Post-damage alkaloid concentration in sweet and bitter lupin varieties and its effect on subsequent herbivory

Abstract:  While most lupin species possess quinolizidine alkaloids, sweet (low alkaloid) varieties are more palatable but at the same time more susceptible to herbivory. Nevertheless, as they are not totally devoid of alkaloids, it may be possible that their alkaloid levels increase after damage. The aim of this study was to compare inductive responses to herbivory in sweet and bitter varieties of Lupinus albus (L.) and Lupinus angustifolius (L.), and to assess if these responses were effective to stop subsequent herbivory. Two experiments were carried out; in the first, Anticarsia gemmatalis (Hübner; Lep., Noctuidae) caterpillars were introduced in field-growing lupin plants and allowed to feed for 72 h, after which leaves were collected and analysed for alkaloid content and composition. The second experiment was a bioassay, in which leaves collected from experiment 1, from treated and control plants, were offered to another set of Anticarsia caterpillars, and consumption was recorded after 24 h. We found that both L. albus varieties (sweet and bitter) had an increase in their alkaloid concentration after damage, while none of the L. angustifolius varieties had. The sweet L. albus variety, Rumbo, had a greater inductive response than the bitter variety. When leaves were offered to caterpillars (bioassay), this variety showed the greatest difference between consumption of controls and previously eaten leaves, implying that alkaloid levels reached after damage were effective to deter subsequent herbivores as a result of plants probably overcoming a 'palatability threshold'.     

Synthesis, transport and accumulation of quinolizidine alkaloids in Lupinus albus L. and L. angustifolius L

Each of the principal quinolizidine alkaloids (QA) found in both xylem and phloem exudates together with extracts from all component organs collected from bitter (cv. Lupini) and sweet (cv. Ultra) cultivars of Lupinus albus L. were quantified by gas chromatographic analyses throughout reproductive development. In addition to establishing the major translocated QA species estimates for fluxes of QA to developing fruits based on their sap composition and water economy showed that around half of the QA that accumulated in fruit tissues was due to synthesis in situ and half to translocation principally by phloem. Detailed analyses of QA in transport fluids and component organs were extended to reciprocal homo- and hetero-grafts using bitter (cv. Fest) and sweet (cv. Danja) cultivars of L. angustifolius L. These data confirmed that the majority of QA were synthesized in shoot tissues. In both lupin species feeding and analysis of deuterated QA (lupanine and 13-hydroxylupanine) were used as tracers to demonstrate direct redistribution of alkaloids by translocation from mature leaves in phloem.     

Nitrate uptake,nitrate reductase distribution and their relation to proton release in five nodulated grain legumes

Nitrate uptake, nitrate reductase activity (NRA) and net proton release were compared in five grain legumes grown at 0.2 and 2 mM nitrate in nutrient solution. Nitrate treatments, imposed on 22-d-old, fully nodulated plants, lasted for 21 d. Increasing nitrate supply did not significantly influence the growth of any of the species during the treatment, but yellow lupin (Lupinus luteus) had a higher growth rate than the other species examined. At 0.2 mM nitrate supply, nitrate uptake rates ranged from 0.6 to 1.5 mg N g(-1) d(-1) in the order: yellow lupin > field pea (Pisum sativum) > chickpea (Cicer arietinum) > narrow-leafed lupin (L angustifolius) > white lupin (L albus). At 2 mM nitrate supply, nitrate uptake ranged from 1.7 to 8.2 mg N g(-1) d(-1) in the order: field pea > chickpea > white lupin > yellow lupin > narrow-leafed lupin. Nitrate reductase activity increased with increased nitrate supply, with the majority of NRA being present in shoots. Field pea and chickpea had much higher shoot NRA than the three lupin species. When 0.2 mM nitrate was supplied, narrow-leafed lupinreleased the most H+ per unit root biomass per day, followed by yellow lupin, white lupin, field pea and chickpea. At 2 mM nitrate, narrow-leafed lupin and yellow lupin showed net proton release, whereas the other species, especially field pea, showed net OH- release. Irrespective of legume species and nitrate supply, proton release was negatively correlated with nitrate uptake and NRA in shoots, but not with NRA in roots.     

