Does phenotypic plasticity in carboxylate exudation differ among rare and widespread Banksia species (Proteaceae)?

Banksia species (Proteaceae) occur on some of the most phosphorus (P)-impoverished soils in the world. We hypothesized that plasticity in the exudation of P-mobilizing carboxylates would be greater in widespread than in rare Banksia species. Glasshouse experiments were conducted to identify and quantify carboxylate exudation in three widespread and six narrowly distributed Banksia species. High concentrations of carboxylates (predominantly malate, citrate, aconitate, oxalate) were measured in the rhizosphere of all nine species of Banksia on six different soils, but widespread species did not have greater plasticity in the composition of exuded carboxylates. Based on the evidence in the present study, rarity in Banksia cannot be explained by limited phenotypic adjustment of carboxylate exudation.     

The pattern of carboxylate exudation in Banksia grandis (Proteaceae) is affected by the form of phosphate added to the soil

Roots of a wide range of plant species exude carboxylates, e.g. citrate, into the rhizosphere, to mobilise sparingly available phosphate. We investigated the carboxylates in root exudates of Banksia grandisWilld. (Proteaceae), which occurs on severely phosphate-impoverished soils in Western Australia. Plants were grown in pots with a nutrient-poor quartz sand, with phosphate, at 25 g P g^–1, added as either K-phosphate, glycerol phosphate, Fe-phosphate or Al-phosphate.Plants grown on Fe-phosphate or Al-phosphate formed `proteoid' or `cluster' roots, and exuded significant amounts of carboxylates. Plants grown on K-phosphate did not form cluster roots; their leaves were chlorotic, and some of these plants died during the experiment. Plants grown on glycerol phosphate did have cluster roots, but their leaves also became chlorotic, albeit later in the experiment.Tri- and dicarboxylates (citrate, 60%; malate, 25%; trans-aconitate, 14%) were the major carboxylates in root exudates when P was supplied as Al-phosphate. The same tri- and dicarboxylates were also exuded when P was supplied as Fe-phosphate (31, 14 and 12%, respectively). In addition, these plants exuded monocarboxylates (lactate, 30%; acetate, 12%). We analysed the effect of the different carboxylates on the mobilisation of phosphate and Fe in two different types of soils. The ecological significance of the difference in exudate spectrum for the mobilisation of nutrients and for the detoxification of aluminium is discussed.Because the leaves of plants grown with K-phosphate or glycerol-phosphate appeared chlorotic, we analysed the concentrations of P, Fe, Zn, Mn and Cu in these leaves. Only the concentration of total P was considerably higher in leaves of plants grown with K- or glycerol-phosphate than that in leaves of plants grown with Fe- or Al-phosphate. Both the concentration of total Fe and that of reduced Fe was the same in chlorotic leaves as that in leaves of plants grown with Fe- or Al-phosphate, which had a healthy appearance. It is concluded that P-induced chlorosis was not due to a lack of total or reduced Fe; it may have been due to precipitation of Fe by phosphate.     

The effect of manganese supply on exudation of carboxylates by roots of lucerne (Medicago sativa)

Some low-molecular-weight carboxylates commonly found in plant root exudates have the potential to increase the availability of Mn in the rhizosphere. Release of various compounds into the rhizosphere by plant roots may also be a mechanism by which certain species and genotypes are able to tolerate conditions of low Mn availability better than others. Lucerne (Medicago sativa L.) plants of Salado, a genotype tolerant to Mn deficiency, and Sirosal, an intolerant genotype, were grown in solution culture with 0, 5 or 500 nM Mn (Mn-0, Mn-5 and Mn-500). Exudates of whole root systems were collected at 14, 24 and 36 d and analysed by HPLC. Oxalate, tartarate, L-malate, lactate, malonate, maleate, citrate and succinate were detected and quantified in exudates under all Mn treatments. Malonate, citrate and succinate accounted for the majority of carboxylates in the exudates. Exudation increased with plant age, but amounts of individual carboxylates remained constant in proportion to the total amount exuded. A significant increase in exudation of all carboxylates other than malonate and maleate resulted from omission of Mn from nutrient solutions. Salado exuded more oxalate, tartarate, L-malate, lactate, citrate and succinate than Sirosal at Mn-0, and more citrate and succinate than Sirosal at Mn-5. Genotypic differences in carboxylate exudation under Mn-0 were associated with production of roots with diameter <100 μm. Plant Mn concentrations and growth rates suggested carboxylate exudation differences were not the sole factor responsible for differential tolerance to Mn deficiency in the lucerne genotypes.     

