Time maters: Exploring the dynamics of bioactive compounds content, bioaccessibility and antioxidant activity during Lupinus angustifolius germination.

Germination is a process that enhances the content of health-promoting secondary metabolites. However, the bioaccessibility of these compounds depends on their stability and solubility throughout the gastrointestinal tract. The study aimed to explore how germination time influences the content and bioaccessibility of γ-aminobutyric acid and polyphenols and antioxidant capacity of lupin (Lupinus angustifolius L.) sprouts during simulated gastrointestinal digestion. Gamma-aminobutyric acid showed a decrease following gastrointestinal digestion (GID) whereas phenolic acids and flavonoids exhibited bioaccessibilities of up to 82.56 and 114.20%, respectively. Although the digestion process affected the profile of phenolic acids and flavonoids, certain isoflavonoids identified in 7-day sprouts (G7) showed resistance to GID. Germination not only favored antioxidant activity but also resulted in germinated samples exhibiting greater antioxidant properties than ungerminated counter parts after GID. Intestinal digests from G7 did not show cytotoxicity in RAW 264.7 macrophages, and notably, they showed an outstanding ability to inhibit the production of reactive oxygen species. This suggests potential benefit in mitigating oxidative stress. These findings contribute to understand the dynamic interplay between bioprocessing and digestion in modulating the bioaccessibility of bioactive compounds in lupin, thereby impacting health.

Characterisation and beneficial effects of a Lupinus angustifolius protein hydrolysate obtained by immobilisation of the enzyme alcalase®.

Bioactive peptides have been considered potential components for the future functional foods and nutraceuticals generation. The enzymatic method of hydrolysis has several advantages compared to those of chemical hydrolysis and fermentation. Despite this fact, the high cost of natural and commercial proteases limits the commercialization of hydrolysates in the food and pharmacological industries. For this reason, more efficient and economically interesting techniques, such as the immobilisation of the enzyme, are gaining attention. In the present study, a new protein hydrolysate from Lupinus angustifolius was generated by enzymatic hydrolysis through the immobilisation of the enzyme alcalase® (imLPH). After the chemical and nutritional characterization of the imLPH, an in vivo study was carried out in order to evaluate the effect of 12 weeks treatment with imLPH on the plasmatic lipid profile and antioxidant status in western-diet-fed apolipoprotein E knockout mice. The immobilisation of alcalase® generated an imLPH with a degree of hydrolysis of 29.71 ± 2.11%. The imLPH was mainly composed of protein (82.50 ± 0.88%) with a high content of glycine/glutamine, arginine, and aspartic acid/asparagine. The imLPH-treatment reduced the amount of abdominal white adipose tissue, total plasma cholesterol, LDL-C, and triglycerides, as well as the cardiovascular risk indexes (CRI) -I, CRI-II, and atherogenic index of plasma. The imLPH-treated mice also showed an increase in the plasma antioxidant capacity. For the first time, this study demonstrates the beneficial in vivo effect of a lupin protein hydrolysate obtained with the alcalase® immobilised and points out this approach as a possible cost-effective solution at the expensive generation of the hydrolysate through the traditional batch conditions with soluble enzymes.

Intercropping of Narrow-Leafed Lupin (Lupinus angustifolius L.) and Barley (Hordeum vulgare L.) Affects the Flavonoid Composition of Both Crops.

Barley (Hordeum vulgare L.) is a common cereal crop in agricultural production and is often included in legume-cereal intercropping. Flavonoids, a major class of secondary metabolites found in barley, are involved in plant defense and protection. However, the effect of intercropping on barley flavonoids remains unknown. Herein, an intercropping system involving barley and lupin (Lupinus angustifolius L.) was studied. Intercropping increased the level of luteolin in lupin roots. Lupin-barley intercropping considerably increased genistein, rutin, and apigenin in barley shoots. Genistein and apigenin were also detected in intercropped barley roots and rhizosphere soil. The three flavonoids have been reported as defense compounds, suggesting that lupin triggers a defense response in barley to strengthen its survival ability.

