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Background and Aims Initiation of cluster roots in white lupin (Lupinus albus) under phosphorus (P) deficiency requires auxin signalling, whereas flavonoids inhibit auxin transport. However, little information is available about the interactions between P deficiency and flavonoids in terms of cluster-root formation in white lupin. Methods Hydroponic and aeroponic systems were used to investigate the role of flavonoids in cluster-root formation, with or without 75 μm P supply. Key Results Phosphorus-deficiency-induced flavonoid accumulation in cluster roots depended on developmental stage, based on in situ determination of fluorescence of flavonoids and flavonoid concentration. LaCHS8, which codes for a chalcone synthase isoform, was highly expressed in cluster roots, and silencing LaCHS8 reduced flavonoid production and rootlet density. Exogenous flavonoids suppressed cluster-root formation. Tissue-specific distribution of flavonoids in roots was altered by P deficiency, suggesting that P deficiency induced flavonoid accumulation, thus fine-tuning the effect of flavonoids on cluster-root formation. Furthermore, naringenin inhibited expression of an auxin-responsive DR5:GUS marker, suggesting an interaction of flavonoids and auxin in regulating cluster-root formation. Conclusions Phosphorus deficiency triggered cluster-root formation through the regulation of flavonoid distribution, which fine-tuned an auxin response in the early stages of cluster-root development. These findings provide valuable insights into the mechanisms of cluster-root formation under P deficiency.

The low bioavailability of nutrients, especially nitrogen (N) and phosphorus (P), is one of the most limiting factors for crop production. In this study, under N- and P-free nutrient solution (-N-P), nodulating white lupin plants developed some nodules and analogous cluster root structures characterized by different morphological, physiological, and molecular responses than those observed upon single nutrient deficiency (strong acidification of external media, a better nutritional status than -N+P and +N-P plants). The multi-elemental analysis highlighted that the concentrations of nutrients in white lupin plants were mainly affected by P availability. Gene-expression analyses provided evidence of interconnections between N and P nutritional pathways that are active to promote N and P balance in plants. The root exudome was mainly characterized by N availability in nutrient solution, and, in particular, the absence of N and P in the nutrient solution triggered a high release of phenolic compounds, nucleosides monophosphate and saponines by roots. These morphological, physiological, and molecular responses result from a close interplay between N and P nutritional pathways. They contribute to the good development of nodulating white lupin plants when grown on N- and P-free media. This study provides evidence that limited N and P availability in the nutrient solution can promote white lupin-Bradyrhizobium symbiosis, which is favourable for the sustainability of legume production.

Indexación: Scopus. Aims: Embothrium coccineum is a pioneer tree that produces cluster roots (CR) induced by phosphorus (P) or nitrogen (N) deficiency, but the role which both N and P play in CR formation for different E. coccineum populations is still unknown. We hypothesized that in E. coccineum, N influences the CR formation response, primarily induced by P limitation. However, this response depends on local adaptations to their specific conditions of geographic origin. Methods: Seedlings from three contrasting edaphoclimatic conditions (Northern, Central and Southern populations) were grown in hydroponic culture under high and low N supply and at five different P supply. Morphophysiological responses, including CR number, growth, biomass, P and N plant concentration, were studied. Results: Our results showed that at high N supply, CR formation was stimulated with increased P supply, but just until it reaches a maximum of 5 μM, above this concentration CR formation decreases. However, at low N supply, a constitutive response was observed regardless of P supply. Moreover, each of these responses depends on the populations. Conclusions: Our study suggests that N drives CR formation induced by P supply and that a differential response exists among E. coccineum populations, that could be related to their origin edaphoclimatic conditions. © 2020, Springer Nature Switzerland AG. https://link-springer-com.recursosbiblioteca.unab.cl/article/10.1007%2Fs11104-020-04622-0

Phosphorus deficiency often limits crop productivity, while phosphate rock, which is used to produce phosphorus fertilisers, is a non-renewable resource. Formation of cluster roots is an adaptation to nutrient-poor soils in Lupinus species, including L. albus. Lupinus species also produce nodules, which require a large investment of phosphorus. Our aim is to test whether nodulation promotes cluster-root formation in L. albus. Seedlings of L. albus, either inoculated with rhizobia or non-inoculated, were grown in nutrient solution with a low phosphorus supply. Non-inoculated plants were provided with the same amount of nitrogen in the form of nitrate as the nodulated ones acquired, from both air and nutrient solution, based on preliminary experiments. We measured biomass, phosphorus and nitrogen concentrations as well as photosynthesis just prior to each harvest. Nodulated plants and non-nodulated control plants produced the same amount of biomass. Nodulated plants had, on average, 86% more cluster roots than non-nodulated ones at the four harvests. As hypothesised, nodulation significantly promoted cluster-root formation; it also enhanced rates of photosynthesis. Nodulation promoted cluster-root formation and photosynthesis, presumably because nodules are significant sinks for phosphorus and photosynthates. Our results do not provide evidence for a trade-off between investment of resources in nodules and cluster roots.

Many plants have been found to show preferential root growth allocation to soil patches with locally increased nutrient availability. Little is known about the influence of the plants’ nutrient status on these responses. Addressing this question, we conducted a climate chamber experiment to investigate how foliar phosphorus (P) and zinc (Zn) application (alone or in combination) would affect root allocation and nutrient uptake by white lupin (Lupinus albus L.) seedlings growing on soil with constructed heterogeneity in P and Zn distribution. The same soil packing was used in all pots. One quarter section of each packing was enriched with Zn, one with P, one with Zn and P, while neither Zn nor P was added to the fourth section (opposite to the section with the combined Zn and P amendment). Neither foliar P nor foliar Zn application had a fertilization effect on plant growth. The allocation of root length growth showed a clear preference for P-enriched soil sections, when no foliar fertilizer was applied. With foliar P or Zn application these preferences disappeared. Cluster root allocation and root P concentration showed a similar behavior. In the treatments with foliar Zn application, root length growth was also preferentially allocated in Zn-enriched soil, while without foliar Zn there was only a tendency for such a response. Root Zn concentrations were always much higher in soil sections with than without Zn amendment. The foliar treatments had no effect on root Zn concentration. While the responses to foliar P application can be understood in terms of an optimization strategy in root allocation for P foraging, the effects of foliar Zn application on root growth allocation cannot be interpreted in this way and require further investigation.

