Comparative Metabolite Profiling of Solanum tuberosum against Six

Article pubs.acs.org/JAFC

Comparative Metabolite Profiling of Solanum tuberosum against Six Wild Solanum Species with Colorado Potato Beetle Resistance Helen H. Tai,*,† Kraig Worrall,†,§ Yvan Pelletier,† David De Koeyer,† and Larry A. Calhoun§ †

Agriculture and Agri-Food Canada Potato Research Centre, P.O. Box 20280, 850 Lincoln Road, Fredericton, New Brunswick, Canada E3B 4Z7 § Department of Chemistry, University of New Brunswick, P.O. Box 4400, Fredericton, New Brunswick, Canada E3B 5A3 S Supporting Information *

ABSTRACT: The Colorado potato beetle Leptinotarsa decemlineata (Say) (CPB) is a coleopteran herbivore that feeds on the foliage on Solanum species, in particular, potato. Six resistant wild Solanum species were identified, and two of these species had low levels of glycoalkaloids. Comparative analysis of the untargeted metabolite profiles of the foliage using UPLC-qTOF-MS was done to find metabolites shared between the wild species but not with Solanum tuberosum (L.) to identify resistance-related metabolites. It was found that only S. tuberosum produced the triose glycoalkaloids solanine and chaconine. Instead, the six wild species produced glycoalkaloids that shared in common tetrose sugar side chains. Additionally, there were non-glycoalkaloid metabolites associated with resistance including hydroxycoumarin and a phenylpropanoid, which were produced in all wild species but not in S. tuberosum. KEYWORDS: Colorado potato beetle resistance, untargeted metabolite profiling, potato, Solanum, glycoalkaloid, phenylpropanoid

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CPB resistance.19 Introgression of Solanum chacoense Hawkes, which is enriched in leptine glycoalkaloids, has also resulted in potato germplasm with increased resistance.20 The demonstrated ability of CPB populations to develop resistance to chemical insecticides21 has made developing new germplasm resources for breeding CPB resistance an increasingly important strategy. Plant resistance to herbivores can be constitutive or induced.22 Constitutive defenses are produced in plants even in the absence of the herbivore and can prevent insect attack. Induced defenses, on the other hand, are produced after herbivore feeding damage has been sustained. Defense metabolites can be used as selectable chemical markers for breeding CPB resistance. Metabolite marker screening can increase the efficiency of breeding through decreasing the need for field testing of CPB defoliation to screen for resistance. The present study examines the differences in metabolite profiles between six wild Solanum species with high levels of CPB resistance and S. tuberosum. The goal was to find common resistance-related metabolites in the wild species that have potential application as selectable chemical markers for breeding.

INTRODUCTION Colorado potato beetle (CPB) feeding on potato foliage causes yield losses of 30−50%.1 CPB selection of host plants is determined by the choice of the mobile adults of plants for feeding and oviposition.2 Successful establishment of first-instar larvae on foliage follows on susceptible host plants.3 Many factors contribute to CPB−host interactions including metabolites produced by the host plant that can function as antifeedants and semiochemicals.4 Glycoalkaloids are a class of potato metabolites that are under regulatory control due to their toxicity in humans.5 In plants, glycoalkaloids can deter herbivore feeding.6 Solanine and chaconine are the major glycoalkaloids of Solanum tuberosum;7,8 however, there is diversity in glycoalkaloids in other Solanum species relatives8−10 including species bearing resistance to CPB. Foliar leptine and α-tomatine glycoalkaloids in Solanum have been associated with decreased adult feeding and increased preimaginal mortality of CPB.11,12 Nonglycoalkaloid metabolites also contribute to CPB resistance. For example, the sesquiterpenes13 and sucrose fatty acid esters14 present in exudates of glandular trichomes are associated with CPB resistance in the wild potato relative, Solanum berthaultii Hawkes. Macrocypins also inhibit CPB and function as protease inhibitors.15 Most commercial potato varieties are susceptible hosts for CPB. Furthermore, the narrow genetic base of the domesticated potato S. tuberosum has made breeding for resistance a challenge. However, there are a number of wild potato species with resistance to CPB, including some that can be introgressed with S. tuberosum.16−18 Breeding for resistance to CPB is an important part of the integrated pest management plan for this insect.17,18 Successful introgression of the glandular trichomecontaining species, S. berthaultii, with S. tuberosum has been achieved, and germplasm resources generated have increased © 2014 American Chemical Society

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MATERIALS AND METHODS

Plant Material for Metabolite Analysis. Wild Solanum plants from the species S. tarijense, S. oplocense, S. piurae, S. acroglossum, S. chomatophilum, and S. paucissectum were propagated from true botanical seed and the domesticated species, S. tuberosum cv. Shepody, was propagated from seed tubers. Plants for each species were grown Received: Revised: Accepted: Published: 9043

May 29, 2014 August 18, 2014 August 21, 2014 August 21, 2014 dx.doi.org/10.1021/jf502508y | J. Agric. Food Chem. 2014, 62, 9043−9055

