Search

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.

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.

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.

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.

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.

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 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.

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.

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.

Physaria fendleri (syn. Lesquerella) is a Brassicaceae producing lesquerolic acid, a highly valued hydroxy fatty acid that could be used for several industrial applications, such as cosmetics, lubricating greases, paints, plastics and biofuels. Free of toxins, Physaria oil is an attractive alternative to imported castor (Ricinus communis) oil, and is hence on the verge of commercialization. Gas chromatography-mass spectrometry analysis of fatty acid methyl esters revealed that lesquerolic acid was synthesized and accumulated in the embryos, reaching 60% (w/w) of the total fatty acids. The sequential extraction and characterization of biomass compounds revealed that Physaria embryo metabolism switched from protein to fatty acid biosynthesis between 18 and 24 days post-anthesis (DPA). In order to unravel the metabolic pathways involved in fatty acid synthesis, a targeted metabolomics study was conducted on Physaria embryos at different stages of development. For this purpose, two novel high-throughput liquid chromatography-tandem mass spectrometry methods were developed and validated to quantify sugars, sugar alcohols and amino acids. Specificity was achieved using multiple reaction monitoring, and the limits of quantification were in the pmole-fmole range. The comparative metabolomic study underlined that: (i) the majority of the metabolites accumulate in Physaria embryos between 18 and 27 DPA; (ii) the oxidative pentose phosphate pathway, glycolysis, the tricarboxilic acid cycle and the anaplerotic pathway drain a substantial amount of carbon; and (iii) ribulose-1,5-bisphosphate is present, which specifically indicates that the Calvin cycle is occurring. The importance and the relevance of these findings regarding fatty acid synthesis were discussed.

Progress has been made towards the commercialization of Lesquerella fendleri (Gray) Wats. as a fall-planted oilseed crop for the southwestern United States. Research to develop suitable cultural practices for commercial production of lesquerella has been underway since the late 1980s; however, information is limited on the plant’s nitrogen (N) requirement. Field experiments were conducted during two growing seasons to determine the optimum amount and timing of N applications for lesquerella production. Yield results indicate that lesquerella grown on desert soils is strongly influenced by N fertilization. Nitrogen rates as high as 180 kg N ha−1 increased seed yield. Seed oil content decreased as the N rate was increased. The increase in seed yield at the higher N rates generally offsets the decrease in oil content, but oil yields appeared to be near maximum at 180 kg N ha−1. Split-applications of N in the Spring increased seed and oil yields in one of two seasons. Nitrogen had no influence on 1000-seed weight or lesquerolic acid content of the oil. No Spring leaf blade total N level was identified as being related to maximum seed yield. A suggested N management program for lesquerella production is to apply a small portion of N at planting time and the major portion of N in several applications during the period from onset of bloom through full bloom (February–April).

Lesquerella synthesizes unique hydroxy fatty acids (HFA) with potential industrial and consumer applications. However, information about its water use requirement and yield response to irrigation is limited. Detailed irrigation studies were conducted with Lesquerella fendleri (Gray) Wats. on a sandy loam in Arizona during the 1991–1992 and 1992–1993, fall–spring seasons to determine its water requirement and yield. In 1991–1992, dry matter yield was linearly related to the total evapotranspiration (ET). The highest dry matter yield was obtained for a control irrigation treatment with seven post-emergence irrigations. Four limited water treatments were given either three or four irrigations, and yielded 26–36% less dry matter than the control. Total ET for the control was 634 mm, whereas ET for the limited water treatments varied from 460 to 500 mm. Total seed yield in 1991–1992, was not determined. However, seed oil content was significantly higher for the control than for the limited water treatments. In 1992–1993, Lesquerella was grown under eight irrigation treatments: weekly (W; 12 post-emergence irrigations), biweekly (B; 7), weekly with two supplemental irrigations in early winter (WS; 14), biweekly with two supplemental irrigations in early winter (BS; 9), and four treatments that were irrigated like treatment B, except that irrigation was withheld during early flowering (B1; 5), withheld during mid-flowering (B2; 6), withheld at full bloom (B3; 6), and withheld during seed formation and ripening (B4; 5). Irrigation treatments affected both the dry matter and seed yield, but not the seed oil content and lesquerolic acid content of the oil. Withholding irrigation on the biweekly application during mid-flower and during seed formation and ripening resulted in the lowest seed yields. The BS treatment had the highest dry matter (7020 kg/ha) and seed yield (888 kg/ha), suggesting a possible yield benefit from the early winter irrigations. Total ET for treatments varied from 535 to 767 mm, and both dry matter and seed yield were related to total ET (although not by linear relationships). Total ET corresponding to the maximum yield was 668 mm. A water management that allows ≈50% depletion of the available soil water from the onset of flowering through seed ripening can result in maximum growth and yield. Providing irrigation every 14 days during this period may be optimum for Lesquerella grown on sandy loam soils.

