Cluster-root formation, carboxylate exudation and proton release of Lupinus pilosus Murr. as affected by medium pH and P deficiency

The study examined the interactive effect of pH and P supply on cluster-root formation, carboxylate exudation and proton release by an alkaline-tolerant lupin species (Lupinus pilosus Murr.) in nutrient solution. The plants were exposed to 1 (P₁, deficient) and 50 μM P (P₅₀, adequate) for 34 days in nutrient solution at either pH 5.6 or 7.8. Plant biomass was not influenced by pH at P₁, but at P₅₀ shoot and root dry weights were 23 and 18% higher, respectively, at pH 7.8 than at pH 5.6. There was no significant difference in plant biomass between two P treatments regardless of medium pH. Phosphorus deficiency increased significantly the number of the second-order lateral roots compared with the P₅₀ treatment. Both total root length and specific root length of plants grown at pH 5.6 were higher than those at pH 7.8 regardless of P supply. Cluster roots were formed at P₁, but cluster-root number was 2-fold higher at pH 7.8 than pH 5.6. Roots released 16 and 31% more protons at pH 5.6 and 7.8, respectively, in P₁ than in P₅₀ treatments, and the rate of proton release followed the similar pattern. At pH 5.6, citrate exudation rate was 0.39 μmol g⁻¹ root DW h⁻¹ at P₁, but was under the detection limit at P₅₀; at pH 7.8, it was 2.4-fold higher in P₁ than in P₅₀ plants. High pH significantly increased citrate exudation rate in comparison to pH 5.6. The uptake of anions P and S was inhibited at P₁ and high pH increased cations Na, Mg and Ca uptake. The results suggested that enhanced cluster-root formation, proton release and citrate exudation may account for the mechanism of efficient P acquisition by alkaline-tolerant L. pilosus well adapted to calcareous soils. Cluster-root formation and citrate exudation in L. pilosus can be altered by medium pH and P deficiency. Phosphorus deficiency-induced proton release may be associated with the reduced anion uptake, but high pH-induced proton release may be partly attributed to increased cation uptake.     

Spatial and temporal variation in citrate and malate exudation and tissue concentration as affected by P stress in roots of white lupin

Exudation of carboxylates represents one the most efficient strategies used by P-starved white lupin (Lupinus albus L.) to acquire phosphorus from sparingly soluble sources. This exudation occurs through proteoid root clusters, with citrate being the predominant organic acid released. The occasional detection of malate in whole root exudates suggests that this acid would also be released, but from tissues other than root clusters. To investigate the spatial and temporal pattern of exudation, citrate and malate exudation and concentration were measured in whole roots and root sections of white lupin, from seedling emergence to plant senescence due to P starvation. Both organic acids were detected in whole root exudates of P-stressed plants, and they were released at similar rates throughout the experiment. Malate was predominantly exuded from apices of both seedling taproots and proteoid roots, whereas citrate exudation was restricted to proteoid root clusters. Studies directed to address the association between carboxylate exudation and concentration in proteoid root clusters showed a non-linear response for citrate, within the range of 7 to 23 mol g^–1 fresh weight. This association was further assessed by altering citrate concentration in the whole root. Adding P to 24-day-old P-starved plants reduced citrate concentration and exudation to the level of the control P-fed plants, demonstrating that citrate exudation and concentration are associated. Malate exudation and concentration did not correlate significantly. Results indicate that citrate release by P-starved white lupin would occur whenever a certain threshold of citrate concentration is attained, and that the sites, the rates and the span of transient exudation depend on the physiological age of the tissue.     

A comparison of structure,development and function in cluster roots of Lupinus albus L. under phosphate and iron stress

Cluster roots are adaptations for nutrient acquisition, found throughout the world in many different plant families and habitats. They arise from changes in root initiation, meristem maintenance and physiology. In Lupinus albus cluster roots form under low internal plant phosphate and low internal plant iron levels. In this study, we compare morphology, structure and physiology of cluster roots formed under –P and –Fe conditions. –Fe cluster roots had a lower density of shorter rootlets than –P roots, and were yellow in colour, probably because of increased phenolics due to down-regulation of peroxidase. Rootlet length and width was reduced in –Fe conditions. The change in exudation of citrate, over time, of –P and –Fe cluster roots shared identical temporal dynamics, with an exudative burst occurring in day 3. However, the –Fe cluster roots displayed much higher rates of exudation than the –P cluster roots. Results are discussed within the context of structural and functional control.     

