Author for correspondence: Cameron W. S. Playsted Tel: +61 (7) 5460 1240 Fax: +61 (7) 5460 1112 Email: firstname.lastname@example.org
• Caustis blakei produces an intriguing morphological adaptation by inducing dauciform roots in response to phosphorus (P) deficiency. We tested the hypothesis that these hairy, swollen lateral roots play a similar role to cluster roots in the exudation of organic chelators and ectoenzymes known to aid the chemical mobilization of sparingly available soil nutrients, such as P.
• Dauciform-root development and exudate composition (carboxylates and acid phosphatase activity) were analysed in C. blakei plants grown in nutrient solution under P-starved conditions. The distribution of dauciform roots in the field was determined in relation to soil profile depth and matrix.
• The percentage of dauciform roots of the entire root mass was greatest at the lowest P concentration ([P]) in solution, and was suppressed with increasing solution [P], while in the field dauciform roots were predominately located in the upper soil horizons, and decreased with increasing soil depth. Citrate was the major carboxylate released in an exudative burst from mature dauciform roots, which also produced elevated levels of acid phosphatase activity. Malonate was the dominant internal carboxylate present, with the highest concentration in young dauciform roots.
• The high concentration of carboxylates and phosphatases released from dauciform roots, combined with their prolific distribution in the organic surface layer of nutrient-impoverished soils, provides an ecophysiological advantage for enhancing nutrient acquisition.
One of the earliest reports on dauciform roots in members of the Cyperaceae was that by Selivanov & Utemova (1969), which was written in Russian. These roots were then further described by Davies et al. (1973) as swollen lateral roots with an abundance of dense, long root hairs, presumed to play a role in nutrient absorption based on their dominance in the organic surface layer of calcareous dune slacks very low in P. Considering the ‘striking resemblance’ (Davies et al., 1973) of swollen lateral roots of Cyperaceae to proteoid (or cluster roots) in the Proteaceae, Lamont (1974) conducted a comparative study, which revealed sufficient differences in growth and anatomy to merit a new classification as ‘dauciform’ (= carrot-shaped) roots. It was proposed that dauciform roots play an analogous role to that of cluster roots as an adaptation to low soil fertility (Lamont, 1982; Dinkelaker et al., 1995). It is now well established that cluster roots release large amounts of carboxylates (anions of organic acids) (Gardner et al., 1983; Dinkelaker et al., 1995), and subsequent studies have shown that many species that produce cluster roots also have increased extracellular acid phosphatase activity (Dinkelaker et al., 1997; Neumann et al., 1999; Grierson & Comerford, 2000), which confers the ability to hydrolyse poorly available organic phosphates (Duff et al., 1994). However, unlike the cluster roots of Proteaceae (Roelofs et al., 2001; Shane et al., 2004) or of the well-studied crop species L. albus (Keerthisinghe et al., 1998; Neumann et al., 2000), the biological role of dauciform roots remains largely undescribed.
Following the discovery of dauciform roots, the impact of varied nitrogen supply on dauciform root formation has been tested (Lamont, 1974), and their anatomy well documented (Davies et al., 1973; Lamont, 1974). However, there is a lack of information on the physiological aspects of dauciform root function and development. A recent study by Shane et al. (2005a) provides evidence that dauciform roots are widespread in members of the Cyperaceae occurring in south-west Australia. These authors found that the formation of dauciform roots is closely dependent on P supply, as these roots are most abundant when plants are grown in solution culture at low concentrations (0–1 µm) of solution P. The low-P induction of dauciform roots in many sedges (Bakker et al., 2005; Shane et al., 2005a) suggests that they may function to increase P mobilization under P deficiency. Previous research on dauciform root development of Schoenus unispiculatus Benth. (Cyperaceae) revealed high levels of citrate released in an exudative burst from mature dauciform roots (Shane et al., 2005b). This has particular relevance in the Australian context, where much of the naturally occurring phosphate in acid and alkaline soils is retained by iron (Fe) or aluminium (Al), and calcium (Ca) oxides, respectively, as a result of their high sorption energy for phosphate (Norrish & Rosser, 1983). The exudation of carboxylates, particularly citrate, by roots plays a crucial role in mobilization of P via ligand exchange from these largely fixed sources of soil P (Jones, 1998; Hinsinger, 2001; Lambers et al., 2002).
