw materials used in many industries. Physaria lindheimeri accumulates over 80% HFA, in the form of lesquerolic acid (20:1OH), in its seed oil. Understanding the seed development of Physaria lindheimeri is an important step to utilizing this unique wild species as a genetic source of HFAs biosynthesis. The changes of seed growth, lipid accumulation and fatty acid composition during seed development of P. lindheimeri were examined from 14 days after pollination (DAP) to desiccation (56 DAP). The seed development could be divided into three periods. During the early period (14 and 21 DAP), seed rapidly increased in size and fresh weight. In mid-maturation period (28, 35, and 42 DAP), lipids and dry weights accumulated steadily. When seeds developed to late-maturation/desiccation stages (49 and 56 DAP), fresh weight dropped significantly due to water loss, and the dry weight and lipid accumulation reached their maximums. Seed color remained green up to 42 DAP and turned to orange-brown at 49 and 56 DAP. The major fatty acid 20:1OH started accumulation when seeds developed into mid-maturation stage (28 DAP) and the accumulation continued thereafter up to 56 DAP, eventually reaching up to 77% of the total seed oil. The HFA accumulation indicates embryonic storage tissue formation, thus 28 DAP defines a critical time point for seed development entering reserve synthesis and accumulation. The information and knowledge obtained from this study are essential to the success of HFA production using metabolic pathway engineering approaches in commodity oilseed crops.

Tetraacylglycerols (triacylglycerol estolides) contain an acylacyl chain (one fatty acid attached to the hydroxyl group of another fatty acid attached to the glycerol backbone) and have different physical properties from those of triacylglycerols. Tetraacylglycerols can be used in industry such as the viscosity improver and pour point depressant of lubricants. The structures, contents and the ratios of the contents of regioisomers of the molecular species of tetraacylglycerols affect the physical properties of the mixture of tetraacylglycerols. They were elucidated by electrospray ionization mass spectrometry of the lithium adducts of tetraacylglycerols from the HPLC fractions of lesquerella oil. The ratios of the contents of acyl and acylacyl chains at the sn−2 position of the molecular species of tetraacylglycerols containing one normal FA (LsLsLsA type, Ls is lesquerolic acid. A is normal fatty acid and without hydroxyl group) in lesquerella oil were as: normal FA 58–98%), LsLs 0–35%), Ls 2–19%). For the molecular species of tetraacylglycerols containing two normal FA, 100% of the chains at the sn−2 position were normal FA, and the Ls, LsLs and LsA chains were all at the sn−1,3 positions. Acyl and acylacyl chains at the sn−2 position of the molecular species of tetraacylglycerols in lesquerella oil were predominately normal FA. Tetraacylglycerols are biodegradable and renewable, of low contents in seed oils, and can be produced through biotechnology focusing on triacylglycerol acyltransferase.

11 Páginas; 6 Figuras, The castor oil plant represents a promising platform to produce oils with industrial applications. However, its use in biotechnology is limited by the absence of a well-established procedure to transform it, and a poor understanding of gene regulation and promoter use in this species. As such, a method has been developed to express proteins or hairpin-RNA in this plant, a method based on the direct injection of Agrobacterium into the developing endosperm of castor oil fruit, enabling different constructs and promoters to be tested. This method produces a high rate of transformation and a good proportion of viable seeds that express reporter genes for up to 20 days after infiltration (DAI). Gene expression under the control of different promoters was tested by quantitative real-time polymerase chain reaction and by directly assaying the activity of the galactouronidase reporter gene, which proved to be strongest when driven by the glycinin promoter. Constructs expressing a fatty acid elongase from Lesquerella fendleri were tested, the expression of which provoked an important increase in the lesquerolic acid in the castor oil endosperm at 5 and 10 DAI, although this fatty acid did not accumulate significantly in the final mature seeds. The nature of this response could reflect the poor availability of substrates for this enzyme. In the light of this data, the potential of this technique to test promoters and different constructs in castor oil plants and other oilseeds is discussed., This work was funded by the Junta de Andalucía project P12-AGR-543, Spain (2012), and AS- Á was supported by a Junta de Andalucía predoctoral fellowship.

