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     

Acid phosphatase activity in phosphorus-deficient white lupin roots

White lupin ( Lupinus albus L.) develops proteoid roots when grown in phosphorus (P)-deficient conditions. These short, lateral, densely clustered roots are adapted to increase P availability. Previous studies from our laboratory have shown proteoid roots have higher rates of non-photosynthetic carbon fixation than normal roots and altered metabolism to support organic acid exudation, which serves to solubilize P in the rhizosphere. The present work indicates that proteoid roots possess additional adaptations for increasing P availability and possibly for conserving P in the plant. Roots from P-deficient (–P) plants had significantly greater acid phosphatase activity in both root extracts and root exudates than comparable samples from P-sufficient (+P) plants beginning 10 d after emergence. The increase in activity in –P plants was most pronounced in the proteoid regions. In contrast, no induction of phytase activity was found in –P plants compared to +P plants. The number of proteoid roots present was not affected by the source of phosphorus supplied, whether organic or inorganic forms. Adding molybdate to the roots increased the number of proteoid roots in plants supplied with organic P, but not inorganic P. Increased acid phosphatase activity was detected in root exudates in the presence of organic P sources. Native-polyacrylamide gel electrophoresis demonstrated that under P-deficient conditions, a unique isoform of acid phosphatase was induced between 10 and 12 d after emergence. This isoform was found not only within the root, but it comprised the major form exuded from proteoid roots of –P plants. The fact that exudation of proteoid-root-specific acid phosphatase coincides with proteoid root development and increased exudation of organic acids indicates that white lupin has several coordinated adaptive strategies to P-deficient conditions.     

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.     

Whole-plant gas exchange and reductive biosynthesis in white lupin

Simultaneous measurements of CO(2) (CER) and O(2) (OER) exchange in roots and shoots of vegetative white lupin (Lupinus albus) were used to calculate the flow of reducing power to the synthesis of biomass that was more reduced per unit of carbon than carbohydrate. On a whole-plant basis, the diverted reductant utilization rate (DRUR which is: 4 x [CER + OER]) of shoot tissue was consistently higher than that of roots, and values obtained in the light were greater than those in the dark. An analysis of the biomass being synthesized over a 24-h period provided an estimate of whole-plant DRUR (3.5 mmol e(-) plant(-1) d(-1)), which was similar to that measured by gas exchange (3.2 mmol e(-) plant(-1) d(-1)). Given that nitrate reduction to ammonia makes up about 74% of whole-plant DRUR, root nitrate reduction in white lupin was estimated to account for less than 43% of whole-plant nitrate reduction. The approach developed here should offer a powerful tool for the noninvasive study of metabolic regulation in intact plants or plant organs.     

Determination of lead in white lupin by anodic stripping voltammetry

An anodic stripping voltammetry method for the simultaneous determination of trace heavy metals in nutrient solutions, soils and plants has been developed at a hydrodynamic electrochemical sensor. Several parameters were optimized in order to enhance sensitivity. Calibration curves in different media are presented. The study of the uptake of lead by white lupin (Lupinus albus L.) was carried out. Toxicity symptoms were observed and compared with levels of lead measured in roots and leaves.     

Arsenate-induced phytochelatins in white lupin: influence of phosphate status

White lupin ( Lupinus albus L.) is adapted to environments of low pH and low available phosphorus through the development of proteoid roots. The high-affinity phosphate/arsenate uptake system is much less sensitive to downregulation by phosphate in white lupin than in other plants. Arsenate is a phosphate analogue and its toxicity to plants is intimately linked to phosphate nutrition. The synthesis of phytochelatins (PCs) has been proposed as a detoxification mechanism for arsenic (As) in plants. The aim of this research was to study PC production by lupin plants in response to As, and the impact of the arsenate–phosphate interaction on PC production. PCs were the most abundant thiols in white lupin under high As exposure, reaching levels higher than in other plants tested. Together, glutathione (GSH) and PCs were able to complex the majority of As in shoots, while an additional PC-independent mechanism might function in roots. P deficiency increased As concentrations in plant tissues, causing an increase in PC accumulation and an increase in the average size of PCs. A direct relationship was observed between PC concentrations and the level of stress caused by As, i.e. the degree of growth inhibition in plants. This study suggests a key role for PCs and GSH in As detoxification by white lupin, especially in shoots. PC analysis may be useful as an early indicator of As exposure and as a tool to assess the degree of As stress of plants, even under P deficiency.     

Burkholderia species are major inhabitants of white lupin cluster roots

The formation of cluster roots by plants represents a highly efficient strategy for acquisition of sparingly available phosphate. This particular root type is characterized by a densely branched structure and high exudation of organic acids and protons, which are likely to influence the resident bacterial community. Until now, the identity of the bacterial populations living in cluster roots has not been investigated. We applied cultivation-dependent and cultivation-independent methods to characterize the dominant bacterial genera inhabiting the growing cluster roots of white lupin. We observed a high relative abundance of Burkholderia species (up to 58% of all isolated strains and 44% of all retrieved 16S rRNA sequences) and a significant enrichment with increasing cluster root age. Most of the sequences retrieved clustered together with known plant- or fungus-associated Burkholderia species, while only one of 98 sequences was affiliated with the Burkholderia cepacia complex. In vitro assays revealed that Burkholderia strains were much more tolerant to low pH than non-Burkholderia strains. Moreover, many strains produced large amounts of siderophores and were able to utilize citrate and oxalate as carbon sources. These features seem to represent important traits for the successful colonization and maintenance of Burkholderia species in white lupin cluster roots.     

