Orbitide Composition of the Flax Core Collection - ACS Publications

Article pubs.acs.org/JAFC

Orbitide Composition of the Flax Core Collection (FCC) Peta-Gaye Gillian Burnett,*,† Clara Marisa Olivia,‡ Denis Paskal Okinyo-Owiti,† and Martin John Tarsisius Reaney*,†,§ †

Department of Plant Sciences, College of Agriculture and Bioresources and ‡Department of Food and Bioproduct Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada § Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering, Jinan University, Guangzhou, Guangdong 510632, China S Supporting Information *

ABSTRACT: The flax (Linum usitatissimum L.) core collection (FCC) was regenerated in Saskatoon, Saskatchewan and Morden, Manitoba in 2009. Seed orbitide content and composition from successfully propagated plants of 391 accessions were analyzed using high-throughput analyses employing high-performance liquid chromatography (HPLC) with reverse-phase monolithic HPLC columns and diode array detection (HPLC−DAD). Seed from plants regenerated in Morden had comparatively higher orbitide content than those grown in Saskatoon. Concentrations of orbitides encoded by contig AFSQ01016651.1 (1, 3, and 8) were higher than those encoded by AFSQ01025165.1 (6, 13, and 17) for most accessions in both locations. The cultivar ‘Primus’ from Poland and an unnamed accession (CN 101580 of unknown origin) exhibited the highest ratio of sum of [1,3,8] to a sum of [6,13,17]. Conversely, the lowest orbitide concentrations and ratio of [1,3,8] to [6,13,17] were observed in cultivars ‘Hollandia’ and ‘Z 11637’, both from The Netherlands. Orbitide expression did not correlate with flax morphological and other chemical traits. KEYWORDS: Linum usitatissimum L., orbitides, chromatography, cyclolinopeptides, plant genetic resources, germplasm characterization

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INTRODUCTION The domestication of flax (Linum usitatissimum L.) for fiberand oil-production dates back to 10 000 BC.1 Flax fiber and oilseed types are distinguished by their phenotypic characteristics such as height, stem branching, and seed size. The total number of flax germplasm accessions preserved in national genebanks, international research collections, and other public genebanks around the world is estimated to be about 48 000.2 The Plant Gene Resources of Canada (PGRC) has assembled more than 3000 flax accessions from 76 countries, representing historic or present flax-cultivation regions.3 PGRC has selected 381 accessions, referred to as the flax core collection (FCC) that embodies much of the genetic diversity found in the entire collection.3 Diederichsen et al. comprehensively details how the core collection was assembled on the basis of characterization and evaluation data.3 Specifically, six different methods were employed that followed (i) seven qualitative characters (capsule dehiscence, seed color, petal color, anther color, filament color, style color, and ciliation of capsule septa); (ii) quantitative characters (earliness of flowering, plant height, petal width, 1000 seed weight, seed oil content, proportion of the six major fatty acids, seed coat mucilage, and petal measurements);4,5 and (iii) stem fiber content, disease ratings, seed vigor, and drought tolerance.6−9 The additional three methods involved inclusion of subsets containing (i) 40 genetically pure lines having extreme low and high values for 1000 seed weight, seed oil content, and fatty acid profiles;10 (ii) 57 most-distinct accessions based on RAPD markers;11 and (iii) important fiber-flax cultivars and linseed cultivars from Europe and Canada, respectively.12 Morphological traits and oil composi© 2016 American Chemical Society

tion of each accession are accessible through the PGRC Web site.13 Flaxseed contains biologically active hydrophobic orbitides or cyclolinopeptides, comprising eight to ten amino acid residues with molecular weights of approximately 1 kDa.14,15 On the basis of their synthesis from longer peptide precursors, flaxseed orbitides are categorized as RiPPs (ribosomally synthesized and post-translationally modified peptides).16,17 Due to the ribosomal origin of flaxseed orbitides, their sequences are available through curated flax transcriptome and genome databases.18,19 Since the discovery of the first flaxseed orbitide, 1, in 1959,20 24 more flaxseed orbitides have been characterized by various analytical methods (Figure 1 and Table 1).14,21−28 These 25 orbitides can be classified on the basis of fundamental parent structures comprising 1, 2, 5, 7, 10, 14, 18, 20−22 and 24, with the remaining 14 resulting from post-translational oxidation of methionine residue(s). The National Center for Biotechnology Information (NCBI) GenBank database contains embedded genome sequences for linear precursors of 1, 2, 7, 18, 21, 22, 24 (in contig AFSQ01016651.1), 5, 10, 14 (in contig AFSQ01025165.1), and 20 (in contigs AFSQ01011783.1 and AFSQ01009065.1).14,26,29 Orbitides exhibit a wide range of biological activity22,24,25,30−33 and have been identified in members of the families Annonaceae, Caryophyllaceae, Euphorbiaceae, LamReceived: Revised: Accepted: Published: 5197

May 4, 2016 June 1, 2016 June 3, 2016 June 3, 2016 DOI: 10.1021/acs.jafc.6b02035 J. Agric. Food Chem. 2016, 64, 5197−5206

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of flaxseed orbitides.