Castor lysophosphatidic acid acyltransferase 2 (RcLPAT2) increases hydroxy fatty acids (HFA) content at the sn-2 position of seed triacylglycerols (TAG). Determining TAG species and their regioisomers from transgenic lesquerella seeds expressing RcLPAT2 provides information for understanding the mechanisms of RcLPAT2 in HFA-containing TAG biosynthesis. Using high-performance liquid chromatography and electrospray ionization mass spectrometry, four molecular species of 3-HFA-TAG (tri-HFA-TAG), five of 2-HFA-TAG, and nine of 1-HFA-TAG were revealed, and the ratios of the regioisomers of these molecular species were quantified. HFA were identified as lesquerolic acid (Ls, OH20:1), ricinoleic acid (R, OH18:1), auricolic acid (Au, OH20:2), and densipolic acid (De, OH18:2). Ls was predominately at the sn-1,3 positions in 1-, 2- and 3-HFA-TAG. RcLPAT2 increased significantly all four kinds of HFA at the sn-2 position of the 3-HFA-TAG, and the content of regioisomer with R at the sn-2 in LsLsR alone counted about 91–94% of the total R in the transgenics. RcPLAT2 slightly decreased each 2-HFA-TAG species. There was no Ls, R and De, but trace amount of Au found at the sn-2 position of 2-HFA-TAG. RcLPAT2 also elevated each 1-HFA-TAG species with Ls found to be the only HFA, and likely at the sn-3 position.

The ratios of regioisomers of 72 molecular species of triacylglycerols (TAG) in lesquerella oil were estimated using the electrospray ionization mass spectrometry of the lithium adducts of TAG in the HPLC fractions of lesquerella oil. The ratios of ion signal intensities (or relative abundances) of the fragment ions from the neutral losses of fatty acids (FA) as α-lactones at the sn-2 position (MS3) of the molecular species of TAG were used as the ratios of the regioisomers. The order of the preference of FA incorporation at the sn-2 position of the molecular species of TAG in lesquerella was as: normal FA > OH18 (monohydroxy FA with 18 carbon atoms) > diOH18 > OH20 > diOH20, while in castor was as: normal FA > OH18 > OH20 > diOH18 > triOH18. Elongation (from C18 to C20) was more effective than hydroxylation in lesquerella to incorporate hydroxy FA at the sn-1/3 positions. The block of elongation in lesquerella may be used to increase the content of hydroxy FA, e.g., ricinoleate, at the sn-2 position of TAG and to produce triricinolein (or castor oil) for industrial uses. The content of normal FA at the sn-2 position was about 95 %, mainly oleate (38 %), linolenate (31 %) and linoleate (23 %). This high normal FA content (95 %) at the sn-2 position was a big space for the replacement of ricinoleate to increase the hydroxy FA content in lesquerella oil. The content of hydroxy FA at the sn-1/3 positions was 91 % mainly lesquerolic acid (85 %) and the content of normal FA was 6.7 % at the sn-1/3 position in lesquerella oil.

Co-expression of a lesquerella fatty acid elongase and the castor fatty acid hydroxylase in camelina results in higher hydroxy fatty acid containing seeds with normal oil content and viability. Producing hydroxy fatty acids (HFA) in oilseed crops has been a long-standing goal to replace castor oil as a renewable source for numerous industrial applications. A fatty acid hydroxylase, RcFAH, from Ricinus communis, was introduced into Camelina sativa, but yielded only 15 % of HFA in its seed oil, much lower than the 90 % found in castor bean. Furthermore, the transgenic seeds contained decreased oil content and the germination ability was severely affected. Interestingly, HFA accumulation was significantly increased in camelina seed when co-expressing RcFAH with a fatty acid condensing enzyme, LfKCS3, from Physaria fendleri, a native HFA accumulator relative to camelina. The oil content and seed germination of the transgenic seeds also appeared normal compared to non-transgenics. LfKCS3 has been previously characterized to specifically elongate the hydroxylated ricinoleic acid to lesquerolic acid, the 20-carbon HFA found in lesquerella oil. The elongation reaction may facilitate the HFA flux from phosphatidylcholine (PC), the site of HFA formation, into the acyl-CoA pool for more efficient utilization in triacylglycerol (TAG) biosynthesis. This was demonstrated by increased HFA accumulation in TAG concurrent with reduced HFA content in PC during camelina seed development, and increased C20-HFA in HFA-TAG molecules. These effects of LfKCS3 thus may effectively relieve the bottleneck for HFA utilization in TAG biosynthesis and the feedback inhibition to fatty acid synthesis, result in higher HFA accumulation and restore oil content and seed viability.

