Mass Spectrometry-Based Metabolite Profiling

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Omics Technologies Applied to Agriculture and Food

GC/MS-based Metabolites Profiling of Nutrients and Antinutrients in Eight Lens and Lupinus Seeds (Fabaceae) Mohamed A. Farag, Amira R. Khattab, Anja Ehrlich, Matthias Kropf, Andreas G. Heiss, and Ludger A. Wessjohann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00369 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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Journal of Agricultural and Food Chemistry

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For submission to: JAFC

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GC/MS-based Metabolites Profiling of Nutrients and Anti-

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nutrients in Eight Lens and Lupinus Seeds (Fabaceae)

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Mohamed A. Farag1,2*, Amira R. Khattab3, Anja Ehrlich4, Matthias Kropf5, Andreas G. Heiss6,

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Ludger A. Wessjohann4

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Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, Egypt, Kasr el Aini St., P.B. 11562;

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Chemistry Department, School of Sciences & Engineering, The American University in Cairo, New Cairo 11835, Egypt;

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Academy for Science, Technology and Maritime Transport, Alexandria, Egypt, P.B.1029;

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Institute for Integrative Nature Conservation Research, University of Natural Resources and Life Sciences Vienna (BOKU), Austria;

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Institute for Integrative Nature Conservation Research, University of Natural Resources and Life Sciences Vienna (BOKU), Austria;

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Pharmacognosy Department, Division of Pharmaceutical Sciences, College of Pharmacy, Arab

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Department for Bioarchaeology, Austrian Archaeological Institute (ÖAI), Austrian Academy of Sciences (ÖAW), Austria.

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*Corresponding author: Mohamed A. Farag

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E-mail: [email protected], [email protected]

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Tel: +011-202-2362245, Fax: +011-202-25320005

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ACS Paragon Plus Environment


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ABSTRACT

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Lens culinaris and several Lupinus species are two legumes regarded as potential protein

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resources aside from their richness in phytochemicals. Consequently, characterization of their

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metabolite composition seems warranted to be considered as a sustainable commercial

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functional food. This study presents a discriminatory-holistic approach for metabolites

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profiling in accessions of four lentil cultivars and four lupinus species via gas

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chromatography-mass spectrometry. A total of 107 metabolites were identified encompassing

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organic and amino acids, sugars and sterols along with anti-nutrients viz. alkaloids and sugar

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phosphates. Among the examined specimens, four nutritionally-valuable accessions ought to

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be prioritized for future breeding to include Lupinus hispanicus, enriched in organic

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(ca.11.7%) and amino acids (ca.5%), and L. angustifolius, rich in sucrose (ca.40%), along

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with two dark-colored lentil cultivars ‘verte du Puy’ and ‘Black Beluga’ enriched in peptides.

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Anti-nutrient chemicals were observed in Lupinus polyphyllus owing to its high alkaloids

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content. Several species-specific markers were also revealed using multivariate data analyses.

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KEYWORDS: Functional foods; GC/MS; Lens culinaris; Lupinus; Metabolite profiling;

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Nutrients; OPLS-DA; PCA.

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1. Introduction

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Increasing demand for protein resources owing to the growing human population along

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with the reported health risk of animal protein consumption warrants the development of

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other plant protein as in soybean derived food. Such interest towards the incorporation of

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plant proteins in human diet derived the development of effective analytical methods for the

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screening of plant foods.1

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Leguminous seeds, viz. lupines, soybeans, lentils, beans, and peas are valued as

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promising alternative supply of proteins for human and animal consumption worldwide.

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Among these, lupin (Lupinus L.) is one of the richest protein resources, almost as high as that

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of soybean (~35-40% of the dry weight) followed by lentil (Lens culinaris Medik.), which is

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known as “poor man’s meat”, with a protein content of ca. 20–25%. It should be noted that

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compared to animal protein, plants present a less complete resource of amino acids which

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warrants mixing different plant protein diet in what is known typically as complementary

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protein. 2-3

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Both Lens culinaris and Lupinus seeds provide a well-balanced source of essential amino

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acids, carbohydrates, fiber, minerals, and vitamins as well as a myriad of bioactive

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phytochemicals viz., quinolizidine alkaloids, polyphenols and saponins of value for the

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management and prevention of ailments.4 However, these seeds contain a number of anti-

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nutrients, i.e. tannins, oligosaccharides, saponins, phytates, and protease inhibitors, well

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reported to cause detrimental effects to human nutritional status by hindering the uptake and

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utilization of minerals, vitamins, proteins and other key nutrients. 3

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Lupinus species (subfamily Faboideae; tribus Genisteae), such as L. albus (white lupin),

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L. angustifolius (blue lupin), L. luteus (yellow lupin), L. mutabilis (pearl lupin) and L.

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hispanicus (Spanish lupin), have been domesticated owing to their nutritive value and ease of



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cultivation under different climatic conditions.1 These species are widely distributed and,

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according to Gladstones (1998) are grouped into “Old World Species”, located in the

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Mediterranean-African region, while “New World Species” include the species found in

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North and South America such as L. polyphyllus. For the three “Old World Species” under

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study (i.e. Lupinus angustifolius, L. hispanicus, and L. luteus; Table 1) a phylogenetic close

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relationship has been demonstrated (cf. Käss & Wink 1997).5 Overall, lupines are

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characterized by a diverse geographical distribution, morphological variation and

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chromosomal polymorphisms.6

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The health benefits of lupin have been extensively reported to include hypoglycemic,

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hypotensive, cholesterol lowering, anticancer and anti-inflammatory effects aside from their

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protection against menopausal symptoms and osteoporosis.1, 7

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All Lupinus species produce quinolizidine alkaloids such as lupinine, lupanine and α-

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isolupanine, albeit with qualitative and quantitative differences as revealed

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multivariate statistical tools.8 The phenolic secondary metabolites in Mediterranean lupin

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species have been reported mostly using LC/MS8-10, particularly considering their value as

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chemotaxonomic markers of Lupinus spp. than the classically employed morphological and

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cytological features.11 Being also a member of the Fabaceae family (subfamily Faboideae),

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lentil seeds (tribus Fabeae) possess a comparable bioactive composition to lupin with

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biological effects to include antioxidant, anticarcinogenic, antihyperglycemic and

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hypocholestrolemic. Lentil is classified into several market classes i.e., extra small red, small

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red, large red, small green, medium green, large green, Spanish brown, zero tannin and Puy.12

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The profiling of phenolic secondary metabolites of the various lentil genotypes was

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reported in relation to its antioxidant capacity.13-14 Phenotypic selection of lentils with


using

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improved nutritional attributes was further achieved based on the selection of a wide range of

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molecular markers.15

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With an increasing interest in a rather holistic view for metabolites composition in

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legumes, the development of large scale analytical methods now follows. Plant metabolomics

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is valued as a comprehensive profiling technology employed to obtain snapshots of all low

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molecular weight molecules (the metabolome) in a given organism. Such approach

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commonly utilizes rigorous hyphenated mass spectrometry techniques such as gas or liquid

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chromatography mass spectrometry (GC/MS) and (LC/MS), applied in food and

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nutraceuticals analysis. The complexity and richness of metabolomics data can only be

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harnessed using a diverse set of chemometric tools, such as classification, dimensionality

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reduction, visualization, pattern recognition, and or modelling.16 Such platforms can be

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employed to confirm the identity of taxonomically-related plant species, monitor its quality

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or purity attributes and/or to standardize plant-derived products.17-19 The untargeted GC/MS-

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based metabolomics is routinely used to record the rich matrix of plants, made up of several

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low molecular weight metabolites.

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There were scarce reports for multivariate data analyses application for the classification

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of examined seeds, except for the application of artificial neural network and PCA for the

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discrimination of two Lupinus species, L. albus and angustifolius.

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the current study was to profile Lens and Lupinus seed accessions metabolites to provide

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better insight into their compositional differences and/or nutritional traits. Considering the

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complexity of acquired data, unsupervised and supervised multivariate data analyses viz.

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principal component analysis (PCA) and orthogonal partial least squares (OPLS), were

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employed for classification of seed samples, and to ensure good analytical rigorousness.

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2. MATERIALS AND METHODS


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The main objective of


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2.1. Plant material

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Lens culinaris and Lupinus seeds were obtained from two main sources: seed exchange

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with four Botanical Gardens (BAS, HOH, IB, COI), as well as two Austrian organic food

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vendors. Details are listed in Table 1.

