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.

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.

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.

1H NMR-based metabolomics and UHPLC-ESI-MS/MS for the investigation of bioactive compounds from Lupinus albus fractions.

Lupinus albus or white lupine has recently received increase attention for its medicinal values. Several studies have described the hypoglycemic effect of the white lupine, which is known as a food plant with potential value for treatment of diabetes. This study provides useful information for the identification and quantification of compounds in L. albus fractions by proton nuclear magnetic resonance (1H NMR) spectroscopy. In total, 35 metabolites were identified from L. albus fractions.Principal component analysis (PCA) was used as a multivariate projection method for visualizing the different composition of four different fractions. The bioactivities of fractions with different polarity obtained from the extract of L. albus seeds are reported. Among the fractions studied, the chloroform fraction (CF) exhibits a high free radical scavenging (DPPH) and α-glucosidase inhibitory activities with IC50 values of 24.08 and 20.08 µg/mL, respectively. A partial least-squares analyses (PLS) model had been successfully performed to correlate the potential active metabolites with the corresponding biological activities. Metabolites containing proline, caprate, asparagine, lupinoisolone C, hydroxyiso lupalbigenin and some unknown compounds show high correlation with the bioactivities studied. Moreover, the structural identification in the active fraction was supported by ultrahigh-performance-liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) analysis. A total of 21 metabolites were tentatively identified from MS/MS data by comparison with previously reported data. Most of these compounds are isoflavonoids without known biological activity. This information may be useful for developing functional food from L. albus with potential application in the management of diabetes.

Effects of root morphology, respiration and carboxylate exudation on carbon economy in two non-mycorrhizal lupines under phosphorus deficiency.

Under phosphorus (P) deficiency, Lupinus albus develops cluster roots that allow efficient P acquisition, while L. angustifolius without cluster roots also grows well. Both species are non-mycorrhizal. We quantitatively examined the carbon budgets to investigate the different strategies of these species. Biomass allocation, respiratory rates, protein amounts and carboxylate exudation rates were examined in hydroponically-grown plants treated with low (1 µM; P1) or high (100 µM; P100) P. At P1, L. albus formed cluster roots, and L. angustifolius increased biomass allocation to the roots. The respiratory rates of the roots were faster in L. albus than in L. angustifolius. The protein amounts of the non-phosphorylating alternative oxidase and uncoupling protein were greater in the cluster roots of L. albus at P1 than in the roots at P100, but similar between the P treatments in L. angustifolius roots. At P1, L. albus exuded carboxylates at a faster rate than L. angustifolius. The carbon budgets at P1 were surprisingly similar between the two species, which is attributed to the contrasting root growth and development strategies. L. albus developed cluster roots with rapid respiratory and carboxylate exudation rates, while L. angustifolius developed a larger root system with slow respiratory and exudation rates.

The Protective Effects of Lupine (Lupinus albus) Fruit Extract Against to Destructive Effects of Hyperglycemia in Type I Diabetic Rats.

BACKGROUND: Lupinus albus is a member of the Fabaceae family. As a natural or cultivated plant, Lupinus albus is distributed in Europe, the Balkans and Turkey, especially in Marmara and Aegean regions. The lupine is a nutritious and protective plant against diabetes. OBJECTIVE: In the present study, the effects of Lupinus albus fruits on malondialdehyde (MDA), reduced glutathione (GSH), total protein, ADEK vitamins, and cholesterol values, which are the indicators of oxidative damage and antioxidant defense. In this regard, muscle, liver, renal, and brain tissues of STZ-induced type I diabetes rats were studied. METHODS: The analyzes of ADEK vitamins and cholesterol levels in tissues were performed via Shimadzu HPLC device. The lipid peroxidation levels were measured at 532 nm in spectrophotometer. Determination of GSH was read at 412 nm against blank, and for the total protein levels Lowry method was applied. RESULTS: According to the results obtained, it was determined that among the rats with induced type I diabetes, the group applied lupine fruit extract was found to have increased GSH level and decreased MDA levels in all the tissues. The protein values were increased in liver tissues but decreased in the other tissues. The level of vitamins was significantly increased in almost all the tissues in the diabetic group. CONCLUSION: In the present study, it was shown that the lupine reduced the devastating effects of type I diabetes by decreasing the fasting blood glucose and lipid peroxidation values and increasing the glutathione level in comparison to the diabetic group.

