A comprehensive metabolomics and lipidomics atlas for the legumes common bean, chickpea, lentil and lupin.

Legumes represent an important component of human and livestock diets; they are rich in macro- and micronutrients such as proteins, dietary fibers and polyunsaturated fatty acids. Whilst several health-promoting and anti-nutritional properties have been associated with grain content, in-depth metabolomics characterization of major legume species remains elusive. In this article, we used both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) to assess the metabolic diversity in the five legume species commonly grown in Europe, including common bean (Phaseolus vulgaris), chickpea (Cicer arietinum), lentil (Lens culinaris), white lupin (Lupinus albus) and pearl lupin (Lupinus mutabilis), at the tissue level. We were able to detect and quantify over 3400 metabolites covering major nutritional and anti-nutritional compounds. Specifically, the metabolomics atlas includes 224 derivatized metabolites, 2283 specialized metabolites and 923 lipids. The data generated here will serve the community as a basis for future integration to metabolomics-assisted crop breeding and facilitate metabolite-based genome-wide association studies to dissect the genetic and biochemical bases of metabolism in legume species.

Pleistocene diversification of unifoliolate-leaved Lupinus (Leguminosae: Papilionoideae) in Florida.

The importance and prevalence of recent ice-age and post-glacial speciation and species diversification during the Pleistocene across many organismal groups and physiographic settings are well established. However, the extent to which Pleistocene diversification can be attributed to climatic oscillations and their effects on distribution ranges and population structure remains debatable. In this study, we use morphologic, geographic and genetic (RADseq) data to document Pleistocene speciation and intra-specific diversification of the unifoliolate-leaved clade of Florida Lupinus, a small group of species largely restricted to inland and coastal sand ridges across the Florida peninsula and panhandle. Phylogenetic and demographic analyses alongside morphological and geographic evidence suggest that recent speciation and intra-specific divergence within this clade were driven by a combination of non-adaptive allopatric divergence caused by edaphic niche conservatism and opportunities presented by the emergence of new post-glacial sand ridge habitats. These results highlight the central importance of even modest geographic isolation and short periods of allopatric divergence following range expansion in the emergence of new taxa and add to the growing evidence that Pleistocene climatic oscillations may contribute to rapid diversification in a myriad of physiographic settings. Furthermore, our results shed new light on long-standing taxonomic debate surrounding the number of species in the Florida unifoliate Lupinus clade providing support for recognition of five species and a set of intra-specific variants. The important conservation implications for the narrowly restricted, highly endangered species Lupinus aridorum, which we show to be genetically distinct from its sister species Lupinus westianus, are discussed.

The LaCLE35 peptide modifies rootlet density and length in cluster roots of white lupin.

White lupin (lupinus albus L.) forms special bottlebrush-like root structures called cluster roots (CR) when phosphorus is low, to remobilise sparingly soluble phosphates in the soil. The molecular mechanisms that control the CR formation remain unknown. Root development in other plants is regulated by CLE  (CLAVATA3/ EMBRYO SURROUNDING REGION (ESR)-RELATED) peptides, which provide more precise control mechanisms than common phytohormones. This makes these peptides interesting candidates to be involved in CR formation, where fine tuning to environmental factors is required. In this study we present an analysis of CLE peptides in white lupin. The peptides LaCLE35 (RGVHy PSGANPLHN) and LaCLE55 (RRVHy PSCHy PDPLHN) reduced root growth and altered CR in hydroponically cultured white lupins. We demonstrate that rootlet density and rootlet length were locally, but not systemically, impaired by exogenously applied CLE35. The peptide was identified in the xylem sap. The inhibitory effect of CLE35 on root growth was attributed to arrested cell elongation in root tips. Taken together, CLE peptides affect both rootlet density and rootlet length, which are two critical factors for CR formation, and may be involved in fine tuning this peculiar root structure that is present in a few crops and many Proteaceae species, under low phosphorus availability.

Genome-wide association study revealed significant SNPs for anthracnose resistance, seed alkaloids and protein content in white lupin.