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.     

Spatial and temporal variation in organic acid anion exudation and nutrient anion uptake in the rhizosphere of Lupinus albus L.

We investigated in situ the temporal patterns and spatial extent of organic acid anion exudation into the rhizosphere solution of Lupinus albus, and its relation with the nutrient anions phosphate, nitrate and sulfate by means of a rhizobox micro suction cup method under P sufficient conditions. We compared the soil solution in the rhizosphere of cluster roots with that in the vicinity of normal roots, nodules and bulk soil. Compared to the other rhizosphere and soil compartments, concentrations of organic acid anions were higher in the vicinity of cluster roots during the exudative burst (citrate, oxalate) and nodules (acetate, malate), while concentrations of inorganic nutrient anions were highest in the bulk soil. Both active cluster roots and nodules were most efficient in taking up nitrate and phosphate. The intensity of citrate exudation by cluster roots was highly variable. The overall temporal patterns during the lifetime of cluster roots were overlaid by a diurnal pattern, i.e. in most cases, the exudation burst consisted of one or more peaks occurring in the afternoon. Multiple exudation peaks occurred daily or were separated by 1 or 2 days. Although citrate concentrations decreased with distance from the cluster root apex, they were still significantly higher at a distance of 6 to 10 mm than in the bulk soil. Phosphate concentrations were extremely variable in the proximity of cluster roots. While our results indicate that under P sufficient conditions cluster roots take up phosphate during their entire life time, the influence of citrate exudation on phosphate mobilization from soil could not be assessed conclusively because of the complex interactions between P uptake, organic acid anion exudation and P mobilization. However, we observed indications of P mobilization concurrent with the highest measured citrate concentrations. In conclusion, this study provides semiquantitative in situ data on the reactivity of different root segments of L. albus L. in terms of root exudation and nutrient uptake under nutrient sufficient conditions, in particular on the temporal variability during the lifetime of cluster roots.     

Spatial variability of symbiotic N2 fixation in grass-white clover pastures estimated by the 15N isotope dilution method and the natural 15N abundance method

Aiming at estimating the spatial variability in N₂ fixation, and to evaluate the appropriateness of the ¹⁵N isotope dilution (ID) method and the natural¹⁵N abundance (NA) method in reflecting spatial variability under the influence of cattle grazing, the symbiotic N₂ fixation in grass–white clover mixture was studied. At the Foulum site, where the ID method was used, differences in the climatic conditions between the two years of investigations caused a considerable difference in plant growth rates and proportion of clover. Consequently, the total N₂ fixation in ungrazed reference plots was significantly less in 1998 than in 1997, being 5.9 and 12.5 g N m^–2, respectively. In both years there was a wide range in concentration of inorganic N in the soil with coefficients of variance of approximately 60–190% for ammonium and 70–340% for nitrate. Significant negative correlations between pNdfa, determined by the ID method, and the log-transformed values of inorganic N and total N in grass were found. The NA method was applied on three nearby commercial dairy farms. They also showed high coefficients of variation. The coefficient of variance for NO₃ ^–-N ranged from 37 to 282% and for NH₄ ⁺-N from 29 to 237%. Average estimates of pNdfa values, which in the NA method were calculated using apparent B values ranging from –2.10 to –2.59, were generally lower (0.7–0.87) for these farms than for the Foulum site (0.89–0.95) using the ID method. For the NA method the ¹⁵N values, i.e. deviation in ¹⁵N concentration from atmospheric N₂, ranged from –7.0 to 5.7 for the grass N, which in several cases was lower than for clover N. Due to this high variability of the ¹⁵N values, probably caused by deposition and plant assimilation of ¹⁵N depleted urinary N in the pastures, the NA method was marginal for accurate determination of pNdfa. Consequently no significant correlation between the pNdfa determined by this method, and the log-transformed values of inorganic N in soil or total N in grass were found.     

