Annals of Botany 89: 341-349, 2002
© 2002 Annals of Botany Company
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
1IACR-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, UK, 2Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark, 3INRA, Unite Genetique et Amélioration des Plantes Fourragères, 86600 Lusignan, France and 4Südwestdeutche Saatzucht, Im Rheinfeld 1–13, D-76437 Rastatt, Germany
* For correspondence. Fax +44 (0) 1582 760981, e-mail simon.kerley{at}bbsrc.ac.uk
Received: 23 July 2001; Returned for revision: 6 November 2001; Accepted: 4 December 2001.
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ABSTRACT |
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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 Giza1. In a limed soil, Giza1 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. Giza1 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.
Key words: Lupinus albus, white lupin, Lupinus pilosus, limed soil, calcium, iron, photosynthesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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White lupin (Lupinus albus L.) is a well-established crop in mainland Europe. With the introduction of determinate-growth cultivars (Julier et al., 1995), L. albus is a potentially new protein crop for the UK (Milford and Shield, 1996). Current agronomic cultivars of L. albus in Europe grow optimally in well-drained soils of acid to neutral pH. A major constraint to their extensive cultivation is their intolerance of limed or calcareous soil. In the UK, calculations made using a land use suitability map of England and Wales (Siddons et al., 1994) indicate that a cultivar tolerant of naturally calcareous soils would increase the potential area of cultivation from 130 000 ha to in excess of 220 000 ha annually, based on a 6–7 year arable rotation.
In limed and calcareous soils, intolerant cultivars of L. albus grow poorly. The major visual effects are a reduction in shoot dry matter, the development of lime-induced chlorosis (White and Robson, 1989) and possibly plant death under more extreme conditions. This poor growth can result from various soil conditions such as high soil pH (Tang et al., 1993), high bicarbonate (HCO3–) concentration (Brand et al., 2000) or free calcium carbonate (as demonstrated in L. angustifolius L.; Jessop et al., 1990).
To facilitate the breeding of cultivars tolerant of limed or calcareous soil, the physiological mechanisms that cause this growth retardation need to be elucidated. As yield evaluations are unreliable indicators of tolerance (Kerley et al., 2001), physiological screening strategies need to be developed. To be most effective, a screen must involve the physiological evaluation of the major nutrient changes in the shoot that are associated with either tolerance or intolerance. The nutrients most suitable for screening are Fe forms, Ca and C (CO2 uptake).
Although the scoring of chlorosis is a simple screen, its absence in many field experiments makes it an unreliable method. Improvements include using a chlorophyll meter to assess greenness quantitatively (e.g. Kerley et al., 2001), or measuring photosynthetic efficiency by altering gas exchange and light response parameters. Although scientifically valuable, the application of these parameters to large-scale screening would be impracticable.
Iron deficiency is a major cause of lime-induced chlorosis (Chaney et al., 1992). However, whole-plant Fe concentrations are not always deficient when L. albus is grown in calcareous soil (Kerley, 2000). Mengel (1994) suggested that differences in leaf tissue Fe2+ and Fe3+ concentrations might explain the induction of lime-induced chlorosis. Thus, the partitioning of the forms of Fe in the shoot might provide a valuable screen.
One of the few elements to show an increased shoot concentration when plants are grown in limed or calcareous soil is Ca (Tyler and Olson, 2001). Under conditions of high rhizospheric Ca, the calcifuge L. luteus L. is unable to control the distribution of Ca from its xylem, resulting in poor growth and reduced CO2 assimilation (De Silva et al., 1994). Within the shoot, the control of Ca is a major tolerance mechanism of calcicole species (Lee, 1999) and its partitioning might provide another potential screen.
Lupinus albus has long been cultivated in soils with high Ca content along the Nile Valley, Egypt. Christiansen et al. (1999) collected a range of genotypes grown in soils of pH 7·5–9·4 and suggested that they may possess better tolerance to calcareous soils than current European cultivars.