Lupin as a Source of Bioactive Antioxidant Compounds for Food Products.

Four species of lupin (white lupin, yellow lupin, blue lupin and Andean lupin) are widely cropped thanks to the excellent nutritional composition of their seeds: high protein content (28-48 g/100 g); good lipid content (4.6-13.5 g/100 g, but up to 20.0 g/100 g in Andean lupin), especially unsaturated triacylglycerols; and richness in antioxidant compounds like carotenoids, tocols and phenolics. Particularly relevant is the amount of free phenolics, highly bioaccessible in the small intestine. However, the typical bitter and toxic alkaloids must be eliminated before lupin consumption, hindering its diffusion and affecting its nutritional value. This review summarises the results of recent research in lupin composition for the above-mentioned three classes of antioxidant compounds, both in non-debittered and debittered seeds. Additionally, the influence of technological processes to further increase their nutritional value as well as the effects of food manufacturing on antioxidant content were scrutinised. Lupin has been demonstrated to be an outstanding raw material source, superior to most crops and suitable for manufacturing foods with good antioxidant and nutritional properties. The bioaccessibility of lupin antioxidants after digestion of ready-to-eat products still emerges as a dearth in current research.

Functional Association between Storage Protein Mobilization and Redox Signaling in Narrow-Leafed Lupin (Lupinus angustifolius L.) Seed Germination and Seedling Development.

(1) Background: Seed storage mobilization, together with oxidative metabolism, with the ascorbate-glutathione (AsA-GSH) cycle as a crucial signaling and metabolic functional crossroad, is one of the main regulators of the control of cell morphogenesis and division, a fundamental physiological process driving seed germination and seedling growth. This study aims to characterize the cellular changes, composition, and patterns of the protein mobilization and ROS-dependent gene expression of redox metabolism in Lupinus angustifolius L. (narrow-leafed lupin, NLL) cotyledons during seed germination. (2) Methods: We performed gene expression analyses via RT-qPCR for conglutins α (1, 2, and 3), ß (1, 2, and 5), γ (1, 2), and δ (2 and 4), including a ubiquitin gene as a control, and for redox metabolism-related genes; GADPH was used as a control gene. A microscopic study was developed on cotyledon samples from different germination stages, including as IMB (imbibition), and 2-5, 7, 9, and 11 DAI (days after imbibition), which were processed for light microscopy. SDS-PAGE and immunocytochemistry assays were performed using an anti-ß-conglutin antibody (Agrisera), and an anti-rabbit IgG Daylight 488-conjugated secondary antibody. The controls were made while omitting primary Ab. (3) Results and Discussion: Our results showed that a large amount of seed storage protein (SSP) accumulates in protein bodies (PBs) and mobilizes during germination. Families of conglutins (ß and γ) may play important roles as functional and signaling molecules, beyond the storage function, at intermediate steps of the seed germination process. In this regard, metabolic activities are closely associated with the regulation of oxidative homeostasis through AsA-GSH activities (γ-L-Glutamyl-L-cysteine synthetase, NOS, Catalase, Cu/Zn-SOD, GPx, GR, GS, GsT) after the imbibition of NLL mature seeds, metabolism activation, and dormancy breakage, which are key molecular and regulatory signaling pathways with particular importance in morphogenesis and developmental processes. (4) Conclusions: The knowledge generated in this study provides evidence for the functional changes and cellular tightly regulated events occurring in the NLL seed cotyledon, orchestrated by the oxidative-related metabolic machinery involved in seed germination advancement.

Abscisic acid- and ethylene-induced abscission of yellow lupine flowers is mediated by jasmonates.