White lupin (Lupinus albus L.) is a high-protein grain legume alternative to soybean in Central Europe, but its cultivation is risky due to the fungal disease anthracnose that can cause severe yield damage. In addition, management of seed alkaloids is critical for human nutrition and animal feed. We report on a white lupin collection of genebank accessions, advanced breeding lines and cultivars that was genotyped and phenotypically characterized for anthracnose resistance and seed alkaloids and protein levels. Using genotyping by sequencing (GBS), SeqSNP-targeted GBS, BiomarkX genotyping and Sanger sequencing, a genetic resource of genome-wide SNPs for white lupin was established. We determined anthracnose resistance in two years field trials at four locations with infection rows and measured seed alkaloids and protein levels by near-infrared spectroscopy (NIRS). Few white lupin breeding lines showed anthracnose resistance comparable or better than Celina and Frieda, currently the best commercial cultivars in Germany. NIRS estimates for seed alkaloids and protein levels revealed variation in the white lupin collection. Using genome-wide association studies (GWAS), we identified SNPs significantly associated with anthracnose resistance in the field representing known and new genomic regions. We confirmed the pauper locus and detected new SNP markers significantly associated with seed alkaloids. For the first time, we present loci associated with total grain protein content. Finally, we tested the potential of genomic prediction (GP) in predicting the phenotype of these three quantitative traits. Application of results and resources are discussed in the context of fostering breeding programs for white lupin.

The development of yellow lupin anthers depends on the relationship between jasmonic acid and indole-3-acetic acid.

The main purpose of this study was to demonstrate that the course of anther development, including post-meiotic maturation, dehiscence and senescence, is ensured by the interdependencies between jasmonic acid (JA) and indole-3-acetic acid (IAA) in yellow lupin (Lupinus luteus L.). The concentration of JA peaked during anther dehiscence when IAA level was low, whereas the inverse relationship was specific to anther senescence. Cellular and tissue localization of JA and IAA, in conjunction with broad expression profile for genes involved in biosynthesis, signalling, response, and homeostasis under different conditions, allowed to complete and define the role of studied phytohormones during late anther development, as well as predict events triggered by them. The development/degeneration of septum and anther wall cells, dehydration of epidermis, and rupture of stomium may involve JA signalling, while the formation of secondary thickening in endothecial cell walls is rather JA independent. The IAA is involved in programmed cell death (PCD)-associated processes during anther senescence but does not exclude its participation in the anther dehiscence processes, mainly related to cell disintegration and degeneration. A detailed understanding of these multistage processes, especially at the level of phytohormonal interplay, can contribute to the effective control of male fertility, potentially revolutionizing the breeding of L. luteus.

Selection of adjunct cultures for the ripening of plant cheese analogues.

Fermentation contributes to the taste and odor of plant cheeses. The selection of functional cultures for the fermentation of plant cheeses, however, is in its infancy. This study aimed to select lactic acid bacteria for ripening of soy and lupin cheese analogues. Bacillus velezensis and B. amyloliquefaciens were used for germination of seeds to produce proteolytic enzymes; Lactococcus lactis and Lactiplantibacillus plantarum served as primary acidifying cultures. Levilactobacillus hammesii, Furfurilactobacillus milii, or Lentilactobacillus buchneri were assessed as adjunct cultures for the ripening of plant cheese. Growth of bacilli was inhibited at low pH. Both Lc. lactis and Lp. plantarum were inactived during plant cheese ripening. Cell counts of Lv. hammesii remained stable over 45 d of ripening while Ff. milii and Lt. buchneri grew slowly. Sequencing of full length 16S rRNA genes confirmed that the inocula the plant cheeses accounted for more than 98% of the bacterial communities. HPLC analysis revealed that Lt. buchneri metabolized lactate to acetate and 1,2-propanediol during ripening. Bacilli enhanced proteolysis as measured by quantification of free amino nitrogen, and the release of glutamate. LC-MS/MS analysis quantified kokumi-active dipeptides. The concentrations of γ-Glu-Leu, γ-Glu-Ile, and γ-Glu-Ala, γ-Glu-Cys in unripened cheeses were increased by seed germination but γ-Glu-Phe was degraded. Lt. buchneri but not Lv. hammesii or Ff. milii accumulated γ-Glu-Val, γ-Glu-Ile or γ-Glu-Leu during ripening, indicating strain-specific differences. In conclusion, a consortium of bacilli, acidification cultures and adjunct cultures accumulates taste- and kokumi-active compounds during ripening of plant cheeses.