Biotransformation of long-chain fatty acids into medium-chain α,ω-dicarboxylic acids or ω-aminocarboxylic acids could be achieved with biocatalysts. This study presents the production of α,ω-dicarboxylic acids (e.g., C9, C11, C12, C13) and ω-aminocarboxylic acids (e.g., C11, C12, C13) directly from fatty acids (e.g., oleic acid, ricinoleic acid, lesquerolic acid) using recombinant Escherichia coli-based biocatalysts. ω-Hydroxycarboxylic acids, which were produced from oxidative cleavage of fatty acids via enzymatic reactions involving a fatty acid double bond hydratase, an alcohol dehydrogenase, a Baeyer–Villiger monooxygenase and an esterase, were then oxidized to α,ω-dicarboxylic acids by alcohol dehydrogenase (ADH, AlkJ) from Pseudomonas putida GPo1 or converted into ω-aminocarboxylic acids by a serial combination of ADH from P. putida GPo1 and an ω-transaminase of Silicibacter pomeroyi. The double bonds present in the fatty acids such as ricinoleic acid and lesquerolic acid were reduced by E. coli-native enzymes during the biotransformations. This study demonstrates that the industrially relevant building blocks (C9 to C13 saturated α,ω-dicarboxylic acids and ω-aminocarboxylic acids) can be produced from renewable fatty acids using biocatalysis.

Hydroxy fatty acids (HFAs) have found a number of uses in today's market, with uses ranging from industrial materials to pharmaceuticals. Castor oil, which is obtained from castor seeds, has served as a source of a versatile HFA; its principle component, ricinoleic acid, can be isolated from castor oil and has been modified extensively for a number of applications. Unfortunately, castor seeds also contain several undesirable compounds which pose severe health risks, including ricin, an unusually stable, deadly protein; ricinine, a poisonous alkaloid; and several allergens. Lesquerella oil, obtained from seeds of the Lesquerella fendleri species, has been identified as the most promising alternative source of HFAs. Lesquerolic acid, the primary HFA found in lesquerella oil, is also most homologous to ricinoleic acid; the only structural difference between the two is that lesquerolic acid possesses two additional methylene groups on the carboxyl end of the molecule. This structural and chemical similarity further suggests lesquerella oil and its derivatives may function as a competitive alternative to castor oil. Unfortunately, the desired HFA, lesquerolic acid, constitutes approximately 50% of lesquerella oil where castor oil contains approximately 90% of the desired HFA. This difference in desired HFA abundance in lesquerella oil required more extensive purification measures and careful identification of the correct component. Additionally, α-hydroxy phosphonates and their corresponding phosphonic acids are functional moieties that have been shown to display a wide variety of biological activities, as enzyme inhibitors, pesticides, antibiotics and anti-cancer therapeutics. We previously reported the synthesis of two families of α-hydroxy phosphonic acids based on ricinoleic acid: a family that retains the cis alkene found in ricinoleic acid and one where the alkene has undergone hydrogenation to produce a saturated α-hydroxy phosphonic acid. We now report the purification of the desired lesquerolic acid component of lesquerella oil and synthesis of lesquerolic acid-derived α-hydroxy phosphonates, both the unsaturated and saturated families. As with the ricinoleic acid-based compounds, the lesquerolic acid-based compounds have been produced in high yields and high purity, and the synthesis of these compounds is reported in this manuscript.

Tetraacylglycerol (an acylglycerol estolide) contains an acyl chain attached to the hydroxyl group of another acyl chain attached to the glycerol backbone. Lesquerolic acid (Ls, OH1420:111) is the main fatty acid in lesquerella oil and may be used industrially for the manufacture of biodegradable industrial products. Electrospray ionization mass spectrometry of the lithium adducts of acylglycerols in the high-performance liquid chromatography fractions from the seed oil of lesquerella (Physaria fendleri) was used to identify thirteen tetraacylglycerols. They were LsLsLsLn, LsLsLsL, LsLs-OH20:2-O, LsLsLsO, LsLsLnLn, LsLsLLn, LsLsOLn, LsLsLL, LsLsOL, LsLsOP, LsLsOO, LsLsLS and LsLsOS. The OH20:2 was auricolic acid (OH1420:211,17). For the four tetraacylglycerols containing one normal fatty acid (non-hydroxy fatty acid), LsLsLsLn, LsLsLsL, LsLs-OH20:2-O and LsLsLsO, the normal fatty acids were all directly attached to the glycerol backbone, not to the hydroxyl group of fatty acids. We propose that the biosynthetic precursors (triacylglycerol acyltransferase) of these four tetraacylglycerols were LsLsLn, LsLsL, LsLsO (Ls-OH20:2-O) and LsLsO individually. LsLsO and Ls-OH20:2-O were equally active as the biosynthetic precursors for LsLs-OH20:2-O. For LsLsLS, linoleate were all attached to the glycerol backbone and LsLsL was proposed to be the biosynthetic precursor. For LsLsOS, stearate were all attached to the glycerol backbone and LsLsS was proposed to be the biosynthetic precursor. For the other seven tetraacylglycerols containing two normal fatty acids, LsLsAB, the biosynthetic precursors could be both LsLsA and LsLsB.