Antioxidant, α-Glucosidase, and Nitric Oxide Inhibitory Activities of Six Algerian Traditional Medicinal Plant Extracts and 1H-NMR-Based Metabolomics Study of the Active Extract.

Claims of effective therapy against diabetes using plants including Peganum harmala L., Zygophyllum album, Anacyclus valentinus L., Ammodaucus leucotrichus, Lupinus albus, and Marrubium vulgare in Algerian empirical medicine prompted our interest in evaluating their antidiabetic activity by screening their free radical scavenging (DPPH), α-glucosidase, and nitric oxide (NO) inhibitory activities as well as the total phenolic content (TPC). Extracts of the selected plants were prepared using different ratios of ethanol (0, 50, 80, and 100%). In this study, 100%, and 80% ethanol extracts of L. albus were found to be the most potent, in inhibiting α-glucosidase activity with IC50 values of 6.45 and 8.66 µg/mL, respectively. The 100% ethanol extract of A. leucotrichus exhibited the highest free radical scavenging activity with an IC50 value of 26.26 µg/mL. Moreover, the highest TPC of 612.84 µg GAE/mg extract was observed in M. vulgare, extracted with 80% ethanol. Metabolite profiling of the active extract was conducted using 1H-NMR metabolomics. Partial least square analysis (PLS) was used to assess the relationship between the α-glucosidase inhibitory activity of L. albus and the metabolites identified in the extract. Based on the PLS model, isoflavonoids (lupinoisoflavone G, lupisoflavone, lupinoisolone C), amino acids (asparagine and thiamine), and several fatty acids (stearic acid and oleic acid) were identified as metabolites that contributed to the inhibition of α-glucosidase activity. The results of this study have clearly strengthened the traditional claim of the antihyperglycemic effects of L. albus.

Journey in the Polyphenol Research World with Ragai Ibrahim.

Dr. Ragai K. Ibrahim, Professor Emeritus at Concordia University, Montréal, Canada, passed away on the November 19, 2017 at the age of 88 years. Dr. Ibrahim dedicated his entire professional life to polyphenols and spent most of his academic career (1967-1997) at the Department of Biology of Concordia University in Montréal. He has been an active member of the Groupe Polyphénols since the beginning. This paper is a tribute to Dr. Ibrahim from some of his former students. An overview of the evolution of polyphenol research since the late 1950s and the outstanding contribution that Dr. Ibrahim had to this topic is given. The input of Dr. Ibrahim's research to the enzymology and genetics of polyphenol biosynthesis is discussed. Furthermore, the links between Dr. Ibrahim's work and some aspects of modern studies on the health benefits of polyphenols are presented.

Influence of root exudation of white lupine (Lupinus albus L.) on uranium phytoavailability in a naturally uranium-rich soil.

Mechanisms of uranium (U) transfer from soil to plants remain poorly understood. The kinetics of supply of U to the soil solution from solid phases could be a key point to understand its phytoavailability and implications for environmental risk assessment. Root activity, particularly the continuous release of organic acids in the rhizosphere, could have an effect on this supply. We tested the impact of citrate exudation by roots of Lupinus albus, either P-sufficient (P+) or P-deficient (P-), on the phytoavailability of U from a naturally contaminated soil (total content of 413 mg U kg-1) using a rhizotest design. Combined effects of P (P-/P+ used to modulate plant physiology) and citrate (model exudate) on the solubilization of U contained in the soils were tested in closed reactors (batch). The batch experiment showed the existence of a low U available pool (0.4% total U) and high accessibility (kd' around 20 L kg-1) which was not significantly affected by P treatment or citrate concentrations. Analysis of U, Fe, Ca, P and citrate concentrations in the batches suggested a complex combination of mechanisms and factors including desorption, resorption, precipitation, co-sorption. On rhizotest, L. albus plants extracted 0.5-0.75% of the total U and between 25 and 40% of the estimated available U present in the rhizotest in 5 days. Uranium accumulation at the whole plant level (20 mg U kg-1d.w., shoot to root ratio around 10-3) seemed to be dependent neither on the plant P nutrition status nor citrate exudation level, possibly in relation with the equivalent accessibility of U whatever the growth conditions. Yet differential translocation to shoots seemed to be positively correlated to citrate exudation.

