Fatty acid composition of developing sea buckthorn (Hippophae rhamnoides L.) berry and the transcriptome of the mature seed

Abstract

Background: Sea buckthorn (Hippophae rhamnoides L.) is a hardy, fruit-producing plant known historically for its medicinal and nutraceutical properties. The most recognized product of sea buckthorn is its fruit oil, composed of seed oil that is rich in essential fatty acids, linoleic (18:2 ω-6) and α-linolenic (18:3 ω-3) acids, and pulp oil that contains high levels of monounsaturated palmitoleic acid (16:1 ω-7). Sea buckthorn is fast gaining popularity as a source of functional food and nutraceuticals, but currently has few genomic resources; therefore, we explored the fatty acid composition of Canadian-grown cultivars (ssp. mongolica) and the sea buckthorn seed transcriptome using the 454 GS FLX sequencing technology.

Results: GC-MS profiling of fatty acids in seeds and pulp of berries indicated that the seed oil contained linoleic and α-linolenic acids at 33-36% and 30-36%, respectively, while the pulp oil contained palmitoleic acid at 32-42%. 454 sequencing of sea buckthorn cDNA collections from mature seeds yielded 500,392 sequence reads, which identified 89,141 putative unigenes represented by 37,482 contigs and 51,659 singletons. Functional annotation by Gene Ontology and computational prediction of metabolic pathways indicated that primary metabolism (protein>nucleic acid>carbohydrate>lipid) and fatty acid and lipid biosynthesis pathways were highly represented categories. Sea buckthorn sequences related to fatty acid biosynthesis genes in Arabidopsis were identified, and a subset of these was examined for transcript expression at four developing stages of the berry.

Conclusion: This study provides the first comprehensive genomic resources represented by expressed sequences for sea buckthorn, and demonstrates that the seed oil of Canadian-grown sea buckthorn cultivars contains high levels of linoleic acid and α-linolenic acid in a close to 1:1 ratio, which is beneficial for human health. These data provide the foundation for further studies on sea buckthorn oil, the enzymes involved in its biosynthesis, and the genes involved in the general hardiness of sea buckthorn against environmental conditions.

Figure 1. Fatty acid composition of total lipids in A) seed oil, B) pulp oil and C) whole berry oil of four cultivars RC-4, E6590, FR-14 and Harvest Moon.

Results represent the mean ± SD of three biological replicates. Minor fatty acids (accounting in total for <3% of the fatty acid composition) are not shown. 16∶0, palmitic acid; 16∶1, palmitoleic acid; 18∶0, stearic acid; 18∶1c9, oleic acid; 18∶1c11, cis-vaccenic acid; 18∶2, linoleic acid; 18∶3, α-linolenic acid.

Figure 2. Fatty acid composition of total lipids during berry development stages of RC-4 cultivar.

The oil samples were extracted from A) seed, B) pulp and C) whole berries of RC-4 cultivar harvested at different stages. Results represent the mean ± SD of three biological replicates. Minor fatty acids (accounting in total for <5% of the total fatty acid composition) are not shown. D) The accumulation of oil in whole berries, seeds and pulp over four developmental stages described by fruit color: G: green, G/Y: green/yellow, Y/O: yellow/orange, O/R: orange/red. 16∶0, palmitic acid; 16∶1, palmitoleic acid; 18∶0, stearic acid; 18∶1c9, oleic acid; 18∶1c11, cis-vaccenic acid; 18∶2, linoleic acid; 18∶3, α-linolenic acid.

Figure 3. Size distribution of sea buckthorn 454 reads and assembled contigs.

Figure 4. Species distribution of top BLAST hits of sea buckthorn sequences with other plant species.

Figure 5. Gene Ontology annotation of sea buckthorn unigenes.

A) Biological process; B) Cellular component; C) Molecular function.

Figure 6. Sea buckthorn sequences associated with fatty acid A) and triacylglycerol B) biosynthetic pathways.

Enzymes with names in full are represented on the left, and with names in short are represented on the right. Enzymes known to function at specific steps of the pathway are represented in black. Enzymes for which sea buckthorn sequences have been identified are shown in green. EAR, enoyl-acyl-ACP-reductase; HAD, 3-hydroxyacyl-ACP dehydratase. Enzymes responsible for phosphatidylcholine acyl editing in TAG assembly (LPCAT, CPT/PDCT, PDAT) are shown in the shaded box in figure 6B.

Figure 7. RT-PCR analysis of genes involved in fatty acid and triacylglycerol biosynthesis in seed and pulp tissues at different developmental stages of fruit from RC-4 cultivar.

G, G/Y, Y/O and O/R represent fruits harvested at green (6 August), green/yellow (17 August), yellow/orange (31 August) and orange/red (22 October) stages, respectively. Ubiquitin5 (UBQ5) was used as loading control.

Figure 8. Sea buckthorn sequences associated with isoprenoid biosynthesis by A) cytosolic MVA pathway and B) plastidial MEP pathway.

Enzymes for which sea buckthorn sequences have been identified are shown in green. Abbreviations used for intermediates are: 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA); mevalonic acid (MVA); mevalonate-5-phosphate (MVAP); MVAPP mevalonate-5-diphosphate (MVAPP); Isopentenyl diphosphate (IPP); Dimethylallyl-diphosphate (DMAPP); d-glyceraldehyde-3-phosphate (GAP); 1-deoxy-D-xylulose-5-phosphate (DXP); 2- C-methyl-D- erythritol-4- phosphate (MEP); 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME); 2-phospho-4-(cytidine 5'C-diphospho) 2-C-methyl-D-erythritol (CDP-ME2P); 2-C- methyl-D- erythritol 2,4-cyclodiphosphate (MECDP); 1-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate (HMBPP); Abbreviations used for enzymes are: Acetoacetyl-CoA thiolase (AACT); 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS); HMG-CoA reductase (HMGR); mevalonate kinase (MVK); diphosphomevalonate kinase (PMK); diphosphomevalonate decarboxylase (PMD); isopentenyl diphosphate isomerase (IPP/IPI); 1-deoxy-d-xylulose-5-phosphate synthase (DXS); 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR); 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (ISPD/CMS); 4-(cytidine 5′-diphospho-2-C-methyl-d-erythritol kinase (ISPE/CMK); 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (ISPF/MCS); 4-hydroxy-3-methyl but-2-enyl diphosphate synthase (ISPG/HDS); 4-hydroxy-3-methyl but-2-enyl diphosphate reductase (ISPH/HDR).

Figure 9. Distribution of Gene Ontology terms within the category “response to stress.”