Castor oil has many industrial uses because of its high content (90 %) of the hydroxy fatty acid, ricinoleic acid (OH1218:19). Lesquerella oil containing lesquerolic acid (Ls, OH1420:111) is potentially useful in industry. Ten molecular species of diacylglycerols and 74 molecular species of triacylglycerols in lesquerella (Physaria fendleri) oil were identified by electrospray ionization mass spectrometry as lithium adducts of acylglycerols in the HPLC fractions of lesquerella oil. Among them were: LsLsO, LsLsLn, LsLsL, LsLn–OH20:2, LsO–OH20:2 and LsL–OH20:2. The structures of the four new hydroxy fatty acid constituents of acylglycerols were proposed by the MS of the lithium adducts of fatty acids as (comparing to those in castor oil): OH1218:29,14 (OH1218:29,13 in castor oil), OH1218:39,14,16 (OH18:3 not detected in castor oil), diOH12,1318:29,14 (diOH11,1218:29,13 in castor oil) and diOH13,1420:111 (diOH20:1 not detected in castor oil, diOH11,1218:19 in castor oil). Trihydroxy fatty acids were not detected in lesquerella oil. The differences in the structures of these C18 hydroxy fatty acids between lesquerella and castor oils indicated that the polyhydroxy fatty acids were biosynthesized and were not the result of autoxidation products.

Lesquerella is a potential industrial oilseed crop that makes hydroxy fatty acid (HFA). Unlike castor its seeds are not poisonous but accumulate lesquerolic acid mostly at the sn-1 and sn-3 positions of triacylglycerol (TAG), whereas castor contains ricinoleic acid (18:1OH) at all three positions. To investigate whether lesquerella can be engineered to accumulate HFAs in the sn-2 position, multiple transgenic lines were made that express castor lysophosphatidic acid acyltransferase 2 (RcLPAT2) in the seed. RcLPAT2 increased 18:1OH at the sn-2 position of TAGs from 2% to 14%–17%, which resulted in an increase of tri-HFA-TAGs from 5% to 13%–14%. Our result is the first example of using a LPAT to increase ricinoleic acid at the sn-2 position of seed TAG. This work provides insights to the mechanism of HFA-containing TAG assembly in lesquerella and directs future research to optimize this plant for HFA production.

The search for alternative feedstocks for biodiesel as a partial replacement for petrodiesel has recently extended to castor oil. In this work, the castor oil methyl esters were prepared and their properties determined in comparison to the methyl esters of lesquerella oil, which, in turn, is seen as an alternative to castor oil. Both oils contain high amounts of hydroxy fatty acids, castor oil containing ricinoleic acid, lesquerella oil containing lesquerolic acid as C20 homolog of ricinoleic acid. Lesquerella oil, however, contains higher amounts of conventional C18 fatty acids than castor oil. The methyl esters of lesquerella oil exhibit a higher cetane number (45. 6 vs. 37.55) and lower kinematic viscosity (11.22 vs. 14.82 mm2/s) than the methyl esters of castor oil, both values for lesquerella methyl esters being closer to specifications in biodiesel standards, while oxidative stability of castor methyl esters is higher although this issue appears complex. Both lesquerella and castor oil methyl esters do not meet biodiesel standards requirements in terms of cetane number, kinematic viscosity, and density. Overall, however, lesquerella methyl esters appear to have more favorable fuel properties than castor methyl esters although sulfur content is elevated. Both lesquerella and castor methyl esters have a greater tendency to exceed free glycerol and water specifications in biodiesel standards. The 1H and 13C NMR spectra of lesquerella methyl esters are reported.

Lesquerella fendleri is a developing oilseed crop in the U.S. The seed oil of L. fendleri is rich in lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid: 20:1OH), a hydroxy fatty acid (HFA) with potential uses in industrial materials. Although the synthesis pathway of HFA is extensively studied, the regulatory mechanism underlying synthesis and accumulation of 20:1OH is largely unknown. In this study, we investigated the fatty acid composition and lipid gene expression pattern in developing seeds of L. fendleri, from 7 days after pollination (DAP) to desiccation (49 DAP). The results showed that accumulation of 20:1OH started at 21 DAP, increased quickly between 21 and 35 DAP and reached a plateau, about 50–55% of total lipids, at 35–49 DAP. In addition, two other HFAs, ricinoleic (12-hydroxy-octadec-cis-9-enoic acid: 18:1OH) and auricolic (14-hydroxy-eicos-cis-11,17-enoic acid: 20:2OH) acids were present at low levels during various stages of seed development. Ricinoleic acid could be detected as early as 14 DAP whereas 20:2OH began to accumulate at 21 DAP or later. Using real-time polymerase chain reaction, we quantified the transcript level of three lipid genes, LFAH12 (bifunctional oleate 12-hydroxylase:desaturase), LfKCS3 (3-ketoacyl-CoA synthase) and LfFen1 (oleate 12-desaturase). While all of these genes displayed a bell-shaped expression pattern with a peak at 35 DAP and a sharp decline at 42–49 DAP, they had different expression levels during early seed development and maximum inductions. Based on the defined time course of seed development, the relationship between gene expression and HFA accumulation is discussed.