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2.2. Chemicals

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All solvents used were of LC/MS grade purchased from J. T. Baker (The Netherlands).

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Umbelliferone was procured from ChromaDex (LGC Standards, Wesel, Germany). All other

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chemicals and standards were purchased from Sigma Aldrich (St. Louis, MO, USA).

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2.3. Sample preparation for GC/MS analysis

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Dried seeds were ground using pestle and mortar under liquid nitrogen. The obtained

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powder (30 mg) was then homogenized with 2.5 mL 100% MeOH containing 5 µg/mL

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umbelliferone (an internal standard for relative quantification) using a Turrax mixer operated

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at 11.000 rpm for 20 s for 5 periods, with 1 min of recession between each mixing period to

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guard against excess heat generated during mixing. For the removal of plant debris, the

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extracts were vortexed vigorously and centrifuged at 3000 g for 30 min.

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2.4. GC/MS analysis of silylated primary metabolites

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For analysis of primary metabolites (viz. amino acids, organic acids, and sugars), 100 µL

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of extract (prepared as described in section 2.3) was evaporated to dryness under nitrogen.

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Derivatization with 150 µL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA)

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was performed at 60◦C for 45 min. Samples were equilibrated at 28◦C prior to analysis using

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Shimadzu GC-17A gas chromatograph interfaced with Shimadzu QP5050A mass

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chromatograph. Separation of silylated derivatives was carried out on Rtx-5MS (30 m length,

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0.25 mm inner diameter, and 0.25 µm film) column. Injections were made in the split mode

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with a split ratio of 1:15 under the following conditions: injector 280˚C, column oven 80˚C


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for 2 min, then programmed at a rate of 5˚C/min to 315˚C, kept at 315˚C for 12 min. He

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carrier gas at 1 mL min-1. The transfer line and ion–source temperatures were adjusted at

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280°C and 180˚C, respectively. The HP quadrupole mass spectrometer was operated in the

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electron ionization mode (EI, 70 eV), with a scan range of 50-650 m/z. The identification of

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the silylated compounds was performed according to the procedure described in 21 and peaks

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were first deconvoluted using AMDIS software (www.amdis.net) and identified by its

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retention indices (RI) relative to n-alkanes (C8-C40), mass spectrum matching to NIST,

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WILEY library database and with authentic standards whenever available.

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2.5. Multivariate data analysis

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Principal component analysis (PCA) and orthogonal partial least squares-discriminate

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analysis (OPLS-DA) were performed with the program SIMCA-P Version 13.0 (Umetrics,

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Umeå, Sweden). The PCA was run for obtaining a general overview of the variance of

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metabolites in the different seed accessions under study, and for obtaining information on

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differences in the metabolite composition among species was revealed by OPLS-DA.

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Performance of chemometric models was evaluated using the two parameters Q2 and R2,

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where R2 is employed to quantify the goodness-of-fit of the model; whereas the model

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predictability is determined from Q2 values. The distance to the model (DModX) was used

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for detecting outliers. Iterative permutation test was further done to omit the non-randomness

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of separation between groups.

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2.6. Statistical Analysis

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Statistical analyses were performed by SPSS (Statistical Package for Social Science) for

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Windows software, version 23.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA followed

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by post hoc Tukey's test was used at significance level less than 0.05 for multiple



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comparisons between mean values of the seed metabolites from three biological replicates to

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determine significant differences among the examined seed accessions.

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3. RESULTS AND DISCUSSION

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The nutritive value of legume seeds depends on its nutrient as well anti-nutrient

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chemicals composition. Seeds richness in a wide range of macronutrients viz. proteins,

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sugars, fatty acids, is rather limited by their anti-nutritive content viz. alkaloids and phytic

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acid that might have a negative impact on the digestibility and bioavailability of other

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nutrient elements. Development of analytical methods for measuring both classes is thus

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warranted in legume seeds. In the current study, two legumes seeds “Lens & Lupinus” were

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subjected to metabolite profiling using GC/MS targeting its primary and secondary low

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molecular weight chemicals.

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3.1.

GC/MS peaks identification in Lens & Lupinus seeds

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GC/MS analysis was employed for profiling primary metabolites viz. sugars, organic and

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amino acids, phosphorylated compounds, fatty acids along with low molecular weight or non-

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polar secondary metabolites exemplified in alkaloids and steroids. The biological variance

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within each seed accession was assessed by analyzing three independent biological replicates

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analysed under the same conditions.

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Chromatograms (Fig. S1) display a representative profile of Lens and Lupinus seed

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accession metabolome. A total of 107 silylated metabolites were detected belonging to

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different metabolite classes including sugars, alkaloids, steroids, and amino as well as fatty

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acids (Table 2).

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ANOVA analysis revealed significant differences in the percentile levels of phosphate, fatty

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acids, viz. linoleic, oleic and stearic acids, and sugars, viz. α-D-glucopyranoside and


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glucopyranose 4-O-β-D-galactopyranosyl, among the different seed accessions with a

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confidence level of 95%. Lupinus hispanicus Boiss. & Reutt. showed a significantly higher

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lupinine content than other seed accessions, whereas, α-isolupanine and its 13-hydroxy

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derivative were significantly higher in L. polyphyllus Lindl. ‘Russell’ at p < 0.05. Differences

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in organic acids composition viz. succinic acid and its methyl derivative, carbamic and

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acetic acids, nitrogenous compounds viz. ethanolamine, hydroxylamine, phenylethanolamine,

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α-Aminoisobutyric acid and cadaverine

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Medik. subsp. culinaris ‘Petite Rouge d’Egypte’compared to other accessions.”

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were statistically significant in Lens culinaris

3.1.1. Nutritients in Lens and Lupinus seeds

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Sugars (mono- and disaccharides) amounted for the major metabolite class in all

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examined Lens and Lupinus seeds (Table 2), making up to ca.75 % of the total metabolite

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content except for the two Lupinus species, namely L. hispanicus Boiss. & Reutt. and L.

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polyphyllus Lindl. ‘Russell’ totaling ca. 48%. A bar chart depicting the major metabolite

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class percentile levels in Lens and Lupinus seed accessions are represented in Fig. 1,

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revealing seeds enrichment in sugars followed by organic acids.

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3.1.1.1. Sugars and sugar alcohols

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Disaccharides presented the most abundant sugar subclass, with sucrose (G95)

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amounting for ca.30% of the total monitored peaks except for Lens culinaris Medik. subsp.

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culinaris ‘Petite Rouge d’Egypte’, with trace sucrose levels, being instead enriched in

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melibiose (G103, ca.46%) followed by Lupinus luteus L. (ca.18%). Melibiose is formed in

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legume seeds via an invertase-mediated hydrolysis of the oligosaccharide raffinose with the

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production of fructose. Raffinose family oligosaccharides “α-galactosyl derivatives of

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sucrose” are ubiquitous seeds metabolites reported to accumulate during seed development

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and disappear rapidly post germination concurrent with an increase in sucrose levels.22 Such



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correlation between sucrose and raffinose was also observed in our seed specimens.

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Melibiose health benefits include enhanced minerals absorption,

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microbiota via promoting the growth of beneficial flora, particularly Bifidobacterium

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and Lactobacillus strains while inhibiting the growth of pathogenic bacteria aside from its

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immunostimulant and anti-allergic effect..23-25

modulation of gut

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Sucrose, a dimer of glucose and fructose, was not reported to promote glucose-

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intolerance; hence, its intake from sucrose-enriched foods in moderate amounts is favored

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due to the evidenced potentiation of insulin release by fructose that occur in the presence of a

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stimulatory glucose level.26 Accordingly, the consumption of sucrose rich seed accession

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under study “L. angustifolius L. Boltensia” is unlikely to cause increase in post prandial

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glucose levels.27

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It should be noted that although sugar alcohols were represented by eleven peaks viz. L-

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Threitol (G74), Meso-Erythritol (G75), Xylitol (G77), D-Arabitol (G78), D-Pinitol (G81), D-

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Sorbitol (G82 & G83) and Galactinol (G97, G99, G102 & G104), they were present at trace

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levels comparable to cyclic sugars, and being most abundant in Lens culinaris Medik. subsp.

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culinaris ‘Petite Rouge d’Egypte’ and Lupinus luteus L.

