Cluster Root Formation Research Articles

Root growth and Zn uptake of three common crop plants in response to heterogeneity in soil texture and Zn distribution

The responses of root growth to non-uniformities in soil conditions can be of great importance for the acquisition of soil nutrients by plants. Here we monitored the root growth response of white lupin, cucumber and spring wheat to constructed horizontal heterogeneity in soil texture (and thereby also soil moisture) and zinc (Zn) distribution in rhizoboxes filled with sandy low-Zn soil using neutron radiography imaging. We investigated how the position of Zn-enriched soil relative to the heterogeneity in soil texture and moisture distribution affected Zn uptake, as compared to a control treatment with homogeneous texture. In all treatments the same total amount of water was added to the rhizoboxes. In cucumber heterogeneity in texture and moisture increased root length, root biomass and, independent of the position of Zn enrichment, also Zn uptake. In lupin it decreased cluster root formation and root biomass and in wheat specific root length, but in both of them it had no effect on total root length and shoot Zn uptake. The effects on root traits were associated with increased shoot biomass in wheat and cucumber, but not in lupin. Spatial differences in root growth between opposite sections in the heterogeneous packings were limited to the treatment in which coarse texture was combined with Zn enrichment. The results indicate that the responses in root system development were dominated by systemic factors with little modification by local responses and that the morphological root traits studied here were not limiting plant Zn uptake.

Cluster root allocation of white lupin (Lupinus albus L.) in soil with heterogeneous phosphorus and water distribution

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.

Interactions among cluster-root investment, leaf phosphorus concentration, and relative growth rate in two Lupinus species.

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. 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. 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. 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.

Cluster-root formation and carboxylate release in Euplassa cantareirae (Proteaceae) from a neotropical biodiversity hotspot

Background and Aims Euplassa cantareirae is a Neotropical Proteaceae, native to the Restinga forest in Brazil and it experiences a wide array of adverse environmental conditions, especially seasonal flooding and low nutrient availability. We aimed to investigate the effects of phosphorus (P) supply on plant growth and cluster root formation, as well as to characterize the main carboxylates released by the cluster roots of this species.

Root morphology and cluster root formation by seabuckthorn (Hippophaë rhamnoides L.) in response to nitrogen, phosphorus and iron deficiency

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.

The southern South American Proteaceae, Embothrium coccineum exhibits intraspecific variation in growth and cluster-root formation depending on climatic and edaphic origins

Background and aims Cluster root (CR) functioning has been studied mainly in Proteaceae species from (P)-deficient old soils. However, in southern South America, six species occur in young P rich soils. The aims were: i) to study the growth and CR formation of Embothrium coccineum seedlings from populations contrasting in edaphic and climatic conditions and, ii) to study the effect of P availability on CR formation.

The acquisition of phosphate by higher plants: Effect of carboxylate release by the roots. A critical review.

AbstractPhosphorus 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.

A re‐assessment of sucrose signaling involved in cluster‐root formation and function in phosphate‐deficient white lupin (Lupinus albus)

Apart from substrate functions, a signaling role of sucrose in root growth regulation is well established. This raised the question whether sucrose signals might also be involved in formation of cluster-roots (CRs) under phosphate (Pi) limitation, mediating exudation of phosphorus (P)-mobilizing root exudates, e.g. in Lupinus albus and members of the Proteaceae. Earlier studies demonstrated that CR formation in L. albus was mimicked to some extent by external application of high sucrose concentrations (25 mM) in the presence of extremely high P supply (1-10 mM), usually suppressing CR formation. In this study, we re-addressed this question using an axenic hydroponic culture system with normal P supply (0.1 mM) and a range of sucrose applications (0.25-25 mM). The 2.5mM sucrose concentration was comparable with internal sucrose levels in the zone of CR initiation in first-order laterals of P-deficient plants (3.4 mM) and induced the same CR morphology. Similar to earlier studies, high sucrose concentrations (25 mM) resulted in root thickening and inhibition of root elongation, associated with a 10-fold increase of the internal sucrose level. The sucrose analog palatinose and a combination of glucose/fructose failed to stimulate CR formation under P-sufficient conditions, demonstrating a signal function of sucrose and excluding osmotic or carbon source effects. In contrast to earlier findings, sucrose was able to induce CR formation but had no effect on CR functioning with respect to citrate exudation, in vitro activity and expression of genes encoding phosphoenolpyruvate carboxylase, secretory acid phosphatase and MATE transporters, mediating P-mobilizing functions of CRs.