A root trait accounting for the extreme phosphorus sensitivity of Hakea prostrata (Proteaceae)

Proteaceae are adapted to acquire P from nutrient‐impoverished soils; many function at very low leaf P levels, but are killed by P fertilization. Phosphorus toxicity develops at a remarkably low external P concentration. Previous studies have described P toxicity in Proteaceae, but the physiological basis for it remained unclear. The aim of the present study was to elucidate the physiological basis of P toxicity in Hakea prostrata R. Br. (Proteaceae). Triticum aestivum L. (Gramineae), Medicago truncatula Gaertn., Lupinus albus L. (both Fabaceae) and Hakea prostrata R.Br. were grown in solution at a range of P concentrations (0–1000 mmol P m^?3), and determined net P‐uptake rates at 5 (all species) and 50 mmol P m^?3 (H. prostrata only). With the exception of H. prostrata, net P‐uptake rates were fastest for plants grown without added P. Down‐regulation occurred for T. aestivum, M. truncatula and L. albus when the P concentration during growth was increased from 0 to 0.8 mmol P m^?3, whereas in H. prostrata rates decreased only for plants grown at 10 mmol P m^?3 or more. The leaf [P] at which P toxicity occurred in H. prostrata exceeded 10 mg g^?1 dry matter, similar to that for crop species. The low capacity to reduce P uptake in response to increased supply offers a physiological explanation for the extreme sensitivity to P supply in H. prostrata, and possibly other Proteaceae.     

Carboxylate release of wheat, canola and 11 grain legume species as affected by phosphorus status

The capacity of plant roots to increase their carboxylate exudation at a low plant phosphorus (P) status is an adaptation to acquire sufficient P at low soil P availability. Our objective was to compare crop species in their adaptive response to a low-P availability, in order to gain knowledge to be used for improving crop P-acquisition efficiency from soils that are low in P or that have a high capacity to retain P. In the present screening study we compared 13 crop species, grown in sand at either 3 or 300 μM of P, and measured root mass ratio, cluster-root development, rhizosphere pH and carboxylate composition of root exudates. Root mass ratio decreased with increasing P supply for Triticum aestivum L., Brassica napus L., Cicer arietinum L. and Lens culinaris Medik., and increased only for Pisum sativum L., while the Lupinus species and Vicia faba L. were not responsive. Lupinus species that had the potential to produce root clusters either increased or decreased biomass allocation to clusters at 300 μM of P compared with allocation at 3 μM of P. All Lupinus species acidified their rhizosphere more than other species did, with average pH decreasing from 6.7 (control) to 4.3 for Lupinus pilosus L. and 5.9 for Lupinus atlanticus L.; B. napus maintained the most alkaline rhizosphere, averaging 7.4 at 300 μM of P. Rhizosphere carboxylate concentrations were lowest for T. aestivum, B. napus, V. faba, and L. culinaris than for the other species. Exuded carboxylates were mainly citrate and malate for all species, with the exception of L. culinaris and C. arietinum, which produced mainly citrate and malonate. Considerable variation in the concentration of exuded carboxylates and protons was found, even with a genus. Cluster-root forming species did not invariably have the highest concentrations of rhizosphere carboxylates. Lupinus species varied both in P-uptake and in the sensitivity of their cluster-root development to external P supply. Given the carbon cost of cluster roots, a greater plasticity in their formation and exudation (i.e. reduced investment in cluster roots and exudation at higher soil P, a negative feedback response) is a desirable trait for agricultural species that may have variable access to readily available P.     