Aphids do not avoid resistance in Australian lupin (Lupinus angustifolius, L. luteus) varieties

Laboratory bioassays and field trials were used to characterize resistance to three aphid species (Myzus persicae (Sulzer), Acyrthosiphon kondoi Shinji, Aphis craccivora (Koch) in two aphid-resistant varieties (Kalya, Tanjil) and one susceptible variety (Tallerack) of Lupinus angustifolius L., and in one resistant variety (Teo) and one susceptible variety (Wodjil) of L. luteus L. Host selection tests in the glasshouse showed that alates of all three species preferred L. luteus to L. angustifolius, but provided no evidence that alates selected susceptible varieties over resistant. These results were supported by a field trial, which showed no difference in the number of colonizing A. kondoi alates collected from the resistant and susceptible lines of each lupin species, but there were significantly more late-instar nymphs and apterous adults on the susceptible lines. In laboratory host suitability experiments, there was much greater suppression of aphid growth and survival on Teo than on Kalya and Tanjil. In field trials, the numbers of aphids were generally lower on resistant compared to susceptible lines of both lupin species with one notable exception: M. persicae numbers were not lower on the resistant variety Tanjil compared to the susceptible variety Tallerack (L. angustifolius). These results suggest that the resistance mechanisms in both lupin species do not affect the selection of hosts by colonizing aphids, but rather are affecting the growth, survival and possibly reproduction of aphids after settling.     

Studies on the distribution of Na and CI in two species of lupin (Lupinus luteus and Lupinus angustifolius) differing in salt tolerance

The distribution of K, Na and CI in various tissues was studied in two species of lupin, Lupinus luteus L. (ex. Portugal) and L. angustifolius L. (cv. Kubesa) under conditions of NaCl-induced salinity stress. L. luteus appeared more tolerant to salt and less effective in excluding Na and Cl from its above-ground parts than L. angustifolius . Electron probe X-ray microanalysis of vacuolar contents of individual root cells of salt-treated L. luteus showed a decreasing gradient of Na and CI contents from the epidermis inwards, but in the inner cortical cells adjacent to the pericycle/endodermis, Na levels were again high while Cl remained low. L. luteus may be inefficient in restricting entry of Na into the shoot because of a limited capacity for Na storage in the vacuoles of root cells. In addition, this species appears to exclude Cl from the vacuoles of inner cortical root cells but not from its symplastic pathway. In the leaves of L. luteus , Cl levels were particularly high towards the lower surface (abaxial side), while Na accumulated in the adaxial side of the leaf. Likewise, different cells of the petiole appeared to place unequal demands on Na and Cl for osmotic adjustment. It is suggested that excess Na in the adaxial side of the leaf and the generally high levels of Na and CI in the foliage may explain the 50% fresh weight stimulation which was observed in L. luteus but not in L. angustifolius at 50 m M NaCl. However, the halophytic response of L. luteus is limited to moderate concentrations of NaCl since 100 m M NaCl caused severe necrosis and leaf abscission.     

Cross compatibility between European and American Lupinus species

The results of attempted hybridization between European species of large-grained species of Lupinus, L. albus, L. angustifolius and L. luteus and between these and the South American species L. mutabilis are described. Pollen-tube growth in interspecific combinations differed significantly from those after self-pollination only when L. luteus , was the female parent. All combinations Involving L. mutabilis pollen exhibited unilateral compatible pollen-tube growth. Substantial evidence of ovule stimulation and enhanced pod retention was recorded in crosses between the European species and L. mutabilis , but viable seeds were not obtained. The indications, from pollen-tube growth and pod retention in certain crosses, are that genetical isolation between some of the species could be overcome.     

Rapid separation of tyrosine-specific tRNA from white lupin.

During isolation of total ribonucleic acids from white lupin (Lupinus albus) and their subsequent separation by 10% polyacrylamide gel electrophoresis, a fast migrating RNA band is very well separated. The nucleotide sequence analysis of 76 nucleotide long sequence with many modified nucleosides was found to be identical with that of tyrosine specific tRNA of yellow lupin seeds (Lupinus luteus) and wheat germ (Triticum aestivum). Also this tRNA(Tyr) is identical with plant amber suppressor tRNA. The presented approach offers a very rapid method of purification of plant tRNA with UAG suppressor activity.     

Studies on shoot regeneration of lupins (Lupinus spp.)

Tests have been made to regenerate plants from different lupin species using leaf, leaf petiole and hypocotyl tissue of L. polyphyllus, L. hartwegii, L. angustifolius and L. luteus. Callus induction rates have been high with all plant parts from all tested lupin species. Regeneration success has been rather limited. One plant could be regenerated from hypocotyl material of L. polyphyllus. Only shoots appeared when using leaf explants from L. luteus or L. hartwegii as well as from leaf petiole tissue of L. hartwegii. With L. angustifolius one regeneration of unknown character occurred.     