LFAH12, an oleate 12-hydroxylase gene from Lesquerella fendleri (L.) was isolated on the basis of nucleotide sequence similarity to an oleate hydroxylase gene from Ricinus communis (L.). Transgenic Arabidopsis plants containing the Lesquerella gene under transcriptional control of the cauliflower mosaic virus 35S promoter accumulated ricinoleic, lesquerolic and densipolic acids in seeds, but not in leaves or roots. However, hydroxylase activity was detectable in crude extracts of vegetative tissues. The discrepancy between the presence of activity and the lack of hydroxy fatty acids suggests selective removal and breakdown of hydroxy fatty acids in vegetative organs. High levels of LFAH12 mRNA accumulation did not lead to correspondingly high levels of protein accumulation, suggesting that accumulation of the hydroxylase may be controlled post-transcriptionally. Expression of the L. fendleri gene in transgenic plants of a fad2 mutant of Arabidopsis, which is deficient in cytoplasmic oleate delta 12 desaturase activity, resulted in partial suppression of the mutant phenotype in roots. Thus, unlike the hydroxylase from R. communis, the L. fendleri enzyme has both hydroxylase and desaturase activities. Fusion of the 5' flanking region of the LFAH12 gene to the beta-glucuronidase coding sequence resulted in a high level of early seed-specific expression of beta-glucuronidase activity in transgenic Arabidopsis plants.

Little information is available on the response of lesquerella grown under saline conditions. This field study was initiated to determine the responses of lesquerella to salinity relative to growth, seed yield and oil content. Six salinity treatments were imposed on a Holtville silty clay (clayey over loamy, montmorillonitic (calcareous), hyperthermic Typic Torrifluvent). The plots were irrigated with Colorado River water artificially salinized with NaCl and CaCl 2 (1:1 by weight). Electrical conductivities of the irrigation treatments in both years were 1.4, 2.2, 4.0, 6.0, 8.0, and 10.0 dS m −1 . Vegetative growth, leaf-ion content, seed yield, oil content and fatty acid composition of the seed were measured. Seed yield decreased from 2100 kg ha −1 under nonsaline control conditions to 650 kg ha −1 at the highest salinity level. Analysis of the combined two-year data showed a 19% reduction in seed yield for each unit increase in soil salinity above a threshold of 6.1 dS m −1 . Based on these results, lesquerella can be classified as a salt tolerant crop. In response to increases in salinity, leaf Ca and Cl increased, whereas Mg decreased. Sodium was effectively excluded from leaf tissue at all levels of salinity. Concentrations of Na were generally an order of magnitude lower in lesquerella leaves than has been reported in leaves of other cruciferous crops grown under saline field conditions. Total oil content of the seeds increased slightly, but significantly, with increases in soil salinity. The fatty acid composition showed a minor, but again significant, increase in linolenic acid (C18:3) content as salinity increased. With this exception, oil composition did not change with salinity level, including the content of lesquerolic acid in the oil.