flax orbitides that impart health benefits will lead to searches for these compounds within flax germplasm collections and, possibly, breeding efforts to enhance them. Orbitide compositions of FCC accessions regenerated in Saskatoon, Saskatchewan (SK) and Morden, Manitoba (MB) in 2009 were determined, and these data are presented here. We also examined flax orbitide content in relation to morphological traits including plant height, stem branching, petal color, seed color, seed weight, seed oil content, and α-linolenic acid content. A high-throughput orbitide screening method was developed that employed a 96 well plate format for extraction,

iaceae (Labiatae), Linaceae, Phytolaccaceae, Rutaceae, Schizandraceae, and Verbenaceae.16 These compounds are found in flaxseed oil and meal at approximately 0.1%. Isolated flaxseed orbitides are anti-inflammatory compounds and might contribute to beneficial effects of consuming flaxseed products, including lowering cholesterol and blood pressure.34 Mucilage, stem fiber, and oil content have been assessed previously for many flax germplasm accessions6,35,36 because knowledge regarding chemical composition among flax cultivars is crucial for understanding genetic variation, if any, and potentially developing value added uses for flax.37 Likewise, awareness of 5198

DOI: 10.1021/acs.jafc.6b02035 J. Agric. Food Chem. 2016, 64, 5197−5206

Article

Journal of Agricultural and Food Chemistry Table 1. Calculated Flaxseed Orbitide Masses and Sequences mass ([M + H]+, Da) a

structure no.

orbitide name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

[1−9-NαC]-linusorb B3 [1−9-NαC]-linusorb B2 [1−9-NαC],[1-MetOd]-linusorb B2 [1−9-NαC],[1-MetO2f]-linusorb B2 [1−8-NαC]-linusorb A2 [1−8-NαC],[1-MetO]-linusorb A2 [1−8-NαC]-linusorb B1 [1−8-NαC],[1-MetO]-linusorb B1 [1−8-NαC],[1-MetO2]-linusorb B1 [1−8-NαC]-linusorb A3 [1−8-NαC],[3-MetO]-linusorb A3 [1−8-NαC],[1-MetO]-linusorb A3 [1−8-NαC],[1,3-MetO]-linusorb A3 [1−8-NαC]-linusorb A1 [1−8-NαC],[1-MetO]-linusorb A1 [1−8-NαC],[3-MetO]-linusorb A1 [1−8-NαC],[1,3-MetO]-linusorb A1 [1−9-NαC]-linusorb C1 [1−9-NαC],[1-MetO]-linusorb C1 [1−9-NαC]-linusorb D1 [1−10-NαC]-linusorb E3 [1−10-NαC]-linusorb E2 [1−10-NαC],[2-MetO]-linusorb E2 [1−9-NαC]-linusorb E1 [1−9-NαC],[2-MetO]-linusorb E1

molecular formula

experimental

calculated

amino acid sequenceb

C57H86N9O9 C56H84N9O9S C56H84N9O10S C56H84N9O11S C57H78N9O8S C57H78N9O9S C51H77N8O8S C51H77N8O9S C51H77N8O10S C55H74N9O8S2 C55H74N9O9S2 C55H74N9O9S2 C55H74N9O10S2 C56H76N9O8S2 C56H76N9O9S2 C56H76N9O9S2 C56H76N9O10S2 C66H88N11O9S C66H88N11O10S C54H77N10O10 C59H89N10O10 C58H87N10O10S C58H87N10O11S C53H80N9O9S C53H80N9O10S

1040.6672c 1058.6236c 1074.6201c 1090.5943g 1048.5838c 1064.5681c 961.5705c 977.5665c 993.5303g 1052.5191c 1068.5185c 1068.5022i 1084.4951i 1066.5387c 1082.5329c 1082.5330c 1098.5299c 1210.6638c 1226.6581c 1025.5946c 1097.6746j 1115.6307j 1131.6229j 1018.5873j 1034.5787j

1040.6543 1058.6107 1074.6056 1090.6006 1048.5689 1064.5638 961.5580 977.5529 993.5478 1052.5096 1068.5045 1068.5045 1084.4995 1066.5253 1082.5202 1082.5202 1098.5151 1210.6482 1226.6431 1025.5819 1097.6758 1115.6322 1131.6271 1018.5794 1034.5743

[1−9-NαC]-ILVPPFFLI [1−9-NαC]-MLIPPFFVI [1−9-NαC]-OeLIPPFFVI [1−9-NαC]-JhLIPPFFVI [1−8-NαC]-MLLPFFWI [1−8-NαC]-OLLPFFWI [1−8-NαC]-MLVFPLFI [1−8-NαC]-OLVFPLFI [1−8-NαC]-JLVFPLFI [1−8-NαC]-MLMPFFWV [1−8-NαC]-MLOPFFWV [1−8-NαC]-OLMPFFWV [1−8-NαC]-OLOPFFWV [1−8-NαC]-MLMPFFWI [1−8-NαC]-OLMPFFWI [1−8-NαC]-MLOPFFWI [1−8-NαC]-OLOPFFWI [1−9-NαC]-MLKPFFFWI [1−9-NαC]-OLKPFFFWI [1−9-NαC]-GIPPFWLTL [1−10- NαC]-GILVPPFFLI [1−10- NαC]-GMLIPPFFVI [1−10- NαC]-GOLIPPFFVI [1−9- NαC]-GMLVFPLFI [1−9- NαC]-GOLVFPLFI