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Lens plants were found to minimize sugar alcohol levels acting as humectant upon

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exposure to high moisture so as to guard against water saturation and decomposition of

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mature seeds.28

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Nevertheless, these metabolites inhibit pathogens growth in the gastrointestinal tract and

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promote the growth of the probiotic strains that possess many health-promoting and

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immunomodulatory properties.29


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Galactosyl cyclitols (galactinol) and cyclitols (D-pinitol) are found to accumulate in the

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legume seeds, functioning as reserve carbohydrates during germination, and for seeds

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viability.30 In that regard, Lens culinaris Medik. subsp. culinaris ‘Petite Rouge d’Egypte’

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encompassed the highest galactinol and pinitol levels (16%), followed by Lupinus luteus L.

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(10%).

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3.1.1.2. Organic and inorganic acids

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Organic acids, the odor-imparting compounds to lupin flour, constituted the second most

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abundant class comprising (5-14%) of the total identified metabolites. Citric acid (G19) is the

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major identified organic acid (4-7%) followed by malic (G13, 1-3%) and malonic acids (G5,

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0.1-2%). Among examined seed specimens, Lupinus hispanicus Boiss. & Reutt. and L.

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polyphyllus Lindl. ‘Russell’ were most enriched in organic acids (ca.12-14%). These acids

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are most probably produced as a result of amino acids degradation caused by microorganisms

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present on the seed hulls.29

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Citric acid exhibits antimicrobial properties due to its acidulation properties in addition

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to antioxidant effect via metal ions chelation, whereas malic acid is commonly found in

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unripe fruits and contributes to its sour taste.31-32 It should be noted that the health hazard

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organic acids viz. oxalic acid was not detected in any of the examined seed extracts.33

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Phosphoric acid (G55) was found most abundant in Lupinus hispanicus Boiss. & Reutt.

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(9.7%) and Lens culinaris Medik. subsp. culinaris ‘Black Beluga’ (5.8%) likely derived from

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phospholipids degradation reported to actively function under high seed moisture content as

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observed in stored soybean seeds.34 Phosphoric acid can synergize the antioxidant potential

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of phenolic antioxidants such as flavonoids in legume seeds.32, 34 Inorganic phosphate was

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also detected in peak (G56), found more abundant in “Old World lupine Species” with the

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highest level in Lupinus hispanicus Boiss. & Reutt. (11%). Phosphate is an essential nutrient



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required for vital biological reactions that maintain cell homeostatic aside from serving as a

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component of genetic material, that is DNA.35

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3.1.1.3. Amino acids/peptides

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Examined specimens of both genera were found to be enriched in free essential amino

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acids, i.e., valine, lysine, threonine, and phenylalanine needed for protein synthesis, growth

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and maintenance of whole-body homeostasis. Nevertheless, examined specimens all lacked

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sulfur-containing essential amino acids in accordance with previous reports.36 Compared to

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lentil amino acid profile, lupine seeds appeared to be more enriched, with L. hispanicus

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Boiss. & Reutt. encompassing the highest levels at ca. 5% of all detected metabolites.

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Consequently, Lupinus species can be regarded as a better nutritive source of plant-based

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protein compared to lens based on free amino acids analysis. Such conclusion needs be

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however further verified by comparing its crude protein content using proteomic tools.

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Free amino acids are reported to exist at small levels in non-germinated Lens seeds, with

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alanine, glutamic, asparagine and aspartic acids the major protein amino acids and in

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agreement with our results.37 γ-Aminobutyric acid (GABA), previously reported to be found

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in germinated Lens culinaris seeds at a concentration of 1.64mg/g dry weight37 and to exhibit

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hypertensive activity38, was detected in all examined seed accessions except L. culinaris

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‘Medik. subsp. culinaris ‘Petite Rouge d’Egypte’.

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Legume seeds are reported to contain peptides with higher antioxidant capacity than their

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intact proteins.39 Two Lens accessions, namely L. culinaris Medik. subsp. culinaris ‘verte du

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Puy’ (brown colored) and L. culinaris Medik. subsp. culinaris ‘Black Beluga’ were found to

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contain the highest peptide levels exemplified in Cys-Gly (G68), Glycyl-l-glutamic acid

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(G69), Ser-Leu (G70) and Ala-Thr (G71).


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3.1.1.4. Sterols/triterpenes

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The two dark colored Lens culinaris seed accessions, namely L. culinaris Medik. subsp.

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culinaris ‘verte du Puy’ (brown colored) and L. culinaris Medik. subsp. culinaris ‘Black

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Beluga’ were found to be the most abundant in the triterpene i.e., α-amyrin (G73), with

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reported anti-inflammatory activity40, present at 6 and 3%, respectively. β-sitosterol (G72),

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major phytosterol in legume seeds41, was detected though at trace levels (0.1%) in most of

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accessions except Lupinus polyphyllus Lindl. ‘Russell’ and Lens culinaris Medik. subsp.

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culinaris ‘Petite Rouge d’Egypte’. β-sitosterol present in lentils is likely to mediate in part for

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its hypocholesterolaemic effect.41

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3.1.2. Anti-nutrient phytochemicals in Lens and Lupinus seeds 3.1.2.1.

Phytic acid

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Phytic acid “myo-inositol-1, 2, 3, 4, 5, 6 hexabisphosphates”, represented by peaks G86

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& G91, was the major detected source of phosphorous in seeds, likely to provide a source of

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inorganic phosphorus during seed development42 and was interestingly found most abundant

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in the three phylogenetically closely related Old World lupine Species “Lupinus angustifolius

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L. ‘Boltensia’, L. luteus L. and L. hispanicus Boiss. & Reutt. with the latter species

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containing the highest levels (ca. 3%). In contrast, only Lens culinaris accession with yellow

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colored seeds (sample code LCY, Table 1) was found to be enriched in phytic acid at 3%

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among Lens specimens.

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Phytic acid is regarded as anti-nutrient forming chelates with mineral and protein and

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thereby decreasing their bioavailability. Albeit, recent research suggests that phytic acid and

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other inositol phosphates provide health benefits such as amelioration of heart diseases, and

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reduced risk of colon cancer.43 Variation in phytic acid levels exists among seed genotype in

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addition to cultivation i.e., climate and type of soil.43



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3.1.2.2. Alkaloids/nitrogenous compounds

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Lupin seeds are generally classified as either bitter or sweet based on their levels of toxic

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quinolizidine alkaloids.8 From a nutritional point of view, the alkaloid profile is crucial as

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significant adverse effects could result such as protein indigestibility and neurological

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disorders.44

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Two bitter Lupinus seeds were identified in this study mainly L. polyphyllus Lindl.

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‘Russell’ with highest alkaloid levels i.e., 13-hydroxy-lupanine (11%) and α-isolupanin (8%),

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followed by L. hispanicus Boiss. & Reutt. encompassing lupinine at ca. 14%. In contrast, L.

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angustifolius L. ‘Boltensia’ is regarded based on GC/MS results as a sweet lupin as it

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contains alkaloids at trace levels (0.2%). Interestingly, cadaverine a toxic diamine produced

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by the decarboxylation of lysine, and serving as precursor for quinolizidine alkaloids,45 was

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detected at its highest levels in Lens culinaris Medik. subsp. culinaris ‘Petite Rouge

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d’Egypte’, though with no downstream effect on the accumulation of lupanine alkaloids.

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Metabolic fate of cadaverine in Lens culinaris has yet to be revealed or its physiological role

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in its seeds.

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Eleven other nitrogenous compounds were detected in examined seed accessions

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amounting for 1-12% of the total monitored peaks. The major identified nitrogenous

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compound was ethanolamine, accounting for ca. 2.3% in the ‘Petite Rouge d’Egypte’ cultivar

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of Lens culinaris. Ethanolamines are derived from N-acylethanolamines, plant lipids with

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anti-inflammatory activity.46 Interestingly, another nitrogenous compound with potential anti-

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inflammatory activity detected include indole-3-acetamide, reported to inhibit phospholipase

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A2, was detected at considerable level (9%) in Lupinus hispanicus.47

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3.2. GC/MS- based multivariate data analysis of Lens & Lupinus seeds


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Untargeted GC/MS metabolite profiling was further performed in order to unveil the

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relative metabolites variation embedded within different Lens and Lupinus seed accessions

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employing multivariate data analyses tools viz. hierarchical cluster analysis (HCA), principal

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component analysis (PCA) and orthogonal projection to latent structures-discriminant

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analysis (OPLS-DA). The un-decoded similarities and variabilities among specimens were

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revealed first via (HCA) using the Ward's algorithm and evaluated based on the distances

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between the clusters (the squared Euclidean distance). The reproducibility of the extraction

335

and analysis conditions were clearly evident from the tight clustering of the independent

336

biological triplicates within same seed accession as seen in Fig. 2-A.