Lesquerolic and auricolic acids were obtained from hydrolyzed lesquerella oil by a low-temperature crystallization procedure. The lesquerolic and auricolic fatty acid fraction was enriched from 55–59% to 85–99% with high yields (94%). Washing the free fatty acids with pH 6.0 buffer provided reproducible crystallizations of those hydroxy fatty acids. In contrast, when hydrolyzed oil from Lesquerella fendleri was not buffer-washed, there was, in most cases, no separation of hydroxy fatty acids by crystallization. This crystallization procedure is suitable for a large-scale separation process of the hydroxy fatty acids from nonhydroxy fatty acids obtained from hydrolyzed lesquerella oil.

Members of the genusLesquerella, native to North America, have oils containing large amounts of hydroxy fatty acids and are under investigation as potential new crops. The triglyceride structure of oils from twenty-fiveLesquerella species in the seed collection at our research center has been examined after being hydrolysis-catalyzed by reverse micellar-encapsulated lipase and alcoholysis-catalyzed by immobilized lipase. These reactions, when coupled with supercritical-fluid chromatographic analysis, provide a powerful, labor-saving method for oil triglyceride analysis. A comprehensive analysis of overall fatty acid composition of these oils has been conducted as well.Lesquerella oils (along with oils from two other Brassicaceae:Physaria floribunda andHeliophilia amplexicaulis) have been grouped into five categories: densipolic acid-rich (Class I); auricolic acid-rich (Class II); lesquerolic acid-rich (Class III); an oil containing a mixture of hydroxy acids (Class IV); and lesquerolic and erucic acid-rich (Class V). The majority of Class I and II triglycerides contain one or two monoestolides at the 1- and 3-glycerol positions and a C18 polyunsaturated acyl group at the 2-position. Most Class III and IV oil triglycerides contain one or two hydroxy acids at the 1- and 3-positions and C18 unsaturated acid at the 2-position. A few of the Class III oils have trace amounts of estolides. The Class V oil triglycerides are mostly pentaacyl triglycerides and contain monestolide and small amounts of diestolide. Our triglyceride structure assignments were supported by1H nuclear magnetic resonance data and mass balances.

Considerable research and developmental efforts are being devoted by both the public and private sectors to commercialize Lesquerella fendleri as a new industrial oilseed crop for the production of hydroxy fatty acids. Selection and breeding research initiated in 1984 at the USDA-ARS, US Water Conservation Laboratory, Phoenix, Arizona has made progress in increasing seed, oil and lesquerolic acid yields. Yields of 1700 kg/ha of seed, 30% oil and 59% lesquerolic acid have been obtained in replicated trials. Single plant selections developed during the interium are traceable to two populations: one to a single collection made in Arizona, PI 331165, and the other to nine different accessions collected from various sites in Texas. Variation among selections within each population exhibited relatively the same amount of genetic variability. Neither population appeared to be superior as a source of variation. The need for acquisition and utilization of new sources of germplasm is indicated. Topcross yield performance of six selected lines appeared to be superior to that of comparable halfsib family progeny, validating the existence of heterosis, which is expected due to the high degree of natural cross pollination observed. These data should serve as a benchmark upon which future progress in varietal and population development can be measured.

The seed oil of Lesquerella and the closely related genus Physaria (Brassicaceae) is rich in hydroxy fatty acids (HFAs). HFAs and their derivatives are used to produce a variety of industrial products including lubricants, nylon-11, plastics, drying agents, protective coatings, surfactants, cosmetics, and pharmaceuticals. Lesquerella fendleri is being developed as a new crop for arid regions of the southwestern United States as an alternative source of HFAs. Between 1995 and 2001, 66 accessions from 28 species of Lesquerella were collected in the United States, 33 accessions from four species were collected in Mexico, and 41 accessions from 15 species of Physaria were collected from the southwestern United States. Mean seed mass ranged from 0.54 to 2.30 mg for Lesquerella compared to 1.70 to 5.80 mg for Physaria. Seed oil content ranged from a high of 32.2% in Lesquerella to a high of 35.4% in Physaria. The fatty acid profile of all species of Physaria and most of the lesquerolic-acid-rich species of Lesquerella contained from 30 to 55% lesquerolic acid, although several species contained >60%. These collections of wild germplasm provide a diverse gene pool that should enhance our breeding program in developing a domestic source of HFAs.