The formation,morphology and anatomy of cluster root of Lupinus albus L. as dependent on soil type and phosphorus supply

The effect of phosphorus supply on the formation, morphology and anatomy of cluster roots of Lupinus albus L. cv Ultra grown in a loam and two sandy soils was examined relative to its effect on total root length, shoot weight and the phosphorus concentration of the shoots. The loam soil was most conducive to the formation of cluster roots. Cluster roots growing in the sandy soils developed to a lesser extent on plants of an equivalent phosphorus status, suggesting that some biotic or abiotic factors independent of phosphorus supply were also operating. The presence of mature cluster rootlets on a length of lateral root increased the root surface area by 14–22 times of an equal length of lateral roots not bearing cluster rootlets. The application of phosphorus decreased cluster-root length, whereas total root length showed a steady increase. There was an inverse relationship between cluster-root production and phosphorus concentration in shoots ranging from 2 to 8.5 mg g^–1 with the critical phosphorus level for maximum shoot growth being around 2.5 mg g^–1. Cluster roots formed in solution culture were not well developed in comparison with those grown in the loam soil or nutrient solution with added loam soil. The organisation of the cluster rootlet was similar to that of the lateral roots. Mature rootlets lacked an apical meristem and a vascular cambium with a reduced root cap and cortical tissue.     

Shoot P status regulates cluster-root growth and citrate exudation in Lupinus albus grown with a divided root system

The present study was carried out to investigate whether the P concentration in the roots or the shoots controls the growth and citrate exudation of cluster roots in white lupin (Lupinus albus L). Foliar P application indicated that low P concentration in the shoots enhanced cluster‐root growth and citrate‐exudation rate more so than low P concentration in the roots. In the split‐root study, the P concentration in the shoots increased with increased P supply (1, 25 or 75 mmol m^?3 P), to the ‘privileged’ root halves. Roots ‘deprived’ of P invariably had the same low P concentrations, whereas those in the ‘privileged’ roots increased with increasing P supply (1, 25 or 75 mmol m^?3 P). Nevertheless, the proportion of the total root mass allocated to cluster roots, and the citrate‐exudation rates from the root halves were always similar on both root halves, irrespective of P supply, and decreased with increasing shoot P concentrations. Peak citrate exudation rates from developing cluster roots were significantly faster from cluster roots on the ‘deprived’ root halves when the ‘privileged’ half was exposed to 1 mmol m^?3 P as compared with 25 or 75 mmol m^?3 P. The possibility that changes in the concentrations of P fractions in the root halves influenced cluster‐root growth and citrate exudation was discounted, because there were no significant differences in insoluble organic P, ester‐P and inorganic P among all ‘deprived’ root halves. The results indicate that cluster‐root proportions and citrate exudation rates were regulated systemically by the P status of the shoot, and that P concentrations in the roots had little influence on growth and citrate exudation of cluster roots in L. albus.     

缺磷白羽扇豆排根与非排根区根尖分泌有机酸的比较

采用根系分泌有机酸原位收集方法及高效液相色谱技术分析了供磷及缺磷后不同时间白羽扇豆(LupinusalbusL .)非排根区根尖和排根分泌有机酸的种类和数量 ,以及相应的根尖、排根组织 ,茎木质部、韧皮部汁液中有机酸含量的变化。结果表明 :(1)缺磷能够诱导白羽扇豆根系产生大量排根 ,根系的有机酸分泌量也明显增加。 (2 )无论在供磷或缺磷条件下 ,排根与非排根区根尖组织中的有机酸种类相同 ,但排根主要分泌柠檬酸和苹果酸 ,而非排根区根尖主要分泌苹果酸和乙酸。 (3)缺磷后非排根区根尖分泌苹果酸的量增加 ,至第 17天达到高峰 ;排根开始分泌柠檬酸的时间相对较晚。缺磷后排根分泌柠檬酸的量随缺磷时间的延长不断增加。 (4 )在缺磷的排根与非排根区根尖组织和茎木质部伤流液中含有大量柠檬酸和苹果酸 ,但在茎韧皮部汁液中则几乎检测不到这两种有机酸。上述结果表明 ,尽管排根和非排根区根尖组织中的有机酸种类相同 ,但它们向外分泌的有机酸种类不同。缺磷后排根及非排根区根尖增加向外分泌的有机酸主要在根中合成     

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.     