337

HCA-driven dendrogram (Fig. 2-A) portrayed two main clusters; cluster “1a”, composed

338

of two species which are the sweet Lupinus angustifolius L. ‘Boltensia’ (LU_A) and Lens.

339

culinaris Medik. subsp. culinaris ‘Petite Rouge d’Egypte’ and cluster “1b” in which the rest

340

of the seed accessions was grouped altogether. The sample grouping in 1a cluster could be

341

ascribed to their diminished alkaloidal content concurrent with an enriched sugar profile.

342

Asides, the three lentil accessions LCBK, LCB, and LCY (Table 1) were distinctly clustered

343

in one sub-branch in cluster “1b” signifying their quite compositional resemblance. It should

344

be noted that overall HCA analysis failed to provide clear discrimination between the

345

specimens of the two genera Lens and Lupinus.

346

PCA model, another unsupervised ordination method but with different graphical

347

representation, was generated using same data matrix for discrimination between the different

348

seed accessions. The established model (Fig. 2-B & C) resulted in the formation of two

349

orthogonal PCs, which accounted for 53% of the total variance using only the first two

350

components, i.e., PC1, accounted for 36% of the variance versus 17% for PC2. The PC1/PC2

351

score plot (Fig. 2-B) shows that triplicates of Lens culinaris Medik subsp. culinaris ‘Petite



352

Rouge d’Egypte’ and one of the three replicates of Lupinus hispanicus Boiss. & Reutt. were

353

positioned on the far right side of the plot (positive PC1 values), whereas on the far left side,

354

most of specimens namely “Lens culinaris Medik. subsp. culinaris ‘verte du Puy’ (brown

355

colored), L. culinaris Medik. subsp. culinaris (yellow colored), L. culinaris Medik. subsp.

356

culinaris ‘Black Beluga’ and Lupinus angustifolius” were located (negative PC1 values). The

357

separation observed in PCA can be explained in terms of the annotated metabolites, using the

358

loading plot (Fig 2-C). MS signals for melibiose, having a positive effect on PC1, contributed

359

the most to the discrimination between examined seed accessions which appeared to be more

360

enriched in the LCO lentil samples, Table 1. The separation of Lupinus hispanicus Boiss. &

361

Reutt. samples (Fig. 2-B) along PC2 can be explained in terms of its enrichment in 1H-

362

indole-3-acetamide and lupinine alkaloid, contributing (negatively) to PC2. The segregation

363

observed in the score plot can mostly be ascribed for seeds enrichment in sugars and

364

alkaloids. However, it should be noted that this PCA model was still not efficient in neither

365

capturing the maximal variability embedded within the studied specimens nor discriminating

366

between Lens and Lupinus samples at the genus level.

367

Supervised orthogonal projection to latent structures discriminant analysis (OPLS-DA)

368

was thus employed by modelling all seed accession replicates in one group to build a

369

classification model for achieving better (species) separation. OPLS-DA also has a greater

370

potential in the identification of markers by providing the most relevant variables for the

371

differentiation between two sample groups. The developed model (Fig. S2) showed a better

372

samples separation, though with low R2 (0.279) and Q2 (0.063) values indicating the weak

373

prediction power of this model.


Page 16 of 42

Page 17 of 42


374

OPLS-DA score plot (Fig S2-A) revealed that Lens accession LCO (Table 1) are

375

clustered in one distinct group. Whereas, other three Lens accessions were closely grouped on

376

the left side. Such scattering is attributable to their enrichment in α-D-glucose and sucrose as

377

depicted from their loading plot (Fig. S2-B), and is in agreement with HCA results (Fig. 2-

378

A). The OPLS model suggests that melibiose enrichment can be regarded as a marker for

379

discriminating LCO from the other lentil accessions. Another improvement in samples

380

classification was observed in the dendrogram derived from OPLS analysis (Fig. S2-C),

381

being successful in grouping all the Lupinus species in one cluster “1c”, that is separately

382

from Lens specimens.

383

Another OPLS-DA modelling was performed in which the Lens and Lupinus species

384

were modelled against each other and with the derived score plot showing a clear separation

385

between both samples this time with optimal prediction parameters “R2 (0.9149) and Q2

386

(0.794)”. The derived score plot (Fig. 3-A) depicted a clear discrimination between both

387

species.

388

The observed sample segregation was ascribed to sugars viz. α-D-glucose, D-

389

Glucopyranose, 4-O-[β-D-galactopyranosyl] and α-amyrin enrichment in Lens versus the

390

abundance of citric acid and lupinine alkaloid in Lupinus specimens as revealed from the

391

corresponding S-plot (Fig. 3-B). The alkaloid lupinine can be regarded as a chemotaxonomic

392

marker for Lupinus being almost absent in Lens specimens examined. The positive

393

correlation of citric acid and lupinine in Lupinus accessions suggest that it functions to

394

solubilize it forming a lupinine citrate salt inside the seeds.

395

In conclusion, the subtle compositional heterogeneity and nutritional quality traits of the

396

examined seed accessions of Lens and Lupinus were explicated through a holistic untargeted



397

approach. The study revealed the most nutritionally valuable seed accessions as follows: the

398

two “Old World lupin Species” Lupinus hispanicus Boiss. & Reutt. (due to its rich

399

compositional profile with free amino, organic acids, and nitrogenous compounds viz. indole-

400

3-acetamide with the latter reported anti-inflammatory activity), and L. angustifolius L.

401

‘Boltensia’ with trace levels of the anti-nutrient quinolizidine alkaloids concurrent with a

402

rather moderate level of amino acids and peptides, and highest sucrose levels. With regards to

403

Lens seeds, the two dark colored Lens culinaris cultivars ‘verte du Puy’ and ‘Black Beluga’

404

were found to possess the most abundant peptide levels and “α-amyrin” triterpene, posing

405

them to likely exhibit antioxidant and anti-inflammatory effects and lacking any alkaloids.

406

It should be noted that the only one examined “New World lupin Species” Lupinus

407

polyphyllus Lindl. ‘Russell’ was revealed to possess the highest levels of toxic alkaloids and

408

least nutritive and deprioritizing it for further breeding programs. The presence of such anti-

409

nutrients in large quantities might have a negative impact on seeds palatability and moreover

410

decrease their human consumption, aside from their reported neurotoxicity.

411

Chemometric tools employed herein were found efficient in pointing out to the species-specific

412

metabolite markers, for example triterpene was regarded as being differentiating metabolites for

413

Lens culinaris specimens versus lupinine alkaloid abundance in Lupinus.

414

Differentiation of the “New World lupine Species” L. polyphyllus Lindl. ‘Russell’ from the

415

other three closely related Old World members of the genus Lupinus, was not achieved

416

suggesting that the metabolic differences arising from the geographical distribution might be

417

obscured due to the recent domestication and/or cultivation traits. We do admit that our

418

selection of Lupinus and Lens resources only covers a small subset of their worldwide

419

diversities, but our approach is certainly feasible for analyzing samples from such further

420

sources. Our approach can also be further applied for exploring other factors on legume


Page 18 of 42

Page 19 of 42


421

metabolites composition, as for instance, seasonal variation, processing method and or

422

storage conditions.

423

ACKNOWLEDGMENTS

424

Prof. Mohamed A. Farag wishes to thank the American University of Cairo Research Support

425

Grant (RSG1-18) and Alexander von Humboldt foundation, Germany for the financial

426

support.

427

CONFLICT OF INTEREST

428

Authors declare no actual or potential conflict of interest including any financial, personal or

429

other relationships with other people or organizations.