Developmental physiology of cluster-root carboxylate synthesis and exudation in harsh hakea. Expression of phosphoenolpyruvate carboxylase and the alternative oxidase

Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrient-impoverished soils in southwest Australia. When phosphorus is limiting, harsh hakea develops proteoid or cluster roots that release carboxylates that mobilize sparingly soluble phosphate in the rhizosphere. To investigate the physiology underlying the synthesis and exudation of carboxylates from cluster roots in Proteaceae, we measured O2 consumption, CO2 release, internal carboxylate concentrations and carboxylate exudation, and the abundance of the enzymes phosphoenolpyruvate carboxylase and alternative oxidase (AOX) over a 3-week time course of cluster-root development. Peak rates of citrate and malate exudation were observed from 12- to 13-d-old cluster roots, preceded by a reduction in cluster-root total protein levels and a reduced rate of O2 consumption. In harsh hakea, phosphoenolpyruvate carboxylase expression was relatively constant in cluster roots, regardless of developmental stage. During cluster-root maturation, however, the expression of AOX protein increased prior to the time when citrate and malate exudation peaked. This increase in AOX protein levels is presumably needed to allow a greater flow of electrons through the mitochondrial electron transport chain in the absence of rapid ATP turnover. Citrate and isocitrate synthesis and accumulation contributed in a major way to the subsequent burst of citrate and malate exudation. Phosphorus accumulated by harsh hakea cluster roots was remobilized during senescence as part of their efficient P cycling strategy for growth on nutrient impoverished soils.     

Shoot P status regulates cluster-root growth and citrate exudation in Lupinus albus grown with a divided root system

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 75 mmol m^?3 P. The possibility that changes in the concentrations of P fractions in the root halves influenced cluster‐root 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.     

Chickpea and white lupin rhizosphere carboxylates vary with soil properties and enhance phosphorus uptake

Chickpea and white lupin roots are able to exude large amounts of carboxylates, but the resulting concentrations in the rhizosphere vary widely. We grew chickpea in pots in eleven different Western Australian soils, all with low phosphorus concentrations. While final plant mass varied more than two-fold and phosphorus content almost five-fold, there were only minor changes in root morphological traits that potentially enhance phosphorus uptake (e.g., the proportion of plant mass allocated to roots, or the length of roots per unit root mass). In contrast, the concentration of carboxylates (mainly malonate, citrate and malate, extracted using a 0.2 mM CaCl₂ solution) varied ten-fold (averaging 2.3 mol g^–1 dry rhizosphere soil, approximately equivalent to a soil solution concentration of 23 mM). Plant phosphorus uptake was positively correlated with the concentration of carboxylates in the rhizosphere, and it was consistently higher in soils with a smaller capacity to sorb phosphorus. Phosphorus content was not correlated with bicarbonate-extractable phosphorus or any other single soil trait. These results suggest that exuded carboxylates increased the availability of phosphorus to the plant, however, the factors that affected root exudation rates are not known. When grown in the same six soils, three commonly used Western Australian chickpea cultivars had very similar rhizosphere carboxylate concentrations (extracted using a 0.2 mM CaCl₂ solution), suggesting that there is little genetic variation for this trait in chickpea. Variation in the concentration of carboxylates in the rhizosphere of white lupin did not parallel that of chickpea across the six soils. However, in both species the proportion of citrate decreased and that of malate increased at lower soil pH. We conclude that patterns of variation in root exudates need to be understood to optimise the use of this trait in enhancing crop phosphorus uptake.     

Cluster Roots: A Curiosity in Context

Cluster roots are an adaptation for nutrient acquisition from nutrient-poor soils. They develop on root systems of a range of species belonging to a number of different families (e.g., Proteaceae, Casuarinaceae, Fabaceae and Myricaceae) and are also found on root systems of some crop species (e.g., albus, Macadamia integrifoliaandCucurbita pepo). Their morphology is variable but typically, large numbers of determinate branch roots develop over very short distances of main root axes. Root clusters are ephemeral, and continually replaced by extension of the main root axes. Carboxylates are released from cluster roots at very fast rates for only a few days during a brief developmental window termed an ‘exudative burst’. Most of the studies of cluster-root metabolism have been carried out using the crop plant L. albus, but results on native plants have provided important additional information on carbon metabolism and exudate composition. Cluster-root forming species are generally non-mycorrhizal, and rely upon their specialised roots for the acquisition of phosphorus and other scarcely available nutrients. Phosphorus is a key plant nutrient for altering cluster-root formation, but their formation is also influenced by N and Fe. The initiation and growth of cluster roots is enhanced when plants are grown at a very low phosphate supply (viz. ≤1 μM P), and cluster-root suppression occurs at relatively higher P supplies. An important feature of some Proteaceae is storage of phosphorus in stem tissues which is associated with the seasonality of cluster-root development and P uptake (winter) and shoot growth (summer), and also maintains low leaf [P]. Some species of Proteaceae develop symptoms of P toxicity at relatively low external P supply. Our findings with Hakea prostrata (Proteaceae) indicate that P-toxicity symptoms result after the capacity of tissues to store P is exceeded. P accumulation in H. prostrata is due to its strongly decreased capacity to down-regulate P uptake when the external P supply is supra-optimal. The present review investigates cluster-root functioning in (1) L.albus (white lupin), the model crop plant for cluster-root studies, and (2) native Proteaceae that have evolved in phosphate-impoverished environments.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

