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.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

Citrate exudation from white lupin induced by phosphorus deficiency differs from that induced by aluminum

Both phosphorus (P) deficiency and aluminum (Al) toxicity induce root exudation of carboxylates, but the relationship between these two effects is not fully understood. Here, carboxylate exudation induced by Al in Lupinus albus (white lupin) was characterized and compared with that induced by P deficiency. Aluminum treatments were applied to whole root systems or selected root zones of plants with limited (1 microM) or sufficient (50 microM) P supply. Aluminum stimulated citrate efflux after 1-2 h; this response was not mimicked by a similar trivalent cation, La(3+). P deficiency triggered citrate release from mature cluster roots, whereas Al stimulated citrate exudation from the 5- to 10-mm subapical root zones of lateral roots and from mature and senescent cluster roots. Al-induced citrate exudation was inhibited by P limitation at the seedling stage, but was stimulated at later growth stages. Citrate exudation was sensitive to anion-channel blockers. Al treatments did not affect primary root elongation, but inhibited the elongation of lateral roots. The data demonstrate differential patterns of citrate exudation in L. albus, depending on root zone, developmental stage, P nutritional status and Al stress. These findings are discussed in terms of possible functions and underlying mechanisms.     

The development of potential screens based on shoot calcium and iron concentrations for the evaluation of tolerance in Egyptian genotypes of white lupin (Lupinus albus L.) to limed soils

European cultivars of white lupin (Lupinus albus L.) grow poorly in limed or calcareous soils. However, Egyptian genotypes are grown successfully in highly calcareous soil and show no stress symptoms. To examine their physiological responses to alkaline soil and develop potential screens for tolerance, three experiments were conducted in limed and non-limed (neutral pH) soil. Measurements included net CO2 uptake, and the partitioning of Fe2+ and Fe3+ and soluble and insoluble Ca in stem and leaf tissue. Intolerant plants showed clear symptoms of stress, whereas stress in the Egyptian genotypes and in L pilosus Murr. (a tolerant species) was less marked. Only the intolerant plants became chlorotic and this contributed to their reduced net CO2 uptake in the limed soil. In contrast, Egyptian genotypes and L pilosus showed no change in net CO2 uptake between the soils. The partitioning of Ca and Fe either resulted from the stress responses, or was itself a stress response. L pilosus and some Egyptian genotypes differed in soluble Ca concentrations compared with the intolerant cultivars, although no significant difference was apparent in the Ca partitioning of the Egyptian genotype Giza 1. In a limed soil, Giza 1 maintained its stem Fe3+ concentration at a level comparable with that of plants grown in non-limed soil, whereas stem [Fe3+] of an intolerant genotype increased. Gizal increased the percentage of plant Fe that was Fe2+ in its leaf tissue under these conditions; that of the intolerant genotype was reduced. The potential tolerance of the Egyptian genotypes through these mechanisms and the possibility of nutritional-based screens are discussed.     

Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.)

We investigated (1) the effect of constant and altered inorganic phosphate (P_i) supply (1–100 mmol m^–3) on proteoid root production by white lupin ( Lupinus albus L.); and (2) the variation in citrate efflux, enzyme activity and phosphate uptake along the proteoid root axis in solution culture. Proteoid root formation was greatest at P_i solution concentrations of 1–10 mmol m^–3 and was suppressed at 25 mmol m^–3 P_i and higher. Except at 1 mmol m^–3 P_i, the formation of proteoid roots did not affect plant dry matter yields or shoot to root dry matter ratios, indicating that proteoid roots can form under conditions of adequate P supply and not at the expense of dry matter production. Plants with over 50% of the root system as proteoid roots had tissue P concentrations considered adequate for maximum growth, providing additional evidence that proteoid roots can form on P-sufficient plants. There was an inverse relationship between the P_i concentration in the youngest mature leaf and proteoid root formation. Citrate efflux and the activities of enzymes associated with citric acid synthesis (phosphoenolpyruvate carboxylase and malate dehydrogenase) varied along the proteoid root axis, being greatest in young proteoid rootlets of the 1–3 cm region from the root tip. Citrate release from the 0–1 and 5–9 cm regions of the proteoid root was only 7% (per unit root length) of that from the 1–3 cm segment. Electrical potential and ³²P_i uptake measurements showed that P_i uptake was more uniform along the proteoid root than citrate efflux.     

The effect of soil freezing on the survial of winter-sown white lupins (Lupinus albus L.)