Comparative analysis of the contribution of phytochelatins to cadmium and arsenic tolerance in soybean and white lupin

The biosynthesis of phytochelatins (PCs) plays a crucial role in the detoxification and homeostasis of heavy metals and metalloids in plants. However, in an increasing number of plant species metal(loid) tolerance is not well correlated with the accumulation of PCs: tolerant ecotypes frequently contain lower levels of PCs than non-tolerant ecotypes. In this study we have compared the responses of soybean (Glycine max L. cv. Resnik) and white lupin (Lupinus albus L. cv. Marta) to cadmium and arsenate in order to assess the role of homophytochelatins (hPCs) in the tolerance of soybean to these toxic elements. Soybean plants treated with Cd and As showed a high contribution of homo-glutathione (hGSH) to the pool of thiols in shoots in comparison to white lupin. Higher levels of hPCs in Cd-treated soybeans compared to PCs in lupins did not prevent growth inhibition. In contrast, the role of hPCs in the detoxification mechanism to arsenate in soybean seems to be clearer, showing higher thiol concentrations and lower growth reductions than those present in lupin plants.     

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.     

Citrate-permeable channels in the plasma membrane of cluster roots from white lupin

White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (-P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3- than Cl- (PCit/PCl = 26.3), while ORAC was selective for Cl- over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of -P plants, as well as from normal (noncluster) roots of plants grown with 25 microm phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of -P plants. However, the IRAC current from +P plants occurred less frequently than in the -P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux.     

Cadmium-stress in white lupin: effects on nodule structure and functioning

Cadmium effects on nodule structure and changes in organic and amino acids, proteins, nutrients and some stress indicators were studied in nodules of white lupin (Lupinus albus L., cv. Multolupa). Plants were grown hydroponically on perlite for 49 d with (18 μM) or without Cd in the nutrient solution. Cadmium-treated plants showed decreases in leaf chlorophyll and shoot sucrose concentrations, but sucrose did not change in nodules. Cadmium application produced alterations in nodule cortex and infected zone structure. Furthermore, Cd supply caused a marked decrease in P, K, leghemoglobin, N–amino compounds, malate, succinate and soluble protein in the nodules. Conversely, the levels of lipid peroxidation and total thiols increased strongly. Results obtained suggest that white lupin nodules are Cd sensitive, in spite of Cd sequestering by cell walls and thiols. The main phytotoxic effects of Cd on nodule structure and function were the occlusion with glycoprotein of intracellular spaces of nodule cortex, alterations in symbiosomes, enrichment in Cd of cell walls and oxidative stress. Glycoprotein accumulation and leghemoglobin depletion may be considered useful indicators of Cd stress in white lupin nodules.     

Cadmium in white lupin nodules: Impact on nitrogen and carbon metabolism

The aims of this work were to investigate the microlocalisation of cadmium (Cd) in Lupinus albus L. cv. Multolupa nodules, and to determine its effects on carbon and nitrogen metabolism. Nodulated white lupin plants were grown in a growth chamber with or without Cd (150 μM). Energy-dispersive X-ray microanalysis showed the walls of the outer nodule cortex cells to be the main area of Cd retention, helping to reduce the harmful effect Cd might have on the amount of N₂ fixed by the bacteroids. Sucrose synthase activity declined by 33% in the nodules of the Cd-treated plants, and smaller reductions were recorded in glutamine synthetase, aspartate aminotransferase, alkaline invertase and NADP-dependent isocitrate dehydrogenase activities. The Cd treatment also sharply reduced nodule concentrations of malate, succinate and citrate, while that of starch doubled, but that of sucrose experienced no significant change. In summary, the present results show that white lupins accumulate significant amounts of Cd in their root nodules. However, the activity of some enzymes involved in ammonium assimilation did decline, promoting a reduction in the plant N content. The downregulation of sucrose synthase limits the availability of carbon to the bacteroids, which might interfere with their respiration. Carbon metabolism therefore plays a primary role in the impaired function of the white lupin root nodule caused by Cd, while N metabolism appears to have a more secondary involvement.     

Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin

Release of large amounts of citric acid from specialized root clusters (proteoid roots) of phosphorus (P)-deficient white lupin (Lupinus albus L.) is an efficient strategy for chemical mobilization of sparingly available P sources in the rhizosphere. The present study demonstrates that increased accumulation and exudation of citric acid and a concomitant release of protons were predominantly restricted to mature root clusters in the later stages of P deficiency. Inhibition of citrate exudation by exogenous application of anion-channel blockers such as ethacrynic- and anthracene-9-carboxylic acids may indicate involvement of an anion channel. Phosphorus-deficiency-induced accumulation and subsequent exudation of citric acid seem to be a consequence of both increased biosynthesis and reduced metabolization of citric acid in the proteoid root tissue, indicated by increased in-vitro activity and enzyme protein levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31), and reduced activity of aconitase (EC 4.2.1.3) and root respiration. Similar to citric acid, acid phosphatase, which is secreted by roots and involved in the mobilization of the organic soil P fraction, was released predominantly from proteoid roots of P-deficient plants. Also ³³Pi uptake per unit root fresh-weight was increased by approximately 50% in juvenile and mature proteoid root clusters compared to apical segments of non-proteoid roots. Kinetic studies revealed a K _m of 30.7 μM for Pi uptake of non-proteoid root apices in P-sufficient plants, versus K _m values of 8.5–8.6 μM for non-proteoid and juvenile proteoid roots under P-deficient conditions, suggesting the induction of a high-affinity Pi-uptake system. Obviously, P-deficiency-induced adaptations of white lupin, involved in P acquisition and mobilization of sparingly available P sources, are predominantly confined to proteoid roots, and moreover to distinct stages during proteoid root development. Received: 10 September 1998 / Accepted: 22 December 1998