Lentil (Lens culinaris Medikus) is a nutrient-rich, cool-season food legume produced in dry regions worldwide. Lentil has been cultivated for over 10,000 years and was domesticated in the fertile crescent (Cubero, 1981). Lentil is an herbaceous, self-pollinating diploid with seven chromosomes (2n = 14). Total global production has been increasing over the last two decades (Kaale et al., 2023), with recent production at 6.16 ± 0.47 million tons annually (5-year mean ± standard deviation: 2018-2022), concentrated in Canada (36% of global production), India (22%), Australia (11%), Turkey (6%), and the United States (4%) (FAO, 2024). Production is expected to continue rising in the coming decades to help feed the growing human population, as lentil is high in protein (20 – 25%), rich in carbohydrates (60 – 63%) and many micronutrients, and low in fat (1.5 – 3%) (Johnson et al., 2020; Sen Gupta et al., 2013; Tahir et al., 2011; Thavarajah et al., 2011; Wang and Daun, 2006; Zhang et al., 2014). Lentil seeds contain high concentrations of prebiotic carbohydrates (11 – 25%), which are not directly digested by humans but are fermented in the gastrointestinal tract by beneficial microorganisms, and are associated with a healthy gut microbiome and other health benefits (Beserra et al., 2015; Gibson et al., 2017; Johnson et al., 2021). Lentil prebiotic carbohydrates consist of three basic carbohydrate classes: oligosaccharides (0.9 – 6.1 g/100 g), sugar alcohols (SAs; 0.25 – 1.7 g/100 g), and resistant starch (3.7 – 22.1 g/100 g) (Johnson et al., 2015, 2021). There are two classes of oligosaccharides in lentil: raffinose family oligosaccharides (RFOs; 0.9 – 6.0 g/100 g) and fructooligosaccharides (FOS; 0.06 – 0.09 g/100 g), with RFOs constituting the vast majority (ca. 97 – 99%) of oligosaccharides in lentil ( Table 1 ).

Oligosaccharides and SAs are critical throughout the lifecycle of many plant species, including legumes, as they improve tolerance to abiotic stress, such as high temperatures, drought, saline conditions, and oxidative stress (Benkeblia, 2022; Merchant and Richter, 2011; Yan et al., 2022). Sucrose and other disaccharides also improve tolerance to abiotic stress (Guy et al., 1992; Koster, 1991; Morelli et al., 2003). RFOs, FOS, SAs, and mono- and disaccharides are referred to in this review article as low molecular weight carbohydrates (LMWCs) and are defined in the following section. While the role of LMWCs in stress tolerance has not been explicitly studied in lentil, recent research showed LMWCs in lentil seeds varied significantly across nine environments, with the total LMWC concentration positively correlated with growing season temperature (Johnson et al., 2015). This research suggests that higher temperatures can lead to more LMWC accumulation in seeds, possibly in response to heat or water-deficit stress. Thus, because of its favorable nutrient profile and potential stress tolerance, lentil is a good candidate for adaptation to a changing climate.

Higher temperatures and frequent droughts will put unprecedented pressure on crop production systems within the next century (Coughlan de Perez et al., 2023; Heino et al., 2023; Robinson et al., 2021). This pressure will be intensified by the expanding nutritional needs of the global human population (UN-DESA, 2024). Many have called for wide-ranging efforts to meetthese increasing food and fiber needs without further degrading the environment (Foley et al., 2011; Godfray et al., 2010; Hunter et al., 2017). Understanding how staple crops such as lentil and other pulses cope with heat and drought stress is critical to ensure global food security. Given the importance of LWMCs for stress tolerance in crops, understanding the genetic underpinnings of LMWC biosynthesis is an essential step towards developing new climate change-resilient cultivars. However, the genetic basis of LMWC biosynthesis has not been well characterized in lentil, especially related to stress tolerance. A better understanding of the genetic basis of LMWC biosynthesis will enable breeders to make more targeted selections to hasten the release of stress-tolerant lentil cultivars. The objectives of this review are to i) describe the role of LMWCs in abiotic stress tolerance, ii) summarize current knowledge of the genes involved in LMWC biosynthesis, especially in response to abiotic stress, and iii) demonstrate how LMWCs can be targeted using diverse genomic resources and markers to accelerate lentil breeding efforts for improved stress tolerance.