Specially constructed soil-freezing growth boxes were used to study the effects of the intensity and duration of soil freezing on root injury and the survival of white lupin seedlings of different ages under controlled conditions. The extent of root damage depended on both the intensity of soil freezing and the stage of seedling development (measured as the extent of lignification of the central stele of the primary root). Seedlings whose secondary root development was well advanced, and in which the endodermis was completely lignified, survived intense soil freezing intact. Young seedlings with weakly lignified roots were damaged by moderate soil freezing (> 5 days at ?1°C) and killed by more intense freezing (5 days at ?2°C). The extent of root development and ligmfkation was correlated with the number of leaf primordia produced at the shoot apex so that the susceptibility to soil freezing damage could be accurately predicted by a simple physiological/leaf production model.     

Citric acid excretion and precipitation of calcium citrate in the rhizosphere of white lupin (Lupinus albus L.)

Abstract. White lupin ( Lupinus albus L.) was grown for 13 weeks in a phosphorus (P) deficient calcareous soil (20% CaCO₃, pH(H₂O)7.5) which had been sterilized prior to planting and fertilized with nitrate as source of nitrogen. In response to P deficiency, proteoid roots developed which accounted for about 50% of the root dry weight. In the rhizosphere soil of the proteoid root zones, the pH dropped to 4.8 and abundant white precipitates became visible. X-ray spectroscopy and chemical analysis showed that these precipitates consisted of calcium citrate. The amount of citrate released as root exudate by 13-week-old plants was about 1 g plant⁻¹, representing about 23% of the total plant dry weight at harvest. In the rhizosphere soil of the proteoid root zones the concentrations of available P decreased and of available Fe, Mn and Zn increased. The strong acidification of the rhizosphere and the cation/anion uptake ratio of the plants strongly suggests that proteoid roots of white lupin excrete citric acid, rather than citrate, into the rhizosphere leading to intensive chemical extraction of a limited soil volume. In a calcareous soil, citric acid excretion leads to dissolution of CaCO₃ and precipitation of calcium citrate in the zone of proteoid roots.     

The effect of modifying plant structure on the yield and maturity of the white lupin Lupinus albus

Experiments at Rothamsted in the UK and Lusignan in France examined the effect of artificially modifying plant structure on the yield and date of harvest of indeterminate autumn-sown cultivars of Lupinus albus. Experiments in 1989/90 were done on the cv. Lugel at Rothamsted and those in 1990/91 on the cvs Lugel and Lunoble in both the UK and France. At Rothamsted in 1990, when the summer was warm and dry, the indeterminate cv. Lugel ripened at the end of August and yielded 5.2 t grain/ha. At Lusignan in 1991, where summer weather was also warm and dry, the two unpruned indeterminate cultivars ripened in late July and yielded c. 4.9 t grain/ ha. However, at Rothamsted in 1991, in a season which was predominantly cool and wet, the two indeterminate cultivars were not harvested until late October and yielded only 1.5 and 2.4 t/ha. Plants pruned to a semi-determinate form consisting of the mainstem plus the 1^st-order branches yielded approximately the same as the unpruned inde-terminates at Rothamsted and slightly less than the indeterminates at Lusignan. The semi-determinates ripened 10 days earlier than the indeterminates at Rothamsted in 1990 and Lusignan in 1991, and 6 weeks earlier in the UK in 1991. Epigonal plants with only a mainstem inflorescence, ripened even earlier than the semi-determinates but, without branch inflorescences, produced less yield and had poorer yield stability. Where disease was not a factor, as at Rothamsted in 1990 and Lusignan in 1991, epigonal plants ripened 3 weeks earlier than the indeterminates and yielded between 1.6 and 2.6 t/ha. When the pruned epigonal plants were badly infested with Pleiochaeta, as at Rothamsted in 1991, they yielded less than 0.4 t/ha. It is concluded that indeterminate genotypes of L. albus are well adapted to the climatic conditions of Lusignan but lack yield stability further north. For the cooler northern regions of Europe, an autumn-sown, florally-determinate genotype with one order of branching would produce adequate yield and have an acceptably early harvest date and yield stability. Such a genotype would have agricultural and economic value as a new protein break crop with a low requirement for nitrogen fertiliser.     

Effects of cold shock on the susceptibility of white lupins to fungal diseases

Experiments in controlled environments tested interactions between freezing soil (a compost-vermiculite mixture) and below-ground infection of white lupins with each of three pathogenic fungi (Botrytis cinerea, Fusarium avenaceum or Pleiochaeta setosa) on plant disease and death. Whilst soil freezing (up to 4 days at – 1^oC) caused slight necrosis and increased the severity of disease symptoms, incidence of plant death was increased only after inoculation, before freezing, of the lower hypocotyl of the youngest plants (soil frozen at less than 17 days old) with P. setosa. It is concluded that the contribution of below-ground infection by pathogenic fungi to overwinter losses in autumn-sown white lupin crops is exacerbated to a negligible extent by soil freezing, the main primary cause of such losses.     

