Changes of Seed Weight, Fatty Acid Composition, and Oil and Protein

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Agricultural and Environmental Chemistry

Changes of Seed Weight, Fatty Acid Composition, Oil and Protein Content from Different Peanut FAD2 Genotypes at Different Seed Developmental and Maturation Stages Ming Li Wang, Charles Y. Chen, Brandon Tonnis, David Pinnow, Jerry Davis, YQ An, and Phat Dang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01238 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018

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Journal of Agricultural and Food Chemistry

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Changes of Seed Weight, Fatty Acid Composition, Oil and Protein Content from

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Different Peanut FAD2 Genotypes at Different Seed Developmental and

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Maturation Stages

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Ming Li Wang,*,† Charles Y. Chen,╪ Brandon Tonnis,† David Pinnow,† Jerry Davis,§ Yong-

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Qiang Charles An,£ and Phat Dang,¥

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†

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╪

USDA-ARS, Plant Genetic Resources Conservation Unit, Griffin, Georgia 30223 Department of Crop, Soil and Environmental Sciences, Auburn University, Auburn, Alabama

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36849

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§

Department of Experimental Statistics, University of Georgia, Griffin, Georgia 30223

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£

USDA-ARS, Plant Genetics Research Unit, Donald Danforth Plant Science Center, St. Louis,

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Missouri 63132

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¥

USDA-ARS, National Peanut Research Laboratory, Dawson, Georgia 39842

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*Corresponding author. Tel: 001 770 229 3342. FAX: 001 770 229 3323.

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Email addresses: [email protected] (M.L. Wang), [email protected] (C.Y. Chen),

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[email protected] (B. Tonnis), [email protected] (D. Pinnow, retired),

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[email protected] (J. Davis), [email protected] (Y. An), [email protected] (P. Dang).

ACS Paragon Plus Environment


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ABSTRACT: The level of oleic acid in peanut seed is one of the most important factors in

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determining seed quality and is controlled by two pairs of homeologous genes (FAD2A and

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FAD2B). The genotypes of eight F8 breeding lines were determined as AABB, aaBB, AAbb, and

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aabb by real-time PCR and sequencing. Fresh seeds were collected from five seed

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developmental stages and, after drying, were used for chemical analysis. Our results showed (1)

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as seeds developed, seed weight, oil content, and oleic acid level significantly increased, whereas

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four other fatty acid levels decreased, but protein content and another four fatty acid levels did

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not significantly change. (2) FAD2A/FAD2B significantly affected fatty acid profiles, but not oil

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and protein content. (3) The data were consistent across two years. The variability of seed quality

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traits revealed here will be useful for peanut breeders, farmers, processers, and consumers.

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KEYWORDS: Arachis hypogaea, cultivated peanut, fatty acid desaturase 2 (FAD2) genotype,

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seed developmental stage, seed chemical composition, nuclear magnetic resonance (NMR),

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nitrogen analyzer (NA), gas chromatography (GC)

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INTRODUCTION

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Peanut is one of the most important oilseed crops worldwide. Peanut seeds contain about 50%

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oil, 25% protein, 15% carbohydrates, 2% fiber, 2% ash, and 6% water. They also contain minor

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bioactive compounds such as folates, minerals, vitamin E, resveratrol, and flavonoids which have

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antioxidant activities. Since oil is the major component of peanut seed, the fatty acid composition

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is a key factor for determining the seed quality. Oleic acid (monounsaturated acid, C18:1) is a

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major fatty acid in cultivated peanut seeds with an average of 45%, ranging from 31% to 82%

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(unpublished data from screening 8,800 accessions in the U.S. cultivated peanut collection).

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Consuming peanut oil containing about 80% oleic acid (similar to olive oil containing about 75%

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oleic acid) can benefit human health by lowering cardiovascular disease (CVD) risk by 15%.1

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Peanut seeds containing a high level of oleic acid (monounsaturated acid) and a low level of

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linoleic acid (polyunsaturated acid) are less susceptible to rancidification resulting in a longer

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shelf life of stored peanut or peanut products. Therefore, developing peanut cultivars containing

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high oleic acid is one of the main objectives in peanut breeding programs.