Journal of Agricultural and Food Chemistry

Article

Figure 1. Solanum plants grown in the field: (a) S. tuberosum cv. Shepody (TUB); (b) S. tuberosum cv. Shepody treated with imidacloprid (TUB + pesticide); (c) S. chomatophilum (CHM); (d) S. oplocense (OPL); (e) S. paucissectum (PCS); (f) S. piurae (PUR); (g) S. acroglossum (ACG); (h) S. tarijense (TAR). Plants were grown in the absence of imidacloprid pesticide treatment except as indicated. in the greenhouse in pots with potting mix and weekly fertilizer application of 20−20−20. Sampling of foliage was done on fully grown plants at 12 weeks after planting. The apical leaflets from five leaves for each plant were pooled in 15 mL conical tubes and flash frozen in liquid nitrogen. Samples were stored at −80 °C prior to extraction. A minimum of three replicate plants were sampled for all species. Growth of Plants in the Field. Four plants from each wild Solanum species and S. tuberosum were grown in pots in the greenhouse in May 2008 and transplanted to a field at the Potato Research Centre 4 weeks after planting. The field was not treated with pesticide to allow for CPB infestation. For comparison, S. tuberosum was also planted in a field with imidacloprid (Admire) pesticide treatment in the soil prior to planting and two foliar treatments in July. The presence/absence of CPB and defoliation was noted during July and August, and photographs were taken. Extraction. The frozen foliar samples were ground into a fine powder using a mortar and pestle while immersed in liquid nitrogen. One hundred milligrams of frozen ground tissue was placed in a 1.5 mL polyethylene screw-cap tube and kept frozen in a liquid nitrogen holding station (SPEX Sampleprep, Metuchen, NJ, USA) until all samples were ground. The ground powder was extracted with 400 μL

of extraction solution (92% methanol, 0.1% formic acid LC-MS grade v/v) (Sigma-Aldrich, Oakville, ON, Canada). The samples were briefly vortexed and placed on ice until all samples were prepared. Samples were then sonicated in a Branson sonicator bath for 15 min and filtered through a 0.2 μm syringe filter into an LC-MS autosampler vial. Sample dilutions were optimized to ensure peak intensities were in the linear range and to avoid detector saturation. Wild Solanum and S. tuberosum samples were diluted 4-fold with extraction buffer. The samples were allowed to equilibrate at 23 °C in the dark for 1 h prior to analysis and were maintained under these conditions for the duration of the LC-MS analysis. UPLC-qTOF-MS. Metabolite analysis was carried out using Acuity ultraperformance liquid chromatography− Xevo quadrupole time-offlight mass spectrometry (UPLC-qTOF-MS) (Waters, Milford, MA, USA). Using a 5 μL loop, 0.75 μL injections were made for all samples in the study. The same volume of test mixture (rutin hydrate, caffeic acid, benzoic acid, p-coumaric acid, quercetin, L-phenylalanine, resveratrol, ferulic acid, L-tryptophan, sinapic acid, naringenin, transcinnamic acid, and isorhamnetin) (Sigma-Aldrich) was injected. All components of the test mixture were present at approximately 35 μg/ mL in 60:40 acetonitrile/water v/v. All chromatographic separations 9044

dx.doi.org/10.1021/jf502508y | J. Agric. Food Chem. 2014, 62, 9043−9055


Article

were carried out on a 1 mm × 100 mm BEH C18 reverse phase column. The mobile phase was composed of LC-MS grade water with 0.1% formic acid (phase A) and LC-MS grade acetonitrile with 0.1% formic acid (phase B). The linear gradient consisted of six segments as follows: initial segment, 95% A, 5% B; 13:33 min, 25% A, 75% B; 13:53 min, 5% A, 95% B; 18:00 min, 5% A, 95% B; 18:01 min, 95% A, 5% B; and 20:00 min, 95% A, 5% B. The flow rate was 45.0 μL/min for all segments. The autosampler bed was maintained at 23 °C and the column at 35 °C. Samples were injected in a randomized fashion. Each sample was injected in triplicate with the exception of the test mixture which was injected after every six samples to evaluate the stability of retention time and mass accuracy over the duration of the experiment. Mass spectrometry data were collected over the duration of the LC ramp from 0 to 800 s. Mass-to-charge ratios (m/z) between 100 and 1500 were detected by electrospray ionization in positive ionization mode. A lockmass solution of dilute leucine enkephalin (LE) in acetonitrile/water (50:50 v/v) was introduced via the lockspray probe at 25 μL/min as directed by the MS manufacturer. Data Processing. Mass spectrometry data were processed using Waters Markerlynx XS software. The UPLC-MS data were detected and noise-reduced in both the UPLC and MS domains such that only true analytical peaks were further processed by the software. Each peak was a feature that was identified using the chromatographic retention time from the chromatogram and m/z of the positive molecular ion. The molecular ion adduct most commonly observed was [M + H]+. No retention time correction was used as retention time stability was sufficient for UPLC. Quantification of peak intensity was done by integrating peaks with a mean retention time in the window of 100− 800 s. The retention time window was selected on the basis of visual evaluation of chromatograms to exclude column void and washout. Features were tentatively assigned compound identities through matching m/z against theoretical masses of compounds in the MetLin (http://metlin.scripps.edu/metabo_search_alt2.php) and KNApSAcK v1.200.3 (http://kanaya.naist.jp/KNApSAcK/) databases. Metabolites present in plant species with molecular masses within 6 ppm of the features were listed. Statistical Analysis. The Multi-Experiment Viewer (MeV 4.7.4) software (http://www.tm4.org/mev/) was used for statistical analysis of the data. Samples for the biological replicates for each species were each injected three times for each species. The data was log10 transformed after adding 1 × 10−12 to all data to remove zeros. The log10-transformed peak intensity data for all of the features were averaged for each of the species, and hierarchical clustering of the Solanum species was done using Euclidean distance. Additionally, oneway analysis of variance (ANOVA) was used to find features with variation between species at p ≤ 0.01. Bonferroni correction was applied. The null hypothesis tested was that there were no differences between the mean peak intensities between species; the alternative hypothesis was that there were differences between species. The features with significant species variation were further analyzed. The untransformed peak intensity data for these features were averaged and further filtered as follows: (a) features with average peak intensity 100fold increased in each of the wild species over S. tuberosum and with a minimum untransformed peak intensity of 10 in at least two wild species; (b) features with peak intensity data 100-fold increase in S. tuberosum over all of the wild species and with a minimal untransformed peak intensity of 10 in S. tuberosum.

