Fungitoxic dihydrofuranoisoflavones and related compounds in white lupin,Lupinus albus

Chromatographic investigation of a methanolic extract of white lupin roots has revealed the presence of six new dihydrofuranoisoflavones (lupinisoflavones A-F). Three monoprenylated (3,3-dimethylallyl-substituted) isoflavones (wighteone, luteone and licoisoflavone A), two diprenylated isoflavones [6,3′-di(3,3-dimethylallyl)genistein (lupalbigenin) and 6,3′-di(3,3-dimethylallyl)-2′-hydroxygenistein (2′-hydroxylupalbigenin)] and two pyranoisoflavones (parvisoflavone B and licoisoflavone B) have also been isolated from the same source. In addition to genistein, leaf extracts of L. italbus contain 3′-O-methylorobol which is presumed to be the precursor of lupisoflavone [5,7,4′-trihydroxy-3′-methoxy-6-(3,3-dimethylallyl)isoflavone]. Probable biogenetic relationships between the prenylated, and dihydrofurano-and pyrano-substituted isoflavones in roots and leaves of L. albus are briefly discussed.     

Biosynthesis of antifungal isoflavonoids in Lupinus albus. Enzymatic prenylation of genistein and 2'-hydroxygenistein.

A 45,000g particulate fraction from hypocotyls of Lupinus albus catalyzes the prenylation of genistein 1 and 2′-hydroxygenistein 2 with dimethylallyl pyrophosphate to the antifungal 6-isopentenylgenistein 3 (wighteone) and 6-isopentenyl-2′-hydroxygenistein 4 (luteone), respectively.     

The Distribution of Nine Elements in Shoots of Lupinus albus L. and Lupinus angustifolius L. Compared with that of Silicon as a Measure of Passive Transport

The Ca, Mg, K, Na, P, Fe, Mn, Zn, Cu and opaline-Si contentsof leaves, stems and inflorescences from each order of shootaxis was determined in Lupinus albus L. and Lupinus angustifoliusL. The distribution of Si was used as a base for passive transportin the transpiration stream. All of the elements investigatedwere redistributed among the leaves, stems and inflorescencesof either L. albus or L. angustifolius. Most of the elementsinvestigated were enriched in the inflorescences and depletedin either the leaves or the stems. Sodium was enriched in thestems whereas Ca and Mn were redistributed only in L. albus.The pattern of element redistribution was similar in each ofthe lateral shoot axes except for the youngest. For the elementsenriched in the inflorescences, more than half was suppliedby redistribution. Calcium redistribution was similar to thatfor K, which is regarded as phloem mobile, but a mechanism forCa redistribution is uncertain. Lupinus albus L., Lupinus angustifolius L., mineral transport, leaves, inflorescences, transpiration, silicon, calcium     

A New Isoflavone with Antifungal Activity from Immature Fruits of Lupinus luteus

A new isoflavone having antifungal activity was isolated from immature fruits of Lupinus luteus (Leguminosae), and named luteone. The structure was shown to be 5,7,2′,4′-tetrahy-droxy-6-(3,3-dimethylallyl)-isoflavone by degradative and spectroscopic studies.     

Acifluorfen-induced isoflavonoids and enzymes of their biosynthesis in mature soybean leaves : whole leaf and mesophyll responses

Mature soybean (Glycine max L. cv Harosoy 63) leaves normally contain kaempferol-3-glycosides but they accumulate no other flavonoids. Whole leaves sprayed with the diphenyl ether herbicide Acifluorfen and maintained in the light developed small necrotic lesions and accumulated isoflavone aglycones, isoflavone glucosides, and pterocarpans. Isoflavonoid accumulation was preceded by induced activity for chalcone synthase (CHS) and by increased activity for phenylalanine ammonia-lyase (PAL) and UDP-glucose:isoflavone 7-O-glucosyl transferase (IGT). PAL and CHS activity was highest between 24 and 30 hours after treatment, isoflavone aglycones and pterocarpans at 48 hours, IGT at 72 hours, and isoflavone glucosides at 96 hours.     

