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Comparative metabolite profiling of foxglove aphids (Aulacorthum solani Kaltenbach) on leaves of resistant and susceptible soybean strains† Dan Sato,‡*a Masahiro Sugimoto,a Hiromichi Akashi,ab Masaru Tomitaa and Tomoyoshi Sogaa Aphid infestations can cause severe decreases in soybean (Glycine max [L.] Merr.) yield. Since planting aphid-resistant soybean strains is a promising approach for pest control, understanding the resistance mechanisms employed by aphids is of considerable importance. We compared aphid resistance in seven soybean strains and found that strain Tohoku149 was the most resistant to the foxglove aphid, Aulacorthum solani Kaltenbach. We subsequently analyzed the metabolite profiles of aphids cultured on the leaves of resistant and susceptible soybean strains using capillary electrophoresis-time-of-flight mass spectrometry. Our findings showed that the metabolite profiles of several amino acids, glucose 6-phosphate, and components of the tricarboxylic acid cycle were similar in aphids reared on Tohoku149 leaves and in aphids maintained under conditions of starvation, suggesting that Tohoku149 is more resistant to aphid feeding. Compared to susceptible strains, we also found that two methylated
Received 16th December 2013, Accepted 29th January 2014
metabolites, S-methylmethionine and trigonelline, were either not detected or decreased in aphids reared on Tohoku149 plants. Since these metabolites function as important sulfur transporters in
DOI: 10.1039/c3mb70595a
phloem sap and osmoprotectants involved in salt and drought stress, respectively, aphid-resistance is considered to be related to sulfur metabolism and methylation. These results contribute to an increase
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in our understanding of soybean aphid resistance mechanisms at the molecular level.
Introduction Increasing the efficiency of agricultural production in limited areas of arable land is becoming increasingly important as the human population increases.1 Agricultural pests have long been a major problem affecting agricultural production, and pest control accounts for approximately US$2 trillion per annum.2 Although the application of pesticides facilitates agricultural crop production, the repeated application of these chemical agents has a negative effect on humans and the environment, and it also increases the cost of the agricultural products themselves. To date, a variety of approaches have been employed to control aphids, including the introduction of biocontrol a
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan. Fax: +81-235-29-0574; Tel: +81-235-29-0528 b Yamagata Shonai Area General Branch Administration Office, Regional Promotion Division, Tsuruoka, Yamagata 997-1392, Japan. Fax: +81-235-64-2382; Tel: +81-235-64-2100 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3mb70595a ‡ Current address: Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan. E-mail: [email protected], [email protected]; Fax: +81-75-724-7532, Tel: +81-75-724-7532.
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agents in the form of natural predators,3 cultivation of transgenic plants with increased insect resistance,4 and using aphidresistant plant strains.5 However, most of these methods have been relatively ineffective and new pest control methods need to be developed so that the aim of effective, safe, and affordable pest control can be realized. Together with rice and corn, soybeans (Glycine max (L.) Merr.) are among the most valuable cash crops in the world. Soybeans are used in a variety of products, primarily as protein and oil sources for humans, feed for livestock and aquaculture, and as a feedstock in biofuel production.6 Although soybean production has expanded three-fold in the last 20 years,7 the actual yield is 26.3% lower than the potentially attainable yield due to the impact of weeds, animal pests, and pathogens.8 The foxglove aphid (Aulacorthum solani Kaltenbach) is a polyphagous insect pest that damages the plants it feeds upon by sucking plant sap and depriving the plant of nutrients, and by transmitting viruses.9 Foxglove aphids cause extensive economic losses, not only in Europe and North America, but also elsewhere throughout the world.10,11 In Japan, infection by this aphid causes premature leaf senescence in soybean, which is considered to have reduced crop yield by 70 to 90% in 2000.12,13 At present, the control of these aphids is primarily by insecticides.14
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In terms of energy efficiency and economic considerations, the development of aphid-resistant soybean strains is a promising strategy for pest control.15 Previous studies have reported that the Dowling and Jackson soybean strains are both resistant to the aphid, Aphis glycines Matsumura.16,17 Several resistance genes, Rag1, Rag2, Rag3, and Rag4, have been identified in these strains,18 but the physiological functions and defense mechanisms associated with these transcripts at the molecular level have yet to be clarified. We previously demonstrated that the soybean strain, Tohoku149, is highly resistant to foxglove aphid inoculation.19 We also compared the metabolic profiles of leaves of different soybean strains after aphid inoculation. Our results suggested that (1) citrate, amino acids, and the concentrations of their intermediates were all intrinsically higher in strain Tohoku149 than they were in a susceptible strain; (2) several of the metabolites involved in the synthesis of flavonoids and alkaloids were drastically changed after 6 h; and (3) concentrations of tricarboxylic acid (TCA) cycle metabolites increased after 48 h in strain Tohoku149.19 Clarifying the metabolite profiles of aphids will allow us to better understand the molecular mechanisms underlying aphid resistance in plants because aphids ingest phloem sap as their sole nutritional resource and, consequently, the aphid metabolites are expected to reflect the components of phloem sap in resistant soybean strains. To our knowledge, this is the first study on aphid metabolomics. It is considered that comparative metabolite profiling of aphids reared on the aphid-resistant and -susceptible soybean strains will provide valuable information for pest control.
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Fig. 1 Changes in the soybean leaf color after aphid inoculation. Representative pictures of seven soybean strains (A; Tohoku149, B; Suzuyutaka, C; Tachiyutaka, D; Enrei, E; Ryuho, F; Adams, and G; Jackson) 96 h after aphid inoculation. The two pictures at top-left (Tohoku149 and Suzuyutaka) are reproduced from our previous paper.19
Results and discussion Phenotypic observation of soybean leaves after aphid inoculation We previously reported that Tohoku149 leaves remained almost asymptomatic following aphid inoculation, as only tiny spots were evident after the aphids were introduced (Fig. 1, panel A); this was in marked contrast to the leaves of the susceptible Suzuyutaka strain, which had numerous uneven brownish blotches.19 We also assessed aphid resistance in five additional soybean strains (Tachiyutaka, Enrei, Ryuho, Adams, and Jackson), and leaves of these representative soybean strains at 96 h postaphid inoculation are shown in Fig. 1. The leaves of these five strains had yellow-brown blotches surrounding the points where aphids fed on phloem juice after inoculation (Fig. 1, panels C to G); similar symptoms were observed on leaves of the Suzuyutaka strain (Fig. 1, panel B). Despite having been reported in a previous field study,20 we were unable to confirm aphid resistance in the Adams soybean strain (Fig. 1, panel F). However, since the extent of damage observed in soybeans varies between genetic polymorphisms and biotypes,21,22 the observed discrepancy in resistance may have resulted from differences in genetic diversity. The Jackson strain is resistant to the aphid, Aphis glycines Matsumura, a serious soybean pest in North America,17 but it is vulnerable to attacks from the
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Fig. 2 Aphid survival rate on soybean leaves. Curves (a) to (g) indicate strains Tohoku149, Ryuho, Adams, Tachiyutaka, Enrei, Jackson, and Suzuyutaka, respectively. Survival rates for Tohoku149 and Suzuyutaka (curves a and g) were published in our previous paper.19 The aphid survival rate without soybean leaves indicates starvation conditions (curve h).