which concurs with previous observations.17,18,23,24 An objective of the study was to find metabolites that can be used as markers for screening CPB resistance in potato germplasm grown in greenhouses. Therefore, comparative analysis of foliar metabolites in S. tuberosum and the wild species was done with greenhouse-grown plants. The total ion chromatograph of extracted metabolites in positive ionization mode (Figure 2) and processed peak intensity data (Supporting Information Table S1) demonstrated metabolite variation between species. The Solanum species were grouped using hierarchical clustering of all feature peak intensities from Table S1 (Figure

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RESULTS AND DISCUSSION Comparative Analysis of Wild Species and S. tuberosum. Studies have shown that the wild species S. acroglossum, S. tarijense, S. chomatophilum, S. oplocense, S. paucisectum, and S. piurae have CPB resistance.17,18,23,24 These wild species along with S. tuberosum were grown in the field in the absence of the pesticide imidacloprid. CPB defoliation was observed on S. tuberosum in the absence (Figure 1a), but not in the presence, of imidacloprid (Figure 1b). The wild species showed low levels of defoliation in the field without imidacloprid (Figure 1c−h),

Figure 2. Total ion chromatograms for (a) S. acroglossum (4.87\307.1725), (b) S. tarijense (1.89\121.0636), (c) S. chomatophilum (7.56\1046.5571), (d) S. oplocense (7.56\1046.5571), (e) S. paucisectum (7.56\1046.5571), (f) S. piurae (7.77\578.4045), and (g) S. tuberosum (7.60\868.5004). The base peak features are given in parentheses. One hundred percent peak intensity for each was set at the intensity of the base peak. Each chromatogram is for a single representative sample for each species. 9045



Article

not shown). 7.60\868.5004 could also be assigned to βsolamarine, solamargine and leptinine I; however, these glycoalkloids were not found to be at high levels in S. tuberosum by others.7,9 These glycoalkaloids could also be assigned to features 8.32\868.5086 and 7.96\868.5099, which were not found at high levels in S. tuberosum, consistent with published studies.7,9 Feature 7.46\884.5049 had an m/z that matched glycoalkaloids leptinine II, solasonine, and solamarine. Table 1 shows that 7.46\884.5049 was found at high levels in S. tuberosum, which was not consistent with published information showing that these glycoalkaloids were not found in S. tuberosum.8 An alternative explanation is that 7.46\884.5049 is the [M + CH3OH + H]+ molecular ion adduct of chaconine (theoretical mass of 884.5366), which can form in positive ionization mode in ESI. Assignment of the feature to the [M + CH3OH + H]+ adduct would concur with the high levels in S. tuberosum observed for 7.46\884.5049. S. oplocense had the next highest intensity for 7.46\884.5049 followed by four other wild species at lower levels (Table 1). The remaining glycoalkloids in Table 1 were all not detectable in S. tuberosum but were produced in wild species. Feature 7.56\1046.5571 was found in the largest number of wild species (four) and was assigned to the glycoalkaloid dehydrocommersonine, which had been previously characterized from S. oplocense.29−31 The peak intensity for dehydrocommersonine was highest in S. oplocense and was the highest peak intensity of all the features in any species. High levels were also found in S. paucisectum, S. chomatophilum, and S. piurae. Feature 7.94\1034.5581 was assigned to tomatine, which is a tomato glycoalkaloid associated with CPB resistance.11 It was detectable in S. piurae at lower levels than dehydrocommersonine and was at very low levels in S. chomatophilum. Removal of the xylose sugar converts tomatine to β-tomatine. The feature 7.80\902.5148 had a m/z matching β-tomatine and, like tomatine, was also found in S. piurae and S. chomatophilum. The feature 7.73\1064.568 was assigned to the glycoalkloid neotomatine, which had highest levels in S. piurae followed by S. chomatophilum and very low detection in S. paucisectum. 7.72\1016.5479 was assigned to dehydrodemissine, which was previously described in Solanum commeronii Dun ex. Poir.32 Dehydrodemissine was found in S. piurae at levels over 300-fold lower than dehydrocommersonine. 8.01\1018.5629 and 6.92\1032.5429 were assigned to glycoalkaloids demissine and dehydrotomatine, respectively. These glycoalkloids had very low peak intensities. The chemical structures of chaconine and solanine were compared with glycoalkaloids from the wild species. Figure 4 shows that the glycoalkaloids found in S. tuberosum had triose sugar side chains compared to wild species, where there were tetrose side chains. The results suggest that the larger tetrose side chain glycoalkaloids were associated with resistance to CPB. These findings correlate with studies showing that CPB has a preference for potato over tomato, which produces the tetrose glycoalkaloid tomatine.33 Other studies have shown that decreasing the sugar side chain on glycoalkaloids reduces their toxicity34 and capacity to disrupt membranes.35 Non-glycoalkaloid Metabolites Associated with CPB Resistance. Previous studies on S. berthaultii have demonstrated that resistance was associated with glandular trichomes.16 However, it was also observed that CPB feeding remains low in S. tarijense after the mechanical removal of trichomes, indicating that there are other components of the leaf involved in resistance.36 Our observations indicate that S.

