Article pubs.acs.org/JAFC
Volatile Profiles of Members of the USDA Geneva Malus Core Collection: Utility in Evaluation of a Hypothesized Biosynthetic Pathway for Esters Derived from 2‑Methylbutanoate and 2‑Methylbutan-1-ol Nobuko Sugimoto,† Philip Forsline,§ and Randolph Beaudry*,† †
Department of Horticulture, Michigan State University, East Lansing, Michigan 48824, United States Plant Genetic Resources Unit, Agricultural Research Service, U.S. Department of Agriculture, Cornell University, Ithaca, New York 14456-0462, United States
§
S Supporting Information *
ABSTRACT: The volatile ester and alcohol profiles of ripening apple fruit from 184 germplasm lines in the USDA Malus Germplasm Repository at the New York Agricultural Experiment Station in Geneva, NY, USA, were evaluated. Cluster analysis suggested biochemical relationships exist between several ester classes. A strong linkage was revealed between 2methylbutanoate, propanoate, and butanoate esters, suggesting the influence of the recently proposed “citramalic acid pathway” in apple fruit. Those lines with a high content of esters formed from 2-methylbutan-1-ol and 2-methylbutanoate (2MB) relative to straight-chain (SC) esters (high 2MB/SC ratio) exhibited a marked increase in isoleucine and citramalic acid during ripening, but those lines with a low content did not. Thus, the data were consistent with the existence of the hypothesized citramalic acid pathway and suggest that the Geneva Malus Germplasm Repository, appropriately used, could be helpful in expanding our understanding of mechanisms for fruit volatile synthesis and other aspects of secondary metabolism. KEYWORDS: ester, citramalate, isoleucine, threonine, aroma, flavor
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INTRODUCTION
breeding and/or the creation or testing of hypotheses related to plant secondary metabolism. In ripening apple fruit, esters are the primary impact compounds that influence aroma and normally account for 80−95% of the total volatile emission.4 Typical apple esters include hexyl acetate, butyl acetate, and 2-methylbutyl acetate and are autonomously produced in abundance during ripening.4 Consumers perceive these esters as ‘fruity’ and ‘floral’.5 Specific esters or ester classes can dominate the ester profile or be unusually abundant in some cultivars. For example, acetate esters predominate in ‘Calville blanc’ and ‘Golden Delicous’, butanoate esters are found in high amounts in ‘Bell de Boskoop’ and ‘Canada blanc’, propanoate esters are abundant in ‘Richard’ and ‘Reinette du Mans’,4 ethyl esters are plentiful in ‘Starking’, ‘Delicious’, and ‘Bisbee Delicious’,4,6−8 and pentyl esters in ‘Redchief Delicious’, although low in amount, are higher than is typically found in most apple cultivars.9 The esters of intact apple fruit are composed of alkyl (alcohol-derived) and alkanoate (acid-derived) groups, typically one to eight carbons in length. The alkanoate groups enter into the ester-forming reaction as CoA thioesters in a reaction catalyzed by alcohol acyl-CoA transferase (AAT, EC 2.3.1.84).10 These alkyl and alkanoate groups can be straight
The genus Malus is genetically diverse, but cultivated apples have a very narrow genetic base.1 A narrow genetic base has the potential to make commercial apple production vulnerable to catastrophic losses and limits the potential diversity of desirable traits that might be manipulated through selection. For this reason, the U.S. Department of Agriculture (USDA) has made increasing the genetic diversity of apples a programmatic goal and in 1987 developed the wild apple germplasm collection at the New York Agricultural Experiment Station at Geneva.2 The genetic diversity of the collection was intended to be broad enough to act as a source for a number of desirable horticultural traits including resistance to biotic and abiotic stresses, fruit quality, and other attributes useful for the development of new apple cultivars.2 Phenotypic data for the collection can be accessed on the Germplasm Resources Information Network (GRIN) http://www.ars.usda.gov/Aboutus/docs.htm?docid= 6169. The trees and fruit from the trees are described with 25 priority descriptors such as morphology (e.g., shape, size, and color), phenology, production, and growth (e.g., tree vigor and habit) as listed in Forsline et al.2 These categories provide useful information for breeding to reduce production costs, increase marketability, and improve traits such as disease resistance.3 Until now, however, the database has lacked information relating to biochemical/metabolomic traits, such as volatile profiles. Properly used, it seems reasonable to expect that analysis of such traits across the diverse Malus sp. population in this collection could be useful information for © XXXX American Chemical Society
Received: November 28, 2014 Revised: January 29, 2015 Accepted: February 4, 2015
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DOI: 10.1021/jf505523m J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