The aim of this study was to compare a range of Egyptian genotypes with European cultivars and genotypes on neutral pH and limed soils, and to assess them using the physiological parameters of photosynthesis and Fe and Ca form and partitioning. Results were used to determine some physiological causes of intolerance and to see whether these studies could be used as potential screens in breeding programmes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For all three experiments, pots of 4 l capacity were filled with a soil mix comprising 80 % (by volume) sieved Kettering loam and 20 % horticultural grit (0·2–0·5 mm diameter gravel), which was added to facilitate drainage. Kettering loam is a fertile silty/sandy soil, sieved to 3 mm, resulting in a fine texture and minimal structure. Although lupins are generally grown on lighter soils in the UK, this soil was not detrimental to root development as they are grown successfully on heavier soils at Rothamsted, UK. The total nutrient concentration (µg g–1 d. wt), determined by inductively coupled plasma atomic emission spectrophotometry (ICP-AES) after digestion in aqua regia (HCl and HNO3 mix) (McGrath and Cunliffe, 1985), was: P, 1266; K, 3771; Ca, 8313; Mg, 3396; Na, 218; Fe, 49 308; Mn, 337; and Zn, 37. Exchangeable ions were also determined (µg g–1 d. wt): Olsen P, 68 (HCO3– extracted; Olsen et al., 1954) and K, 135; Ca, 6250; Mg, 316; Na, 56 (all extracted in 1 M NH4Ac; Metson, 1965). The soil pH was 6·8 when measured in deionized water (1 : 2·5). This nutrient status was greater than that of many field soils as it was intended to be non-limiting.
To create a limed soil, powdered calcium oxide was added to the soil mix at 1·5 % by soil dry weight. This raised the soil pH to 7·8 when stable and the total Ca content to 51 x 103 µg g–1 soil. The Olsen P concentration was reduced to 35 µg g–1 soil and exchangeable Ca was 20 000 µg g–1 (determined by extraction in NH4NO3; Stuanes et al., 1984). Although bio-available Fe was not determined, the concentration would be lowered by the addition of lime. The soil was left in the limed state for 3 d prior to planting.
No micronutrients were added to either treatment. The plants formed sufficient numbers of visually active nodules with naturally occurring rhizobium in the soil. This indicated that the plants were not N-limited. The soil was wetted to approx. 20 % moisture content (w/w) using deionized water before being added to the pots. The limed vs. non-limed soils were the two soil treatments in the experiments below.
A range of genotypes or cultivars was selected for the experiments. The genotypes Egypt 121, Egypt 99 and Giza1 (J. Christiansen) were selected to determine their potential tolerance to limed soils. The cultivars Lucyanne and Lublanc were selected as control plants as they have been shown in previous pot and field trials to be intolerant of calcareous or limed soil. The genotypes La 668 and La 675 (C. Huyghe) were also used because they were shown to possess slightly more tolerance to limed soil than Lucyanne or Lublanc in UK field trials. However, they are still considered intolerant plants. A genotype of L. pilosus Murr. (Ld 124; C. Huyghe) was selected for the known tolerance of this species to calcareous soil.
Plants were grown in controlled environment conditions of 20 °C/16 h day, 18 °C/8 h night, at 70 % relative humidity and a light intensity of 500 µmol m–2 s–1 (tungsten lamps), and were watered to water holding capacity every 2–3 d with deionized H2O and allowed to drain freely.
Experiment 1. Establishing the potential tolerance of Egyptian genotypes to limed soil
Seven cultivars or genotypes were selected for this experiment. The Egyptian genotypes Egypt 121, Egypt 99 and Giza1 were selected to assess their potential tolerance. The cultivars Lucyanne and Lublanc and genotype La 668 were included as intolerant controls. The L. pilosus genotype was included as a tolerant benchmark for the Egyptian material.