430

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Page 24 of 42

570

571

Tables & Figures

572

Table 1: Origin of Lens and Lupinus seed accessions used for metabolites analyses. All accessions

573

are permanently stored at the Department of Bioarchaeology at the Austrian Archaeological

574

Institute (ÖAI), and are accessible on request. Seed providers: Botanischer Garten der Universität

575

Basel, Switzerland (BAS), Jardim Botânico da Universidade de Coimbra, Portugal (COI),

576

Hohenheimer Gärten, Germany (HOH), Alpengarten Patscherkofel und Botanischer Garten

577

Innsbruck, Austria (IB), MARAP HandelsgesmbH, Vienna, Austria (MRP), and Vollkraft

Country of

ÖAI

origin

accession

Sample Species and cultivar

Provider

code number LCB

Lens culinaris Medik. subsp. culinaris ‘verte du Puy’ (brown

Austria VKR

2827

colored seed)

LCO

Lens culinaris Medik. subsp. culinaris ‘Petite Rouge d’Egypte’

MRP

Austria

2376

LCY

Lens culinaris Medik. subsp. culinaris (yellow colored seed)

BAS

Switzerland

555

Lens culinaris Medik. subsp. culinaris ‘Black Beluga’

MRP

Austria

2375

LU_PO

Lupinus polyphyllus Lindl. ‘Russell’

HOH

Germany

510

LU_A

Lupinus angustifolius L. ‘Boltensia’

IB

Austria

649

LU_L

Lupinus luteus L.

IB

Austria

1325

LU_H

Lupinus hispanicus Boiss. & Reutt.

COI

Portugal

2872

LCBK

578

Naturnahrung Handels und Produktions GmbH, Grimmenstein, Austria (VKR).


Page 25 of 42


579 580



Page 26 of 42

Table 2: Relative percentile of silylated primary and secondary metabolites in Lens and Lupinus seed accessions as analyzed via GC/MS n= 3. Different letters indicate significant differences between seed specimens according to least significant difference analysis (LSD)

Peak

RT

#

(min)

L.culinaris, KI

Metabolites

brown colored (a)

L. culinaris, Petite Rouge d’Egypte (b)

L.culinaris,

L.culinaris

yellow

Black

colored (c)

Beluga (d)

0.6

0.1

L.polyphyllus (e)

L.angustifolius (f)

L.luteus (g)

L.hispanicus (h)

0.1

0.4

0.1

Acids

G1

5.65

1019

Carbamic acid (TMS) Lactic

G2

6.39

1055

acid(2TMS)oxy -, ester Methyl 2-ethyl

G3

6.66

1067

malonate (TMS)

G4

6.84

1076

G5

9.87

1203

G6

10.77

1238

G7

12.73

1315

Acetic acid (2TMS) Malonic acid

0.3

0.1 (0.01)

b

(0.04)a,c,d,e,f,g, h

(0.6)

b

(0.01)

0.1 b

(0.02)

b

(0.03)

b

b

(0.2)

(0.01)b

0.2

0.3

0.2

0.2

0.2

0.2

0.3

0.2

(0.1)g

(0.02)

(0.1)g

(0.02)g

(0.04)g

(0.0)

(0.2)a,c,d,e,h

(0.01)g

tr.

tr.

tr.

tr.

0.2

tr.

tr.

tr.

e

e

e

e

e

-e

-

-

1.2

0.1 (0.01)

-

b

(1.01)a,c,d,e,f,g, h

b,c,d.f.g.h

-

0.1

(0.1)

0.1

(0.03)

b

(0.01)

0.1 b

-

-

0.1

(0.01)

b

0.2

b

0.1 b

(0)

(0.1)

(0.01)b

0.4

0.2

0.14

0.3

1.8

0.2

0.2

0.6

(0.1)e

(0)e

(0.1)e

(0.1)e

(0.8)a,b,c,d,f,g,h

(0.03)e

(0.04)e

(0.3)e

0.2

2.1

0.3

0.4

0.4

0.3

0.2

0.2

oxy (3TMS)

(0.1)

(0.3)

(0.1)

(0.03)

(0.1)

(0.1)

(0.04)

(0.01)

Succinic acid

0.1

1.0

0.3

0.1

0.2

0.3

0.6

0.5

(2TMS) Carbamic acid,

(2TMS)

(0.04)

b

(0.4)

a,c,d,e,f,g,h

(0.2)

b

(0.01)

b


b

(0.2)

(0.01)

b

b

(0.3)

(0.04)b

Page 27 of 42


G8

12.99

1324

G9

13.64

1350

G10

14.92

1399

Methyl succinic

0.1

0.8

0.1

0.1

0.4

0.1

0.2

0.1

acid (2TMS)

(0.01)b

(0.1)a,c,d,f,h

(0.04)b

(0.01)b

(0.6)

(0)b

(0.2)

(0.02)b

Fumaric acid

tr.

tr.

tr.

tr.

tr.

tr.

tr.

-

-

-

-

-

-

-

-

tr.

tr.

-

-

(2TMS)

-

-

Methylmaleic

tr.

acid (2TMS)

-

tr.

-

-

0.2

0.51

tr.

0.2

tr.

tr.

0.2

0.3

(0.04)

(0.4)e,c,f

-b

(0.01)

-b

-b

(0.1)

(0.1)

tr.

0.1

tr.

0.1

tr.

tr.

0.1

0.1

-

g

-

3.3

1.8

-

3,4G11

15.72

1431

Dihydroxybuta noic acid (3TMS)

G12

16.60

1467

G13

17.08

1486

G14

18.40

1541

G15

18.85

1560

Hydroxymaloni c acid (3TMS) Malic acid (3TMS) L-Threonic acid (3TMS) L-Threonic acid (3TMS) α-

G16

19.19

1574

Hydroxyglutari c acid (3TMS)

G17

19.78

1598

-

(0.01)

1.4 (0.1)

c,f

0.9 e

tr. 0.2 (0.01) tr. -

(0.1)

-

b,g

(0)

1.1 e

-

-

-

(0.3)

1.4 e

(0.2)

e

(1.1)

a,b,c,d,f,g

(0.3)

(0.04)

c,f

0.8 e

(0.5)

(0.01) 2.3

e

(0.3)

tr.

tr.

tr.

tr.

tr.

tr.

-

-

-

-

-

-

0.2

0.3

0.1

0.1

0.1

(0.03)

(0.14)

(0)

0.1 (0.1)

e

c

(0.01)

(0.13)

tr.

tr.

0.1

tr.

0.1

0.1

-

-

(0.02)

-

(0.2)

(0.01)

3-Hydroxy-3-

0.1

0.3

0.1

0.1

0.1

0.1

0.5

0.12

methylglutaric

(0.04)

(0.2)

(0.02)

(0.01)

(0.1)

(0.02)

(0.8)

(0.1)



Page 28 of 42

acid (3TMS) G18

23.11

1748

G19

24.56

1815

Trans-Aconitic acid (3TMS) Citric acid (4TMS)

Total acids

0.1 (0.03)

h

3.8

0.1

-

(0.1)

0.01

(0.5)

(0)

0.1 h

1.6

e,h

(1.4)

e,h

0.1

0.1

(0.02)

(0.1)

(0.03)

3.0

6.7

5.1

(0.1)

b,c,g

(1.4)

h

tr.

0.2

-

(0.03)a,c,f

1.18

(0.01)

(2.03)

e,h

6.9 (0.68)b,c,g

6.9

7.6

4.6

6.3

14

8.5

4.9

11.7

tr.

tr.

1.4

tr.

tr.

tr.

tr.

tr.

-

-

(0.3)f

-

-

-c

-

-

0.5

1.3

0.1

0.1

0.3

0.6

1.2

0.3

(0.4)g

(0.1)c

(0.1)g,b

(0.1)

(0.04)g

(0.5)g

(1.4)a,c,e,f,h

(0.1)g

tr.

tr.

tr.

tr.

tr.

tr.

c

b

Alcohols G20

5.28

1001

G21

7.88

1122

G22

G23

10.88

11.66

1242

1273

1,2-Propanediol (2TMS) Ethylene glycol (2TMS) Diethylene

0.1

tr.

(0.01)a,c,d,e,f,g,

b,c

glycol (2-TMS)

-

Glycerol

0.2

0.1

(3TMS)

(0.1)

(0)

h

a,b,d,f,h

-

b,c

-

b

-

-b,c

-

-

1.1

0.3

0.2

0.3

0.6

1.6

(0.4)

(0.01)

(0.04)

(0.1)

(0.5)

(0.3)

0.4

0.03

0.1

0.1

0.1

0.2

1,2G24

22.02

1698

Propanediol-1phosphate

0.1 (0.03)

c

-

(0.4)

a,e,f,g,h

(0)

c

(0.1)

c

(0.01)

c

(0.2)

c

(0.04)c

(3TMS) GlycerylG25

33.85

2284

glycoside (TMS)

1.4 b,c,f,g,h

(0.2)

0.04 (0)

a,d

0.5 (0.4)

a,d

1.1 (0.01)

b,e,f,g,h


0.1 (0.03)

0.1 a,d

(0)

a,d

0.1 (0.1)

a,d

0.1 (0.01)a,d

Page 29 of 42


Total alcohols

2.2

1.5

3.5

1.9

0.7

1.2

2.0

2.2

Lupinine

-

-

-

-

tr.

tr.