Ecophysiological role of Embothrium coccineum, a proteaceae species bearing cluster roots, at increasing p availability in its rhizosphere

Native forests in southern South America are constantly subjected to natural disasters such as volcanic eruptions. Soil affected by volcanic ash contain large amounts of total P but low P availability, as this element is strongly adsorbed to soil colloids (i.e. allophane). This lack of available P is one of the main limitations to plant growth. In this context, it is necessary an in-deep study of plant species that have developed some root physiological strategies for P acquisition. An example of this is the formation of cluster roots by Proteaceae species. Recently, information has been reported that aids to our understanding of the functioning of Proteaceae species growing in volcanic soils. The aim of this review is to discuss the ecophysiological role of Proteaceae species growing in young volcanic soils, with a special emphasis on Embothrium coccineum, a pioneer species of extremely disturbed environments. In summary, we reveal here that E. coccineum has several features that make it suitable for recovering degraded soils in south-central Chile. Some of these characteristics include its ability to survive and successfully establish in poor soils due to its specialized roots adaptation and its ability to shed its leaves under stressful conditions. According to recent evidence, E. coccineum has relatively low foliar nutrient resorption leaving at least half of the nutrients in its senescent leaves; this, in turn, promotes nutrient cycling via mineralization of its leaves. Finally, we conclude that the cluster roots of E. coccineum promote P solubilization and mineralization in the rhizosphere soil allowing for increased P availability for the plant itself and potentially also for neighbouring species.

Root architecture responses: in search of phosphate.

Soil phosphate represents the only source of phosphorus for plants and, consequently, is its entry into the trophic chain. This major component of nucleic acids, phospholipids, and energy currency of the cell (ATP) can limit plant growth because of its low mobility in soil. As a result, root responses to low phosphate favor the exploration of the shallower part of the soil, where phosphate tends to be more abundant, a strategy described as topsoil foraging. We will review the diverse developmental strategies that can be observed among plants by detailing the effect of phosphate deficiency on primary and lateral roots. We also discuss the formation of cluster roots: an advanced adaptive strategy to cope with low phosphate availability observed in a limited number of species. Finally, we will put this work into perspective for future research directions.

Increasing nitrogen supply stimulates phosphorus acquisition mechanisms in the fynbos species Aspalathus linearis.

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 100μM, 300μM, 500μM and 700µM N at a low P level of 10µM and a high P level of 100µM. In solution, plants received 200μM and 500µM N at a low P level of 5µM and a high P level of 15µM. Cluster roots formed only in plants with low P supply. Roots showed greater citrate and malate production and phosphatase activity at 5µM P than at 15µM P. At 10µM 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 15µM, 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.

The regulatory network of cluster‐root function and development in phosphate‐deficient white lupin (Lupinus albus) identified by transcriptome sequencing

Lupinus albus serves as model plant for root-induced mobilization of sparingly soluble soil phosphates via the formation of cluster-roots (CRs) that mediate secretion of protons, citrate, phenolics and acid phosphatases (APases). This study employed next-generation sequencing to investigate the molecular mechanisms behind these complex adaptive responses at the transcriptome level. We compared different stages of CR development, including pre-emergent (PE), juvenile (JU) and the mature (MA) stages. The results confirmed that the primary metabolism underwent significant modifications during CR maturation, promoting the biosynthesis of organic acids, as had been deduced from physiological studies. Citrate catabolism was downregulated, associated with citrate accumulation in MA clusters. Upregulation of the phenylpropanoid pathway reflected the accumulation of phenolics. Specific transcript expression of ALMT and MATE transporter genes correlated with the exudation of citrate and flavonoids. The expression of transcripts related to nucleotide degradation and APases in MA clusters coincided with the re-mobilization and hydrolysis of organic phosphate resources. Most interestingly, hormone-related gene expression suggested a central role of ethylene during CR maturation. This was associated with the upregulation of the iron (Fe)-deficiency regulated network that mediates ethylene-induced expression of Fe-deficiency responses in other species. Finally, transcripts related to abscisic acid and jasmonic acid were upregulated in MA clusters, while auxin- and brassinosteroid-related genes and cytokinin receptors were most strongly expressed during CR initiation. Key regulations proposed by the RNA-seq data were confirmed by quantitative real-time polymerase chain reaction (RT-qPCR) and some physiological analyses. A model for the gene network regulating CR development and function is presented.

Legume genomics: understanding biology through DNA and RNA sequencing.