Seeds of each plant were imbibed for 8 h in deionized H2O. Those of L. pilosus were scarified to allow imbibition. After 3 d incubation in the dark at 20 °C on damp filter paper in a Petri dish, a single seedling was planted into each pot. Plants were grown in a randomized block design consisting of two blocks, each with three individuals of each cultivar for each soil treatment.
Leaf greenness was recorded 35 d after sowing (DAS) using a chlorophyll meter (SPAD-502; Minolta Camera Co. Ltd). Greenness was measured as SPAD units on the three largest leaflets of the youngest fully expanded leaf (to limit the effect of age differences), and averaged to give a score for the plant. Measurements were taken from one side of the central section of each leaflet to ensure there was no error due to the midrib. A measure of 50–65 SPAD units indicated a dark green leaf, whereas a value below 40 indicated interveinal chlorosis. As plants could become less green but remain non-chlorotic, a visual observation of the presence or absence of chlorosis (inter-veinal yellowing on the youngest emerged leaves) was made at the same time. No graded scoring system was used as such analysis was provided by the SPAD data.
At 35 DAS, CO2 exchange measurements were made on the youngest emerged leaf of each plant. To determine the response of leaf photosynthesis to changes in radiation, a CIRAS-1 portable infra-red gas analysis system (PP systems, Hitchin, UK) fitted with a broad leaf cuvette (exposed leaf area 250 mm2) was used (Parsons et al., 1997). Light was applied uniformly across the leaf at levels that would ensure the creation of a response curve, including the asymptote. The radiation levels used were comparable with those of early summer in the UK. In Egypt, plants are sown as a winter crop and the radiation levels are unlikely to exceed the values used. CO2 was applied at ambient concentration. A radiation response curve of CO2 fixed (µmol m–2 s–1) against photosynthetically active radiation (PAR) was generated and an exponential function was fitted. The photosynthetic capacity (light saturated CO2 assimilation) of the leaf (Pmax) was determined from the curve asymptote. The light compensation point (no net efflux of CO2) was calculated as the PAR at which CO2 fixation was zero. The efficiency of light utilization by photosynthesis (apparent maximum quantum yield) was calculated from the initial slope of the curve.
At 45 DAS the number of fully expanded leaves was determined and the plants were harvested. The shoots were removed at the stem base and partially dried for 2–4 min in a microwave oven at 750 W to minimize the repartitioning of nutrients (Bollons and Barraclough, 1997). Dry weight was determined after drying at 80 °C for 24 h. Dried plant matter was divided into leaf and stem material and ground to pass through a 1-mm sieve.
The concentration of soluble Ca in the leaf material was determined by extracting a known dry weight of material (up to 0·25 g) in 25 ml deionized H2O for 30 min on a rolling bed shaker. The extract was filtered through a Whatman® No. 6 filter paper and the filtrate analysed for Ca using a Jenway PFP7 Flame Photometer.
The soil was removed from the pots, the root system was washed on a 1·4 mm sieve under running tap water to remove all adhering soil and the dry weight was determined after drying for 24 h at 80 °C. As growth in a confined pot greatly influences root formation and activity, no further analyses of root form were made.
Experiment 2. The role of stem and leaf partitioning and form of calcium in the tolerance mechanism of an Egyptian genotype
The Egyptian genotype Giza1 was selected for this study and compared with the intolerant genotype La 675 and cultivar Lublanc. The plants were germinated as above and sown singly into pots. The plants were arranged in two blocks, each containing five replicates of each soil and plant treatment, and were maintained under controlled environment conditions, as described above, for 30 d.
Plants were harvested at 30 DAS by cutting the stems at the soil surface and dividing each plant into leaflet and stem (including petiole) tissue. After determining the fresh weight, the tissues were partially dried for 2–4 min in a microwave oven at 750 W. Dry weight was then determined after drying further at 80 °C for 24 h, and the plant matter ground through a 1-mm sieve. The concentration of soluble Ca in each tissue was determined as above. After extraction, the plant material was removed from the filter paper and re-dried. To determine the insoluble Ca fraction, the Ca concentration of the re-dried sample was determined using ICP-AES after digestion by nitric/perchloric acid (Zarcinas et al., 1987).