New systematic nomenclature proposed by Shim et al.42 b[1−#-NαC] describes N-to-C linkage through the α-amino group between amino acid 1 and amino acid ‘#’. cExperimental masses reported by Owiti et al.26 dDesignation MetO describes methionine S-oxide. eAmino acid symbol O used for methionine S-oxide. fDesignation MetO2 describes methionine S,S-dioxide. gExperimental masses reported by Jadhav et al.50 hAmino acid symbol J used for methionine S,S-dioxide. iExperimental masses unpublished by Reaney, M. J. T. jExperimental masses reported by Burnett et al.14 a

black chernozemic.35 Some accessions did not set seed and, as such, the current analysis was conducted in duplicate only for accessions that yielded seed. Specifically, HPLC analyses were conducted on 314 accessions in Saskatoon and 345 accessions in Morden, including the three check cultivars. Sample Preparation. Effect of Sample Size, Temperature, and Grinding on Orbitide Yield. Preliminary experiments to examine the effects of sample size, degumming, temperature, and grinding on orbitide yield were conducted on the check flax cultivar ‘CDC Bethune’, grown in Saskatoon, SK in 2008. A total of three flaxseed samples of 8 (0.0460 g), 25 (0.1422 g), and 200 (1.1988 g) seeds were directly weighed in 2 mL microcentrifuge tubes. Seeds were ground with three 2 mm steel beads at a frequency of 25 per s (10 min) in a Mixer Mill MM 300 bead mill (F. Kurt Retsch GmbH & Co. KG, Haan, Germany) and then incubated with methanol−water (70:30, v/ v) at a seed-to-solvent ratio of 1:10 (w/v). After 2 h of incubation at 60 °C, mixtures were briefly vortexed and then centrifuged at 10612g (10 min). Resulting supernatants were filtered through 0.45 μm PTFE membranes before HPLC−DAD analyses. The effects of grinding and incubation temperature on orbitide extraction efficiency were simultaneously investigated. Seeds were degummed by 2 h of incubation in H2O at 60 °C using a seed−water ratio of 1:10 (w/v). Following degumming, one set of seeds was ground while the second set was left intact. Ground and intact seeds were extracted as above, employing two extraction conditions: 2 h of incubation at either ambient temperature or 60 °C. Extracts were treated as detailed above for HPLC−DAD analyses. Effect of Oxidation and Quenching on Orbitide Stability. Fresh flaxseed extracts comprise methionine-containing orbitides as the major orbitide components and low concentrations of methionine Soxide-containing orbitides.26,27 Therefore, it was important to convert all orbitides to the same oxidation state for composition analysis (that

reverse-phase monolithic high-performance liquid chromatography (HPLC) columns, and diode array detection (DAD). High-throughput screening approaches are widely used in plant-breeding programs for selection of improved accessions. Our investigation uncovers flax orbitide diversity that may benefit breeding programs in improving orbitide content and composition, in addition to functional properties of cultivated flax.

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MATERIALS AND METHODS

Flaxseed. The FCC seeds were regenerated in Saskatoon, SK and Morden, MB in 2009 and were kindly donated to us by Dr. Scott Duguid (Morden Research Station, MB) and Drs. Helen Booker and Gordon Rowland (Crop Development Centre, Saskatoon, SK). The collection consisted of 381 accessions (selected by PGRC) that represented the widest variation of flax-gene resources and an additional 10 accessions selected by plant breeders involved in this research. Thus, a total of 391 flax accessions (of which 13 had duplicated Canadian National (CN) serial numbers) were regenerated for these studies in 500 plots. Flax cultivars included as checks were ‘CDC Bethune’, ‘Macbeth’, and ‘Hanley’, in which the repeated check ‘CDC Bethune’ was systematically positioned at every fifth plot (totalling 100 plots), while ‘Macbeth’ and ‘Hanley’ were each repeated randomly in six plots. Accessions were described as medium-late maturing oilseed flax with moderate resistance to Fusarium oxysporum f. sp. lini induced wilt, and immunity to Melampsora lini induced rust.38−40 The FCC regenerated in Saskatoon was seeded and harvested on May 26 and October 14, 2009, respectively, whereas at Morden, seeding and harvesting occurred on June 2 and October 13, 2009, respectively. The soil in Saskatoon was described as loamy, dark brown chernozemic, and that in Morden was described as clay, loamy, 5199