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In most peanut cultivars (Arachis hypogaea, 2n = 4x = 40, AABB), the oleic acid level is

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mainly controlled by two pairs of homeologous genes. These two genes encoding fatty acid

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desaturase enzymes (FAD) are located on genomes A and B, and are designated FAD2A and

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FAD2B, respectively.2,3 The fatty acid desaturase enzyme converts oleic acid (C18:1) into

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linoleic acid (C18:2). The functional mutations in the genes for the enzyme can greatly reduce

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fatty acid desaturase activity which causes the levels of oleic and linoleic acids to increase and

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decrease, respectively.3,4 Sequence analysis of FAD2A and FAD2B genes in the double mutant

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identified a base substitution G448A on genome A and an insertion 442insA on genome B,

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leading to D150N (a missense amino acid substitution from aspartic acid to asparagine) and a



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premature stop codon, respectively. Based on these mutations, there are four homozygous

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genotypes identified: wild type (AABB, no mutation on FAD2A and FD2B), a single mutation on

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FAD2A (aaBB) on genome A, a single mutation on FAD2B (AAbb) on genome B, and a double

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mutation on both FAD2A and FAD2B (aabb). So far, there have been no mutants identified

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naturally which only contained single functional mutation on FAD2B but normal function on

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FAD2A. All of the single mutants (AAbb) on FAD2B were created by the selection of segregation

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progenies from the crosses generated between high oleic acid parent (aabb) and wild type

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(AABB). Different FAD2 genotypes can significantly affect the level of oleic acid in peanut seed.

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Peanut is one of the unique legume species producing pods underground and thus also called

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“groundnut”. When the plant is reaching harvest time (physiological maturity stage), different

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pods harvested from the same plant can be at different seed developmental and maturation stages.

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Based on pod colors, seed development and maturation were described as 14 stages.5 But for

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seed collection purposes, a Peanut Profile Board (jointly described by USDA-ARS, Georgia

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Peanut Commission, and the University of Georgia), classifies pods into six developmental and

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maturation stages (white, yellow 1, yellow 2, orange, brown, and black). In early reports,6-8 some

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seed quality characters (such as sugars, starch, fatty acid composition) at different developmental

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stages had been documented; but comprehensive and in-depth characterization of seed chemical

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composition and its dynamic changes in different FAD2 genotypes over the course of seed

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developmental and maturation stages is not available. This information will be very useful for

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peanut breeders to determine their selection strategies, for peanut farmers to determine their

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harvesting time, for food processors to determine their chosen seed types in food production, and

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for consumers to choose their preferred nutritional products.


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In order to fill this knowledge gap, we grew four peanut FAD2 genotypes for two years. After

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harvesting and high pressure blasting the pod skin, the peanut pods were sorted into five seed

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developmental and maturation stages. The seeds from different developmental stages were used

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for chemical analysis. Therefore, the objectives of this study were to (i) determine the changes in

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seed oil and protein content, seed weight, and fatty acid composition during seed development

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and maturation; (ii) compare the effect of genotype (AABB, aaBB, AAbb, and aabb) in

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FAD2A/FAD2B genes on seed oil and protein content, seed weight, and fatty acid composition;

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(iii) determine the correlations among investigated seed quality traits; (iv) determine whether

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enhancing the level of oleic acid can significantly reduce the oil and protein content.

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MATERIALS AND METHODS Selection of Four Genotypes by Real-time PCR and Sequence Analysis of FAD2A/FAD2B

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Genes. The breeding lines of progeny F8 for developing high oleic acid cultivars from the peanut

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breeding program at Auburn University were first screened by genotyping using real-time PCR.

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Based on the field agronomic performance and real-time PCR screening results, eight lines were

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selected. Then FAD2A/FAD2B genes from these eight lines were amplified by PCR and

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sequenced. Based on the sequence results, these eight lines were classified into four genotypes:

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AA/BB, aa/BB, AA/bb, and aa/bb. Lines 1 and 2 belonged to AA/BB genotype. Lines 3 and 4

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belonged to aa/BB genotype. Lines 5 and 6 belonged to AA/bb genotype. Lines 7 and 8 belonged

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to aa/bb genotype.

