Unlocking the nutritional potential of chickpea: strategies for biofortification and enhanced multinutrient quality

Chickpea (Cicer arietinum L.) is a vital grain legume, offering an excellent balance of protein, carbohydrates, fats, fiber, essential micronutrients, and vitamins that can contribute to addressing the global population’s increasing food and nutritional demands. Chickpea protein offers a balanced source of amino acids with high bioavailability. Moreover, due to its balanced nutrients and affordable price, chickpea is an excellent alternative to animal protein, offering a formidable tool for combating hidden hunger and malnutrition, particularly prevalent in low-income countries. This review examines chickpea’s nutritional profile, encompassing protein, amino acids, carbohydrates, fatty acids, micronutrients, vitamins, antioxidant properties, and bioactive compounds of significance in health and pharmaceutical domains. Emphasis is placed on incorporating chickpeas into diets for their myriad health benefits and nutritional richness, aimed at enhancing human protein and micronutrient nutrition. We discuss advances in plant breeding and genomics that have facilitated the discovery of diverse genotypes and key genomic variants/regions/quantitative trait loci contributing to enhanced macro- and micronutrient contents and other quality parameters. Furthermore, we explore the potential of innovative breeding tools such as CRISPR/Cas9 in enhancing chickpea’s nutritional profile. Envisioning chickpea as a nutritionally smart crop, we endeavor to safeguard food security, combat hunger and malnutrition, and promote dietary diversity within sustainable agrifood systems.


Introduction
Chickpea (Cicer arietinum L.), a nutritionally dense pulse crop, is widely consumed by humans and cultivated annually, predominantly in semiarid and temperate climates under rainfed conditions (Gao et al., 2015).Global chickpea cultivation spans 14.56 million hectares (Mha), producing approximately 15 million tons (Mt) annually (FAOSTAT, 2021).Major producers include India, Australia, Pakistan, Central America, and East Africa (Knights and Hobson, 2016), with India leading production at 11.4 Mt from 9.9 Mha (FAOSTAT, 2021).Chickpea varieties are categorized as 'desi' and 'kabuli' based on seed shape and color (Knights and Hobson, 2016), with desi types predominant in Australia, Central America, East Africa, and India, while kabuli types thrive in the Mediterranean, Middle East, North Africa, and North America (Knights and Hobson, 2016;Grasso et al., 2022).
Global climate change and burgeoning human populations threaten food and nutritional security.Despite increased agricultural productivity, over 820 million people globally suffer from food insecurity, and at least 2 billion face nutritional insecurity (Jha et al., 2024).Approximately 3 billion people in Asia, Africa, and Latin America face micronutrient deficiencies, particularly zinc (Zn) and iron (Fe) (Welch and Graham, 2004;Darnton-Hill et al., 2006), crucial for optimal growth and development (Brown et al., 2002;Welch, 2002).Chickpea, inherently abundant in these micronutrients and vitamins, can help address 'hidden hunger,' particularly among infants and women of childbearing age in lowincome countries (Broughton et al., 2003;Ufaz and Galili, 2008;Burchi et al., 2011).Moreover, chickpea contributes to human disease prevention, including diabetes, hyperlipidemia, kwashiorkor, and anemia (Younis et al., 2015).
Recognizing that staple cereals alone cannot meet diverse micronutrient needs, supplementation with grain legumes like chickpea can provide essential micronutrients, fiber, and low glycemic index foods to combat nutritional insecurity and other health-related problems, particularly diabetes and obesity (Rebello et al., 2014;Ramani et al., 2021;Arya and Kumar, 2022;Gupta et al., 2023a).This review examines chickpea's nutritional components, genetic determinants, and genomic regions contributing to improved nutrition.It also explores how emerging breeding tools and the CRISPR/Cas9 approach could enhance chickpea biofortification to help sustain global food security, address micronutrient deficiencies, mitigate malnutrition, and diversify food resources while supporting modern cropping systems and agricultural sustainability.

