JXB Advance Access originally published online on December 15, 2005
Journal of Experimental Botany 2006 57(2):413-423; doi:10.1093/jxb/erj004
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RESEARCH PAPER |
Systemic suppression of cluster-root formation and net P-uptake rates in Grevillea crithmifolia at elevated P supply: a proteacean with resistance for developing symptoms of ‘P toxicity’
School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, WA 6009, Australia
* Present address and to whom correspondence should be addressed. Department of Botany, University of Cape Town, Private Bag, Rondebosch, 7701, South Africa. E-mail: mshane{at}cyllene.uwa.edu.au
Received 31 May 2005; Accepted 8 September 2005
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Abstract |
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Grevillea crithmifolia R. Br. is a species of Proteaceae that is resistant to developing P-toxicity symptoms at phosphorus supplies in the root environment that induce P-toxicity symptoms in the closely related Hakea prostrata (Proteaceae). It was discovered previously that development of P-toxicity symptoms in H. prostrata is related to its low capacity to down-regulate net P-uptake rates (i.e. its low plasticity). The plasticity of net P-uptake rates and whole-plant growth responses in G. crithmifolia has now been assessed in two separate experiments: (i) a range of P, from 0 to 200 µmol P d–1, was supplied to whole root systems; (ii) using a split-root design, one root half was supplied with 0, 3, 75, or 225 µmol P d–1, while the other root half invariably received 3 µmol P d–1. Fresh mass was significantly greater in G. crithmifolia plants that had received a greater daily P supply during the pretreatments, but symptoms of P toxicity were never observed. Cluster-root growth decreased from about half the total root fresh mass when the leaf [P] was lowest (c. 0.1 mg P g–1 DM) to complete suppression of cluster-root growth when leaf [P] was 1–2 mg P g–1 DM. Split-root studies revealed that cluster-root initiation and growth, and net P-uptake rates by roots were regulated systemically, possibly by shoot P concentration. It is concluded that, in response to higher P supply, G. crithmifolia does not develop symptoms of P toxicity because of (i) greater plasticity of its net P-uptake capacity, and (ii) its greater plasticity for allocating P to growth and P storage in roots. This ecologically important difference in plasticity is most probably related to a slightly higher nutrient availability in the natural habitat of G. crithmifolia when compared with that of H. prostrata.
Key words: Hakea prostrata, net P uptake, phosphorus toxicity, plant growth, proteoid roots, split-root design, systemic regulation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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The Proteaceae comprise some 1800 species in 80 genera. The largest numbers of Proteaceous species occur in the South West Botanical Province of Western Australia (c. 650–682) and the Cape Floristic Region of South Africa (c. 331 species) (Cowling and Lamont, 1998
; Pate et al., 2001); both areas have a Mediterranean climate, and are recognized as ‘Global Biodiversity Hotspots’ (Myers et al., 2000).
Common factors linking species from this diverse group are a well-known ability for growth on oligotrophic soils, low in available phosphorus, and a remarkable plasticity of their roots: the acclimation and functioning of their root systems has been a major focus of ecophysiological investigations (Grundon, 1972; Lamont, 1982; Dinkelaker et al., 1995; Neumann and Martinoia, 2002; Shane and Lambers, 2005). Many essential nutrient resources (e.g. phosphorus and manganese) in soils from south-western Western Australia occur mostly in a chemically bound (fixed) form (Pate and Dell, 1984; Foulds, 1993), and are thus only sparingly accessible to plants not adapted to these conditions (Handreck, 1997a, b). Almost all of the Proteaceae form very dense clusters (termed ‘proteoid’ roots; Purnell, 1960) of short, determinate (branch) rootlets, at discrete regions along the growing root axis (Lamont, 1982; Watt and Evans, 1999). At low external P supplies, cluster roots may comprise half or more of the total mass of the root system, whereas higher P supplies suppress the proportion of total root mass invested in cluster roots (Lamont, 2003; Shane and Lambers, 2005). Furthermore, when P is limiting growth, the carbon metabolism of the cluster roots is geared toward synthesis and exudation of large quantities or carboxylates (e.g. citrate) (Dinkelaker et al., 1989; Neumann and Martinoia, 2002; Shane et al., 2004a). The released carboxylates solubilize sparingly available nutrients, especially mineral-bound phosphorus (Ryan et al., 2001; Vance et al., 2003). Cluster roots also exude phosphatases that release P from organic P sources (Adams and Pate, 1992; Grierson and Comerford, 2000; Vance et al., 2003).
