Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius

Following uptake of [(3)H]zeatin riboside and [(3)H]dihydrozeatin riboside by girdled lupin (Lupinus angustifolius L.) stems via the transpiration stream, rapid lateral movement of the radioactivity from xylem to bark was observed. Short-term studies with intact stems, and other studies with excised stem tissues, revealed that the ribosides and/or the corresponding nucleotides were the cytokinin forms which actually moved into the bark tissues. Relative to cytokinin metabolism in xylem plus pith, metabolism in bark was both more rapid and more complex. Riboside cleavage and formation of the O-acetylzeatin and O-acetyldihydrozeatin ribosides and nucleotides were almost completely confined to bark tissues. Exogenous (3)H-labelled O-acetylzeatin riboside was converted to zeatin riboside in bark tissue, but the presence of the acetyl group suppressed degradation to adenine metabolites. The sequestration and modification of xylem cytokinins by stem tissues probably contributes significantly to the cytokinin status of the shoot. New cytokinins identified by mass spectrometry in lupin were: O-acetyldihydrozeatin 9-riboside, a metabolite of exogenous dihydrozeatin riboside in stem bark; O-methylzeatin nucleotide and O-methyldihydrozeatin 9-riboside, metabolites of endogenous cytokinins in stem bark; O-methylzeatin nucleotide and O-methylzeatin 9-riboside, metabolites of exogenous zeatin riboside in excised pod walls.     

Kinship rivalry does not trigger specific allocation strategies in Lupinus angustifolius

Background and Aims

Research on the ability of plants to recognize kin and modify plant development to ameliorate competition with coexisting relatives is an area of very active current exploration. Empirical evidence, however, is insufficient to provide a sound picture of this phenomenon.

Methods

An experiment was designed to assess multi-trait phenotypic expression in response to competition with conspecifics of varied degrees of genealogical relatedness. Groups of siblings, cousins and strangers of Lupinus angustifolius were set in competition in a pots assay. Several whole-plant and organ-level traits, directly related to competition for above- and below-ground resources, were measured. In addition, group-level root proliferation was measured as a key response trait to relatedness to neighbours, as identified in previous work.

Key Results

No major significant phenotypic differences were found between individuals and groups that could be assigned to the gradient of relatedness used here. This occurred in univariate models, and also when multi-trait interactions were evaluated through multi-group comparisons of Structural Equation Models. Root proliferation was higher in phenotypically more heterogeneous groups, but phenotypic heterogeneity was independent of the relatedness treatments of the experiment, and root proliferation was alike in the neighbourhoods of siblings, cousins and strangers.

Conclusions

In contrast to recent findings in other species, genealogical relatedness to competing neighbours has a negligible impact on the phenotypic expression of individuals and groups of L. angustifolius. This suggests that kin recognition needs further exploration to assess its generality, the ecological scenarios where it might have been favoured or penalized by natural selection, and its preponderance in different plant lineages.     

High pH in the nutrient solution impairs water uptake in Lupinus angustifolius L.

Root growth in Lupinus angustifolius is greatly decreased when the nutrient solution has a pH above 6.0. This study examined the water relations in this species (cv. Yandee) in response to high pH in solution culture in a glasshouse.The dry weight of roots, the length of taproots and lateral roots and the number of lateral roots were significantly reduced at day 12 after transfer to solution with a pH of 7.5 compared to pH 5.2. This resulted in a marked reduction of total root surface area. However, shoot growth and total leaf area were not affected. In comparison with pH 5.2, plants grown at pH 7.5 in the nutrient solution had a 14–38% more-negative leaf water potential, and their stomatal resistance had increased by 67%.The observations indicate that the impairment of the water relations by high pH is mainly caused by decreased root growth.     

Flavone C-glycosides from Lupinus hartwegii

From the aerial parts of Lupinus hartwegii, two new flavone C-glycosides apigenin-7-O-beta-apiofuranosyl-6, 8-di-C-beta-glucopyranoside (1) and apigenin-7-O-beta-apiofuranosyl-6-C-beta-glucopyranosyl-8-C-(6z.qprime;-O-E-feruloyl)- beta-glucopyranoside (2) have been isolated together with two known isoflavonoid glucosides genistein-7-O-beta-glucopyranoside (3) and genistein-7, 4'-di-O-beta-glucopyranoside (4) as well as two known compounds ferulic acid 4-O-beta-glucopyranoside (5) and sparteine (6). The structures of the isolated compounds were verified by means of MS and NMR spectral analyses.     