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.     

Structure,ecology and physiology of root clusters – a review

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

Cluster-root production and organic anion exudation in a group of old-world lupins and a new-world lupin

We examined the capacity of several Old-World lupin species (Lupinus luteus L., L. hispanicus Boiss. et Reuter and L. angustifolius L.) and one species of a New-World lupin (L. mutabilis Sweet) to form cluster roots under a range of conditions in solution culture. The effect of the synthetic auxin, IBA (indole-3-butyric acid), on cluster-root development in L. luteus and L. albus L. provided with an adequate phosphorus (P) supply was also investigated. In addition, the effect of a high nitrate-N (NO₃-N) supply on the efflux of citrate and malate from roots of L. angustifolius was examined to determine if specific regions of the root system exuded these organic anions. When P-deficient, L. hispanicus, L. luteus and L. mutabilis formed cluster roots that secreted organic anions. Citrate was generally the dominant organic anion exuded, although succinate was also exuded in large quantities from L. luteus. Citrate efflux by L. hispanicus and L. luteus was at least comparable to that reported for P-deficient L. albus[up to 1.092 nmol g^–1 fresh weight (FW) s^–1], but was over an order of magnitude lower in L. mutabilis (0.036 nmol g^–1 FW s^–1). Citrate and malate were not detected in significant amounts from either the lateral roots or the root tips of any species grown under P-sufficient or -deficient conditions. Citrate efflux from cluster roots of L. luteus showed a diurnal pattern, similar to that reported for L. albus, with maximum efflux during the day, and declining to a minimum before dawn. IBA added to the nutrient solution induced cluster-root formation on both L. albus and L. luteus at concentrations of P that would normally suppress the production of these roots. However, the IBA-induced cluster roots did not exude significant amounts of citrate. Although L. angustifolius did not produce cluster roots when P-deficient, it produced cluster-like root structures that exuded citrate (0.053 nmol g^–1 FW s^–1) when grown at a high nitrate-N (NO₃-N) supply. L. angustifolius did not exude significant citrate or malate from lateral roots or root tips when grown at either high or low NO₃-N supply. Our findings for L. hispanicus and L. luteus are the first reports of cluster-root formation in response to P deficiency for these Old-World species, and for L. mutabilis, it is the first report of cluster roots for a New-World lupin species. These reports indicate that evolutionary and biogeographical aspects of cluster-root formation in the genus Lupinus need to be revised. Furthermore, investigation is warranted to determine the capacity of species of the large group of New-World lupins to form cluster roots in soils of their native habitats.     

Differences in investment and functioning of cluster roots account for different distributions of Banksia attenuata and B. sessilis,with contrasting life history

Aims

Banksia attenuata is a resprouting species growing in deep sand, while B. sessilis is a fire-killed species occurring in shallow sand over laterite or limestone. We aimed to discover the ecophysiological basis for their different distributions by exploring their investment in deep non-cluster roots and shallow cluster roots, and their cluster-root functioning.

Methods

Deep-pot (1 m), shallow-pot (400 mm), hydroponic experiments and phosphorus (P)-extraction experiment were carried out. Biomass allocation, cluster-root exudation, plant P and leaf manganese (Mn) concentrations were measured.

