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.     

Profiling isoflavone conjugates in different organs of Lupinus exaltatus Zucc

The profiles of isoflavone conjugates in extracts obtained from different parts of Lupinus exaltatus Zucc. grown in Mexico were compared using HPLC-UV and HPLC-ESI/MSn. Collision-induced dissociation-MSn experiments were performed using an ion trap analyser during HPLC-ESI/MS analyses. Nineteen isoflavone conjugates were identified in samples obtained from air-dried roots, leaves, stems and inflorescences of lupin plants. It was possible to determine the structures of the studied compounds on the basis of the MS recorded. The compounds identified were di- and mono-glucosides of genistein and 2'-hydroxygenistein with a different pattern of C- and O-glycosylation. Some glucosides were acylated with malonic acid. It was not possible to establish the glycosylation sites on the basis of MS alone; however, it was possible to differentiate isoflavone C- and O-glucosides. The highest levels of isoflavones and their conjugates were detected in roots and the lowest in stems. Free aglycones were identified in roots and inflorescences but they were not found in stems and leaves.     

Iron deficiency induces changes in metabolism of citrate in lateral roots and cluster roots of Lupinus albus

This paper describes the first measurement of enzyme activities in cluster roots under –Fe stress, at different stages of cluster root development and function. In Lupinus albus L., Cluster roots are produced both under iron- and phosphorus-deficient conditions. In both cases the structure is similar, but the level of exudation is much greater in iron-deficient plants. Much work has been done on the enzyme kinetics of P-deficient cluster roots, but none on enzyme activities of Fe-deficient cluster roots. The enzymes investigated were citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), isocitrate dehydrogenase [IDH(NAD) (EC 1.1.1.41) and IDH (NADP) (EC 1.1.1.42)] and lactate dehydrogenase (LDH) (EC 1.1.1.27). In cluster roots, citrate synthase activity was initially lower than in lateral roots but, after 5 days, recovered to the lateral root level. Cluster root aconitase levels initially increased, but fell sharply on day 3, and no activity was detected after day 5. IDH (NAD) levels were much lower in cluster roots than in laterals, dropping to a low on day 3, and then rising throughout development. IDH (NADP) levels were always higher in cluster roots than in lateral roots, increasing throughout development. LDH levels in cluster roots fell throughout development. Internal tissue concentrations of citrate were markedly higher in –Fe laterals than in +Fe lateral roots and in cluster roots. Cluster root levels of citrate increased dramatically after day 3. Results are discussed within the context of previous work on enzyme kinetics under –P, and the importance of a block in aconitase activity is highlighted.     

Cell wall-associated isoflavonoids and β-glucosidase activity in Lupinus albus plants responding to environmental stimuli

Plants respond to various biotic or abiotic stimuli with different mechanisms and the mobilization of phenolic compounds is one of them. In white lupin (Lupinus albus L.) plants, isoflavones and their glucosides were localized in cell walls where the high constitutive activity of β‐glucosidase (EC 3·2·1·21) was also identified. The enzyme was partially purified from root cell walls. Its polymeric active form has 180 or 200 kDa as determined by non‐denaturing electrophoresis and gel filtration, respectively, and the isoelectric point is at pH 6·9. The enzyme is an exoglucosidase, preferentially hydrolysing conjugates of phenolic compounds with β anomers of glucose. It is not active against purified lupin cell walls. The specific β‐glucosidase activity varies in different tissues with the highest one in roots, and always higher in cell walls than in protoplast. The cell wall location of the enzyme was confirmed biochemically by its activity in intercellular washing fluids. Both aglycones and glycosides were also present in these fluids. The specific β‐glucosidase activity correlated well with the isoflavonoid aglycone/glycoside ratios in various tissues: the higher β‐glucosidase activity, the higher relative aglycone content. β‐glucosidase activity was found to be inducible under conditions of yeast elicitor treatment. Induction of the enzyme was accompanied by changes in the isoflavone secretion and accumulation, suggesting the regulatory role of β‐glucosidase activity in root exudation of isoflavonoids. No correlation however, was found between changes in β‐glucosidase activity and the presence of isoflavonoids in root exudates of plants grown under various nitrogen nutrition regimes.     

Effect of drought and rewatering on the metabolism of Lupinus albus organs

Alterations in the metabolism of Lupinus albus organs that result from and subsequently follow a period of severe water deficit (WD) are described. By means of 13C-nuclear magnetic resonance (NMR), changes in the major metabolites were monitored in several plant organs (leaflets and petiole, roots, stem stele and cortex). During the stress, most of the leaves were lost and the stem functioned as a storage repository of sugars (glucose and sucrose) and amino acids (asparagine and proline). Upon rewatering, lupin plants rapidly re-established the relative water content (RWC) and produced new leaves. However, at the metabolic level, the events seem to be more complex, since proline (a stress related metabolite) disappeared rapidly while sugars and asparagine reached the initial pattern more slowly, particularly in the stem.     

