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.     

ATP citrate lyase: cloning, heterologous expression and possible implication in root organic acid metabolism and excretion

In order to cope with phosphate deficiency, white lupin produces bottle‐brushed like roots, so‐called cluster or proteoid roots which are specialized in malate and citrate excretion. Young, developing cluster roots mainly excrete malate whereas mature cluster roots mainly release citrate. Mature proteoid roots excrete four to six times more carboxylates compared with juvenile proteoid roots. Using a cDNA‐amplified restriction fragment length polymorphism (AFLP) approach we identified a gene coding for a putative ATP‐citrate lyase (ACL) up‐regulated in young cluster roots. Cloning of the lupin ACL revealed that plant ACL is constituted by two polypeptides (ACLA and ACLB) encoded by two different genes. This contrasts with the animal ACL, constituted of one polypeptide which covers ACLA and ACLB. The ACL function of the two lupin gene products has been demonstrated by heterologous expression in yeast. Both subunits are required for ACL activity. In lupin cluster roots, our results suggest that ACL activity could be responsible for the switch between malate and citrate excretion in the different developmental stages of cluster roots. In primary roots of lupin and maize, ACL activity was positively correlated with malate exudation. These results show that ACL is implicated in root exudation of organic acids and hence plays a novel role in addition to lipid synthesis. Our results suggest that in addition to lipid biosynthesis, in plants, ACL is implicated in malate excretion.     

Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress

White lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to -P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA(i))-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under -P stress. Here, we show that LaMATE had high expression in proteoid roots not only under -P, but also under -Fe, -N, -Mn and +Al stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein. LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA(i)-based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.     

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.     

Interactions between atmospheric CO2 concentration and phosphorus nutrition on the formation of proteoid roots in white lupin (Lupinus albus L.)

Atmospheric [CO₂] affects photosynthesis and therefore should affect the supply of carbon to roots. To evaluate interactions between carbon supply and nutrient acquisition, the [CO₂] effects on root growth, proteoid root formation and phosphorus (P) uptake capacity were studied in white lupin (Lupinus albus L.) grown hydroponically at 200, 410 and 750 µmol mol^?1 CO₂, under sufficient (0·25 mm P) and deficient (0·69 µm P) phosphorus. Plant size increased with increasing [CO₂] only at high P. Both P deficiency and increasing [CO₂] increased the production of proteoid clusters; the increase in response to increased [CO₂] was proportionally greater from low to ambient [CO₂] than from ambient to high. The activity of phosphoenol pyruvate carboxylase in the proteoid root, the exudation of organic acids from the roots, and the specific uptake of P increased with P deficiency, but were unaffected by [CO₂]. Increasing [CO₂] from Pleistocene levels to those predicted for the next century increased plant size and allocation to proteoid roots, but did not change the specific P uptake capacity per unit root mass. Hence, rising [CO₂] should promote nutrient uptake by allowing lupins to mine greater volumes of soil.     

Structure,ecology and physiology of root clusters – a review

Hairy rootlets, aggregated in longitudinal rows to form distinct clusters, are a major part of the root system in some species. These root clusters are almost universal (1600 species) in the family Proteaceae (proteoid roots), with fewer species in another seven families. There may be 10–1000 rootlets per cm length of parent root in 2–7 rows. Proteoid roots may increase the surface area by over 140× and soil volume explored by 300× that per length of an equivalent non-proteoid root. This greatly enhances exudation of carboxylates, phenolics and water, solubilisation of mineral and organic nutrients and uptake of inorganic nutrients, amino acids and water per unit root mass. Root cluster production peaks at soil nutrient levels (P, N, Fe) suboptimal for growth of the rest of the root system, and may cease when shoot mass peaks. As with other root types, root cluster production is controlled by the interplay between external and internal nutrient levels, and mediated by auxin and other hormones to which the process is particularly sensitive. Proteoid roots are concentrated in the humus-rich surface soil horizons, by 800× in Banksia scrub-heath. Compared with an equal mass of the B horizon, the A₁ horizon has much higher levels of N, P, K and Ca in soils where species with proteoid root clusters are prominent, and the concentration of root clusters in that region ensures that uptake is optimal where supply is maximal. Both proteoid and non-proteoid root growth are promoted wherever the humus-rich layer is located in the soil profile, with 4× more proteoid roots per root length in Hakea laurina. Proteoid root production near the soil surface is favoured among hakeas, even in uniform soil, but to a lesser extent, while addition of dilute N or P solutions in split-root system studies promotes non-proteoid, but inhibits proteoid, root production. Local or seasonal applications of water to hakeas initiate non-proteoid, then proteoid, root production, while waterlogging inhibits non-proteoid, but promotes proteoid, root production near the soil surface. A chemical stimulus, probably of bacterial origin, may be associated with root cluster initiation, but most experiments have alternative interpretations. It is possible that the bacterial component of soil pockets rich in organic matter, rather than their nutrient component, could be responsible for the proliferation of proteoid roots there, but much more research on root cluster microbiology is needed.     