during the first season. Lines were selected on the basis of their having high or low emission of esters derived from 2-methylbutan-1-ol or 2methylbutanoate (2MB) relative to SC esters as described below. Handling was essentially the same as for the first year of the study except that the fruits were obtained on two separate dates: prior to the onset of the ethylene autocatalytic burst and after ripening was well advanced. Maturity and volatile analyses performed were the same as for the 184 lines in 2005. Additional analyses included the metabolites citramalic acid and the BCAAs isoleucine, leucine, and valine and the isoleucine precursor, threonine, as described subsequently. Four replicate fruits nearest the IEC median were used for volatile profile measurements, and two replicates (four fruits each) were used for metabolite analyses. Measurement of IEC. The IEC was determined by withdrawing a 1 mL gas sample from the interior of the apples and subjecting the gas sample to GC (Carle series 400 AGC; Hach Co., Loveland, CO, USA) analysis as previously described.20 Due to limitations on the interior gas space, the IEC could be accurately determined only on fruit with a mass >5 g. The ethylene detection limit was approximately 0.005 μL L−1. The ethylene concentration was calculated relative to a certified standard (Matheson Gas Products Inc., Montgomeryville, PA, USA) containing 0.979 μL L−1 ethylene, 4.85% CO2, and 1.95% O2, balanced with N2. Measurement of Starch Index and °Brix. The starch index at harvest was determined by cutting fruit in half through the seed cavity along the plane perpendicular to the longitudinal axis. The cut flesh was dipped into an iodine solution containing 10 g of KI and 40 g of I2 per 4 L of water. Color development was allowed to proceed for at least 1 min. Starch index (1−8) was determined by comparison to the Cornell Starch Chart.21 °Brix was measured on a drop of juice expressed from the fruit cortex using a hand-held refractometer (Atago N1, Atago Co. Ltd., Tokyo, Japan). Measurement of Firmness. Firmness was determined only on those lines with a fruit mass >20 g using a drill-stand-mounted Effegi penetrometer (FT-327; McCormick Fruit Tree Inc., Yakima, WA, USA) fitted with an 11 mm diameter probe. The penetrometer was calibrated at 53.4 N (12 lb) using a top-loading balance. Two skin disks (2−3 cm in diameter) and 3−5 mm of underlying cortex tissue were removed from opposite sides of each fruit. The penetrometer probe was pressed into the tissue of the cut surface to a depth of 8−9 mm in a single smooth motion requiring approximately 1 s. Data were recorded as pounds and converted to newtons by multiplying by 4.45 N/lb. Measurement of Respiration. Respiration was measured as described by Sugimoto et al.,19 using a 1 L Teflon chamber (Savillex Corp., Minnetonka, MN, USA) and a 20 min holding time at 20 °C. CO2 concentration was measured on 0.1 mL gas samples withdrawn from a sampling port sealed with a Teflon-lined half-hole septum (Supelco Co., Bellefonte, PA, USA) using an insulin-type 0.5 mL plastic syringe. The gas sample was injected into an infrared gas analyzer (model 225-MK3; Analytical Development Co., Hoddesdon, UK) operated in a flow-through mode with N2 as the carrier gas and a flow rate of 100 mL min−1. The CO2 concentration was calculated relative to the certified standard noted previously. Measurement of Volatiles. For volatile measurement, a GC (HP6890, Hewlett-Packard Co., Wilmington, DE, USA) was used in conjunction with a time-of-flight mass spectrometer (TOFMS) (Pegasus III, LECO Corp., St. Joseph, MI, USA). Fruit were placed in a 1-L Teflon container (Savillex Corp., Minnetonka, MN, USA) fitted with a gas-sampling port. The number of apples held in the container varied from 1 to 20 depending on the fruit size, and the analysis was performed on a minimum of 10 g of total fruit mass. Sampling ports were sealed with a Teflon-lined half-hole septum (Supelco Co., Bellefonte, PA, USA), and apples were incubated at 22 °C for 20 min. Headspace volatiles were sampled using a 1 cm long, solid-phase microextraction (SPME) fiber (65 μm PDMS-DVB, Supelco Co.) and a 3 min sorption period.22 After sampling, the SPME fiber was immediately transferred to the GC for desorption and separation of volatiles. The injection port was operated in splitless mode and held isothermally at 230 °C; the desorption time was 2 min.
or branched chain. Several pathways for ester biosynthesis have been proposed, but conclusive evidence is lacking for some of the pathways. Precursors of straight-chain (SC) esters have been suggested to be formed from fatty acids by a combination of β-oxidation and lipoxygenase pathways,11−13 and precursors of branched-chain (BC) esters have been suggested to be formed from the metabolic pathways associated with branchedchain amino acid (BCAA) formation14−18 and via the recently proposed “citramalic acid pathway”.19 Identification of apple lines in the USDA Geneva Malus Germplasm Repository that differ markedly in the composition of their volatile profile should prove useful in the dissection of the various pathways of synthesis for ester precursors. Hypothetically, if the volatile profile data are of adequate quality and detail, statistical associations between classes of alcohols and/or esters should permit the selection of apple lines sufficiently divergent in their volatile profile to enable testing of proposed biochemical pathways. The objective of this research was to investigate the diversity of volatile esters produced by ripe fruit from each line in the four-copy USDA Geneva Malus Core Collection and to use those data to investigate a hypothetical pathway implicated in the formation of odor-active esters. The information will serve to expand the GRIN system database and act as a resource for selection for volatile phenotype in breeding efforts.