Results

Banksia attenuata allocated more biomass to deep roots and less biomass to cluster roots than B. sessilis did in deep pots. The two Banksias released similar carboxylates in all experiments, with similar carboxylate-exudation rates in hydroponics. The carboxylate amount per unit cluster root of B. sessilis grown in shallow pots was greater than that of B. attenuata, and B, sessilis acquired more P than B. attenuata did in limestone substrate.

Conclusions

Greater investment in deep roots for water uptake accounts for the presence of B. attenuata in deep sand, and vice versa for the absence of B. sessilis. A larger investment in cluster roots, which released greater amounts of carboxylates, likely accounts for B. sessilis occurring over limestone. Trade-offs in investment and cluster-root functioning support the species’ distribution patterns and life histories. Leaf Mn concentration was a good proxy for the plant capacity to acquire P.

     

Phosphorus-mobilization ecosystem engineering: the roles of cluster roots and carboxylate exudation in young P-limited ecosystems

Background

Carboxylate-releasing cluster roots of Proteaceae play a key role in acquiring phosphorus (P) from ancient nutrient-impoverished soils in Australia. However, cluster roots are also found in Proteaceae on young, P-rich soils in Chile where they allow P acquisition from soils that strongly sorb P.

Scope

Unlike Proteaceae in Australia that tend to proficiently remobilize P from senescent leaves, Chilean Proteaceae produce leaf litter rich in P. Consequently, they may act as ecosystem engineers, providing P for plants without specialized roots to access sorbed P. We propose a similar ecosystem-engineering role for species that release large amounts of carboxylates in other relatively young, strongly P-sorbing substrates, e.g. young acidic volcanic deposits and calcareous dunes. Many of these species also fix atmospheric nitrogen and release nutrient-rich litter, but their role as ecosystem engineers is commonly ascribed only to their diazotrophic nature.

Conclusions

We propose that the P-mobilizing capacity of Proteaceae on young soils, which contain an abundance of P, but where P is poorly available, in combination with inefficient nutrient remobilization from senescing leaves allows these species to function as ecosystem engineers. We suggest that diazotrophic species that colonize young soils with strong P-sorption potential should be considered for their positive effect on P availability, as well as their widely accepted role in nitrogen fixation. Their P-mobilizing activity possibly also enhances their nitrogen-fixing capacity. These diazotrophic species may therefore facilitate the establishment and growth of species with less-efficient P-uptake strategies on more-developed soils with low P availability through similar mechanisms. We argue that the significance of cluster roots and high carboxylate exudation in the development of young ecosystems is probably far more important than has been envisaged thus far.     

Tight control of nitrate acquisition in a plant species that evolved in an extremely phosphorus‐impoverished environment

Hakea prostrata (Proteaceae) has evolved in an extremely phosphorus (P)‐limited environment. This species exhibits an exceptionally low ribosomal RNA (rRNA) and low protein and nitrogen (N) concentration in its leaves. Little is known about the N requirement of this species and its link to P metabolism, despite this being the key to understanding how it functions with a minimal P budget. H. prostrata plants were grown with various N supplies. Metabolite and elemental analyses were performed to determine its N requirement. H. prostrata maintained its organ N content and concentration at a set point, independent of a 25‐fold difference nitrate supplies. This is in sharp contrast to plants that are typically studied, which take up and store excess nitrate. Plants grown without nitrate had lower leaf chlorophyll and carotenoid concentrations, indicating N deficiency. However, H. prostrata plants at low or high nitrate availability had the same photosynthetic pigment levels and hence were not physiologically compromised by the treatments. The tight control of nitrate acquisition in H. prostrata retains protein at a very low level, which results in a low demand for rRNA and P. We surmise that the constrained nitrate acquisition is an adaptation to severely P‐impoverished soils.     

Spatial and temporal variation in organic acid anion exudation and nutrient anion uptake in the rhizosphere of Lupinus albus L.