foxglove aphid (Fig. 1, panel F), possibly because of specific differences among host species. We quantitatively evaluated aphid resistance by examining survival rate, which was calculated by assessing the number of living aphids on target leaves (Fig. 2). As in our previous study,19 the median survival time (i.e. 50% survival on a Kaplan–Meier plot) of aphids on Tohoku149 leaves was 4 days (Fig. 2, curve a). In contrast, most aphids survived on the leaves of the other six strains and the survival rate never decreased below 50% (Fig. 2, curves b to g). Taken together, these findings confirm that Tohoku149 is highly resistant to the foxglove aphid. Overview of metabolomic analysis In order to understand the overall metabolomic profiles of aphids inoculated on each soybean strain, we conducted a PCA
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Fig. 3 Score plots of the principal component (PC) analysis for metabolomic profiles of aphids reared on leaves of each soybean strain. The contribution ratios of the 1st, 2nd, and 3rd PCs were 26.9, 18.1, and 11.6, respectively. The triangle indicates the control, and the rectangle and the circle indicate 6 h and 12 h post-inoculation, respectively. Blue, red, green, pink, orange, brown, light blue, and black indicate starvation, Tohoku149, Suzuyutaka, Tachiyutaka, Enrei, Ryuho, Adams, and Jackson strains, respectively.
using the control data as well as the profiles at 6 h and 12 h and visualized the score plots (Fig. 3). All of the 6 h post-inoculation data formed a robust cluster close to the control plot, but the 12 h post-inoculation data were more diverse. The small size of the cluster at 6 h implies that the metabolic characteristics of Suzuyutaka metabolites from pre-inoculation feeding have a marked effect on the metabolomic profiles of aphids at 6 h. Meanwhile, the effect from pre-inoculation diminished with each soybean strain, and their effects on the metabolomic profiles of the aphids are increasingly apparent. Interestingly, the 12 h post-inoculation plots obtained for starvation and feeding on Tohoku149 were most similar, i.e. the similarity between their metabolomic profiles was the highest. To clarify patterns in the metabolites of aphids inoculated on the six soybean strains, those soybean metabolites exhibiting large standard deviations (S.D.) at 12 h post-inoculation were visualized as a heat map (Fig. 4). Fig. 4 shows most of the amino acids and the oxidized form of glutathione (GSSG). Heat maps of all metabolites measured at 6 h and 12 h postinoculation are shown in Fig. S1 (ESI†). The dendrograms shown in Fig. 4 show that the metabolomic profiles obtained for the starvation and Tohoku149 treatments were the most similar. Most of the metabolite concentrations in the aphids reared on Adams, Jackson, and Ryuho soybean strains were higher than those in aphids reared on strain Tohoku149. Since aphid survival rates on strain Tohoku149 were better than under starvation conditions (Fig. 2), the observed resistance is considered to be due to uptake inhibition of metabolites rather than the deleterious effects associated with metabolites produced by Tohoku149. Although the metabolomic profiles obtained after rearing on Tohoku149 and after starvation were not clustered the closest at 6 h post-inoculation (Fig. S1, ESI†),
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Fig. 4 Heat map showing the relative metabolite concentrations in aphids at 12 h post-inoculation. The average concentration of six duplicates measured for each strain was used. Using these average concentrations, only those metabolites exhibiting large standard deviations among the eight strains (within top 30 ranking) were used for this analysis. Averaged concentrations of each strain were converted to a Z-score. Red and blue reflect higher and lower concentrations, respectively, compared to the average (white). The Euclidean distance was used to evaluate the similarity of both metabolites and samples.
several metabolites, such as Gln, Asn, lactate, and Lys, exhibited marked differences among all strains, suggesting a possible association with the differences in the survival rates of these strains. Energy and amino acid metabolism We previously demonstrated that citrate, amino acids, and their intermediate concentrations were higher in leaves of strain Tohoku149 than they were in leaves of strain Suzuyutaka before aphid inoculation, and also that TCA cycle metabolite concentrations increased in Tohoku149 leaves at 48 h after inoculation.19 These differences between Tohoku149 and Suzuyutaka directly affected aphid metabolism because the aphids rely completely on the phloem sap of the host plant for all of their nutrition. In this study, we compared the metabolite profiles of aphid extracts prepared from aphids reared on Tohoku149 leaves with extracts prepared from aphids reared on six susceptible soybean strains. Although the most interesting finding was that the citrate content of the soybean extract from Tohoku149 was 34 times higher than it was in the extract from Suzuyutaka,19 no significant difference was observed in the citrate concentrations of aphid extracts prepared from aphids reared on these soybean strains. Conversely, in aphids reared on Tohoku149, malate and succinate, which are components of the aphid TCA cycle, increased 2.5-fold and 1.7-fold from 6 h to 12 h, respectively. Other than G6P, no glycolytic metabolites were observed in the aphid extracts, possibly due to their low concentrations. G6P concentrations in aphids were equivalent
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among soybean strains at 6 h post-inoculation, but G6P was not detected in aphid extracts reared on Tohoku149 or under starvation conditions. The free amino acid profiles revealed that branched-chain amino acids (Val, Leu, and Ile), Thr, homoserine, and Phe, in aphids reared on Tohoku149 were significantly lower than they were in aphids reared on susceptible soybean strains. Interestingly, these amino acids are synthesized by endosymbiotic bacteria, e.g. Buchnera,23 suggesting that some factor(s) contained in the phloem sap can influence amino acid synthesis in the endosymbiotic bacteria of the aphids; this effect on amino acid biosynthesis may provide a clue on how to develop pestresistant crops. S-Methylmethionine and trigonelline We identified two metabolites, S-methylmethionine (SMM) and trigonelline, with unique profiles in Tohoku149 (Fig. 6). SMM was not detected in aphids reared on Tohoku149 leaves, and only slightly detected in aphids at 6 h and 12 h post-inoculation. Conversely, in aphids reared on susceptible strains, SMM was detected at both time points, and had increased 2.5-fold at 6 h (Fig. 6A). SMM, which accounts for approximately 2% of the free amino acids in wheat phloem sap, is a major transporter of sulfur in the phloem sap from the leaves to consumption and storage tissues (e.g., flowers, stems, roots, and seeds) in wheat, canola, Arabidopsis, legumes, and other plant species.24 Trigonelline concentrations in aphids reared on Tohoku149 were also lower than they were in aphids reared on the susceptible soybean strains (Fig. 6B). Trigonelline is an osmoprotectant employed in
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the metabolism of salt and drought stress in soybeans, although the mechanism of action is currently unknown.25 SMM and trigonelline are both synthesized through the methylation of Met and nicotinic acid using S-adenosyl methionine (SAM). It is therefore conceivable that the SAM concentrations are attenuated in Tohoku149, and/or that SAM is consumed by other methylation reactions, implying that methylation may be involved in aphid resistance. Flux analysis is a robust technique to identify the target metabolites, whereas the choice of label influences the result of analysis. 13C-methylation can be a potential marker to identify the specific metabolite(s) for aphid-infection. It is likely that aphids acquire Met from SMM using homocysteine S-methyltransferase, and consequently, that the SMM concentration in the sap is associated with sulfur-containing amino acid metabolism in aphids. However, Met concentrations did not differ between aphids reared on resistant or susceptible strains (Fig. 5). We proposed that Met is supplemented by the de novo cysteine synthetic pathway after the transsulfuration pathway in Buchnera (Fig. 5).26 The shortage of cystathionine, an intermediate of the transsulfuration pathway, may be due to the activation of Met synthesis in this pathway. This unique feature of sulfur-containing amino acid metabolism will likely be helpful in developing new aphid-resistant soybean strains in the future. Limitations and perspective We validated and described the metabolite profiles of foxglove aphids reared on leaves of aphid-resistant and -susceptible
Fig. 5 Changes in the level of representative primary metabolites after aphid inoculation. Metabolites per mg of aphids (mean SEM) at 6 h and 12 h after inoculation are shown. The black, hatched, and white bars indicate aphids inoculated on Tohoku149 (T149), sensitive strains (SS), and exposed to starvation (ST), respectively. The letters a, b, and c indicate significant differences (P o 0.05) between T149 and SS, T149 and ST, and SS and ST, respectively (Bonferroni multiple-comparison test after one-way ANOVA analysis).