Experiment 3. The role of stem and leaf partitioning and form of iron in the tolerance mechanism of an Egyptian genotype
As with the previous experiment, the genotype Giza1 was selected for this study and was compared with the intolerant genotype La 675 and cultivar Lublanc. The plants were germinated and pots established as described previously. The plants were arranged in two blocks, each containing five replicates of each soil and plant treatment, and were maintained for 30 d under the controlled environment conditions used in the previous experiments.
At 30 DAS, leaf greenness was recorded using a chlorophyll meter. The shoots were then removed at soil level and divided into leaflet and stem (including petiole) material. Each tissue was placed into a foil envelope and rapidly frozen in liquid N2 to reduce the possible change in Fe form, and stored at –80 °C. To extract Fe2+ from the tissues, the procedure of Katyal and Sharma (1980) was followed. A 2 g tissue sample was placed into a sample tube containing 20 ml of 1·5 % (w/w) 1,10-Phenanthroline (in H2O, adjusted to pH 3 with 1 M HCl). After 16 h at room temperature, the samples were filtered through a Whatman® No. 1 filter paper and the concentration of Fe2+ in the filtrate was determined spectrophotometrically at 510 nm using a ferrous chloride standard curve. The tissue remaining on the filter paper was then re-dried, ground in a mortar and pestle and the remaining Fe content (Fe3+) determined by ICP-AES analysis following digestion in nitric acid and hydrogen peroxide.
Statistical analyses
Data from all experiments were subjected to two-way ANOVA using Genstat 5 (fourth edition). Differences were considered significant at a probability level of P 
0·05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experiment 1. Establishing the potential tolerance of Egyptian genotypes to limed soil
The shoot dry weight of all plants (even L. pilosus) was approx. 50 % lower when grown in limed compared with neutral-pH soil (Table 1). When grown in limed soil, all plants had fewer fully expanded leaves than when grown in neutral-pH soil. However, the reduction in leaf number in the limed compared with the neutral-pH soil was less for the Egyptian genotypes than for the other genotypes or cultivars.
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In the limed soil, most of the Lublanc plants were chlorotic (Table 1), as were half of the Lucyanne and two-thirds of the La 668 plants; neither the Egyptian nor L. pilosus genotypes were chlorotic. Leaf greenness (SPAD analysis) confirmed this result; cultivars Lublanc and Lucyanne had lower SPAD values in the limed soil than the Egyptian or L. pilosus genotypes or when grown in the neutral-pH soil. Although not chlorotic when grown in limed soil, the Egyptian genotypes were less green compared with plants grown in neutral-pH soil. In contrast, the leaf greenness of L. pilosus was unchanged.
In Lublanc, the concentration of soluble Ca increased when grown in limed compared with neutral-pH soil (Table 1). In contrast, L. pilosus and Egypt 121 had a reduced leaf soluble-Ca concentration, whereas that of the remaining genotypes did not vary between soil treatments.
The root to shoot ratio differed between the genotypes (Table 1); Egypt 121 had the highest and L. pilosus the lowest ratio. All plants had a higher ratio in limed than in neutral-pH soil.
Net CO2 uptake against radiation is shown for each genotype in Table 2. A genotype by soil interaction was present at all except the lowest radiation levels. In the limed soil, the response of Lucyanne and Lublanc to increasing light radiation was small whereas, when grown in neutral-pH soil, CO2 uptake was greater. In comparison, the CO2 uptake of L. pilosus appeared slightly higher in the limed compared with non-limed soil, although not at the highest radiation level. Genotype La 668 was the only one in which CO2 uptake did not differ between soil treatments at any radiation level. Of the Egyptian genotypes, Egypt 121 differed in response between the soil types only at the highest radiation intensity, at which CO2 uptake was higher in limed than in neutral-pH soil. Giza1 showed a slight reduction in CO2 uptake in limed compared with neutral-pH soil, and Egypt 99 responded in a manner comparable to that of L. pilosus.