Compositional changes in (iso)flavonoids and estrogenic activity of three edible Lupinus species by germination and Rhizopus-elicitation.

The effects of germination and elicitation on (iso)flavonoid composition of extracts from three edible lupine species (Lupinus luteus, Lupinus albus, Lupinus angustifolius) were determined by RP-UHPLC-MS(n). The total (iso)flavonoid content of lupine increased over 10-fold upon germination, with the total content and composition of isoflavonoids more affected than those of flavonoids. Glycosylated isoflavones were the most predominant compounds found in lupine seedlings. Lesser amounts of isoflavone aglycones, including prenylated ones, were also accumulated. Elicitation with Rhizopus oryzae, in addition to germination, raised the content of isoflavonoids further: the total content of 2'-hydroxygenistein derivatives was increased considerably, without increasing that of genistein derivatives. Elicitation by fungus triggered prenylation of isoflavonoids, especially of the 2'-hydroxygenistein derivatives. The preferred positions of prenylation differed among the three lupine species. The change in isoflavone composition increased the agonistic activity of the extracts towards the human estrogen receptors, whereas no antagonistic activity was observed.

Hormonal interactions during cluster-root development in phosphate-deficient white lupin (Lupinus albus L.).

This study addresses hormonal interactions involved in cluster-root (CR) development of phosphate (Pi)-deficient white lupin (Lupinus albus), which represents the most efficient plant strategy for root-induced mobilisation of sparingly soluble soil phosphorus (P) sources. Shoot-to-root translocation of auxin was unaffected by P-limitation, while strong stimulatory effects of external sucrose on CR formation, even in P-sufficient plants, suggest sucrose, rather than auxins, acts as a shoot-borne signal, triggering the induction of CR primordia. Ethylene may act as mediator of the sucrose signal, as indicated by moderately increased expression of genes involved in ethylene biosynthesis in pre-emergent clusters and by strong inhibitory effects of the ethylene antagonist CoCl2 on CR formation induced by sucrose amendments or P-limitation. As reported in other plants, moderately increased production of brassinosteroids (BRs) and cytokinin, in pre-emergent clusters, may be required for the formation of auxin gradients necessary for induction of CR primordia via interference with auxin biosynthesis and transport. The well-documented inhibition of root elongation by high doses of ethylene may be involved in the growth inhibition of lateral rootlets during CR maturation, indicated by a massive increased expression of gene involved in ethylene production, associated with a declined expression of transcripts with stimulatory effects (BR and auxin-related genes).

Cluster-root formation and carboxylate release in three Lupinus species as dependent on phosphorus supply, internal phosphorus concentration and relative growth rate.