Acid phosphatase activity in phosphorus-deficient white lupin roots

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

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.     

Factors affecting formation of cluster roots in Myrica gale seedlings in water culture

The effect of nutrient deficiency, aeration, phosphorus supply, and nitrogen source on the formation of cluster (proteoid) roots was examined in Myrica gale seedlings growing in water culture. Only the omission of phosphorus resulted in the formation of significant numbers to cluster roots when plants were grown in a number of 1/4 strength Hoagland's solutions, each lacking one mineral nutrient. Aeration shortened the time required for cluster root formation and increased the percentage of plants forming cluster roots. The proportion of the root system comprised of cluster roots decreased as the phosphorus concentration in the solution increased and no cluster roots formed in solutions containing 8 mg P/L. Phosphorus supply also affected total plant biomass, proportion of biomass comprising nitrogen-fixing nodules, shoot:root ratio, phosphorus concentration in the leaves and phosphorus content of the plants. The plants showed luxury consumption of phosphorus and were able to produce large amounts of biomass utilizing only stored phosphorus.Nitrogen source also affected cluster root formation. Urea-fed plants produced cluster roots more quickly and devoted a substantially larger proportion of root growth to cluster roots than did nitrate-fed plants. The longest cluster root axes were produced in nitrate-fed plants supplied with no phosphorus and the shortest were in urea-fed plants at 4 mg P L^–1.Four methods for expressing the extent of cluster root formation were examined and it was concluded that cluster roots as a proportion of total fine root dry weight is preferable in many cases. Formation of cluster roots in response to phosphorus deficiency coupled with previously demonstrated traits allows Myrica gale to adapt to a wide range of soil conditions.     

Proteoid Root Development of Phosphorus Deficient Lupin is Mimicked by Auxin and Phosphonate

White lupin (Lupinus albus L.) develops proteoid (cluster) rootsin response to phosphorus deficiency. Proteoid roots are composedof tight clusters of rootlets that initiate from the pericycleopposite protoxylem poles and emerge from every protoxylem polewithin the proteoid root axis. Auxins are required for lateralroot development, but little is known of their role in proteoidroot formation. Proteoid root numbers were dramatically increasedin P-sufficient (+P) plants by application of the syntheticauxin, naphthalene acetic acid (NAA), to leaves, and were reducedin P-deficient (-P) plants by the presence of auxin transportinhibitors [2,3,5-triiodobenzoic acid (TIBA) and naphthylphthalamicacid (NPA)]. While ethylene concentrations in the root zonewere 1.5-fold higher in -P plants, there was no effect on proteoidroot numbers of the ethylene inhibitors aminoethoxyvinvylglycine(AVG) and silver thiosulphate. Phosphonate, which interfereswith plant perception of internal P concentration, dramaticallyincreased the number of proteoid root segments in +P plants.Activities of phosphoenolpyruvate carboxylase (PEPC), malatedehydrogenase (MDH) and exuded acid phosphatase in proteoidroot segments were not different from +P controls when NAA wasapplied to +P lupin plants, but increased to levels comparableto -P plants in the phosphonate treatment. Addition of TIBAor NPA to -P plants reduced PEPC and MDH activity of -P proteoidroots to levels found in +P or -P normal root tissues, but didnot affect acid phosphatase in root exudates. These resultssuggest that auxin transport from the shoot plays a role inthe formation of proteoid roots during P deficiency. Auxin-stimulatedproteoid root formation is necessary, but not sufficient, tosignal the up-regulation of PEPC and MDH in proteoid root segments.In contrast, phosphonate applied to P-sufficient white lupinelicits the full suite of coordinated responses to P deficiencyCopyright2000 Annals of Botany Company Lupinus albus L., white lupin, proteoid roots, auxin, ethylene, phosphonate, phosphorus deficiency     