Cluster roots are structures formed by many plants adapted to phosphorus (P)-deficient soils. We investigated the combined influence of spatial heterogeneity in soil water and P distribution on the allocation of cluster root formation in white lupin (Lupinus albus L.). In this study, single plants were grown at a low or a high rate of water supply in containers filled with a P-poor sand to which either no P was added or which was fertilized homogeneously or heterogeneously. Furthermore, heterogeneous soil water distribution was established in half of the containers by using a finer instead of a coarser sand in a lateral third of the containers. Plant growth increased with water supply rate, but P fertilization had no influence on shoot biomass production. Although overall cluster root production decreased with increasing homogeneous P supply, localized P fertilization had no effect on cluster root allocation. However, cluster roots were preferentially allocated in the soil sections with lower water availability when overall water supply rate was low. The results suggest that overall cluster root production was a systemic response to initial plant P status, while cluster root growth was stimulated locally in drier patches when overall water supply was limiting plant growth.

PREMISE OF THE STUDY Cluster-root (CR) formation is a desirable trait to improve phosphorus (P) acquisition as global P resources are dwindling. CRs in some lupine species are suppressed at higher P status. Whether increased growth rate enhances CR formation due to a "dilution" of leaf P concentration is unknown. We investigated interactive effects of leaf P status and relative growth rate (RGR) on CR formation in two Lupinus species, which differ in their CR biomass investment. Variation in RGR was imposed by varying day length. METHODS Lupinus albus and L. pilosus were grown hydroponically with KH2PO4 at a day length of 6, 10, or 14 h. We used a slightly higher P supply at longer day lengths to avoid a decline in leaf P concentration, which would induce CRs. Cluster-root percentage, leaf P concentrations, and RGR were determined at 22, 38, and 52 d after sowing. KEY RESULTS Lupinus species grown at similar root P availability, but with a faster growth rate, as dependent on day length, showed a greater CR percentage. Because our aim to achieve exactly the same leaf P concentrations at different day lengths was only partially achieved, we carried out a multiple regression analysis. This analysis showed the CR percentage was strongly and negatively correlated with plant P status and only marginally and positively correlated with RGR. CONCLUSIONS The two Lupinus species invariably formed fewer cluster roots at higher leaf P status, irrespective of RGR. Differences in RGR or leaf P concentration cannot explain the species-specific variation in cluster-root investment.

The aims were to investigate effects of availability of nitrogen (N), phosphorus (P) and iron (Fe) on root properties of seabuckthorn (Hippophae rhamnoides) and to test the hypothesis that seabuckthorn is able to form cluster roots (CRs). Two sources of seabuckthorn were used: the seabuckthorn cultivar BHi10726 originating from a breeding programme based on H.r. ssp mongolica and carried out in rich agricultural field soil in the black earth (chernozem) region of Russia and the seabuckthorn accession named Pk originating in a natural population of H.r. ssp turkestanica in the mountainous region of northern Pakistan. Three cultivation systems giving different water availabilities were used at two levels each of N, P and Fe. Root morphology of seedlings and clones was characterized and metabolite content in extracts of young and old CRs of Pk was analyzed by proton nuclear magnetic resonance spectroscopy. Availability of N affected growth and distribution of biomass between shoot and root, while P and Fe deficiency modified root system architecture towards more lateral roots. Densely positioned rootlets with a determinate type of growth consistent with the definition of CR were observed under low P and low Fe. Pk formed on average 12 CRs per plant, which was 3 to 4-fold higher compared to BHi10726 also when normalized per root length. Malate and glycine were most abundant of the organic acids and amino acids, respectively, and decreased in old CRs. Seabuckthorn has the ability to form cluster roots especially in Pk and under deficiency of P and Fe. The two sources of seabuckthorn with different histories showed distinctly different root system architectures. The high contents of malate and glycine and their decrease in old CRs may reflect roles in CR metabolism.

Cluster roots have a profound effect on their rhizosphere. Our aim was to determine the effect of cluster roots of Embothrium coccineum, growing under natural conditions, on soil enzyme activities and phosphorus (P) lability in its rhizosphere. We determined enzyme activities: acid phosphatase (P-ase), dehydrogenase and β-glucosidase, and the rate of hydrolysis of fluorescein diacetate (FDA), as well as P fractions in the cluster root rhizosphere at different cluster-root developmental stages (juvenile, mature, semi-senescent, senescent), in the non-cluster root rhizosphere, and in bulk soil. In addition, the concentrations of total P and manganese Mn in roots was measured. The rhizosphere of senescing cluster roots presented the highest P-ase, β-glucosidase and dehydrogenase activities, and fastest rate of FDA hydrolysis, being 2.6-, 4.6-, 3.3- and 25.8-fold greater, respectively, than those in the rhizosphere of mature cluster roots. The P fractionation showed that the inorganic P (Pi) fraction was 15 % greater in the rhizosphere of mature cluster roots than in that of other stages. Mature cluster roots showed the highest total [P], suggesting the fastest P uptake. Cluster roots of E. coccineum modified their rhizosphere depending on their developmental stage, presenting lower soil enzyme activities at the mature stage than at other development stages. In addition, mature cluster roots increased the Pi fraction in their rhizosphere, allowing the highest total root [P] at this developmental stage.

Phosphorus is one of the most limiting macronutrients for plant productivity in agriculture worldwide. The main reasons are the limited rock phosphate reserves and the high affinity of phosphate (P) to the soil solid phase, restricting the P availability to the plant roots. Plants can adapt to soils low in available P by changing morphological or/and physiological root features. Morphological changes include the formation of longer root hairs and a higher root : shoot ratio both parameters increasing the root surface which provides the shoot with P. This may be successful if the P availability in soil, i.e., the P concentration of the soil solution is not extremely low (> 1–2 µM P). If the P concentration of the soil solution is lower, the diffusive flux to the root surface will be very low and may not satisfy the P demand of the shoots. Under these conditions plants have developed strategies to increase the rhizosphere soil solution concentration by secreting mobilizing agents. The most effective way of P mobilization is the release of di- and tricarboxylic acid anions, especially oxalate and citrate. Citrate can accumulate in the rhizosphere up to concentrations up to 80 µmol g−1 soil. Cluster root formation is an efficient way of carboxylate accumulation in the cluster root rhizosphere improving P mobilization. Cluster roots strongly improve the acquisition of the mobilized P. Considering a single root, around 80–90% of the mobilized P diffuses away from the root. From the rhizosphere of cluster roots, most of the mobilized P is taken up by the cluster roots. Both, the strong accumulation of carboxylates in and the effective P uptake from the cluster-root rhizosphere are the basis of the unique ability of P acquisition by cluster root-forming plants. Plants that do not form cluster roots, e.g., red clover, can also accumulate carboxylates in the rhizosphere. Red clover accumulates high quantities of citrate in the rhizosphere soil. Model calculations show that the release of citrate by red clover roots and its accumulation in the rhizosphere strongly improve P acquisition by this plant species in various soils. Similar results are obtained with alfalfa. In sugar beet, oxalate release can strongly contribute to P acquisition. In summary, P acquisition can be strongly improved by the release of carboxylates and should be taken as a challenge for basic and applied research.