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Journal of Agricultural and Food Chemistry is, to all methionine S-oxide-containing forms). Toward this end, we explored oxidizing reagents, including hydrogen peroxide (H2O2), sodium periodate (NaIO4), and potassium iodate (KIO3) added in solution to seed extracts. Oxidants were evaluated at varying concentrations (1.5, 3, 4.5, 6, and 12%) to determine the concentration required for optimal orbitide oxidation. Additionally, we evaluated sodium metabisulphite (NaHSO3) and sodium thiosulfate (Na2S2O3) as potential quenching agents to prevent orbitide overoxidation to methionine S,S-dioxide. Thus, NaHSO3 (0.3 M) was prepared in 50% aq MeOH and was added to oxidized extracts at varying volume ratio to H2O2 (0.5:1, 1:1, 1.5:1, and 2:1). However, Na2S2O3 (0.3 M) was prepared in 70% aq MeOH and added similarly to oxidized extracts. Orbitide Composition of the FCC. Peptide extraction was optimized for a 120 mg sample size due to limited amounts of seed available and the limited volume of 96 well plate wells. Approximately 120 mg of flaxseed from each accession was weighed and placed directly into wells of 96 well plates. Preheated H2O (60 °C) was added to each well at a seed−water ratio of 1:10 (w/v), and wells were covered with 96 well plate-sealing mats (square cap). Mat-covered plates were incubated at 60 °C for 2 h, and then gum extracts were removed via pipet. Degummed seeds were ground at 1400 strokes per min for 10 min using a 2010 Geno/Grinder (SPEX CertiPrep, Inc., Methucen, NJ) and spherical 5 mm zirconia grinding media (one per well). A 100 μL aliquot of internal standard [1−9-NαC],[1-Abu]linusorb B2 (0.1 mg/mL in MeOH), prepared as previously reported and then referred to as 1-Abu-CLB,41 was added to each well. Orbitide nomenclature is described in Shim et al.42 Subsequently, 860 μL of methanol−water (78:22, v/v) was added to each well to make a final dilution of 1:8 (w/v). Samples were mixed for 2 min at 1400 strokes per min with a 2010 Geno/Grinder and were subsequently incubated at 60 °C for 2 h. Samples were centrifuged at 1760g for 20 min, and 400 μL of resulting supernatants were transferred to 96 well filter plates. Filter plates were covered and placed on top of receiving 96 well plates and assembled plates were placed in a centrifuge for filtration at 1760g for 10 min. Filtered extracts (100 μL aliquots) were transferred to 96 well HPLC trays and subsequently oxidized with H2O2 (50 μL, 4.5% in H2O, v/v) for 1 h at ambient temperature. Reactions were quenched with Na2S2O3 (150 μL, 0.2 M) in 70% aq. MeOH and mat-covered 96 well trays were placed in an HPLC−DAD with an autosampler for sample analyses. HPLC−DAD Analyses. Analyses of FCC orbitide composition were performed on a 1200 series HPLC system (Agilent Technologies Canada, Mississauga, ON) equipped with a quaternary pump, autosampler, photodiode-array detector (wavelength range 190−300 nm), and a degasser. Chromatographic separations were carried out on 50 mm × 4.6 mm i.d. reverse-phase Chromolith SpeedRod RP-18e columns (Merck KGaA, Darmstadt, Germany) equipped with in-line filters. The linear-gradient mobile phase consisted of (A) H2O and (B) acetonitrile (CH3CN) with the following gradient: 70 to 30% A in 4 min, followed by 10% A in 0.5 min, then 70% A in 0.5 min, held for equilibration for 1 min, at a flow rate of 2 mL/min.41 All analyses were conducted at 23 °C, with injections of 10 μL per sample and absorbance recorded over the entire UV spectrum. Peak-area integration was conducted at a wavelength of 214 nm with a 10 nm bandwidth. High-resolution high-performance liquid chromatography−electrospray ionization−mass spectrometry (HR-HPLC−ESI−MS) and highperformance liquid chromatography−electrospray ionization−tandem mass spectrometry (HPLC−ESI−MS/MS) analyses were performed on an Agilent 1200 series HPLC system connected directly to a micrOTOF-Q II hybrid quadrupole time-of-flight MS/MS (Bruker Daltonik GmbH, Bremen, Germany) with Apollo II electrospray ionization ion source at a capillary voltage of −4500 V, nebulizer gas at 4.0 bar, and drying gas temperature held at 200 °C. Chromatographic separation for MS analyses was achieved at ambient temperature using 50 mm × 2.0 mm i.d. Chromolith FastGradient RP-18e columns (Merck KGaA, Darmstadt, Germany). The mobile phase consisted of (A) 0.1% formic acid in H2O and (B) 0.1% formic acid in CH3CN with the following linear gradient: 60% A for 2 min, followed by 10% A

in 8 min, then 60% A in 0.5 min, held for equilibration for 5.5 min, at a flow rate of 0.40 mL/min.26 HPLC−ESI−MS/MS analyses were conducted on the same mass spectrometer using identical parameters as described for HR-HPLC−ESI−MS. Statistical Data Analysis. Orbitide content was proportional to peak area of each orbitide observed at 214 nm. We have reported data in terms of integrated peak areas and not absolute concentration because the purpose of this study was to identify atypical flax cultivars with unique orbitide compositions. To measure absolute concentrations of compounds in complex matrixes requires determination of the matrix effects on quantitation. This was not feasible on large numbers of diverse small samples arising from the core collection. Average values for check accessions were used in statistical analyses, and any accessions with duplicated CN numbers were excluded from these analyses. Student’s t test was used to evaluate significance at α = 0.01. The relationship between total orbitide contents and phenotypic traits (plant height, seed weight, total seed oil content, and α-linolenic acid content) were determined using Pearson product−moment correlation coefficients. Relations of seed color, petal color, and stem branching to total orbitide content were evaluated by descriptive statistics (minimum, maximum, mean, standard deviation, and coefficient of variation).