Differential metabolic response of narrow leafed lupine (Lupinus angustifolius) leaves to infection with Colletotrichum lupini

Flavones and isoflavones are a major group of phenolic secondary metabolites which occur in leaves of narrow leafed lupine (Lupinus angustifolius) either as free aglycones or in a form of glycosides and malonyl-glycosides. Profiles of phenolic compounds in leaves of seedlings infected with anthracnose causing fungus Colletotrichum lupini were compared to those of healthy plants. A HPLC with diode array UV detector was used as the analytical method and identification of these secondary metabolites was confirmed with a HPLC/MSⁿ instrument. Isomers of several target compounds differing in the glycosilation and/or malonylation pattern were detected in the studied samples. However, the application of standard HPLC with C18 columns resulted in the co-elution of several glyconjugates in single chromatographic peaks whereas for isoflavonoid aglycones complete resolution was achieved. Lupine plants grown in a greenhouse were either sprayed with the C. lupini spore suspension or the suspension was spotted on to wounded leaves. Profiles of the isoflavones were altered in result to infection with both methods. In particular, the concentration of isoflavone free aglycones detected in extracts from diseased plants was substantially increased in all of the studied samples. However, the pattern of these compounds depended on the age of lupine leaves as well as on the method of infection. Synthesis of luteone and 2′-hydroxygenistein was enhanced in the youngest leaves of plants sprayed with spores as well as in wound-infected leaves. Wighteone synthesis was induced mainly in older leaves of plants sprayed with the spore suspension.     

The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds

 A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants). Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related (PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against fungal infection, though our results do not exclude other functions for this protein. Received: 15 March 1999 / Accepted: 12 July 1999     

Effects of phosphorus supply on growth, phosphate concentration and cluster-root formation in three Lupinus species

Background and Aims

In some lupin species, phosphate deficiency induces cluster-root formation, which enhances P uptake by increasing root surface area and, more importantly, the release of root exudates which enhances P availability.

Methods

Three species of Lupinus, L. albus, L. atlanticus and L. micranthus, with inherently different relative growth rates were cultivated under hydroponics in a greenhouse at four phosphate concentrations (1, 10, 50 and 150 µm) to compare the role of internal P in regulating cluster-root formation.

Key Results

The highest growth rate was observed in L. atlanticus, followed by L. albus and L. micranthus. At 1 µm P, cluster-root formation was markedly induced in all three species. The highest P uptake and accumulation was observed in L. micranthus, followed by L. atlanticus and then L. albus. Inhibition of cluster-root formation was severe at 10 µm P in L. atlanticus, but occurred stepwise with increasing P concentration in the root medium in L. albus.

Conclusions

In L. atlanticus and L. albus cluster-root formation was suppressed by P treatments above 10 µm, indicating a P-inducible regulating system for cluster-root formation, as expected. By contrast, production of cluster roots in L. micranthus, in spite of a high internal P concentration, indicated a lower sensitivity to P status, which allowed P-toxicity symptoms to develop.     

2,3-dimethylnonacosane and tropane alkaloids from Hyoscyamus albus

In addition to hyoscine and hyoscyamine, a new compound isolated from the leaves and stems of Hyoscyamus albus has been characterized as 2,3-dimethylnonacosane by spectral studies.     

Biosynthesis of White Lupin Isoflavonoids from [U-C]l-Phenylalanine and Their Release into the Culture Medium

Pulse-labeling experiments of white lupin (Lupinus albus L.) cell cultures with [U-¹⁴C]l-phenylalanine for 72 h resulted in the incorporation of the radioactivity into the isoflavone aglucones, glucosides, and prenylated derivatives. Both the aglucones genistein and 2′-hydroxygenistein and their 7-O-glucosides accounted for 85% of the total isoflavonoids identified in the cultured cells and contained 35% of the radioactivity, whereas the prenylated derivatives comprised 15 and 65%, respectively. Almost 20% of the labeled isoflavones of the cellular pool was recovered from the culture medium, 90% of which were monoprenylated and diprenylated derivatives containing 80% of the radioactivity. These results clearly demonstrate the release into the culture medium of a substantial amount of the endogenously synthesized isoflavonoids, especially the prenylated derivatives.     