3). S. tuberosum was found to be the most distant from the other species. S. chomatophilum and S. oplocense were found to

Figure 3. Hierarchical clustering of species based on metabolite profiles. TUB, S. tuberosum cv. Shepody; CHM S. chomatophilum; OPL, S. oplocense; PCS, S. paucissectum; PUR, S. piurae; ACG, S. acroglossum; TAR, S. tarijense. The scale is the Euclidean distance.

be closely related as were S. paucisectum and S. piurae. S. acroglossum and S. tarijense were more distant from these wild species. The results from hierarchical clustering were compared to published taxonomic groupings for species. All of the species studied were members of Solanum section Petota Dumortier, including S. tuberosum. Using morphological and genetic markers S. piurae, S. acroglossum, S. paucisectum, and, more recently S. chomatophilum were all placed in series Piurana.25 Interestingly, our results showed that S. acroglossum did not cluster with the other three species on the basis of foliar metabolite profiles. S. tarijense has been recently found to be synonymous with S. berthaultii.26 S. tarijense and S. berthaultii were designated as belonging to series Tuberosa III and Yungasensa by Hawkes. S. oplocense is part of the series Tuberosa, and more recently this species has been classified as belonging to the S. brevicaule complex, which encompasses some 20 species.27 The clustering of metabolites places S. oplocense close to the Piurana species S. chomatophilum and, to a lesser extent, S. paucisectum and S. piurae. The results show that metabolite diversity occurs within a taxonomic grouping, and similarities occur between species described as more distantly related. Chemotaxonomic analysis of sapogenins from wild Solanum species also demonstrated clustering of species not found to be grouped together taxonomically.28 Variability between accessions of the same species was also noted. Glycoalkaloid Variation between Species. Features with m/z corresponding to known glycoalkaloids found in S. tuberosum and other Solanum species7−10 were identified from Table S1 and are listed in Table 1. The glycoalkaloid peaks in the chromatographic spectra were found at retention times between 7.00 and 8.50 min. The major peaks for S. tuberosum, S. chomatophilum, S. oplocense, S. paucisectum, and S. piurae were glycoalkaloids (Figure 2 and Table 1). S. tarijense and S. acroglossum had major peaks outside the range for glycoalkaloids (Figure 2) and lower levels of known glycoalkaloids (Table 1). S. acroglossum, in particular, did not have high peak intensity in the range between 7.00 and 8.50 min. The total peak intensity for all features across all species was similar (Table S1). The differences were in the distribution of peaks in the spectra (Figure 2). The feature 8.05\852.514 had a m/z that was a match with chaconine. 8.05\852.514 was barely detectable in the wild species but had high levels of production in S. tuberosum. Solanine, the other glycoalkaloid found in S. tuberosum, was a match with 7.60\868.5004 and had high peak intensity S. tuberosum but not the wild species (Table 1). These results were similar to those reported by others.7,9 The assignment of chaconine and solanine was also verified using standards (data 9046


9047

902.5108 1016.5425 1018.5581 1032.5374 1034.5531 1046.5531 1064.5636

884.5366c

884.5002

4 5 4 5 4 3 4

5 5 5 35

0 0 0

5 5 5 5

3 3 3 3

5 5 5 5

4

Δ ppm

0 0.0025 0.0058 0.0062 0.0147 0 0

0

0.0056

0.0085

0

0

0

ACG

0 0.0050 0.0170 0.0117 0 0 0.0096

0.0042

0.0068

0

0

0

0

TAR

7.2080 7.8641 2.0795 0 0.8863 858.3495 153.6252

10.0907

0

0

0.0231

0.0691

0.0054

CHM

0.0592 0 1.6633 0 0 2445.9540 0

70.9600

0

0.0033

0.0709

0.0009

0

OPL

49.6026 0.5620 2.4713 0.0104 1.3943 947.8109 187.6920

10.5427

0

0

0.0098

0.0041

0.0008

PCS

26.4639 14.4214 0 4.7287 89.0285 266.7079 558.2309

4.6792

0.0020

0

0

0.1245

0.1038

PUR

0 0.0076 0 0.0013 0.0691 0 0

1320.1321

7.8517

532.7175

17.5432

2135.0593

1266.9750

TUB

Metabolites with m/z matching with known glycoalkaloids are shown. The feature ID is the retention time\m/z combination. Compounds in the MetLin database that matched the m/z are listed along with their molecular formulas and theoretical mass of the [M + H]+ molecular ion. Differences between m/z of the feature and theoretical masses of matching compounds are indicated as Δ ppm. TUB, S. tuberosum cv. Shepody; CHM, S. chomatophilum; OPL, S. oplocense; PCS, S. paucissectum; PUR,S. piurae; ACG, S. acroglossum; TAR, S. tarijense. The average Markerlynx processed peak intensity across the replicates for each species is presented. bTheoretical mass is for the [M + H]+ molecular ion unless otherwise indicated. cTheoretical mass is for the [M + CH3OH + H]+ molecular ion.

a

C45H75NO17 C50H81NO20 C50H83NO20 C50H81NO21 C50H83NO21 C51H83NO21 C51H85NO22

β-tomatine dehydrodemissine demissine dehydrotomatine tomatine dehydrocommersonine neotomatine

7.80\902.5148 7.72\1016.5479 8.01\1018.5629 6.92\1032.5429 7.94\1034.5581 7.56\1046.5571 7.73\1064.568

C45H73NO16 C45H73NO16 C45H73NO16 C45H73NO14

leptinine II solasonine solamarine chaconine

7.46\884.5049

884.5002

C45H73NO16 C45H73NO16 C45H73NO16

leptinine II solasonine solamarine

8.54\884.5002

868.5053


solanine β-solamarine solamargine leptinine I

7.96\868.5099

868.5053



8.32\868.5086

868.5053



852.5104

C45H73NO14

7.60\868.5004

theor massb

formula

chaconine

glycoalkaloid

8.05\852.514

feature ID

Table 1. Glycoalkaloidsa

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Figure 4. Chemical structures of glycoalkaloids. Chaconine and solanine were produced in S. tuberosum; dehydrocommersonine, tomatine, and neotomatine were produced in the CPB-resistant wild Solanum species. Triose and tetrose sugar side chains are indicated.