European yellow lupine, Lupinus luteus, and narrow-leaf lupine, Lupinus angustifolius, as hosts for the pea aphid, Acyrthosiphon pisum

The pea aphid, Acyrthosiphon pisum Harris (Homoptera: Aphididae), fed, developed, and reproduced on yellow lupine, Lupinus luteus L. (Fabaceae: Genisteae). No clear preferences for any variety within L. luteus were found. Acyrthosiphon pisum showed negative values of relative growth rate and no aphid completed development on any variety of narrow-leaf lupine Lupinus angustifolius L. Aphids did not ingest phloem sap while probing on L. angustifolius and the probes were very short. All varieties of L. angustifolius were rejected by aphids during an early stage of probing in peripheral tissues, that is, epidermis or mesophyll. There were qualitative and quantitative differences in alkaloid and soluble sugar content between the two lupine species. Within species, the relative content of individual compounds differed among the varieties. Lupinus angustifolius contained four quinolizidine alkaloids (13-hydroxylupanine, dehydrolupanine, lupanine, and angustifoline), while L. luteus contained two (lupanine and sparteine). Lupanine occurred in all varieties of both lupine species. The total content of soluble carbohydrates was similar in L. luteus and L. angustifolius . The following cyclitols were found in both lupine species: myo -inositol, D-ononitol, and D-pinitol. Lupinus angustifolius also contained D- chiro -inositol. The study of aphid probing behaviour, development, and reproduction demonstrated that L. luteus is a suitable host plant for A. pisum while L. angustifolius is not. It is likely that the rejection of L. angustifolius by A. pisum was caused by chemical factors detected by aphids at the epidermis and mesophyll level.     

Screening of cytoplasmic DNA diversity between and within Lupinus mutabilis Sweet and Lupinus albus sensu lato by restriction fragment length polymorphism (RFLP)

Seven populations and five mutant lines of the Andean lupin and four species from the section Albus were screened for their mitochondrial and chloroplast polymorphisms. For this purpose the RFLP method with EcoRI as a restriction enzyme was used. Lupinus luteus, Lupinus albus and Phaseolus vulgaris organellar clones as well as amplified fragments were used as probes. We found that mitochondrial probes were more suitable than chloroplast probes for identification of inter- and intra-specific variations within the examined material. Most mitochondrial probes differentiate the two species investigated. A high level of mitochondrial polymorphism was observed among the populations of L. mutabilis in contrast to monomorphism among the species in the section Albus. A limited polymorphism was detected between the mutant lines of L. mutabilis. We conclude from this study that the mitochondrial RFLP analysis is a valuable tool for identification of variability among Andean lupin populations.     

Lupinus luteus cv. Wodjil takes up more phosphorus and cadmium than Lupinus angustifolius cv. Kalya

Plant and Soil - A field experiment on an acidic lateritic ironstone gravel sand in south-western Australia compared how Lupinus luteus L. cv. Wodjil and L. angustifolius L. cv. Kalya used...     

Characterization and benefits of selenium uptake by an Astragalus hyperaccumulator and a non-accumulator

Plant and Soil - The benefits of biological nitrogen fixation (BNF) for blue lupin (Lupinus angustifolius L.) are well known. However, it is unclear how multiple microorganisms, other than plant...     

Genetic diversity analysis for narrow-leafed lupin (Lupinus angustifolius L.) by SSR markers

Molecular Biology Reports - Narrow-leafed lupin (Lupinus angustifolius L.) is used as grain legumes, fodder for livestock and green manure in the world and has a great potential to be developed as...     

The interaction of phosphorus and potassium with seed alkaloid concentrations, yield and mineral content in narrow-leafed lupin (Lupinus angustifoliusL.)

We tested the impact of P deficiency, K deficiency, and their interaction on seed alkaloid concentrations and profile, yield and mineral content in sweet (low-alkaloid) and bitter (high-alkaloid) varieties of narrow-leafed lupin (Lupinus angustifolius L.). P deficiency reduced seed alkaloid concentrations in sweet, but not in bitter, varieties. Under P deficiency, the alkaloid profile in harvested seed of sweet varieties mimicked that of the bitter variety Fest, with 13-hydroxylupanine dominating over lupanine. With adequate or abundant P, lupanine was the predominant alkaloid in sweet varieties. K deficiency was associated with an 8-fold increase of seed alkaloid concentrations in the sweet variety Danja (from 1000 to 8000 mg kg^–1 DM), mostly due to the stimulation of lupanine production. There was a significant interaction between P and K that affected seed alkaloid concentrations in two ways: (i) the inhibitory effect of P deficiency was only apparent under K deficiency and (ii) the lowest seed alkaloid concentrations occurred with abundant K (240 mg K kg^–1) and P (60 mg P kg^–1). Seed yield of all varieties increased asymptotically with increasing P and reached a maximum at adequate P (30 mg P kg^–1). There was no impact of K deficiency on seed yield. In sweet and bitter varieties P supply increased seed N, P and Zn concentrations, but not K. In contrast, seed K concentrations increased and P concentrations decreased with increasing K supply. These findings suggest that P fertiliser should be supplemented with K, to avoid high seed alkaloid concentrations stimulated by asymptomatic K deficiency at high P levels.     