The appropriate timing of organ abscission determines plant growth, development, reproductive success, and yield in relation to crop species. Among these, yellow lupine is an example of a crop species that loses many fully developed flowers, which limits the formation of pods with high-protein seeds and affects its economic value. Lupine flower abscission, similarly to the separation of other organs, depends on a complex regulatory network functioning in the cells of the abscission zone (AZ). In the present study, genetic, biochemical, and cellular methods were used to highlight the complexity of the interactions among strong hormonal stimulators of abscission, including abscisic acid (ABA), ethylene, and jasmonates (JAs) precisely in the AZ cells, with all results supporting that the JA-related pathway has an important role in the phytohormonal cross-talk leading to flower abscission in yellow lupine. Based on obtained results, we conclude that ABA and ET have positive influence on JAs biosynthesis and signaling pathway in time-dependent manner. Both phytohormones changes lipoxygenase (LOX) gene expression, affects LOX protein abundance, and JA accumulation in AZ cells. We have also shown that the signaling pathway of JA is highly sensitive to ABA and ET, given the accumulation of COI1 receptor and MYC2 transcription factor in response to these phytohormones. The results presented provide novel information about the JAs-dependent separation of organs and provide insight and details about the phytohormone-related mechanisms of lupine flower abscission.

The causal mutation leading to sweetness in modern white lupin cultivars.

Lupins are high-protein crops that are rapidly gaining interest as hardy alternatives to soybean; however, they accumulate antinutritional alkaloids of the quinolizidine type (QAs). Lupin domestication was enabled by the discovery of genetic loci conferring low QA levels (sweetness), but the precise identity of the underlying genes remains uncertain. We show that pauper, the most common sweet locus in white lupin, encodes an acetyltransferase (AT) unexpectedly involved in the early QA pathway. In pauper plants, a single-nucleotide polymorphism (SNP) strongly impairs AT activity, causing pathway blockage. We corroborate our hypothesis by replicating the pauper chemotype in narrow-leafed lupin via mutagenesis. Our work adds a new dimension to QA biosynthesis and establishes the identity of a lupin sweet gene for the first time, thus facilitating lupin breeding and enabling domestication of other QA-containing legumes.

Regulatory Effects of ABA and GA on the Expression of Conglutin Genes and LAFL Network Genes in Yellow Lupine (Lupinus luteus L.) Seeds.

The maturation of seeds is a process of particular importance both for the plant itself by assuring the survival of the species and for the human population for nutritional and economic reasons. Controlling this process requires a strict coordination of many factors at different levels of the functioning of genetic and hormonal changes as well as cellular organization. One of the most important examples is the transcriptional activity of the LAFL gene regulatory network, which includes LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) and ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEC2 (LEAFY COTYLEDON2), as well as hormonal homeostasis-of abscisic acid (ABA) and gibberellins (GA) in particular. From the nutritional point of view, the key to seed development is the ability of seeds to accumulate large amounts of proteins with different structures and properties. The world's food deficit is mainly related to shortages of protein, and taking into consideration the environmental changes occurring on Earth, it is becoming necessary to search for a way to obtain large amounts of plant-derived protein while maintaining the diversity of its origin. Yellow lupin, whose storage proteins are conglutins, is one of the plant species native to Europe that accumulates large amounts of this nutrient in its seeds. In this article we have shown the key changes occurring in the developing seeds of the yellow-lupin cultivar Taper by means of modern molecular biology techniques, including RNA-seq, chromatographic techniques and quantitative PCR analysis. We identified regulatory genes fundamental to the seed-filling process, as well as genes encoding conglutins. We also investigated how exogenous application of ABA and GA3 affects the expression of LlLEC2, LlABI3, LlFUS3, and genes encoding ß- and δ-conglutins and whether it results in the amount of accumulated seed storage proteins. The research shows that for each species, even related plants, very specific changes can be identified. Thus the analysis and possibility of using such an approach to improve and stabilize yields requires even more detailed and extended research.

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.

Sugar Starvation Disrupts Lipid Breakdown by Inducing Autophagy in Embryonic Axes of Lupin (Lupinus spp.) Germinating Seeds.