acroglossum did not have trichomes, which agrees with previous descriptions of the species as glabrous.37 Hence, resistance in S. acroglossum is not associated with high levels of either glycoalkaloids or trichomes. An analysis of foliar metabolites shared between the wild species in the study but not S. tuberosum was done to identify non-glycoalkaloid and nontrichome metabolites associated with resistance to CPB. An ANOVA analysis was done to find features with significant variation between the species. Eight hundred and sixty-seven features had significant variation in peak intensity between the species (see Supporting Information Table S2). The 867 features were screened to identify features with peak intensities 100 times increased over S. tuberosum for all of the wild species (Table 2). Additionally, features had to have untransformed peak intensities of at least 10 in two of the wild species. The criteria were used to select for features highly associated with CPB resistance and to eliminate features that were produced at

very low levels or only in a single wild species. Features 3.60\163.0387 and 4.02\163.0393 had an m/z that was matched to hydroxycoumarin. This feature was present in all of the wild species. There are six isomers of hydroxycoumarin, each with the hydroxyl group in a different position. 7Hydroxycoumarin or umbelliferone is a precursor to other coumarins, including bergamottin, which is ovicidal to CPB.38 4-Hydroxycoumarin itself can act as a blood anticoagulant in humans but is often used as a precursor for synthesis of more powerful anticoagulants such as warfarin.39 Derivatives of 4hydroxycoumarin also have larvicidal activity against Aedes aegyti.40 Hydroxycoumarin produced in the wild species may directly affect CPB or may be a common precursor to other metabolites that are antifeedants. 5.80\319.0417 was assigned to 3-deoxy-D-manno-octulosonate 8-phosphate (KDOP). Plants synthesize KDOP for polysaccharide cross-links of pectin.41 KDOP is a precursor 9048


3-hexenedioic acid allylmalonic acid (E)-3-methylglutaconic acid (E)-hex-2-enedioic acid 2,3-dimethylmaleate methylitaconate 2-methyleneglutarate triacetate, 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one 1,5-Aanhydro-4-deoxy-D-glycero-hex-3-en-2-ulose 1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose 2E-methylglutaconic acid 3-hydroxyadipic acid 3,6-lactone dimethyl fumarate ethyl hydrogen fumarate (E)-2-methylglutaconic acid

3-hydroxycoumarin 7-hydroxycoumarin umbelliferone 7-hydroxy-2H-chromen-2-one 4-hydroxycoumarin 5-hydroxycoumarin 8-hydroxycoumarin 6-hydroxycoumarin

3-hydroxycoumarin 7-hydroxycoumarin umbelliferone 7-hydroxy-2H-chromen-2-one 4-hydroxycoumarin 5-hydroxycoumarin 8-hydroxycoumarin 6-hydroxycoumarin

herniarin 7-methoxychromon E 2-propenal, 3-(1,3-benzodioxol-5-yl)1,3,8-naphthalenertriol 4-methylumbelliferone 10-hydroxy-8E-decene-2,4,6-triynoic acid 10-hydroxy-2,8-decadiene-4,6-diynoic acid 1,4,5-naphthalenetriol

3.60\163.0387

4.02\163.0393

4.84\177.0546

putative ID

7.84\145.0502

feature ID

Table 2. Metabolites Increased in All Wild Species over S. tuberosuma

9049

C10H8O3

C9H6O3

C9H6O3

C6H8O4

formula

179.0473

163.0393

163.0387

145.0502

theor mass [M + H]+

0

2

1

4

Δ ppm

180.2685

49.8817

1.4602

0.3426

ACG

8.7228

141.2625

30.7208

2.2907

TAR

CHM

19.7829

40.4365

16.5359

37.2655

OPL

0.5979

19.9633

7.8594

1.8167

PCS

2.3425

98.6877

60.0625

21.5554

PUR

6.9256

95.3974

50.8605

80.3951

0

0

0

0

TUB



unknown unknown unknown unknown unknown unknown unknown

pelargonidin 3-O-[2-O-(6-O-(E)-feruloyl-β-D-glucopyranosyl)-β-Dglucopyranoside]-5-O-(β-D-glucopyranoside) pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-β-D-glucopyranosyl-β-Dglucopyranoside]-5-O-(β-D-glucopyranoside) peonidin 3-p-coumarylsophoroside-5-glucoside peonidin 3-caffeylrutinoside-5-glucoside petanin isovitexin 2′′-O-(6′′′-(E)-feruloyl)glucoside 4′-O-glucoside kaempferol 3-neohesperidoside-7-(2′′-ferulylglucoside) kaempferol 3-[2Gal-(6′′′-feruloylglucosyl)robinobioside] petunidin-3-(p-coumaroyl)-rutinoside-5-glucoside

5.50\470.2281 7.84\534.7624 7.80\562.4093 6.81\576.3895 7.63\576.3906 7.77\578.4045 6.11\592.3855

5.99\933.2699

demissidine oblonginine veramiline

7.81\400.3566

marioside

unknown

2.11\399.0485

7.83\459.2213

3-deoxy-D-manno-octulosonate 8-phosphate

5.80\319.0417

tomatidine soladucidine

unknown

6.71\261.1699

7.78\416.3512

N,N-diisopropyl-3-nitrobenzamide NH-Phe(NMe)-Gly-OMe heptabarbital 4-coumaroyl-2-hydroxyputrescine caffeoylputrescine