We investigated in situ the temporal patterns and spatial extent of organic acid anion exudation into the rhizosphere solution of Lupinus albus, and its relation with the nutrient anions phosphate, nitrate and sulfate by means of a rhizobox micro suction cup method under P sufficient conditions. We compared the soil solution in the rhizosphere of cluster roots with that in the vicinity of normal roots, nodules and bulk soil. Compared to the other rhizosphere and soil compartments, concentrations of organic acid anions were higher in the vicinity of cluster roots during the exudative burst (citrate, oxalate) and nodules (acetate, malate), while concentrations of inorganic nutrient anions were highest in the bulk soil. Both active cluster roots and nodules were most efficient in taking up nitrate and phosphate. The intensity of citrate exudation by cluster roots was highly variable. The overall temporal patterns during the lifetime of cluster roots were overlaid by a diurnal pattern, i.e. in most cases, the exudation burst consisted of one or more peaks occurring in the afternoon. Multiple exudation peaks occurred daily or were separated by 1 or 2 days. Although citrate concentrations decreased with distance from the cluster root apex, they were still significantly higher at a distance of 6 to 10 mm than in the bulk soil. Phosphate concentrations were extremely variable in the proximity of cluster roots. While our results indicate that under P sufficient conditions cluster roots take up phosphate during their entire life time, the influence of citrate exudation on phosphate mobilization from soil could not be assessed conclusively because of the complex interactions between P uptake, organic acid anion exudation and P mobilization. However, we observed indications of P mobilization concurrent with the highest measured citrate concentrations. In conclusion, this study provides semiquantitative in situ data on the reactivity of different root segments of L. albus L. in terms of root exudation and nutrient uptake under nutrient sufficient conditions, in particular on the temporal variability during the lifetime of cluster roots.     

Carbon input from plant to soil through root exudation inDigitaria adscendens andAmbrosia artemisiifolia

The amount of carbon released into soil through root exudation byDigitaria adscendens Herrm. andAmbrosia artemisiifolia L. var.elatior Desc., which often predominate at the early stages of secondary succession, was evaluated in a laboratory experiment conducted over a 60-day period. Differences in the amount of exuded carbon between these species and between the developmental stages were examined. The amount of carbon exuded increased with growth in both species. The percentage of exuded carbon to photosynthetically net fixed carbon, which was higher at younger stages (13%) inD. adscendens, decreased to 3.1% with time. On the other hand, no reduction in the amount of carbon exuded was observed inA. artemisiifolia (4.7–8.1% range). The total amount of carbon released through root exudation inD. adscendens andA. artemisiifolia was estimated at 3.1% and 6.9% of photosynthetically net fixed carbon, respectively. These results suggest the possibility that wild plants exude a considerable amount of carbon from their roots to the soil, and emphasizes the necessity for considering root exudation in the carbon cycle.     

Impact of phosphorus mineral source (Al–P or Fe–P) and pH on cluster-root formation and carboxylate exudation in Lupinus albus L.

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.     

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

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

Post‐fire seed predation: Does distance to unburnt vegetation matter?

Human‐induced changes to fire regimes result in smaller, more patchy fires in many peri‐urban areas, with a concomitant increase in potential edge effects. In sclerophyll vegetation, many structurally dominant serotinous plants rely on the immediate post‐fire environment for recruitment. However, there is little information about how fire attributes affect seed predation or recruitment for these species. We examined the influence of distance to unburnt vegetation on post‐dispersal seed predation for five serotinous species from sclerophyll vegetation in the Sydney region, south‐eastern Australia; Banksia serrata L.f., Banksia spinulosa Sm. var. spinulosa, Hakea gibbosa (Sm.) Cav., Hakea teretifolia (Salisb.) Britten (all Proteaceae) and Allocasuarina distyla (Vent.) L. Johnson (Casuarinaceae). We used cafeteria trials and differential exclusion of vertebrates and invertebrates to test whether rates of seed removal for these five species differed among (i) unburnt, (ii) burnt‐edge (approx. 10 m from unburnt vegetation) and (iii) burnt‐interior (approx. 100 m from unburnt vegetation) locations. When all animals had access to seeds, seeds were removed at lower rates from burnt‐interior areas than from other locations. Vertebrates (small mammals) showed this pattern markedly the first time the experiment was run, but in a repeat trial this effect disappeared. Rate of seed removal by invertebrates differed among plant species but we did not detect any such differences for removal by vertebrates. Overall rates of seed removal also differed significantly between the two fires studied. Our results indicate that small mammal seed predation can be substantial for large‐seeded serotinous shrubs, and that differences in the perimeter: area ratio, severity or size of a fire are likely to affect seed predation.