Biosynthesis, accumulation and secretion of isoflavonoids during germination and development of white lupin (Lupinus albus L.)

A combination of ¹⁴C labelling experiments of white lupin seedlings(Lupinus albus L.) and high pressure liquid chromatography oftissue extracts indicated active biosynthesis of isoflavonoidsduring the first 2–4 d of seed germination. These weresynthesized mostly as glucosides and to a lesser extent as aglycones,with trace amounts of prenylated derivatives. There was a generaldecrease in total isoflavonoids during later stages of germination(c. until d 13), which may be ascribed to their turnover and/orexudation into the rhizosphere. In addition, exudation of isoflavonoidsby lupin seeds germinated under sterile conditions continuesfor 12 d with a preferential release of monoprenylated compounds.The relationship between the specific developmental events duringseed germination and the accumulation of certain groups of isoflavonoidsis discussed in relation to their possible role in the growthprocesses of lupin. Key words: Isoflavonoids, glucosides, aglycones, prenylated derivatives, root exudates, white lupin     

Activation of phenylpropanoid pathway in legume plants exposed to heavy metals. Part II. Profiling of isoflavonoids and their glycoconjugates induced in roots of lupine (Lupinus luteus) seedlings treated with cadmium and lead

We examined changes in profiles of isoflavonoids in roots of lupine (Lupinus luteus L. cv. Juno) seedlings in response to treatment with two heavy metals: cadmium (at 10 mg/l) and lead (at 150 mg/l). Overall, 21 flavonoid conjugates were identified in root extracts, some of them with up to six positional isomers. The total amount of all isoflavonoids increased by about 15 % in cadmium-treated plants and by 46 % in lead-treated ones. Heavy metals markedly increased the content of two compounds: 2'-hydroxygenistein glucoside and 2'-hydroxygenistein 7-O-glucoside malonylated. Possible functions of the identified isoflavonoids in yellow lupine exposed to heavy metal stress are discussed.     

Organogenesis from hypocotyl thin cell layers of Lupinus mutabilis and Lupinus albus

When thin cell layers (TCLn), isolated from the nodalregion of the hypocotyl of 5 day old seedlings, werecultured in medium M1 with IAA and BA, entire plantswere obtained from L. mutabilis explants andprolific shoots from L. albus explants.Histological studies carried out on L. mutabilisTCLn showed that meristematic cells, present in theexplants at the time of inoculation, gave rise to theformation of a primary meristem resulting inorganogenesis. In TCLn where apical or axillarymeristematic cells were absent, we observed theformation of a nodular structure with a cambium-likelayer at its periphery which divided to form a primarymeristem leading to organogenesis. Plant regenerationfrom TCLn may be useful in genetic transformationassays.     

Characterization of isoperoxidase-B2 inactivation in etiolated Lupinus albus hypocotyls

One basic peroxidase isoenzyme, with a pI of 8.8, is present in the intercellular washing fluid in the aerial part of 6-day-old Lupinus albus hypocotyl seedlings. This isoenzyme, called LuP-B2, is the principal soluble component secreted into the apoplastic space and it is a constitutive enzyme along the whole length of etiolated hypocotyl. The enzymatic inactivation process which this apoplastic peroxidase undergoes is described for the first time. The kinetic constants which describe its inactivation by H(2)O(2) in the absence of reductant substrates are determined. LuP-B2 is inactivated in situ and in vitro in a time- and concentration-dependent manner. H(2)O(2) acts as a suicide substrate according to a model previously proposed by us. The constant values calculated are similar to those calculated for the basic isoenzyme of horseradish roots, HRP-C. LuP-B2 presents a k(inact) value of 7.5 x 10(-3) s(-1) and a k(cat) of 6.7 s(-1). This isoenzyme makes 889 catalytic cycles for each inactivation event. The similarity in behavior and the constant values, together with other situations (both are excreted, soluble and constitutive isoenzymes) suggest that the inactivation process could play an important role in plant development and stress situations.     

Alterations in carbon and nitrogen metabolism induced by water deficit in the stems and leaves of Lupinus albus L.