Phosphorus (P), in its ionized form (Pi), is one of the most limiting nutrients for plant growth and development. White lupin is a dinitrogen (N2)-fixing legume that can increase Pi availability in soils. Under Pi deficiency, white lupin develops cluster roots, also known as proteoid roots. Cluster roots are densely packed lateral roots, resembling bottlebrushes. The resulting increase in root surface, together with coordinated biochemical responses, releases bound Pi and makes it available for plant uptake. The most noticeable biochemical responses that increase Pi availability include excretion of organic anions and phosphatases. As a consequence, white lupin can grow without addition of Pi fertilizer, and its ability to fix N2 is less inhibited by Pi deficiency, compared to other legumes. However, formation of cluster roots requires additional carbon (C) and nitrogen (N). Thus, white lupin needs to carefully balance C use with the formation of cluster roots and nodules. High-throughput approaches, particularly RNA-seq, have revealed many of the genes involved in cluster root formation and function and are beginning to reveal networks that regulate Pi acquisition and use. A better understanding of white lupin’s responses to low nutrients may help to overcome inhibition of N2 fixation by low Pi availability in other legumes.

Summary 1. Proteaceae species in south-western Australia thrive on phosphorus-impoverished soils, employing a phosphorus-mining strategy involving carboxylate-releasing cluster roots. Some develop symptoms of phosphorus toxicity at slightly elevated soil phosphorus concentrations, due to their low capacity to down-regulate phosphorus uptake. In contrast, Proteaceae species in Chile, e.g. Embothrium coccineum J.R. Forst. & G. Forst., occur on volcanic soils, which contain high levels of total phosphorus, but phosphorus availability is low. 2. We hypothesised that the functioning of cluster roots of E. coccineum differs from that of south-western Australian Proteaceae species, in accordance with the difference in soil phosphorus status. With more phosphorus to be gained from the soil with high levels of total phosphorus, we expect less investment in biomass and more release of carboxylates. Furthermore, we hypothesised that E. coccineum regulates its phosphorus-uptake capacity, avoiding phosphorus toxicity when grown at elevated phosphorus levels. To test these hypotheses, E. coccineum seedlings were grown at a range of phosphorus supplies in nutrient solution. 3. We show that E. coccineum allocated at least five times less biomass to cluster roots that released at least nine times more carboxylates per unit cluster root weight compared with south-western Australian species (e.g. Banksia, Hakea). The highest phosphorus supply caused a growth inhibition and high leaf phosphorus concentration, without symptoms of phosphorus toxicity. We accept our hypotheses on the functioning of cluster roots and the high capacity to reduce the net phosphorus uptake in plants grown at a high-phosphorus supply. 4. This novel combination of traits indicates divergent functioning of Proteaceae species from southern South America, exposed to frequent phosphorus input due to volcanic activity, in contrast with the functioning of south-western Australian Proteaceae species that thrive on severely phosphorus-impoverished soils. These traits could explain the functioning of E. coccineum on soils that are rich in total phosphorus, but with a low concentration of available phosphorus.

Some Lupinus species produce cluster roots in response to low plant phosphorus (P) status. The cause of variation in cluster-root formation among cluster-root-forming Lupinus species is unknown. The aim of this study was to investigate if cluster-root formation is, in part, dependent on different relative growth rates (RGRs) among Lupinus species when they show similar shoot P status.Three cluster-root-forming Lupinus species, L. albus, L. pilosus and L. atlanticus, were grown in washed river sand at 0, 7·5, 15 or 40 mg P kg(-1) dry sand. Plants were harvested at 34, 42 or 62 d after sowing, and fresh and dry weight of leaves, stems, cluster roots and non-cluster roots of different ages were measured. The percentage of cluster roots, tissue P concentrations, root exudates and plant RGR were determined.Phosphorus treatments had major effects on cluster-root allocation, with a significant but incomplete suppression in L. albus and L. pilosus when P supply exceeded 15 mg P kg(-1) sand. Complete suppression was found in L. atlanticus at the highest P supply; this species never invested more than 20 % of its root weight in cluster roots. For L. pilosus and L. atlanticus, cluster-root formation was decreased at high internal P concentration, irrespective of RGR. For L. albus, there was a trend in the same direction, but this was not significant.Cluster-root formation in all three Lupinus species was suppressed at high leaf P concentration, irrespective of RGR. Variation in cluster-root formation among the three species cannot be explained by species-specific variation in RGR or leaf P concentration.

Embothrium coccineum (R. et J. Forst.) is a Proteaceae species from the southern part of South America. South-central Chilean soils are younger and contain more phosphorus (P) than soils in Australia and South Africa, where Proteaceae are common. Phosphorus deficiency is the main factor promoting cluster-root formation in Proteaceae. It is not known, however, whether this also applies to E. coccineum, which grows on soils with higher P content. Four-month-old seedlings were grown for 4 weeks in hydroponic cultures with 1 μM P or 50 μM P. The number of cluster roots, relative height increment, biomass distribution, cluster root/total root biomass ratio, foliar P concentration, root acid phosphatase activity and root carboxylate-exudation rates were determined. Seedlings growing at 50 μM P showed a 10−, 1.3− and 3.3-fold greater increase in relative height, total dry mass and foliar P concentration, respectively, compared with those grown at1 μM P. However, seedlings grown at 1 μM P showed a 5−, 16−, 1.7− and 1.3-fold greater number of cluster roots, cluster root/total root biomass ratio, phosphatase activity and total carboxylate exudation, respectively, as compared with those grown at 50 μM P. A low P supply promotes the initiation, growth and metabolic activity of cluster roots which is in accordance with reports on Proteaceae species occurring in ancient and highly weathered soils.