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MATERIALS AND METHODS
Plant Material. Apple fruit from 172 lines of the four-copy Geneva Malus Core Collection and an additional 12 Kazakhstan lines (all M. sieversii) were harvested on nine dates between August 24, 2005, and Oct 24, 2005, from the USDA Plant Genetic Resources Unit’s Malus Germplasm Repository at Cornell University, Geneva, NY, USA. Several lines bore no fruit during the harvest season and so were omitted from the study. The 184 lines consisted of 39 taxa: 20 taxa were primary species (48 accessions), 8 were cultivated species (88 accessions), 9 were secondary species (12 accessions), 1 consisted of unidentified species (2 accessions), and 1 consisted of uncharacterized hybrid species (34 accessions). Harvest times were initially estimated on the basis of the GRIN harvest code, which classified lines relative to the timing for ‘Delicious’ maturation: (1) extreme early, >60 days before ‘Delicious’; (2) very early, 50−60 days before ‘Delicious’; (3) early, 30−50 days before ‘Delicious’; (4) midearly, 20−30 days before ‘Delicious’; (5) mid, 10 days before ‘Delicious’; (6) midlate, same time as ‘Delicious’; (7) late, 10 days later than ‘Delicious’; (8) very late, 20− 30 days later than ‘Delicious’; (9) extremely late, >30 days later than ‘Delicious’. The harvested apples were packed and secured in a specialized corrugated cardboard box with polyurethane padding containing 7 cm diameter by 5 cm deep cylindrical cavities for individual apples to avoid bruising during shipping. The packed apples were shipped overnight to Michigan State University for evaluation. Samples for each of the 184 lines consisted of 3−50 fruits depending on fruit size and availability on the tree. On each occasion, fruit were held overnight in the laboratory to equilibrate to laboratory temperature (22 ± 1 °C) while covered with ventilated, black, 4 mil (0.1016 mm) thick plastic bags to avoid desiccation and responses to intermittent laboratory light before harvest maturity analysis. In addition to volatile profile, measurements included fresh weight, internal ethylene concentration (IEC), starch index, firmness, background color, and °Brix. Volatile compounds were analyzed after fruits were judged to be ripe according to IEC, starch index, °Brix, background color, and/or perceived intensity of aroma. If the fruit received were not yet ripening, they were re-sent at a later date or held for a short period to enable ripening to commence and re-evaluated. Ten lines of apple fruit were also obtained from USDA Plant Genetic Resources Unit’s Malus Germplasm Repository in the second season (2008) of the study based on ester composition determined B
DOI: 10.1021/jf505523m J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
1:1:1 (v/v/v) ratio containing 10 μM deuterated methionine (Met-d3) (Cambridge Isotope Laboratories Inc., Andover, MA, USA) as an internal standard, mixed vigorously, incubated at 70−80 °C for 5 min, and immediately transferred in ice. After centrifugation at 5000gn for 10 min at 4 °C, the supernatant was filtered through a 0.22 μm filter (Millex GS, Millipore, Billerica, MA, USA). The cleared filtrate was diluted to one-fifth of its original concentration with water containing the internal standard and used for amino acid analysis. The amino acids threonine, isoleucine, leucine, and valine (SigmaAldrich Corp., St. Louis, MO, USA) were dissolved in water containing 10 μM Met-d3 to make individual stock solutions of 1 mM. A master mixture was created by mixing amino acids to a final concentration of 50 μM each. A series of six working standards ranging from 0.5 to 40 μM, each containing 10 μM Met-d3, was prepared by serial dilutions from the 50 μM master mixture using water containing 10 μM Met-d3. Amino acid samples were quantified by calibration curves obtained from the six working standards using linear regression, plotting amino acid concentration as a function of ratio of the amino acid peak area to the Met-d3 peak area. Amino acids were analyzed using a tandem mass spectrometer (Waters Quattro micro, Waters Inc., Milford, MA, USA) coupled to a high-pressure liquid chromatograph (LC-20AD HPLC, Shimadzu, Columbia, MD, USA) equipped with an autosampler (SIL-5000, Shimadzu). The 2.1 × 50 mm column was packed with a 2.7 μm diameter C18 stationary phase (Ascentis Express C18, Sigma-Aldrich) and held isothermally at 30 °C. Injection volume was 10 μL, and solvents used were 1 mM perfluoroheptanoic acid (mobile phase A) and acetonitrile (mobile phase B); the flow rate was held constant at 0.3 mL min−1. The gradient program was as follows: 98% mobile phase A and 2% mobile phase B at start, 10% mobile phase B after 0.1 min, increasing