Once P is in soluble inorganic form, roots acquire it by the energy-dependent uptake of the inorganic phosphate (Pi) from the soil (Abel et al., 2002; Epstein and Bloom, 2004). The active transport of Pi across the root epidermal plasmalemma is mediated by both high- and low-affinity transport systems (Smith et al., 2003). The high-affinity Pi transporters are H+-dependent, and are encoded by Pht1 multigene families (Raghothama, 1999). It is essential that the uptake of phosphate be regulated in order to prevent P toxicity when soil P is not limiting and Pht1 gene transcript amounts are generally greatly suppressed when P in the root zone is abundant, and enhanced when P is limiting (Raghothama, 1999; Smith et al., 2003).
Many species of Proteaceae, either soil-grown or grown in hydroponics, are extremely sensitive to the amount of P supplied for growth, and develop P-toxicity symptoms at [P] in the root environment that are harmless to other species (e.g. Banksia ericifolia, Handreck, 1991; Parks et al., 2000; B. grandis, Lambers et al., 2002; H. prostrata, Shane et al., 2004b). However, many other species of Proteaceae tolerate far greater amounts of P in their root environment (e.g. Grevillea crithmifolia, Handreck, 1997a, b). Previous findings have uncovered the mechanisms involved in this ‘P-sensitivity’ by showing that a ‘P-sensitive’ species, H. prostrata, had a very low capacity to reduce its net P-uptake rates. The low capacity to down-regulate net P-uptake rates in H. prostrata was interpreted as an adaptation to allow for continued P acquisition and storage during the wet season (Shane et al., 2004b, c). In the Mediterranean climate in the south-west of Western Australia, cluster-root initiation and functioning occur exclusively in the wet winter season (Jeschke and Pate, 1995), whereas shoot growth predominantly occurs in spring and summer (into mid-summer for H. prostrata; Lamont, 1976). In the dry season, stored P is remobilized for growth.
In the present study, the susceptibility of G. crithmifolia R.Br. (Proteaceae), a species that is in the sister genus to that of H. prostrata (Briggs, 1998), to develop ‘P-toxicity’ symptoms at a range of P supplies for growth was tested. G. crithmifolia occurs naturally along a narrow strip of coastal land in the south-west of Western Australia near Perth (Fig. 1A). This species ‘inhabits calcareous sand and coastal limestone soil in coastal scrub, eucalypt and banksia woodland and in open coastal heath’ (Olde and Marriott, 1995). Like most natural environments in Western Australia, the habitat of G. crithmifolia is considered relatively nutrient poor, but not quite as nutrient-impoverished as that of H. prostrata, which is ‘a common species of south-western Australia and found on white or grey sand, gravel or sandy clay in low dense heath, Eucalyptus woodland, coastal heath or jarrah woodland’ (Barker et al., 1999) (Fig. 1B). The ‘P-insensitive’ G. crithmifolia was grown in nutrient solutions in a similar manner as done previously for ‘P-sensitive’ H. prostrata; that is, supplied with external [P] from severely limiting for growth to [P] that is toxic to H. prostrata. Plasticity to P supply was determined for its growth response, cluster-root formation, internal [P], and net P-uptake rates.