†Background and Aims The Australian legume species Viminaria juncea forms both cluster roots and mycorrhizal associations. The aim of this study was to identify if these root specializations are expressed at differential supplies of phosphorus (P) and at different shoot P concentrations [P]. †Methods Seedlings were planted in sand and provided with a mycorrhizal inoculum and basal nutrients plus one of 21 P treatments, ranging from 0 to 50 mg P kg 21 dry soil. Plants were harvested after 12 weeks, and roots, shoots and cluster roots were measured for length and fresh and dry weight. The number of cluster roots, the percentage of mycorrhizal colonization, and shoot [P] were determined. †Key Results Shoot biomass accumulation increased with increasing P supply until a shoot dry weight of 3 g was reached at a P supply of approx. 27. 5m g Pk g 21 dry soil. Neither cluster-root formation nor mycorrhizal colonization was fully suppressed at the highest P supply. Most intriguingly, shoot [P] did not differ across treatments, with an average of 1. 4m g Pk g 21 shoot dry weight. †Conclusions The almost constant shoot [P] in V. juncea over the very wide range of P supplies is, to our knowledge, unprecedented. To maintain these stable values, this species down-regulates its growth rate when no P is supplied; conversely, it down-regulates its P-uptake capacity very tightly at the highest P supplies, when its maximum growth rate has been reached. It is proposed that the persistence of cluster roots and mycorrhizal colonization up to the highest P treatments is a consequence of its tightly controlled shoot [P]. This unusual P physiology of V. juncea is surmised to be related to the habitat of this N2-fixing species. Water and nutrients are available at a low but steady supply for most of the year, negating the need for storage of P which would be metabolically costly and be at the expense of metabolic energy and P available for symbiotic N2 fixation.

Background Phosphorus (P) is an essential element for plant growth and development but it is often a limiting nutrient in soils. Hence, P acquisition from soil by plant roots is a subject of considerable interest in agriculture, ecology and plant root biology. Root architecture, with its shape and structured development, can be considered as an evolutionary response to scarcity of resources. Scope This review discusses the significance of root architecture development in response to low P availability and its beneficial effects on alleviation of P stress. It also focuses on recent progress in unravelling cellular, physiological and molecular mechanisms in root developmental adaptation to P starvation. The progress in a more detailed understanding of these mechanisms might be used for developing strategies that build upon the observed explorative behaviour of plant roots. Conclusions The role of root architecture in alleviation of P stress is well documented. However, this paper describes how plants adjust their root architecture to low-P conditions through inhibition of primary root growth, promotion of lateral root growth, enhancement of root hair development and cluster root formation, which all promote P acquisition by plants. The mechanisms for activating alterations in root architecture in response to P deprivation depend on changes in the localized P concentration, and transport of or sensitivity to growth regulators such as sugars, auxins, ethylene, cytokinins, nitric oxide (NO), reactive oxygen species (ROS) and abscisic acid (ABA). In the process, many genes are activated, which in turn trigger changes in molecular, physiological and cellular processes. As a result, root architecture is modified, allowing plants to adapt effectively to the low-P environment. This review provides a framework for understanding how P deficiency alters root architecture, with a focus on integrated physiological and molecular signalling.

Exudation of organic acid anions is one of the mechanisms responsible for aluminum (Al) tolerance. The plasma membrane (PM) H+-ATPase is involved in the exudation of organic acid anions. However, the relationship between organic acid exudation and PM H+-ATPase under Al toxicity remains unclear. This study aims to investigate the correlation among Al-induced citrate exudation, PM H+-ATPase activity and counterions for citrate release from cluster roots of phosphorus-deficient (−P) white lupin. Al and various pharmacological agents were applied to incubate the cluster roots of P-deficient white lupin; the citrate exudation rate, PM H+-ATPase activity and ion exudation of cluster roots were examined. Citrate exudation from cluster roots of P-deficient white lupin was induced by 50 μM Al treatment within 1.5 h, but no extra increase was found when the duration of Al treatment increased. The PM H+-ATPase activity of cluster roots was insensitive to Al treatment, irrespective of Al concentration and duration of Al treatment. Al treatment increased K+ efflux but not H+ efflux from cluster roots. After application of pharmacological agents to P-deficient cluster roots under Al treatment or not, vanadate decreased H+ efflux and increased K+ efflux, but had no inhibitory effect on citrate exudation; fusicoccin increased H+ efflux and citrate exudation, but decreased K+ efflux; tetraethylammonium (TEA) chloride, a K+-channel inhibitor, inhibited K+ efflux and increased H+ efflux. These results indicate that citrate exudation induced by combined treatment with P-deficiency and Al is independent of PM H+-ATPase activity, and is coupled with K+ efflux, which may compensate H+ efflux for keeping the charge balance for Al-induced citrate exudation from cluster roots of P-deficient white lupin.

Phosphorus (P) is one of 17 essential elements (nutrients) required for plant growth ([Tiessen, 2008][1]; [Cordell et al., 2009][2]). Although bound P is quite abundant in many soils, it is largely unavailable for uptake. As such, P is frequently the most limiting element for plant growth and

Summary • White lupin (Lupinus albus) forms specialized cluster roots characterized by exudation of organic anions under phosphorus (P) deficiency. Here, the role of nitric oxide (NO) in P deficiency-induced cluster-root formation and citrate exudation was evaluated. • White lupin plants were treated with the NO donor sodium nitroprusside (SNP) and scavenger or inhibitor of NO synthase under conditions of P deficiency (0 lM) or P sufficiency (50 lM). • Phosphorus deficiency enhanced NO production in primary and lateral root tips, with a greater increase in cluster roots than in noncluster roots. NO concentrations decreased with cluster root development from the pre-emergent stage, through the juvenile stage, to the mature stage. The P deficiency-induced increase in NO production was inhibited by antagonists of NO synthase and xanthine oxidoreductase, suggesting the involvement of these enzymes in NO production. SNP markedly increased the number of cluster roots. Citrate exudation from different root segments in P-deficient roots was positively correlated with endogenous root NO concentrations. • These findings demonstrate differential patterns of NO production in white lupin, depending on root zone, developmental stage and P nutritional status. NO appears to play a regulatory role in the formation of cluster roots and citrate exudation in white lupin under conditions of P deficiency.