Under nutrient deficiency or starvation conditions, the mobilization of storage compounds during seed germination is enhanced to primarily supply respiratory substrates and hence increase the potential of cell survival. Nevertheless, we found that, under sugar starvation conditions in isolated embryonic axes of white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet) cultured in vitro for 96 h, the disruption of lipid breakdown occurs, as was reflected in the higher lipid content in the sugar-starved (-S) than in the sucrose-fed (+S) axes. We postulate that pexophagy (autophagic degradation of the peroxisome-a key organelle in lipid catabolism) is one of the reasons for the disruption in lipid breakdown under starvation conditions. Evidence of pexophagy can be: (i) the higher transcript level of genes encoding proteins of pexophagy machinery, and (ii) the lower content of the peroxisome marker Pex14p and its increase caused by an autophagy inhibitor (concanamycin A) in -S axes in comparison to the +S axes. Additionally, based on ultrastructure observation, we documented that, under sugar starvation conditions lipophagy (autophagic degradation of whole lipid droplets) may also occur but this type of selective autophagy seems to be restricted under starvation conditions. Our results also show that autophagy occurs at the very early stages of plant growth and development, including the cells of embryonic seed organs, and allows cell survival under starvation conditions.

ß-Conglutins' Unique Mobile Arm Is a Key Structural Domain Involved in Molecular Nutraceutical Properties of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Narrow-leafed lupin (NLL; Lupinus angustifolius L.) has multiple nutraceutical properties that may result from unique structural features of ß-conglutin proteins, such as the mobile arm at the N-terminal, a structural domain rich in α-helices. A similar domain has not been found in other vicilin proteins of legume species. We used affinity chromatography to purify recombinant complete and truncated (without the mobile arm domain, tß5 and tß7) forms of NLL ß5 and ß7 conglutin proteins. We then used biochemical and molecular biology techniques in ex vivo and in vitro systems to evaluate their anti-inflammatory activity and antioxidant capacity. The complete ß5 and ß7 conglutin proteins decreased pro-inflammatory mediator levels (e.g., nitric oxide), mRNA expression levels (iNOS, TNFα, IL-1ß), and the protein levels of pro-inflammatory cytokine TNF-α, interleukins (IL-1ß, IL-2, IL-6, IL-8, IL-12, IL-17, IL-27), and other mediators (INFγ, MOP, S-TNF-R1/-R2, and TWEAK), and exerted a regulatory oxidative balance effect in cells as demonstrated in glutathione, catalase, and superoxide dismutase assays. The truncated tß5 and tß7 conglutin proteins did not have these molecular effects. These results suggest that ß5 and ß7 conglutins have potential as functional food components due to their anti-inflammatory and oxidative cell state regulatory properties, and that the mobile arm of NLL ß-conglutin proteins is a key domain in the development of nutraceutical properties, making NLL ß5 and ß7 excellent innovative candidates as functional foods.

Metabolomics and chemometrics depict the changes in the chemical profile of white lupine (Lupinus albus L.) bioactive metabolites during seed germination.

The current study attempts to illustrate how the chemical and biological profile of white lupine seeds varies throughout the course of various germination days using UHPLC-QqQ-MS combined to chemometrics. Abscisic acid showed maximum level in the un-germinated seeds and started to decline with seed germination accompanied by an increase in the levels of gibberellins which were undetectable in un-germinated seeds. Coumaronochromones were the most prevalent constituents detected in un-germinated seeds while day 2 sprouts showed significant accumulation of flavones. The levels of alkaloids showed significant increase upon germination of the seeds reaching its maximum in day 14 sprouts. The OPLS model coefficients plot indicated that lupinalbin D and F, apigenin hexoside, kaempferol hexoside, albine, and hydoxylupanine showed strong positive correlation to the alpha amylase inhibitory activity of the tested samples while lupinalbin A, lupinisoflavone, lupinic acid and multiflorine were positively correlated to the inhibition of alpha glycosidase activity. The results obtained indicated that seed germination has a profound effect on the chemical profile as well as the in-vitro antidiabetic activity of lupine seeds.