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.     

Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls

The longitudinal distribution of unaltered radioactive indole-3-acetic acid (IAA), after application of [5-³H]-IAA to decapitated etiolated lupin hypocotyls. exhibited a wave-like pattern similar to that obtained with endogenous IAA. Waves of radioactive IAA were localizated both in the elongation zone and in the non-growing basal region of the hypocotyl. These IAA waves were transient because of basipetal polar transport and metabolism of IAA.
The level of endogenous IAA in different zones of the hypocotyl varied with age, following a wave-like pattern. During the elongation period of each zone, IAA was parallel to the bell-shaped curve of the growth rate. In addition, a role in secondary cell wall deposition is suggested for the other IAA wave that appeared after the cell elongation period, since an electron microscopic morphometric analysis of the cell wall showed that the cell wall thickness increased once the cell elongation ceased.
As the oscillation of endogenous IAA level occured in both space (distribution along the hypocotyl) and time (variation with age), it is suggested that the level of IAA really depended on the growth status of the cells. The response of the cells to the positional information submitted by the auxin waves as regards the growth status of the cell is discussed.     

Heavy metals in white lupin: uptake, root-to-shoot transfer and redistribution within the plant

The translocation of manganese (Mn), nickel (Ni), cobalt (Co), zinc (Zn) and cadmium (Cd) in white lupin (Lupinus albus cv. Amiga) was compared considering root-to-shoot transport, and redistribution in the root system and in the shoot, as well as the content at different stages of cluster roots and in other roots. To investigate the redistribution of these heavy metals, lupin plants were labelled via the root for 24 h with radionuclides and subsequently grown hydroponically for several weeks. 54Mn, 63Ni and 65Zn were transported via the xylem to the shoot. 63Ni and 65Zn were redistributed afterwards via the phloem from older to younger leaves, while 54Mn remained in the oldest leaves. A strong retention in the root was observed for 57Co and 109Cd. Cluster roots contained higher concentrations of all heavy metals than noncluster roots. Concentrations were generally higher at the beginning of cluster root development (juvenile and immature stages). Mature cluster roots also contained high levels of 54Mn and 57Co, but only reduced concentrations of 63Ni, 65Zn and 109Cd.     

The first genetic and comparative map of white lupin (Lupinus albus L.): identification of QTLs for anthracnose resistance and flowering time, and a locus for alkaloid content.

We report the first genetic linkage map of white lupin (Lupinus albus L.). An F8 recombinant inbred line population developed from Kiev mutant x P27174 was mapped with 220 amplified fragment length polymorphism and 105 gene-based markers. The genetic map consists of 28 main linkage groups (LGs) that varied in length from 22.7 cM to 246.5 cM and spanned a total length of 2951 cM. There were seven additional pairs and 15 unlinked markers, and 12.8% of markers showed segregation distortion at P < 0.05. Syntenic relationships between Medicago truncatula and L. albus were complex. Forty-five orthologous markers that mapped between M. truncatula and L. albus identified 17 small syntenic blocks, and each M. truncatula chromosome aligned to between one and six syntenic blocks in L. albus. Genetic mapping of three important traits: anthracnose resistance, flowering time, and alkaloid content allowed loci governing these traits to be defined. Two quantitative trait loci (QTLs) with significant effects were identified for anthracnose resistance on LG4 and LG17, and two QTLs were detected for flowering time on the top of LG1 and LG3. Alkaloid content was mapped as a Mendelian trait to LG11.     

The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.)

The narrow-leafed lupin possesses valuable traits for environment-friendly agriculture and for the production of unconventional agricultural products. Despite various genetic and environmental studies, the breeding of improved cultivars has been slow due to the limited knowledge of its genomic structure. Further advances in genomics require, among other things, the availability of a genomic DNA library with large inserts. We report here on the construction of the first DNA library cloned in a BAC (bacterial artificial chromosome) vector from diploid Lupinus angustifolius L. cv. Sonet. The high molecular weight DNA used for its preparation was isolated from interphase nuclei that were purified by flow cytometry. The library comprises 55,296 clones and is ordered in 144×384-well microtitre plates. With an average insert size of 100 kb, the library represents six haploid genome equivalents. Thanks to the purification of the nuclei by flow cytometry, contamination with chloroplast DNA and mitochondrial DNA was negligible. The availability of a BAC library opens avenues for the development of a physical contig map and positional gene cloning, as well as for the analysis of the plant’s genome structure and evolution.     