One of the main factors that favours the formation of cluster roots is a low supply of phosphorus (P). The soils of southern Chile are mainly formed from volcanic ash, characterized by low levels of available P. Embothrium coccineum, a Chilean Proteaceae species produces cluster roots (CR). The factors that control CR formation in Chilean Proteaceae have not been extensively studied. The objective of this work was to assess the effects of P on the growth and cluster-root formation of E. coccineum. Plants were produced from seeds collected at two different locations: Valdivia and Pichicolo both at 39oS. They were cultured under similar greenhouse conditions, from June to September, watered twice a week using: distilled water (W), full strength Hoagland’s nutrient solution (H) or Hoagland without P (H-P). At the end of the experiment, height, total dry biomass, number of cluster roots (CR) per plant, CR /total root weight, were measured. Also acid exudation of CR was assayed using bromocresol purple on sterile agar plates. Treatments significantly affected growth and proportion of CR, the highest growth was observed with H. Under all treatments plants produced a similar number of CR. However, the proportion of CR biomass was higher with W and H-P than with H. Plants under W exhibited the lowest growth and low shoot/root ratio. Acid exudation of CR was not detectable in our experiment. These results are discussed comparing CR formation in low P conditions on Lupinus albus and other Proteaceae species, and the possible role of CR formation in E. coccineum considering its wide geographical distribution.

BACKGROUND AND AIMS In some lupin species, phosphate deficiency induces cluster-root formation, which enhances P uptake by increasing root surface area and, more importantly, the release of root exudates which enhances P availability. METHODS Three species of Lupinus, L. albus, L. atlanticus and L. micranthus, with inherently different relative growth rates were cultivated under hydroponics in a greenhouse at four phosphate concentrations (1, 10, 50 and 150 microm) to compare the role of internal P in regulating cluster-root formation. KEY RESULTS The highest growth rate was observed in L. atlanticus, followed by L. albus and L. micranthus. At 1 microm P, cluster-root formation was markedly induced in all three species. The highest P uptake and accumulation was observed in L. micranthus, followed by L. atlanticus and then L. albus. Inhibition of cluster-root formation was severe at 10 microm P in L. atlanticus, but occurred stepwise with increasing P concentration in the root medium in L. albus. CONCLUSIONS In L. atlanticus and L. albus cluster-root formation was suppressed by P treatments above 10 microm, indicating a P-inducible regulating system for cluster-root formation, as expected. By contrast, production of cluster roots in L. micranthus, in spite of a high internal P concentration, indicated a lower sensitivity to P status, which allowed P-toxicity symptoms to develop.

The influence of phosphorus (P) availability on growth and P uptake was investigated in South African Proteaceae: (1) Protea compacta R.Br., endemic on severely nutrient-impoverished colluvial sands; (2) Protea obtusifolia Bueck ex Meissner; and (3) Leucadendron meridianum I. J. Williams, the latter both endemic on comparatively fertile limestone-derived soils. Plants were grown hydroponically in 1000 L tanks at 0.01, 0.1 or 1.0 microm P for 14 weeks. Biomass accumulation was influenced by P availability, doubling as [P] increased from 0.1 to 1.0 microm. Total biomass was greatest for P. compacta, but L. meridianum and P. obtusifolia had two to four times greater relative biomass accumulation at 0.1 and 1.0 microm [P]. Proteoid root clusters developed at both 0.01 and 0.1 microm[P], but were suppressed at 1.0 microm [P]; this was a 10-fold lower [P] than previously reported to inhibit cluster root formation. Rates of net P uptake at 5 microm P decreased in response to increased P availability from 0.01 to 1.0 microm P. Significant between-species differences in rates of P uptake and capacity to down-regulate P uptake were observed: P. compacta < P. obtusifolia < L. meridianum. The species responses are discussed in terms of adaptation to mosaics in soil P availability and the high beta diversity in the natural habitat.

Proteoid roots are reportedly an adaptation to soils with either low phosphorus (P) or iron (Fe) or both. Since macadamia (Macadamia integrifolia, a member of the family Proteaceae) is an important crop in Hawaii, a factorial experiment with soil P levels of 0, 150, and 500 mg/kg and Fe levels of 0, 5, and 10 mg/kg was conducted to evaluate the effects of P and Fe fertilizations and possible Fe x P interactions on proteoid root development and macadamia growth. The soil was a highly weathered Oxisol having 0.015 mg P/L and 0.03 mg Fe/L in the soil solution in its unamended state. Proteoid roots were reduced in number and as a percentage of the total root mass at the highest levels of P and Fe. Phosphorus, however was a major controlling factor. Optimum dry matter associated with at least 10% proteoid roots, corresponding to a P concentration approximately of 0.035 mg/L in soil solution and 0.10% P in leaves. Total Fe in leaves or the amount of Fe applied was not a good predictor of plant growth. In contrast, chlorophyll content, a surrogate of biologically active Fe, was. Soil-solution ratio of Fe 1/3/ P (molar basis) could be used to predict the response of macadamia growth to P and Fe fertilizations.

Lupinus albus L. were grown in rhizoboxes containing a soil amended with sparingly available Fe–P or Al–P (100 μg P g−1 soil/resin mixture). Root halves of individual plants were supplied with nutrient solution (minus P) buffered at either pH 5.5 or 7.5, to assess whether the source of mineral-bound P and/or pH influence cluster-root growth and carboxylate exudation. The P-amended soil was mixed 3:1 (w/w) with anion-exchange resins to allow rapid fixation of carboxylates. Treatments lasted 10 weeks. Forty percent and 30% of the root mass developed as cluster roots in plants grown on Fe–P and Al–P respectively, but cluster-root growth was the same on root-halves grown at pH 5.5 or 7.5. Mineral-bound P source (Al– or Fe–P) had no influence on the types of carboxylates measured in soil associated with cluster roots—citrate (and trace amounts of malate and fumarate) was the only major carboxylate detected. The [citrate] in the rhizosphere of cluster roots decreased with increased shoot P status (suggesting a systemic effect) and also, only for plants grown on Al–P, with decreased pH in the root environment (suggesting a local effect). In a separate experiment using anion exchange resins pre-loaded with malate or citrate, we measured malate (50%) and citrate (79%) recovery after 30 days in soil. We therefore, also conclude that measurements of [citrate] and [malate] at the root surface may be underestimated and would be greater than the 40- and 1.6-μmol g−1 root DM, respectively estimated by us and others because of decomposition of carboxylates around roots prior to sampling.

Summary • Both phosphorus (P) deficiency and aluminum (Al) toxicity induce root exudation of carboxylates, but the relationship between these two effects is not fully understood. Here, carboxylate exudation induced by Al in Lupinus albus (white lupin) was characterized and compared with that induced by P deficiency. • Aluminum treatments were applied to whole root systems or selected root zones of plants with limited (1 µ M ) or sufficient (50 µ M ) P supply. • Aluminum stimulated citrate efflux after 1‐2 h; this response was not mimicked by a similar trivalent cation, La 3 + . P deficiency triggered citrate release from mature cluster roots, whereas Al stimulated citrate exudation from the 5- to 10-mm subapical root zones of lateral roots and from mature and senescent cluster roots. Al-induced citrate exudation was inhibited by P limitation at the seedling stage, but was stimulated at later growth stages. Citrate exudation was sensitive to anion-channel blockers. Al treatments did not affect primary root elongation, but inhibited the elongation of lateral roots. • The data demonstrate differential patterns of citrate exudation in L. albus , depending on root zone, developmental stage, P nutritional status and Al stress. These findings are discussed in terms of possible functions and underlying mechanisms.