Three types of reaction systems, all batch-mode, were employed for production of hydroxy (lesquerolic and auricolic) fatty acidsvia 1,3-specific lipolysis ofLesquerella fendleri oil: “Free”Rhizopus arrhizus or immobilizedRhizomucor miehei lipase (Lipozyme™) in reverse micelles (System 1), Lipozyme suspended in lesquerella oil/isooctane mixture (System 2) and a suspension of water and freeR. miehei lipase in lesquerella oil/isooctane (System 3). The objective was to find the system that best maximized yield (i.e., percent hydrolysis), the proportion of hydroxy acids among the free acids liberated (hydroxy acid “purity”), and recovery/reuse of lipase activity, and that could be easily adapted into a large-scale process. System 1 provided the largest percent hydrolysis (55%) and hydroxy acid purity (85%), but of the three systems would be the most difficult to scale up. Thus, System 1 would be the most desirable reaction system only when small batch sizes are to be processed. System 3 yielded 47.2% hydrolysis, but the hydroxy acid purity was at most 73%, making it the least desirable of the three systems to employ. System 2 yielded moderate extents of hydrolyses (30–40%) and large hydroxy acid purity initially (80–83%), but the purity decreased slightly in the latter stages of the reaction due to acyl migration. System 2 was the system most easily adaptable to a large-scale process, making it the method of choice. For System 2 reactions, only when the medium was initially saturated with water and water consumed by the reaction was continuously replaced could 30–40% hydrolysis be achieved. External mass transfer limitations for Lipozyme-catalyzed reactions were not present when the solution’s water content was not above saturation, and its kinematic viscosity, controlled by the temperature and the proportion of isooctane, was below 41 centistokes.

A lesquerella species (Lesquerella fendleri) being investigated as a domestic source of seed oil containing hydroxy fatty acids shows good agronomic properties and is being tested in semi-commercial production.Lesquerella fendleri seeds contain 25% oil, of which 55% is lesquerolic acid (14-hydroxy-cis-11-eicosenoic). Oils produced in pilot-plant quantities by screw press, prepress-solvent extraction and extrusion-solvent extraction processes have been refined in the laboratory by filtering, degumming and bleaching. Two American Oil Chemists’ Society (AOCS) standard bleaching earths and two commercial earths were compared for effectiveness in bleaching these dark, yellow-red, crude lesquerella oils. Free fatty acids (1.3%), iodine value (111), peroxide value (

Lesquerella fendleri (Gray) Wats. is a promising new crop for cultivation in the western United States. Its oil-bearing seed contains high amounts of the hydroxy fatty acid (HFA), lesquerolic acid (C20:1-OH), suitable as a raw material for many types of industrial applications. Currently the U.S. imports castor seed oil as its chief source of HFA, used primarily in the production of lubricants and plastics. Lesquerella could complement castor oil imports as well as provide new applications and products. Lesquerella meal is being tested as a feed source. Other lesquerella species contain varying amounts and types of HFA. L. fendleri appears to be highly cross-pollinated, and as a consequence, considerable genetic variation exists for traits such as seed oil content, HFA, yield, flowering time, and growth habit. Rapid progress is being made toward full commercialization of lesquerella with private companies and government agencies working together in parallel studies on yield improvement and on finding new uses for the seed oil. Cooperative plantings began in 1990 and included 12 ha in central Arizona and Texas. Planting for 1991 has been increased to 30 ha and covered a wider range of climatic and soil conditions. The seed oil from these plantings is being used to formulate and test lubricant and cosmetics products. Agronomic management practices being investigated include planting methods, time of harvest, and irrigation scheduling. Fortunately, existing farm equipment with minor modification can be used for planting and harvesting the crop. Genetic and germplasm improvement studies are also being conducted and covers germplasm evaluation, selection, and hybridization.