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full expansion of the third or fourth trifoliolate. The photosynthetic photon flux density was approximately 100 mmol m 2 s 1. A foxglove aphid clone was established from a single nymph collected from a tomato seedling in Tsuruoka, Japan, on May 2011. The aphid strain was maintained on a continuous supply of Suzuyutaka soybean seedlings under the temperature and photoperiod conditions described above. All of the experiments were repeated six times. Measurement of the aphid survival rate
Fig. 6 Changes in the amount of S-methylmethionine and trigonelline in the aphid extract after inoculation. Aphids were inoculated on Tohoku149 (T149), sensitive strains (SS), and exposed to starvation (ST) for either 6 h or 12 h. Aphid metabolite concentrations = mean SEM. Asterisks (*, ***, and ****) denote statistical significance with P o 0.05, P o 0.001, and P o 0.0001, respectively (Bonferroni multiple comparison test after oneway ANOVA analysis).
soybean plants. To the best of our knowledge, this paper and our previous report are the first metabolomic studies on soybeans and aphids.19 Although microarray analysis of resistant and susceptible soybean strains has been reported,27 the genetic changes that arise in aphids in response to feeding on aphidresistant and -susceptible soybean leaves are poorly understood. Integrated analyses of metabolomic and transcriptomic data will likely increase our understanding of the associated aphidresistance mechanism(s). Although CE-TOFMS is well suited to metabolite analysis, application of the method for profiling secondary metabolites is limited by the fact that it can only be used to assay charged and hydrophilic metabolites.28 Plants produce a huge variety of secondary metabolites, many of which have various physiological activities. Therefore it is conceivable that secondary metabolites play a role in aphid resistance. To identify a greater variety of metabolites, several techniques including tandem-MS and/or other platforms (e.g., liquid chromatography-MS and gas chromatography-MS) need to be employed to profile non-polar and volatile metabolites in future.29–31 Flux analysis using labeling techniques is also necessary to identify the transition of key metabolites that contribute to the resistance feature.
Experimental Insect and plant materials Five soybean strains (Tohoku149, Suzuyutaka, Tachiyutaka, Ryuho, and Enrei) were gifted by the Yamagata Prefectural Rice Breeding and Crop Science Experiment Station (Tsuruoka, Japan). Two strains (Jackson and Adams) were purchased from the National Institute of Agrobiological Sciences (NIAS) Genebank (http://www.gene.affrc.go.jp/). Experimental plants were prepared by planting one seed in a plastic pot (+ 9 cm, depth 4.5 cm) containing sterile culture soil. The plants were grown in a growth chamber at 22 1C under a photoperiod of 16 h light: 8 h dark, and all experiments were performed after
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Thirty first instar nymphs were gently and evenly put onto the adaxial surface of a first trifoliolate leaf of each soybean strain (five plants per strain) using a fine-tipped paintbrush. The target leaf attached to the plant was sealed in a clear vinyl bag (85 mm 120 mm) which had tiny pinholes for ventilation. The vinyl bags were sealed to prevent the aphids from leaving the leaf. The number of survivors was counted 6 and 12 h after application, and the survival rate was plotted on a Kaplan– Meier plot. This experiment was repeated five times using the first trifoliolate leaves of five plants. The number of survivors in a vinyl bag that did not contain a leaf was also counted as a control. Extraction of aphid metabolites Approximately 100–200 nymphs (first instar) were gently placed onto soybean leaves and sealed in vinyl bags as described above. Aphids were also placed in sealed vinyl bags without leaves to simulate starvation conditions. Aphids were collected after 6 h and 12 h, frozen in liquid nitrogen, and metabolites were extracted as described previously19 with minor modifications. The frozen aphid homogenate (5–15 mg) was dissolved in 500 mL methanol and centrifuged at 15 000 g for 15 min at 4 1C. Ten microliters of the supernatant was mixed with 390 mL methanol and 160 mL of water solution containing internal standards (see below), and then vortexed with 400 mL of chloroform. Insoluble materials and hydrophobic metabolites were removed by centrifugation at 15 000 g for 10 min. The aqueous solutions containing hydrophilic metabolites (200 mL each) were then filtered using an Amicon Ultrafree-MC ultrafilter (cut off = 3000 Da, Millipore Co., Billerica, MA) with centrifugation at 9100 g at 4 1C for approximately 3 h. The filtrates were dried and stored at 80 1C until use. The internal standard solution consists of 20 mmol L 1 methionine sulfone, 20 mmol L 1 2-(N-morpholino)ethanesulfonic acid, and 40 mmol L 1 D-camphor10-sulfonic acid. These compounds were added to correct migration times in the electropherograms and to normalize peak areas to eliminate unexpected systematic bias among runs. Instrumentation and measurement conditions The instrumentation and measurement conditions for capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) have been described elsewhere.32 All capillary electrophoresiselectrospray ionization-mass spectrometry (CE-ESI-MS) experiments were performed using an Agilent CE capillary electrophoresis system, an Agilent G6220A LC/MSD TOF system, an Agilent 1100 series isocratic HPLC pump, a G1603A Agilent CE-MS
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adapter kit, and a G1607A Agilent CE-ESI-MS sprayer kit (Agilent Technologies, Santa Clara, CA). The CE-MS adapter kit included a capillary cassette to facilitate thermostating of the capillary. The CE-ESI-MS sprayer kit simplified coupling of the CE system with the MS systems, and was equipped with an electrospray source. For system control and data acquisition, we used G2201AA Agilent ChemStation software for CE and Agilent MassHunter software for TOF-MS. Detailed analysis conditions for the CE-TOFMS are described.19
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Acknowledgements We would like to thank the Yamagata Prefectural Rice Breeding and Crop Science Experiment Station for providing us with soybean seeds, and the Lowland Farming Research Division of the Tohoku Agricultural Research Center for their valuable advice regarding the characteristics of Tohoku149. This work was supported by research funds from the Yamagata Prefectural Government and the city of Tsuruoka.