Quantitation of Lupinus spp. Quinolizidine Alkaloids by qNMR and Accelerated Debittering with a Resin-Based Protocol.

Quinolizidine alkaloids (QAs) are toxic secondary metabolites of the Lupinus species, the presence of which limits the expansion of lupin beans consumption, despite their high protein content. Evaluation of the level of alkaloids in edible Lupinus species is crucial from a food safety point of view. However, quantitation of QAs is complicated by the fact that not all important alkaloids used for quantitation are commercially available. In this context, we developed a method for the simultaneous quantitation of eight major lupin alkaloids using quantitative NMR spectroscopy (qNMR). Quantitation and analysis were performed in 15 different seed extracts of 11 Lupinus spp. some of which belonged to the same species, with different geographical origins and time of harvest, as well as in all aerial parts of L. pilosus. The mature seeds of L. pilosus were found to be a uniquely rich source of multiflorine. Additionally, we developed a protocol using adsorption or ionic resins for easy, fast, and efficient debittering of the lupine seeds. The protocol was applied to L. albus, leading to a decrease of the time required for alkaloids removal as well as water consumption and to a method for QA isolation from the debittering wastewater.

Effect of different oligomerization assemblies of γ-conglutin on its interaction behavior with vitexin.

BACKGROUND: Several different factors underlie the molecular mechanisms of phenolic compound-protein interactions. They include the environmental conditions. In the case of γ-conglutin, pH conditions translate directly into the adoption of two distinct oligomeric assemblies, i.e. hexameric (pH 7.5) or monomeric (pH 4.5). This paper reports research on the pH-dependent oligomerization of γ-conglutin in terms of its ability to form complexes with a model flavonoid (vitexin). RESULTS: Fluorescence-quenching thermodynamic measurements indicate that hydrogen bonds, electrostatic forces, and van der Waals interactions are the main driving forces involved in the complex formation. The interaction turned out to be a spontaneous and exothermic process. Assessment of structural composition (secondary structure changes and arrangement/dynamics of aromatic amino acids), molecular size, and the thermal stability of the different oligomeric forms showed that γ-conglutin in a monomeric state was less affected by vitexin during the interaction. CONCLUSION: The data show precisely how environmental conditions might influence phenolic compound-protein complex formation directly. This knowledge is essential for the preparation of food products containing γ-conglutin. The results can contribute to a better understanding of the detailed fate of this unique health-promoting lupin seed protein after its intake. © 2023 Society of Chemical Industry.

Looking for a Novel Vegan Protein Supplement from Faba Bean, Lupine, and Soybean: a Dietary and Industrial Standpoint.

Global population growth poses a threat to sustainable development. Meanwhile, the use of plant proteins as healthy and sustainable alternatives to animal proteins needs further research. Therefore, this investigation was designed to study the nutritive, structural, and thermal properties of isolated protein fractions from different legumes, i.e., faba bean (FPI), soybean (SPI), and lupine (LPI). As a prospective plant-based protein powder, an equal mixture (MPI) of the three prior legume samples was formulated to study its properties compared to each sole sample. The alkaline extraction and isoelectric precipitation (AE-IP) technique was used for protein isolation. Results showed that all protein isolates had reasonable levels of protein with maximum protein content in SPI (96.15%). The MPI sample, however, came out on top in terms of amino acid profile followed by FBI. Compared to SPI and LPI, it had the highest isoleucine content and higher methionine, valine, leucine, phenylalanine, and lysine. Moreover, MPI showed a median particle charge (-37.1 mV) compared to FPI, SPI, and LPI samples. MPI sample peak showed resistance to heat denaturation at a temperature greater than 200 °C when the DSC test was conducted. With respect to its rheological characteristics, it outperformed the other three protein isolates and exhibited the highest values of storage modulus G' and loss modulus G". Consequently, our study suggests that pulse-derived protein isolate mixture can be used as a unique type of nutritious dietary protein supplement. It could be a good nutritional alternative to proteins derived from animals.

