Phosphorus Stress-Induced Proteoid Roots Show Altered Metabolism in Lupinus albus

Proteoid roots develop in Lupinus albus L. in response to nutrient stress, especially P. Proteoid roots excrete citrate and thus increase the availability of P, Fe, and Mn in the rhizosphere. In an effort to understand citrate synthesis and organic acid metabolism in proteoid roots of lupin, we have evaluated in vitro enzyme activities of citrate synthase (CS), malate dehydrogenase (MDH), and phosphoenolpyruvate carboxylase (PEPC) in proteoid and normal roots of plants grown with or without P. Organic acid concentrations, respiration rates, and dark 14CO2-labeling patterns were also determined. The in vitro specific activities of CS, MDH, and PEPC and in vivo dark 14CO2 fixation were higher in proteoid roots compared to normal roots, particularly under P stress. Western blot analysis showed that PEPC enzyme protein was more highly expressed in -P proteoid roots compared to other tissues. The majority of the fixed 14C was found in organic acids, predominantly malate and citrate. A larger fraction of citrate was labeled in P- stressed proteoid roots compared to other root tissue. Respiration rates of proteoid roots were 31% less than those of normal roots. The data provide evidence for increased synthesis of citrate in proteoid roots compared to normal roots, particularly under P stress. A portion of the carbon for citrate synthesis is derived from nonautotrophic CO2 fixation via PEPC in proteoid roots.     

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.     

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.     

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.     

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     

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.     

缺磷条件下白羽扇豆排根发育与生长素及miR164的关系

以缺磷条件下白羽扇豆为材料,观察了外源生长素NAA和生长素运输的抑制剂NPA 对白羽扇豆排根形成及其活性的影响,同时运用基因芯片与RT-PCR的方法分析了生长素信号转导途径中转录因子NAC1以及调控NAC1表达的上游microRNA164(miR164)在不同发育阶段排根中的表达变化,以探讨白羽扇豆在缺磷时排根形成与发育的调控机制.结果表明,缺磷胁迫下排根大量形成与生长素及其运输有关,排根NAC1的表达在初生阶段上调,成熟后下调,并受其上游的miR164的负调控,而排根衰老后则上述基因的表达都减弱.研究发现,在缺磷诱导的排根发生至发育成熟过程中,miR164、NAC1、生长素与排根发育之间很可能组成了一个级联系统,从而控制排根的发生与发育.     

White Lupin (Lupinus albus) response to phosphorus stress: evidence for complex regulation of LaSAP1

Proteoid roots are a unique adaptation that allow white lupin (Lupinus albus L. var Ultra) to survive under extreme phosphorus (P) deficient conditions. The cascade of events that signals P-deficiency induced gene expression in proteoid roots remains unknown. Through promoter::GUS analysis we showed that expression of acid phosphatase (LaSAP1) in P-deficient proteoid roots depends on DNA located from ?465 bp to ?345 bp 5′ of the ATG start codon and that the P1BS (PHR1 Binding Site) element, located at ?160 bp, also contributes regulatory control. DNA located within the ?414 bp to ?250 bp region of the LaSAP1 promoter was bound by nuclear proteins isolated from P-sufficient normal roots in electrophoretic mobility shift assays (EMSA), suggesting negative regulation. Competition experiments were performed with unlabeled oligonucleotides to further delineate the region of the LaSAP1 promoter bound by P-sufficient normal root nuclear proteins to a motif spanning ?361 bp to ?346 bp. The promoter motif characterized through EMSA spanning ?361 bp to ?345 bp was used as “bait” in a yeast one-hybrid (Y1H) experiment and 31 putative DNA binding proteins were isolated. Taken together, our results increase understanding of P-deficiency signaling by identifying regulatory regions and putative regulatory proteins for LaSAP1 expression.     

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.     

Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency

White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.     

Physiological Aspects of Cluster Root Function and Development in Phosphorus-deficient White Lupin (Lupinus albus L.)

Cluster root formation in white lupin (Lupinus albus L.) isinduced mainly by phosphorus (P) starvation, and seems to beregulated by the endogenous P status of the plant. Increasedformation of cluster roots, when indole acetic acid is suppliedto the growth medium of P sufficient plants, and inhibitoryeffects of kinetin application suggest the involvement of endogenousphytohormones (auxins and cytokinins), which may act in an antagonisticmanner in the P-starvation response. Phosphorus deficiency-inducedadaptations of white lupin, involved in P acquisition and mobilizationof sparingly available P sources, are predominantly confinedto the cluster roots, and moreover to distinct stages duringtheir development. Increased accumulation and exudation of citrateand a concomitant release of protons were found to be mainlyrestricted to mature root clusters after prolonged culture (3–4weeks) under P-deficient conditions. Inhibition of citrate exudationby exogenous application of anion channel antagonists such asethacrynic- and anthracene-9-carboxylic acids may indicate involvementof an anion channel. Phosphorus deficiency-induced accumulationand subsequent exudation of citric acid seems to be a consequenceof both enhanced biosynthesis and reduced turnover of citricacid in the cluster root tissue, indicated by enhanced expressionof sucrose synthase, fructokinase, phosphoglucomutase, phosphoenol-pyruvatecarboxylase, but reduced activity of aconitase and slower rootrespiration. The release of acid phosphatase and of phenoliccompounds (isoflavonoids) as well as the induction of a putativehigh-affinity P uptake system was more highly expressed in juvenile,mature and even senescent cluster regions than in apical zonesof non-proteoid roots. An AFLP-cDNA library for cluster root-specificgene expression was constructed to assist in the identificationof further genes involved in cluster root development. Copyright2000 Annals of Botany Company Acid phosphatase, auxin, citric acid, cluster roots, cytokinin, Lupinus albus L., P acquisition, P uptake, root exudates     

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.     