Lupinus species differ in their requirements for iron

The effect of iron supply on the growth and nodulation ofLupinus angustifolius L. (Gungurru),Lupinus luteus L. (R-1171) andLupinus pilosus Murr. (P20957) was studied in acid solutions. Plants of the three species were grown together in the same solution and inoculated withBradyrhizobium (Lupinus) WU 425. Plants were then grown with or without applied NH₄NO₃. The lupin species differed greatly in their sensitivity to low iron concentrations in solution withL. pilosus being most tolerant andL. luteus most sensitive.L. pilosus had the highest iron concentration in tissues and had a higher ratio of iron concentration in the youngest fully expanded leaf blades (YEB) to that in roots than the other two species.L. luteus had higher iron concentrations in roots but lower iron concentration in YEB and shoots than didL. angustifolius. The requirements of internal iron for the maximal chlorophyll synthesis in YEB were 65 μg g^-1 forL. angustifolius andL. luteus, and 52 μg g^-1 forL. pilosus. In contrast to effects on growth, the three species had similar external iron requirements for nodule formation in roots and for maximal nitrogen concentrations in shoots. The results indicate that iron tolerant lupin species require lower internal and external iron supply and have a greater ability than sensitive species to translocate iron from roots to shoots.     

Role of phosphorus nutrition in development of cluster roots and release of carboxylates in soil-grown Lupinus albus

The present study examined the effect of phosphorus (P) limitation on cluster root formation and exudation of carboxylates by N₂-fixing white lupin (Lupinus albus L. cv. Kiev) grown in a P-deficient sandy soil. Plants received 10 (limited P) or 200 g P g^–1 soil as FePO₄ (adequate P) and were grown in a phytotron at 20/12 °C (12/12 h) for 76 days in soil columns. Cluster root formation was assessed and root exudates were collected at 9-day intervals. Shoot and root dry weights were higher in plants grown in the adequate-P compared to the limited-P treatment for 67 days. No clear difference in the total root length was observed between two P treatments before day 58. However, the specific root length increased rapidly from 17 m g^–1 DW at day 40 to 28 m g^–1 at day 49 in the P-limited plants, but decreased in the P-adequate plants. The effect of P limitation on enhancement of cluster root formation was observed from day 40 and reached the maximum at day 58. The number of cluster roots was negatively correlated with the P concentration in both roots and shoots. Phosphorus limitation increased exudation of citrate from day 40. The exudation of citrate displayed a cyclic pattern throughout the experiment, and appeared related to internal P concentration in plants, particularly P concentration in shoots. The sorption of exogenously added citrate in the soil was also examined. The amount of extractable citrate remained unchanged for 2 h, but decreased thereafter, suggesting that the soil had a low capacity to sorb citrate, and the rate of its decomposition by microorganisms was slow. Collecting solution leached through a soil column is a simple and reliable method to acquire root exudates from white lupin grown in soil. The results suggest that formation of cluster roots and exudation of citrate in white lupin are regulated by P concentration in shoots.     

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.     

Growth Medium and Phosphorus Supply Affect Cluster Root Formation and Citrate Exudation by Lupinus albus Grown in a Sand/Solution Split-Root System