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RESULTS AND DISCUSSION Effect of Sample Size, Temperature, and Grinding on Orbitide Yield. Aqueous methanolic extracts of flaxseed yielded known orbitides 1, 2, 5, 7, 10, and 14 as major orbitides corroborating previous observations.27 Although orbitide composition appeared similar regardless of the number of seeds extracted (data not shown), a 2−3 fold increase in orbitide concentration was observed as sample size increased from 8 to 25 seeds. However, there was no significant difference between orbitide yield from 25 seeds and that from 200 seeds. These observations indicated that efficient and reproducible orbitide extraction is achievable with a sample size ranging between 25 and 200 seeds. From parameters investigated, orbitide extraction was most efficient when seeds were ground and then extracted by incubation at 60 °C (data not included). Specifically, orbitide concentrations in ground flaxseed extracts were 3.4−4.5 fold higher than those from whole seeds, and incubation at 60 °C increased orbitide recovery from 1.4 to 2 fold. Effect of Oxidation and Quenching on Orbitide Stability. Reduced orbitides (Figure 1), typically observed in fresh aqueous methanolic extracts, were undesirable analytical targets because they are not fully separable using the Chromolith SpeedRod column. As previously stated, our chromatographic separation of unoxidized flaxseed extracts showed six major orbitides (1, 2, 5, 7, 10, and 14) eluting within a 1 min period and with coelution of 1, 7 and 10 (Figure 2). The coelution of 1, 7 and 10 was confirmed by HPLC− ESI−MS and HPLC−ESI−MS/MS analyses (data not shown). Aladedunye et al. also reported the coelution of 1 and 7 on a Kinetex C18 column using a 45 min HPLC gradient.43 Although other authors have demonstrated the separation of 1 and 7 using a Kinetex Phenyl-Hexyl column, the HPLC run time was 25 min,21 which is not suitable for high-throughput screening. To overcome the coelution of 1, 7 and 10, and thereby enable orbitide composition analysis by HPLC−DAD analyses, we oxidized methionine (Met) residues to methionine S-oxide (MetO). Orbitide 1 does not contain Met and, therefore, is unaffected by oxidation. Attempts to oxidize orbitide extracts with KIO3 were not successful under our experimental conditions, whereas NaIO 4 oxidations led to loss of 5200


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on the same column and solvent gradient, with retention times of approximately 2.4, 2.6, 2.7, 3.1, and 3.4 min for 13, 17, 3, 8, and 6, respectively (Figure 2). Orbitide Composition of FCC Accessions. From the current investigation, we determined flax orbitide composition of 301 and 332 FCC accessions planted in Saskatoon, SK and Morden, MB in 2009. Following extract oxidation, each accession was found to contain orbitides 1, 3, 6, 8, 13, and 17. The internal standard [1−9-NαC],[1-Abu]-linusorb B2 was used to account for extraction efficiency and reproducibility and to verify orbitide composition analysis of accessions. The three check accessions (‘CDC Bethune’, ‘Hanley’, and ‘Macbeth’) from two locations showed consistent orbitide composition (Table 2) on the basis of mean, standard deviation, and number of samples. For example, the coefficient of variation for total orbitide content from ‘CDC Bethune’ was less than 10% at either site, suggesting low dispersion (minimal scatter) in that variable. Considering the number of samples (n = 94 for Saskatoon and n = 88 for Morden), the low dispersion indicated extraction day-to-day consistency and may be attributed to reliability of the extraction procedure. Interestingly, flaxseed orbitide composition of check accessions were similar for both locations, with Morden yielding approximately 10% more orbitide content than Saskatoon suggesting minimal environmental impact on orbitides in accessions that are welladapted to Canada. The concentrations of orbitides observed in individual accessions at the two locations were examined to identify outliers. Total orbitide content of FCC samples planted in Saskatoon (n = 300) ranged from 71.4 to 226.9 milli absorbance units (mAU), with a mean value of 155.3 ± 25.0 mAU (x̅ ± SD), while that of Morden samples (n = 324) ranged from 96.2 to 243.6 mAU, with a mean value of 172.2 ± 25.3 mAU (x̅ ± SD). The higher concentrations of total orbitide content from accessions regenerated in Morden compared to those in Saskatoon were consistent with those observed in check cultivars. Total orbitide distribution in both locations resembled a normal distribution (Figure 3) with about 70% of the values within 1SD from the mean. Overall, total orbitide content of the FCC planted in the two locations showed a positive (r = 0.5839) and significant (p < 0.01) correlation (Figure 4). The accession ‘AC Watson’ (CN 18973) was identified as the flax accession expressing highest orbitide content in both locations (Table 3). In comparison to the mean orbitide concentration of seed extracts from the respective locations, the total orbitide concentration in ‘AC Watson’ reached more than 2SD greater than the mean of all accessions. However, accessions ‘Hollandia’ (CN 98056) and ‘Z 11637’

Figure 2. HPLC−DAD chromatograms of flaxseed (L. usitatissimum) ‘CDC Bethune’ (A) unoxidized orbitide extract and (B) oxidized orbitide extract.