There are many Australian native sedges that thrive in the nutrient-poor conditions of Australian plant communities (Pate & Bell, 1999; Specht & Specht, 1999), and these provide an opportunity for discovery and elucidation of low-P adaptations in the Cyperaceae. The aim of the present study was to characterize the possible role of dauciform roots of the Australian sedge Caustis blakei (Cyperaceae) in the accumulation and exudation of carboxylates and/or acid phosphatases according to their developmental stage. The formation of dauciform roots at low solution P concentrations of ≤ 1 µm (Playsted et al., 2005) suggests that these specialized roots may be an adaptation for survival of C. blakei in low-P ecosystems (Johnston et al., 1996).
Materials and Methods
The experiments were conducted in a glasshouse at The University of Queensland Australia, Gatton Campus nursery beginning in summer, from 20 February 2004 through winter to 17 September 2004. Young Caustis blakei Kük (Cyperaceae) plants, raised in nursery potting media, were transferred to 22-l containers with nutrient solution when they reached approx. 20 cm in height. Upon transfer, the roots were rinsed carefully with deionized water to remove any residual media, and the plants were then grown under 50% shade cloth in normal daylight conditions, where the temperature ranged between 15 and 35°C.
In the first experiment, basal nutrients were added to 22 l of deionized water (electric conductivity (EC) 0.008 dS m−1), to achieve the following composition (µm): nitrogen (N) 1000 (as NO3−), Ca 750, sulphur (S) 700, potassium (K) 250, magnesium (Mg) 100, chlorine (Cl) 10, silicon (Si) 5, boron (B) 3, Fe 2, zinc (Zn) 0.5, manganese (Mn) 0.25, copper (Cu) 0.1, cobalt (Co) 0.04, and molybdenum (Mo) 0.025. The solutions were changed each week, and the pH was maintained as close as possible to pH 6.0 by addition of 1 m HCl or 0.1 m NaOH. Solution culture vessels were continuously aerated. The P concentration of the mixed-bed resin-purified water used to make the solutions was assayed (≤ 0.08 µm P) using the malachite green assay as described by Van Veldhoven & Mannaerts (1987), and no P was added to any of the solutions. C. blakei plants were grown for 28 wk in the above conditions before exudate sampling of dauciform roots.
In a second experiment, a nutrient solution of identical composition was added to 2.5-l pots and C. blakei seedlings were exposed to four different solution P concentrations of 0.01, 0.1, 1.0 and 10 µm. Seedlings in this experiment were grown for 20 wk under glasshouse conditions as described in the first paragraph of this section, except that the nutrient solution was replaced daily and dauciform root development was monitored weekly.
Dauciform root exudate sampling
After 28 wk in solution culture, 16 plants of uniform size from the first experiment were transferred to individual 22-l containers for exudate sampling over a 2-wk period. Nine replicate plants were sampled, and the dauciform roots from each plant were selected on a developmental basis, as recently initiated, juvenile, mature or senescent dauciform roots. Nondauciform roots from the same plants were also selected, to quantify their exudate composition, which served as a control for comparison with dauciform root exudates.
Exudates were collected for a period of 3 h only, to avoid microbial breakdown of any exuded carboxylates. Collection was made in 5 ml of complete nutrient solution (composition as described in the section ‘Plant growth’), made on ultrapure water (18.2 MΩ cm−1). Circular Perspex collection cuvettes were designed after the method of Keerthisinghe et al. (1998), and made watertight with a layer of silicon grease over a group of dauciform roots supported on a flat Perspex stage. To avoid damage to the root, a notch was made at the junction of the Perspex and the axial and distal portions of the lateral root. Following completion of the exudate collection, the nutrient solution was removed and immediately filter-sterilized through a 0.22-µm Millipore filter (Millipore, Billerica, MA, USA). Each of the root segments sampled was excised from the plant and quickly blotted dry, and its fresh weight (FW) was recorded. The primary central axis of dauciform and nondauciform roots (Fig. 1a) was included in all FW calculations. The root and exudate samples were then snap-frozen in liquid nitrogen, and stored at −80°C until required for further analysis.