Evidence of phloem boron transport in response to interrupted boron supply in white lupin (Lupinus albus L. cv. Kiev Mutant) at the reproductive stage

The present study investigates whether previously acquired boron(B) in mature leaves in white lupin can be retranslocated intothe rapidly growing young reproductive organs, in response toshort-term (3 d) interrupted B supply. In a preliminary experimentwith white lupin in soil culture, B concentrations in phloemexudates remained at 300–500 µM, which were substantiallyhigher than those in the xylem sap (10–30 µM). Thehigh ratios of B concentrations in phloem exudates to thosein the xylem sap were close to values published for potassiumin lupin plants. To differentiate ‘old’ B in theshoot from ‘new’ B in the root, an experiment wascarried out in which the plants were first supplied with 20µM ¹¹B (99.34% by weight) in nutrient solution for 48d after germination (DAG) until early flowering and then transferredinto either 0.2 µM or 20 µM ¹⁰B (99.47% by weight)for 3 d. Regardless of the ¹⁰B treatments, significant levelsof ¹¹B were found in the phloem exudates (200–300 µMin 20 µM ¹⁰B and 430 µM in 0.2 µM ¹⁰B treatment)and xylem sap over the three days even without ¹¹B supply tothe root. In response to the 0.2 µM ¹⁰B treatment, thetranslocation of previously acquired ¹¹B in the young (the uppermostthree leaves), matured, and old leaves was enhanced, coincidingwith the rise of ¹¹B in the xylem sap (to >15 µM) andphloem exudates (430 µM). The evidence supports the hypothesisthat previously acquired B in the shoot was recirculated tothe root via the phloem, transferred into the xylem in the root,and transported in the xylem to the shoot. In addition, somepreviously acquired ¹¹B in the leaves may have been translocatedinto the rapidly growing inflorescence. Phloem B transport resultedin the continued net increment of ¹¹B in the flowers over 3d without ¹¹B supply. However, it is still uncertain whetherthe amount of B available for recirculation is adequate to supportreproductive growth until seed maturation. Key words: ¹⁰B, ¹¹B, B recirculation, Lupinus albus L., phloem exudate, xylem sap Received 9 October 2007; Revised 28 November 2007 Accepted 30 November 2007     

Lupin peroxidases. II. Binding of acidic isoperoxidases to cell walls

Extracellular acidic isoperoxidases (EC 1.11.1.7), isolated from both the cell walls and intercellular spaces of lupin ( Lupinus albus L. cv. multolupa) hypocotyls, bound to water-insoluble pectins of wall fragments also isolated from the hypocotyls. The binding was sáturable by increasing the isoenzyme concentration in the assay medium and it was dependent on the pH; neutral pH (6.0–7.0) favoured release, while acidic pH (4.0–5.0) favoured the attachment to the cell wall. Binding of acidic isoperoxidases to wall fractions was correlated with the in vitro acid-induced growth of hypocotyl segments, and both were modulated in the same direction by the Ca²⁺/H⁺ ratio in the incubation media, although the two responses were clearly separated when the Ca²⁺/H⁺ ratio varied. Binding of acidic isoperoxidases of cell walls could operate as a fine control of the activity of these cell wall enzymes, although its physiological role in the cell wall stiffening remains unclear. Some aspects of Ca²⁺ on the control of peroxidase activity at this level are also discussed.     

Triticum aestivum shows a greater biomass response to a supply of aluminium phosphate than Lupinus albus, despite releasing fewer carboxylates into the rhizosphere

The relationship between carboxylate release and the ability of plants to access phosphorus from AlPO4 and to detoxify aluminium was studied by comparing species with a low and high rate of carboxylate release, Triticum aestivum (wheat) and Lupinus albus (white lupin), respectively. Species were supplied with P at 10, 20, 40 or 100 mg P kg-1 sand in the form of sparingly soluble AlPO4 or soluble KH2PO4; control plants did not receive any P. Triticum aestivum was significantly better than L. albus at accessing P from AlPO4, despite accumulating fewer carboxylates in its rhizosphere. Rhizosphere pH of L. albus did not vary with form or level of P supply, while the rhizosphere pH of T. aestivum increased with the level of P supplied. Based on the evidence in the present study, a model is proposed to explain the poor performance of L. albus, whereby the release of carboxylates and associated protons reduces the chelating ability of exuded carboxylates, thus reducing P acquisition and increasing Al toxicity.