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Lipid metabolites in seeds of diverse Gossypium accessions: molecular identification of a high oleic mutant allele

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Abstract

Main conclusion

Genetically diverse cottonseeds show altered compositions and spatial distributions of phosphatidylcholines and triacylglycerols. Lipidomics profiling led to the discovery of a novel FAD2 - 1 allele, fad2 - 1D - 1 , resulting in a high oleic phenotype.

The domestication and breeding of cotton for elite, high‐fiber cultivars have led to reduced variation of seed constituents within currently cultivated upland cotton genotypes. However, a recent screen of the genetically diverse U.S. National Cotton Germplasm Collection identified Gossypium accessions with marked differences in seed oil and protein content. Here, several of these accessions representing substantial variation in seed oil content were analyzed for quantitative and spatial differences in lipid compositions by mass spectrometric approaches. Results indicate considerable variation in amount and spatial distribution of pathway metabolites for triacylglycerol biosynthesis in embryos across Gossypium accessions, suggesting that this variation might be exploited by breeders for seed composition traits. By way of example, these lipid metabolite differences led to the identification of a mutant allele of the D-subgenome homolog of the delta-12 desaturase (fad2-1D-1) in a wild accession of G. barbadense that has a high oil and high oleic seed phenotype. This mutation is a 90-bp insertion in the 3′ end of the FAD2-1D coding sequence and a modification of the 3′ end of the gene beyond the coding sequence leading to the introduction of a premature stop codon. Given the large amounts of cottonseed produced around the world that is currently not processed into higher value products, these efforts might be one avenue to raise the overall value of the cotton crop for producers.

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Abbreviations

MALDI:

Matrix assisted laser desorption/ionization

MS:

Mass spectrometry

ESI:

Electron spray ionization

TD‐NMR:

Time-domain 1H nuclear magnetic resonance

PC:

Phosphatidylcholine

TAG:

Triacylglycerol

TLE:

Total lipid extract

P:

Palmitic acid (16:0)

O:

Oleic acid (18:1)

L:

Linoleic acid (18:2)

Numerical designation of lipids indicates number of carbons in acyl chains:

Number of double bonds

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Acknowledgements

This research was supported in part by grants from Cotton Incorporated (Agreement# 08‐395) to screen cotton germplasm. We thank the Hoblitzelle Foundation for the support of MS imaging and cryostat instrumentation. We thank Drs. Vladimir Shulaev and Guido Verbeck, University of North Texas, for ongoing analytical advice. We also thank Drs. Kerstin Strupat and Mari Prieto Conaway of Thermo‐Fisher Scientific for technical support in MS imaging experiments. Patrick Horn was supported through the UNT Doctoral Fellowship program.

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  1. Patrick Horn

    Present address: U.S. Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824, USA

Authors and Affiliations

  1. Department of Biological Sciences, Center for Plant Lipid Research, BioDiscovery Institute, University of North Texas, 1155 Union Circle #305220, Denton, TX, 76203-5217, USA

    Drew Sturtevant, Patrick Horn, Christopher Kennedy & Kent Chapman

  2. USDA/ARS, Southern Plains Agricultural Research Center, College Station, TX, 77845, USA

    Lori Hinze & Richard Percy

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  1. Drew Sturtevant
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Correspondence to Kent Chapman.

Electronic supplementary material

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Supplementary material 1 (DOCX 11 kb)

425_2016_2630_MOESM2_ESM.png

Supplemental Fig. 1. Fatty acid composition from extracts of Gossypium accessions. Relative quantification of fatty acids on a mol % were measuring palmitic (16:0; P), steric (18:0; S), oleic (18:1: O), and linoleic (18:2; L) fatty acids. Measurements were taken in triplicate with 5 seeds each in each replicate (PNG 107 kb)

425_2016_2630_MOESM3_ESM.png

Supplemental Fig. 2. Images of lipid metabolites in cross-sections of embryos (middle) from G. barbadense (GB-0331). Ion maps are generated from MALDI-MS for selected PC (a) and TAG (b) molecular species containing one cyclic fatty acid (either sterculic (19:1) or dihydrosterculic (19:0); malvalic acid, also a cyclic fatty acid in cottonseed cannot be resolved from oleic acid in these analyses and so species with malvalic acid are not shown). The intensity scale in all rows are set to the same values so direct comparison can be made for all PC and TAG molecular species respectively. PC and TAG molecular species are denoted as total number of acyl carbons and number of total double bonds. Images are converted from mol % of class with red as highest relative amount. For orientation, a brightfield image of the longitudinal section prior to matrix application and MS imaging is provided in (c) (PNG 2552 kb)

425_2016_2630_MOESM4_ESM.png

Supplemental Fig. 3. PCR amplification of FAD2-1A and FAD2-1D in G. barbadense accessions. (a) Agarose gel electrophoresis of genomic DNA fragments with gene specific primers of the A-homolog of FAD2-1 in G. barbadense backgrounds. (b) Agarose gel electrophoresis of genomic DNA fragments with gene specific primers for long-read PCR spanning FAD2-1 (5′) + Gorai.013G248700 (3′) in G. barbadense backgrounds.. The long-read PCR product in GB-0331 was ~ 10 Kb, while in Pima-S6 it was ~ 7 Kb. Gene specific primers used for amplification can be found in Supplementary Table 1. (c) Agarose gel electrophoresis of RT-PCR products of FAD2-1D and Actin control common coding sequence gene specific primers in G. barbadense backgrounds. Primers for FAD2-1 were “FAD2-1D common coding seq” (Table S1). Pima-S7 was used here as an additional negative control against GB-0331. (d) Schematic of the long-read primer design spanning FAD2-1D and Gorai.013G248700-D (PNG 746 kb)

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Sturtevant, D., Horn, P., Kennedy, C. et al. Lipid metabolites in seeds of diverse Gossypium accessions: molecular identification of a high oleic mutant allele. Planta 245, 595–610 (2017). https://doi.org/10.1007/s00425-016-2630-3

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