Interactive effect of cytokinin and potassium on sink-source relationships in Lupinus angustifolius

This study examined whether increased K supply in conjunction with BAPcould increase lupin seed yield and harvest index by enlarging sink volume (podnumber), increasing assimilate and improving assimilate partitioning to filltheadditional pods induced by BAP treatment. Narrow-leafed lupin(Lupinusangustifolius, cv. Danja abs^– mutant) was grown inaglasshouse, in pots containing sandy soil with four K treatments (0, 15, 60 and120 mg K/kg soil). BAP (2 mM) was applied daily toallmain stem flowers throughout the life of each flower from opening to senesced.BAP application did not affect assimilate production (as measured by totalabove-ground biomass), but changed assimilate partitioning. On BAP-treatedplants, there were greater proportions of seed to pod wall dry weight on themain stem but smaller proportions on the branches, and an increased weightratioof seed to pod wall overall which meant more assimilate was used for seedgrowthrather than pod wall growth. BAP increased the number of pods per plant by35% and this more than compensated for the decreases in seeds per podandseed weight. Therefore, there was an increased harvest index (+11%)and seed yield per plant (+13%) in BAP-treated plants. BAP alsoincreased the number of pods with filled seeds (146%) on the main stemand main stem seed K⁺ concentration (from 0.81% to0.87%). Added K increased biomass but only slightly affected assimilatepartitioning. As applied K increased, relatively more assimilate was used forpod wall growth rather than seed growth. Added K increased seed yield per plantby about 14% due to increases in seed weight and the number of pods onthe main stem. Moreover, K⁺ concentration in seeds and shootsincreased with increasing level of applied K. Seed yield was enhanced more byBAP when K was supplied at high levels. Increasing K supply interactedpositively with added BAP by increasing narrow-leaf lupin seed yield andharvestindex through increases in assimilate supply and its partitioning into seeds.     

Triterpenoidal lupin saponins from the Chilean legume Lupinus oreophilus Phil

Two lupin saponins, 3beta,21alpha,22beta,24-tetrahydroxyolean-12-en-3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl-21-O-alpha-L-arabinopyranoside and 3beta,21alpha,22beta,24-tetrahydroxyolean-12-en-3-O-beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl-21-O-alpha-L-rhamnopyranoside, along with eight other saponins and one triterpene previously reported from other legumes, were isolated from the aerial parts of Lupinus oreophilus collected in northern Chile. The structures of the isolated compounds were established with the help of extensive spectroscopic techniques.     

Triterpene glycosides with in vitro anti-inflammatory activity from Cyclamen repandum tubers

A new triterpene glycoside, repandoside (1) was isolated together with six known saponins (2-7) from the methanol extract of Cyclamen repandum tubers. The isolated saponins were characterized by high resolution mass spectrometry and both 1D and 2D NMR experiments. The in vitro effect of saponins on LPS-induced IL-8 and TNF-α mRNA level (by quantitative RT-PCR) and protein release (by ELISA) was evaluated in human THP-1 macrophages. We found that compounds 1 (repandoside), 2 (deglucocyclamin) and 4 (anagalloside B) at 100 μM inhibited the LPS-induced IL-8 and TNF-α expressions.     

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.     

The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase

Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage. Peptides were purified and sequenced. An oligonucleotide probe was constructed for the sequence MPGQW. This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322. pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (>90%) and nucleotide (>80%) level. Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner. Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.     

The signal peptide NPFSD fused to ricin A chain enhances cell uptake and cytotoxicity in Candida albicans

Microorganisms possess stringent cell membranes which limit the cellular uptake of antimicrobials. One strategy to overcome these barriers is to attach drugs or research reagents to carrier peptides that enter cells by passive permeation or active uptake. Here the short endocytosis signal peptide NPFSD was found to efficiently deliver both FITC and GFP into Saccharomyces cerevisiae and Candida albicans with uptake into the majority of cells in a population. The NPFSD signal is itself non-toxic, but when fused to the ricin A chain toxin (RTA) the peptide enhanced both cell uptake and toxicity against C. albicans, which like other yeasts is resistant to naked RTA. Cell entry required at least 1 h incubation, temperatures above 4 degrees C, and an energy source, and uptake was out-competed with free peptide. Therefore, the NPFSD peptide can carry a range of compounds into yeasts and this delivery route holds promise to enhance the activity of antifungals.     