Metabolite identification, quantification and data analysis Metabolite identification and quantification were carried out as described previously.32,33 Metabolites detected in more than four samples among six replicates were used for the PCA analysis. For data visualization, heat maps were constructed using the averaged concentration of repeated experiments (n = 6). Principal component analysis (PCA), heat map visualization, and Kaplan–Meier analysis were performed using JMP (ver. 9.0.2. http://www.jmp.com/), Mev (ver. 4.8.1. http://www.tm4. org/mev/), and Graphpad Prism ver. 5.0.4 (Graphpad Software, San Diego, CA), respectively.
Conclusion We successfully characterized aphid resistance in seven soybean strains and compared the metabolomic profiles of aphids reared on leaves of Tohoku149, a resistant soybean strain, to the profiles of six susceptible strains in an attempt to elucidate the mechanism(s) underlying aphid resistance. The PCA results revealed that the metabolomic profiles obtained at 12 h postinoculation were highly diverse, while those of aphids reared on Tohoku149 and under starvation conditions were most similar. Comparative metabolomic analyses revealed that malate and succinate, components of the TCA cycle, increased from 6 h to 12 h, whereas G6P decreased drastically in aphids reared on Tohoku149 leaves. Free amino acid profiles revealed the presence of branched-chain amino acids (Val, Leu, and Ile), Thr, homoserine, and Phe, in aphids reared on Tohoku149 leaves, even while non-essential amino acids were being synthesized by endosymbiotic bacteria. These results suggest that Tohoku149 phloem sap contains factor(s) that inhibit amino acid synthesis pathways, particularly those in endosymbiotic bacteria. We also identified two unique metabolites, SMM and trigonelline, both of which occurred at lower or marginal levels in aphids cultured on Tohoku149 leaves. Since both metabolites are synthesized by methylation using SAM, aphid resistance is considered to be linked to the methylation reaction. These findings will likely facilitate the discovery of new markers for screening resistant strains and for developing novel insecticides.
Financial support This work was supported by research funds from the Yamagata Prefectural Government and the City of Tsuruoka.
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Notes and references 1 M. Tester and P. Langridge, Science, 2010, 327, 818–822. 2 D. Pimentel, in Integrated Pest Management: InnovationDevelopment Process, ed. R. Peshin and A. Dhawan, Springer, Netherlands, 2009, ch. 3, pp. 83–87, DOI: 10.1007/978-14020-8992-3_3. 3 T. Nakata, Appl. Entomol. Zool., 1995, 30, 129–138. 4 T. H. Schuler, G. M. Poppy, B. R. Kerry and I. Denholm, Trends Biotechnol., 1998, 16, 168–175. 5 S. Ohnishi, N. Miyake, T. Takeuchi, F. Kousaka, S. Hiura, O. Kanehira, M. Saito, T. Sayama, A. Higashi, M. Ishimoto, Y. Tanaka and S. Fujita, Breed. Sci., 2012, 61, 618–624. 6 T. Masuda and P. D. Goldsmith, Int. Food Agribus. Man., 2009, 12, 143–161. 7 G. L. Hartman, E. D. West and T. K. Herman, Food Sec., 2011, 3, 5–17. 8 E.-C. Oerke, J. Agric. Sci., 2005, 144, 31. 9 D. W. Ragsdale, D. A. Landis, J. Brodeur, G. E. Heimpel and N. Desneux, Annu. Rev. Entomol., 2011, 56, 375–399. 10 R. E. Down, A. M. R. Gatehouse, W. D. O. Hamilton and J. A. Gatehouse, J. Insect Physiol., 1996, 42, 1035–1045. 11 S. E. Jandricic, S. P. Wraight, K. C. Bennett and J. P. Sanderson, Environ. Entomol., 2010, 39, 1631–1642. 12 K. Ueno, H. Takatori and T. Saito, Annu. Rep. Soc. Plant Prot. North Jpn., 2001, 2001, 183–184. 13 T. Nagano, Y. Umetsu, N. Hoshi and T. Kidokoro, Annu. Rep. Soc. Plant Prot. North Jpn., 2001, 52, 175–177. 14 T. Ono, J. Oyama and T. Kidokoro, Annu. Rep. Soc. Plant Prot. North Jpn., 2006, 2006, 148–150. 15 R. Karban and J. H. Myers, Annu. Rev. Ecol. Syst., 1989, 20, 331–348. 16 C. B. Hill, Y. Li and G. L. Hartman, Crop Sci., 2006, 46, 1601–1605. 17 C. B. Hill, Y. Li and G. L. Hartman, Crop Sci., 2006, 46, 1606–1608. 18 C. B. Hill, A. Chirumamilla and G. L. Hartman, Euphytica, 2012, 186, 635–646. 19 D. Sato, H. Akashi, M. Sugimoto, M. Tomita and T. Soga, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2013, 925, 95–103. 20 H. Jinno, K. Araki, K. Kira and Y. Shinada, Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan, 1997, 112–113.
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21 M. Kobayashi and T. Murai, Jpn. J. Appl. Entomol. Zool., 2012, 56, 49–55. 22 C. B. Hill, L. Crull, T. K. Herman, D. J. Voegtlin and G. L. Hartman, J. Econ. Entomol., 2010, 103, 509–515. 23 S. Shigenobu, H. Watanabe, M. Hattori, Y. Sakaki and H. Ishikawa, Nature, 2000, 407, 81–86. 24 F. Bourgis, S. Roje, M. L. Nuccio, D. B. Fisher, M. C. Tarczynski, C. J. Li, C. Herschbach, H. Rennenberg, M. J. Pimenta, T. L. Shen, D. A. Gage and A. D. Hanson, Plant Cell, 1999, 11, 1485–1497. 25 T. H. Phang, G. Shao and H. M. Lam, J. Integr. Plant Biol., 2008, 50, 1196–1212. 26 The International Aphid Genomics Consortium, PLoS Biol., 2010, 8, e1000313. 27 Y. Li, J. Zou, M. Li, D. D. Bilgin, L. O. Vodkin, G. L. Hartman and S. J. Clough, New Phytol., 2008, 179, 185–195.
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28 M. R. Monton and T. Soga, J. Chromatogr. A, 2007, 1168, 237–246. 29 R. Ramautar, E. Nevedomskaya, O. A. Mayboroda, A. M. Deelder, I. D. Wilson, H. G. Gika, G. A. Theodoridis, G. W. Somsen and G. J. de Jong, Mol. Biosyst., 2011, 7, 194–199. 30 G. Graça, B. J. Goodfellow, A. S. Barros, S. Diaz, I. F. Duarte, K. Spagou, K. Veselkov, E. J. Want, J. C. Lindon, I. M. Carreira, E. Galhano, C. Pita and A. M. Gil, Mol. Biosyst., 2012, 8, 1243–1254. 31 H. G. Gika, G. A. Theodoridis and I. D. Wilson, J. Sep. Sci., 2008, 31, 1598–1608. 32 M. Sugimoto, D. T. Wong, A. Hirayama, T. Soga and M. Tomita, Metabolomics, 2010, 6, 78–95. 33 M. Sugimoto, H. Goto, K. Otomo, M. Ito, H. Onuma, A. Suzuki, M. Sugawara, S. Abe, M. Tomita and T. Soga, J. Agric. Food Chem., 2010, 58, 8418–8425.