Acid phosphatase activity
At the conclusion of experiment 1, a preliminary study on acid phosphatase activity from nondauciform and mature dauciform roots of C. blakei was conducted. Duplicate samples of nondauciform root tips and mature dauciform roots were excised from the plant and placed in 0.5 mm CaCl2. The acid phosphatase activity of intact roots was measured by incubating the roots in 4 ml of 15 mm 2-[N-Morpholino]ethanesulfonic acid (MES)/0.5 mm CaCl2 buffer at pH 5.5 containing 10 mm p-nitrophenyl Pi (p-NPP) as previously described by Richardson et al. (2000) with minor modifications. Root samples were incubated at 27°C for 30 min until the reaction was terminated by the addition of 0.25 m NaOH, and acid phosphatase activity was measured in a spectrophotometer at 412 nm relative to standard solutions of pNP.
Extraction of internal carboxylates from dauciform roots was based on the method of Keerthisinghe et al. (1998). Freeze-dried dauciform roots were ground into a fine powder under liquid nitrogen, and solubilised by the addition of 1.0 ml of ice-cold 0.6 m perchloric acid, and thoroughly mixed by vortexing. The samples were then centrifuged for 15 min at 15 000 g at 4°C, before removing 750 µl of the supernatant, and adding 50 µl of 5 m K2CO3 in order to neutralize the solution. The aliquot was then centrifuged a second time for 15 min at 15 000 g at 4°C, and 700 µl of the supernatant was collected for analysis. The final pH was adjusted within the range 1.0–2.5 by the addition of 85%[volume/volume (v/v)] orthophosphoric acid.
Confirmation of the peaks determined by HPLC was achieved with gas chromatography–mass spectrometry (GC-MS), using a Hewlett Packard 5890 gas chromatograph (Hewlett Packard, Palo Alta, CA, USA) interfaced with a 5970B mass selective detector (Hewlett Packard) (70 eV, electron impact mode), with a DB-5 ms capillary column (30 m × 0.25 mm i.d., 0.25-µm film; J & W Scientific, Folsom, CA, USA). Data acquisition and processing were performed using HP Chemstation G1034C (HP 59970C) version 2.00 software (Hewlett Packard). A 100-µl aliquot of selected tissue extracts was evaporated to dryness using a SVC100H Speed Vac (Savant Instruments, Farmingdale, NY, USA), then redissolved in 100 µl of pyridine (Pierce, Rockford, IL, USA) containing 50 µl of MTBSTFA (Pierce) and subsequently heated at 70°C for 30 min. One microlitre of the derivatized solution was injected in either a split or splitless mode, depending on the expected concentration. Both retention time and mass spectral fragmentation patterns were used for the positive identification of the carboxylates using pure standards as a reference. Conditions of the GC-MS were as follows: injector at 280°C, transfer line 305°C, with the column held initially at 60°C for 2 min, then increased to 150°C at 15°C min−1, followed by a further increase from 150 to 300°C at 6°C min−1 and held for 0.5 min. Data acquisition for the MS was from 50 to 550 amu at a rate of 1.3 scans s−1. The method described here for the GC-MS analyses was an adaptation of that by Chen et al. (1998).
Field sampling of C. blakei roots
Soil cores were taken from a stand of C. blakei plants by hammering a 6-cm-diameter cylinder into the ground to a depth of 35 cm at a distance of 15 cm from the centre of each plant. Three soil cores were taken from four replicate plants, which were then divided into four respective horizons. Horizon A0 was taken as the top 5 cm of the decomposing organic matter directly beneath the loose leaf litter. The deeper A1, A2 and B1 horizons were separated at depths of 5–15, 15–25 and 25–35 cm, respectively. Samples from each of the horizons were washed and sieved to remove the soil, before separating the roots into nondauciform and dauciform root portions for FW determination.
Data were analysed using one-way analysis of variance (ANOVA) for comparison of means of single variables. In this context ANOVA was used to test for differences in internal carboxylate concentrations from each root development stage; differences in growth data and the proportion of dauciform and nondauciform roots from each solution P treatment and differences in the proportion of dauciform and nondauciform roots in each of the soil horizons from field samples. Differences between means at the 5% significance level were assessed using pairwise t-testing (Tukey test). As the exudate data did not conform to normality and homogeneity of residuals and variances, the nonparametric Kruskal–Wallis test was used for comparison of mean carboxylate concentrations from exudates of dauciform and nondauciform roots.