While Lesquerella fendleri Gray (Wats.) is not yet a commercial crop, its history serves as a model for new crop development. The most important characteristic is the absence of any significant biological barriers to commercialization. Other potential crops may have valuable, high-demand products but possess traits difficult to overcome such as seed shattering or poor yield capacity. Lesquerella has a distinctive plant architecture that is conducive to seed productivity under a variety of conditions, and the trait could be further exploited. The plant also has high amounts of within-species and interspecific genetic diversity allowing breeding improvements in traits including oil quantity and quality. The unique seed oil is predominately composed of a hydroxy fatty acid, lesquerolic acid (C20:1OH), that is similar to ricinoleic acid (C18:1OH) found in castor oil. Improvements in agronomics will help increase seed yields, water use efficiency, while reducing crop production costs. New tools offered by remote sensing will help plant breeders and growers assess crop development. Defining herbicides and obtaining registrations for use in lesquerella appears to be the biggest obstacle for commercialization of this crop. The improvements in agronomics, breeding, genetics, and the expansion of new markets started in the 1980's, and has made lesquerella a viable potential crop that could utilize thousands of hectares in arid climates of the world provided research continues.

Seeds of Lesquerella (Brassicaceae) contain oil rich in hydroxy fatty acids (HFA) that may be used in several industrial products such as motor oils, greases, plastics, cosmetics and pharmaceuticals. One of the most abundant HFA in the seed oil is lesquerolic acid, which is chemically similar to ricinoleic acid from castor (Ricinus communis L.). Hence, lesquerolic oil derived from a domestically grown crop could reduce the import of castor oil. In nature, Lesquerella grows in open and arid habitats, and therefore it may be an alternative arid-land crop. Domestication and breeding efforts warranted establishing a Lesquerella germplasm collection by the USDA-ARS, National Plant Germplasm System (NPGS). Seeds of 195 accessions (32 species) from the collection were evaluated for content of four different HFA (lesquerolic, densipolic, auricolic and ricinoleic) and seven other fatty acids (palmitic, palmitoleic, stearic, oleic, linolenic, linoleic and arachidic) by gas chromatography. The highest content of lesquerolic acid (C20:1 OH) was found in seeds of L. pallida and L. lindheimeri (average of >80%); the highest content of densipolic acid (C18:2 OH) was in seeds of L. perforata, L. stonensis, L. densipila, L. lyrata and L. lescurii (average>40%); the highest content of auricolic acid (C20:2 OH) was in L. auriculata and L. densiflora (average>30%), and the highest ricinoleic acid (C18:1 OH) content was in seeds of L. densipila, L. lescurii, L. lyrata and L. perforata (>10%). The highest percentages of the seven other fatty acids evaluated were oleic 23.8 (L. lasiocarpa), linoleic 18.7 (L. ludoviciana), linolenic 11.4 (L. cinerea), stearic 4.9 (L. densiflora), palmitic 4.7 (L. stoniensis), palmitoleic 2.8 (L. angustifolia), and arachidic 1.7% (L. gordonii and L. gracilis). Fatty-acid composition varied across species and accessions, and was likely influenced by the location and year the seeds were harvested, and by seed color. The NPGS Lesquerella collection could potentially serve as a valuable source of germplasm for the crop improvement.

Lesquerella fendleri is a developing oilseed crop suitable for temperate growing regions in the US. The seed oil is rich in lesquerolic acid (57%) and could serve as a domestic source of hydroxy fatty acids. A method for the analysis of fatty acids and total oil content of a half or single lesquerella seed was developed. Lesquerella seeds are small with 1000 seed weights around 0.6 g (half seed mass of 200–500 μg). Conventional analytical balances provide mass accuracy to ±40 μg which fails to provide sufficient accuracy/precession (4–40% mass error) for the initial seed mass. A microbalance which measures to ±2 μg provided good reproducibility in initial seed weights but was not suitable for mass balance of the extracted oil. A normal phase HPLC coupled to an evaporative light scattering detector gave good response for oil in the mass range of 22 μg to 110 μg/mL. Therefore, micelle concentrations from single or half seed extractions could be determined with good reproducibility. This method was then evaluated on bulk seed that had been fractionated on a gravity table. Gravity table fractionation of L. fendleri seeds obtained from a large field plot provided seven fractions with increasing bulk density. These fractions were then analyzed in sets of 30 individual seeds and as an aggregate of 50 seeds. Oil content for individual seeds varied widely (15.6–44.2%) as did lesquerolic acid content (42.2–63.7%). The mean oil content increased (27–33%) with increasing bulk density (684–745 g/L). The mean lesquerolic acid content did not correlate with bulk density.