Two hydroxy acids, lesquerolic (53 wt%) and auricolic (4%), are present at significant quantities inLesquerella fendleri seed oil. Results reported here indicate the selective release of hydroxy fatty acids during hydrolysis of this oil catalyzed byRhizopus arrhizus lipase. For example, hydroxy acids composed 85–90 wt% of the free fatty acids released during lipolysis, as compared to 54% present overall in the oil. In addition, over 80% of the lesquerolic acid is released from the triglycerides. The reason for this lipase’s success was determined to be its 1,3-positional specificity. The vast majority of lesquerella oil’s hydroxy acids is at the 1- and 3-positions of its triglycerides, as confirmed by the compositional analysis of partial glycerides formed during lipolysis.

Lesquerella species contain a seed oil which is approximately 55% lesquerolic acid, a 20-carbon long fatty acid with a single hydroxyl group and double bond, and has a similar hydroxy fatty acid (HFA) profile as castor oil. Large markets exist for hydroxylated oils as feedstocks for lithium greases, polymers in paints and coatings, base stocks for lubricants, nylon-11, hydraulic fluids, and applications in the personal care industry (Roetheli et al. 1992). The hydroxyl group of these oils makes them prime candidates as additives to diesel fuel to improve lubricity (Naughton 1992). Goodrum and Geller (2005) demonstrated that lesquerella oil has superior performance compared to castor, soybean, and rapeseed methyl esters at concentrations as low as 0.25% in reducing wear and damage to diesel engines, primarily with fuel injection systems. Castor oil also contains high amounts of HFAs, but the main HFA, ricinoleic acid, is two carbons shorter than lesquerolic acid, which imparts slightly different physicochemical properties to the oil. Lesquerella could be established as a reliable, domestic oilseed supply of HFAs for a variety of industrial applications (Roetheli et al. 1992) and at the same time provide an alternative crop for farmers and increase local profits. Lesquerella will not replace current commodity crops but instead will be placed in a rotation with these crops, e.g., a 2-year, 3-crop rotation of lesquerella, grain sorghum, and cotton.

Attention is being focused onLesquerella species as a source of hydroxy acids to replace imported castor oil. Genetic and agronomic improvement and utilization of the seed oil and meal are being studies. We have conducted laboratory experiments to extract oil fromL. fendleri seed in preparation for extracting large quantities of seed.L. fendleri is a member of the Cruciferae family, and when seeds are crushed glucosinolates release isothiocyanates by the action of a thioglucosidase enzyme system. Therefore, our experiments included moist heat treatment of whole seeds to inactivate this enzyme. The seed was then flaked in a Wolf mill, and the flakes were exhaustively extracted with hexane. The oil was degummed and bleached, and then analyzed for hydroxyl (103), saponification (174), and iodine values (107), and for unsaponifiables (1.5%), FFA (1.13%) and P (10 ppm) contents. Hydroxy fatty acids, 55% lesquerolic (14-hydroxy-cis-11-eicosenoic) and 3% auricolic (14-hydroxy-cis-11,cis-17-eicosadienoic), and total fatty acid distribution were determined by gas chromatography of the methyl esters. The defatted meal was analyzed for residual oil (1%), protein (29.8%), non-protein nitrogen (0.7%), ash (6.45%). crude fiber (12.9%), and for distribution of amino acids. DefattedL. fendleri meal has an excellent distribution of amino acids, including favorable levels of lysine, methionine and threonine compared with soybean meal.