The influence of P availability on growth and dauciform root development
The term ‘dauciform root’ as coined by Lamont (1974) was applied to secondary carrot-shaped roots (Fig. 1a–d) containing clusters of densely packed, long root hairs (Fig. 1a). Dauciform root development was strongly dependent on the P concentration ([P]) in the solution, and dauciform roots were most abundant in P-deficient plants with reduced growth when supplied with 0.01 and 0.1 µm solution [P], comprising 37 and 31% of the entire root mass, respectively (Fig. 2). The ratio of dauciform to nondauciform roots showed a steady decline in response to increasing solution [P]. While total FW peaked in plants supplied with 1.0 µm[P], the proportion of dauciform roots to the entire root mass dropped to 5% and dauciform roots were completely absent in plants grown at 10 µm solution [P], which showed symptoms of P toxicity. Dauciform roots were present in C. blakei plants receiving 0.01, 0.1 and 1.0 µm[P] after 15 wk of growth, and showed a steady turnover for the remaining 5 wk. Under our growth conditions, dauciform roots lasted up to 20 d before senescence occurred.
Exudation of carboxylates and acid phosphatase
The fastest exudation rate of total carboxylates was found from mature dauciform roots that were approx. 15–20 d old, while exudation rates from recently initiated and juvenile dauciform roots as well as from nondauciform roots were considerably slower, and negligible amounts of carboxylates were released from senescent dauciform roots (Fig. 3a). Citrate was the major carboxylate released from mature dauciform roots, reaching a peak rate of 0.12 nmol g−1 FW s−1 (Fig. 3a), which was significantly faster than rates obtained from nondauciform roots. Malate exudation was also significantly faster in mature dauciform roots than in nondauciform roots (Fig. 3a). Acetate was present in the root exudates of mature dauciform roots, and released at a similar rate to that from nondauciform roots, where it was the dominant carboxylate. Malonate was present at a low concentration in the exudate from both mature dauciform and nondauciform roots (Fig. 3a).
Acid phosphatase (APase) activity was determined for intact nondauciform root tips and mature dauciform roots. There was a substantial 4.5-fold increase in APase activity in mature dauciform roots when compared with that in nondauciform roots tips (Fig. 4).
There was a build-up of internal carboxylates in dauciform root tissue, where total carboxylate concentrations were significantly higher in recently initiated and juvenile dauciform roots than in nondauciform and senescent dauciform roots (Fig. 3b). In contrast to findings for the composition of exuded carboxylates, inside the recently initiated dauciform roots malonate was the dominant carboxylate, with a peak concentration of 17 µmol g−1 FW (Fig. 3b); this was significantly higher than that in mature and senescent dauciform roots (Fig. 3b).
A similar trend was found for both citrate and malate, whose concentrations were highest in juvenile and recently initiated dauciform roots, respectively (Fig. 3b). The internal concentration of citrate continued to increase in both recently initiated and juvenile dauciform roots, in contrast to malonate, which peaked in the recently initiated dauciform roots and then declined during the later stages of dauciform root development. The peak concentrations of citrate and malate dropped significantly, to relatively low levels, in mature dauciform roots. The steep decline in internal carboxylate concentration in mature dauciform roots occurred concomitantly with the release of carboxylates in root exudates. Citrate and malate were only present in minute amounts in senescent dauciform roots (Fig. 3b).
Soil profile and dauciform root distribution
Field analysis of a stand of C. blakei from Landsborough in south-east Queensland, Australia (Fig. 5a), revealed an abundant proliferation of dauciform roots within the surface soil layers, which were high in humus and organic matter (Fig. 5b). Floristic associations of C. blakei included Proteaceaous Banksia spp. (Fig. 5c), with cluster roots occasionally found in the soil samples analysed. The concentration of dauciform roots was greatest in the thick layer of decomposing litter (Fig. 5d–e) present in the A0 horizon and in the A1 humic layer. These two horizons represented the top 15 cm of the soil profile where the percentage of dauciform roots (> 30%) was approx. 2.5 times (Fig. 6) that of the highly bleached A2 horizon which contained little organic matter relative to the A0 and A1 soil horizons (Fig. 5b). The percentage of dauciform roots continued to decline rapidly with increasing soil depth, such that no dauciform roots were present in the B1 horizon 25–35 cm below the soil surface (Fig. 6).