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Comparative metabolite profiling of foxglove aphids (Aulacorthum solani Kaltenbach) on leaves of resistant and susceptible soybean strains† Dan Sato,‡*a Masahiro Sugimoto,a Hiromichi Akashi,ab Masaru Tomitaa and Tomoyoshi Sogaa Aphid infestations can cause severe decreases in soybean (Glycine max [L.] Merr.) yield. Since planting aphid-resistant soybean strains is a promising approach for pest control, understanding the resistance mechanisms employed by aphids is of considerable importance. We compared aphid resistance in seven soybean strains and found that strain Tohoku149 was the most resistant to the foxglove aphid, Aulacorthum solani Kaltenbach. We subsequently analyzed the metabolite profiles of aphids cultured on the leaves of resistant and susceptible soybean strains using capillary electrophoresis-time-of-flight mass spectrometry. Our findings showed that the metabolite profiles of several amino acids, glucose 6-phosphate, and components of the tricarboxylic acid cycle were similar in aphids reared on Tohoku149 leaves and in aphids maintained under conditions of starvation, suggesting that Tohoku149 is more resistant to aphid feeding. Compared to susceptible strains, we also found that two methylated
Received 16th December 2013, Accepted 29th January 2014
metabolites, S-methylmethionine and trigonelline, were either not detected or decreased in aphids reared on Tohoku149 plants. Since these metabolites function as important sulfur transporters in
DOI: 10.1039/c3mb70595a
phloem sap and osmoprotectants involved in salt and drought stress, respectively, aphid-resistance is considered to be related to sulfur metabolism and methylation. These results contribute to an increase
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in our understanding of soybean aphid resistance mechanisms at the molecular level.
Introduction Increasing the efficiency of agricultural production in limited areas of arable land is becoming increasingly important as the human population increases.1 Agricultural pests have long been a major problem affecting agricultural production, and pest control accounts for approximately US$2 trillion per annum.2 Although the application of pesticides facilitates agricultural crop production, the repeated application of these chemical agents has a negative effect on humans and the environment, and it also increases the cost of the agricultural products themselves. To date, a variety of approaches have been employed to control aphids, including the introduction of biocontrol a
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan. Fax: +81-235-29-0574; Tel: +81-235-29-0528 b Yamagata Shonai Area General Branch Administration Office, Regional Promotion Division, Tsuruoka, Yamagata 997-1392, Japan. Fax: +81-235-64-2382; Tel: +81-235-64-2100 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3mb70595a ‡ Current address: Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan. E-mail: [email protected], [email protected]; Fax: +81-75-724-7532, Tel: +81-75-724-7532.
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agents in the form of natural predators,3 cultivation of transgenic plants with increased insect resistance,4 and using aphidresistant plant strains.5 However, most of these methods have been relatively ineffective and new pest control methods need to be developed so that the aim of effective, safe, and affordable pest control can be realized. Together with rice and corn, soybeans (Glycine max (L.) Merr.) are among the most valuable cash crops in the world. Soybeans are used in a variety of products, primarily as protein and oil sources for humans, feed for livestock and aquaculture, and as a feedstock in biofuel production.6 Although soybean production has expanded three-fold in the last 20 years,7 the actual yield is 26.3% lower than the potentially attainable yield due to the impact of weeds, animal pests, and pathogens.8 The foxglove aphid (Aulacorthum solani Kaltenbach) is a polyphagous insect pest that damages the plants it feeds upon by sucking plant sap and depriving the plant of nutrients, and by transmitting viruses.9 Foxglove aphids cause extensive economic losses, not only in Europe and North America, but also elsewhere throughout the world.10,11 In Japan, infection by this aphid causes premature leaf senescence in soybean, which is considered to have reduced crop yield by 70 to 90% in 2000.12,13 At present, the control of these aphids is primarily by insecticides.14
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In terms of energy efficiency and economic considerations, the development of aphid-resistant soybean strains is a promising strategy for pest control.15 Previous studies have reported that the Dowling and Jackson soybean strains are both resistant to the aphid, Aphis glycines Matsumura.16,17 Several resistance genes, Rag1, Rag2, Rag3, and Rag4, have been identified in these strains,18 but the physiological functions and defense mechanisms associated with these transcripts at the molecular level have yet to be clarified. We previously demonstrated that the soybean strain, Tohoku149, is highly resistant to foxglove aphid inoculation.19 We also compared the metabolic profiles of leaves of different soybean strains after aphid inoculation. Our results suggested that (1) citrate, amino acids, and the concentrations of their intermediates were all intrinsically higher in strain Tohoku149 than they were in a susceptible strain; (2) several of the metabolites involved in the synthesis of flavonoids and alkaloids were drastically changed after 6 h; and (3) concentrations of tricarboxylic acid (TCA) cycle metabolites increased after 48 h in strain Tohoku149.19 Clarifying the metabolite profiles of aphids will allow us to better understand the molecular mechanisms underlying aphid resistance in plants because aphids ingest phloem sap as their sole nutritional resource and, consequently, the aphid metabolites are expected to reflect the components of phloem sap in resistant soybean strains. To our knowledge, this is the first study on aphid metabolomics. It is considered that comparative metabolite profiling of aphids reared on the aphid-resistant and -susceptible soybean strains will provide valuable information for pest control.
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Fig. 1 Changes in the soybean leaf color after aphid inoculation. Representative pictures of seven soybean strains (A; Tohoku149, B; Suzuyutaka, C; Tachiyutaka, D; Enrei, E; Ryuho, F; Adams, and G; Jackson) 96 h after aphid inoculation. The two pictures at top-left (Tohoku149 and Suzuyutaka) are reproduced from our previous paper.19
Results and discussion Phenotypic observation of soybean leaves after aphid inoculation We previously reported that Tohoku149 leaves remained almost asymptomatic following aphid inoculation, as only tiny spots were evident after the aphids were introduced (Fig. 1, panel A); this was in marked contrast to the leaves of the susceptible Suzuyutaka strain, which had numerous uneven brownish blotches.19 We also assessed aphid resistance in five additional soybean strains (Tachiyutaka, Enrei, Ryuho, Adams, and Jackson), and leaves of these representative soybean strains at 96 h postaphid inoculation are shown in Fig. 1. The leaves of these five strains had yellow-brown blotches surrounding the points where aphids fed on phloem juice after inoculation (Fig. 1, panels C to G); similar symptoms were observed on leaves of the Suzuyutaka strain (Fig. 1, panel B). Despite having been reported in a previous field study,20 we were unable to confirm aphid resistance in the Adams soybean strain (Fig. 1, panel F). However, since the extent of damage observed in soybeans varies between genetic polymorphisms and biotypes,21,22 the observed discrepancy in resistance may have resulted from differences in genetic diversity. The Jackson strain is resistant to the aphid, Aphis glycines Matsumura, a serious soybean pest in North America,17 but it is vulnerable to attacks from the
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Fig. 2 Aphid survival rate on soybean leaves. Curves (a) to (g) indicate strains Tohoku149, Ryuho, Adams, Tachiyutaka, Enrei, Jackson, and Suzuyutaka, respectively. Survival rates for Tohoku149 and Suzuyutaka (curves a and g) were published in our previous paper.19 The aphid survival rate without soybean leaves indicates starvation conditions (curve h).