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     

Higher rates of nitrogen fertilization decrease soil enzyme activities, microbial functional diversity and nitrification capacity in a Chinese polytunnel greenhouse vegetable land

White lupin (Lupinus albus L.) is considered a model system for understanding plant acclimation to nutrient deficiency. It acclimates to phosphorus (P) and iron (Fe) deficiency by the development of short, densely clustered lateral roots called proteoid (or cluster) roots; proteoid-root development is further influenced by nitrogen (N) supply. In an effort to better understand proteoid root function under various nutrient deficiencies, we used nylon filter arrays to analyze 2,102 expressed sequence tags (ESTs) from proteoid roots of P-deficient white lupin. These have been previously analyzed for up-regulation in ?P proteoid roots, and were here analyzed for up-regulation in proteoid roots of N-deprived plants. We identified a total of 19 genes that displayed up-regulation in proteoid roots under both P and N deprivation. One of these genes showed homology to putative formamidases. The corresponding open reading frame was cloned, overexpressed in E. coli, and the encoded protein was purified; functional characterization of the recombinant protein confirmed formamidase activity. Though many homologues of bacterial and fungal formamidases have been identified in plants, to our knowledge, this is the first report of a functional characterization of a plant formamidase.     

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.     

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

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

子叶磷在白羽扇豆缺磷适应性反应中的作用

实验用液体培养的方法,对比分析了在不同供磷条件下,白羽扇豆子叶中的磷对植物生长发育的影响,以及排根和根尖中有机酸积累和分泌的作用,结果表明,子叶中的磷能使白羽扇豆在完全缺磷２３d的环境中,不仅没有使干物质的积累减少,反而使干物质的积累略有增加,相反,如果没有子叶磷的供给,则使白羽扇豆在缺磷环境中产生强烈的抗胁迫反应,表现在干物质的积累明显下降,根系能产生大量的排根,排根能积累和分泌大量的柠檬酸,而根尖能积累和分泌萍果酸,在整个缺磷反应过程中,根尖中苹果酸的分泌要早于排根可柠檬酸的积累和分泌。     

Root Cluster Formation and Citrate Exudation of White Lupin （Lupinus albus L.） as Related to Phosphorus Availability

A split-root system was used to investigate whether the external or internal P concentration controls root cluster formation and citrate exudation in white lupin (Lupinus albus L.) grown under controlled conditions. In spite of low P concentrations in the shoots and roots of the -P plant, its dry weight was not reduced compared with the P plant. Supplying external P (0.25 mmol/L) to one root half resulted in an increase in P concentration not only in the shoot, but also in the P-deprived root half, indicating P cycling within the plants. Omitting P from both split-root pots stimulated root cluster formation in both root halves,whereas P supply to one root half stimulated root cluster formation at the beginning of the treatment. Neither P supply to just one root half continuously nor resupply of P to one root half after 19 d of P starvation inhibited root cluster formation on the P-deprived side, although the concentration of P in this root half and shoot increased markedly. The results indicate that root cluster formation in L. albus is controlled by both shoot and root P concentrations. The rates of citrate exudation by both root halves with P deficiency were higher than those of the one root half supplied with P only. In the treatment with one root half supplied with P, the rates of citrate exudation by either the P-supplied or -deprived root halves were almost the same,regardless of P concentration in the roots. The results suggest that internal P concentration controls root cluster formation and citrate exudation in white lupin, but these processes may be regulated by different mechanisms.     

Root Cluster Formation and Citrate Exudation of White Lupin (Lupinus albus L.) as Related to Phosphorus Availability

A split-root system was used to investigate whether the external or internal P concentration controls root cluster formation and citrate exudation in white lupin (Lupinus albus L.) grown under controlled conditions. In spite of low P concentrations in the shoots and roots of the -P plant, its dry weight was not reduced compared with the P plant. Supplying external P (0.25 mmol/L) to one root halfresulted in an increase in P concentration not only in the shoot, but also in the P-deprived root half, indicating P cycling within the plants. Omitting P from both split-root pots stimulated root cluster formation in both root halves,whereas P supply to one root halfstimulated root cluster formation at the beginning of the treatment. Neither P supply to just one root half continuously nor resupply of P to one root half after 19 d of P starvation inhibited root cluster formation on the P-deprived side, although the concentration of P in this root half and shoot increased markedly. The results indicate that root cluster formation in L. albus is controlled by both shoot and root P concentrations. The rates of citrate exudation by both root halves with P deficiency were higher than those of the one root half supplied with P only. In the treatment with one root half supplied with P, the rates of citrate exudation by either the P-supplied or -deprived root halves were almost the same,regardless of P concentration in the roots. The results suggest that internal P concentration controls root cluster formation and citrate exudation in white lupin, but these processes may be regulated by different mechanisms.     