0.2

6.2

-

-

(0.2)

(0.7)

Lupinine

tr.

tr.

h

-

-

h

tr.

tr.

tr.

4.3

7.3

h

-

h

-

h

-

(4.3)

tr.

tr.

tr.

tr.

7.9

tr.

0.1

tr.

-e

-e

-e

-e

(2.2)a,b,c,d,f,g,h

-e

(0.2)e

-e

tr.

tr.

0.3

-

-e

-e

(0.2)a,c,d

-

-

Alkaloids G26

17.08

1476

G27

17.47

1502

G28

32.63

2222

G29

34.99

2342

G30

35.76

2381

isomer α-Isolupanine

-

37.80

2485

(0.8)a,b,d,e,f,g

α-Isolupanine

tr.

isomer

-e

13-Hydroxy-

tr.

tr.

tr.

tr.

10.3

0.1

tr.

tr.

lupanine (TMS)

-e

-e

-e

-e

(3.2)a,b,c,d,f,g,h

(0.1)e

-e

-e

-

-

-

-

0.4

tr.

(0.2)

-

-

-

tr.

tr.

tr.

tr.

18.9

0.2

4.6

13.5

0.2

0.5

-

13-HydroxyG31

h

lupanine (TMS) isomer

Total alkaloids

Amino acids G32

7.42

1103

G33

10.13

1213

L-Alanine, N(2TMS) L-Valine, N(2TMS)

0.3 (0.1)

0.2 h

(0.04)

0.1

tr.

(0.04)

h,d

-

0.3 h

(0.2)

0.3 h

0.1 (0.1)

(0.01)

h

0.1 (0)

b


(0.1)

h

(0.1)

0.2 h

(0.2)

1.2 h

(0.2)a,b,c,d,e,f,g

0.1

0.1

0.1

0.1

(0.03)

(0)

(0.1)

(0.03)b


G34

11.21

1255

G35

11.46

1265

G36

12.14

1291

G37

12.42

1302

G38

14.49

1383

G39

15.48

1422

G40

15.53

1424

G41

17.84

1517

Serine (2TMS) Pipecolic acid

18.44

1543

19.40

1583

G44

19.66

1594

tr.

tr.

tr.

tr.

0.1

0.1

-

(0.2)

-

-

-

-

(0.1)

(0.01)

tr.

tr.

tr.

tr.

0.8

0.1

0.1

0.8

e,h

e,h

e,h

e,h

L-Threonine

Glycine (3TMS)

tr.

tr.

tr.

tr.

0.1

tr.

0.1

g,h

h

-

h

-

h

-

h

b

-

(0.02)a,b,c,d,e,f

0.2

0.2

0.1

0.3

0.2

0.5

(0.01)

(0.2)

(0.2)b,e

0.1 (0.03)

L-Threonine

tr.

(3TMS)

-

l-Aspartic acid

0.4

(2TMS)

(0.1)

L-lysine

tr.

(3TMS)

-

L-Proline, 5oxo-1-(2TMS)

Phenylalanine

L-Asparagine (2TMS) L-Asparagine (2TMS)

f

h

(0) h

(0.1)

(0.1)a,b,c,d,f,g

h

0.2

(0.02)

h

tr.

-

(0.1)

e,h

-

-

-

a,b,c,d,f

-

(0.1)

(TMS) G43

0.3

-

LG42

tr.

(TMS)

(TMS)

Page 30 of 42

(0.1)

(0.02)

(0.04)

tr.

tr.

tr.

tr.

tr.

tr.

tr.

-

-

-

-

-

-

-

tr.

0.7

h,d

(0.3)c,e,f,g

0.2

-

(0.3)

-

0.5 h

(0.02)

0.2 g

(0.2)

0.2 h

(0.02)

h

-

tr.

tr.

tr.

tr.

tr.

0.1

-

-

-

-

-

(0.02)

1.4

0.01

0.3

0.7

0.4

1.7

0.2

0.4

(1.2)

(0)

(0.3)

(0.1)

(0.2)

(0.4)

(0.2)

(0.1)

0.1

tr.

tr.

tr.

tr.

0.5

h

h

h

h

-

(0.4)a,c,d,e,f,g

0.1

0.1

(0.1)

(0.1)b

tr.

0.2

a

(0.3)

0.1 (0.01)

h

0.1 (0.01) 0.2 (0.13)

b,e,g

-

(0.1)

0.6 (0.2)

c,d,e,f,h

0.01 (0)

a

h

-

-

0.1

0.1

b

b

(0)

0.1 (0.1)

(0)

0.1 (0.02)

0.2

tr.

(0.01)

a


-

-

0.1 b

(0.01) -

b

-

Page 31 of 42


G45

20.21

1618

G46

21.30

1666

Glutamic acid (3TMS)-I L-Asparagine (2TMS)

Total amino acids

0.6

-

(0.6) 0.2

-

(0.2)

0.2

1.04

0.2

0.2

(0.2)d

(0.2)c,e,f,h

(0.2)d

(0.2)d

tr.

0.2

-

(0.01)

-

-

tr.

-

-d

tr.

tr.

-

-

3.4

1.2

1.6

3.3

2.2

3.3

0.9

4.8

0.5

0.8

0.1

0.2

0.1

0.2

1

0.2

(0.6)

(0.1)

(0.1)

(0.01)

(0.01)

(0.03)

(0.9)

(0.04)

tr.

tr.

tr.

tr.

tr.

tr.

a

-

a

-

a

-

-

a

a

-

-

0.1

0.2

0.2

0.2

1

0.2

0.5

0.3

0.3

0.4

0.4

Aromatic Benzaldehyde, G47

13.81

1357

4-hydroxy, oxime (2TMS) p-

G48

20.34

1623

Hydroxybenzoi c acid (TMS)

Total aromatics

0.1 (0.04)

-

c,d,e,f,g

0.6

0.8

0.2

0.1

Fatty acids Hexadecanoic G49

29.12

2043

acid (TMS) Linoleic acid

G50

32.11

2199

(TMS) Oleic acid

G51

32.22

2201

(TMS) Stearic acid

G52

32.69

2224

(TMS)

G53

34.36

2310

Oxylipin

(0.04)

(0.01)

0.1 (0.02)

tr. g,h

c,g,h

-

0.2 (0.02)

tr. g,h

0.1 (0.01) -

g,h

g,h

(0.4)

(0.03)

(0.1)

(0)

(0.4)

(0.04)b

0.3

0.2

0.2

0.2

0.3

0.7

b,h

(0.2)

0.5

c,g,h

(0.3)

0.04

0.1

-

(0) -

g,h

0.7

0.2 b

(0.03) -

(0.01)

h

0.3

(0.02)

h

0.1 g,h

(0.01)

(0.03)

g,h

(0.1)

0.3

g,h

0.1 g,h

-


(0.04) 0.4

(0.1)

g,h

(0.1)

g,h

0.1 g,h

(0) -

g,h

b

(0.2)

a,b,e,f

0.4 (0.3)

a,b,e,f

0.2 (0.2)

a,b,c,d,e,f

-

(0.04)a,b,c,d,e,f 0.6 (0.04)a,b,d,e,f 0.3 (0.02)a,b,c,d,e,f -


Page 32 of 42

(0.2) 1Monopalmitin G54

38.27

2508

(TMS)

Total fatty acids

0.1 (0.01)

b

0.6

tr.

tr.

0.1

a,c,d,e

b,f

-

(0)

b

0.2

1.4

0.9

1.3

tr.

0.5

2.2

0.5

-

0.1 (0.02)

0.1 b

tr.

tr.

-

-

1.04

1.4

2.3

1.1

0.4

(0.02)

c

Inorganics Phosphoric acid G55

9.18

1175

(2TMS)

d

(0.2)

(0.6)

(0.3)b

5.8

1.5

2.4

5.7

9.7

(3.4)a,b,d,e,f

(0.2)c,g

(0.8)b,c,g

(0.03)c,h

(7.2)b,d,h

(2)a,b,e,f,g

0.1

4.1

8.01

2

3.5

6.03

11.1

0.3

2.3

0.3

0.7

0.3

1.1

0.9

0.6

(2TMS)

(0.3)b

(0.7)a,c,d,e,f,h

(0.3)b

(0.1)b

(0.1)b

(0.3)b

(1.01)

(0.1)b

(3TMS)

0.1

2.2

0.1

0.2

0.2

0.42

0.3

0.3

(0.1)b

(0.4)a,c,d,e,f,g,h

(0.1)b

(0.02)b

(0.12)b

(0.2)b

(0.5)b

(0.02)b

tr.

tr.

tr.

