Effect of high external NaCl concentrations on ion transport within the shoot of Lupinus albus. I. Ions in xylem sap

Abstract. Xylem sap was collected from individual leaves of intact transpiring lupin plants exposed to increasing concentrations of NaCl by applying pneumatic pressure to the roots. Concentrations of Na⁺ and Cl⁻ in the xylem sap increased linearly with increases in the external NaCl concentration, averaging about 10% of the external concentration. Concentrations of K⁺ and NO₃⁻, the other major inorganic ions in the sap, were constant at about 2.5 and 1.5 mol m⁻³, respectively. There was no preferential direction of Na ⁺ or Cl⁻ to either young or old leaves: leaves of all ages received xylem sap having similar concentrations of Na⁺ and Cl⁻, and transpiration rates (per unit leaf area) were also similar for all leaves. Plants exposed to 120–160 mol m⁻³ NaCl rapidly developed injury of oldest leaves; when this occurred, the Na⁺ concentration in the leaflet midrib sap had increased to about 40 mol m⁻³ and the total solute concentration to 130 osmol m⁻³. This suggests that uptake of salts from the transpiration stream had fallen behind the rate of delivery to the leaf and that salts were building up in the apoplast.     

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.     

Stress-induced changes in the root architecture of white lupin (Lupinus albus) in response to pH, bicarbonate, and calcium in liquid culture

Current agronomic cultivars of white lupin (Lupinus albus) are intolerant of calcareous or limed soils. In these soils, high pH, bicarbonate (HCO₃^?), and calcium (Ca) concentrations are the major chemical stresses to the root system. To determine the responses of the root system to these factors, evaluate root architecture, and compare genotypes for tolerance, a series of liquid culture experiments was completed using root chambers that allowed the study of the root system in two dimensions. Each stress condition caused changes in different parts of the root system and there was no generalised stress response. HCO₃^? (5 mM) had the greatest effect on cultivars intolerant of calcareous soil; it decreased the dry weight of the shoot and caused the highest percentage of tap root deaths. HCO₃^? also discriminated between short (determinate) and long (indeterminate) roots, as it decreased the number and density of the determinate roots only. Calcium (3 mM) affected all parts of the root system. The tap root was shortened and showed an increased tortuousness in its path compared with 1 mM Ca, although no plants suffered tap root death. The numbers and densities of the two lateral root forms were also decreased, as were the lengths of the indeterminate roots. Stress from alkaline pH (7.5) media caused a lower number and density of determinate lateral roots to be produced than at pH 6.5. The experiments demonstrated that each culture condition elicited a definable stress response. Stress conditions altered the root architecture of genotypes reported to be tolerant of calcareous soil less than in intolerant genotypes. Although soil is more complex than liquid culture, it is possible that in a calcareous or limed soil each stress condition examined may affect the overall stress of the plant, and increased tolerance may result from tolerance to a single stress.     

Active chitinases in the apoplastic fluids of healthy white lupin (Lupinus albus L.) plants

Acidic exocellular class III chitinase (EC 3.2.1.14) was previously identified in healthy white lupin (Lupinus albus L.) plants and suspension-cultured cells by N-terminal microse-quencing. In this study, the detection of chitinase activity with Remazol Brilliant Violet 5R (RBV)-labelled chitin derivatives is described. Chitinase activity was observed in protein fractions of cytoplasmic or exocellular origin from roots, hypocotyls, cotyledons, and leaves of healthy white lupin plants. Using isoelectrofocusing followed by a new overlay technique with carboxymethyl chitin-RBV conjugate-containing gel, up to six different chitinase isoforms were visualised. Their activity was distributed fairly evenly within a plant with acidic isoforms predominating in cell walls and basic (or neutral) ones found intracellularly. Exocellular location of some chitinase isoforms were also confirmed by detection of their activities in intercellular washing fluids from white lupin tissues. Chitinase activity was demonstrated in culture filtrates and cell walls of suspension-cultured white lupin cells.     

A simple system for determining the phloem mobility of compounds using excised pods of lupin (Lupinus albus L.)