Feeding preferences of the weevils Sitona gressorius and Sitona griseus on different lupin genotypes and the role of alkaloids

The weevils Sitona gressorius and Sitona griseus are specialist herbivores on lupins in Europe. The adult weevils feed on the leaves, and the larvae on the root nodules of the plants. This causes severe damage to lupin crops. In the present study, the feeding preferences of lupin weevil adults on different lupin genotypes were examined with respect to a possible effect of lupin alkaloids on host selection. A total of 12 genotypes from the species Lupinus albus, L. angustifolius, L. luteus, and L. nanus were grown in a field experiment and the feeding damage on the leaves caused by naturally occurring lupin weevil adults was estimated. Additionally, a feeding choice test with S. gressorius adults was performed to examine feeding preferences under laboratory conditions. A gas chromatographic analysis provided information on the alkaloid content and profiles in the leaves of the tested lupin genotypes. In the field experiment, significant differences in the extent of the feeding damage within the 12 lupin genotypes were observed. The dual-choice feeding bioassay did not show discrimination of lupin species, but two L. angustifolius genotypes were significantly less affected than the standard L. luteus “Bornal”. The alkaloid analysis revealed large contrasts in alkaloid concentrations and profiles in the leaves of the tested genotypes. Correlation analysis with the results from the field and laboratory did not indicate a significant influence of the total foliar alkaloid content on the extent of weevil feeding.     

Genetic diversity of rhizobia associated with root nodules of white lupin (Lupinus albus L.) in Tunisian calcareous soils

With a view to introducing white lupin (Lupinus albus L.) for cultivation in Tunisian calcareous soils, compatible indigenous rhizobia for nitrogen-fixing symbiosis were investigated and characterized. Two L. albus varieties, Mekna and Lumen, were used to trap rhizobia in soil samples collected from 56 sites with high active lime contents (0–49%). Nodulation occurred in only 15 soils. The local variety, Mekna, developed significantly more root nodules and had a trapping capacity in more soils than the imported variety Lumen. A phylogenetic analysis based on the partial 16S-23S ribosomal RNA internal transcribed spacer region (ITS) and multi-locus sequence analysis (MLSA) of three chromosomal housekeeping genes, recA, atpD and dnaK, showed that strains were affiliated to Agrobacterium, Rhizobium, and Neorhizobium, with large internal diversity, including separate lineages. Infectivity tests highlighted some nodulation specificity at the plant variety level, since the strains originating from Mekna could only nodulate this variety, while strains trapped in Lumen could nodulate both varieties. When inoculated, almost all strains resulted in a significant increase in plant shoot dry weight on L. albus. Although Agrobacterium sp. strains isolated from L. albus could nodulate and had a plant growth promoting effect, no nodA and nodC genes could be amplified. This is discussed together with the absence of bradyrhizobia and the general infrequency of L. albus–nodulating rhizobia in Tunisian soils. The adapted and efficient rhizobial strains reported here were promising candidates for inoculant development and represent a contribution towards successful cultivation of L. albus in Tunisia, especially the most promising Mekna variety.     

Variability for protein and oil quality in Lupinus albus

Amino acid composition and fatty acid composition were determined on seed samples of a range of white lupin (Lupinus albus) cultivars and accessions grown in either of two environments.Variability between genotypes was found for lysine, arginine and glutamic acid content, but not for the concentrations of other amino acids. The deficiency in sulphurcontaining amino acids, typical of legume proteins, was evident, with methionine and cyst(e)ine totalling only 2.2% of the protein. Variability was limited, indicating that improvement by breeding would be impracticable. Lupinus albus differed slightly from other lupin species in amino acid composition, having higher levels of threonine, tyrosine and isoleucine, but a lower level of glutamic acid than both L. angustifolius and L. luteus. Four low-alkaloid lines of L albus each had higher lysine content than the high-alkaloid line, but ‘Kiev Mutant’, despite earlier claims, had a lysine level no higher than the other three low-alkaloid lines.Fatty acid composition of the seed oil varied considerably between genotypes. Oleic acid ranged from 43.6 to 54.4% and linolenic acid from 6.7 to 15.2%, these two fatty acids being negatively correlated at one site. Linoleic acid content varied between 17.2 and 26.9% and was not correlated with other fatty acids. Total oil content averaged 9.6% with little variability between lines.It is concluded that, relative to other lupin species, L. albus has a more favourable amino acid profile for its utilisation in cereal-based diets for animals, particularly if the energy source is wheat, which is deficient in threonine. The higher oil content would be an important energy benefit to such diets and may allow their protein/energy balance to be maintained at higher levels of incorporation of L. albus seed meal than is possible with other lupin species.     