It is well known that lateral roots of some plants can respond to heterogeneous nutrient supply, but it is unknown how the cluster roots of white lupin, the special root structure induced by phosphorus (P) deficiency, respond to P-rich patches of different P sources in soil. The present study examined the effect of localized P supply on cluster root formation and root exudation by white lupin (Lupinus albus L. cv. Kiev Mutant) grown in a P-deficient sandy soil. Phosphorus was applied only to one compartment in a four-compartment split-root system as either hydroxyapatite (Ca-P), iron phosphate (Fe-P), phytate (Phy-P) or KH2PO4 (K-P). The proportion of dry root biomass allocated to cluster roots in P-supplemented compartments was greater than that in the compartments without P application. The shoot P concentration of Phy-P plants was much lower than that of Fe-P ones, but there was no difference in the proportion of cluster roots in these two treatments. In comparison with Phy-P and Fe-P, there was a lower proportion of cluster roots in Ca-P and K-P plants. Citrate exudation rate increased significantly in the Fe-P-supplemented compartment, but did not respond to other P sources, compared with the compartment without P application. It is concluded that localized P application significantly stimulates cluster root formation by white lupin, and cluster root formation and citrate exudation are greatly dependent on the form of P supplied.

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.

White lupine (Lupinus albus L.) was used as a phosphorus (P)-efficient model plant to study the effects of elevated atmospheric CO(2) concentrations on (i) P acquisition, (ii) the related alterations in root development and rhizosphere chemistry, and (iii) the functional and structural diversity of rhizosphere microbial communities, on a P-deficient calcareous subsoil with and without soluble P fertilization. In both +P (80 mg P kg(-1)) and -P treatments (no added P), elevated CO(2) (800 micromol mol(-1)) increased shoot biomass production by 20 to 35% and accelerated the development of cluster roots, which exhibit important functions in chemical mobilization of sparingly soluble soil P sources. Accordingly, cluster root formation was stimulated in plants without P application by 140 and 60% for ambient and elevated CO(2) treatments, respectively. Intense accumulation of citrate and increased activities of acid and alkaline phosphatases, but also of chitinase, in the rhizosphere were mainly confined to later stages of cluster root development in -P treatments. Regardless of atmospheric CO(2) concentrations, there was no significant effect on accumulation of citrate or on selected enzyme activities of C, N, and P cycles in the rhizosphere of individual root clusters. Discriminant analysis of selected enzyme activities revealed that mainly phosphatase and chitinase contributed to the experimental variance (81.3%) of the data. Phosphatase and chitinase activities in the rhizosphere might be dominated by the secretion from cluster roots rather than by microbial activity. Alterations in rhizosphere bacterial communities analyzed by denaturing gradient gel electrophoresis (DGGE) were related with the intense changes in root secretory activity observed during cluster root development but not with elevated CO(2) concentrations.

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.

White lupin ( Lupinus albus L. cv. Amiga) plants were grown in nutrient solution, using a split-root system with two compartments to examine the effect of localized phosphorus (P) supply on nutrient uptake, cluster root formation and root exudation. Phosphorus was supplied at 0 and 250 μM P in the −P and +P compartments, respectively, with three treatments of −P+P, −P−P and +P+P. Shoot dry weight in −P+P was higher than that in −P−P, but not significantly different from that in +P+P. Shoot P concentration [P] in −P+P was lower than that in +P+P, but higher than that in −P−P. In the −P+P treatment, root [P] was lower in the −P than in the +P root half and the similar trend was observed for root dry weight. Compared with −P−P, 19% of P uptake in the +P root half in −P+P was translocated to the −P root half. Phosphorus deficiency decreased K, Mg and S uptake in −P−P, and localized P deficiency reduced S uptake in the −P root half in −P+P. The number of cluster roots and exudation rates of protons, citrate and acid phosphatase (APase) in both root halves were higher in −P−P than in +P+P. The localized P supply increased the total number of cluster roots in the +P root half compared with the −P root half in −P+P. Compared with −P−P, the localized P supply significantly increased the shoot [P] and decreased the exudation rate of citrate and APase in both root halves in −P+P. Exudation rate of citrate in the −P root half in −P+P was higher than that of the +P root half in +P+P. Localized P supply reduced proton release in the +P and even −P root halves in −P+P, but the pH was lower in the −P than +P compartments for the individual plant, indicating a significant effect of the localized P supply on proton release. The results suggest that cluster root formation and citrate exudation are regulated by the shoot [P] and affected by localized supply of external P, and that proton release is inhibited by localized P supply by altering the balance of anion and cation uptake.

White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (−P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3− than Cl− (PCit/PCl = 26.3), while ORAC was selective for Cl− over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of −P plants, as well as from normal (noncluster) roots of plants grown with 25 μm phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of −P plants. However, the IRAC current from +P plants occurred less frequently than in the −P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux.

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 - 3 P. The possibility that changes in the concentrations of P fractions in the root halves influenced clusterroot 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 .

Although there has been speculation that cluster roots play a role in plant nitrogen (N) acquisition there have been few experimental studies, demonstrating this. We grew the subtropical wet heathland plant Hakea actities in sand and in axenic vermiculite-sand cultures to investigate its use of different N sources. Seedlings produced greater quantities of cluster roots when grown with NO3−, glutathione or protein as N sources than with NH4+, glutamine or without N addition, suggesting that N source influences cluster root initiation and development. Axenically grown seedlings acquired more N when grown with 4.5 mM than with 0.5 mM glutathione or protein as N sources suggesting that seedlings are able to assimilate complex organic N. However, control seedlings that did not receive N other than seed storage N (approximately 0.2 mg N) acquired 2 mg N from an unknown source, so that data are not unequivocal. Peptidase activity in extracts derived from non-axenic cluster root tissue changed with age of cluster roots. Developing and mature cluster roots had higher peptidase activity than young and senescing cluster roots. To determine whether peptidases are associated with the outer surface of cluster roots, entire cluster roots were gently centrifuged and the resulting solution analysed for peptidase activity using gelatine-containing PAGE. Different peptidases were active at different times of cluster root development, suggesting that plant or microbially derived peptidases could play a role in organic N acquisition by cluster roots. Roots and cluster roots expressed a putative amino acid transporter (HaAAT4-1) and peptide transporter (HaPepT1) as determined by northern blot analysis. Expression of HaPepT1 in cluster roots increased throughout cluster root development although further studies need to confirm this.