0.1

tr.

tr.

-

d

d

-

d

-

(3TMS) 11.57

1269

phosphate

Total inorganics

d,h

(0.4)

3.7

0.1

3.6

(0.6)c,h

(0)c,e,g,h

4.8

(0.5)

-

d

(0.1)

a,b,c,e,f,g

d,b

d

1.4

(0.3)

monomethyl

G56

1.1

d

Nitrogenous compounds G57

7.17

1091

G58

7.65

1112

G59

10.99

1247

Ethanolamine

hydroxylamine Urea (2TMS) Silanamine

G60

11.40

1262

trimethyl-N(TMS)-N-[2-

d

-

0.1 (0.01)h

-

-

(0.01)

a,c,e,f,h

-

tr. -d

tr.

0.1

0.1

0.2

0.2

0.6

-h

(0)h

(0.04)h

(0.01)h

(0.3)h

(0.2)a,c,d,e,f,g


Page 33 of 42


[(TMS)oxy]eth yl]G61

13.39

1340

G62

13.90

1359

Phenylethanola mine (3TMS) Pipecolinic acid (2TMS) α-

G63

14.56

1385

Aminoisobutyri c acid (3TMS)

G64

19.52

1588

0.1

0.1

0.4 b

(0.1)

(0.1)

a,c,d,e,f,g

(0.1)

0.01

-

(0)

0.1 b,g

(0.1)

-

h

a,c,d,e,f,g,h

(0.02)

0.1 b

b,g

(0.1)

-

0.1 b

22.91

1739

(0.1)

(0.1)

(0.01)

0.01

0.1

(0)

0.2 b

0.1 b

h

(0.01)b,e,g

0.2

(0.02)

b

(0.2)

0.1

a,b,c

(0.01)b

Cadaverine

tr.

0.3

tr.

0.1

tr.

0.1

0.1

0.1

(4TMS)

-b

(0.1)a,c,d,e,f,h

-b

(0.01)b

-b

(0.02)b

(0.1)

(0.01)b

-

-

-

-

-

-

0.2

0.2

1H-Indole-3G65

(0.02)

0.1 b

-

h

0.1 (0.01)

(0.03)

0.2 b

tr.

-

0.1

0.4 (0.1)

0.2 b

acetamide

0.1 (0.02)

(TMS) G66

38.18

2504

Uridine (TMS)

G67

18.03

1525

GABA (3TMS)

Total nitrogenous compounds

(0) 0.6 (0.4) 1.5

(0.1)

0.3 h

5.9

(0.04)

d,f,g,h

0.1 (0.01)

h

0.2 c

(0.02)

0.2 h

(0.1)

9.3 (1.3)e

0.1 c

(0.03)

0.1 c

(0.01)b,c,e

0.3

0.7

0.4

0.3

0.5

1.1

(0.3)

(0.02)

(0.32)

(0.01)

(0.8)

(0.2)

1.2

2.2

1.4

2.6

2.4

12.4

tr.

tr.

tr.

-a,d

-c,e

-a,d

-

-

-

0.5

1.3

0.7

0.5

0.3

0.2

Peptides G68

17.73

1513

G69

18.15

1531

Cys-Gly

tr.

(3TMS)

-c,e

Glycyl-l-

1.1

0.01



glutamic acid

Page 34 of 42

(0.8)

(0)d

(0.5)

(0.1)b

(0.6)

(0)

(0.42)

(0.04)

0.6

tr.

0.4

0.5

tr.

tr.

0.1

0.2

(2TMS) G70

21.82

1689

G71

25.69

1870

Ser-Leu (2TMS)

(0.1)

c,d,g,h

-

(0.3)

a

(0.02)

a,e,g

-

-

(0.04)

a,d

(0.1)a

Ala-Thr

0.1

tr.

0.1

0.2

tr.

tr.

tr.

tr.

(2TMS)

(0.03)

-

(0.1)

(0)

-

-

-

-

1.7

0.1

1.02

1.9

0.7

0.6

0.4

0.4

0.1

tr.

0.1

0.1

tr.

0.1

0.1

0.1

(0.01)b

-a,c,d,g

(0)b

(0.02)b,e,f,g

-d,g

(0.03)d

(0.03)b,d,e

(0.01)

5.8

2.2

3.2

4.1

2.03

0.9

3.3

1.3

(0.3)e,f,g,h

(0.2)

(2.9)

(0.4)

(1.6)a

(0.2)a

(2.9)a

(0.01)a

5.9

2.2

3.3

4.2

2.1

1

3.4

1.3

tr.

0.06

tr.

tr.

tr.

tr.

tr.

0.1

b,h

b,h

-

h

-

h

-

(0.01)a,c,d,f,g

Total peptides Sterols/triterp enes G72

48.63

3036

G73

49.82

3096

β-Sitosterol (TMS) α-Amyrin (TMS)

Total sterols/triterpenes Sugars G74

17.38

1498

G75

17.56

1506

G76

19.29

1579

G77

21.91

1693

L-Threitol

b,h

(4TMS)

-

Meso-Erythritol

tr.

(4TMS)

-

Xylulose

tr.

(4TMS) Xylitol (5TMS)

e

-

tr. -

(0.03)

a,c,d

-

-

0.06

tr.

tr.

0.1

tr.

tr.

0.1

(0.03)

h

-

-

(0.03)

-

-

(0.01)c

-

-

-

0.1

tr.

tr.

e

-e

tr.

tr.

0.03

tr.

tr.

tr.

-

-

(0.02)

-

-

-

-


(0.01)

a,f,g

-

-

Page 35 of 42

G78


22.42

1717

D-Arabitol

tr.

tr.

tr.

tr.

0.5

tr.

tr.

0.1

(5TMS)

-

-

-

-

(0.8)

-

-

(0.02)

tr.

tr.

tr.

tr.

-

-

-

-

2-Keto-lG79

23.09

1747

gluconic acid (5TMS)

tr.

-

-

tr.

-

-

α-DG80

24.63

1818

Glucopyranosid e, methyl

17.03 (1.2)

tr.

e,f,g,h

-

13.3 (3.2)

e,f,g,h

17.6 (0.4)

e,g,h

1.2 (0.8)

4.6

a,c,d

(0.3)

a,c

1.8 (1.8)

1.2

a,c,d

(0.2)a,c,d

(4TMS) G81

24.76

1825

G82

26.67

1923

G83

26.89

1930

G84

26.97

1933

G85

27.16

1943

G86

27.24

1947

G87

27.89

1980

D-Pinitol

1.7

(5TMS)

(0.03)

D-Sorbitol

tr.

(6TMS)

-

D-Sorbitol (6TMS) Fructose oxime (6TMS) Fructose oxime (6TMS) Myo-Inositol (6TMS) DGlucoseoxime

-

-

1.1

2.5

1.2

0.3

1.8

0.7

(0.2)g

(0.1)f,h

(0.3)

(0.04)d,g

(2)c,f,h

(0.1)d,g

tr.

tr.

0.3

tr.

0.6

tr.

-

-

(0.5)

-

(1)

-

0.1

0.02

0.1

0.1

0.2

0.1

0.3

0.1

(0.01)

(0)

(0.03)

(0)

(0.2)

(0.01)

(0.4)

(0.1)

tr.

0.1

0.2

tr.

0.2

0.1

0.1 (0.02)

-

g

0.1

-

(0.01) 0.1 (0.02)

-

f,g

tr.

0.3 b

(0.1)

a,g

-

-

(0)

g

(0.3)

0.1

0.1

0.2

0.4

0.4

0.1

(0.02)

(0)

(0.3)

(0.01)

(0.2)

(0.01)

0.1

0.2

0.1

0.7

0.7

0.3

(0)

f,g

0.1 (0.03)

(0)

f,g

0.2 (0.02)


g

f,g

(0.1)

0.2 (0.2)

-

(0.04)

a,c,d,e,g,h

0.1 (0)

(0.2)

(0.4)

a,d,e

a,c,e,f,h

0.2

(0.01)

(0.01)f,g tr.

b

(0.2)

-


Page 36 of 42

(6TMS) G88

28.05

1988

G89

28.43

2008

D-Gluconic acid (6TMS) Glucose oxime (6TMS)

0.1

tr. -

b,f

a,d,g

-

-

-

(0.02) 0.1 (0.02)

0.04

h

g,h

(0.03)

tr.

tr.