Multielemental, Nutritional, and Proteomic Characterization of Different Lupinus spp. Genotypes: A Source of Nutrients for Dietary Use.

Among grain pulses, lupins have recently gained considerable interest for a number of attractive nutritional attributes relating to their high protein and dietary fiber and negligible starch contents. The seeds of Lupinus albus (cv. Multitalia and Luxor, and the Modica ecotype); L. luteus (cv. Dukat, Mister, and Taper); and L. angustifolius (cv. Sonet) analyzed in this study were deposited within the germplasm collection of the Research Centre for Cereal and Industrial Crops of Acireale and were sowed in East Sicily in 2013/14. The collected seeds were analyzed for their multielemental micro- and macronutrient profiles, resulting in a wide variability between genotypes. Lupin seed flour samples were subjected to a defatting process using supercritical CO2, with oil yields dependent on the species and genotype. We determined the fatty acid profile and tocopherol content of the lupin oil samples, finding that the total saturated fatty acid quantities of different samples were very close, and the total tocopherol content was about 1500.00 µg/g FW. The proteomic analysis of the defatted lupin seed flours showed substantial equivalence between the cultivars of the same species of Lupinus albus and L. luteus. Moreover, the L. angustifolius proteome map showed the presence of additional spots in comparison to L. albus, corresponding to α-conglutins. Lupin, in addition to being a good source of mineral elements, also contributes vitamin E and, thanks to the very high content of gamma-tocopherols, demonstrates powerful antioxidant activity.

Yield and quality of Dega white lupine grain (Lupinus Albus) and yubileynaya 80 spring wheat (Triticum Aestivum L.) depending on the application method of sodium selenite.

In 2017-2019, we conducted the field and vegetation experiments at the field station of Russian State Agrarian University, Moscow Timiryazev Agricultural Academy to study the effect of sodium selenite on the yield and grain quality indicators of white lupine, Dega variety, and spring wheat, Yubileynaya-80 variety. The best way found to use selenium is to spray vegetative plants with 0.01% aqueous sodium selenite solution. The studies have shown an increase in grain yield by 15-17%, crude protein content by 9-15% and crude fat content by 5-7% when treated with sodium selenite. The obtained grain yield of white lupine has a higher feed and nutritional value and is suitable for feeding animals and preparing various types of feed and feed additives. The optimal way to use selenium is spraying vegetative plants before shooting. Treatment with sodium selenite contributes to an increase in wheat yield by 1.5 times. We have established the positive effect of sodium selenite on the quality indicators of wheat grain. An increase in the content of raw gluten and glassiness of grain has been noted, which determines high bread-making qualities.

The vacuolar transporter LaMTP8.1 detoxifies manganese in leaves of Lupinus albus.

Manganese (Mn) is an essential microelement, but overaccumulation is harmful to many plant species. Most plants have similar minimal Mn requirements, but the tolerance to elevated Mn varies considerably. Mobilization of phosphate (P) by plant roots leads to increased Mn uptake, and shoot Mn levels have been reported to serve as an indicator for P mobilization efficiency in the presence of P deficiency. White lupin (Lupinus albus L.) mobilizes P and Mn with outstanding efficiency due to the formation of determinate cluster roots that release carboxylates. The high Mn tolerance of L. albus goes along with shoot Mn accumulation, but the molecular basis of this detoxification mechanism has been unknown. In this study, we identify LaMTP8.1 as the transporter mediating vacuolar sequestration of Mn in the shoot of white lupin. The function of Mn transport was demonstrated by yeast complementation analysis, in which LaMTP8.1 detoxified Mn in pmr1∆ mutant cells upon elevated Mn supply. In addition, LaMTP8.1 also functioned as an iron (Fe) transporter in yeast assays. The expression of LaMTP8.1 was particularly high in old leaves under high Mn stress. However, low P availability per se did not result in transcriptional upregulation of LaMTP8.1. Moreover, LaMTP8.1 expression was strongly upregulated under Fe deficiency, where it was accompanied by Mn accumulation, indicating a role in the interaction of these micronutrients in L. albus. In conclusion, the tonoplast-localized Mn transporter LaMTP8.1 mediates Mn detoxification in leaf vacuoles, providing a mechanistic explanation for the high Mn accumulation and Mn tolerance in this species.