tryptophan-containing orbitides, perhaps due to decomposition. However, oxidation of all Met residues to MetO was successfully achieved by direct addition of 4.5% H2O2 in H2O to flaxseed methanol extracts, thereby converting 2, 5, 7, 10, and 14 to 3, 6, 8, 13, and 17, respectively (Figure 1). Preventing overoxidation of these orbitides from their MetO to methionine S,S-dioxide (MetO2) forms required quenching of H2O2 to stop further oxidation. The quenching reagents explored, NaHSO3 and Na2S2O3, effectively stopped H2O2 oxidation of extracts. Because NaHSO3 was only soluble in 50% aq MeOH, further analyses were conducted with Na2S2O3 because of its solubility in 70% aq MeOH. Moreover, oxidized and quenched orbitides in extract mixtures remained stable over a 7 h period, allowing for analyses to be completed. Orbitide overoxidation was undesirable because of the coelution of 4 and 8,44 as well as that of 6 and 9. Unlike overlapping Met-containing orbitides, MetO-containing orbitides were chromatographically resolved eluting within 2 min

Table 2. Orbitide Distribution in Check Accessions of the FCC Grown in Saskatoon, SK and Morden, MB in 2009a Saskatoon, SK orbitide 1 3 6 8 13 17 Total orbitide [1,3,8]/[6,13,17] a

‘CDC Bethune’b 40.8 38.7 15.2 40.9 12.1 37.0 184.7 1.9

± ± ± ± ± ± ± ±

3.8 3.7 2.4 4.3 1.6 5.5 18.4 0.2

‘Hanley’c 38.3 36.8 15.6 42.4 12.6 38.5 184.1 1.8

± ± ± ± ± ± ± ±

6.9 8.9 4.2 9.9 4.1 12.0 44.8 0.2

Morden, MB ‘Macbeth’d 44.3 41.4 17.0 48.7 15.0 42.6 209.0 1.8

± ± ± ± ± ± ± ±

4.2 7.7 3.0 7.5 3.2 6.8 30.4 0.2

‘CDC Bethune’e 45.3 44.4 16.3 47.6 14.4 42.0 210.0 1.9

± ± ± ± ± ± ± ±

3.0 3.6 1.9 3.8 1.7 5.6 14.3 0.2

‘Hanley’d 41.8 41.7 16.1 44.4 13.0 40.5 197.5 1.8

± ± ± ± ± ± ± ±

1.1 3.7 1.9 1.8 0.9 3.4 8.6 0.1

‘Macbeth’c 48.8 47.6 17.8 53.3 15.3 46.1 228.9 1.9

± ± ± ± ± ± ± ±

2.6 2.7 1.5 4.4 1.4 4.6 10.7 0.1

All values are expressed as peak area in mAU. bn = 94. cn = 5. dn = 6. en = 88. 5201


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Table 4. Correlation Coefficients of Orbitide Concentrations from the FCC between Flaxseed Accessions Planted in Saskatoon, SK and Morden, MB in 2009 (n = 283, p < 0.01)

Figure 3. Histogram showing total orbitide content expressed by the FCC planted in Saskatoon, SK (n = 300) and Morden, MB (n = 324) in 2009.

orbitide

r

1 3 6 8 13 17 [1,3,8] [6,13,17]

0.6924 0.6854 0.7108 0.5801 0.4964 0.4832 0.6562 0.5012

Contig AFSQ01016651.1 contained three orbitide-encoding domains with one copy each for the linear precursor orbitides of 1, 2 and 7 (precursors to mature orbitides 1, 3 and 8). The concentration ratio of orbitide concentrations 1:3:8 expressed in ‘CDC Bethune’ was 1:1:1, suggesting that a single linear precursor protein with three orbitide-encoding domains produced these three orbitides. As such, we examined the concentration ratios of 8-to-1 and 8-to-3 (first precursor orbitide sequence in the gene held constant) to identify variants from the 1:1:1 ratio for these orbitides within the core collection. Anomalous ratios were observed only in the two accessions ‘Hollandia’ and ‘Z 11637’. These accessions lack 1 and 3 (traces detectable by HPLC−ESI−MS), while the concentration of 8 was comparable to the mean of the core collection. These outcomes were consistent with the lowexpression concentration of total orbitide exhibited by these accessions. Additionally, these observations indicated that ‘Hollandia’ and ‘Z 11637’ may contain different orbitide encoding sequences in the region of contig AFSQ01016651.1 compared to other flaxseed accessions. These accessions may differ in their transcription, translation, or post-translational modification with respect to other accessions. Unlike contig AFSQ01016651.1 that encodes a single copy for each of orbitides 1, 3, and 8, AFSQ01025165.1 encodes a linear precursor peptide with five orbitide domains, including a single copy each of 6 and 13 and three copies for that of 17. The concentration ratios of 17 to 6 and 17 to 13 expressed in ‘CDC Bethune’ were approximately 3.0:1 and 2.5:1, respectively, supporting the hypothesis that each translated precursor protein yielded five orbitides. Due to the observed high correlation between orbitide concentrations of 17 and 13 encoded within the same gene, we examined only the FCC for potential concentration variants of 17 to 13. A total of three accessions were identified that contained unusually high concentration ratios of 17 to 13, namely ‘Sorth Behbehan’ (CN 97180), ‘Bjelo Katjacs’ (CN 100864), and an unnamed accession (CN 100837) (Table 6). Another three accessions had unusually low concentration ratios of 17 to 13 (CN 97129; CN 101279; and CN 101595) (Table 6). These potential

Figure 4. Histogram showing the concentrations of orbitides encoded by GenBank contig AFSQ01016651.1 ([1, 3, 8]) compared to that encoded by contig AFSQ01025165.1 ([6, 13, 17]) expressed by the FCC planted in Saskatoon, SK (n = 300) and Morden, MB (n = 324) in 2009.