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Materials and methods |
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In all experiments 2-month-old soil-grown G. crithmifolia R.Br. were used. Seedlings were obtained from Kings Park and Botanic Garden (Perth, Western Australia). Soil was gently washed from root systems using tap water, and each plant was grown in aerated nutrient solution (pH 5.8) containing (in mmol m–3): 400
200 Ca2+, 200 K+, 154 
54 Mg2+, 20 Cl–, 2.0 Fe-EDTA, 0.24 Mn2+, 0.10 Zn2+, 0.02 Cu2+, 2.4 H3BO3, and 0.3 Mo4+ made up in deionized water. Plants were grown in a glasshouse at min/max 20/32 °C, and root temperature was maintained at 18–20 °C with the pots in a temperature-controlled tank (Fig. 2A, B).
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1 µmol P d–1. (D) Split-root G. crithmifolia plant grown with one root half receiving 3 µmol P d–1 (left side of photograph) while the other root half received 225 µmol P d–1 (right side of the photograph). Cluster roots were not produced on either root half. (E) Split-root G. crithmifolia plant grown where each root half received 3 µmol P d–1. Cluster roots were produced on both root halves but their initiation and growth was not synchronized, i.e. the root half shown on the left side of the photograph had young (white) developing cluster roots, while the most recently developed cluster roots on the other root half (supplied with the same amount of P) were already senesced.Experiment 1: growth at a range of phosphorus supplies
Twenty-three plants of uniform size were selected, and each was grown in 2.0 l of nutrient solution as above, supplemented with P (supplied as KH2PO4) to give six P supplies: 0, 0.4, 0.8, 2, 20, and 200 µmol P d–1 (n=4 plants in each P treatment, except the zero P treatment, where n=3) (Fig. 2A). The P treatments were carried out for 8 weeks (December, 2003 to January, 2004), and nutrient solutions containing the daily P supply were replaced every day.
Experiment 2: growth in a split-root design
Seedlings were prepared for growth in a split-root design, as described for H. prostrata (Shane et al., 2003a). Twenty-four plants of uniform size and with equal numbers of lateral roots on each ‘root half’ were transferred to split-root containers, and shoots were supported in the centre of a rectangular, grey-foam lid. Each root half was supplied with 3.0 l of continuously aerated nutrient solution as above (Fig. 2B, D, E). All plants had one root half supplied with 3 µmol P d–1, while the other half was either: (i) deprived of P, or supplied with (ii) 3 µmol P d–1, (iii) 75 µmol P d–1, or (iv) 225 µmol P d–1 (n=6). The complete nutrient solution in each pot was changed daily for the duration of the experiment (12 weeks).
Measurements of net P-uptake rates
Net P-uptake rates were determined for whole root systems as described in Shane et al. (2004c). Briefly, P depletion was measured from an external solution having an initial [P] of 5 mmol P m–3 over 3 h. It was determined, in preliminary experiments, that 6.0 l of nutrient solution containing 5 mmol P m–3 was required to give a linear P-depletion rate over 3 h. Plants remained in the glasshouse in which they were grown during all P-uptake measurements. A small volume of concentrated KH2PO4 solution (less than 100 µl) was added to each pot containing fresh nutrient solution minus P (i.e. one plant per 6.0 l pot) to give a final concentration of 5 mmol P m–3. After 120 s of mixing by vigorous aeration in each pot, a 1 ml sample (time zero) was taken from each pot, and subsequent samples taken at 30-min intervals, between approximately 10.30 h and 13.30 h.
Harvests and determination of phosphorus concentrations
Plants were harvested after an 8- (P-range experiment) or a 12-weeks (split-root experiment) treatment, and immediately after P-depletion measurements. Plants were separated into roots (cluster roots and non-cluster roots separately) and shoots (young: still expanding leaves, and mature leaves: fully expanded, and stems). Root [P] was measured only in young growing roots, therefore non-cluster roots were further subdivided into regions 30 mm proximal to the root tip, and young (white) cluster roots only were used for analysis of root [P]. Samples were weighed fresh, and again after drying for 7 d at 80 °C. Dried samples were digested in concentrated HNO3:HClO4 (3:1. v:v) at 175 °C. Total [P] in tissue digests and in solutions collected from P-depletion studies were determined using the Malachite green colorimetric method (Motomizu et al., 1983).