7-hydroxy-6-methyl-2H-1-benzopyran-2-one 3-(3,4-methylenedioxyphenyl)propenal

putative ID

3.61\251.1384

feature ID

Table 2. continued

9050

C43H48O23

C22H34O10

C27H45NO2

C27H45NO

C8H15O11P

C13H18N2O3

formula

933.2659

459.2225

415.3450

400.3574

319.0425

250.1317

theor mass [M + H]+

4

2

2

1

2

2

Δ ppm

81.7991

0.0691 4.0082 0.0350 0.0257 0.0310 0.0500 0.0045

0.5093

0.0280

0.0183

64.7065

14.6945

472.2803

0.6402

ACG

0.4922

0.0536 3.7564 1.6845 0.0141 0.0255 0.0794 0.0120

417.1292

0.0306

0.2617

0.0079

0.2787

0.1759

0.0380

TAR

5.6224

0.1472 6.1707 493.5220 5.3507 63.2127 316.2244 32.1651

1.0307

123.9308

163.9837

0.0013

0.1642

0.0594

28.6526

CHM

27.5091

27.8406 92.9919 87.1664 15.1207 4.9227 37.9129 1.0518

12.4345

13.9041

35.2965

1.3982

13.5001

19.1877

8.1334

OPL

49.0876

231.5718 40.1914 492.2046 7.4583 51.6607 306.7277 11.2982

1.6100

121.7996

169.8858

16.4110

0.2783

110.2894

105.1551

PCS

169.9223

45.4547 45.4547 188.1997 128.4867 263.4704 766.7678 11.3511

0.0918

315.8535

65.2168

7.6426

3.1046

152.1236

88.2894

PUR

0

0 0 0 0 0 0 0

0

0

0

0

0

0

0

TUB



0 95.5583 30.6382 0.4790 43.2917 0.0156 0.0028 unknown 7.60\1062.5525

amurenoside A amurenoside B

for KDO, which is component of rhamnogalactouronan II, a borate-binding cross-linked component of pectin in cell walls.42 S. acroglossum and S. oplocense produce the highest levels of KDOP among the species. The results suggest that these two species may have altered cell wall structures. 7.81\400.3566 was assigned to demissidine, a glycoalkaloid aglycon; additionally, the feature could also be assigned to veramiline, an alkaloid with the same chemical formula. The levels were highest in S. chomatophilum and S. piurae. Demissidine could be a fragment ion of the glycocoalkaloid demissine, which was detected in S. chomatophilum and S. paucisectum at low levels (Table 1). Demissine is the major glycoalkloid of Solanum demissum Lindl., another species with resistance to CPB, and is an antifeedant for CPB.43 However, the feature identified as demissine, 8.01\1018.5629, had a retention time slower than 7.81\400.3566, which is inconsistent with a parent molecular ion for a demissidine aglycon fragment ion. It is possible that demissidine serves as an aglycon to an unknown glycoalkaloid. Tomatidine and soladucidine were aglycons that were both assigned to 7.78\416.3512. Tomatine, a glycoalkaloid with a tomatidine aglycon, was identified as feature 7.94\1034.5581 (Table 1). However, 7.94\1034.5581 had a slower retention time than 7.78\416.3512, indicating that 7.78\416.3512 was unlikely a fragment ion. Alternatively, 7.78\416.3512 may be the aglycon of an unknown glycoalkaloid. It is also possible that 7.78\416.3512 is an unknown non-glycoalkaloid metabolite with a mass similar to that of tomatidine and soladucidine. 7.83\459.2213 was assigned to marioside, a norcarotane sesquiterpene glucoside found in ferns.44 This metabolite had high peak intensity in S. tarijense. There is very little information on the biological functions of marioside. 7.84\145.0502 and 4.84\177.0546 were assigned to multiple metabolites that were not previously associated with plant insect interactions. 5.99\933.2699 had highest peak intensity in S. piurae followed by S. acroglossum. 5.99\933.2699 had a large number of m/z matches in the chemical databases. Chief among these metabolites were anthocyanins and other flavonoid glycosides in the phenylpropanoid biosynthetic pathway. 3.61\251.1384 was assigned to the polyamine-conjugated phenylpropanoid, caffeyolputrescine, in addition to other metabolites. Phenylpropanoids have been noted for their role in plant defense against pathogens, herbivores, and environmental stress. The loss of flavonoids in tomatoes was associated with increased herbivory by coleopterans.45 Increased expression of phenylpropanoid biosynthetic enzyme genes were also noted with CPB herbivory.46 The results of the current study demonstrate that phenylpropanoids were increased in CPB-resistant wild species. Flavonoids also have health benefits for humans,47 as opposed to glycoalkaloids that are toxic.5 Several of the features in Table 2 were unknowns with no metabolites in the MetLin and KNApSAcK databases with matching masses. Metabolites Increased in S. tuberosum. The metabolites with increased levels in S. tuberosum relative to the wild species would not be associated with CPB resistance. Metabolites highly enriched in S. tuberosum over each of the wild species were screened by selecting features with 100-fold increased peak intensity in S. tuberosum over each of the wild species (Table 3). Features with m/z matching chaconine and solanine, the two major glycoalkaloids for S. tuberosum,8 were found, which concurs with the analysis of glycoalkloids (Table 1).

The feature ID is the retention time\m/z combination. Compounds in the MetLin database that matched the m/z are listed along with their molecular formulas and theoretical mass of the [M + H]+ molecular ion. Differences between m/z of the feature and theoretical masses of matching compounds are indicated as Δ ppm. TUB, S. tuberosum cv. Shepody; CHM, S. chomatophilum; OPL, S. oplocense; PCS, S. paucissectum; PUR, S. piurae; ACG, S. acroglossum; TAR, S. tarijense. The average Markerlynx processed peak intensity across the replicates for each species is presented.

