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Cluster roots – an underground adaptation for survival in extreme environments Günter Neumann and Enrico Martinoia Cluster roots are a characteristic of members of the Proteaceae and of several other plant species that are adapted to habitats of extremely low soil fertility, usually without formation of mycorrhizal associations. Functionally linked with intense mobilization of nutrients (P, Fe, Zn, Mn) by root-induced chemical changes (pH, root exudates, redox potential) in the rhizosphere, cluster-rooted plant species can serve as model plants to study rhizosphere processes and regulatory aspects of plant adaptations for chemical mobilization of nutrients in the rhizosphere. Published online: 14 March 2002

Günter Neumann* Institut für Planzenernährung (330), Universität Hohenheim, 70593 Stuttgart, Germany. *e-mail: gd.neumann@ t-online.de Enrico Martinoia Laboratoire de Physiologie Végétale, Institut de Botanique, Université de Neuchâtel, Rue Emile Argand 13, CH-2007 Neuchâtel, Switzerland.

Cluster roots [1] are bottlebrush-like clusters of rootlets with limited growth that arise from the pericycle opposite the protoxylem poles along the lateral roots in many species of the Proteaceae [2]. In many cases, members of this family are slowgrowing sclerophyllous shrubs and trees, and a major component of the Mediterranean flora in Western Australia and South Africa. They are adapted to habitats of extremely low soil fertility, such as highly leached sands, sandstones and laterites, with phosphorus (P) as a major limiting nutrient for plant growth [3]. Cluster roots have been functionally linked with an efficient chemical mobilization of sparingly soluble soil P sources by organic chelators (e.g. citrate, malate and phenolics) and ectoenzymes (acid phosphatase) released into the rhizosphere of root clusters in extraordinarily high quantities (Figs 1 and 2) [4–7]. Slow growth rates and an efficient internal P use, with seasonal separation of P uptake and storage mainly during the winter rain period and P use for shoot growth during spring and summer [8], are considered to be additional important determinants for P efficiency in members of the Proteaceae. Apart from the 236 species in 27 genera of the Proteaceae, cluster roots are also formed in some members of the Betulaceae, Casuarinaceae, Cucurbitaceae, Cyperaceae, Eleagnaceae, Leguminosae, Moraceae, Myricaceae and Restionaceae, which are all adapted to low fertility soils [2]. Cluster-rooted plant species frequently exhibit N2 fixation via Rhizobia and Frankia symbiosis but, in most cases, mycorrhizal associations are lacking. Chemical mobilization of nutrients by root exudates released in huge amounts from individual root clusters over a limited time period of 1–3 days http://plants.trends.com

(Fig. 3) [4,9,10] might be an alternative strategy to nutrient acquisition via mycorrhizal associations. The rapid development of cluster roots within several days could be an advantage, particularly in seasonally arid climates where the establishment of functionally active mycorrhizae, which usually requires longer periods of time, might be biased by limited periods of rainfall. Therefore, along with mycorrhizae and N2-fixing nodules, cluster roots are postulated to be the third major adaptation for nutrient acquisition in terrestrial vascular sporophytes [2]. Induction and development of cluster roots

Formation of cluster roots appears to be mainly induced by a shortage of P and, at least in some plant species, by Fe deficiency [3,11–13]. Experiments with foliar and split-root P application have shown that cluster root formation is at least partially triggered by a low internal P status of the plant [14,15]. However, in many cases, a high and sometimes even phytotoxic P supply is necessary for complete depression of cluster root development [3,6]. Nevertheless, cluster root formation seems to be stimulated in nutrient-rich patches [1,16] and in upper soil layers rich in organic matter [17], suggesting that external factors are also involved. This has been attributed to the proliferation of lateral roots as a response to localized nutrient supply, which are the sites of cluster root initiation [16]. Additionally, certain constituents of dissolved organic matter, the pH of the growth medium, as well as microbial factors, might exert some stimulatory effects but are probably not obligatory for the development of cluster roots [3,18,19]. Also, differences in the pattern of cluster root formation between plant species cannot be excluded. In addition, auxin–cytokinin interactions have been implicated in cluster root formation, with auxin application having a promoting effect and auxin antagonists and cytokinins having an inhibitory effect [10,20,21]. Accordingly, characterization of cluster root expressed sequence tags (ESTs) associated with plant hormones revealed down-regulation of an EST with homology to indole acetic acid (IAA) glycosyltransferase and up-regulation of a zeatin glycosyltransferase [22], both enzymes involved in the inactivation of auxins (e.g. IAA) and cytokinins (e.g. zeatin), respectively. Moreover, enhanced expression of an EST with

