Lentils (Lens culinaris) have nutritional, economical, and environmental advantages as an excellent source of protein-based human diet (Kumar et al., 2016). Lentils have twice the level of proteins than cereals and are rich in minerals, polyphenols, and vitamins (Yu et al., 2023). Lentil proteins have been a source of novel food formulations in milk substitute, curd-like products, meat products, extruded products, and baked goods (Boye et al., 2010). Notwithstanding its superb nutritional characteristics, consumption of lentil proteins causes food allergy among some individuals, particularly in pediatric population.

Lentils are the major causes of IgE-mediated allergic reactions in children of the Mediterranean population (Pascual et al., 1999). In Spain, allergy to lentils ranks the fifth most common cause of IgE-mediated food allergy in children (Martínez San Ireneo et al., 2008; Valenta et al., 2015). In Turkey, where most of the populations consume legumes as staple food, lentils are the sixth most common food allergen in the pediatric population as well (Akarsu et al., 2021). Similarly, in India, where legumes are essential protein sources of vegetarian diet, lentils and chickpeas are recognized as key contributing factors to legume allergy (Patil et al., 2001). Three major allergen groups from lentils have been identified. Len c1 is derived from vicilin, the most abundant component of seed storage protein (López-Torrejón et al., 2003). Len c2 is a 66-kDa biotinylated protein (Sánchez-Monge et al., 2000), and Len c3 is a non-specific lipid transfer protein (LTP) of approximately 9 kDa (Akkerdaas et al., 2012). Len c3 was extracted from germinated lentils seeds and verified as immunologically potential allergens using immunoblot analysis (Akkerdaas et al., 2012). LTPs can easily bind to multiple types of lipid molecules such as fatty acids and phospholipids (Shenkarev et al., 2017). Although Len c3 is less abundant compared with Len c1 and Len c2, LTPs are highly cross-reactive and are considered as one of the main plant allergens (Shenkarev et al., 2017; Halima et al., 2022).

While crop breeding and agronomic advances have greatly increased crop yield, food security on a global scale urgently demands improvements in nutritional quality. Natural variations harbor numerous mutations and abundant historical recombination and are cost effective for population development phenotyping and repeated phenotyping (Liang et al., 2021). A major challenge is to identify and utilize the advantageous traits in a breeding program (Gur and Zamir, 2004). Molecular tools for lentil breeding such as genome sequencing and transcriptome profiling are being developed rapidly (Fedoruk et al., 2013; Haile et al., 2020; Song et al., 2022; Yu et al., 2023), which have rendered this relatively facile for identifying the target genes. In this study, we identified the LTP gene that encodes Len c3 in lentil and performed genetic screening via gene sequencing for LTP mutation from a collection of natural lentil accessions. We further conducted transcriptome analysis of developing seeds to investigate gene expression of vicilin in the identified natural variation. Lastly, the identified natural LTP mutation was introduced into two cultivated lines, CDC Redmoon and CDC Gold, to generate an allergen-less germplasm pool.

Allergens in lentil have been a significant concern due to increased consumption in parts of the world (Sackesen et al., 2020). Numerous efforts have been made to identify and qualify lentil allergens, but lentil allergy management through modifications in food systems requires further development (Halima et al., 2022). One goal in crop breeding for better seed nutrition quality is to reduce levels of anti-nutritional factors, such as allergens (Song et al., 2022). In this study, we took a non-GMO approach, focusing on exploring natural variations to search for lentil accessions that harbor natural mutated allergen-encoding genes.

Up to date, three major allergens are known in lentil seeds. Although Len c3 is a rather minor component of lentils’ seed protein portfolio, its interacting IgE is detected in 9 out of 10 patients’ sera, indicating its prevalence in invoking allergic responses (Akkerdaas et al., 2012). LTPs are a group of plant proteins initially defined by their ability to bind polar lipids in a non-specific manner in vitro (Salminen et al., 2016). Voluminous literature indicates that sensitization to LTPs can lead to cross-reaction to homologous food allergens (Rial and Sastre, 2018). From a plant productivity point of view, LTPs represent a minuscule portion of total seed proteins in lentils. Hence, eliminating LTPs will not affect seed protein yield or nutritional profiles. In this study, Len c3-encoding gene LcLTP3b was first identified and used as a target for genetic screening to identify natural mutations in a large collection of natural accessions ( Figures 1A, B ). The screening process identified one natural variation, named M11, harboring mutation at LcLTP3b, naming lcltp3b ( Figure 1C ). A previous study has shown that mutations at Thr41, Arg45, and/or Tyr80 significantly affect the ligand-binding capacity and the allergenic potential of Len c3 (Melnikova et al., 2021). The Lcu.2RBY.4g013600 gene encodes a functional 118-aa (amino acid) protein, while the mutated gene only encodes a 56-aa abnormal protein with substitutions at sites 40/45 ( Figure 1D ), suggesting that the mutation of Lcu.2RBY.4g013600 reduces allergens in M11. Moreover, the identified lentil variation M11 has a much lower vicinlin (7S seed storage protein) level, which might be due to a lower transcription activity ( Figures 1F , 2 ) compared to two commercial cultivars, CDC Redmoon and CDC Gold. Taken together, the genetic evidence suggests that M11 appear to produce seeds containing less allergens.

For breeding purpose, we introduced the mutated allele lcltp3b from M11 into Redmoon and Gold, which are in different genetic backgrounds through genetic crosses. We have obtained lcltp3b homozygous lines from the two crosses, five lines from the Redmoon × M11 crosses and four lines form the Gold × M11 crosses ( Figure 3B ). The F2 generation lines produced seeds varying in seed size, weight, and seed coat color ( Table 1 ). However, these agronomic traits need continuous breeding process to be fixed. It would be useful to develop a haploid inducer line to shorten the stabilization process of these lcltp3b homozygous lines (Gilles et al., 2017; Chen et al., 2023). The lcltp3b homozygous lines are the results of different genetic combinations of M11 and other cultivars; thus, they can be directly used as breeding materials in searching for favorable agronomic traits in in the allergen-less background. The findings of this study reveal the potential of lentil natural variation M11 as a breeding material for reducing allergy risk.

The original contributions presented in the study are included in the article/ Supplementary Materials , further inquiries can be directed to the corresponding authors.

JS: Investigation, Methodology, Writing – original draft. IM: Formal Analysis, Methodology, Software, Writing – review & editing. WS: Methodology, Writing – review & editing. HY: Methodology, Writing – review & editing. JZ: Conceptualization, Project administration, Supervision, Writing – original draft.