Topsoil analysis of the chemical characteristics from the A0 and A1 layers (0–15 cm) of the Landsborough site (Table 1) was conducted to aid the ecological interpretation of dauciform root exudate results. Notable findings from the full soil analysis revealed quite low levels of organic carbon (1%) and P (2 mg kg−1, bicarbonate-extractable) in the acidic soil samples (pH 4.5, 1 : 5 water).
Table 1. Chemical characteristics of soil profile horizons A0–A1 from a stand of Caustis blakei at Landsborough, south-east Queensland, Australia (adapted from Johnston, 1994)
Carboxylate accumulation and exudation of dauciform roots
Similar to other cluster-forming species (Dinkelaker et al., 1995; Keerthisinghe et al., 1998; Roelofs et al., 2001; Shane & Lambers, 2005), dauciform roots of C. blakei built up substantial internal concentrations of carboxylates that enable their rapid release in an exudative burst during the mature stage of development. Both the quantity and composition of carboxylates released by dauciform roots varied according to stage of development (Fig. 3a). In particular, an increase in the ratio of citrate to malate was apparent with increasing age of dauciform roots approaching maturity. By sampling dauciform root exudates at each of their developmental stages, our study has shown an 8-fold increase in the amount of citrate released during the mature stage compared with the early stages of development. This parallels exudation of carboxylates from L. albus (Neumann et al., 1999) and reinforces the common finding that citrate is often the most abundant carboxylate released by root clusters; citrate has the added advantage of being a tricarboxylate, which is highly effective in mobilizing nonlabile soil P from metal cations (Lambers et al., 2002; Shane & Lambers, 2005). The transitory release of carboxylates coincided with the mature development of dauciform roots, after which only trace amounts of carboxylates were detected in senescent dauciform roots, similar to the pattern found in cluster roots of L. albus (Watt & Evans, 1999) and members of the Proteaceae (Dinkelaker et al., 1995; Shane et al., 2004). It is likely that the internal build-up of carboxylates, particularly citrate, in initiated and juvenile dauciform roots supplied the majority of the carboxylates released during the exudative burst in mature dauciform roots.
For clusters of Hakea prostrata, Shane et al. (2004) suggested that the internal build-up of isocitrate in cluster roots is followed by a decarboxylation event to produce malate, which is released with citrate during the exudative burst, but, unlike isocitrate, is not accumulated prior to the exudative burst. The findings of the present study are similar to those obtained in the research on clusters of H. prostrata, because internal malonate concentrations peaked in young dauciform roots, but were largely absent in the root exudates. Malonate is an analogue of succinate, and acts as a competitive inhibitor of succinate hydrogenase, preventing the conversion of succinate in the tricarboxcylic acid (TCA) cycle. For this reason, malonate accumulation in roots has been postulated to play a role in defence against soil pathogens and/or herbivores (Li & Copeland, 2000). However, recent findings demonstrate that malonate may play a role in the carboxylation pathway of cytosolic acetyl-CoA metabolism, because it rescues an ATP-citrate lyase- (ACL) deficient phenotype in Arabidopsis thaliana (Fatland et al., 2005). Exogenous malonate was able to restore the depletion of cuticular waxes and the reduced growth rates associated with the A. thaliana ACL-deficient phenotype (Fatland et al., 2005). Reduced ACL expression in L. albus favours the production and subsequent release of citrate by mature cluster roots (Langlade et al., 2002). If reduced expression of ACL is necessary for the internal build-up of citrate in mature dauciform roots, then decreased cytosolic production of acetyl-CoA would be expected, along with a reduction in the amount of malate produced (Shane & Lambers, 2005), as observed in this study. We speculate that high malonate concentrations play a role in root carboxylate metabolism, where high concentrations of internal malonate may compensate for a deficiency in ACL-derived cytosolic acetyl-CoA, thereby allowing low expression of ACL to continue, resulting in the build-up of citrate before its release in an exudative burst. In addition, the inhibition of the TCA cycle by malonate may facilitate citrate accumulation as a result of decreased citrate catabolism.