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The light saturated rate of CO2 uptake (Pmax), photosynthetic efficiency on a quantum yield basis, and the compensation PAR irradiance for each plant and soil type are presented in Table 3. Pmax of Giza1 was comparable with that of L. pilosus and was greater than that of the other L. albus genotypes. The lowest Pmax values were found when Lublanc and Lucyanne were grown in the limed soil. The quantum yields of Lublanc and Lucyanne were lower in the limed compared with the neutral-pH soil, whereas those of the other genotypes did not differ between soil types. There were no genotype or soil treatment differences in compensation PAR.
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Experiment 2. The role of stem and leaf partitioning and form of calcium in the tolerance mechanism of an Egyptian genotype
When shoot Ca was partitioned into soluble or insoluble forms and into stem or leaf tissue, there was no significant interaction between genotype and soil type (Table 4). However, differences were apparent between the soil types in all analyses. All genotypes had a higher shoot total Ca concentration in the limed compared with the neutral-pH soil. Genotype Giza1 had a lower total Ca concentration than the other genotypes in both soils. When whole-shoot soluble Ca was expressed as a percentage of total Ca, that of La 675 was greater than that of Giza1 or Lublanc. However, in limed compared with non-limed soil, all plants showed an increase in the proportion of total Ca that was in a soluble form.
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Total Ca concentration in the stems was lowest in Giza1 and highest in Lublanc, and increased in all plants when grown in limed compared with neutral-pH soil. When stem soluble Ca was calculated as a percentage of whole-plant Ca, it was highest in the limed treatment; no differences between genotypes were apparent. However, when calculated as a percentage of stem Ca concentration, that of La 675 was greater than that of Giza1 and Lublanc, although it was higher in all plants in limed compared with neutral-pH soil.
No genotype differences were apparent in the partitioning of Ca into leaf tissue. However, in limed soil the leaf total Ca concentration was nearly twice that in the neutral-pH soil. Leaf soluble Ca in plants grown in limed soil accounted for a significantly higher percentage of the whole-plant and leaf total Ca compared with neutral-pH soil grown plants.
Experiment 3. The role of stem and leaf partitioning and form of iron in the tolerance mechanism of an Egyptian genotype
As germination of Lublanc was very poor in this experiment, it was not included in the analyses. However, it was still possible to compare the potential tolerance mechanism of the Egyptian genotype Giza1 with that of the intolerant European genotype La 675. When comparing Giza1 and La 675 in neutral-pH and limed soils, interaction effects were present in selected Fe forms and tissue partitioning (Table 5). Leaf greenness of both La 675 and Giza1 was reduced from 63 SPAD units in the neutral-pH soil, to 44 and 22 units for Giza1 and La 675, respectively, in the limed soil. The difference in response was visually apparent; Giza1 only became less green, whereas La 675 became chlorotic.
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When grown in limed compared with neutral-pH soil, there was no change in the whole-shoot Fe concentration of La 675, whereas that of Giza1 declined by more than 30 %. The concentration of stem total Fe differed between soil types in La 675 only; it was over twice the concentration when grown in limed soil compared with neutral-pH soil. In contrast, there was no genotype difference in leaf total Fe concentration, although plants grown in limed soil had lower concentrations than those grown in neutral-pH soil.
No genotype or soil treatment differences were apparent in the whole-shoot Fe2+ concentration. However, an interaction occurred in whole-shoot Fe3+ concentration: that of Giza1 declined in limed compared with neutral-pH soil, whereas that of La 675 remained unchanged.
Interaction effects were apparent when whole-shoot Fe was partitioned into tissue or Fe form. Giza1 contained less Fe2+ in its stem than did La 675, but Fe2+ accounted for less than 1 % of stem Fe in both plants and soil treatments. Giza1 also had comparable stem Fe3+ concentrations in both soil treatments; that of La 675 more than doubled when grown in limed soil. Nearly 40 % of the whole-shoot Fe in La 675 was accounted for in the stem; this was greater than in Giza1.