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Root cluster AI– aluminon complex (red) Fig. 1. (a) Chemical changes in the rhizosphere of cluster roots in different plant species. Rhizosphere acidification cluster roots of Hakea undulata (Proteaceae) demonstrated by application of agar sheets with pH indicator (yellow, pH 4.0; red, pH 5.5). Scale bar = 5 cm. (b) Reduction of MnIV-oxide applied on filter paper in the rhizosphere of cluster roots of Hakea undulata. Scale bar = 4 cm. (c) Reduction of Fe3+ at the surface of cluster roots of Lupinus albus. Formation of a red complex between Fe2+ and and the Fe2+ chelator bathophenantrolinedisulfonic acid (BPDS) after reduction of Fe3+, applied in agar sheets. Scale bar = 1 cm. (d) Aluminium complexation along the root system of Lupinus luteus demonstrated after root-induced splitting of a red Al–aluminon complex applied with agar sheets. Scale bar = 1 cm. (e) Precipitation of Ca-citrate in the rhizosphere of root clusters of white lupin (Lupinus albus), grown in a calcareous soil. Scale bar = 0.1 cm. (f) Demonstration of acid phosphatase activity in the rhizosphere of root clusters of Hakea undulata by application of filter papers with naphtylphosphate/fast-red TR (4-chloro-2-methylbenzenediazonium salt) as an artificial substrate for acid phosphatase. Scale bar = 1 cm.

homology to 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase), involved in biosynthesis of ethylene was observed [22]. Although cluster root initiation in Lupinus albus was not affected by application of ethylene inhibitors [20], ethylene might play a role in the dense formation of root hairs [23] during maturation of cluster roots. However, the mechanisms for the coordinated induction and termination of cluster root growth are largely unknown. Mechanisms of nutrient mobilization

Early studies showed P mobilization from Fe- and Al-P fractions in acidic soils [24,25], and from acid-soluble Ca-P fractions [26] in the rhizosphere of cluster roots, with a concomitant solubilization of Fe, Mn, Zn, Al and P bound to Fe- and Al-humic acid complexes. This was attributed to the exudation of large amounts of carboxylates (mainly citrate and malate, but also of others, such as trans-aconitate and malonate [4,7]) from cluster roots, frequently associated with rhizosphere acidification [6,25,26]. Accordingly, many Proteaceae are characterized by the ability to accumulate high concentrations of Mn and Al in the shoot tissues, probably reflecting http://plants.trends.com

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mechanisms to counteract excessive uptake by internal detoxification [3,27]. Mobilization of sparingly soluble P forms in the rhizosphere soil seems to be mediated by ligand exchange, solubilization and occupation of P sorption sites at the soil matrix [25], and also by enzymatic hydrolysis of soluble organic P esters by root secretory phosphohydrolases [5,6,10,28] (Figs 1 and 2), but the exact mechanisms are unclear. According to the stability constants of complexes with Fe, Al, and Ca, citrate and oxalate are among the most effective compounds with respect to P mobilization [29]. However, in P-deficient soils, significant inorganic phosphorus (Pi) desorption usually requires the accumulation of large amounts of these carboxylates in the rhizosphere (e.g. >10 µmol citrate g −1 rhizosphere soil, corresponding with millimolar concentration levels in the rhizosphere soil solution). To date, comparable carboxylate concentrations have been detected predominantly in the rhizosphere of cluster-rooted plant species [4,9,26,28,30]. In intercropping experiments, root-induced P mobilization from rock phosphate by Lupinus albus was even sufficient to increase P uptake in wheat with a low inherent capacity for mobilization of sparingly soluble P sources in soils [31]. The special morphology of cluster roots, characterized by many closely spaced lateral root tips (50–1000 rootlets cm−1 root axis), densely covered with root hairs (Fig. 3), might contribute to highlevels of P-mobilizing root exudates accumulating in the rhizosphere because of an increased root surface area with secretory activity. By contrast, a function of root clusters to increase the surface for nutrient absorption seems to be of minor importance because the high density of lateral rootlets and root hairs would lead to overlapping zones of nutrient depletion [3]. Mature root clusters with the highest secretory potential have no more growth activity [32]. Thus, P-mobilizing root exudates can be released over an extended period of time (2–3 days) into the same rhizosphere soil compartment [4,9] (Fig. 3). This is not possible in apical root zones of normal lateral roots, which frequently exhibit the highest secretory activity. Assuming root growth rates of up to 1.5–2.5 cm per day [33], in most cases, this time period seems to be much too short to account for a significant accumulation of P-mobilizing root exudates in the rhizosphere of apical root zones. In cluster roots, the secretory activity decreases after 2–3 days and the cluster is decomposed within several weeks (Fig. 3), reflecting a strategy of efficient step-by-step extraction of small soil compartments. Because root clusters can comprise up to 60% of the whole root system [3], frequently forming dense mats of clusters in the upper soil layers that have the highest availability of nutrients, effective nutrient mobilization is also achieved on a larger scale. Moreover, mats of cluster roots have been implicated in binding soil particles, thereby counteracting soil erosion [34].