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.     

Investigations into the exploitation of heterogeneous soils by Lupinus albus L. and L. pilosus Murr. and the effect upon plant growth

In calcareous soils, genotypes of Lupinus albus L. generally grow poorly, resulting in stunted plants that often develop lime-induced chlorosis. In contrast, some genotypes of L. pilosus Murr. occur naturally in calcareous soils without developing any visible symptoms of stress. Some genotypic variation for tolerance to calcareous soil does exist in L. albus and the tolerance mechanisms need to be determined. The adaptation through root system morphological plasticity of L. albus and L. pilosus, to heterogeneous limed soil profiles (pH 7.8) containing either patches of acid (non-limed) soil, or vertically split between acid and limed soil, was investigated. When grown in the presence of patches of acid soil, L. albus had a 52% greater shoot dry weight and visibly greener leaves compared with plants grown in the homogeneous limed soil. Total root dry matter in the acid-soil patches was greater than in the control limed-soil patches. This was due to a four-fold increase in the cluster root mass, accounting for 95% of the root dry matter in the acid-soil patch. Although these cluster roots secreted no more citric acid per unit mass than those in the limed soil did, their greater mass resulted in a higher citrate concentration in the surrounding soil. L. pilosus responded to the patches of acid soil in a manner comparable with L. albus. When grown in the homogeneous limed soil, L. pilosus had a greater maximum net CO₂ assimilation rate (P_max) than L. albus, however, the P_max of both species increased after they had accessed a patch of acid soil. Differences were apparent between the L. albus genotypes grown in soil profiles split vertically into limed and acid soil. A genotype by soil interaction occurred in the partitioning between soils of the cluster roots. The genotype La 674 was comparable with L. pilosus and produced over 11% of its cluster roots in the limed soil, whereas the other genotypes produced only 1–3% of their cluster roots in the limed soil. These results indicate L. pilosus is better adapted to the limed soil than L. albus, but that both species respond to a heterogeneous soil by producing mainly cluster roots in an acid-soil patch. This revised version was published online in June 2006 with corrections to the Cover Date.     

Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium

In this work we analysed different chromosomal and symbiotic markers in rhizobial strains nodulating Lupinus albus (white lupin) in several continents. Collectively the analysis of their rrs and atpD genes, and 16S-23S intergenic spacers (ITS), showed that they belong to at least four chromosomal lineages within the genus Bradyrhizobium. Most isolates from the Canary Islands (near to the African continent) grouped with some strains isolated on mainland Spain and were identified as Bradyrhizobium canariense. These strains are divided into two ITS subgroups coincident with those previously described from isolates nodulating Ornithopus. The remaining strains isolated on mainland Spain grouped with most isolates from Chile (American continent) forming a new lineage related to Bradyrhizobium japonicum. The strains BLUT2 and ISLU207 isolated from the Canary Islands and Chile, respectively, formed two new lineages phylogenetically close to different species of Bradyrhizobium depending on the marker analyzed. The analysis of the nodC gene showed that all strains nodulating L. albus belong to the biovar genistearum; nevertheless they form four different nodC lineages of which lineage C is at present exclusively formed by L. albus endosymbionts isolated from different continents.     