Vitamin E
Chickpea oil is particularly rich in tocopherols, with alphatocopherol the highest among pulses, reaching up to 13.7 mg 100 g -1 (Wood and Grusak, 2007;Pittaway et al., 2008).The oil comprises four different forms of tocopherols-alpha, beta, gamma, and delta (Zia-Ul- Haq et al., 2009), with gamma-tocopherol recognized as a natural seed antioxidant (Gul and Egesel, 2008;Boschin and Arnoldi, 2011).The total tocopherol in chickpea oil ranges from 20.69-52.44 mg kg -1 (Gul and Egesel, 2008), with higher amounts of vitamin E in uncooked chickpea (0.82 mg 100 g -1 ) than cooked chickpea (0.35 mg 100 g -1 ) (Wallace et al., 2016).The National Health and Nutrition Examination Survey reported that chickpea consumers intake 10.1 mg day -1 more vitamin E than non-chickpea consumers (Wallace et al., 2016).The significant genetic variability observed for these vitamins underscores the potential of chickpea for addressing nutritional deficiencies, especially in regions where vitamin-related deficiencies are prevalent.

Bioactive compounds
Chickpea is rich in bioactive compounds, including antioxidants, phenolic acids, flavonoids, and condensed tannins, which offer numerous health benefits (Heiras-Palazuelos et al., 2013).These compounds play significant roles in physiological and metabolic processes and contribute to reducing the risk of various diseases.Antioxidants in small quantities prevent the formation of free radicals or reactive oxygen species by retarding the oxidation of unsaturated fats, which are easily oxidized.Desi and kabuli chickpea exhibit varying antioxidant activities (Heiras-Palazuelos et al., 2013).
Phytic acid forms complexes with proteins, impeding the absorption of micronutrients such as Fe, Ca, Zn, Cu, and Mg in the gastrointestinal tract (Tiwari and Singh, 2012).Studies have reported phytic acid levels ranging from 3.49-11.52mg g -1 in desi types and 3.45-12.35mg g -1 in kabuli types (Mondor et al., 2009), 11.33 mg g -1 in whole chickpeas, 11.53 mg g -1 in split chickpeas, and 14 mg g -1 in desi types (Shi et al., 2018), and 9.43-13.67mg g -1 in kabuli types, 8.48-18.39mg g -1 in desi types, and 4.24-8.48mg g -1 in wild species (Kaur et al., 2019).Phytic acid has a negative association with most minerals except Zn; an increase in phytic acid suggests a negative impact on the absorption of these minerals (Kaur et al., 2019).
Trypsin inhibitors hinder digestive enzymes, specifically trypsin and chymotrypsin, affecting the utilization of sulfur amino acids in the body.Genetic variability for trypsin inhibitors in chickpea ranges from 111.5-218.4trypsin inhibitor units (TIU) g -1 (Gupta et al., 2017), 38.53-64.47TIU g -1 in kabuli types, 32.91-112.32TIU g -1 in desi types, and 122.73-150.18TIU g -1 in wild species (Kaur et al., 2019).Thus, minimizing anti-nutrient contents through breeding, genomics, and other innovative approaches could improve the bioavailability of essential micronutrients, vitamins, phosphorus, and other nutrients, enhancing their nutritional values and health benefits (Table 2).
A recombinant inbred lines-based bi-parental mapping population approach using an ICC 4958 × ICC 8261 population identified eight QTLs governing seed Fe and Zn on six chromosomes, explaining a combined 39.4% PVE (Upadhyaya et al., 2016a).Furthermore, genotyping 92 sequenced desi and kabuli accessions with 24,620 SNPs identified 16 genomic loci/ genes contributing to seed Fe and Zn, accounting for a combined 29% PVE (Upadhyaya et al., 2016a).Subsequently, a GWAS on a diverse panel of 147 chickpea genotypes phenotyped for two years and genotyped with an "Axiom ® 50K CicerSNP array" identified 35 significant marker-trait associations (MTAs) contributing to grain Zn, Fe, Cu, and Mn, with five MTAs consistently identified in different environments (stable), six explaining more than 15% of the phenotypic variation (major), and three both stable and major MTAs (Fayaz et al., 2022).Likewise, over two years, SNP204 on LG1 and SNP9478 on LG5 showed significant MTAs for Fe content at Sanliurfa, and SNP8254 and SNP8255 on LG4 showed significant MTAs for Fe content at Bornova (Karaca et al., 2020).A GWAS on 258 chickpea genotypes using 318,644 SNPs derived from whole genome sequencing revealed 62 significant MTAs for 12 important nutritional traits, including crude protein, b-carotene, seed Ca, and folate content, on chromosomes Ca1, Ca3, Ca4, and Ca6, explaining up to 29% PVE (Roorkiwal et al., 2022).A GWAS on a reference set of 280 chickpea genotypes using a 5k SNP array and the FarmCPU and BLINK models identified seven significant SNPs for grain protein, 12 SNPs for Fe, and one SNP for Zn on chromosomes 1, 4, 6, and 7 (Srungarapu et al., 2022b).Another GWAS analysis identified 181 MTAs for grain protein, Zn, and Fe content in 140 diverse chickpea genotypes under non-stress, drought, and heat stress conditions, with 48 and 63 MTAs significantly associated with drought stress and heat stress, respectively (Samineni et al., 2022).Thus, targeting the identified overlapping/common genomic regions controlling these micronutrients for cloning could help elucidate the precise functions of candidate genes associated with nutrient content.