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Fig. 2. Model for root-induced chemical phosphate mobilization in the rhizosphere by exudation of carboxylates, protons and root secretory phosphohydrolases.

Adaptive alterations in cluster root physiology

There is strong evidence for metabolic changes during cluster root development, leading to increased accumulation of carboxylates in the root tissue and finally to a transient release of these compounds into the rhizosphere (Fig. 4). After cluster root initiation, lateral rootlets begin to grow after 3–4 days. After reaching the final length of 3–4 mm, no more meristematic activity is detectable in mature root clusters [32]. During this developmental stage, large amounts of carboxylates, protons, phenolics and acid phosphatases are released into the rhizosphere over a period of 1–3 days [3,4,9,32], with a diurnal rhythm reported for citrate exudation from cluster roots of Lupinus albus [32]. This pattern of cluster root development is associated with increased carboxylate concentrations in the root tissue and a shift from malate to citrate accumulation before the pulse of exudation [6]. Accordingly, enhanced gene expression and in vitro activities of enzymes involved in catabolism of carbohydrates (sucrose synthase, phosphoglucomutase, fructokinase), biosynthesis of organic acids [phosphoenolpyruvate (PEP) carboxylase, malate dehydrogenase, citrate synthase] and hydrolysis of organic P esters (root secretory acid phosphatase) have been reported in cluster roots [10,35,36] (Fig. 4). Many of these enzymes are supposed to represent metabolic bypass reactions to circumvent Pi-dependent reaction steps of the conventional carbohydrate catabolism, and thus contributing to a more efficient metabolic Pi use under conditions of P starvation [37]. Additionally, PEP carboxylase-mediated non-photosynthetic CO2 fixation provides a substantial proportion of carbon (>30%) to carboxylate production in cluster roots [38–40]. This anaplerotic (replenishing) carbon supply might http://plants.trends.com

at least partially compensate for carbon losses associated with carboxylate exudation and rapid root turnover. However, the shift from malate production to accumulation of extraordinarily high levels of citrate during cluster root maturation (up to 30 µmol g−1 fresh weight) before the pulse of exudation, does not seem to be closely related to the activity of PEP carboxylase [10,32]. There is increasing evidence that this specific accumulation of citrate might be linked instead with the reduced activity of several metabolic sequences involved in citrate turnover (Fig. 4), such as (1) P deficiency-induced depression of root respiration [6,36], (2) inhibition of NO3− uptake and assimilation [10], and (3) inhibition of the ATP-citrate lyase reaction, which has putative functions for anaplerotic production of acetyl-CoA and malate by cleavage of citrate under P-deficient conditions (N. Langlade et al., unpublished). This might lead to citrate accumulation in the root tissue and finally to increased citrate exudation. The lower internal P and energy (ATP) status of mature root clusters compared with other parts of the root system [10,36] might be related to the limited longevity of cluster roots, which probably requires continuous redistribution of P from senescent clusters to the juvenile and actively growing parts of the root with a high P demand. Accordingly, cluster root development from the juvenile to the senescent stage in Lupinus albus was associated with a 90% loss of total RNA, probably involved in Pi recycling [10,36]. This might explain the intense expression of physiological P-deficiency responses in mature root clusters, even when the P supply for the remaining plant tissues is adequate. The transient release of carboxylates from cluster roots is probably mediated by a controlled transport mechanism. In Lupinus albus, inhibitor studies have suggested the involvement of an anion channel for citrate exudation coupled with a concomitant release of protons to maintain charge balance [6]. Similarly, root excretion of malate and citrate via anion channels as a detoxification mechanism in response to toxic levels of Al has been shown recently by patch clamp studies with wheat and maize [41–43]. Proton extrusion is stimulated by increased specific activity of H+-ATPase, detected in plasma membrane vesicles isolated from cluster roots [44,45], and can be at least partially attributed to enhanced steady-state levels of plasma membrane-ATPase enzyme protein (A. Kania et al., unpublished) and, in part, to alterations of kinetic properties of the enzyme [45]. High uptake rates of 14C-citrate into inside-out plasma membrane vesicles from cluster roots and a stimulation of ATP-dependent intravesicular H+ accumulation in the presence of external carboxylate anions further support the hypothesis of a carboxylate transport mechanism located in the plasma membrane of cluster roots, and coupled with ATP-dependent H+ extrusion [44]. ATP-limitation of the plasma membrane-H+-ATPase in later stages of cluster root development might