Lupinus albus γ-Conglutin: New Findings about Its Action at the Intestinal Barrier and a Critical Analysis of the State of the Art on Its Postprandial Glycaemic Regulating Activity.

γ-Conglutin (γ-C) is the glycoprotein from the edible seed L. albus, studied for long time for its postprandial glycaemic regulating action. It still lacks clear information on what could happen at the meeting point between the protein and the organism: the intestinal barrier. We compared an in vitro system involving Caco-2 and IPEC-J2 cells with an ex vivo system using pig ileum and jejunum segments to study γ-C transport from the apical to the basolateral compartment, and its effects on the D-glucose uptake and glucose transporters protein expression. Finally, we studied its potential in modulating glucose metabolism by assessing the possible inhibition of α-amylase and α-glucosidase. RP-HPLC analyses showed that γ-C may be transported to the basolateral side in the in vitro system but not in the pig intestines. γ-C was also able to promote a decrease in glucose uptake in both cells and jejunum independently from the expression of the SGLT1 and GLUT2 transporters.

Investigations on the Transfer of Quinolizidine Alkaloids from Lupinus angustifolius into the Milk of Dairy Cows.

Lupin varieties with a low content of quinolizidine alkaloids (QAs) like blue sweet lupin (BSL) have long been used as a protein source for dairy cows. A health concern for humans may arise from the transfer of acute toxic QAs from feed into cow's milk. This study is the first to quantify the transfer of QAs from BSL into cow's milk with experimental and modeling methods. Four lactating dairy cows were subjected to two 7 day feeding periods with 1 and 2 kg/d BSL, respectively, each followed by a depuration period. BSL contained 1774 mg/kg dry matter total QAs. Individual milk samples were taken twice daily and QA contents in feed and milk determined with liquid chromatography-tandem mass spectrometry. Transfer of QAs into the milk was already seen with the administration of 1 kg/d BSL, with differences in transfer rates (TRs) between individual QAs. A toxicokinetic model was derived to quantify and predict QA feed-to-food transfer. For the four most prominent QAs, our model shows an α-half-life of around 0.27 d. TRs were obtained for six QAs and were between 0.13 (sparteine) and 3.74% (multiflorine). A toxicological assessment of milk containing QAs as measured in this study indicated a potential health concern.

Elevated CO2 and biochar differentially affect plant C:N:P stoichiometry and soil microbiota in the rhizosphere of white lupin (Lupinus albus L.).

Biochar application is a potent climate change mitigation strategy in agroecosystems. However, little is known about the interactive effects of elevated CO2 (eCO2) and biochar on plant nutrient uptake and soil microbial processes. A pot experiment was conducted to investigate the effects of eCO2 and biochar addition on plant C:N:P stoichiometry and rhizobacterial community for better management of nutrient balance and use efficiency in a future climate scenario. White lupin (Lupinus albus L.) was grown for 30 days in topsoil and subsoil with or without 2% corn-stubble biochar under ambient CO2 (aCO2: 390 ppm) or eCO2 (550 ppm). Elevated CO2 increased, but biochar decreased, plant biomass and shoot N and P uptake, with no interactions in either soil layer. Elevated CO2 decreased shoot N concentration by 16% and biochar decreased shoot P concentration by 11%. As a result, eCO2 increased shoot C:N ratio by 20% and decreased the N:P ratio by 11%. Biochar decreased shoot C:N ratio by 8% in the subsoil under eCO2. However, biochar increased shoot C:P ratio by an average of 13% and N:P ratio by 23% in the subsoil. Moreover, plants grown in the subsoil showed lower shoot N (35%) and P (70%) uptake compared to the topsoil. The results indicate that N and P are the more limiting factors that regulate plant growth under eCO2 and biochar application, respectively. Elevated CO2 and biochar oppositely affected dominant rhizobacterial community composition, with the eCO2 effect being greater. The microbiota in the subsoil held a greater diversity of contrasting species than the topsoil, which were associated with nutrient cycling, hydrocarbon degradation and plant productivity. These results enrich our understanding of potential soil nutrient cycling and plant nutrient balance in future agroecosystems.