Statistics
Data were analysed with one-way analysis of variance (GenStat 7.1, Lawes Agricultural Trust; Rothamsted Experimental Station). Tukey's pair-wise multiple comparison tests were used to determine which levels differed significantly (
=0.05). To ensure normality and homogeneity of variances, data were log transformed when necessary.
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Results |
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Experiment 1: Plants grown at a range of phosphorus supplies
Symptoms of P toxicity (i.e. premature leaf senescence, chlorosis and necrosis, and stunted growth) were never observed in G. crithmifolia, not even at a P supply of 200 µmol P d–1. There was no significant influence of the lowest three P-supply rates (0, 0.4, and 0.8 µmol P d–1) on root, stem, and leaf fresh mass (Fig. 3A) and the fresh mass invested in cluster roots, as a percentage of total root fresh mass, was approximately 50%. The ratio of total plant mass to total root mass (i.e. root mass ratio) decreased with increasing P supply, from 0.48 (no P supplied) to about 0.27 (20 or 200 µmol P d–1) (Fig. 3A). When the external P-supply rate was 2 µmol P d–1, the fresh mass of non-cluster roots, stems, and leaves was almost double, whereas that of cluster roots was about c. 25% of the total root mass (Fig. 3A). Higher P-supply rates, either 20 or 200 µmol P d–1, were associated with c. 3-fold higher fresh mass of non-cluster roots, stems, and leaves, whereas cluster-root fresh mass was c. 6% of the total root mass at a P supply rate of 20 µmol d–1, and cluster roots were completely suppressed on plants supplied with 200 µmol P d–1.
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Leaf [P] increased from 0.15 mg P g–1 DM (no P supplied) to 2.5 mg P g–1 DM at the highest external P-supply rate of 200 µmol P d–1 (Fig. 3B). Leaf [P] was always less than that of roots, but leaf and root [P] were most similar (c. 1.6 and 2.1 mg P g–1 DM, respectively) in plants grown at 20 µmol P d–1. When the P supply for growth was 200 µmol P d–1, root [P] was sharply higher at 7.7 mg P g–1 DM, whereas that of leaves was not significantly higher (Fig. 3B). Water content of mature leaves was approximately 65% in plants supplied with 0, 0.4, and 0.8 µmol P d–1, whereas it was c. 72% when P was supplied at rates of 2, 20, and 200 mmol m–3 d–1 (data not shown).
The fastest net P-uptake rate (i.e. 0.09 nmol P g–1 root FM s–1) at a standard external concentration of 5 mmol P m–3 was measured for G. crithmifolia plants that had been deprived of P (Fig. 4), and these plants also had the lowest leaf and root [P] (Fig. 3B). Net P-uptake rates decreased linearly (r2=0.93) to 0.047 nmol P g–1 FM s–1 as external P-supply rates supplied for growth during the 12 weeks in the pre-treatment increased from 0 to 200 µmol P d–1 (Fig. 4). However, the net P-uptake rates, at a standard external concentration of 5 mmol P m–3, were the same for G. crithmifolia plants that had been grown at 2, 20, and 200 µmol P d–1 (Fig. 4), despite further significant increases in root and leaf [P].
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Experiment 2: plants grown in a split-root design
All plants grew well in the split-root design, and none showed symptoms of P toxicity, even at the highest P supply of 225 µmol P d–1 to one root half. Plants that had one root half deprived of P, while the other root half was supplied with 3 µmol P d–1 were small (c. 38 g FM plant–1, Fig. 5A), whereas the fresh mass of G. crithmifolia plants nearly doubled (c. 66 g plant–1) for plants that had both root halves supplied with 3 µmol P d–1; the higher mass resulted mostly from larger shoot biomass (Fig. 5A). When the P-supply rate to one root half was either 75 or 225 µmol P d–1, the fresh mass of G. crithmifolia was nearly 3-fold greater (c. 200 g), accompanied by a significant increase in root as well as shoot biomass. The root mass ratio (using the combined mass of each root half on individual plants) was 0.54 for plants with one root half deprived of P, but lower (0.22) for plants with one root half supplied with either 75 or 225 µmol P d–1 (Fig. 5A).