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(ii) Premature pH 5–6 Exudation [nmol h–1 g–1 FW] Citrate: 300–700 Malate: 500–600 Juvenile pH 5–6 Exudation [nmol h–1 g–1 FW] Citrate: 300–400 Malate: 500–600

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Proteaceae, root exudation of carboxylates and phenolics from proteoid roots was found to be associated with rhizosphere acidification [3,4], but this finding could not be confirmed for carboxylate exudation in other studies [7], and a relationship with K+ extrusion has been reported [46]. Experiments with a varying external pH of the root medium suggest that cluster roots might respond with rhizosphere acidification in more alkaline environments and even with alkalinization when the external pH is low [7] (G. Neumann, unpublished). In spite of energy limitation, mature and even senescent root clusters of white lupin (Lupinus albus) are able to take up significant amounts of Pi at a higher rate than non-proteoid roots [6,10]. Phosphate uptake is probably mediated by a high-affinity Pi uptake system, which seems to be particularly expressed in root clusters of P-deficient plants [10]. Analysis of P uptake by cluster roots in different species of the Proteaceae revealed contradictory results [3]. However, this might be attributed to the examination of different stages in cluster root development. Open questions and perspectives for future research

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Fig. 3. Spatial and temporal variation in root morphology and root exudation during cluster root development. (a) Stages of cluster root development in Lupinus albus. Root-induced pH changes monitored by application of agar sheets with pH-indicator. Scale bar = 1 cm. (i) Juvenile: initial stage with emerging laterals. Scale bar = 500 µm. (ii) Juvenile: root hair development on a lateral rootlet, predominant exudation of malate. Scale bar = 260 µm. (iii) Mature: lateral rootlets without further growth activity, completely covered with root hairs; burst of citrate exudation and release of H+. Scale bar = 250 µm. (iv) Transversal section of (iii) Scale bar = 260 µm. (v) Protozoa (Vorticella) living on the surface of root hairs in mature root clusters Scale bar = 40 µm. (b) Temporal changes during cluster root development and root exudation in Proteaceae and Lupinus albus.

explain the inhibition of citrate exudation in senescent root clusters in spite of high internal citrate concentrations [6,44]. Similarly, in members of the http://plants.trends.com

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There is an increasing interest in investigating the function and development of cluster roots at the physiological and molecular genetic level, with the final goal of identifying putative targets for transformation of important crop plants to improve nutrient acquisition and tolerance to adverse soil chemical conditions. Enhanced P acquisition and Al tolerance in response to transgenic overexpression of citrate synthase has been reported recently for tobacco and Arabidopsis [47,48]. However, Emmanuel Delhaize et al. [49] were unable to confirm the results obtained with tobacco and concluded that overexpression of CS genes is unlikely to be a robust and easy to reproduce strategy for enhancing Al tolerance and P acquisition of crop and pasture species. Similar to the metabolic changes observed in cluster roots, increased intracellular accumulation of carboxylates active in nutrient mobilization or detoxification of toxic elements might be also achieved by upregulation (PEPC, malate dehydrogenase) or downregulation (aconitase, isocitrate dehydrogenase) of other enzymes involved in organic acid metabolism [50]. However, the finding that in some plant species, intense carboxylate exudation is triggered by Al toxicity, but not in response to P deficiency [51], suggests that understanding the regulation of transport mechanisms is of similar importance. A further open question is the development of cluster roots. It is well established that auxins and cytokinins play a role in cluster root formation. However, nothing is known about the pattern formation leading to the bottlebrush-like clusters of rootlets. This structure requires densely packed primordia for lateral root formation, as well as a signal limiting the elongation of the lateral rootlets.