We examined cluster root formation and root exudation by white lupin (Lupinus albus L. cv. Kiev Mutant) in response to growth medium and phosphorus supply in a sand/solution split-root system. The split-root system consisted of a nutrient solution compartment and a siliceous sand compartment. Phosphorus was applied at 1 (low-P plants) or 50 (high-P plants) μM as KH₂PO₄ to the solution compartment and at 10, 50 or 250 mg P kg⁻¹ as hydroxyapatite (Ca-P) to the sand compartment. In contrast to the high-P plants, P concentration and P uptake in the low-P plants increased with increasing P supply to the sand compartment. The NaHCO₃-extractable P was lower in the rhizosphere of the low-P plants than the high-P ones. The proton extrusion rate by the solution-grown roots of the low-P plants was higher than that of the high-P plants at the early growth stage. For the low-P plants, the proportion of dry root biomass allocated to cluster roots was higher in the solution compartment than that in the sand compartment. The citrate exudation increased in the sand compartment and decreased in the solution compartment with time, showing a lack of synchronization in citrate exudation by two root halves grown in different media. The cluster root proportion and citrate exudation in both compartments decreased with increasing shoot P concentration. An additional experiment with no P added to either root compartment showed that the proportion of cluster roots was about 9% lower in the sand than solution compartments. The results suggest that cluster root formation and citrate exudation can be significantly affected by the root growth medium in addition to being regulated by shoot P status. More P can be exploited from sparingly available Ca-P by the low-P plants than the high-P ones due to greater citrate exudation under P deficiency.     

pH above 6.0 reduces nodulation in Lupinus species

Lupinus angustifolius and Lupinus pilosus differ substantially in root growth in response to high solution pH with L. angustifolius showing much greater sensitivity to pH above 6. This study examined the effect of pH above 6 on nodulation of these two species in buffered solution. Shoot weight and root weight and length in L. pilosus was not significantly affected by pH, whereas the growth of shoots and roots of L. angustifolius was markedly impaired by increasing pH. Total root length, the number of lateral roots, and the length of individual lateral roots were greatly decreased, resulting in decreased uptake of iron and phosphorus. In addition, L. angustifolius had a higher internal requirement for iron than L. pilosus. A solution pH above 6 decreased the number of nodule initials and nodules similarly in both species but decreased nodule mass much more in L. angustifolius. The effect of high pH on nodule formation occurred prior to that on host shoot growth. High pH also decreased nitrogen concentration and content in both species but to a greater extent for L. angustifolius. The results suggested that pH above 6 has a specific effect in the impairment of nodulation in lupins.     

Acclimation of white lupin to phosphorus deficiency involves enhanced expression of genes related to organic acid metabolism

White lupin (Lupinus albus L.) acclimates to phosphorus deficiency (–P) by the development of short, densely clustered lateral roots called proteoid (or cluster) roots. These specialized plant organs display increased exudation of citric and malic acid. The enhanced exudation of organic acids from P stressed white lupin roots is accompanied by increased in vitro phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activity. Here we report the cloning of full-length white lupin PEPC and MDH cDNAs. RNA blot analysis indicates enhanced expression of these genes in –P proteoid roots, placing higher gene expression at the site of organic acid exudation. Correspondingly, macroarray analysis of about 1250 ESTs (expressed sequence tags) revealed induced expression of genes involved in organic acid metabolism in –P proteoid roots. In situ hybridization revealed that PEPC and MDH were both expressed in the cortex of emerging and mature proteoid rootlets. A C₃ PEPC protein was partially purified from proteoid roots of P deficient white lupin. Native and subunit Mr were determined to be 440 kD and 110 kD, respectively. Citrate and malate were effective inhibitors of in vitro PEPC activity at pH 7. Addition of ATP partially relieved inhibition of PEPC by malate but had little effect on citrate inhibition. Taken together, the results presented here suggest that acclimation of white lupin to low P involves modified expression of plant genes involved in carbon metabolism.     

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.     

Time and substrate dependent exudation of carboxylates by Lupinus albus L. and Brassica napus L.

Root exudates influence significantly physical, chemical and biological characteristics of rhizosphere soil. Their qualitative and quantitative composition is affected by environmental factors such as pH, soil type, oxygen status, light intensity, soil temperature, plant growth, nutrient availability and microorganisms. The aim of the present study was to assess the influence of growth substrate and plant age on the release of carboxylates from Lupinus albus L. and Brassica napus L.Both plant species were studied in continuously percolated microcosms filled with either sand, soil or sand + soil (1:1) mixture. Soil solution was collected every week at 7, 14, 21, 28 and 35 days after planting (DAP). Carboxylate concentrations were determined by reversed-phase liquid chromatography - electrospray ionization - time of flight mass spectrometry (LC-ESI-TOFMS).Oxalate, citrate, succinate, malate and maleate were detected in soil solutions of both plant species. Their concentrations were correlated with the physiological status of the plant and the growth substrate. Oxalate was the predominant carboxylate detected within the soil solution of B. napus plants while oxalate and citrate were the predominant ones found in the soil solutions of L. albus plants.The sampling determination of carboxylates released by plant roots with continuous percolation systems seems to be promising as it is a non-destructive method and allows sampling and determination of soluble low molecular weight organic compounds derived from root exudation as well as the concentration of soluble nutrients, which both might reflect the nutritional status of plants.     