In contrast to the stem response, no genotype or soil differences were apparent in leaf Fe2+ concentrations. However, when expressed as a percentage of whole-shoot Fe, the leaf Fe2+ content increased in Giza1 grown in limed compared with non-limed soils, whereas it decreased in La 675. Although no genotype differences were apparent in leaf Fe3+, the concentration was lower in plants grown in limed compared with neutral-pH soil.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The results presented in this study demonstrate that a level of tolerance to limed soil is present in the Egyptian L. albus genotypes that is more comparable with the calcareous-soil tolerant L. pilosus than with European cultivars or genotypes. The study also shows differences in Fe and, potentially, Ca nutrition that demonstrate stress-related physiology in the European plants, and indicates potential tolerance mechanisms in the Egyptian genotypes.
Physiological responses to a limed soil
Although a limed soil differs from one that is naturally calcareous, it does provide a medium for growing tolerant plants in sub-lethal stress conditions, allowing examination of selected calcareous soil-related stress responses and identification of potential screens. The European cultivars Lucyanne and Lublanc demonstrated calcifuge-type behaviour that was characterized by the partitioning of Ca and Fe in the shoot.
A major response of a calcicole to calcareous soil is the regulation of Ca in the shoot tissue, specifically that of free Ca2+ (Kinzel and Lechner, 1992). On limed compared with neutral-pH soil, the greater uptake of Ca by L. albus resulted in more soluble and insoluble forms of Ca in the plant. However, the soluble fraction showed the greatest increase and contrasted with the reduction in concentration present in L. pilosus shoot tissue. Calcicoles regulate their concentration of soluble Ca; they possess mechanisms to sequester it into forms such as Ca-oxalate in trichomes (De Silva et al., 1996) or bound to Ca-specific proteins in the cytoplasm (Le Gales et al., 1980). From this pot study and after field experiments (Kerley et al., 2001), L. pilosus appears to possess mechanisms to regulate the concentration of soluble Ca. These mechanisms are not present in Lucyanne or Lublanc, indicating an inability to restrict uptake, or sequester Ca in an insoluble form.
A potential process that could restrict Ca uptake at the root was observed in L. albus by Dinkelaker et al. (1989). They observed the precipitation of Ca by citrate secreted into the rhizosphere by cluster roots. Whether this is an external sequestration mechanism or a confounding of the P uptake system is unclear. However, in a limed soil, L. pilosus secreted more citrate than L. albus (Kerley and Huyghe, 2002) and this could impact on the uptake of Ca between the species.
A deficiency in Fe concentration through uptake inhibition by HCO3– is a major cause of intolerance to calcareous soil (Chaney et al., 1992) and prevention of HCO3 uptake may be a mechanism of tolerance in L. pilosus (Brand et al., 2000). Although Fe concentrations declined in the shoots when the soil was limed, important changes in form and partitioning were apparent in this study, and such changes have been used to explain the induction of lime-induced chlorosis in the absence of Fe deficiency (Mengel, 1994). In the genotype La 675, less Fe reached the leaflet tissue due to both reduced uptake of Fe and an increase in the concentration of stem Fe. Within the stem this increase was due to immobilization of Fe as the Fe3+ form, whereas in the leaf tissue there was a lowering in the concentration of the Fe2+ form (as well as Fe3+). Zohlen and Tyler (2000) observed a reduction in Fe2+ in calcifuges and considered immobilization of Fe into ‘less active’ forms within tissues to be an important response of the calcifuge–calcicole behaviour.