A pan-genome and chromosome-length reference genome of narrow-leafed lupin (Lupinus angustifolius) reveals genomic diversity and insights into key industry and biological traits.

Narrow-leafed lupin (NLL; Lupinus angustifolius) is a key rotational crop for sustainable farming systems, whose grain is high in protein content. It is a gluten-free, non-genetically modified, alternative protein source to soybean (Glycine max) and as such has gained interest as a human food ingredient. Here, we present a chromosome-length reference genome for the species and a pan-genome assembly comprising 55 NLL lines, including Australian and European cultivars, breeding lines and wild accessions. We present the core and variable genes for the species and report on the absence of essential mycorrhizal associated genes. The genome and pan-genomes of NLL and its close relative white lupin (Lupinus albus) are compared. Furthermore, we provide additional evidence supporting LaRAP2-7 as the key alkaloid regulatory gene for NLL and demonstrate the NLL genome is underrepresented in classical NLR disease resistance genes compared to other sequenced legume species. The NLL genomic resources generated here coupled with previously generated RNA sequencing datasets provide new opportunities to fast-track lupin crop improvement.

Auxin triggers pectin modification during rootlet emergence in white lupin.

Emergence of secondary roots through parental tissue is a highly controlled developmental process. Although the model plant Arabidopsis has been useful to uncover the predominant role of auxin in this process, its simple root structure is not representative of how emergence takes place in most plants, which display more complex root anatomy. White lupin is a legume crop producing structures called cluster roots, where closely spaced rootlets emerge synchronously. Rootlet primordia push their way through several cortical cell layers while maintaining the parent root integrity, reflecting more generally the lateral root emergence process in most multilayered species. In this study, we showed that lupin rootlet emergence is associated with an upregulation of cell wall pectin modifying and degrading genes under the active control of auxin. Among them, we identified LaPG3, a polygalacturonase gene typically expressed in cells surrounding the rootlet primordium and we showed that its downregulation delays emergence. Immunolabeling of pectin epitopes and their quantification uncovered a gradual pectin demethylesterification in the emergence zone, which was further enhanced by auxin treatment, revealing a direct hormonal control of cell wall properties. We also report rhamnogalacturonan-I modifications affecting cortical cells that undergo separation as a consequence of primordium outgrowth. In conclusion, we describe a model of how external tissues in front of rootlet primordia display cell wall modifications to allow for the passage of newly formed rootlets.

Quantification of root water uptake and redistribution using neutron imaging: a review and future directions.

Quantifying root water uptake is essential to understanding plant water use and responses to different environmental conditions. However, non-destructive measurement of water transport and related hydraulics in the soil-root system remains a challenge. Neutron imaging, with its high sensitivity to hydrogen, has become an unparalleled tool to visualize and quantify root water uptake in vivo. In combination with isotopes (e.g., deuterated water) and a diffusion-convection model, root water uptake and hydraulic redistribution in root and soil can be quantified. Here, we review recent advances in utilizing neutron imaging to visualize and quantify root water uptake, hydraulic redistribution in roots and soil, and root hydraulic properties of different plant species. Under uniform soil moisture distributions, neutron radiographic studies have shown that water uptake was not uniform along the root and depended on both root type and age. For both tap (e.g., lupine [Lupinus albus L.]) and fibrous (e.g., maize [Zea mays L.]) root systems, water was mainly taken up through lateral roots. In mature maize, the location of water uptake shifted from seminal roots and their laterals to crown/nodal roots and their laterals. Under non-uniform soil moisture distributions, part of the water taken up during the daytime maintained the growth of crown/nodal roots in the upper, drier soil layers. Ultra-fast neutron tomography provides new insights into 3D water movement in soil and roots. We discuss the limitations of using neutron imaging and propose future directions to utilize neutron imaging to advance our understanding of root water uptake and soil-root interactions.