In our study, substantial levels of acetate were found in the root exudates of mature dauciform roots. As malonate decarboxylase catalyses the conversion of malonate to acetate (Byun & Kim, 1994; Kim, 2002), it is possible that as the dauciform roots approach maturity some malonate is decarboxylated, yielding acetate and CO2. Alternatively, high microbial activity in the unstirred layers of the densely spaced dauciform root hairs may be responsible for decarboxylation of malonate to acetate.
Ecophysiological advantages of dauciform roots
The large surface area per unit weight of mature dauciform roots is ideal for mobilization of immobile elements such as P, because exuded carboxylates are able to reach the critical concentrations needed to impact rhizosphere chemistry and counteract P sorption in the soil (Shane & Lambers, 2005). The high density of very long dauciform root hairs (Fig. 1a,c) probably creates overlapping P-depletion zones, as found in cluster roots (Gerke et al., 2000), such that any chemically mobilized P that diffuses away from the excreting root will be taken up by neighbouring root hairs.
The present study showing an increased release of citrate and malate from mature dauciform roots offers a possible explanation for the ability of other members of the Cyperaceae that produce dauciform roots, such as Carex lasiocarpa (Davies et al., 1973), to absorb P from insoluble sources, i.e. AlPO4 and FePO4 (Pèrez Corona et al., 1996). Numerous studies have shown citrate and malate to be effective chelators of Fe and Al, both of which form complexes with P in acidic soils (Gardner et al., 1982; Gerke, 1994; Dinkelaker et al., 1995; Lambers et al., 2002). C. blakei is found in acidic sandy soils of open woodland ecosystems with a low P content and a relatively high content of Fe (Johnston, 1994). Our studies provide the first evidence that dauciform roots in C. blakei may be crucial for access to scarcely available soil P, from either adsorbed mineral complexes or organic P in the soil surface layer. Dauciform roots predominantly occur in surface soil horizons, such as the organic layer of calcareous dune slacks (Davies et al., 1973) and weathered sandstone soils described in this study. The nature of the dauciform roots of C. blakei intermingled within the soil organic layer is reminiscent of that of the cluster roots of Banksia species (Lambers et al., 2002) (Pate & Watt, 2002) which form a dense mat, described as a ‘nutrient-trapping organ’ (Jeffery, 1967), in the decomposing litter. Our results demonstrate that mature dauciform roots of C. blakei play a distinct role in the release of APases, which are known to hydrolyse organic P in the soil solution to provide plant-available inorganic P (Duff et al., 1994). The activity of APases released in combination with carboxylates such as citrate by dauciform roots of C. blakei is likely to have a synergistic effect which increases the mobilization of nonlabile P (Neumann et al., 2000), especially in soils where there is low solubility of organic P (Hens et al., 2003).
Our study provides evidence for a specialized P-acquisition strategy in C. blakei where dauciform roots may be important in liberating the largely sorbed P in nutrient-poor Australian soils. We have demonstrated a level of root plasticity in C. blakei, such that dauciform roots are initiated or suppressed in response to changes in the P concentration of nutrient solutions. Like cluster roots, these dauciform roots release a citrate burst which is thought to aid the chemical mobilization of P from its many complex forms in the soil. Increased production of acid phosphatases by mature dauciform roots may facilitate hydrolysis of organic P esters in the soil surface layer where dauciform roots are abundant. The compact morphology of dauciform roots, with copious fine root hairs, allows a concentrated release of carboxylates and acid phosphatases in a coordinated manner. Understanding of the factors governing the initiation and function of dauciform roots is still in its infancy, and, although P limitation appears to be the major trigger, the influence of other nutrient interactions is yet to be studied. There remains much to be learned from the ability of sedge species to survive in some of the most nutrient-poor ecosystems of the world, where dauciform roots provide an alternative adaptation to the mycorrhizal root for maximizing the uptake of soil P.
The authors wish to thank Dr Michael Shane for help with dauciform root carboxylate extraction and Mr Alan Lisle for statistical assistance. This research was supported by a Research Development Grant from The University of Queensland, a Discovery Grant to HL from the Australian Research Council and a Small Grant from The University of Western Australia to HL and Professor John Kuo. CWSP received an Australian Postgraduate Award and a Science and Innovation Award from the Australian Government Department of Agriculture Fisheries and Forestry.