Plant carbohydrates are generally classified by the type, number, and linkage configuration of monosaccharides bonded to form more complex carbohydrates ( Table 2 ). The monosaccharides glucose, fructose, and galactose are bonded in various configurations to form polymers of increasing complexity or are reduced to form SAs. Sucrose is a disaccharide consisting of one glucose molecule and one fructose molecule; it is the primary carbon transport molecule in plants, is used to synthesize many essential compounds, and helps to mitigate abiotic stress (Huber and Huber, 1996; Kühn et al., 1999; Nägele et al., 2012; Peshev et al., 2013). Other disaccharides such as trehalose and maltose are common in plants; trehalose metabolism is tightly linked with sucrose metabolism (Lunn et al., 2014), and maltose is a starch breakdown product that is important in many aspects of carbon metabolism (Fincher, 1989; Lu and Sharkey, 2006). Oligosaccharides are carbohydrates composed of three to 20 polymerized monosaccharides (Cummings and Stephen, 2007) and have diverse physiological roles in plants, such as carbon storage, stress tolerance, and carbon transport in certain taxa (Hannah et al., 2006; Yan et al., 2022). Among oligosaccharides, RFOs and FOS are the two most abundant classes in plants (Van den Ende, 2013). Sugar alcohols such as sorbitol and mannitol are derived from glucose or fructose by one or more chemical reduction steps and have many functions in plants, including carbon transport and storage and stress tolerance (Dumschott et al., 2017; Loescher and Everard, 2000). Starch is referred to as a high molecular weight carbohydrate and is comprised of two highly polymerized carbohydrates: amylopectin (70 – 85% of starch by weight; degree of polymerization ca. 40 – 50) and amylose (15 – 30% of starch; degree of polymerization ca. 30) (Cummings and Stephen, 2007). Starch is the primary form of carbon storage in plants (MacNeill et al., 2017). Resistant starch is a nutritional term referring to starch that is not readily digested because it is i) bound within a food matrix and physically inaccessible to enzyme activity, ii) inaccessible to enzyme activity due to granule type, especially when raw, iii) recrystallized after cooking and cooling (retrograded), iv) structurally modified, or v) complexed with a lipid (Cummings and Stephen, 2007; Dhital et al., 2016; Gutiérrez and Tovar, 2021).

The biosynthetic pathways of sucrose, oligosaccharides, and SAs have been elucidated in many plant species. These begin with fructose, glucose, or galactose, which are combined or modified to form a variety of more complex or reduced carbohydrates ( Figure 1 ) (Dumschott et al., 2017; Johnson et al., 2020; Sanyal et al., 2023; Singh et al., 2017). Sucrose is synthesized from modified forms of glucose and fructose, i.e., uridine diphosphate glucose and fructose-6-phosphate, in a reaction catalyzed by sucrose phosphate synthase to form sucrose-6-phosphate, which is then converted to sucrose by sucrose phosphatase (Winter and Huber, 2000). The biosynthesis of RFOs typically begins with the formation of galactinol from myo-inositol and uridine diphosphate-galactose, the galactose donor, and is catalyzed by galactinol synthase (GolS or GS) (Peterbauer and Richter, 2001). Raffinose is formed from sucrose, and the galactosyl is transferred from galactinol, catalyzed by raffinose synthase (RafS or RS). Stachyose is formed from raffinose and galactinol (galactosyl donor), and is catalyzed by stachyose synthase (StaS or STS). Verbascose is formed from stachyose and galactinol, and may be catalyzed by verbascose synthase or stachyose synthase (Elango et al., 2022; Lahuta et al., 2010). Biosynthesis of FOS begins with the formation of kestose from sucrose and fructose, and is catalyzed by sucrose:sucrose 1-fructosyl-transferase. Nystose is formed from kestose and fructose, catalyzed by fructan:fructan 1-fructosyltransferase (Singh et al., 2017; Vijn and Smeekens, 1999). SA biosynthesis begins, generally, with either fructose or glucose. Mannitol is formed from fructose-6-phosphate, with two intermediate forms (mannose-6-phosphate and mannitol-1-phosphate), catalyzed by mannose-6-phosphate isomerase, mannose-6-phosphate reductase, and mannitol-1-phosphate phosphatase. Sorbitol is formed from glucose-6-phosphate, with sorbitol-6-phosphate as an intermediate step and is catalyzed by aldose-6-phosphate reductase and sorbitol-6-phosphate phosphatase (Dumschott et al., 2017; Loescher and Everard, 2000). Biosynthetic pathways for LMWCs have not yet been elucidated in lentil because these pathways are common among most flowering plants (Benkeblia, 2022; Loescher and Everard, 2000; Salerno and Curatti, 2003; Sengupta et al., 2015).