Differences in cluster-root formation and carboxylate exudation in Lupinus albusL. under different nutrient deficiencies

In contrast with the well document role of proteoid root formation and carboxylate exudation in acclimation to P deficiency in white lupin (Lupinus albus L.), their role under other nutrient deficiencies and their ecological significance are still poorly understood. In the present work, differences in proteoid root formation, exudation of carboxylates by root clusters, non-proteoid and proteoid root tips by using a non-destructive method, and concentrations of organic acids in the tissues of plants grown in the absence of P, Fe or K were studied. Proton release from roots increased soon after withdrawing Fe from the medium; within three days the solution pH decreased from 6 to about 4, and this increased release in protons continued until the end of the experiment. Acidification appeared much later, on the 10th day and the 14th day after withdrawal of P and K, respectively; the extent of the acidification was also weaker than under –Fe (5.2 for –P and 5.7 for control on the 10th day; 6.0 for –K and 6.1 for control on the 14th day). Root clusters formed when plants were grown under –P and –Fe, but not under –K conditions. The root clusters developed sooner under –Fe conditions, but the number of clusters was far less than under –P. Under P deficiency, root clusters released mainly citrate, but also some malate; while the major organic acid released by root tips of both non-proteoid and proteoid roots was malate. However, under Fe deficiency, the majority of the organic acids exuded both by the root clusters and root tips was malate, whereas only a small amount of citrate was detected. The release rate of citrate by – P root clusters was greater than that by – Fe root clusters. Moreover, the release rate of malate was greater in –Fe root clusters than in –P root clusters, but the opposite was found in proteoid root tips, i.e. faster in –P than in –Fe proteoid root tips. The significances of proteoid root formation and release of organic acids in acclimation to different nutrient deficiencies for white lupin plants are discussed.     

The relationship between silicon availability,and growth and silicon concentration of the salt marsh halophyte Spartina anglica

Incorporation of cover crops into cropping systems may contribute to a more efficient utilization of soil and fertilizer P by less P-efficient crops through exudation of P-mobilizing compounds by the roots of P-efficient plant species. The main objective of the present work was to test this hypothesis. First a method has been developed which allows the quantification of organic anion exudation from individual cluster roots formed by P-deficient white lupin (Lupinus albus L.). Lupin plants were grown in nutrient solution at 1 μM P and in a low P loess in small rhizotrons. Organic anions exuded from intact plants grown in nutrient solution were collected from individual cluster roots and root tips sealed in small compartments by an anion-exchange resin placed in nylon bags (resin-bags). Succinate was the dominant organic anion exuded followed by citrate and malate. The mean of citrate exudation-rate was 0.06 pmol mm⁻¹ s⁻¹ with exudation highly dependent on the citrate concentration and on the age of the cluster roots. Exudates from cluster roots and root tips grown at the soil surface (rhizotron-grown plants) were collected using overlayered resin–agar (resin mixed with agar). Citrate exudation from cluster roots was 10 times higher than that from root tips. Fractionation of P in the cluster root rhizosphere-soil indicates that white lupin can mobilize P not only from the available and acid-soluble P, but also from the stable residual soil P fractions. In pot experiments with an acid luvisol derived from loess low in available P, growth of wheat was significantly improved when mixed-cropped with white lupin due to improved P uptake. Both in mixed culture and in rotation wheat could benefit from the P mobilization capacity of white lupin, supporting the hypothesis above. Nine tropical leguminous cover crops and maize were grown in a pot experiment using a luvisol from Northern Nigeria low in available P. All plant species derived most of their P from the resin and bicarbonate-extractable inorganic P. Organic P (P_o) accumulated particularly in the rhizosphere of all plant species. There was a significant negative correlation between the species-specific rhizosphere acid phosphatase activity and P_o accumulation. Growth and P uptake of maize grown in rotation after legumes were enhanced indicating that improved P nutrition was a contributing factor. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.