We investigated new microbial systems for their ability to convert lesquerolic acid (LQA; 14-hydroxy-11(Z)-eicosenoic acid) to value-added products. A strain of Sphingobacterium multivorum (NRRL B-23212) was found previously to convert LQA to 14-oxo-11(Z)-eicosenoic acid (14-OEA) as determined by gas chromatography–mass spectrometry and nuclear magnetic resonance analyses. Conversion of LQA was subsequently extended to examine S. multivorum and closely related species of Pedobacter, Spirosoma, Chryseobacterium, and Flavobacterium. Among 25 of such environmental isolates, a group of bacteria, whose identity was further confirmed by 16S rRNA gene sequence analysis as S. multivorum, was the only species found to conduct LQA conversion to produce 14-OEA. Among these strains, however, NRRL B-14797 was a variant strain devoid of the specific biologic activity. A new culture medium at pH 7.0 was defined to include Fe2+ and Mn2+ mineral ions, glycerol, and EDTA·2Na to improve the production of 14-OEA from the initial yields of 2% to 13% to approximately ≥75% for the reactive S. multivorum strains. These S. multivorum strains represent the first group of bacteria reported to carry out the functional modification of LQA.

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l−1 BA, 1 mg l−1 NAA and 1 mg l−1 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l−1 BA, 0.2 mg l−1 NAA and 100 μmol l−1As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l−1 BA, 0.2 mg l−1NAA, 500 mg l−1 Cef and 10 mg l−1 hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l−1 on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l−1BA, 0.2 mg l−1 NAA, 500 mg l−1 Cef and 25 mg l−1 hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species.

The objective of this study was to explore the extent of microbial conversion of lesquerolic acid (14-hydroxy-cis-11-eicosenoic acid; LQA) by whole cell catalysis and to identify the newly converted products. Among compost isolates including NRRL strains B-23212 (Sphingobacterium multivorum), B-23213 (Acinetobacter sp.), B-23257 (Enterobacter cloacae B), B-23259 (Escherichia sp.) and B-23260 (Pseudomonas aeruginosa) the S.multivorum strain was the only microorganism that converted LQA to produce a new product identified as 14-oxo-cis-11-eicosenoic acid by GC-MS and NMR analyses. The conversion yield was 47.4% in 48 h at 200 rpm and 28°C in small shake flask experiments. In comparison, both Acinetobacter and Pseudomonas strains failed to convert LQA to major new products but used LQA apparently as an energy source during fermentation. For structural analysis, 6.88 g of 14-oxo-cis-11-eicosenoic acid was produced from converting 11 g LQA (a 62% yield) in 72 h at 200 rpm and 28 °C in Fernbach flasks using 18-h-old NRRL B-23212 cultures and an improved medium that also contained EDTA and glycerol in lieu of glucose as carbon source. NRRL B-23212 was further identified by 16S rRNA gene sequence analysis as a unique strain of S. multivorum. Therefore, S. multivorum NRRL B-23212 possesses an enzymatic activity presumably a secondary alcohol dehydrogenase for converting LQA to produce 14-oxo-cis-11-eicosenoic acid, a first report that demonstrates the functional modification of LQA by whole cell catalysis.

The seed oil of Lesquerella fendleri is valued for its use in industrial products including lubricants, methyl esters for diesel fuel additives, greases, drying agents, plastics, nylon-11, surfactants and protective coatings. The predominate fatty acids in the seed oil of all Lesquerella species are hydroxy fatty acids (HFA) and the HFA found in L. fendleri , the species being developed as a new crop for the southwestern U.S., is lesquerolic acid. The amount of variability for this and other fatty acids in its seed oil profile was considered somewhat narrow, limiting the potential progress in breeding for oil quality traits. A half seed method (HSM) of fatty acid analysis adapted for this small-seeded species, where the mean seed weight is 0.006 g, when compared to a bulk seed method (BSM) shows that much diversity actually exists. This variability was masked because the BSM is an average value of up to 50 seeds for each analysis compared to a half of a seed that results from a single pollination event. One thousand HSM seeds from an improved breeding population, WCL-LO3 were analyzed and the same breeding population analyzed by the BSM. A population of 32 unimproved accessions was also included for comparison because it represents the most diverse germplasm available for L. fendleri , originating from several geographical locations. Greater variability was detected with the HSM, especially for lesquerolic acid. Plants with lesquerolic acid values up to 74% and as low as 36% were found by the HSM, compared to an average of 55% by the BSM. These are the highest values reported for lesquerolic acid in this species. Lower values for two acids causing high oxidative instability of the oil, linoleic and linolenic were also detected. Plants with high and low lesquerolic acid values were then selected and S 1 populations produced. The means from these ‘high’ and ‘low’ selected progeny populations were not the same as the respective mean parental values. The variability in these progeny was similar to the range and mean observed in the original population. This is likely due to at least a couple of genes segregating for this trait, requiring further generations for improvement and to determine the genetic inheritance of fatty acid.