Nitrogen accumulation in sole and mixed stands of sweet-blue lupin (Lupinus angustifolius L.), ryegrass and oats

Nitrogen fixation was measured in monocropped sweet-blue lupin (Lupinus angustifolius), lupin intercropped with two ryegrass (Lolium multiflorum) cultivars or with oats (Avena sativa) on an Andosol soil, using the ¹⁵N isotope dilution method. At 117 days after planting and at a mean temperature below 10°C, monocropped lupin derived an average of 92% or 195 kg N ha⁻¹ of its N from N₂ fixation. Intercropping lupin with cereals increased (p<0.05) the percentage of N derived from atmospheric N₂ (% Ndfa) to a mean of 96%. Compared to the monocropped, total N fixed per hectare in intercropped lupin declined approximately 50%, in line with the decrease in seeding rate and dry matter yield. With these high values of N₂ fixation, selection of the reference crop was not a problem; all the cereals, intercropped or grown singly produced similar estimates of N₂ fixed in lupin. It was deduced from the ¹⁵N data that significant N transfer occurred from lupin to intercropped Italian ryegrass but not to intercropped Westerwoldian ryegrass or to oats. Doubling the ¹⁵N fertilizer rate from 30 to 60 kg N ha⁻¹ decreased % Ndfa to 86% (p<0.05), but total N fixed was unaltered. These results indicate that lupin has a high potential for N₂ fixation at low temperatures, and can maintain higher rates of N₂ fixation in soils of high N than many other forage and pasture legumes.     

Triterpenoid glycosides from Bacopa monnieri

Two triterpenoid glycosides have been isolated along with 10 known saponins from Bacopa monnieri. Structures of the compounds have been elucidated as 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl] jujubogenin (1) and 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl] pseudojujubogenin (2) by high resolution NMR spectral data and chemical correlations. Further, the chemical compositions of bacosides A and B have been delineated.     

New pregnane glycosides from Caralluma dalzielii

Twenty-seven new pregnane glycosides were isolated from the whole plant of Caralluma dalzielii, and their structures elucidated from extensive 2D NMR analysis as well as ESI-MS experiments. All isolated compounds were tested for their antiproliferative activity on J774.A1, HEK-293, and WEHI-164 cell lines. Moderate to high potency of cytotoxicities were found in almost all tested compounds, confirming the significant cytotoxic activity of pregnane glycosides.     

Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans

Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.     

Polyunsaturated fatty acids cause apoptosis in C. albicans and C. dubliniensis biofilms

Background

Polyunsaturated fatty acids (PUFAs) have antifungal properties, but the mode by which they induce their action is not always clear. The aim of the study was to investigate apoptosis as a mode of action of antifungal PUFAs (stearidonic acid, eicosapentaenoic acid and docosapentaenoic acid) which are inhibitory towards biofilm formation of C. albicans and C. dubliniensis.

Methods

Candida biofilms were grown in the absence or presence of 1 mM PUFAs (linoleic acid, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid) for 48 h at 37 °C. The effect of these PUFAs on the membrane fatty acid profile and unsaturation index, oxidative stress, mitochondrial transmembrane potential and apoptosis was evaluated.

Results

When biofilms of C. albicans and C. dubliniensis were exposed to certain PUFAs there was an increase in unsaturation index of the cellular membranes and accumulation of intracellular reactive oxygen species (ROS). This resulted in apoptosis, evidenced by reduced mitochondrial membrane potential and nuclear condensation and fragmentation. The most effective PUFA was stearidonic acid.

Conclusions

The resultant cell death of both C. albicans and C. dubliniensis is due to apoptosis.