foxglove aphid (Fig. 1, panel F), possibly because of specific differences among host species. We quantitatively evaluated aphid resistance by examining survival rate, which was calculated by assessing the number of living aphids on target leaves (Fig. 2). As in our previous study,19 the median survival time (i.e. 50% survival on a Kaplan–Meier plot) of aphids on Tohoku149 leaves was 4 days (Fig. 2, curve a). In contrast, most aphids survived on the leaves of the other six strains and the survival rate never decreased below 50% (Fig. 2, curves b to g). Taken together, these findings confirm that Tohoku149 is highly resistant to the foxglove aphid. Overview of metabolomic analysis In order to understand the overall metabolomic profiles of aphids inoculated on each soybean strain, we conducted a PCA
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Fig. 3 Score plots of the principal component (PC) analysis for metabolomic profiles of aphids reared on leaves of each soybean strain. The contribution ratios of the 1st, 2nd, and 3rd PCs were 26.9, 18.1, and 11.6, respectively. The triangle indicates the control, and the rectangle and the circle indicate 6 h and 12 h post-inoculation, respectively. Blue, red, green, pink, orange, brown, light blue, and black indicate starvation, Tohoku149, Suzuyutaka, Tachiyutaka, Enrei, Ryuho, Adams, and Jackson strains, respectively.
using the control data as well as the profiles at 6 h and 12 h and visualized the score plots (Fig. 3). All of the 6 h post-inoculation data formed a robust cluster close to the control plot, but the 12 h post-inoculation data were more diverse. The small size of the cluster at 6 h implies that the metabolic characteristics of Suzuyutaka metabolites from pre-inoculation feeding have a marked effect on the metabolomic profiles of aphids at 6 h. Meanwhile, the effect from pre-inoculation diminished with each soybean strain, and their effects on the metabolomic profiles of the aphids are increasingly apparent. Interestingly, the 12 h post-inoculation plots obtained for starvation and feeding on Tohoku149 were most similar, i.e. the similarity between their metabolomic profiles was the highest. To clarify patterns in the metabolites of aphids inoculated on the six soybean strains, those soybean metabolites exhibiting large standard deviations (S.D.) at 12 h post-inoculation were visualized as a heat map (Fig. 4). Fig. 4 shows most of the amino acids and the oxidized form of glutathione (GSSG). Heat maps of all metabolites measured at 6 h and 12 h postinoculation are shown in Fig. S1 (ESI†). The dendrograms shown in Fig. 4 show that the metabolomic profiles obtained for the starvation and Tohoku149 treatments were the most similar. Most of the metabolite concentrations in the aphids reared on Adams, Jackson, and Ryuho soybean strains were higher than those in aphids reared on strain Tohoku149. Since aphid survival rates on strain Tohoku149 were better than under starvation conditions (Fig. 2), the observed resistance is considered to be due to uptake inhibition of metabolites rather than the deleterious effects associated with metabolites produced by Tohoku149. Although the metabolomic profiles obtained after rearing on Tohoku149 and after starvation were not clustered the closest at 6 h post-inoculation (Fig. S1, ESI†),
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Fig. 4 Heat map showing the relative metabolite concentrations in aphids at 12 h post-inoculation. The average concentration of six duplicates measured for each strain was used. Using these average concentrations, only those metabolites exhibiting large standard deviations among the eight strains (within top 30 ranking) were used for this analysis. Averaged concentrations of each strain were converted to a Z-score. Red and blue reflect higher and lower concentrations, respectively, compared to the average (white). The Euclidean distance was used to evaluate the similarity of both metabolites and samples.
several metabolites, such as Gln, Asn, lactate, and Lys, exhibited marked differences among all strains, suggesting a possible association with the differences in the survival rates of these strains. Energy and amino acid metabolism We previously demonstrated that citrate, amino acids, and their intermediate concentrations were higher in leaves of strain Tohoku149 than they were in leaves of strain Suzuyutaka before aphid inoculation, and also that TCA cycle metabolite concentrations increased in Tohoku149 leaves at 48 h after inoculation.19 These differences between Tohoku149 and Suzuyutaka directly affected aphid metabolism because the aphids rely completely on the phloem sap of the host plant for all of their nutrition. In this study, we compared the metabolite profiles of aphid extracts prepared from aphids reared on Tohoku149 leaves with extracts prepared from aphids reared on six susceptible soybean strains. Although the most interesting finding was that the citrate content of the soybean extract from Tohoku149 was 34 times higher than it was in the extract from Suzuyutaka,19 no significant difference was observed in the citrate concentrations of aphid extracts prepared from aphids reared on these soybean strains. Conversely, in aphids reared on Tohoku149, malate and succinate, which are components of the aphid TCA cycle, increased 2.5-fold and 1.7-fold from 6 h to 12 h, respectively. Other than G6P, no glycolytic metabolites were observed in the aphid extracts, possibly due to their low concentrations. G6P concentrations in aphids were equivalent
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among soybean strains at 6 h post-inoculation, but G6P was not detected in aphid extracts reared on Tohoku149 or under starvation conditions. The free amino acid profiles revealed that branched-chain amino acids (Val, Leu, and Ile), Thr, homoserine, and Phe, in aphids reared on Tohoku149 were significantly lower than they were in aphids reared on susceptible soybean strains. Interestingly, these amino acids are synthesized by endosymbiotic bacteria, e.g. Buchnera,23 suggesting that some factor(s) contained in the phloem sap can influence amino acid synthesis in the endosymbiotic bacteria of the aphids; this effect on amino acid biosynthesis may provide a clue on how to develop pestresistant crops. S-Methylmethionine and trigonelline We identified two metabolites, S-methylmethionine (SMM) and trigonelline, with unique profiles in Tohoku149 (Fig. 6). SMM was not detected in aphids reared on Tohoku149 leaves, and only slightly detected in aphids at 6 h and 12 h post-inoculation. Conversely, in aphids reared on susceptible strains, SMM was detected at both time points, and had increased 2.5-fold at 6 h (Fig. 6A). SMM, which accounts for approximately 2% of the free amino acids in wheat phloem sap, is a major transporter of sulfur in the phloem sap from the leaves to consumption and storage tissues (e.g., flowers, stems, roots, and seeds) in wheat, canola, Arabidopsis, legumes, and other plant species.24 Trigonelline concentrations in aphids reared on Tohoku149 were also lower than they were in aphids reared on the susceptible soybean strains (Fig. 6B). Trigonelline is an osmoprotectant employed in
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the metabolism of salt and drought stress in soybeans, although the mechanism of action is currently unknown.25 SMM and trigonelline are both synthesized through the methylation of Met and nicotinic acid using S-adenosyl methionine (SAM). It is therefore conceivable that the SAM concentrations are attenuated in Tohoku149, and/or that SAM is consumed by other methylation reactions, implying that methylation may be involved in aphid resistance. Flux analysis is a robust technique to identify the target metabolites, whereas the choice of label influences the result of analysis. 13C-methylation can be a potential marker to identify the specific metabolite(s) for aphid-infection. It is likely that aphids acquire Met from SMM using homocysteine S-methyltransferase, and consequently, that the SMM concentration in the sap is associated with sulfur-containing amino acid metabolism in aphids. However, Met concentrations did not differ between aphids reared on resistant or susceptible strains (Fig. 5). We proposed that Met is supplemented by the de novo cysteine synthetic pathway after the transsulfuration pathway in Buchnera (Fig. 5).26 The shortage of cystathionine, an intermediate of the transsulfuration pathway, may be due to the activation of Met synthesis in this pathway. This unique feature of sulfur-containing amino acid metabolism will likely be helpful in developing new aphid-resistant soybean strains in the future. Limitations and perspective We validated and described the metabolite profiles of foxglove aphids reared on leaves of aphid-resistant and -susceptible
Fig. 5 Changes in the level of representative primary metabolites after aphid inoculation. Metabolites per mg of aphids (mean SEM) at 6 h and 12 h after inoculation are shown. The black, hatched, and white bars indicate aphids inoculated on Tohoku149 (T149), sensitive strains (SS), and exposed to starvation (ST), respectively. The letters a, b, and c indicate significant differences (P o 0.05) between T149 and SS, T149 and ST, and SS and ST, respectively (Bonferroni multiple-comparison test after one-way ANOVA analysis).