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 μM P while the other halves were supplied with 0, 1, 25 or 75 μM 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 μM 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 μM P, whereas it continued on the other halves supplied with 1 μM 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 μM P was significantly reduced when the other half was either deprived of P or supplied with 25 or 75 μM 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 μM P were the same, whether the other root half was supplied with 1, 25 or 75 μM 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.

Introduction H. Lambers, et al. The biology of cluster roots and the acquisition of P from the rhizosphere. Structure, ecology and physiology of root clusters a review B.B. Lamont. The evolution of physiology and development in the cluster root: teaching an old dog new tricks? K.R. Skene. Organic acid behaviour in soils misconceptions and knowledge gaps D.L. Jones, et al. Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: A review P. Hinsinger, et al. Strategies to isolate transporters that facilitate organic anion efflux from plant roots P.R. Ryan, et al. Phosphate transport in plants F.W. Smith, et al. Development of the adhesive pad on climbing fig (Ficus punila) stems from clusters of adventitious roots E.P. Groot, et al. Pathways of carbon metabolism related to enhanced exudation of organic acids and acid phosphatase. Acclimation of white lupin to phosphorus deficiency involves enhanced expression of genes related to organic acid metabolism C. Uhde-Stone, et al. Phosphorus deficiency-induced modifications in citrate catabolism and in cystolic pH as related to citrate exudation in cluster roots of white lupin A. Kania. Secreted acid phosphatase is expressed in cluster roots of lupin in response to phosphorus deficiency J. Wasaki, et al. Effects of altered citrate synthase and isocitrate dehydrogenase expression on internal citrate concentrations and citrate efflux from tobacco (Nicotiana tabacum L.) roots E. Delhaize, et al. Characterization of NAPD-isocitrate dehydrogenase expression in a carrot mutant cell line with enhanced citrate excretion T. Kihara, et al. The influence of organic acid exudation and cluster roots on P and N acquisition. Do cluster roots of Hakea actities (Proteaceae) acquire complex organic nitrogen? S. Schmidt, et al. Lupinus luteus cv. Wodjil takes up more phosphorus and cadmium than Lupinus angustifolius cv. Kalya R.F. Brennan, M.D.A. Bolland. Chick pea and white lupin rhizosphere carboxylates vary with soil properties and enhance phosphorus uptake E.J. Veneklaas, et al. Role of phosphorus nutrition in development of cluster roots and release of carboxylates in soil-grown Lupinus albus J. Shen, et al. The influence of plant nutrition on plant growth and development of cluster roots. Effects of external phosphorus supply on internal phosphorus concentration and the initiation, growth and exudation of cluster roots in Hahea prostrata R. Br. M.W. Shane, et al. Differences in cluster root formation and carboxylate exudation in Lupinus albus L. under different nutrient deficiencies R. Liang, C. Li. Is Fe deficiency rather than P deficiency the cause of cluster root formation in Casuarina species? E.H. Zaid, et al. The formation, morphology and anatomy of cluster root of Lupinus albus L. as dependent on soil type and phosphorus supply C.S. Peek, et al. Localized supply of phosphorus induces root morphological and architectural changes of rice in split and stratified soil cultures Y. He, et al. Interaction of nitrogen and phosphorus nutrition in determining growth C.C.de Groot, et al. Implications of root architecture, root-soil interactions and mycorrhiza on plant P nutrition. Phosphorus acquisition from soil by white lupin (Lupinus albus L.) and soybean (Glycine max L.) species with contrasting root development M. Watt, J.R. Evans. Plantago lanceolata L. and Rumex acetosella L. differ in their utilisation of soil phosphorus fractions A.-M. Fransson, et al. Chickpea facilitates phosphorus uptake by intercropped wheat from an organic phosphorus source L. Li, et al. Using competitive and facilitative interactions in intercropping systems enhances crop productivity and nutrient-use efficiency F. Zhang, L. Li. Phosphorus uptake by a community of arbuscular mycorrhizal fungi in jarrah forest M.Z. Solaiman, L.K. Abbott. Relationships between cluster root-bearing taxa and laterite across landscapes in southwest Western Australia: an approach using airborne radiometric and digital elevation models W.H. Verboom, J.S. Pate. Volume contents. Author index.

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 μg 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.

We investigated the physiological basis for tolerance of limiting P supply and for enhanced growth with simultaneous addition of N and P in Aspalathus linearis (Burm. f.) R. Dahlgren. It was hypothesised that increasing N supply would stimulate P acquisition mechanisms and enhance plant growth with high P supply. In sand, plants received 100 μM, 300 μM, 500 μM and 700 µM N at a low P level of 10 µM and a high P level of 100 µM. In solution, plants received 200 μM and 500 µM N at a low P level of 5 µM and a high P level of 15 µM. Cluster roots formed only in plants with low P supply. Roots showed greater citrate and malate production and phosphatase activity at 5 µM P than at 15 µM P. At 10 µM P, greater N supply enhanced cluster root formation to 60% of root biomass, and increased the phosphatase activity of noncluster roots and succinate release by both root types. At a high P supply of 15 µM, greater N supply stimulated phosphatase activity of roots by 50%, increasing P uptake and plant growth. With increased resource partitioning towards P acquisition due to greater P demand, A. linearis is tolerant of low P supply and highly responsive to combined addition of N and P.

▪ Abstract The rhizosphere is the zone of soil immediately surrounding plant roots that is modified by root activity. In this critical zone, plants perceive and respond to their environment. As a consequence of normal growth and development, a large range of organic and inorganic substances are exchanged between the root and soil, which inevitably leads to changes in the biochemical and physical properties of the rhizosphere. Plants also modify their rhizosphere in response to certain environmental signals and stresses. Organic anions are commonly detected in this region, and their exudation from plant roots has now been associated with nutrient deficiencies and inorganic ion stresses. This review summarizes recent developments in the understanding of the function, mechanism, and regulation of organic anion exudation from roots. The benefits that plants derive from the presence of organic anions in the rhizosphere are described and the potential for biotechnology to increase organic anion exudation is highlighted.