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The fresh mass of root halves supplied with 3 µmol P d–1 varied from 14–25 g, depending upon the P supply to the other root half (Table 1). Root halves receiving 75 µmol P d–1 had the greatest mass (61 g), whereas the mass of root halves supplied with 225 µmol P d–1 was similar (21 g) to that of the other half (25 g) supplied with 3 µmol P d–1 (Table 1).
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All plants had one root half that received the same ‘relatively low’ supply of P (i.e. 3 µmol P d–1). Cluster-root formation in G. crithmifolia was completely suppressed on both root halves of plants when either 75 or 225 µmol P d–1 was supplied to one root half (Table 1). When one root half was either deprived of P or supplied with 3 µmol P d–1 (while the other root half was supplied with 3 µmol P d–1) the percentage of root fresh mass allocated to cluster roots was similar (range 32–44%) on each root half of individual plants (Table 1). On plants that developed cluster roots, the timing of cluster-root initiation and growth was not synchronized between root halves on individual plants.
The [P] of old leaves was less than that of young leaves in G. crithmifolia when one root half received either 0 or 3 µmol P d–1 (Fig. 5B). When 75 µmol P d–1 was supplied to one root half, the [P] in old leaves was 6-fold higher than in the absence of P, equalling that of young leaves, in which [P] was 2-fold higher than in the absence of P; on plants with one root half receiving 225 µmol P d–1 the [P] in old leaves was 11-fold higher (c. twice that of young leaves) (Fig. 5B) than in the absence of P. Root [P] was measured only in young white roots (Fig. 5C). The [P] of these roots, for root halves supplied with 3 µmol P d–1, was approximately 1.4 mg P g–1 DM, and increased to 2.1 mg P g–1 DM as the P supply to the other root-half was increased from 0 to 225 µmol P d–1; however, the increase in root [P] (c. 50%) was significant only for those plants whose other root half received 225 µmol P d–1 (Fig. 5C). The [P] of root halves with a variable P supply (i.e. 0, 3, 75, and 225 µmol P d–1) increased significantly from 0.6 to 4.0 mg P g–1 DM (c. 600%).
Net P-uptake rates, measured at a standard external P concentration of 5 mmol P m–3, were the same on both root halves of individual G. crithmifolia plants, regardless of the large difference in P supply to the root halves during growth (Fig. 6). For root-halves supplied with 3 µmol P d–1, the net P-uptake rate was fastest (0.08 nmol P g–1 root FM s–1) when the other half was deprived of P. The net P-uptake rates for both root halves decreased incrementally to 0.02 nmol P g–1 root FM s–1 as the P-supply to one root-half was increased from 0 to 3, 75, and 225 µmol P d–1, respectively (Fig. 6).
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Discussion |
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The results of the present experiments confirm earlier work (Handreck, 1997a
, b) that Grevillea crithmifolia (Proteaceae) is resistant to developing symptoms of P toxicity at external P supplies that cause P toxicity in a ‘P-sensitive’ Proteaceae, for example, Hakea prostrata (Shane et al., 2004b, c). These results are particularly exciting, in that they show a distinct difference in ecophysiological functioning of two species of closely related (paraphyletic; Briggs, 1998) genera. This difference in functioning, in species endemic to the south-western corner of Western Australia, a Global Biodiversity Hotspot (Myers et al., 2000; Hopper and Gioia, 2004), is most likely linked to a subtle difference in habitat characteristics, as discussed below.