A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium.

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

Phosphorus absorption kinetics and exudation strategies of roots developed by three lupin species to tackle P deficiency.

MAIN CONCLUSION: Different lupin species exhibited varied biomass, P allocation, and physiological responses to P-deprivation. White and yellow lupins had higher carboxylate exudation rates, while blue lupin showed the highest phosphatase activity. White lupin (Lupinus albus) can produce specialized root structures, called cluster roots, which are adapted to low-phosphorus (P) soil. Blue lupin (L. angustifolius) and yellow lupin (L. luteus), which are two close relatives of white lupin, do not produce cluster roots. This study characterized plant responses to nutrient limitation by analyzing biomass accumulation and P distribution, absorption kinetics and root exudation in white, blue, and yellow lupins. Plants were grown in hydroponic culture with (64 µM NaH2PO4) or without P for 31 days. Under P limitation, more biomass was allocated to roots to improve P absorption. Furthermore, the relative growth rate of blue lupin showed the strongest inhibition. Under + P conditions, the plant total-P contents of blue lupin and yellow lupin were higher than that of white lupin. To elucidate the responses of lupins via the perspective of absorption kinetics and secretion analysis, blue and yellow lupins were confirmed to have stronger affinity and absorption capacity for orthophosphate after P-deprivation cultivation, whereas white lupin and yellow lupin had greater ability to secrete organic acids. The exudation of blue lupin had higher acid phosphatase activity. This study elucidated that blue lupin was more sensitive to P-scarcity stress and yellow had the greater tolerance of P-deficient condition than either of the other two lupin species. The three lupin species have evolved different adaptation strategies to cope with P deficiency.

Transcriptomic profiling reveals differences in the adaptation of two Tetragenococcus halophilus strains to a lupine moromi model medium.

BACKGROUND: Tetragenococcus (T.) halophilus is a common member of the microbial consortia of food fermented under high salt conditions. These comprises salty condiments based on soy or lupine beans, fish sauce, shrimp paste and brined anchovies. Within these fermentations this lactic acid bacterium (LAB) is responsible for the formation of lactic and other short chain acids that contribute to the flavor and lower the pH of the product. In this study, we investigated the transcriptomic profile of the two T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine moromi model medium supplied with galactose. To get further insights into which genomic trait is important, we used a setup with two strains. That way we can determine if strain dependent pathways contribute to the overall fitness. These strains differ in the ability to utilize L-arginine, L-aspartate, L-arabinose, D-sorbitol, glycerol, D-lactose or D-melibiose. The lupine moromi model medium is an adapted version of the regular MRS medium supplied with lupine peptone instead of casein peptone and meat extract, to simulate the amino acid availabilities in lupine moromi. RESULTS: The transcriptomic profiles of the T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine peptone-based model media supplied with galactose, used as simulation media for a lupine seasoning sauce fermentation, were compared to the determine potentially important traits. Both strains, have a great overlap in their response to the culture conditions but some strain specific features such as the utilization of glycerol, sorbitol and arginine contribute to the overall fitness of the strain TMW 2.2256. Interestingly, although both strains have two non-identical copies of the tagatose-6P pathway and the Leloir pathway increased under the same conditions, TMW 2.2256 prefers the degradation via the tagatose-6P pathway while TMW 2.2254 does not. Furthermore, TMW 2.2256 shows an increase in pathways required for balancing out the intracellular NADH/NADH+ ratios. CONCLUSIONS: Our study reveals for the first time, that both versions of tagatose-6P pathways encoded in both strains are simultaneously active together with the Leloir pathway and contribute to the degradation of galactose. These findings will help to understand the strain dependent features that might be required for a starter strain in lupine moromi.

Root acid phosphatases and rhizobacteria synergistically enhance white lupin and rice phosphorus acquisition.