Among LMWCs, oligosaccharides and SAs are emerging as key compounds that plants use to manage abiotic stresses, such as extreme temperatures, drought, salinity, and oxidative damage (Cacela and Hincha, 2006; Corbineau et al., 2000; Shimosaka and Ozawa, 2015; Stoyanova et al., 2011) ( Figure 2 ). The main oligosaccharides produced by lentil are RFOs, with only small amounts of FOS produced by these crops (Johnson et al., 2015, 2021) ( Table 1 ).

Much research has been carried out to identify the genes responsible for the biosynthesis of LMWCs in response to abiotic stress. To achieve this, studies have inserted, eliminated, or modified genes responsible for RFO, FOS, and SA biosynthetic pathways in many plant species, with corresponding changes in LMWC concentrations and abiotic stress tolerance (Bie et al., 2012; Egert et al., 2013; Panikulangara et al., 2004; Zhifang and Loescher, 2003).

LMWCs in lentil and other pulse crops have been studied for crop improvement (Johnson et al., 2021; Thavarajah et al., 2022). Within the last decade, several studies have confirmed the genetic basis for variation of RFOs and SAs, suggesting these LMWCs in lentil can be increased with targeted breeding (Johnson et al., 2015, 2021). Broad-sense heritability estimates (H²) for LMWCs in lentil have not been well defined, but Johnson et al. (2021) found values ranged from 0.29 – 0.41 for RFOs and 0.34 – 0.45 for SAs within a diverse population of 143 lentil accessions ( Table 1 ). These values are similar to those reported for other pulse crops: values for RFOs ranged from 0.25 – 0.56 in chickpea (Gangola et al., 2013) and 0.44 – 0.54 in common bean (McPhee et al., 2002). Similarly, Thavarajah et al. (2022) studied the heritability of LMWCs in field peas and found H² values ranging from 0.64 – 0.74 for RFOs and 0.42 – 0.66 for SAs. Such moderate heritability values for LMWCs in lentil and related crops are likely due to the quantitative nature of these diverse traits. These results suggest conventional breeding efforts should be paired with genomic approaches to improve selection accuracy and efficiency to accelerate the development of new stress-tolerant lentil cultivars.

Modern genomic-assisted breeding has improved the quality and quantity of genetic data available to plant breeders to develop more climate change-resilient cultivars. This is a powerful approach because it allows the selection of parents based on higher-resolution genomic data and sophisticated statistical techniques that identify genomic regions associated with desired traits. For example, genome-wide association studies (GWAS) can aid in the identification of genes by first identifying single nucleotide polymorphisms (SNPs) and quantitative trait loci (QTL) associated with specific traits. Confirmed genes, SNPs, and QTL can then be used to improve the accuracy of selecting breeding parents by avoiding selection based solely on phenotypic information. Genomic-assisted breeding is especially useful for complex, quantitative traits that are influenced by environmental factors (Kumar et al., 2016). Thus, breeding programs that utilize genomic-assisted techniques such as GWAS can make more targeted crosses and increase genetic gain more quickly than conventional breeding programs ( Figure 3 ).