Functional genomics approaches for discovering candidate gene(s) contributing to nutrients
Advances in functional genomics, particularly RNA-seq-based transcriptome assembly, have greatly enhanced our ability to identify trait-based candidate genes in chickpea (Kudapa et al., 2018).For instance, quantitative RT-PCR can be used to profile the expression of candidate genes for SPC.Notably, GWAS revealed higher differential upregulatory expression in high SPC-containing mapping individuals (21.5-22.4%)than in low SPC-containing mapping individuals (15.6-16.5%)during the seed development stage (Upadhyaya et al., 2016b).Similarly, functional analysis of the SPC gene ROP1ENHANCER1, identified through combined QTLseq and candidate gene-based association mapping, demonstrated a significant reduction in SPC when this gene was knocked down in chickpea (Chakraborty et al., 2023).Similarly, Upadhyaya et al. (2016a) used qRT-PCR to explore the functional expression of candidate genes related to grain Fe and Zn, reporting high expression in the seeds of parental chickpea genotypes with high Fe and Zn compared to those with low Fe and Zn.
Furthermore, investigating the potential role of various transporter genes ("FRO2, IRT1, NRAMP3, V1T1, YSL1, FER3, GCN2, and WEE1") in Fe metabolism, Jahan et al. (2023) validated their function in Fe uptake, root and stem translocation, and leaf tissue accumulation.Examining the expression patterns of identified genes related to carotenoid content, Rezaei et al. (2016) analyzed the expression of 19 selected genes associated with the carotenoid biosynthesis pathway in five chickpea cultivars, reporting up-regulatory expression in the CDC Jade cultivar.
Functional genomics advancements will potentially uncover more candidate genes associated with quality traits, offering insights into their precise functions.This knowledge could facilitate the cloning and transfer of these genes to elite chickpea cultivars.

Innovative breeding tools for improving nutritional components
Recent advances in breeding approaches, including genomic selection, speed breeding, and high-throughput phenotyping, offer promising avenues for improving the nutritional components of chickpea and developing nutritionally dense or biofortified genotypes.Genomic selection (GS) can harness high-throughput SNP markers derived from chickpea genomics resources to select progenies with superior genetic merit for various nutritional traits using prediction models trained on a large target population (Meuwissen et al., 2001).Speed breeding protocols can expedite the generation of mapping populations, such as recombinant lines and backcross populations, for mapping various nutritional component QTLs/genes (Watson et al., 2018).Advances in highthroughput phenotyping and non-destructive phenotyping, including hyperspectral imaging, Fourier transform near-infrared imaging, and micro-computed tomography imaging, offer efficient means of assessing nutritional components in chickpea (Hacisalihoglu and Armstrong, 2023).Emerging approaches like artificial intelligence and machine learning tools that use convolutional and deep neural networks could predict nutritional quality and the role of novel genes/pathways associated with various nutritional and anti-nutritional components in chickpea (Tachie et al., 2023).By integrating these innovative breeding tools into chickpea breeding programs, researchers can accelerate the development of nutritionally enhanced varieties, contributing to efforts to combat hunger and improve food security worldwide.