The rhizosheath is a belowground area that acts as a communication hub at the root-soil interface to promote water and nutrient acquisition. Certain crops, such as white lupin (Lupinus albus), acquire large amounts of phosphorus (P), owing partially to exudation of acid phosphatases (APases). Plant growth-promoting rhizobacteria also increase soil P availability. However, potential synergistic effects of root APases and rhizosheath-associated microbiota on P acquisition require further research. In this study, we investigated the roles of root purple APases (PAPs) and plant growth-promoting rhizobacteria in rhizosheath formation and P acquisition under conditions of soil drying (SD) and P treatment (+P: soil with P fertilizer; -P: soil without fertilizer). We expressed purple acid phosphatase12 (LaPAP12) in white lupin and rice (Oryza sativa) plants and analyzed the rhizosheath-associated microbiome. Increased or heterologous LaPAP12 expression promoted APase activity and rhizosheath formation, resulting in increased P acquisition mainly under SD-P conditions. It also increased the abundance of members of the genus Bacillus in the rhizosheath-associated microbial communities of white lupin and rice. We isolated a phosphate-solubilizing, auxin-producing Bacillus megaterium strain from the rhizosheath of white lupin and used this to inoculate white lupin and rice plants. Inoculation promoted rhizosheath formation and P acquisition, especially in plants with increased LaPAP12 expression and under SD-P conditions, suggesting a functional role of the bacteria in alleviating P deficit stress via rhizosheath formation. Together, our results suggest a synergistic enhancing effect of LaPAP12 and plant growth-promoting rhizobacteria on rhizosheath formation and P acquisition under SD-P conditions.

Differential patterns of within- and between-population genetically based trait variation in Lupinus angustifolius.

BACKGROUND AND AIMS: Within-population genetic and phenotypic variation play a key role in the development of adaptive responses to environmental change. Between-population variation is also an essential element in assessing the evolutionary potential of species in response to changes in environmental conditions. In this context, common garden experiments are a useful tool to separate the genetic and environmental components of phenotypic variation. We aimed to assess within- and between-population phenotypic variation of Lupinus angustifolius L. in terms of its evolutionary potential to adapt to ongoing climate change. METHODS: We evaluated populations' phenotypic variation of foliar, phenological and reproductive traits with a common garden experiment. Patterns of functional trait variation were assessed with (1) mixed model analyses and coefficients of variation (CVs) with confidence intervals, (2) principal component analyses (PCAs) and (3) correlations between pairs of traits. Analyses were performed at the population level (four populations) and at the latitude level (grouping pairs of populations located in two latitudinal ranges). KEY RESULTS: Phenotypic variation had a significant genetic component associated with a latitudinal pattern. (1) Mixed models found lower specific leaf area, advanced flowering phenology and lower seed production of heavier seeds in southern populations, whereas CV analyses showed lower within-latitude variation especially in phenological and reproductive traits in southern populations. (2) PCAs showed a clearer differentiation of phenotypic variation between latitudes than between populations. (3) Correlation analyses showed a greater number of significant correlations between traits in southern populations (25 vs. 13). CONCLUSIONS: Between-population phenotypic variation was determined by contrasting temperature and drought at different latitude and elevation. Southern populations had differential trait values compatible with adaptations to high temperatures and drought. Moreover, they had lower within-population variation and a greater number of trait correlations probably as a result of these limiting conditions, making them more vulnerable to climate change.

High prevalence of lupin allergy among patients with peanut allergy: Identification of γ-conglutin as major allergen.