PUR PCS OPL CHM TAR ACG Δ ppm theor mass [M + H]+ formula putative ID feature ID

Table 2. continued

Article

a

TUB


9051


unknown

solanidine verazine epi-verazine iso-shinonomenine shinonomenine veralinine veralomidine

newbouldiaquinone A unknown unknown blestrianol C unknown chaconine unknown unknown sansevistatin 1 malonylsaikosaponin A malonylsaikosaponin D

unknown

solanine solamargine β-solamarine solanelagnin solanidine 3-O-[α-L-rhamnopyranosyl-(1→2)[β-D-glucopyranosyl-(1→4)]β-D-glucopyranoside leptinine I

solanine solamargine β-solamarine solanelagnin solanidine 3-O-[α-L-rhamnopyranosyl-(1→2)[β-D-glucopyranosyl-(1→4)]β-D-glucopyranoside leptinine I

solanine solamargine β-solamarine

7.98\398.3406

11.34\411.0882 7.95\445.7395 7.42\453.7369 11.34\587.2077 8.08\850.5003 8.05\852.514 7.94\866.4931 7.00\866.4934 9.14\867.479

9.10\868.4968

7.60\868.5004

8.32\868.5086

7.96\868.5099

putative ID

4.89\229.0674

feature ID

Table 3. Metabolites Increased in S. tuberosum over All Wild Speciesa

9052

867.4737

C45H70O16

C45H73NO15

C45H73NO15

868.5053

868.5053

868.5053

852.5104

C45H73NO14

C45H73NO15

587.2064

398.3417

theor mass [M + H]+

C37H30O7

C25H14O6

C27H43NO

formula

2

5

5

5

6

4

4

2

Δ ppm ACG

0.0085

0

0

0.0112

0 0.0034 0 0.0054 0 0 0 0 0

0

0

TAR

0

0

0

0

0.0311 0.0022 0.0117 0.0078 0.0093 0 0.0029 0.0039 0

0

0.0252

CHM

0

0.0231

0.0692

0.0012

0.0127 0 0 0.0107 0 0.0054 0 0 0.0009

3.9729

0.0023

OPL

0.0033

0.0709

0.0010

0

0 0.0042 0 0 0 0 0.0145 0.0019 0

34.8602

0.0080

PCS

0

0.0098

0.0038

0

0.0070 0.0013 0.7788 0.0020 0 0.0007 0 0.0020 0

0.5949

0.0070

PUR

0

0

0.1245

0

0.0091 0 0.0051 0.0039 0 0.1038 0 0 0

0

0.0035

TUB

532.7175

17.5432

2135.0590

10.2539

10.7033 38.4241 82.5877 11.5610 31.1549 1266.9750 15.2681 15.1389 28.6052

185.0466

10.4685



9053

diosgenin 3-[glucosyl-(1→4)-rhamnosyl-(1→4)-glucoside] deltonin gracillin polypodoside A β-chacotriosyllilagen graecunin G melongoside H orbiculatoside B spiroconazole A anguivioside A lyconoside II shatavarin VII pennogenin 3-O-β-chacotrioside mubenoside A

10.54\885.4881

C45H72O17

formula

885.4842

theor mass [M + H]+

4

Δ ppm

0

0 0.0068 0.0089 0

ACG

0

0.0094 0 0 0

TAR

0.0117

0 0 0 0.0159

CHM

0.0066

0.0059 0 0.0033 0

OPL

0.0050

0.0006 0.0022 0.0007 0.0006

PCS

0.0013

0.0014 0.0006 0 0.0034

PUR

11.1546

11.7016 11.3667 26.4519 42.0095

TUB

The feature ID is the retention time\m/z combination. Compounds in the MetLin database that matched the m/z are listed along with their molecular formulas and theoretical mass of the [M + H]+ molecular ion. Differences between m/z of the feature and theoretical masses of matching compounds are indicated as Δ ppm. TUB, S. tuberosum cv. Shepody; CHM, S. chomatophilum; OPL, S. oplocense; PCS, S. paucissectum; PUR, S. piurae; ACG, S. acroglossum; TAR, S. tarijense. The average Markerlynx processed peak intensity across the replicates for each species is presented.

a

unknown unknown unknown unknown

solanelagnin solanidine 3-O-[α-L-rhamnopyranosyl-(1→2)[β-D-glucopyranosyl-(1→4)]β-D-glucopyranoside leptinine I

putative ID

7.59\874.2084 7.56\874.5895 7.52\877.0086 8.99\883.4724

feature ID

Table 3. continued




■

Metabolites from other organisms that had the same m/z as solanine, chaconine, and solanidine that were found in the MetLin database are also shown in Table 3. These metabolites are less likely to occur in S. tuberosum. Feature 11.34\411.0882 had a m/z that matched to newbouldiaquinone A, a metabolite from the medicinal plant Newbouldia laevis that is a naphthoquinone−anthroquinone coupled pigment.48 11.34\587.2077 was matched with blestrianol C, a biphenanthrene isolated from the tubers of the orchid Bletillla striata.49 Alternatively, the latter two features may be derived from the same unknown metabolite as they share the same retention time, indicating that 11.34\411.0882 could be a fragment ion of 11.34\587.2077. 9.14\867.479 and 10.54\885.4881 were assigned to several metabolites with structures corresponding to steroidal glycoside saponins.50 11.34\411.0882, 11.34\587.2077, 9.14\867.479, and 10.54\885.4881 were found at lower levels in S. tuberosum compared with glycoalkaloids. In summary, the comparative analysis of wild species with CPB resistance against CPB-susceptible S. tuberosum done in this study has resulted in the identification of several metabolites correlated with resistance. Glycoalkaloids solanine and chaconine were found to be unique to S. tuberosum, whereas glycoalkaloids with tetrose side chains dehydrocommersonine, tomatine, and neotomatine were associated with CPB-resistant species S. oplocense, S. paucisectum, S. chomatophilum, and S. piurae. This study has added to the evidence that tetrose side chain glycoalklaoids are associated with CPB resistance. The results can also be applied to the development of selection methods for CPB-resistant breeding material derived from intercrosses of S. tuberosum with species enriched in tetrose glycoalkaloids. For example, a high ratio of tetrose glycoalkaloids to solanine and/or chaconine can be used to select for CPB resistance. Non-glycoalkaloids hydroxycoumarin and phenylpropanoid metabolites were also found increased in all wild species and have also application as markers for CPB resistance.