Scope of genome editing for improving nutrient bioavailability
Conventional breeding approaches have significantly increased the global yield and production of chickpea.Before the advent of CRISPR/Cas9 (Wang et al., 2016), other genome editing systems like zinc-finger nucleases (ZFNs) (Urnov et al., 2010), transcription activator-like effector nucleases (TALENs) (Joung and Sander, 2013), and homing endonucleases or meganucleases (Paques and Duchateau, 2007;Daboussi et al., 2015) were used.Zinc-finger nucleases, comprising distinct DNA binding and FokI nuclease DNA cleavage domains, were among the earliest synthetic proteins used for targeted mutagenesis and gene replacement (Li et al., 1992;Carroll, 2011;Davies et al., 2017).However, they suffered from low specificity, limited efficacy, and inability to achieve gene knockout and RNA editing (Wang et al., 2016;Salsman and Dellaire, 2017;Kumar et al., 2022).Similarly, TALENs, which function as nonspecific DNA-cleaving nucleases, tether a restriction nuclease to a DNA-binding protein domain termed TAL effector (Hensel and Kumlehn, 2019) but faced similar drawbacks as ZFNs.Meganucleases, rare cutting enzymes also known as homing endonucleases (Pingoud and Silva, 2007), offered highly specific site cleavage (Khan, 2019) with low cytotoxicity.Despite their efficiency in excising large DNA sequences, challenges in manufacturing and potential off-targeting effects hindered their widespread use (Jin et al., 2016;Kumar et al., 2022).
Recent advances in genome editing, such as base editing and prime editing, further enhance the efficiency and precision of CRISPR/Cas9.Base editing introduces single nucleotide variants into DNA/RNA through programmable base editors (Porto et al., 2020), while prime editing enables insertions, deletions, or base conversions of up to 12 nucleotides without introducing doublestrand breaks (Anzalone et al., 2019;Zong et al., 2022;Ahmad et al., 2023;Zhao et al., 2023).These technologies hold promise for addressing challenges in crop improvement, including developing biofortified chickpea varieties to combat malnutrition and promote global nutritional security.However, overcoming technical hurdles like reliable transformation and regeneration protocols, identifying genomic regions for target traits, and determining the precise metabolic pathways involved (Ku and Ha, 2020) remains crucial for realizing the full potential of genome editing in crop enhancement.

Conclusions and future perspectives
Addressing the challenges of global food security and malnutrition requires concerted efforts to enhance the nutritional quality of crops like chickpea.While progress has been made in increasing chickpea yield, there remains a need to balance this with improved nutritional traits.Thorough studies on the correlation of various quality traits, including carbohydrates, proteins, fats, and micronutrients, are crucial for integrating these factors into efforts aimed at enhancing nutritional characteristics.Recognizing the tradeoff between production and quality traits is crucial, and breeding programs should aim to find the optimal balance to meet both needs.
Using the genetic diversity in chickpea crop wild relatives, landraces, and germplasm resources and leveraging genomic resources such as the chickpea genome sequence and pan-genome assembly can help identify the key genetic determinants/gene(s)/ QTL controlling nutritional traits.Marker-assisted selection can facilitate the transfer of nutrient-dense genomic regions to elite chickpea cultivars (see Figure 2).Furthermore, CRISPR/Cas9 genome editing offers precise editing of genomic regions related to anti-nutrients, enhancing nutrient bioavailability.Efforts to elucidate metabolic pathways associated with quality traits will deepen our understanding of the molecular mechanisms and gene (s) governing these quality traits.Developing improved chickpea cultivars with enhanced nutrition can help meet the rising demand for protein-rich diets and combat malnutrition, contributing to global food and nutrition security, modern cropping system diversity, and agricultural sustainability.

TABLE 1
Genetic variability for seed protein/lipid/carbohydrate content in chickpea from different countries.

TABLE 3
List of QTL/genomic regions contributing to various nutritional components in chickpea.