What accounts for variable development of P-toxicity symptoms amongst species of Proteaceae grown at low P supply?
It is improbable that native plants in their own habitat are ever exposed to levels of soil P that induce P toxicity, because the levels in that habitat are too low for that. The development of P-toxicity symptoms generally only happens on fertilized soils or when, because of regular watering, plants can continually take-up P (Shane et al., 2004b, c). The difference in susceptibilities among species for developing P-toxicity symptoms, at the same external P supply, might result from differences in (i) ‘P sensitivity’ of cells and tissues, (ii) growth responses, (iii) nutrient allocation, and (iv) rates of net P uptake.
The leaf [P] required to cause P toxicity symptoms does not differ greatly among species. P-toxicity symptoms (e.g. reduced growth, leaf chlorosis, and necrosis) are similar among species of Proteaceae (Grundon, 1972; Handreck, 1997b; Silber et al., 1998; Table 2 in Shane et al., 2004b) and crops (Asher and Loneragan, 1967; Marschner, 1995), and generally follow the accumulation of P to approximately 10 mg P g–1 in the leaf DM. In the present study, leaf [P] in G. crithmifolia never exceeded 2.5 mg g–1 DM (Figs 2B, 3B), which is well below that reported to cause P toxicity. Although whole tissue nutrient analysis is useful, it reveals little about the nutrient concentrations in individual cells (Pate and Dell, 1984; Karley et al., 2000). [P] had previously been measured in vacuoles of mesophyll cells of intact leaves of H. prostrata, and it was found that it was similar (millimolar range) to that in other species (Shane et al., 2004b) when whole tissue analysis of leaf [P] exhibited concentrations of less than 1 mg P g–1 dry mass. Thus, enhanced P sensitivity of leaf tissues and cells does not explain why species differ in their susceptibility to developing P toxicity (i.e. does not explain why H. prostrata is very sensitive to P toxicity compared with G. crithmifolia at relatively low external P supply).
In terms of nutrient allocation, G. crithmifolia accumulated more P in young roots (Figs 3B, 4C) compared with bulked root samples of H. prostrata grown at the same range of P supplies (Table 2 in Shane et al., 2004b). The significant accumulation of much higher [P] in roots of G. crithmifolia was accompanied by a non-significant increase in leaf [P] when plants were grown at the highest P supplies. The allocation of ‘excess’ P to roots of G. crithmifolia buffered the leaf [P] (Fig. 3A, B). Several other species of Proteaceae are also known to accumulate or store P in roots and stems (Jeschke and Pate, 1995; Parks et al., 2000; Shane et al., 2004b). Stem [P] was not measured in G. crithmifolia, but quantifying tissue-P-allocation patterns between species with differences in susceptibility to developing symptoms of P toxicity, and assessing their net P-uptake rates, would help to examine further the importance of nutrient-allocation patterns in determining the susceptibility to developing P toxicity.
Fresh mass of G. crithmifolia more than doubled when the P supply for growth increased from 2 to 20 µmol P d–1, but increasing the P supply to 200 µmol P d–1 had no further influence on growth. Growth of H. prostrata also increased in response to a similar range of P supplies (Fig. 1A, D; Shane et al., 2004b, c, respectively), albeit not as strongly as that of G. crithmifolia. Differences between the two species in susceptibility to P toxicity cannot be accounted for entirely by P dilution by growth, because growth saturated before the highest P-supply rate was reached in the P-insensitive G. crithmifolia (Fig. 3A). The same growth pattern was found in P-sensitive H. prostrata (Fig. 1A, B in Shane et al., 2004b; Fig. 2 in Shane et al., 2003a). Because H. prostrata developed P-toxicity symptoms at low external P supplies that had no harmful effects on G. crithmifolia, the toxic effects of P accumulation might be responsible for the decreased growth of H. prostrata, rather than decreased growth leading to P accumulation.