Members of the genus Lesquerella produce seed oil that contains a high proportion of hydroxy fatty acids (HFAs). There are three groups of Lesquerella species that are distinguished by their most abundant seed oil fatty acid: lesquerolic acid (20:1OH; e.g. Lesquerella fendleri), densipolic acid (18:2OH; e.g. Lesquerella kathryn), and auricolic acid (20:2OH; e.g. Lesquerella auriculata). To investigate the biochemistry of HFA production in Lesquerella species, the conversion of putative radiolabeled intermediates of HFA biosynthesis, including 18:1, 20:1,18:1OH, 18:2OH, and 20:1OH, was examined in developing embryos of L. fendleri, L.kathryn, and L. auriculata. The results are consistent with (a) 18:1OH formation by hydroxylation of 18:1, (b) elongation and desaturation of 18:1OH to produce 20:1OH and 18:2OH, respectively, and (c) desaturation of 20:1OH to produce 20:2OH. The desaturation of 20:1OH was also found to occur in developing embryos of high, but not low, linolenic acid flax. This suggests that the desaturation is catalyzed by the extraplastidial linoleate desaturase. Confirming this suggestion, it was notable that 18:1OH and 18:2OH were found in low and high linolenic flax (Linum usitatissimum) seeds, respectively, at levels of 0.2 to 1%.

Members of the genus Lesquerella produce seed oils containing a high proportion of hydroxy fatty acids. There are three types of Lesquerella which are distinguished by the most abundant seed oil fatty acid — lesquerolic acid (20:1OH), densipolic acid (18:2OH) or auricolic acid (20:2OH), as represented by L. fendleri, L. kathryn and L. auriculata, respectively. We have investigated the conversion of radiolabelled intermediates of hydroxy fatty acid biosynthesis in the above three species.

Species of the genus Lesquerella, within the Brassicaceae family, have seed oils containing hydroxy fatty acids. In most Lesquerella species, either lesquerolic (14-hydroxy-eicosa-11-enoic), auricolic (14-hydroxy-eicosa-11,17-dienoic) or densipolic (12-hydroxy-octadeca-9,15-dienoic) acid dominates in the seed oils. Incubations of developing seed from Lesquerella species with 1-14C-fatty acids were conducted in order to study the biosynthetic pathways of these hydroxylated fatty acids. [14C]Oleic (octadeca-9-enoic) acid, but not [14C]linoleic (octadeca-9,12-dienoic) acid, was converted into the hydroxy fatty acid, ricinoleic (12-hydroxy-octadeca-9-enoic) acid, which was rapidly desaturated to densipolic (12-hydroxy-octadeca-9,15-dienoic) acid. In addition, [14C] ricinoleic acid added to Lesquerella seeds was efficiently desaturated at the Δ15 carbon. A pathway for the biosynthesis of the various hydroxylated fatty acids in Lesquerella seeds is proposed. The demonstration of desaturation at position Δ15 of a fatty acid with a hydroxy group at position Δ12 in Lesquerella prompted a comparison of the substrate recognition of the desaturases from Lesquerella and linseed. It was demonstrated that developing linseed also was able to desaturate ricinoleate at position Δ15 into densipolic acid. In addition, the linseed Δ15 desaturase was able to desaturate vernolic (12,13-epoxy-octadeca-9-enoic) acid and safflower microsomal Δ12 desaturase was able to desaturate 9-hydroxy-stearate. Thus, hydroxy and epoxy groups may substitute for double bonds in substrate recognition for oil-seed Δ12 and Δ15 desaturases.

Seed yields of unselected bulk populations of Lesquerella fendleri (Gray) Wats, grown at populations ranging from 100,000 to 1·1 million plants/ha averaged nearly 1200 kg/ha. One cycle of selection increased seed yields about 15%. Mean seed yield of the 10 best half-sib families from single plant selections, from which a second cycle of selection was initiated, was 1573 kg/ha. These yields and increases through selection strongly support the conclusion that lesquerella can be successfully developed as a new desert plant source for hydroxy fatty acids, and can serve as a replacement for castor oil.