A novel hydroxylated primary fatty amide was produced from lesquerolic acid by direct amidation with ammonia catalyzed by immobilized Pseudozyma (Candida) antarctica lipase B (Novozym 435) in organic solvent. The amides of ricinoleic acid and oleic acid were also produced for comparison. The hydroxy FA were transformed at comparable rates to that of oleic acid. The rate of amide formation was greater for the longerchain lesquerolic acid than for ricinoleic acid. All products exhibited characteristic primary-amide mass spectrum peaks with a base peak at m/z 59 and a strong ion fragment at m/z 72. Other peaks present are consistent with cleavage on either side of the hydroxyl position. The mass spectra, together with 1H and 13C NMR data, suggest that the products of lipase-catalyzed direct amidation of ricinoleic acid and lesquerolic acid are 12-hydroxy-9(Z)-octadecenamide and 14-hydroxy-11(Z)-eicosenamide, respectively.

Pseudozyma ( Candida ) antarctica lipase B is known to catalyze the direct amidation of carboxylic acids with ammonia in organic solvents. We tested this system for production of primary fatty amides from hydroxy fatty acids including the naturally occurring mono-hydroxy fatty acids, ricinoleic acid (RA) and lesquerolic acid (LQA) and the novel multihydroxy fatty acids, 7,10-dihydroxy-8( E )-octadecenoic acid (DOD) and 7,10,12-trihydroxy-8( E )-octadecenoic acid (TOD). Reactions were performed at temperatures up to 55 °C. Ricinoleic acid and lesquerolic acid were transformed at initial rates comparable to or better than that of oleic acid, a non-hydroxylated substrate. Transformation percentage at 7 days was better than 95% for all substrates except TOD (93.9%). At 55 °C, most reactions approached completion within 1 day. The primary amides of LQA, DOD, and TOD are novel compounds having melting points of 73, 105, and 100 °C, respectively.

Interspecific hybridization offers potential to improve the hydroxy fatty acid (HFA) content of lesquerella. Lesquerella fendleri is currently being developed for cultivation as a potential new industrial oilseed crop because of its seed productivity. However, it has lower HFA content compared to L. pallida and L. lindheimeri. The objectives of this research were to improve the HFA oil content of L. fenderi through interspecific hybridization and to characterize hybrids and successive generations for seed oil fatty acid profile, fertility, seed set and other morphological traits. In this work, three species were successfully hybridized, self-pollinated, and backcrossed. Ovule culture was used in some cases to produce progeny where interspecific hybrids did not produce viable embryos. The traits measured were petal length, ovules per silique, seeds per silique, and weight of 1000 seed. Patterns of leaf trichomes were used to distinguish between parents and hybrids. Seed per silique indicated that autofertility occurred in L. pallida but not in the other two species. HFA oil content of L. fendleri seed were 50.5% compared to 80 and 84% for L. pallida and L. lindheimeri, respectively. The HFA oil content of the hybrids ranged from 57 to 70% in A2 and A3 generations, and the range of values depended on the parents used in the crosses. These measurements will help predict the value of different interspecific crosses for breeding. Segregation for various yield related traits should allow selection for favorable improvements in the HFA trait and in seed yield.