BACKGROUND: Lupin is a protein-rich legume with a growing presence in the food market worldwide. With increased consumption, lupin allergy (LA) reports are also rising. Uncertainties exist on the cross-reactivity between peanut and lupin, the allergenic potential of different lupin species, and sensitization patterns among different populations. OBJECTIVE: To evaluate the molecular basis of LA and to determine lupin allergens from 3 different species that may be involved in peanut allergy (PA) cross-reactivity. METHODS: A total of 43 subjects with PA, those with LA, or controls without food allergy were evaluated with skin prick tests (SPTs) and specific IgEs (sIgEs). Lupin-sensitized subjects were offered a lupin oral food challenge (OFC). Immunoblots and enzyme-linked immunosorbent assays were performed on sera from lupin-sensitized subjects. RESULTS: In this study, 44% of the PA subjects were confirmed to have LA by OFC. Anaphylaxis was the most frequent manifestation after lupin consumption, with a minimal eliciting dosage of 1 g lupin flour. There was no difference in lupin sIgE or SPT wheal size between lupin-sensitized and confirmed LA subjects or in the severity of symptoms among confirmed LA subjects. Sera from lupin-sensitized subjects uniformly reacted to all 3 different lupin species. Immunoblotting and enzyme-linked immunosorbent assays revealed immunoglobulin E binding to α- and γ-conglutin in all analyzed sera, whereas α- and ß-conglutin recognition was variable. CONCLUSION: Our findings reveal a high prevalence of LA among PA subjects, emphasizing lupin must be labeled as an allergen in foods. Owing to high variability in lupin-sIgE and lupin-SPT results, LA diagnosis may require OFC. In our population, γ-conglutin is the major allergen of lupin.

Adaptation of high-efficiency CRISPR/Cas9-based multiplex genome editing system in white lupin by using endogenous promoters.

White lupin (Lupinus albus L.) is an important crop with high phosphorus (P) use efficiency; however, technologies for its functional genomic and molecular analyses are limited. Cluster regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been applied to gene editing and function genomics in many crops, but its application in white lupin has not been well documented. Here, we adapted the CRISPR/Cas9-based multiplex genome editing system by using the native U3/U6 and ubiquitin (UBQ) promoters to drive sgRNAs and Cas9. Two target sites (T1 and T2) within the Lalb_Chr05g0223881 gene, encoding a putative trehalase, were selected to validate its efficacy in white lupin based on the Agrobacterium rhizogenes-mediated transformation. We found that the T0 hairy roots were efficiently mutated at T1 and T2 with a frequency of 6.25%-35% and 50%-92.31%, respectively. The mutation types include nucleotide insertion, deletion, substitution, and complicated variant. Simultaneous mutations of the two targets were also observed with a range of 6.25%-35%. The combination of LaU6.6 promoter for sgRNA and LaUBQ12 promoter for Cas9 generated the highest frequency of homozygous/biallelic mutations at 38.46%. In addition, the target-sgRNA sequence also contributes to the editing efficiency of the CRISPR/Cas9 system in white lupin. In conclusion, our results expand the toolbox of the CRISPR/Cas9 system and benefit the basic research in white lupin.

Overexpression of LaGRAS enhances phosphorus acquisition via increased root growth of phosphorus-deficient white lupin.

The GRAS transcription factors play an indispensable role in plant growth and responses to environmental stresses. The GRAS gene family has extensively been explored in various plant species; however, the comprehensive investigation of GRAS genes in white lupin remains insufficient. In this study, bioinformatics analysis of white lupin genome revealed 51 LaGRAS genes distributed into 10 distinct phylogenetic clades. Gene structure analyses revealed that LaGRAS proteins were considerably conserved among the same subfamilies. Notably, 25 segmental duplications and a single tandem duplication showed that segmental duplication was the major driving force for the expansion of GRAS genes in white lupin. Moreover, LaGRAS genes exhibited preferential expression in young cluster root and mature cluster roots and may play key roles in nutrient acquisition, particularly phosphorus (P). To validate this, RT-qPCR analysis of white lupin plants grown under +P (normal P) and -P (P deficiency) conditions elucidated significant differences in the transcript level of GRAS genes. Among them, LaGRAS38 and LaGRAS39 were identified as potential candidates with induced expression in MCR under -P. Additionally, white lupin transgenic hairy root overexpressing OE-LaGRAS38 and OE-LaGRAS39 showed increased root growth, and P concentration in root and leaf compared to those with empty vector control, suggesting their role in P acquisition. We believe this comprehensive analysis of GRAS members in white lupin is a first step in exploring their role in the regulation of root growth, tissue development, and ultimately improving P use efficiency in legume crops under natural environments.