Lupin pods were isolated from the plant and the pedicels placed in solutions of either radiolabeled sucrose, glucose, abscisic acid, or gibberellin A₉ (GA₉). Phloem exudate was collected from the cut stylar ends of the pods and both quantitative and qualitative data were obtained on the activity in the sap. Fed sucrose, abscisic acid, and GA₉ were found in phloem exudate, but labeled glucose was immobile as the monosaccharide and activity in exudate appeared in sucrose.Abbreviations GA gibberellic acid - ABA abscisic acid - TLC thin layer chromatography     

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 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 water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit,leaves and phloem exudate

Abscisic acid (ABA) was identified by combined gas liquid chromatography-mass spectrometry in sieve-tube exudate collected from the cut stylar ends of white lupin fruit. Water stress caused an increase in ABA levels in leaf, seed and pod tissues and phloem exudate. When compared with levels in extracts of these tissues, the concentration of ABA in sieve-tube sap was very high. It is suggested that ABA is actively transported out of mature leaves in the phloem and this finding is discussed in terms of the ABA balance of the plant.Abbreviations ABA abscisic acid - GLC gas liquid chromatography     

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.     

Nitric oxide is the shared signalling molecule in phosphorus- and iron-deficiency-induced formation of cluster roots in white lupin (Lupinus albus)

Background and Aims

Formation of cluster roots is one of the most specific root adaptations to nutrient deficiency. In white lupin (Lupinus albus), cluster roots can be induced by phosphorus (P) or iron (Fe) deficiency. The aim of the present work was to investigate the potential shared signalling pathway in P- and Fe-deficiency-induced cluster root formation.

Methods

Measurements were made of the internal concentration of nutrients, levels of nitric oxide (NO), citrate exudation and expression of some specific genes under four P × Fe combinations, namely (1) 50 µm P and 10 µm Fe (+P + Fe); (2) 0 P and 10 µm Fe (–P + Fe); (3) 50 µm P and 0 Fe (+P–Fe); and (4) 0 P and 0 Fe (–P–Fe), and these were examined in relation to the formation of cluster roots.

Key Results

The deficiency of P, Fe or both increased the cluster root number and cluster zones. It also enhanced NO accumulation in pericycle cells and rootlet primordia at various stages of cluster root development. The formation of cluster roots and rootlet primordia, together with the expression of LaSCR1 and LaSCR2 which is crucial in cluster root formation, were induced by the exogenous NO donor S-nitrosoglutathione (GSNO) under the +P + Fe condition, but were inhibited by the NO-specific endogenous scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl- 3-oxide (cPTIO) under –P + Fe, +P–Fe and –P–Fe conditions. However, cluster roots induced by an exogenous supply of the NO donor did not secrete citrate, unlike those formed under –P or –Fe conditions.

Conclusions

NO plays an important role in the shared signalling pathway of the P- and Fe-deficiency-induced formation of cluster roots in white lupin.     

Variability of globulin composition in cultivars and individually tested seeds of yellow lupin (Lupinus luteus L.)

A study on globulins, major storage proteins in yellow lupin seeds, called conglutins, was conducted using SDS polyacrylamide gel electrophoresis. In this paper, an extensive and not yet published list of yellow lupin conglutins is presented. The patterns of subunits of major conglutins in seeds of three yellow lupin cultivars were similar to each other, varying only in the level of some polypeptides. Investigations of seeds of cultivar Parys showed considerable quantitative differences in major subunits. Some minor subunits occurred only in some seeds and were absent in the others. Great differences were shown between single individuals in the amount of subunits of conglutin which is of the most nutritional value due to high content of methionine.     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Interactions Between High pH and Iron Supply on Nodulation and Iron Nutrition of Lupinus albus L. Genotypes Differing in Sensitivity to Iron Deficiency