-

0.1 h

28.99

2040

methyloxime

-

29.81

2079

G92

34.89

2337

Myo-Inositol (6TMS)

35.41

2362

0.01 (0)

2478

39.00

2546

40.66

2630

(0.2)

e

(0.1)

-

a

-

-

0.6

2.1

(0.3)

a,c,d,h

(0.1)

0.1

tr.

b,h

(0.1) tr.

2.2 (1.1)

-g

-

-

c,h

(0.03)a,b,c,e,g

3.1 h

(0.1)e,f,g

0.2

0.1

tr.

0.2

0.1

acid (5TMS)

(0.01)

(0.1)

(0.1)

(0.1)

(0.01)

-

(0.3)

(0.04)

0.2

tr.

tr.

0.1

tr.

0.1

0.1

a,d,e,h

d

a,d

(0.1)

(0)b,d

phosphate

(0.01)

b,c,d,e,f

-

-

0.3 (0.01)a,b,c,e.f, h

(0.02)

a,b,d

-

0.2

0.2

0.2

0.3

0.1

0.1

0.1

0.1

(0.1)e,f,h

(0.1)h

(0.02)

(0)e,f,g,h

(0.02)a,d

(0.03)a,d

(0.03)d

(0.02)a,b,d

Sucrose

20.1

0.1

23.6

17

15.8

40.2

14.5

8.5

(8TMS)

(2.1)

(0.03)f

(4.3)

(0.1)

(5.1)

(0.8)b

(11.5)

(0.5)

6.5

1.1

7.7

8.04

2.5

1.2

1.3

1.8

Mannobiose

DG96

2.7

tr.

(0.2)

0.2

(8TMS) G95

(0.5)

e,f

0.1

0.7

b,h

0.2

3-α37.66

3.01

(0.02)

0.1 h

0.6

(7TMS)

G94

(0.1)

-

e

b

0.1 h

D-Glucuronic

D-Myo-Inositol G93

2.6

(0.01)

(0.01)

(8TMS) G91

(0) 0.1

Maltose G90

0.1

Glucopyranose, 4-O-(4TMS)-β-

b,d,e,f,g,h

(1.1)

(0.4)

a,e,f,g,h

(1)

e,d,f,g,h

(0.1)

a,c,e


(1.1)

a,b,c,d

(0.4)

a,b,c,d

(0.9)

a,b,c,d

(0.4)a,b,c,d

Page 37 of 42


Dgalactopyranos yl] (4TMS) G97

41.81

2689

G98

42.12

2704

G99

42.43

2719

G100

43.11

2755

G101

43.29

2783

G102

43.80

2790

G103

45.76

2889

G104

49.31

3070

G105

49.36

3085

Total sugars

Galactinol (6TMS) D-Turanose (7TMS) Galactinol (9TMS) D-Lactose (8TMS) D-Lactose (8TMS) Galactinol (9TMS) Melibiose (8TMS) Galactinol

1.7

1.3

(0.3)

g

2.1

(0.5)

0.1

0.7

(0.04)

(0.6)

c,e,f

1.4

2.4

f,g,h

(0.1)

(0.03)

tr.

0.1

f,g,h

0.4

(0.6)

(0.2)

0.1

b

-

(0.01)

(0.01)

b

0.5

c,d

(0.2)

0.9

a,c,d

(0.2)c,d

tr.

0.1

0.1

b

-

(0.01)

(0.02)

0.2

1.9

0.3

0.3

0.4

0.3

0.2

0.2

(0.1)b

(0.9)a,c,d,e,f,g,h

(0.02)b

(0.03)b

(0.2)b

(0.1)b

(0.04)b

(0.1)b

0.3

3

0.3

0.3

0.5

4.7

3.1

1.6

(0.1)

(1.8)

(0.01)

(0.02)

(0.3)

(0.6)

(1.5)

(0.2)

0.5

3.2

0.6

0.5

0.5

4.8

3.1

1.6

(0.1)

(2)

(0.04)

(0.03)

(0.3)

(0.5)

(1.5)

(0.2)

0.7

2.5

0.9

0.6

0.3

0.9

2.1

1.3

(0.3)b

(1.4)a,c,d,e,f,h

(0.1)b

(0.02)b

(0.1)b

(0.1)b

(1.8)

(0.2)b

1

45.9

7.7

0.6

6.2

0.5

17.7

b

(0.2)

(3.1)

a,c,d,e,f,g

(7.4)

b

(0.1)

b

(9.03)

b

b

(0.1)

(15.9)

1.3 b

(0.4)

2.4

10.1

2.9

2.8

3.1

2.9

6.04

2.6

(9TMS)

(2.4)

(1.2)

(1.7)

(1.8)

(3.8)

(0.7)

(2.9)

(0.5)

Unknown

15.7

10.1

14.8

13.9

20.6

13.3

15.9

disaccharide

(2.02) 72.4

b

(6)

a,c,d,e,f,g,h

80.3

(1.4)

b

79.1

(0.8)

b

71

Unknowns


b

(5.5)

b

(1.4)

(12.3)

56.6

78.04

73.1

13.5 b

(11.1)b 40.2


G106

13.07

1328

Unknown

G107

22.53

1721

Unknown

0.1

tr.

0.1

tr.

tr.

tr.

tr.

-

(0.03)d

-

(0.01)b,e,f,g,h

-d

-d

-d

-d

tr.

0.03

-

(0)

-

-

-

Total

a

tr.

tr.

Total unknowns

Page 38 of 42

-

tr. -

0.1

0.1

0.1

0.1

tr.

tr.

tr.

0.1

100

100

100

100

100

100

100

100

(LCB) Lens culinaris Medik subsp. culinaris “verte du Puy” brown colored seed; (LCO) L. culinaris Medik subsp. culinaris “Petite Rouge d’Egypte”; (LCY) L.

culinaris Medik subsp. culinaris yellow colored seed; (LCBK) L. culinaris Medik subsp. culinaris “Black Beluga”; (LU_PO) Lupinus polyphyllus Lindl “Russell”; (LU_A) L. angustifolius L. “Boltensia”; (LU_L) L. luteus L.; (LU_H) L. hispanicus Boiss & Reutt. b

-

(-): absent. c Tr.: Present at trace level < 0.05. Bold numbers: indicate the highest percentile levels of the different seed metabolites. Results are average of 3 independent biological replicates ± (std. deviation).

a–h Different letters indicate significant differences between seed specimens according to least significant difference analysis (P < .05; Tukey's test).


Page 39 of 42

581


Figure Captions

582 583

Figure 1: Major metabolite classes’ percentile levels in different Lens and Lupinus seeds as listed in Table 1 analyzed using GC/MS post silylation.

584 585 586 587 588

Figure 2: GC/MS based unsupervised hierarchical clustering and principal component analyses (PCA) of metabolites found in Lens and Lupinus seeds. (A) HCA plot (B) PCA score plot of PC1 vs. PC2 scores. (C) Loading plot for PC1 & PC2 contributing primary metabolites and their assignments. The metabolome clusters are located at the distinct positions in two-dimensional space described by the principal component 1 (PC1) = 36% and PC2 = 17%.

589 590 591 592

(LCB) Lens culinaris Medik subsp. culinaris “verte du Puy” brown colored seed; (LCO) L. culinaris Medik subsp. culinaris “Petite Rouge d’Egypte”; (LCY) L. culinaris Medik subsp. Culinaris yellow colored seed; (LCBK) L. culinaris Medik subsp. culinaris “Black Beluga”; (LU_PO) Lupinus polyphyllus Lindl “Russell”; (LU_A) L. angustifolius L. “Boltensia”; (LU_L) L. luteus L.; (LU_H) L. hispanicus Boiss & Reutt.

593 594 595 596 597 598

Figure 3: GC/MS based OPLS-DA score plot derived from modelling Lupinus seeds against Lens seeds (A). The S-plot (B) shows the covariance p[1] against the correlation p(cor)[1] of the variables of the discriminating component of the OPLS-DA model. Cut-off values of P

Mass Spectrometry-Based Metabolite Profiling

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