In this study, P-toxicity symptoms were not observed in G. crithmifolia, even at the highest P supplies (200 µmol P d–1; Figs 1, 3), whereas in H. prostrata P-toxicity symptoms developed when plants were grown at the same range of P supplies (Shane et al., 2004b). The accumulation of P to toxic levels in leaves of H. prostrata was related to the species' low capacity to down-regulate net P-uptake rates (Shane et al., 2004c). Here, it was shown that net P-uptake rates, measured at a standard external P concentration, decreased linearly in G. crithmifolia plants grown at the same P-supply rates used for H. prostrata (i.e. from 0 to 2 µmol P d–1, Figs 2, 4; cf. Shane et al., 2003a, 2004b, c). The fact that leaf P did not accumulate to toxic concentrations in leaves of G. crithmifolia, and that this species had a strong capacity to down-regulate net P-uptake rates, supports the hypothesis that development of P-toxicity symptoms is related to a low capacity to down-regulate net P-uptake rate (Shane et al., 2004c). It is concluded that susceptibilities for developing P-toxicity symptoms, at the same external P supply, in species of Proteaceae adapted to nutrient-impoverished soils, is related to a species' capacity to down-regulate net P-uptake rates by its roots.
Signalling of P status in ‘P-toxicity sensitive’ and ‘P-toxicity resistant’ Proteaceae
In the split-root experiment with G. crithmifolia, it was shown that suppression of cluster-root growth (Table 1) and net P-uptake rates (Fig. 6) occurred systemically with respect to elevated shoot [P]: i.e. cluster-root growth and net P-uptake rates were similar on both root halves of individual plants despite significant differences in P supply (and [P]) to the root halves. Systemic regulation of cluster-root growth has also been found for white lupin (Lupinus albus) grown in a similar split-root design (Fig. 2; Shane et al., 2003b; Tian et al., 2004; Li and Liang, 2005; Shen et al., 2005). These results contrast strongly with previous observations on H. prostrata, which continues to develop cluster roots on ‘low-P’ root halves despite suppression of cluster-root growth on ‘high-P’ root halves and increased shoot [P] (Table 1; Shane et al., 2003a). This may mean that species that are tolerant of a higher P supply in the root environment (i.e. G. crithmifolia and L. albus), and which do not readily develop P-toxicity symptoms (Keerthisinghe et al., 1998; Shane et al., 2004c), react systemically with a reduction in both cluster-root growth and net P-uptake rates in response to enhanced P supply. This requires further investigation.
It is known that systemic signalling in plants during phosphate starvation is responsible for P-deficiency-induced gene expression in Lycospersicon esculentum, and that the expression of these genes is suppressed by elevated shoot [P] (Burleigh and Harrison, 1999). Many studies have tried to identify components in the signal-transduction pathway between ‘sensing’ shoot [P] status and the observed systemic alterations in root growth and metabolism. For example, auxins and cytokinins are most likely involved in cluster-root growth, but their influence is probably to be at the end of the signal-transduction pathway (Gilbert et al., 2000; Skene and James, 2000; Neumann and Martinoia, 2002). At the cellular level it is known that cytoplasmic [P] often remains relatively constant, even though the external P supply is increased (Schachtman et al., 1998). Therefore, where and how the low [P] in tissues (e.g. young or old leaves) generates intracellular signals, the factors that modify gene expression in the nucleus, are unknown (Schachtman et al., 1998). Recently, levels of sugars and sugar metabolites (i.e. glucose, fructose, and sucrose) were found to stimulate LaPT1 (phosphate transporter) and LaSAP1 (secreted acid phosphatase) gene expression in the cluster-root forming L. albus (Liu et al., 2005). Moreover, expression of these genes in P-deficient plants was able to be manipulated depending upon whether or not the plants were in the light (photosynthesis) (Liu et al., 2005). Furthermore, in Arabidopsis, several phosphate-starvation-inducible genes are also sugar-induced (Müller et al., 2005). Investigations of species with distinct differences (e.g. H. prostrata and G. crithmifolia) in their systemic regulation of (cluster-) root growth and net P-uptake rates may help identify components of the signal-transduction pathway between ‘sensing’ [P] and the regulation of gene expression leading to changes in root growth and P uptake.