The lesquerolic acid in lesquerella seed can be used in industrial applications such as greases, cosmetics, polishes, inks, and coatings. Successful commercialization of lesquerella will depend on the development of improved cultural practices. Lesquerella flowers are bright yellow and are prominently displayed. As a result, many cultural practices could be tied to flowering in a qualitative way. A new method of estimating flowers such as those of lesquerella using digital images has been developed that is rapid, not labor intensive, and can be automated. The purpose of this study was to determine the effects of nitrogen fertilizer and plant density on flowering of lesquerella and to develop relationships between seed yield and flowering. The lesquerella crop was planted on 15 October 1997, at the Maricopa Agricultural Center, approximately 40 km south of Phoenix, Arizona on a variable Mohall sandy loam (fine-loamy, mixed hyperthermic, Typic Haplargid). The experimental design was a complete factorial of three fertilizer rates and four seeding rates. Ammonium sulfate at rates of 0, 60 and 120 kg of N ha−1 was applied at flowering on 18 March 1998. Digital images of the plots were taken periodically from 19 March 1998 to 4 June 1998 using a color digital camera. Images were acquired between 1030 and 1300 h MST. In this experiment, the crop did not respond to seeding rate. Flowers present at initial bloom could be used to estimate stand establishment. The early flowers did not contribute much to final yield, but flowers present in the first 3 weeks of May were a good predictor of yield. Flowering increased with N additions and noticeable peaks in flowering occurred after irrigations. The new method was verified as a viable method for estimating flower number. The method of flower estimation should also be useful for plant breeders for selection of earlier maturing lines, which would increase the potential for use of lesquerella in rotational systems.

Expression of a cDNA encoding the castor bean ( Ricinus communis L.) oleate Delta12-hydroxylase in the developing seeds of Arabidopsis thaliana (L.) Heynh. results in the synthesis of four novel hydroxy fatty acids. These have been previously identified as ricinoleic acid (12-hydroxy-octadec- cis-9-enoic acid: 18:1-OH), densipolic acid (12-hydroxy-octadec- cis-9,15-enoic acid: 18:2-OH), lesquerolic acid (14-hydroxy-eicos- cis-11-enoic acid: 20:1-OH) and auricolic acid (14-hydroxy-eicos- cis-11,17-enoic acid: 20:2-OH). Using mutant lines of Arabidopsis that lack the activity of the FAE1 condensing enzyme or FAD3 ER Delta-15-desaturase, we have shown that these enzymes are required for the synthesis of C20 hydroxy fatty acids and polyunsaturated hydroxy fatty acids, respectively. Analysis of the seed fatty acid composition of transformed plants demonstrated a dramatic increase in oleic acid (18:1) levels and a decrease in linoleic acid (18:2) content correlating to the levels of hydroxy fatty acid present in the seed. Plants in which FAD2 (ER Delta12-desaturase) activity was absent showed a decrease in 18:1 content and a slight increase in 18:2 levels corresponding to hydroxy fatty acid content. Expression of the castor hydroxylase protein in yeast indicates that this enzyme has a low level of fatty acid Delta12-desaturase activity. Lipase catalysed 1,3-specific lipolysis of triacylglycerol from transformed plants demonstrated that ricinoleic acid is not excluded from the sn-2 position of triacylglycerol, but is the only hydroxy fatty acid present at this position.

In a study conducted in 1997–1998 to determine the salt tolerance of Lesquerella fendleri (Gray) S. Wats., saline irrigation waters greater than 21 dS m−1 electrical conductivity were found to cause high plant mortality. Surviving plants were inter-mated under controlled conditions and seeds were collected from these plants. The following season, seeds of the selected salt tolerant full-sibs, designated WCL-SL1, were direct-seeded into 21 outdoor sand tanks, along with two other lines. One line was the original seed planted the previous year called ‘1986 bulk’, and the other was an unselected line, designated ‘line 54’. In an effort to determine whether salt tolerance is a heritable trait in lesquerella, this study compared the salinity response of WCL-SL1 with the two other lines. After seeding into the sand tanks, they were irrigated daily with complete nutrient solutions. Seven salinity treatments were imposed by stepwise additions of mixed salt salinity composed of Na+, SO42−, Cl−, Mg2+ and Ca2+. Final electrical conductivities (ECi) of the irrigation waters were 3, 7, 11, 15, 18, 21 and 24 dS m−1. Two weeks after salinization, significant treatment differences in both plant height and survival were observed among lines. The parental line ‘1986 bulk’ was most sensitive, WCL-SL1 line most tolerant, and ‘line 54’ intermediate. Shoot weights in all lines decreased as a function of increasing salinity and ranked mean differences within lines were consistent across all salinity levels. At 7, 15 and 18 dS m−1 salinity levels, the shoot weights of the WCL-SL1 line were significantly greater than the parental line. Total seed oil and lesquerolic acid contents increased significantly as irrigation water salinity increased from 3 to 18 dS m−1. Improved salt tolerance of line WCL-SL1 was not attributed to differences in mineral ion uptake and accumulation in leaf tissue. Our results indicate that a single cycle of selection with lesquerella in salinized sand cultures resulted in an improved lesquerella line that had higher absolute and relative salt tolerance based on seedling survival, plant height, shoot biomass production, and seed yield than the original line.