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Collection of Peanut Pods under Different Developmental and Maturation Stages. Ten seeds from each of these eight lines were planted in environmentally controlled plots at the USDA-



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ARS, National Peanut Research Laboratory for two years (2015 and 2016). The soil type

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consisted of a Greenville sandy clay loam (fine, kaolinitic, thermic Rhodic Kandiudults). During

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growth, plants were fully irrigated, based on soil moisture sensor measurements (MPS-2,

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Decagon Devices, Pullman, WA). Fungicides, herbicides, insecticides, and soil nutrition

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supplements were applied throughout the growing season in accordance with recommended

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production practices. Single plants were dug out, and pods were harvested by hand. Peanut pods

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were pressure blasted using a lightweight pressure washer (Troy-Bilt 2800-PSI max) with a turbo

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nozzle attachment and sorted into six seed progressive developmental stages (white, yellow 1,

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yellow 2, orange, brown, and black, see Figure 1) based on a modified method originally

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described as hull-scrap method.5 Peanut pods were subjected to mechanical curing until kernel

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moisture was around 10%, using heated air 8º C above ambient temperature.9 After drying,

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peanut pods at five different developmental and maturation stages (the white stage was excluded

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because not enough seeds were collected) were shelled by hand and used for chemical analysis.

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Chemical Analysis of Seed Quality Traits. In total, eleven chemical traits were investigated

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including seed oil and protein content, nine fatty acids, and also 100 seed weight. Oil Content by NMR Analysis. Oil content was quantified by nuclear magnetic resonance

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(NMR) analysis on a Minispec Seed Analyzer (Bruker Optics Inc., Houston, TX, USA)

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following the standard operation procedure. Seed oil and water content were measured, and the

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mass of each measurement was converted to a percentage of the total weight of each sample. All

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samples were measured in triplicate, and the results averaged.

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Fatty Acid Composition by GC Analysis. Fatty acid methyl esters (FAMEs) were prepared

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from seeds by alkaline transmethylation,10 and fatty acid composition was determined using an

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Agilent 7890A GC equipped with a flame ionization detector (FID) and an autosampler. Sample


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preparation, GC operation, and data collection followed the standard methods used by our lab

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routinely.11

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Protein Content by Nitrogen Analyzer. Seed protein content was measured using a Rapid N

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Exceed nitrogen analyzer (Elementar, Hanau, Germany/New Jersey, USA). The sample

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preparation and measurement procedure followed the protocol developed by our laboratory.12

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Total nitrogen content was expressed as a percentage of the sample mass. A protein factor of

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5.46 was used to estimate the protein content in peanut seeds (Protein % = N% x 5.46).

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Seed Weight. In addition to eleven investigated chemical traits, seed weight was also measured

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for each line. Three samples of available seeds from each line were counted and weighed. The

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average weight for each genotype and stage was expressed as grams per 100 seeds.

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Statistical Analysis. Data were recorded by replicates, five developmental stages, and four

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genotypes for two years. An analysis of variance was performed on the data, and means were

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separated using Tukey’s multiple comparison procedure (SAS, 2008, Online Doc® 9.2. Cary, NC:

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SAS Institute Inc.). Correlations between investigated seed traits were determined using Pearson

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correlation coefficients.

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RESULTS AND DISCUSSION Variability of the Investigated Seed Quality Traits. Significant variability was identified in seed

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quality traits among different FAD2 genotypes from seeds collected from different seed

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developmental and maturation stages (Table 1). There was significant variation in 100 seed

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weight with an average of 46.73 g ranging from 19.32 to 83.98 g. There was also significant

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variation in protein and oil content with averages of 23.58% and 47.74% (ranging from 19.84 to



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27.19% and from 37.04 to 53.7%), respectively. Oil content had a wider range of variation than

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protein content. There was significant variation in major fatty acids. Variation in palmitic acid

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(C16:0) ranged from 5.7 to 14.45% with an average 10.41%. Variation in oleic acid (C18:1) and

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linoleic acid (C18:2) ranged from 34.49 to 81.81% and from 2.4 to 43.38% with an average of

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54.45% and 26.08%, respectively. Linoleic acid had a much wider variation than oleic acid.