White lupin (Lupinus albus L.) was grown in hydroponic culture with 1 μm phosphorus to enable the development of proteoid roots to be observed in conjunction with organic acid exudation. Discrete regions of closely spaced, determinate secondary laterals (proteoid rootlets) emerged in near synchrony on the same plant. One day after reaching their final length (4 mm), citrate exudation occurred over a 3-d pulse. The rate of exudation varied diurnally, with maximal rates during the photoperiod. At the onset of citrate efflux, rootlets had exhausted their apical meristems and had differentiated root hairs and vascular tissues along their lengths. Neither in vitro phosphoenolpyruvate carboxylase nor citrate synthase activity was correlated with the rate of citrate exudation. We suggest that an unidentified transport process, presumably at the plasma membrane, regulates citrate efflux. Growth with elevated (700 μL L−1) atmospheric [CO2] promoted earlier onset of rootlet determinacy by 1 d, resulting in shorter rootlets and citrate export beginning 1 d earlier as a 2-d diurnal pulse. Citrate was the dominant organic acid exported, and neither the rate of exudation per unit length of root nor the composition of exudate was altered by atmospheric [CO2].

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.

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 32 P i uptake measurements showed that P i uptake was more uniform along the proteoid root than citrate efflux.

The effect of iron deficiency, phosphorus, NaHCO3, chelator supply and nitrogen source on the formation of cluster (proteoid) roots was investigated in Casuarina glauca growing in water culture. The addition of iron-binding chelators (e.g. EDDHA, DTPA, EDTA) or increase in nutrient solution pH with NaHCO3 resulted in the formation of cluster roots when plants were grown in solution lacking iron. Phosphorus supply even at a concentration of 500 µM did not inhibit cluster root formation if EDDHA was added to the iron-deficient medium. Cluster root formation was influenced significantly by nitrogen source and occurred only in nitrate-fed plants. C. glauca seemed to be very sensitive to iron deficiency as shown by plant chlorosis when grown on alkaline soil. The symptoms of chlorosis decreased as the chlorophyll content in shoots and the number of cluster roots increased, suggesting that the alleviation of iron deficiency in plant tissues was correlated with cluster root formation. It appears that iron deficiency is more important than phosphorus deficiency in inducing the formation of cluster roots in C. glauca.

 Premise of the study: Cluster roots are a characteristic root adaptation of Proteaceae species. In South African and Australian species, cluster roots promote phosphorus (P) acquisition from poor soils. In a South American Proteaceae species, where cluster roots have been scarcely studied and their function is unknown, we tested whether cluster-root formation is stimulated by low soil nutrition, in particular low P-availability.  Methods : Small and large seedlings ( 6-months old, respectively) of Embothrium coccineum and soil were collected across four different sites in Patagonia (Chile). We determined cluster-root number and relative mass, and leaf Pi concentration per mass (Pi mass ) and per area (Pi area ) for each seedling, and tested relationships with Olsen–P (OP), sorbed–P (sP) and total nitrogen (N) using generalized linear mixed-effects models and model selection to assess the relative strength of soil and plant drivers.  Key results: Best-fi t models showed a negative logarithmic relationship between cluster-root number and soil nitrogen (N), and between cluster-root relative mass and both leaf Pi area and soil N, and a positive logarithmic relationship between cluster-root number and leaf Pi area . Cluster-root relative mass was higher in small than in large seedlings.  Conclusions: Contrary to that found in South African and Australian Proteaceae, cluster roots of E. coccineum do not appear to be driven by soil P, but rather by soil N and leaf Pi area . We suggest that cluster roots are a constitutive and functional trait that allows plants to prevail in poor N soils.

The capacity for nitrate reduction, as measured by nitrate reductase activity (NRA), was generally low for a range of plant communities in Australia (coastal heathland, rainforest, savanna woodland, monsoon forest, mangrove, open Eucalyptus forest, coral cay open forest) and only a loose relationship existed between NRA and leaf nitrogen concentration. This suggests that nitrate ions are not the sole nitrogen source in these communities. Based on (15)N labeling experiments, we found a range of tree species exhibiting a pronounced preference for uptake of ammonium over nitrate. Analysis of soil solutions from several forest and heathland communities indicated that ammonium ions were more prevalent than nitrate ions and that soluble forms of organic nitrogen (amino acids and protein) were present in concentrations similar to those of mineral nitrogen. To determine the extent to which root adaptations and associations might broaden nitrogen source utilization to include organic nitrogen, we assessed the effects of various nitrogen sources on seedling growth in sterile culture. Non-mycorrhizal seedlings of Eucalyptus grandis W. Hill ex Maiden and Eucalyptus maculata Hook. grew well on mineral sources of nitrogen, but did not grow on organic sources of nitrogen other than glutamine. Mycorrhizal seedlings grew well on a range of organic nitrogen sources. When offered a mixture of inorganic and organic nitrogen sources at low concentrations, mycorrhizal seedlings derived a significant proportion of their nitrogen budget from organic sources. We also demonstrated that a species of the obligately non-mycorrhizal genus Hakea, a heathland proteaceous shrub possessing cluster roots, had the ability to incorporate (15)N-labeled organic sources (e.g., glycine). We conclude that mycorrhizal associations and root adaptations confer the ability to substantially broaden the nitrogen source base on some plant species.

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.

†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-deficiencyinduced 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 mM P and 10 mM Fe (+P + Fe); (2) 0 P and 10 mM Fe (‐P + Fe); (3) 50 mM 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.

Background In soils with a low phosphorus (P) supply, sugar beet is known to intake more P than other species such as maize, wheat, or groundnut. We hypothesized that organic compounds exuded by sugar beet roots solubilize soil P and that this exudation is stimulated by P starvation. Results Root exudates were collected from plants grown in hydroponics under low- and high-P availability. Exudate components were separated by HPLC, ionized by electrospray, and detected by mass spectrometry in the range of mass-to-charge ratio (m/z) from 100 to 1000. Eight mass spectrometric signals were enhanced at least 5-fold by low P availability at all harvest times. Among these signals, negative ions with an m/z of 137 and 147 were shown to originate from salicylic acid and citramalic acid. The ability of both compounds to mobilize soil P was demonstrated by incubation of pure substances with Oxisol soil fertilized with calcium phosphate. Conclusions Root exudates of sugar beet contain salicylic acid and citramalic acid, the latter of which has rarely been detected in plants so far. Both metabolites solubilize soil P and their exudation by roots is stimulated by P deficiency. These results provide the first assignment of a biological function to citramalic acid of plant origin.

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.

• The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage. • To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS). • The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots. • Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.