Effects of external phosphorus supply on internal phosphorus concentration and the initiation,growth and exudation of cluster roots in Hakea prostrata R.Br.

The response of internal phosphorus concentration, cluster-root initiation, and growth and carboxylate exudation to different external P supplies was investigated in Hakea prostrata R.Br. using a split-root design. After removal of most of the taproot, equal amounts of laterals were allowed to grow in two separate pots fastened together at the top, so that the separate root halves could be exposed to different conditions. Plants were grown for 10 weeks in this system; one root half was supplied with 1 M P while the other halves were supplied with 0, 1, 25 or 75 M P. Higher concentrations of P supplied to one root half significantly increased the P concentration of those roots and in the shoots. The P concentrations in root halves supplied with 1 M P were invariably low, regardless of the P concentration supplied to the other root half. Cluster root initiation was completely suppressed on root halves supplied with 25 or 75 M P, whereas it continued on the other halves supplied with 1 M P indicating that cluster-root initiation was regulated by local root P concentration. Cluster-root growth (dry mass increment) on root halves supplied with 1 M P was significantly reduced when the other half was either deprived of P or supplied with 25 or 75 M P. Cluster-root growth was favoured by a low shoot P status at a root P supply that was adequate for increased growth of roots and shoots without increased tissue P concentrations. The differences in cluster-root growth on root halves with the same P supply suggest that decreased cluster-root growth was systemically regulated. Carboxylate-exudation rates from cluster roots on root halves supplied with 1 M P were the same, whether the other root half was supplied with 1, 25 or 75 M P, but were approximately 30 times faster when the other half was deprived of P. Estimates of root P-uptake rates suggest a rather limited capacity for down-regulating P uptake when phosphate was readily available.     

Soil Phosphorus Uptake by Continuously Cropped Lupinus albus: A New Microcosm Design

When grown in soils with sparingly available phosphorus (P), white lupin (Lupinus albus L.) forms special root structures, called cluster roots, which secrete large amounts of organic acids and concomitantly acidify the rhizosphere. Many studies dealing with the understanding of this P acquisition strategy have been performed in short time experiments either in hydroponic cultures or in small microcosm designs with sand or sand:soil mixtures. In the present study, we applied an experimental design which came nearer to the natural field conditions: we performed a one-year experiment on large microcosms containing 7 kg of soil and allowing separation of rhizosphere soil and bulk soil. We planted six successive generations of lupins and analysed P uptake, organic P desorption, phosphatase activities and organic acid concentrations in different soil samples along a spatio-temporal gradient. We compared the rhizosphere soil samples of cluster (RSC) and non-cluster roots (RSNC) as well as the bulk soil (BS) samples. A total shoot biomass of 55.69 ± 1.51 g (d.w.) y⁻¹ was produced and P uptake reached 220.59 ± 5.99 mg y⁻¹. More P was desorbed from RSC than from RSNC or BS (P < 0.05). RSC and RSNC showed a higher activity of acid and alkaline phosphatases than BS samples and a higher acid phosphatase activity was observed in RSC than in RSNC throughout the one-year experiment. Fumarate was the most abundant organic acid in all rhizosphere soil samples. Citrate was only present in detectable amounts in RSC while malate and fumarate were recovered from both RSC and RSNC. Almost no organic acids could be detected in the BS samples. Our results demonstrated that over a one-year cultivation period in the absence of an external P supply, white lupin was able to acquire phosphate from the soil and that the processes leading to this P uptake took place preferentially in the rhizosphere of cluster roots.