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full expansion of the third or fourth trifoliolate. The photosynthetic photon flux density was approximately 100 mmol m 2 s 1. A foxglove aphid clone was established from a single nymph collected from a tomato seedling in Tsuruoka, Japan, on May 2011. The aphid strain was maintained on a continuous supply of Suzuyutaka soybean seedlings under the temperature and photoperiod conditions described above. All of the experiments were repeated six times. Measurement of the aphid survival rate
Fig. 6 Changes in the amount of S-methylmethionine and trigonelline in the aphid extract after inoculation. Aphids were inoculated on Tohoku149 (T149), sensitive strains (SS), and exposed to starvation (ST) for either 6 h or 12 h. Aphid metabolite concentrations = mean SEM. Asterisks (*, ***, and ****) denote statistical significance with P o 0.05, P o 0.001, and P o 0.0001, respectively (Bonferroni multiple comparison test after oneway ANOVA analysis).
soybean plants. To the best of our knowledge, this paper and our previous report are the first metabolomic studies on soybeans and aphids.19 Although microarray analysis of resistant and susceptible soybean strains has been reported,27 the genetic changes that arise in aphids in response to feeding on aphidresistant and -susceptible soybean leaves are poorly understood. Integrated analyses of metabolomic and transcriptomic data will likely increase our understanding of the associated aphidresistance mechanism(s). Although CE-TOFMS is well suited to metabolite analysis, application of the method for profiling secondary metabolites is limited by the fact that it can only be used to assay charged and hydrophilic metabolites.28 Plants produce a huge variety of secondary metabolites, many of which have various physiological activities. Therefore it is conceivable that secondary metabolites play a role in aphid resistance. To identify a greater variety of metabolites, several techniques including tandem-MS and/or other platforms (e.g., liquid chromatography-MS and gas chromatography-MS) need to be employed to profile non-polar and volatile metabolites in future.29–31 Flux analysis using labeling techniques is also necessary to identify the transition of key metabolites that contribute to the resistance feature.
Experimental Insect and plant materials Five soybean strains (Tohoku149, Suzuyutaka, Tachiyutaka, Ryuho, and Enrei) were gifted by the Yamagata Prefectural Rice Breeding and Crop Science Experiment Station (Tsuruoka, Japan). Two strains (Jackson and Adams) were purchased from the National Institute of Agrobiological Sciences (NIAS) Genebank (http://www.gene.affrc.go.jp/). Experimental plants were prepared by planting one seed in a plastic pot (+ 9 cm, depth 4.5 cm) containing sterile culture soil. The plants were grown in a growth chamber at 22 1C under a photoperiod of 16 h light: 8 h dark, and all experiments were performed after
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Thirty first instar nymphs were gently and evenly put onto the adaxial surface of a first trifoliolate leaf of each soybean strain (five plants per strain) using a fine-tipped paintbrush. The target leaf attached to the plant was sealed in a clear vinyl bag (85 mm 120 mm) which had tiny pinholes for ventilation. The vinyl bags were sealed to prevent the aphids from leaving the leaf. The number of survivors was counted 6 and 12 h after application, and the survival rate was plotted on a Kaplan– Meier plot. This experiment was repeated five times using the first trifoliolate leaves of five plants. The number of survivors in a vinyl bag that did not contain a leaf was also counted as a control. Extraction of aphid metabolites Approximately 100–200 nymphs (first instar) were gently placed onto soybean leaves and sealed in vinyl bags as described above. Aphids were also placed in sealed vinyl bags without leaves to simulate starvation conditions. Aphids were collected after 6 h and 12 h, frozen in liquid nitrogen, and metabolites were extracted as described previously19 with minor modifications. The frozen aphid homogenate (5–15 mg) was dissolved in 500 mL methanol and centrifuged at 15 000 g for 15 min at 4 1C. Ten microliters of the supernatant was mixed with 390 mL methanol and 160 mL of water solution containing internal standards (see below), and then vortexed with 400 mL of chloroform. Insoluble materials and hydrophobic metabolites were removed by centrifugation at 15 000 g for 10 min. The aqueous solutions containing hydrophilic metabolites (200 mL each) were then filtered using an Amicon Ultrafree-MC ultrafilter (cut off = 3000 Da, Millipore Co., Billerica, MA) with centrifugation at 9100 g at 4 1C for approximately 3 h. The filtrates were dried and stored at 80 1C until use. The internal standard solution consists of 20 mmol L 1 methionine sulfone, 20 mmol L 1 2-(N-morpholino)ethanesulfonic acid, and 40 mmol L 1 D-camphor10-sulfonic acid. These compounds were added to correct migration times in the electropherograms and to normalize peak areas to eliminate unexpected systematic bias among runs. Instrumentation and measurement conditions The instrumentation and measurement conditions for capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) have been described elsewhere.32 All capillary electrophoresiselectrospray ionization-mass spectrometry (CE-ESI-MS) experiments were performed using an Agilent CE capillary electrophoresis system, an Agilent G6220A LC/MSD TOF system, an Agilent 1100 series isocratic HPLC pump, a G1603A Agilent CE-MS
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adapter kit, and a G1607A Agilent CE-ESI-MS sprayer kit (Agilent Technologies, Santa Clara, CA). The CE-MS adapter kit included a capillary cassette to facilitate thermostating of the capillary. The CE-ESI-MS sprayer kit simplified coupling of the CE system with the MS systems, and was equipped with an electrospray source. For system control and data acquisition, we used G2201AA Agilent ChemStation software for CE and Agilent MassHunter software for TOF-MS. Detailed analysis conditions for the CE-TOFMS are described.19
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Acknowledgements We would like to thank the Yamagata Prefectural Rice Breeding and Crop Science Experiment Station for providing us with soybean seeds, and the Lowland Farming Research Division of the Tohoku Agricultural Research Center for their valuable advice regarding the characteristics of Tohoku149. This work was supported by research funds from the Yamagata Prefectural Government and the city of Tsuruoka.