(CN 98150) expressed the lowest total orbitide content (Table 3). Individual orbitide concentrations between locations exhibited significant correlations of r ≥ 0.5 (Table 4). The strongest correlations between individual orbitides occurred among those encoded by the same gene, other than for 6 (Table 5). Concentrations of 1 to 3 and 8 were shown to have r values of 0.9538 and 0.8180, respectively. Correlation between concentrations of 3 to 8 was slightly lower, with a r value of 0.8017. The largest correlation of orbitide concentrations was observed between 13 to 17 (r = 0.9184), in agreement with Gui et al.,29 which are known to be orbitides encoded within the same reading frame of contig AFSQ01025165.1. Correlations between concentrations of 6 to 13 and 17 were lower, with r values of 0.3976 and 0.4261, respectively.

Table 3. Flaxseed Accessions with Highest and Lowest Total Orbitide Content (Peak Area in mAU) total orbitide (mAU) CN

accession name

origin

height (cm)

stem branching

seed weight (mg)

seed color

petal color

SK

MB

18973 98056 98150

‘AC Watson’ ‘Hollandia’ ‘Z 11637’

Canada Netherlands Netherlands

51 72 91

1/4 branched 1/5 branched 1/4 branched

6.3 4.2 3.8

brown brown brown

blue lavender white

222.6 71.4 88.1

243.6 101.4 96.2

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Table 5. Correlation Coefficients among Average Orbitide Concentrations in Flaxseed Accessions from the FCC Planted in Saskatoon, SK and Morden, MB in 2009 (n = 283, p < 0.01)

a

orbitide

1

3

6

8

13

3 6 8 13 17 [6,13,17]

0.9538a 0.2228 0.8180 0.3250 0.3007

0.2599 0.8017 0.3377 0.3359

0.2821 0.3976 0.4261

0.4117 0.3599

0.9184

[1,3,8]

0.3821

Not significantly different at p < 0.01.

Table 6. Flaxseed Accessions with Highest and Lowest Concentration Ratios of Orbitides 17 to 13 (Orbitide Variation within Contig AFSQ01025165.1) [17]/[13]

a

CN

accession name

origin

height (cm)

97180 100864 100837 97129 101279 101595

‘Sorth Behbehan’ ‘Bjelo Katjacs’ − − − −

Iran Hungary Turkey Iran −a −a

72 84 80 49 48 54

stem branching 1/4 1/4 1/5 1/3 1/5 1/3

branched branched branched branched branched branched

seed weight (mg)

seed color

petal color

SK

MB

6.1 6.1 5.8 4.8 7.4 10.9

−a mottled brown mixture brown mottled

white white lavender lavender blue white

3.5 4.0 3.8 2.4 2.2 2.3

4.0 3.6 3.4 2.1 2.1 2.1

Not specified on the PGRC Web site.13

variants may reflect differences in the coding sequences of contig AFSQ01025165.1. For check cultivars, the concentrations of orbitides encoded by contig AFSQ01016651.1 (containing embedded sequences for 1, 3, and 8), hereafter referred to as [1,3,8], were higher than the concentrations of those encoded by contig AFSQ01025165.1 (containing embedded sequences for 6, 13, and 17), hereafter referred to as [6,13,17] (Table 1). Additionally, the correlation between orbitide concentrations at the two locations was positive (r = 0.5946, p < 0.01) (Figure 4). The majority of the accessions (98%) expressed higher cumulative concentrations of [1,3,8] than [6,13,17]. Approximately 76% of the accessions in Saskatoon and Morden were within 1SD of the mean of all accessions (2.2 ± 0.5 and 2.1 ± 0.5, respectively) (Figure 5). Accessions ‘Primus’ (CN 98689) and an unnamed accession (CN 101580) expressed the highest ratios of [1,3,8] in comparison to [6,13,17] in both locations (Table 6). Lowest ratios of [1,3,8] to [6,13,17] were observed in accessions ‘Hollandia’ and ‘Z 11637′, which is consistent with the extremely low to negligible expression of 1 and 3. Accessions with the highest and lowest ratios of [1,3,8] to [6,13,17] may represent sequence variants that have altered mRNA-ribosomal binding kinetics or mRNA half-lives (Table 7). Association of Total Orbitide Content with Plant Morphological Traits. As previously stated, ‘CDC Bethune’, ‘Hanley’, and ‘Macbeth’ were included in the FCC as check cultivars against 388 accessions. Generally, checks grown at Morden had higher orbitide concentrations than those grown in Saskatoon, with ‘Macbeth’ expressing higher orbitide concentrations in comparison with the other two checks (Table 2). These observations could be attributed to the larger seed size and higher oil content of ‘Macbeth’.40 ‘CDC Bethune’ and ‘Hanley’ have medium seed size, medium oil content, and high yield when seeded in Prairie brown and black soil zones.38,39 We also investigated orbitide content in relation to quantitative and qualitative morphological plant traits. In

Figure 5. Correlation between (A) total orbitide content and (B) the concentration of orbitides encoded by GenBank contig AFSQ01016651.1 ([1,3,8]) to that encoded by contig AFSQ01025165.1 ([6,13,17]) expressed by 283 accessions of the FCC planted in Saskatoon, SK and Morden, MB in 2009.