Potassium nutrition effects on seed alkaloid concentrations,yield and mineral content of lupins (Lupinus angustifolius)

To ensure that narrow-leafed lupin (Lupinus angustifolius L.) meets feed quality standards, the concentration of alkaloids must be kept under the maximum acceptable limit of 200 mg kg^–1 DM. One of the factors that may affect seed alkaloid concentration is soil nutrient deficiency. In this paper, we report the results of glasshouse and field experiments that tested the effect of potassium (K) deficiency on seed alkaloid concentrations. In the glasshouse, seed alkaloid concentrations increased by 385, 400 and 205% under severe K deficiency in sweet varieties (Danja, Gungurru and Yorrel, respectively) of L. angustifolius. The concentration of alkaloids in Fest, the bitter variety, was always high regardless of soil K status. At all levels of applied K (0–240 mg kg^–1 soil), lupanine was the predominant alkaloid in sweet varieties, whereas 13-hydroxylupanine prevailed in the bitter variety. Seed yield of all varieties increased exponentially with increasing amounts of applied K, reaching a maximum at 60 mg K kg^–1 soil. In the field, application of K to deficient soils decreased seed alkaloid concentration at Badgingarra, Western Australia (WA) but not at Nyabing, WA, in 1996. In both field trials, seed yield and mineral content were not affected by the amounts of K fertiliser applied. These findings highlighted the need for adequate K fertilisation of deficient soils in WA to avoid the risk of producing low quality lupin seed with high alkaloid concentrations. K deficiency is involved in stimulating alkaloid production in sweet varieties of L. angustifolius.     

Root-induced acidification and excess cation uptake by N2-fixing Lupinus albus grown in phosphorus-deficient soil

Plant and Soil - White lupin plants (Lupinus albus L. cv. Kiev) were grown in soil columns under controlled conditions at 20/12?°C (12/12 h) for 76 d to investigate the effect of...     

Quinolizidine alkaloid profiles of Lupinus varius orientalis, L. albus albus, L. hartwegii, and L. densiflorus

Alkaloid profiles of two Lupinus species growing naturally in Egypt (L. albus albus [synonym L. termis], L. varius orientalis) in addition to two New World species (L. hartwegii, L. densiflorus) which were cultivated in Egypt were studied by capillary GLC and GLC-mass spectrometry with respect to quinolizidine alkaloids. Altogether 44 quinolizidine, bipiperidyl and proto-indole alkaloids were identified; 29 in L. albus, 13 in L. varius orientalis, 15 in L. hartwegii, 6 in L. densiflorus. Some of these alkaloids were identified for the first time in these plants. The alkaloidal patterns of various plant organs (leaves, flowers, stems, roots, pods and seeds) are documented. Screening for antimicrobial activity of these plant extracts demonstrated substantial activity against Candida albicans, Aspergillus flavus and Bacillus subtilis.     

Activation of isoflavone biosynthesis in excised cotyledons of Lupinus seedlings by jasmonoids and excess light

Exogenous jasmonic acid (JA) and methyl jasmonate (MJ) induced accumulation of isoflavone constituents in cotyledons prepared from imbibed seeds of white lupin (Lupinus albus L.). Exogenous 0.2 mM MJ enhanced the levels of 7-O-(6"-O-malonyl)glucosylgenistein and 7-O-glucosylgenistein in the cotyledons of etiolated seedlings that had been incubated in the dark for 48 h. Regarding isoflavone induced by excision and slicing in the cotyledons as background level, the effect of light was 2- to 3-fold higher than that of 0.2 mM MJ. Cotyledons exposed to MJ along with a 24-h light period displayed a higher level of isoflavone accumulation than that of light alone. Total molar amounts of isoflavone accumulated in the cotyledons treated with MJ under continuous light were approximately the sum of those induced by MJ alone and light alone, respectively. The additive-like effect of MJ and light on isoflavone accumulation in lupin tissues suggested the presence of two different signaling systems independently responsible for those two stimuli. Excised cotyledons from etiolated yellow lupin (L. luteus L. cv. Topaz) seedlings also supported this hypothesis. The cotyledons could accumulate both an isoflavone and a flavone, and MJ selectively increased some of the isoflavone constituents, whereas light enhanced the levels of both. The selective accumulation mechanism of isoflavonoids in cotyledons, in which jasmonoids are involved, clearly differed from that activated by light.