to 25% mobile phase B at 0.5 min and held until 4 min, decreasing to 2% mobile phase B at 4.1 min for re-equilibration of the column. The total run time was 6 min. Mass spectra were acquired using electrospray ionization in positive ion mode (ESI+). The capillary voltage was 3.17 kV, the extractor voltage was 4 V, the rf lens was held at 0.3 V, the cone gas flow rate was 30 L h−1, the desolvation gas flow rate was 400 L h−1, the source temperature was 110 °C, and the desolvation temperature was 350 °C as described by Gu et al.26 The data acquisition method was split into two functions, the first from 0 to 1.85 min and the second from 1.85 to 6.0 min. Ten multiple reaction monitoring (MRM) transitions were included in function 1 and 11 in function 2. Function number, collision energies, and masses of observed ions for each amino acid are were described by Sugimoto et al.19 Data were collected and quantified with proprietary software (MassLynx 4.0 and QuanLynx; Waters). Citramalic Acid Analysis. Approximately 5 g of frozen apple peel from each of the two four-fruit replicates was ground into fine powder using a liquid nitrogen cooled mortal and pestle. The ground tissue was divided into three technical replicates, each containing 0.5 g of tissue. The powdered tissue was transferred into 1 mL of preheated water, mixed vigorously, and incubated at 90−95 °C for 10 min. After centrifugation at 5000gn for 5 min, the supernatant was filtered through a 0.22 μm filter (Millex GS, Millipore, Billerica, MA, USA), and the cleared filtrate diluted to one-tenth of its original concentration with water was used for analysis. Citramalic acid (Sigma-Aldrich) was dissolved in water to make a stock solution of 1 mM. A master mixture (50 μM) was created for making standard dilutions. A series of five working standards ranging from 1 to 40 μM was prepared by serial dilutions from the 50 μM master mixture and used to create calibration curves. Citramalic acid was analyzed using a mass spectrometer (Quattro Premier XE, Waters Corp., Milford, MA, USA) coupled to an ultraperformance liquid chromatograph (UPLC) (Acquity, Waters Corp.). The column (Thermo Betasil C18, 2.1 × 150 mm, 5 μm particles, Thermo Fisher Scientific Inc., Waltham, MA, USA) was held isothermally at 50 °C. The injection volume was 5 μL; solvents were 1 mM N,N-dimethylhexylammonium acetate (pH 7.7) in water (mobile phase A) and methanol (mobile phase B), and the flow rate was 0.4 mL min−1. The gradient program was as follows: 80% mobile phase A and 20% mobile phase B at start, increasing to 75% mobile phase B
Desorbed volatiles were trapped on-column using a liquid nitrogen cryofocusing trap. Conditions of GC separation and TOFMS analysis were as previously described.22 Identification of compounds was by comparison of the mass spectrum with authenticated reference standards and/or with spectra in the National Institute for Standard and Technology (NIST) mass spectrum library (version 05) when no standard was available. Volatile compound concentrations (nanomoles per liter) in the chamber headspace were quantified by calibration with a known concentration of an authenticated, high-purity, 72-component standard mixture of esters and alcohols as previously described.22 Where no standard was available, quantization was by estimation of the instrument response factor based on the Kovats index for the compound of interest.23 Cluster Analysis for Volatiles. Hierarchical cluster analysis was performed on all of the esters detected in this study for all taxons using Cluster 3.0,24 which was developed from the original program by Eisen et al.25 and TreeView software.25 The uncentered correlation and centroid linkage method was used. Selection of Germplasm Lines for Metabolite Analysis. As noted above, Malus sp. lines from high and low 2MB/SC classes were selected for further characterization in season two. SC esters were defined as those having one, two, or four or more carbons arranged in a straight chain on both the alcohol and acid moieties of the ester. 2MB esters were those derived from 2-methylbutanoate or 2methylbutan-1-ol and having either a branched or straight chain of any length for the complementary alcohol or acid moiety of the ester. The sum of the concentration (nanomoles per liter) of SC and 2MB esters were calculated for each of the 184 lines, and the ratio of 2MB/ SC was determined. Selection of “high” and “low” 2MB/SC lines was based on the concentration of 2MB and SC ester concentrations in the chamber headspace and on the ratio of 2MB to SC esters. Selection required 2MB and SC ester emissions to exceed 3 nmol L−1. In addition, high 2MB/SC lines had to have a 2MB/SC ester ratio >1, and low 2MB/SC esters had to have a ratio of