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There was significant variation in minor fatty acids. Variation in stearic acid (C18:0) ranged

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from 1.15 to 3.04% with an average of 1.97%. Variation in arachidic acid (C20:0) and gadoleic

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acid (C20:1) ranged from 0.83 to 1.51% and from 0.82 to 2.67% with an average of 1.12% and

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1.55%, respectively. There was significant variation in very long-chain (C≥22) fatty acids.

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Variation in behenic acid (C22:0) ranged from 1.92 to 4.02% with an average of 2.79%.

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Variation in lignoceric acid (C24:0) and hexacosanoic acid (C26:0) ranged from 0.87 to 2.70%

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and from 0 to 0.41% with an average of 1.57% and 0.18%, respectively. Peanut seeds contained

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~5% very long-chain saturated fatty acids, but their biological function is still not clear.1

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Changes of Seed Weight, Oil and Protein Content, Fatty Acid Composition at Five Different

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Seed Developmental and Maturation Stages. The results of variability in seed quality traits from

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five seed developmental and maturation stages are listed in Table 2 and shown in Figure 2. As

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seed development proceeded (from Yellow 1 to Black), oil content and seed weight significantly

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increased from 41.84 to 51.29% and from 30.09 to 58.22 g/100 seeds. However, changes in

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protein content (22-24%) from Yellow 1 to Black stages were not statistically significant. In

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general, the protein, oil content, and seed weight results from two years were consistent (Table 2

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and Figure 2A). For major fatty acids, oleic acid level significantly increased (50.53-57.16%)

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whereas linoleic acid level significantly decreased (29.07-23.75%), but palmitic acid level

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remained consistent (10.47-10.59%) from Yellow 1 to Black stages. The results from two years


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were also consistent. During seed development and maturation, as fresh seed weight increased,

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the level of oleic acid increased and the level of linoleic acid decreased. This similar trend was

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also observed in other peanut studies.13 For minor fatty acids, the level of gadoleic acid (C20:1)

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significantly decreased (from 1.96 to 1.30%), but the level of arachidic acid (C20:0) did not have

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a significant change (from 1.06 to 1.21%). There was a difference in the level of stearic acid

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between the two years. For the first year, the level of stearic acid (1.69-2.14%) increased, but its

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level from the second year did not have a significant change (2.02-2.10%). For very long-chain

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fatty acids, both levels of behenic (C22:0) and lignoceric (C24:0) acid significantly decreased

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(3.36-2.36%, 0.32-0.12%), but the level of hexacosanoic acid (C26:0) did not have a significant

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change (0.16-0.21%).

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Changes of Seed Weight, Oil and Protein Content, Fatty Acid Composition among Four

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Different FAD2 Genotypes. The results of variability in seed quality traits from four FAD2

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genotypes are listed in Table 3 and shown in Figure 3. The data from two years did not indicate

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much change in oil and protein content, but highly significant changes in seed weight among the

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four genotypes (Figure 3A). The change orders in seed weight were aaBB (59.73 and 60.78

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g/100 seeds) > AAbb (51.23 and 52.54 g/100 seeds) > aabb (42.99 and 42.07 g/100 seeds) >

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AABB (30.08 and 34.42 g/100seeds). The seed weight can affect the plant yield, but the seed

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number per plant can also contribute to plant yield. The seed weight of the high oleic acid

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genotype (aabb) was significantly higher than the wild type (AABB), but also significantly lower

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than two single mutant genotypes (aaBB and AAbb). The results for oil and protein content from

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two years were slightly different. Results from the first year showed no significant differences in

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oil and protein content among four genotypes. For the second year, the oil content of AAbb

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(49.33%) was slightly higher than the oil content of AABB (47.25%) and aabb (47.95%). The



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protein content of AABB (25.36%) was slightly higher than three other genotypes of aabb

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(23.17%), AAbb (23.78%), and aabb (23.70%). For the differences in oil and protein content

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among the four genotypes from the second year, they were statistically significant, but the

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difference value was only 1-2%. Since the results of oil and protein content from two years were