Poor growth of white lupin (Lupinus albus L.) on alkaline soils may result from its sensitivity to iron deficiency and poor nodulation. This study examined interactive effects of iron supply and high pH on the growth and nodulation of three genotypes differing in their sensitivity to iron deficiency. Three genotypes (P27486, Ultra and WTD180) were grown for 17 days in buffered solutions with Fe supply of 0.2, 2 and 20 μM. Solution pH was adjusted to 5.2, 6.5 or 7.5. Plant growth, nodulation and nutrient concentrations in plants were measured. Decreasing Fe supply decreased chlorophyll concentration in young leaves by up to 92%. Increasing pH decreased chlorophyll concentration by an average of 40% at pH 6.5 and by 47% at pH 7.5. The decrease of chlorophyll was less obvious in P27485 than in Ultra or WTD180. Shoot biomass was reduced by up to 18% by Fe deficiency, with such decrease being less for P27486. Increasing pH exacerbated the effect of Fe deficiency on shoot biomass only of Ultra. Decreasing Fe supply decreased nodule number by an average of 54%, and increasing pH decreased nodule number by 80%. P27486 formed the greatest number of nodules while WTD180 the least. P27486 had high Fe uptake and low internal requirement. Irrespective of genotype, leaf chlorosis positively correlated with cluster root formation. The results suggest that a combination of Fe deficiency and high pH impaired nodulation in L. albus, and that selection of genotypes for both tolerance of iron deficiency and good nodulation at high pH is important for a successful lupin crop on alkaline soils.     

137Cs uptake in spring wheat (Triticum aestivum L.cv. Tonic) at varying K supply

Spring wheat (Triticum aestivum L. cv. Tonic) was grown for 16 days in a sandy loam soil which was contaminated with ¹³⁷Cs. The soil was fertilised with K at three rates (0,1 and 2 mmol K per 950 g dry soil) and with NO₃ ^--N at two rates (0 and 2 mmol per 950 g dry soil) in a factorial design. The ¹³⁷Cs Activity Concentration (AC) in the shoot tissue significantly reduced 8.2-fold (nil N treatment, p<0.001) and 9.3-fold (highest N dose, p<0.001) with increasing K supply. In contrast, the K application increased the ¹³⁷Cs AC in soil solution 1.7 fold (nil N treatment) or had no significant effect (highest N dose). At similar K application, the application of N increased the ¹³⁷Cs AC in the shoot compared to the control. This effect is most probably due to the increased NH₄ ⁺ concentration in soil solution which increased the ¹³⁷Cs AC in soil solution. The soil solution composition (¹³⁷Cs and K concentration) in the rhizosphere was estimated from the average soil solution composition at day 16 and solute transport calculations. The ¹³⁷Cs AC in the shoot tissue was predicted from the estimated soil solution composition in the rhizosphere and the relationship between K concentration and ¹³⁷Cs uptake derived from a nutrient solution experiment. The predictions of ¹³⁷Cs AC's in the shoot are qualitatively correct for the fertiliser effects but underestimate the observations between 1.4 and 9.9 fold.     

Vessel-diameter distribution in roots of grapevines (Vitis vinifera L. cv. Shiraz)

The distribution frequency patterns of diameter of xylem vessels and percentage of total predicted axial conductances were studied in 190-day and 212-day-old main roots of grapevine (Vitis vinifera L. cv. Shiraz) grown under well-watered and stressed conditions. The protoxylem were the first to mature and were responsible for most of the theoretical conductance in root segments between the tip and 2.5 cm from the tip. Some large xylem vessels retained cross walls and protoplasm up to 22.5 cm from the tip. Statistical tests using the Kolmogorov-Smirnov two sample test showed that the pattern of distribution frequency of xylem vessels classified in different diameter classes varied with distance from the root tip. The distribution frequency of xylem vessels was similar in both well-watered and stressed plants from the tip up to 15 cm from the tip. At distances further from the tip the distribution frequency of xylem vessels of well-watered plants was significantly different from that of stressed plants, with the former having more larger vessels than the latter. The pattern of vessel distribution frequency was different from that of percent total axial conductance (K_h) predicted with fewer large vessels carrying most of the axial flow.     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

Changes in the amide and amino acid composition of xylem exudate from perennial ryegrass (Lolium perenne L.) during regrowth after defoliation

The paper presents the results of amino acid analyses in xylem sap during leaf regrowth of ryegrass plants defoliated firstly at the 8th and secondly at the 12th week of culture. The free amino acid composition of leaves, stubble and roots was also determined and some of the results are reported. Prior to defoliation, xylem sap contained a high proportion of amides, particularly glutamine. During regrowth after defoliation, the proportion of asparagine in the xylem sap increased until the third day when the highest ratios of asparagine/glutamine appeared. The results are compared with relative amounts of free amino acids in the different plant parts and discussed in relation to source-sink nitrogen transfer.