Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low \u03b2-L-ODAP grass pea lines

Peter M F Emmrich,Martin Rejzek,Lionel Hill,Anne Edwards,Cathie Martin,Abhimanyu Sarkar,Paul Brett,Trevor L Wang,Rob A Field

doi:10.1186/s12870-019-2091-5

Abstract

BackgroundGrass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties.ResultsA plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for β-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled β-L-ODAP) allowing accurate quantification of β-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any β-L-ODAP in these species. The LCMS method was also used to quantify β-L-ODAP accurately in different tissues of grass pea.ConclusionsThe plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and β-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of β-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new ‘gold standard’ for β-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-β-L-ODAP genotypes.

Highlights

Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change
Despite its potential for food security and its 8000-year history of cultivation [7,8,9,10], grass pea remains greatly underutilised, primarily due to its association with the disease neurolathyrism [11,12,13,14,15,16,17] caused by the toxin β-N-oxalyl-L-α,βdiaminopropionic acid (β-L-ODAP), known as β-Noxalyl-amino-L-alanine (BOAA) or dencichine, which is produced by Lathyrus sativus and closely related species [18]
This assay is the most widely used [25,26,27,28] because of its low setup and running costs but lacks sensitivity and specificity. All variations of this assay are based on the same reaction of 2,3-diaminopropionic acid with βmercaptoethanol and o-phthalaldehyde (OPA) in the presence of a tetraborate buffer system to form a yellow soluble pigment the concentration of which is measured by spectrophotometry at 420 nm

Summary

Introduction

Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. Grass pea (Lathyrus sativus) is a legume crop with exceptional tolerance to environmental stress factors, in particular drought and flooding [1,2,3] This gives the crop considerable potential for improving food security in water-stressed areas of the world [3, 4]. The first assay developed to measure ODAP relied on a spectrophotometric method [22] which was later adapted by many other researchers [23, 24] This assay is the most widely used [25,26,27,28] because of its low setup and running costs but lacks sensitivity and specificity. Distinguishes between the α - and the β-isomers of ODAP and so they are subject to a slight overestimation of the toxin content since grass pea contains a small amount of the α-isomer and only the β-isomer is the active neurotoxin [29]

Methods

Results

Discussion

Conclusion