The alkaloid content of sweet lupin seed used in feeding trials on pigs and rats

The alkaloid content of the seeds of several new cultivars of “sweet” lupins was determined. The alkaloid contents ranged from < 0.01% for L. angustifolius cv. Uniwhite to 0.09% for L. albus cv. Neuland (a). The major alkaloid component (70%) of Neuland (a) was identified as dl-lupanine. This was the sample of L. albus that markedly suppressed feed intake and growth of young pigs; alcohol extraction reduced the alkaloid content to 0.02% and the extracted lupin-seed meal supported growth and food consumption equivalent to Uniwhite.Rats given a diet containing 50% L. albus cv. Neuland (a) grew normally over 13 weeks. Rats given Neuland (a) as the sole source of protein at 10% of the diet, had a protein efficiency ratio of 0.13. Supplementation of the diet with methionine increased the p.e.r. to 2.45. The addition of increasing amounts of lupin alkaloid, up to maximum content of 0.15% of the diet, had no effect on p.e.r. The p.e.r. obtained from diets containing ethanol-extracted Neuland (a), with and without methionine, were 0.94 and 2.61 respectively, as compared to 0.36 and 2.60 for the unextracted Neuland (a) diets, suggesting that lupin alkaloids can reduce protein quality in methionine deficient diets.The conclusion is that, in marked contrast to pigs, rats are more resistant to dietary lupin alkaloids.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

Ultrastructural localization of a diprenylated isoflavone in Rhizobium lupini-Lupinus albus symbiotic association

Polyclonal IgGs raised against the diprenylated isoflavone,2'-hydroxylupalbigenin were used in an immunocytochemical studyof the root and nodule tissues of white lupin (Lupinus albusL.). In the roots, the antigen was detected in the secondarywalls of both xylem vessels and pericycle cells. Examinationof nodules revealed the presence of the antigen in the innercortex, associated with a neoparietal material which is depositedas globules on the cell wall and occluding the intercellularspaces. The discrete location of the diprenylated isoflavonein specialized cytoplasmic organelles suggests that its compartmentationwithin the wall is mediated by membrane vesicles. In the infectedcells, more than half of the bacteroids exhibited a specificlabelling of their inner core associated with a central fibrillarsystem, and sometimes surrounding an electron-dense nucleoidregion. These results suggest that 2'-hydroxylupalbigenin, andpossibly other prenylated isoflavones, may play an importantrole as a biochemical factor in early symbiotic events otherthan nod gene induction or inhibition. Its putative biologicalfunctions in the post-infectional phases of symbiosis are discussed. Key words: Bacteroid, 2'-hydroxylupalbigenin, in situ localization, Lupinus albus, nodules     

Sucrose-induced lupine defense against Fusarium oxysporum. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum.

Defense responses to inoculation with Fusarium oxysporum SCHLECHT f. sp. lupini were studied in embryo axes of Lupinus luteus L. cv. Polo cultured on a medium with sucrose (60 mM) or without it. Exogenous sucrose caused a marked endogenous increase in concentrations of sucrose, glucose and fructose in embryo axes. In axes cultured with sucrose, high performance liquid chromatography (HPLC) revealed generally higher levels of isoflavone glycosides (particularly until 48 h of culture) and free aglycones (genistein, wighteone, luteone). Inoculation resulted in a considerable decline in soluble carbohydrates between 24 and 72 h of culture. Simultaneously, the infection stimulated an increase in the level of free isoflavone aglycones in inoculated embryo axes, as compared to non-inoculated ones. Concentrations of free aglycones (i.e. genistein, wighteone and luteone) after infection were particularly high in inoculated embryo axes fed with sucrose. Genistein was a better inhibitor to F. oxysporum growth than genistein 7-O-glucoside tested. Exogenous sucrose also stimulated the activity of phenylalanine ammonialyase (PAL, EC 4.3.1.5)--an important enzyme initiating phenylpropanoid metabolism. After infection of tissues, a strong increase was observed in the activity of PAL and beta-glucosidase (EC 3.2.1.21)--an enzyme hydrolyzing isoflavone glycosides. Furthermore, the growth of inoculated embryo axes cultured with sucrose was less inhibited as a result of infection than inoculated axes cultured under carbohydrate deficiency conditions. Additionally, it had been reported previously that disease symptoms of embryo axes growing in the presence of sucrose were less intensive [30]. These results suggest that soluble sugars are involved in the mechanism of resistance, as they can stimulate phenylpropanoid metabolism and contribute to the increase in concentration of isoflavonoids, which are important elements of the defense system of legumes.