Water deficit (WD) in Lupinus albus L. brings about tissue-specific responses that are dependent on stress intensity. Carbohydrate metabolism is very sensitive to changes in plant water status. Six days from withholding water (DAW), sucrose, glucose and fructose levels of the leaf blade had already increased over 5-fold, and the activities of SS and INV(A) had increased c. 1.5-2 times. From 9 DAW on, when stress intensity was more pronounced, these effects were reversed with fructose and glucose concentrations as well as INV(A) activity dropping in parallel. The stem (specifically the stele) responded to the stress intensification with striking increases in the concentration of sugars, N and S, and in the induction of thaumatin-like-protein and an increase in chitinase and peroxidase. At 13 DAW, the plants lost most of the leaves but on rewatering they fully recovered. Thus, the observed changes appear to contribute to a general mechanism of survival under drought, the stem playing a key role in that process.     

Changes in lipoxygenase activity during seedling development of Lupinus albus

A lipoxygenase preparation was obtained from Lupinus albus seeds and was shown to differ from previously characterized lipoxygenase. This study describes changes in lipoxygenase activity during seedling development of Lupinus albus. The enzyme activity shows a decrease from 0–6 h postgermination (about 15%), is roughly constant or even rises slightly from 6–30 h and then shows a large increase between 30 and 48 h (about 50%). Enzymatically active proteins from 48 h-old seedlings were isolated and the increase of enzyme activity was mainly due to the presence of two components with maximum activity at pH 6 and pH 8.5, respectively. When arachidonic acid was used as substrate, the two enzymatic activities produce 15 HPETE. The increase in lipoxygenase activity during seedling development was inhibited by cycloheximide. Cordycepin appears to have no direct effect on lipoxygenase synthesis in vivo at the studied doses.     

A Cytokinin Complex in the Developing Fruits of Lupinus albus

The apices of Lupinus albus L. were analysed for cytokinin activity at three stages of development. Little cytokinin activity could be detected in the apices at the time of flowering. However, a considerable amount of activity was detected as the fruits developed. Separate analyses of seed and pod material indicated that there was a high level of cytokinin in both these parts of the fruit. After fractionation of the peaks of activity obtained from paper chromatograms on Sephadex LH-20, four peaks of cytokinin activity were recorded. Two of these co-eluted with zeatin and zeatin riboside. A third peak at an elution volume of 360–440 ml could be hydrolysed with β-glucosidase to give activity at elution volumes corresponding to those of zeatin and zeatin riboside. This strongly suggested that both glucosylated zeatin and glucosylated zeatin riboside were present in the developing fruits of L. albus. The fourth peak at an elution volume of 160–280 ml did not disappear upon hydrolysis with β-glucosidase, and it is possible that it represented a nucleotide cytokinin.     

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.     

In vivo metabolism of labelled indole-3-acetic acid during polar transport in etiolated hypocotyls of Lupinus albus: Relationship with growth

The in vivo metabolism of indole-3-acetic acid (IAA) in etiolated hypocotyls of lupin (Lupinus albus L., from Bari, Italy) was investigated by appliying IAA labelled with two radioisotopes ([1-¹⁴C]-IAA+[5-³H]-IAA) to the apical end of decapitated seedlings, followed by extraction of the radioactivity in the different regions along the hypocotyl. This method allowed detection of IAA decarboxylation in zones distant from the cut surface and, therefore, containing intact cells. When IAA was added directly in solution to the cut surface, decarboxylation was high especially in those hypocotyl regions where transient accumulations characteristic of the polar transport of IAA occurred. In 10-day-old seedlings such accumulations were observed both in the elongation zone (2^nd, 3^rd, and 4^th cm) and in the non elongating basal zone (8^th, 9^th and 10^th cm). When the IAA, instead, was applied with an agar block deposited on the cut surface, IAA metabolism (decarboxylation as well as conjugation) was increased but almost exclusively in tissues within 10 mm of the cut surface. In both kinds of experiment, the increase in IAA decarboxylation seemed to coincide with a decrease in the transport of IAA, since in the assay without agar the transient accumulations of radioactivity were probably due to a decrease in the transport velocity, while in the assay with agar the transport intensity was much lower than in the assay without agar. These results point to a competitive relationship between IAA metabolism and transport. Consequently, it is suggested that hypocotyl regions that probably use auxin for development processes (e.g., cell elongation and differentiation) may have a more intense IAA metabolism in parallel with their higher IAA concentrations.     