Global Identification of White Lupin lncRNAs Reveals Their Role in Cluster Roots under Phosphorus Deficiency.

Phosphorus (P) deficiency heterogeneously affected plant nutritional status and physiological performance, ultimately leading to a severe yield reduction. A few putative long non-coding RNAs (lncRNAs) responding to P-starvation in the model crops Arabidopsis thaliana and Oryza sativa have been characterized. White lupin (Lupinus albus) is of prime importance, and is a legume with increasing agronomic value as a protein crop as it exhibits extreme tolerance to nutrient deficiency, particularly P deficiency. Despite its adapted nature to P deficiency, nothing is known about low P-induced lncRNAs in white lupin roots. To address this issue, we identified 39,840 mRNA and 2028 lncRNAs in the eight developmental stages of white lupin root (S0-S7 and lateral root, LR) grown under P deficiency. From these 2028 lncRNAs, 1564 were intergenic and 464 natural antisense intergenic transcript (NAT) lncRNAs. We further predicted six potential targets of miRNAs with twelve lncRNAs, which may regulate P-deficiency-related processes. Moreover, the weighted gene co-expression network analysis (WGCNA) revealed seven modules that were correlated with the expression pattern of lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed 606 GO terms and 27 different pathways including signal transduction, energy synthesis, detoxification, and Pi transport. In addition, we screened 13 putative lncRNAs that showed a distinct expression pattern in each root, indicating their role in the P deficiency regulatory network. Therefore, white lupin may be a reference legume to characterize P-deficiency-responsive novel lncRNAs, which would highlight the role of lncRNAs in the regulation of plant responses to P deficiency.

Identification and expression analysis of phosphate transporter (PHT) gene family in Lupinus albus cluster root under phosphorus stress.

According to global estimation, 5.7 billion hectares of agricultural land contain limited phosphorus (P) availability leading to insufficient plant growth and productivity. Internal phosphate transporters play an essential role in mediating P mobilization and uptake from the soil. White lupin (Lupinus albus) is a cluster root (CR) forming crop with great potential to survive under P limited soil. However, it is imperative to identify and characterize the phosphate transporter (PHT) gene family in plants to validate their involvement in solving P deficiency problems. The recent availability of white lupin high-quality genome allowed us an exhaustive searches in the whole genome and identified five phosphates transporters subfamilies, including 35 putative genes that are unevenly distributed on 16 chromosomes. The LaPHT1 subfamily contained eight genes, LaPHT2 subfamily have three, LaPHT3 subfamily have eight, LaPHT4 subfamily have nine, and LaPHO subfamily has seven. Gene structure and duplication were also examined in detail. Syntenic analysis revealed that white lupin PHT family members had maximum the collinear relationship with those in L. angustifolius followed by Phaseolus vulgaris but showed the least collinear relationship with those in Arabidopsis. Gene ontology (GO) analysis revealed that the in white lupin PHT genes were enriched in functions regulated P uptake, transport, and recycling mechanisms. RT-qPCR was performed to evaluate the transcript levels of LaPHT genes in different parts of CR under P deficient hydroponic culture. Our study would provide better understanding the genetic evolution and expression phosphate of phosphate transporters in L. albus CR under P deficiency. It will also be helpful for further functional-based studies to solve P deficiency-related issues and mitigate P stress responses.