General significance

Due to the increase in drug resistance, alternative antifungal drugs are needed. A group of natural antifungal compounds is PUFAs. However, understanding their mechanisms of action is important for further use and development of these compounds as antifungal drugs. This paper provides insight into a possible mode of action of antifungal PUFAs.     

Antifungal mechanism of an antimicrobial peptide, HP (2--20), derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans

The antifungal activity and mechanism of HP (2-20), a peptide derived from the N-terminus sequence of Helicobacter pylori Ribosomal Protein L1 were investigated. HP (2--20) displayed a strong antifungal activity against various fungi, and the antifungal activity was inhibited by Ca(2+) and Mg(2+) ions. In order to investigate the antifungal mechanism(s) of HP (2-20), fluorescence activated flow cytometry was performed. As determined by propidium iodide staining, Candida albicans treated with HP (2-20) showed a higher fluorescence intensity than untreated cells and was similar to melittin-treated cells. The effect on fungal cell membranes was examined by investigating the change in membrane dynamics of C. albicans using 1,6-diphenyl-1,3,5-hexatriene as a membrane probe and by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w) and by treating protoplasts of C. albicans with the peptide. The action of peptide against fungal cell membrane was further examined by the potassium-release test, and HP (2-20) was able to increase the amount of K(+) released from the cells. The result suggests that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membrane via pore formation or directly interacts with the lipid bilayers in a salt-dependent manner.     

Alkaloid level in narrow-leafed lupin, Lupinus angustifolius, influences green peach aphid reproductive performance

Two viviparous parthenogenetic clones of the green peach aphid, Myzus persicae (Sulzer), one collected from Rydalmere, New South Wales (NSW), and the other from South Perth (SP), Western Australia, were reared on radish, Raphanus sativus L. cv. Scarlet Globe, under controlled conditions. The NSW clone was fed on simple artificial diets containing alkaloid extracted from narrow-leafed lupin, Lupinus angustifolius L. cv. Fest., and its reproductive performance monitored over 112 h. Forty (40) h into the experiment and thereafter, aphids on the control diet (sucrose solution) produced significantly more offspring (P<0.05) than those on diets containing alkaloid. In a separate experiment, apterae of each clone were caged on three lines (cv. Yorrel, cv. Danja and 84L:441) of narrow-leafed lupin, and allowed to reproduce. The first three offspring were retained, and all developed to 3rd or 4th instar stage. Two nymphs were removed, and the remaining nymph reared through. All three lines produced adults. The number of young produced were counted over 11 days. Fecundity of the SP clone was lower on line 84L:441, but there was no difference in the fecundity of the NSW clone. Phloem exudate and green tissue was concomitantly collected from all lines, and analysed by GC-MS for the alkaloids lupanine and 13-hydroxylupanine. Line 84L:441 contained the highest level of total alkaloids in both phloem and tissue. All experiments indicate that alkaloid level may suppress fecundity of green peach aphids.     

Triterpenoid saponins and phenylethanoid glycosides from stem of Akebia trifoliata var. australis

A detailed phytochemical study on the 70% aqueous ethanol extract of stems of Akebia trifoliata (Thunb.) Koidz. var. australis (Diels) Rehd led to isolation of five compounds, together with 12 known triterpenoid saponins and three known phenylethanoid glycosides. The structures of the five compounds were elucidated on the basis of analysis of spectroscopic data and physicochemical properties as: 2alpha, 3beta, 23-trihydroxy-30-norolean-12-en-28-oic acid beta-D-glucopyranosyl ester (1), 2alpha, 3beta, 23-trihydroxy-30-norolean-12-en-28-oic acid beta-D-xylopyranosyl-(1-->3)-O-alpha-D-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl ester (2), 2alpha, 3beta, 23-trihydroxyurs-12-en-28-oic acid beta-D-xylopyranosyl-(1-->3)-O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl ester (3), 3-beta-[(beta-D-glucopyranosyl-(1-->3)-O-alpha-L-arabinopyranosyl)oxy]-23-hydroxy-30-norolean-12-en-28-oic acid alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl ester (4) and 3-beta-[(alpha-L-xylopyranosyl-(1-->2)-O-alpha-L-arabinopyranosyl)oxy]-30-norolean-12-en-28-oic acid alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl ester (5), named mutongsaponin A, B, C, D and E, respectively.