■

REFERENCES

(1) McLeod, C.; Tolman, J. H. Evaluation of losses in potatoes. In Potato Pest Management in Canada, Proceedings of a Symposium on Improving Potato Pest Protection, Fredericton, NB; Boiteau, G., Singh, R. P., Parry, R. H., Eds.; New Brunswick Department of Agriculture: Fredericton, N. B., 1987; pp 363−373. (2) Hare, J. D. Ecology and management of the Colorado potato beetle. Annu. Rev. Entomol. 1990, 35, 81−100. (3) Pelletier, Y. Recognition of conspeciflc eggs by female Colorado potato beetles (Coleoptera: Chrysomelidae). Environ. Entomol. 1995, 24, 875−878. (4) Pelletier, Y.; King, R. R. Semiochemicals and potato pests: review and perspective for crop protection. In Potato Pest Management in Canada, Proceedings of a Symposium on Improving Potato Pest Protection, Fredericton, NB; Boiteau, G., Singh, R. P., Parry, R. H., Eds.; New Brunswick Department of Agriculture: Fredericton, N. B., 1987; pp 146−172. (5) Mensinga, T. T.; Sips, A. J.; Rompelberg, C. J.; van Twillert, K.; Meulenbelt, J.; van den Top, H. J.; van Egmond, H. P. Potato glycoalkaloids and adverse effects in humans: an ascending dose study. Regul. Toxicol. Pharmacol. 2005, 41, 66−72. (6) Ginzberg, I.; Tokuhisa, J.; Veilleux, R. Potato steroidal glycoalkaloids: biosynthesis and genetic manipulation. Potato Res. 2009, 52, 1−15. (7) Friedman, M. Potato glycoalkaloids and metabolites: roles in the plant and in the diet. J. Agric. Food Chem. 2006, 54, 8655−8681. (8) Shakya, R.; Navarre, D. A. LC-MS analysis of solanidane glycoalkaloid diversity among tubers of four wild potato species and three cultivars (Solanum tuberosum). J. Agric. Food Chem. 2008, 56, 6949−6958. (9) Kozukue, N.; Yoon, K.-S.; Byun, G.-I.; Misoo, S.; Levin, C. E.; Friedman, M. Distribution of glycoalkaloids in potato tubers of 59 accessions of two wild and five cultivated Solanum species. J. Agric. Food Chem. 2008, 56, 11920−11928. (10) Mweetwa, A. M.; Hunter, D.; Poe, R.; Harich, K. C.; Ginzberg, I.; Veilleux, R. E.; Tokuhisa, J. G. Steroidal glycoalkaloids in Solanum chacoense. Phytochemistry 2012, 75, 32−40. (11) Barbour, J. D.; Kennedy, G. G. Role of steroidal glycoalkaloid αtomatine in host-plant resistance of tomato to colorado potato beetle. J. Chem. Ecol. 1991, 17, 989−1005. (12) Rangarajan, A.; Miller, A. R.; Veilleux, R. E. Leptine glycoalkaloids reduce feeding by Colorado potato beetle in diploid Solanum sp. hybrids. J. Am. Soc. Hortic. Sci. 2000, 125, 689−693. (13) Carter, C. D.; Gianfagna, T. J.; Sacalis, J. N. Sesquiterpenes in glandular trichomes of a wild tomato species and toxicity to the Colorado potato beetle. J. Agric. Food Chem. 1989, 37, 1425−1428. (14) King, R. R.; Pelletier, Y.; Singh, R. P.; Calhoun, L. A. 3,4-Di-Oisobutyryl-6-O-caprylsucrose: the major component of a novel sucrose ester comples from the type B glandular trichomers of Solanum berthaultii Hawkes (PI473340). J. Chem. Soc., Chem. Commun. 1986, 1078−1079. (15) Sabotič, J.; Smid, I.; Gruden, K.; Gašparič, M. B.; Koruza, K.; Petek, M.; Pohleven, J.; Brzin, J.; Janko, K.; Zel, J. Inhibition of the growth of Colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom. J. Agric. Food Chem. 2013, 61, 12499−12509. (16) Pelletier, Y.; Smilowitz, Z. Effect of trichome B exudate of Solanum berthaultii Hawkes on consumption by the Colorado potato beetle, Leptinotarsa decemlineata (Say). J. Chem. Ecol. 1990, 16, 1547− 1555. (17) Jansky, S. H.; Simon, R.; Spooner, D. M. A test of taxonomic predictivity: resistance to the Colorado potato beetle in wild relatives of cultivated potato. J. Econ. Entomol. 2009, 102, 422−431. (18) Pelletier, Y.; Tai, G. C. C. Genotypic variability and mode of action of Colorado potato beetle (Coleoptera: Chrysomelidae) resistance in seven Solanum species. J. Econ. Entomol. 2001, 94, 572−578.

ASSOCIATED CONTENT

S Supporting Information *

Table S1: Peak intensity data for all replicates. Table S2: Averaged peak intensities of features with significant species variation in ANOVA. This material is available free of charge via the Internet at http://pubs.acs.org.

■

Article

AUTHOR INFORMATION

Corresponding Author

*(H.H.T.) E-mail: [email protected]. Phone: (506) 4604519. Fax: (506) 460-4377. Funding

The work was funded by Agriculture and Agri-Food Canada Agricultural Bioproducts Innovation Program and the Developing Innovative Agri-products program. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Charlotte Davidson and Catherine Clark, who provided technical assistance. 9054



Article

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Comparative Metabolite Profiling of Solanum tuberosum against Six

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