Metabolite identification, quantification and data analysis Metabolite identification and quantification were carried out as described previously.32,33 Metabolites detected in more than four samples among six replicates were used for the PCA analysis. For data visualization, heat maps were constructed using the averaged concentration of repeated experiments (n = 6). Principal component analysis (PCA), heat map visualization, and Kaplan–Meier analysis were performed using JMP (ver. 9.0.2. http://www.jmp.com/), Mev (ver. 4.8.1. http://www.tm4. org/mev/), and Graphpad Prism ver. 5.0.4 (Graphpad Software, San Diego, CA), respectively.
Conclusion We successfully characterized aphid resistance in seven soybean strains and compared the metabolomic profiles of aphids reared on leaves of Tohoku149, a resistant soybean strain, to the profiles of six susceptible strains in an attempt to elucidate the mechanism(s) underlying aphid resistance. The PCA results revealed that the metabolomic profiles obtained at 12 h postinoculation were highly diverse, while those of aphids reared on Tohoku149 and under starvation conditions were most similar. Comparative metabolomic analyses revealed that malate and succinate, components of the TCA cycle, increased from 6 h to 12 h, whereas G6P decreased drastically in aphids reared on Tohoku149 leaves. Free amino acid profiles revealed the presence of branched-chain amino acids (Val, Leu, and Ile), Thr, homoserine, and Phe, in aphids reared on Tohoku149 leaves, even while non-essential amino acids were being synthesized by endosymbiotic bacteria. These results suggest that Tohoku149 phloem sap contains factor(s) that inhibit amino acid synthesis pathways, particularly those in endosymbiotic bacteria. We also identified two unique metabolites, SMM and trigonelline, both of which occurred at lower or marginal levels in aphids cultured on Tohoku149 leaves. Since both metabolites are synthesized by methylation using SAM, aphid resistance is considered to be linked to the methylation reaction. These findings will likely facilitate the discovery of new markers for screening resistant strains and for developing novel insecticides.
Financial support This work was supported by research funds from the Yamagata Prefectural Government and the City of Tsuruoka.
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Notes and references 1 M. Tester and P. Langridge, Science, 2010, 327, 818–822. 2 D. Pimentel, in Integrated Pest Management: InnovationDevelopment Process, ed. R. Peshin and A. Dhawan, Springer, Netherlands, 2009, ch. 3, pp. 83–87, DOI: 10.1007/978-14020-8992-3_3. 3 T. Nakata, Appl. Entomol. Zool., 1995, 30, 129–138. 4 T. H. Schuler, G. M. Poppy, B. R. Kerry and I. Denholm, Trends Biotechnol., 1998, 16, 168–175. 5 S. Ohnishi, N. Miyake, T. Takeuchi, F. Kousaka, S. Hiura, O. Kanehira, M. Saito, T. Sayama, A. Higashi, M. Ishimoto, Y. Tanaka and S. Fujita, Breed. Sci., 2012, 61, 618–624. 6 T. Masuda and P. D. Goldsmith, Int. Food Agribus. Man., 2009, 12, 143–161. 7 G. L. Hartman, E. D. West and T. K. Herman, Food Sec., 2011, 3, 5–17. 8 E.-C. Oerke, J. Agric. Sci., 2005, 144, 31. 9 D. W. Ragsdale, D. A. Landis, J. Brodeur, G. E. Heimpel and N. Desneux, Annu. Rev. Entomol., 2011, 56, 375–399. 10 R. E. Down, A. M. R. Gatehouse, W. D. O. Hamilton and J. A. Gatehouse, J. Insect Physiol., 1996, 42, 1035–1045. 11 S. E. Jandricic, S. P. Wraight, K. C. Bennett and J. P. Sanderson, Environ. Entomol., 2010, 39, 1631–1642. 12 K. Ueno, H. Takatori and T. Saito, Annu. Rep. Soc. Plant Prot. North Jpn., 2001, 2001, 183–184. 13 T. Nagano, Y. Umetsu, N. Hoshi and T. Kidokoro, Annu. Rep. Soc. Plant Prot. North Jpn., 2001, 52, 175–177. 14 T. Ono, J. Oyama and T. Kidokoro, Annu. Rep. Soc. Plant Prot. North Jpn., 2006, 2006, 148–150. 15 R. Karban and J. H. Myers, Annu. Rev. Ecol. Syst., 1989, 20, 331–348. 16 C. B. Hill, Y. Li and G. L. Hartman, Crop Sci., 2006, 46, 1601–1605. 17 C. B. Hill, Y. Li and G. L. Hartman, Crop Sci., 2006, 46, 1606–1608. 18 C. B. Hill, A. Chirumamilla and G. L. Hartman, Euphytica, 2012, 186, 635–646. 19 D. Sato, H. Akashi, M. Sugimoto, M. Tomita and T. Soga, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2013, 925, 95–103. 20 H. Jinno, K. Araki, K. Kira and Y. Shinada, Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan, 1997, 112–113.
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21 M. Kobayashi and T. Murai, Jpn. J. Appl. Entomol. Zool., 2012, 56, 49–55. 22 C. B. Hill, L. Crull, T. K. Herman, D. J. Voegtlin and G. L. Hartman, J. Econ. Entomol., 2010, 103, 509–515. 23 S. Shigenobu, H. Watanabe, M. Hattori, Y. Sakaki and H. Ishikawa, Nature, 2000, 407, 81–86. 24 F. Bourgis, S. Roje, M. L. Nuccio, D. B. Fisher, M. C. Tarczynski, C. J. Li, C. Herschbach, H. Rennenberg, M. J. Pimenta, T. L. Shen, D. A. Gage and A. D. Hanson, Plant Cell, 1999, 11, 1485–1497. 25 T. H. Phang, G. Shao and H. M. Lam, J. Integr. Plant Biol., 2008, 50, 1196–1212. 26 The International Aphid Genomics Consortium, PLoS Biol., 2010, 8, e1000313. 27 Y. Li, J. Zou, M. Li, D. D. Bilgin, L. O. Vodkin, G. L. Hartman and S. J. Clough, New Phytol., 2008, 179, 185–195.
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28 M. R. Monton and T. Soga, J. Chromatogr. A, 2007, 1168, 237–246. 29 R. Ramautar, E. Nevedomskaya, O. A. Mayboroda, A. M. Deelder, I. D. Wilson, H. G. Gika, G. A. Theodoridis, G. W. Somsen and G. J. de Jong, Mol. Biosyst., 2011, 7, 194–199. 30 G. Graça, B. J. Goodfellow, A. S. Barros, S. Diaz, I. F. Duarte, K. Spagou, K. Veselkov, E. J. Want, J. C. Lindon, I. M. Carreira, E. Galhano, C. Pita and A. M. Gil, Mol. Biosyst., 2012, 8, 1243–1254. 31 H. G. Gika, G. A. Theodoridis and I. D. Wilson, J. Sep. Sci., 2008, 31, 1598–1608. 32 M. Sugimoto, D. T. Wong, A. Hirayama, T. Soga and M. Tomita, Metabolomics, 2010, 6, 78–95. 33 M. Sugimoto, H. Goto, K. Otomo, M. Ito, H. Onuma, A. Suzuki, M. Sugawara, S. Abe, M. Tomita and T. Soga, J. Agric. Food Chem., 2010, 58, 8418–8425.
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