While the genomic regions responsible for the biosynthesis of LWMCs in lentil have received limited study, important progress toward gene identification has been made (Johnson et al., 2021; Kannan et al., 2016, 2021). Specifically, Kannan et al. (2016, 2021) identified putative GolS, RafS, and StaS genes from a cDNA library, and SNPs for mannitol and the sum of raffinose and stachyose have been identified using genome-wide association mapping (Johnson et al., 2021). These findings hold promise for continued elucidation of the genetic basis of LMWC biosynthesis in lentil, especially as more diverse genomic resources are characterized. As genomic resources for lentil are built, increased diversity will improve the predictive ability of statistical techniques used to identify candidate genes, and the diversity of potential parents will improve as well. Once QTL or genes involved in LMWC biosynthesis are better understood in lentil, more research will be required to confirm their function. For example, up- or downregulating GolS and RafS genes in lentil will help to confirm gene function, as determined by differences in RFO concentrations. Further, abiotic stress studies should be conducted in coordination with gene studies to improve our understanding of gene function and the concomitant role of LMWCs in stress tolerance in lentil. Specifically, testing crop performance as abiotic stresses are applied to lentil genotypes with up- or downregulating GolS and RafS genes will help to elucidate gene identify and function.

Within the context of a genomic-assisted breeding program, target LMWC concentrations should be developed. Target LMWC concentrations in lentil should consider their potential benefits for both human and plant health, as well as the potential drawbacks of consuming large amounts of oligosaccharides and SAs, which can lead to gastrointestinal distress in certain populations or individuals (Douglas and Sanders, 2008). While concentrations have not been established to maximize benefits for human or plant health, mean RFO (6.11 g/100 g) and SA (1.68 g/100 g) concentrations of nine commercial cultivars field-grown in six countries were below the recommended daily allowance (RDA) values of 7-30 g/day suggested for oligosaccharides (Coussement, 1999; Johnson et al., 2013; Silk et al., 2009). For sensitive individuals, these concentrations may lead to gastrointestinal discomfort but are at the low end of suggested RDA values and will likely not negatively affect most individuals.

Concentrations for LMWCs such as RFOs and SAs in vegetative tissue, which are important to consider given their critical role throughout the crop’s life cycle, are not often measured because most research has focused on the nutrient content and digestibility of lentil seeds. Thus, target concentrations for LMWCs in lentil vegetative tissues will require further study to identify optimal concentrations under different environmental conditions, especially abiotic stress. Any physiological tradeoffs between LMWC biosynthesis and other aspects of carbon metabolism should also be considered when determining target LMWC concentrations.

To make efficient progress toward new stress-tolerant lentil cultivars, future work must rely on diverse genomic resources and recent advances in genomic techniques such as whole-genome sequencing and complimentary statistical analyses. Yet, more research is needed to better understand the genes responsible for LMWC biosynthesis in lentil, as well optimal quantities for both human and plant health. With this information, genomic-assisted breeding techniques can be employed to hasten the development of stress tolerant lentil cultivars for a more food secure future.

Lentil is a nutrient-dense food crop that is well adapted to the challenging growing conditions of the dry regions where it is primarily produced, and it is well positioned to contribute to global food security in the future. However, more research is needed to take better advantage of LMWCs in lentil to improve tolerance to heat and drought stress, thereby addressing the dual threats posed by climate change and a growing human population. Importantly, LMWCs in lentil also provide health benefits to consumers, which is significant for all global consumers. Broadening and characterizing lentil genomic resources are the first steps toward better understanding the genes responsible for LMWC biosynthesis in lentil, followed by confirming gene function as stress response compounds. From this work, diverse genomic resources and genomic-assisted breeding techniques can be leveraged to develop more climate-resilient lentil cultivars based on LMWCs. Thus, by employing genomic-assisted breeding techniques that focus on LMWCs, lentil yield and nutritional quality can be maintained or improved, helping to ensure food security in a changing world.