Interactive effects of phosphorus deficiency and exogenous auxin on root morphological and physiological traits in white lupin (Lupinus albus L.)

White lupin (Lupinus albus) exhibits strong root morphological and physiological responses to phosphorus (P) deficiency and auxin treatments, but the interactive effects of P and auxin in regulating root morphological and physiological traits are not fully understood. This study aimed to assess white lupin root traits as influenced by P (0 or 250 μmol L^?1) and auxin (10^?8 mol L^?1 NAA) in nutrient solution. Both P deficiency and auxin treatments significantly altered root morphological traits, as evidenced by reduced taproot length, increased number and density of first-order lateral roots, and enhanced cluster-root formation. Changes in root physiological traits were also observed, i.e., increased proton, citrate, and acid phosphatase exudation. Exogenous auxin enhanced root responses and sensitivity to P deficiency. A significant interplay exists between P and auxin in the regulation of root morphological and physiological traits. Principal component analysis showed that P availability explained 64.8% and auxin addition 21.3% of the total variation in root trait parameters, indicating that P availability is much more important than auxin in modifying root responses of white lupin. This suggests that white lupin can coordinate root morphological and physiological responses to enhance acquisition of P resources, with an optimal trade-off between root morphological and physiological traits regulated by external stimuli such as P availability and auxin.     

The Response of Lupinus albus Roots to the Signal from Phosphorus-Deficient Substrate1

The excretion of organic acids into the rhizosphere is induced by low phosphorus content in roots of white lupine (Lupinus albus L.). The aim of this study was to investigate how did the white lupine roots respond to the signals of P-deficiency in the substrate, by using the method of separating the root system into two parts, one part being placed into P-deficient solution and another part into P-sufficient solution. The results showed that the root tips (1-cm-long) accept the signal of P-deficiency in the substrate. This signal can be transmitted from one root tip to another, and can induce the accumulation and excretion of malic acid in the root tips and the development of proteoid roots. In order to investigate whether a phytohormone was involved in the response, the external hormones were used. Indole-3-butyric acid added to P-sufficient solution can induce the development of proteoid roots, but without any accumulation of citric acid in them. On the contrary, 6-benzyladenine (BA) added to the P-deficient solution can inhibit the development of proteoid roots and the accumulation of malic acid in the root tips. The inhibitory effect increases with increasing BA supply.     

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.     

The Role of the Stem in Phloem Loading of Minerals in Lupinus albus L. cv. Ultra

Collections of phloem sap made over a 40-day period from a varietyof locations on nodulated white lupin plants (Lupinus albusL. cv. ultra) showed considerable enrichment with K⁺ and Mg²⁺in the phloem streams destined for the shoot apices or fruitsrelative to the streams arising from the leaflets (up to 5.5times). Sodium showed enrichment in the streams destined forthe roots (up to 2.5 times) but only when present in the watersupply at a high level (3 mM). The stem, in view of its centrallocation in the transport pathway, is seen as an organ capableof redistributing minerals in the phloem independently of photosynthate. Lupinus albus L., lupin, phloem loading, magnesium, potassium, sodium, mineral elements     

Root Carbon Dioxide Fixation by Phosphorus-Deficient Lupinus albus (Contribution to Organic Acid Exudation by Proteoid Roots)

When white lupin (Lupinus albus L.) is subjected to P deficiency lateral root development is altered and densely clustered, tertiary lateral roots (proteoid roots) are initiated. These proteoid roots exude large amounts of citrate, which increases P solubilization. In the current study plants were grown with either 1 mM P (+P-treated) or without P (-P-treated). Shoots or roots of intact plants from both P treatments were labeled independently with 14CO2 to compare the relative contribution of C fixed in each with the C exuded from roots as citrate and other organic acids. About 25-fold more acid-stable 14C, primarily in citrate and malate, was recovered in exudates from the roots of -P-treated plants compared with +P-treated plants. The rate of in vivo C fixation in roots was about 4-fold higher in -P-treated plants than in +P-treated plants. Evidence from labeling intact shoots or roots indicates that synthesis of citrate exuded by -P-treated roots is directly related to nonphotosynthetic C fixation in roots. C fixed in roots of -P-treated plants contributed about 25 and 34% of the C exuded as citrate and malate, respectively. Nonphotosynthetic C fixation in white lupin roots is an integral component in the exudation of large amounts of citrate and malate, thus increasing the P available to the plant.     

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.