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Fig. 4. Model for phosphorus deficiency-induced metabolic changes related to intracellular accumulation and exudation of citrate in cluster roots of Lupinus albus. Stimulation of metabolic reactions and sequences marked in green, inhibition, marked in red. Abbreviations: ACL, ATP-citrate lyase; CS, citrate synthase; HK, hexokinase; MDH, malate dehydrogenase; PEP, phosphoenol pyruvate; PEPC, phosphoenolpyruvate carboxylase; PGM, phosphoglucomutase; Susy, sucrose synthase.

Acknowledgements Work in our laboratories was supported by the German Research Foundation, DFG; Bundesamt für Bildung und Wissenschaft; and the NCCR project Plant Survival of the Swiss National Foundation.

Lupinus albus is the only cluster-rooted plant species of agricultural importance that has been investigated to date. Most of the knowledge about cluster root physiology and the first attempts towards a molecular genetic characterization are based on studies with Lupinus albus grown in hydroponic culture systems with NO3− as a nitrogen source [10,35,36,38–40,52]. This raises the question as to whether these results can be regarded as generally representative of the function of cluster roots and of the natural growth conditions of cluster-rooted plant species. Cluster roots of Hakea have a high capacity for uptake of organic N, and there is increasing evidence that organic N and NH4+ are major N sources in natural habitats of the Proteaceae [53]. However, cluster root function and development seems to be influenced by the form and the amount of external N supply. The effects of nitrogen include the stimulation of cluster root initiation by NH4+ supply (L. Sas et al., unpublished) and by localized

References 1 Purnell, H.M. (1960) Studies of the family Proteaceae. Anatomy and morphology of the roots of some Victorian species. Aust. J. Bot. 8, 38–50 2 Skene, K.R. (2000) Pattern formation in cluster roots. Some developmental and evolutionary considerations. Ann. Bot. 85, 901–908 3 Dinkelaker, B. et al. (1995) Distribution and function of proteoid roots and other root clusters. Bot. Acta 108, 183–200 http://plants.trends.com

N application [16], inhibition of cluster root development by high levels of nitrogen [3,17] and reduced carboxylate exudation in plants supplied with NH4+ as a nitrogen source. [54] Earlier studies suggested that microorganisms play an important role in the induction and function of cluster roots [3], but, to date, there is little experimental evidence to verify this assumption. Inhibition of cluster root formation observed under sterile conditions might be at least partially attributed to the absence of root growth-promoting microbial factors but also to the liberation of rhizotoxic compounds during soil sterilization [3]. Drastic alterations in rhizosphere pH, redox potential, release of carboxylates and phenolics, and a high level of expression of chitinase in cluster roots [3,4,10] suggest a marked influence on microbial communities during cluster root development. This might, in turn, have important consequences for microbial turnover of root exudates and for the production of microbial metabolites involved in nutrient mobilization. Organic acids in root exudates are a preferential carbon source for root-associative diazotrophic bacteria [55] with a capacity for biological N2 fixation and stimulation of root growth by production of phytohormones [56]. Therefore, an enhanced exudation of carboxylates might also be a way to increase root colonization by diazotrophs and thereby improve plant acquisition of nitrogen and other nutrients. Accordingly, particularly intense colonization by N2-fixing bacteria has been reported in cluster roots of Colophospermum mopane [57]. Differences in microbial colonization might at least partially explain the heterogeneity of organic acid patterns reported for root exudates collected from proteoid roots in different species of the Proteaceae [3,4,58] (Z. Rengel, unpublished). Alternatively, Zed Rengel suggests that the heterogeneity of exudate compounds reflects adaptations for exploitation of different nutrient pools to minimize competition in a community of plant species growing in an environment generally low in available nutrients. Therefore, comparative studies on nutrient acquisition by cluster-rooted plant species might not only contribute to a better understanding of nutrient cycles in natural ecosystems, but might also provide a powerful tool to unravel as yet unidentified mechanisms for mobilization of sparingly available nutrients in soils, with the prospect transforming crop plants in the future.

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