Changes in both the Ca and Fe physiology may have direct effects on plant growth, for example both nutrients can affect photosynthesis. Net assimilation rates were reduced in L. luteus through the suppression of stomatal opening by the increase in Ca concentration in guard cells (De Silva et al., 1994). Both nutrients will have impacted on the photosynthetic capacity of Lucyanne and Lublanc, as they were unable to fix as much CO2 when grown in limed compared with neutral-pH soil. These cultivars showed a 40–70 % reduction in Pmax due to a lower efficiency of moles CO2 fixed per quantum incident on the leaf. This would have been directly attributable to reduced chlorophyll or photosynthetic enzyme levels, or possibly the reduced control of the stomata. This reduction in photosynthetic efficiency in limed soil will have contributed to the reduced shoot dry weight and expanded leaf number. In contrast, the maintenance of photosynthetic efficiency and lack of chlorosis in L. pilosus may have resulted from the maintenance of Ca and Fe concentrations when grown in limed soil. However, as L. pilosus growth (shoot dry weight) and development were slowed, a level of intolerance was still apparent.
Egyptian genotype tolerance and potential screens
The responses of the Egyptian genotypes to limed soil differed from those of either Lucyanne or Lublanc. Although leaf greenness is not always consistent within an experiment over time (Brand et al., 1999), the absence of chlorosis in any of the Egyptian material was comparable with tolerant L. pilosus, and indicated that the plants tolerated the stress better than the European material. The chlorophyll-meter analysis discriminated between L. pilosus and the Egyptian genotypes and indicated that the Egyptian genotypes were subjected to a sub-chlorotic level of stress. Leaf emergence was considered by Tang et al. (1995) to be a potential means of genotype evaluation, and did discriminate effectively between the Egyptian and intolerant plants in this study. However, this evaluation will give no insight into the tolerance physiology and was not sufficiently discriminatory in a UK field trial (Kerley et al., 2001).
The above screens measure the results of physiological processes; more definitive screens will measure the processes themselves, e.g. Ca and Fe partitioning and photosynthesis. Differences in the leaf soluble Ca in the first experiment confirmed lower concentrations in Egypt 99 and 121 compared with Lucyanne and Lublanc, indicating that some control over soluble Ca was present. Unfortunately this was not apparent in either experiment in Giza1, indicating that such a process may be minimal in this genotype. However, there was a trend in the second experiment for Giza1 to have lower mean values for soluble Ca than Lublanc, with values for La 675 being intermediate; as such it may possess some form of tolerance that requires further study in a truly calcareous soil.
A potential tolerance mechanism was apparent in the Fe response of Giza1 compared with the intolerant La 675. Giza1 showed a smaller partitioning of its Fe as stem Fe3+ in limed compared with neutral-pH soil and even increased the fraction of active Fe2+ in the leaf tissue, accounting for its lack of chlorosis. This mechanism, considered by Zohlen and Tyler (2000) to facilitate calcicole growth in calcareous soil, might be one explanation for why the Egyptian material can be cropped in highly calcareous soils (Christiansen et al., 1999). This response also explains their maintenance of Pmax values and the absence of a decrease in quantum yield under limed soil conditions.
Tolerance does not, however, imply optimal growth conditions. Destructive shoot dry weight determination demonstrated that both L. pilosus and the Egyptian genotypes did experience considerable limed-induced stress and showed that all genotypes grew optimally on the neutral-pH soil.
Having confirmed tolerance in the Egyptian genotypes, there is a need now to discriminate more effectively between these genotypes, to understand the mechanisms of this tolerance and to investigate the response of the root system to a calcareous soil. Such work will enhance and focus the strategy of breeding programmes designed to develop calcareous soil tolerant agronomic cultivars of L. albus.
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ACKNOWLEDGEMENTS |
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The authors should like to thank Ian Shield for advice and Jenny Swain for technical support. This study has been carried out with financial support from the Department of the Environment, Food and Rural Affairs, and the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD programme, CT96–1965, ‘Creation of varieties and technologies for increasing production and utilisation of high quality protein from the white lupin in Europe’. It does not necessarily reflect its view and in no way anticipates the Commission’s future policy in this area. IACR is grant aided by the Biotechnological and Biological Sciences Research Council.
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