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not consistent, further experiments are needed. Similar results were also observed in double

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mutant soybean (FAD2-1aabb). The level of oleic acid was enhanced to 80%, whereas the oil

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and protein content was not significantly changed.14

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For major fatty acids (Table 3 and Figure 3B), high oleate double mutant (aabb) had a

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significantly lower amount of palmitic acid (6.26% and 6.49%) than the other three genotypes

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(aaBB 10.27% and 10.86%, AAbb 11.06% and 10.73%, and AABB 13.67% and 13.96%). As

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expected, high oleate double mutant (aabb) had the highest and lowest levels of oleic acid and

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linoleic acid (80.1% and 80.11%, 4.39% and 3.46%) among the four genotypes (aaBB 46.18%

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and 48.57%, 33.45% and 30.63%; AAbb 48.04% and 55.13%, 33.45% and 30.63%; AABB 38.05%

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and 39.54%, 40.73% and 38.72%, respectively). The level of oleate enhancement in the double

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mutant mainly came from the decrease of linoleic acid, but also from the decrease of palmitic

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acid. Similar results were also observed in double mutant soybean (FAD2-1aabb). Compared

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with wild type soybean (FAD2-1AABB), the enhanced level of oleic acid in double mutant were

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mainly from the decrease of palmitic acid (from 12.3 to 7.9%) and linoleic acid (from 54.6 to

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4.2%).14 Additionally, as shown in the second year results, the levels of oleic and linoleic acids

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(55.13% and 25.71%) from single mutant AAbb was significantly higher and lower than the ones

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from single mutant aaBB (48.57% and 30.63%). The differences in the levels of oleic and

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linoleic acids between single mutant aaBB and AAbb may be explained by two reasons. One was

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that the insertion mutation in FAD2B may have a larger effect on reducing desaturase activity


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than the substitution mutation in FAD2A. Another was that initially FAD2B was expressed in a

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higher level than FAD2A in the wild type (AABB) during seed development.

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For minor fatty acids (Table 3 and Figure 3C), the high oleate double mutant (aabb) had higher

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amounts of stearic and arachidic acids (2.09% and 2.46%, 1.14% and 1.30%) than other

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genotypes, especially much higher than the single mutant AAbb (1.42% and 1.57%, 0.93% and

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0.98%). The high oleate double mutant also had a higher amount of gadoleic acid (1.83% and

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1.69%) than wildtype AABB (1.03% and 0.99%) and single mutant aaBB (1.60% and 1.45%), but

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slightly lower than or similar to the single mutant AAbb (1.94% and 1.86%). The results for the

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minor fatty acids from two years were consistent. For very long-chain fatty acids (Table 3 and

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Figure 3D), single mutant aaBB contained a significantly higher amount of behenic acid (3.20%

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and 3.18%) than the three other genotypes (AAbb 2.83% and 2.82%, aabb 2.69% and 2.85%, and

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AABB 2.34% and 2.42%). Two single mutants (aaBB and AAbb) contained significantly higher

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amounts of lignoceric acid (1.88% and 1.83%, 1.90% and 1.83%) and hexacosanoic acid (0.29%

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and 0.27%, 0.33% and 0.30%) than the wild type AABB (1.05% and 1.14%, 0% and 0%) and

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double mutant aabb (1.42% and 1.47%, 0.10% and 0.16%). The results for very long-chain fatty

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acids among four different genotypes from two years were also very consistent.

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Correlations between Investigated Seed Quality Traits. The correlations between twelve

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investigated seed quality traits are shown in Table 4 (but only was the correlation with r ≥ 0.8, p

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< 0.0001 mentioned in the text). Palmitic acid was positively correlated with linoleic acid (r =

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0.95, p < 0.0001) but negatively correlated with oleic acid (r = -0.95, p < 0.0001). Stearic acid

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was positively correlated with arachidic acid (r = 0.91, p < 0.0001). Oleic acid was negatively

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correlated with linoleic acid (r = -0.99, p < 0.0001). Gadoleic and behenic acids were positively

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correlated with lignoceric (r = 0.79, r = 0.83 at p

Changes of Seed Weight, Fatty Acid Composition, and Oil and Protein

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