The legume family (Leguminosae) consists of approx. 17 000 species. A few of these species, including, but not limited to, Phaseolus vulgaris, Cicer arietinum and Cajanus cajan, are important dietary components, providing protein for approx. 300 million people worldwide. Additional species, including soybean (Glycine max) and alfalfa (Medicago sativa), are important crops utilized mainly in animal feed. In addition, legumes are important contributors to biological nitrogen, forming symbiotic relationships with rhizobia to fix atmospheric N2 and providing up to 30 % of available nitrogen for the next season of crops. The application of high-throughput genomic technologies including genome sequencing projects, genome re-sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) by the legume research community has provided major insights into genome evolution, genomic architecture and domestication. This review presents an overview of the current state of legume genomics and explores the role that next-generation sequencing technologies play in advancing legume genomics. The adoption of next-generation sequencing and implementation of associated bioinformatic tools has allowed researchers to turn each species of interest into their own model organism. To illustrate the power of next-generation sequencing, an in-depth overview of the transcriptomes of both soybean and white lupin (Lupinus albus) is provided. The soybean transcriptome focuses on analysing seed development in two near-isogenic lines, examining the role of transporters, oil biosynthesis and nitrogen utilization. The white lupin transcriptome analysis examines how phosphate deficiency alters gene expression patterns, inducing the formation of cluster roots. Such studies illustrate the power of next-generation sequencing and bioinformatic analyses in elucidating the gene networks underlying biological processes.

Interactions between light intensity and phosphorus nutrition affect the phosphate-mining capacity of white lupin (Lupinus albus L.).

Light intensity affects photosynthetic carbon (C) fixation and the supply of carbon to roots. To evaluate interactions between carbon supply and phosphorus (P) supply, effects of light intensity on sucrose accumulation, root growth, cluster root formation, carboxylate exudation, and P uptake capacity were studied in white lupin (Lupinus albus L.) grown hydroponically with either 200 µmol m(-2) s(-1) or 600 µmol m(-2) s(-1) light and a sufficient (50 µM P) or deficient (1 µM P) P supply. Plant biomass and root:shoot ratio increased with increasing light intensity, particularly when plants were supplied with sufficient P. Both low P supply and increasing light intensity increased the production of cluster roots and citrate exudation. Transcripts of a phosphoenol pyruvate carboxylase gene (LaPEPC3) in cluster roots (which is related to the exudation of citrate), transcripts of a phosphate transporter gene (LaPT1), and P uptake all increased with increasing light intensity, under both P-sufficient and P-deficient conditions. Across all four experimental treatments, increased cluster root formation and carboxylate exudation were associated with lower P concentration in the shoot and greater sucrose concentration in the roots. It is suggested that C in excess of shoot growth capabilities is translocated to the roots as sucrose, which serves as both a nutritional signal and a C-substrate for carboxylate exudation and cluster root formation.

Soil nitrogen, and not phosphorus, promotes cluster‐root formation in a South American Proteaceae, Embothrium coccineum

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. Small and large seedlings (< 6- and > 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 (Pimass) and per area (Piarea) 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. Best-fit models showed a negative logarithmic relationship between cluster-root number and soil nitrogen (N), and between cluster-root relative mass and both leaf Piarea and soil N, and a positive logarithmic relationship between cluster-root number and leaf Piarea. Cluster-root relative mass was higher in small than in large seedlings. 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 Piarea. We suggest that cluster roots are a constitutive and functional trait that allows plants to prevail in poor N soils.

Phosphorus deficiency affects the allocation of below-ground resources to combined cluster roots and nodules in Lupinus albus

Lupins can rely on both cluster roots and nodules for P acquisition and biological nitrogen fixation (BNF), respectively. The resource allocation (C, N and P) between cluster roots and nodules has been largely understudied during P-deficient conditions. The aim of this investigation was therefore to determine the changes in resource allocation between these organs during fluctuations in P supply. Lupinus albus was cultivated in sand culture for 3 weeks, with either sufficient (2mM high) or limiting (0.1mM low) P supply. Although variation on P supply had no effect on the total biomass, there were significant differences in specialised below-ground organ allocation to cluster roots and nodule formation. Cluster root formation and the associated C-costs increased during low P supply, but at sufficient P-supply the construction and growth respiration costs of cluster roots declined along with their growth. In contrast to the cluster root decline at high P supply, there was an increase in nodule growth allocation and corresponding C-costs. However, this was not associated with an increase in BNF. Since cluster roots were able to increase P acquisition under low P conditions, this below-ground investment may also have benefited the P nutrition of nodules. These findings provide evidence that when lupins acquire N via BNF in their nodules, there may be a trade-off in resource allocation between cluster roots and nodules.

Cluster-root formation and carboxylate release in three Lupinus species as dependent on phosphorus supply, internal phosphorus concentration and relative growth rate

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.

The effect of phosphorus on cluster-root formation and functioning of Embothrium coccineum (R. et J. Forst.)

Background and aims 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.

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.

Viminaria juncea does not vary its shoot phosphorus concentration and only marginally decreases its mycorrhizal colonization and cluster-root dry weight under a wide range of phosphorus supplies.

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]. 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(-1) 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. Shoot biomass accumulation increased with increasing P supply until a shoot dry weight of 3 g was reached at a P supply of approx. 27·5 mg P kg(-1) 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 1·4 mg P kg(-1) shoot dry weight. 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.