Ultrastructural changes associated with the accumulation and secretion of sanguinarine in Papaver bracteatum suspension cultures treated with fungal elicitor

Suspension cultures of Papaver bracteatum Arya II Lindl., grown without hormone in the presence of conidial extracts of Verticillium dahliae Kleb., accumulate millimolar quantities of the benzophenanthridine alkaloid, sanguinarine. Under the fluorescence microscope, the elicitor-treated cells display an orange-yellow fluorescence characteristic of sanguinarine, primarily near the periphery of the cells. Electron-microscopic inspection showed the presence of slightly dilated endoplasmic reticulum and of electron-dense protuberances on the tonoplast of large central vacuoles. These osmiophilic aggregates lining the tonoplast bud into spherical bodies, appear to become detached from the membrane and are released into the vacuole. Upon subcellular fractionation of elicited cells on Renografin step gradients, sanguinarine was found to be distributed in all bands but with 86% concentrated in the gradient pellet. Analysis of the pellet by electron microscopy showed that it contained electron-dense fragments similar to the osmiophilic bodies observed on the tonoplast of intact elicited cells. In elicited cell cultures, most of the sanguinarine was recovered from medium in a 100·g sedimenting, cell-free, particulate fraction accounting for as much as 85% of the media sanguinarine and 62% of the total sanguinarine. The sanguinarine-rich 100·g media pellet was determined to be two-thirds protein, one-third RNA and was essentially devoid of phenolics, phospholipid and DNA. The pellet consisted of electrondense material and cytoplasmic remnants resembling those found in the Renografin pellet and tonoplast aggregates of intact cells. When placed under hypotonic conditions or extracted with aqueous buffer, pH 3–11, the pellet did not release sanguinarine. These observations provide evidence for storage of sanguinarine at electron-dense deposits which occur on the tonoplast and as freely floating bodies in vacuoles.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - ER endoplasmic reticulum - HPLC high-pressure liquid chromatography - MRST Murashige and Skoog's revised tobacco medium     

Release of phenols from Lupinus albus L. roots exposed to Cu and their possible role in Cu detoxification

The mechanisms enabling plants to tolerate high concentrations of available Cu in their rhizosphere are still poorly understood. To better understand the mechanisms involved, Lupinus albus L. (white lupin) was grown over 40 days in a hydroponic system compelling roots to develop under sterile conditions in the presence of a nutrient solution containing 0.5, 20 or 62 M Cu. The following parameters were investigated in detail: low molecular weight phenols in nutrient solution (colorimetric assay), high molecular weight phenols in roots and in solution (HPLC-MS, HPLC-UV), pH, redox potential in solution (electrochemistry) and Cu distribution in the plant (AAS) as well as in apical root sections (EDX microanalysis). Finally, in vitro adsorption studies using voltammetry were conducted to evaluate the Cu adsorption behaviour of different phenolic compounds. When exposed to 62 M Cu, biomass production of white lupin was strongly reduced. Plants grown in the presence of 20 M Cu had a similar dry matter production compared to the control plants grown in a 0.5 M Cu solution. However, an increased release of soluble and high molecular weight phenols into the solution was observed. The concentration of polyphenolic compounds in the roots (particularly isoflavonoids like genistein and genistein-(malonyl)-glucoside) was significantly higher for lupins grown in a 20 M Cu solution compared to the control plants. As shown by an in vitro adsorption study, these phenolic compounds can bind Cu ions. In addition, plants exposed to 20 and 62 M Cu cumulated high Cu amounts in root cell walls whereas only low amounts reached the symplasm. Therefore, it is proposed that the complexation of Cu²⁺ ions in the rhizosphere and in the roots apoplasm by phenolic compounds could alleviate Cu-mediated toxicity.     

Short- and long-term effects of dehydroascorbate in Lupinus albus and Allium cepa roots

Administration of 1 mM dehydroascorbate (DHA) results in a rapid and large increase in cellular ascorbate (AA) content in both Lupinus albus L. and Allium cepa L. root tips. Uptake of DHA from the medium occurs at a high rate within 10-12 h of incubation, and is slowed down thereafter. In the first few h, DHA reduction to AA is apparently correlated to GSH depletion and slightly higher DHA reductase activity. DHA incubation also seems to induce new GSH synthesis. Longer DHA incubation (24 h) affects root growth by inhibiting cell proliferation. At this stage, an apparently generalised oxidation of SH-containing proteins is observed in DHA-treated roots. Treatment with 1 mM L-galactono-gamma-lactone, the last precursor of AA biosynthesis, results in an increase in AA content similar to that obtained with DHA, but stimulates growth and affects the redox state of SH-containing proteins in the opposite way. A possible multi-step mechanism of DHA reduction/removal is suggested and the hypothesis that DHA inhibits cell cycle progression by affecting the redox state of SH-containing proteins is discussed.