BACKGROUND AND AIMS: Some Lupinus species produce cluster roots in response to low plant phosphorus (P) status. The cause of variation in cluster-root formation among cluster-root-forming Lupinus species is unknown. The aim of this study was to investigate if cluster-root formation is, in part, dependent on different relative growth rates (RGRs) among Lupinus species when they show similar shoot P status. METHODS: Three cluster-root-forming Lupinus species, L. albus, L. pilosus and L. atlanticus, were grown in washed river sand at 0, 7·5, 15 or 40 mg P kg(-1) dry sand. Plants were harvested at 34, 42 or 62 d after sowing, and fresh and dry weight of leaves, stems, cluster roots and non-cluster roots of different ages were measured. The percentage of cluster roots, tissue P concentrations, root exudates and plant RGR were determined. KEY RESULTS: Phosphorus treatments had major effects on cluster-root allocation, with a significant but incomplete suppression in L. albus and L. pilosus when P supply exceeded 15 mg P kg(-1) sand. Complete suppression was found in L. atlanticus at the highest P supply; this species never invested more than 20 % of its root weight in cluster roots. For L. pilosus and L. atlanticus, cluster-root formation was decreased at high internal P concentration, irrespective of RGR. For L. albus, there was a trend in the same direction, but this was not significant. CONCLUSIONS: Cluster-root formation in all three Lupinus species was suppressed at high leaf P concentration, irrespective of RGR. Variation in cluster-root formation among the three species cannot be explained by species-specific variation in RGR or leaf P concentration.

Do rhizospheric processes linked to P nutrition participate in U absorption by Lupinus albus grown in hydroponics?

Phosphate (P) is an essential element for plant development but is generally present in limiting amount in the soil solution. Plant species have developed different mechanisms promoting the solubilization of this element in soils to ensure a sufficient supply for their growth. One of these mechanisms is based on the ability of certain species such as L. albus to exude large amounts of citrate through specific tertiary roots called cluster-roots. Uranium (U) is an ubiquitous contaminant known firstly for its chemical toxicity and secondly for its high affinity for P with which it forms low-soluble complexes in soils. We highlight the effects of P-U interaction on the physiology of L. albus and particularly on citrate exudation, and the impact of this root process on the phytoavailability of U and its accumulation in plants in a hydroponic study. Different levels of P (1 and 100 µM) and U (0 and 20 µM) have been tested. Our results show no toxicity of U on the development of L. albus with an adequate P supply, whereas the effects of P starvation are amplified by the presence of U in the growth medium, except for the production of cluster-roots. Citrate exudation is totally inhibited by U in a low-P environment whereas it increases in the presence of U when its toxicity is lowered by the addition of P. The differences observed in terms of toxicity and accumulation are partly explained by the microphotographs obtained by electron microscopy (TEM-EDX): in the absence of P, U penetrates deep into the roots and causes lethal damages, whereas in presence of P, we observe the formation of U-P complexes which limit the internalization of the pollutant and so its toxicity.

Sugar signalling mediates cluster root formation and phosphorus starvation-induced gene expression in white lupin.

Cluster root (CR) formation contributes much to the adaptation to phosphorus (P) deficiency. CR formation by white lupin (Lupinus albus L.) is affected by the P-limiting level in shoots, but not in roots. Thus, shoot-derived signals have been expected to transmit the message of P-deficiency to stimulate CR formation. In this study, it is shown that sugars are required for a response to P starvation including CR formation and the expression of P starvation-induced genes. White lupin plants were grown in vitro on P-sufficient or P-deficient media supplemented with sucrose for 4 weeks. Sucrose supply stimulated CR formation in plants on both P-sufficient and P-deficient media, but no CR appeared on the P-sufficient medium without sucrose. Glucose and fructose also stimulated CR formation on the P-sufficient medium. On the medium with sucrose, a high concentration of inorganic phosphate in leaves did not suppress CR formation. Because sorbitol or organic acid in the media did not stimulate CR formation, the sucrose effect was not due to increased osmotic pressure or enriched energy source, that is, sucrose acted as a signal. Gene transcription induced by P starvation, LaPT1 and LaPEPC3, was magnified by the combination of P limitation and sucrose feeding, and that of LaSAP was stimulated by sucrose supply independently of P supply. These results suggest that at least two sugar-signalling mediating systems control P starvation responses in white lupin roots. One system regulates CR formation and LaSAP expression, which acts even when P is sufficient if roots receive sugar as a signal. The other system controls LaPT1 and LaPEPC3 expression, which acts when P is insufficient.