these studies, no correlations were drawn between total orbitide content and plant height, seed weight, seed oil 5203


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Table 7. Flaxseed Accessions with Highest and Lowest Concentration Ratios of Orbitides [1,3,8] to [6,13,17] (Orbitide Variation between Contigs AFSQ01016651.1 and AFSQ01025165.1) [1,3,8]/ [6,13,17]

a

CN

accession name

origin

height (cm)

101580 98689 98150 98056

− ‘Primus’ ‘Z 11637’ ‘Hollandia’

−a Poland Netherlands Netherlands

47 45 91 72

stem branching 1/2 1/3 1/4 1/5

seed weight (mg)

seed color

petal color

SK

MB

7.6 8.3 3.8 4.2

yellow brown brown brown

pink lavender white lavender

3.7 4.1 0.6 0.7

3.5 3.7 0.7 0.7

branched branched branched branched

Not specified on the PGRC Web site.13

content, or α-linolenic acid concentration. Additionally, the degree of stem branching, petal color, and seed color were not associated with total orbitide content in accessions planted in either location. These results agree with other studies that determined no association between seed mucilage content35 and stem fiber content.6 Long, nonbranched stems, low seed yields, and small seeds are typical traits of fiber flax. However, Diederichsen and Raney10 reported that there was no association found between seed quantitative (plant height, total seed oil, and α-linolenic acid) and qualitative (stem branching, petal color, and seed color) traits. The absence of correlation between total orbitide content with qualitative and quantitative traits indicates that orbitide expression is independent and should not be affected by changes in these traits. Thus, flax breeders face no biological restriction when extreme qualitative and quantitative flax traits are combined with desired orbitide expression. Environment should be considered as a potential impact factor on orbitide content and composition. Lower flax mucilage content has been linked to extreme humidity during harvest.45 Variation in buckwheat rutin content has been associated with cropping season (to some extent, probably due to solar radiation differences)46 and soil types.47 Nahapetian and Bassiri observed a strong influence of seasonal differences on wheat phytate concentration.48 However, Gui and coworkers found no correlation between environment (growth location and climate) and orbitide production in five flaxseed cultivars (‘CDC Bethune’, ‘CDC Valour’, ‘Flanders’, ‘Somme’, and ‘Vimy’) planted in Saskatoon and Floral, SK.29 Oomah et al. proposed that environment may affect accessions differentially, such that metabolite expression is governed by sensitivity of individual accession to environmental changes.49 Although we observed minimal environmental effects on the orbitide content of check accessions, it is impossible to discern environmental impact, if any, on orbitide content of FCC accessions because many are not adapted for growth in Canada. In lieu of that, we postulate that observed anomalous ratios of orbitides may be due to the existence of true flax variants that possess different orbitide precursor genes that affect orbitide expression. The ability to identify genetic variations including both the type and amount of orbitide expressed in flaxseed is critical for potential harnessing of these compounds for their biological activity.

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reagent Na2S2O3 (Figure S2), and (iii) correlation between plant quantitative morphological traits to total orbitide content expressed by FCC accessions planted in both locations (Figure S3). Tables showing a summary of agronomic and chemical variations within the FCC (Table S1), tabulated data of flax orbitide distributions in two sites (Tables S2 and S3), and orbitide content of accessions planted in either location in relation to qualitative morphological plant traits (Table S4). (PDF)

AUTHOR INFORMATION

Corresponding Authors

*P.-G. G. B. tel: +1 306 966 8840; fax: +1 306 966 5015; email: [email protected]. *M. J. T. R. tel: +1 306 966 5027; fax: +1 306 966 5015; e-mail: [email protected]. Funding

Financial support was provided by Genome Canada (TUFGEN project no. 1309) and the Saskatchewan Ministry of Agriculture Agricultural Development Fund (grants 20080205, 20120099, and 20120146). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Vijaya Jadhav, Michelle Nie, and Da Wang for laboratory assistance and Dr. Lester Young for thoughtful discussion while preparing this manuscript.

■

ABBREVIATIONS USED FCC, flax core collection; PGRC, Plant Gene Resources of Canada; CDC, Crop Development Centre; CN, Canadian National; HPLC−DAD, high-performance liquid chromatography with diode array detector; HR-HPLC−ESI-MS, highresolution high-performance liquid chromatography with electrospray ionization mass spectrometry; HPLC−ESI-MS/ MS, high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectrometry; mRNA, messenger ribonucleic acid

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REFERENCES

(1) Zohary, D.; Hopf, M. Domestication of plants in the Old World. The origin and spread of cultivated plants in West Asia, Europe, and the Nile Valley. 3rd ed.; Oxford University Press: New York, 2000. (2) Diederichsen, A. Ex situ collections of cultivated flax (Linum usitatissimum L.) and other species of the genus Linum L. Genet. Resour. Crop Evol. 2007, 54, 661−678. (3) Diederichsen, A.; Kusters, P.; Kessler, D.; Bainas, Z.; Gugel, R. Assembling a core collection from the flax world collection maintained by Plant Gene Resources of Canada. Genet. Resour. Crop Evol. 2013, 60, 1479−1485.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02035. Figures showing variation of qualitative morphological traits within the FCC (Figure S1), orbitide distribution in a mixture of oxidized flaxseed extract and the quenching 5204


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