Searching for an ecophysiological explanation for differences in a species' predisposition to developing symptoms of P toxicity
A more plastic growth response to added nutrients, the enhanced allocation of P to roots, a ‘stronger’ systemic P-signalling system, and a greater capacity to down-regulate net P-uptake rates in G. crithmifolia compared with that in H. prostrata are likely to be adaptive responses of a plant functioning at slightly elevated P concentrations in the rhizosphere. Because Grevillea is paraphyletic with respect to Hakea (Briggs, 1998), an examination of the natural environments in which H. prostrata and G. crithmifolia characteristically occur may provide significant clues as to the ecophysiological significance of these differences in the response to P availability. Both species are endemic to the south-west of Western Australia. H. prostrata is widely distributed (Fig. 1A), invariably on severely phosphorus-impoverished acid soils, whereas G. crithmifolia has a much narrower habitat range (Fig. 1B), occurring along the coast on soils over limestone. Limestone formation is the result of leaching of calcium carbonate; soils over limestone are presumably also enriched in other nutrients that leached towards these sites. In south-western Western Australia, somewhat nutrient-enriched ‘kwongan’ sites are associated with soils over limestone, having a pH of 7.0 or more (Foulds, 1993). In southern South Africa, ‘fynbos’ soils over limestone (pH 7–8) have P, N, and micronutrient levels up to 10 times higher than those in nearby acidic soils (Esler et al., 1989). The present differences between H. prostrata and G. crithmifolia to regulate their P status might have resulted from G. crithmifolia adapting to less P-impoverished soils. It was found that G. crithmifolia grows better when P levels are somewhat enhanced (Fig. 1A), and in its natural habitat it is associated with slightly nutrient-enriched sites over limestone (Olde and Marriott, 1995), which have slightly elevated nutrient levels (Foulds, 1993).
Species-specific differences in ability to prevent excessive uptake of ions at the root surface are related to edaphic factors (Rajakaruna et al., 2003), and to their root growth allocation patterns (Poot and Lambers, 2003). The present discovery that H. prostrata does not, whilst G. crithmifolia does show plasticity in its P-uptake capacity offers an explanation for the question why H. prostrata cannot enter the habitat of G. crithmifolia. However, why G. crithmifolia does not occur in the habitats occupied by H. prostrata remains unknown. In South African ‘fynbos’, distribution of Protea species that are restricted to either soils over limestone (P. obtusifolia) or acid soils (P. susannae) is thought to result, in part, from competitive exclusion (Mustart and Cowling, 1993). It is speculated that this is also related to species differences in the efficiency of nutrient capture or nutrient use. For example, P retranslocation from senescing leaves varies considerably among species adapted to infertile soils (Güsewell, 2005). Although some species of Proteaceae can remove up to 90% of the P from senescing leaves (Chapter 6, Table 24 in Lambers et al., 1998), it is likely that this efficiency is less for other species of Proteaceae, for example, G. crithmifolia. In addition, there might be differences in P-allocation patterns and tissues used for temporary P storage (Dixon et al., 1983; Pate and Dell, 1984; Shane et al., 2004b) amongst species of Proteaceae.
The present findings suggest that physiological aspects of root functioning in Proteaceae, in relation to soil P characteristics are involved in niche preferences. Further examinations of Proteacean species that are restricted to specific soils may yield alternative adaptive mechanisms that allow species to avoid Al or Mn toxicity, and cope with low availability of Ca, Mg, or Mo. Species restricted to calcareous soils may also have adaptations to prevent bicarbonate toxicity, and to tolerate excessive Ca and the limited availability of Fe, Mn, and Zn. These aspects deserve to be studied in greater detail and may provide alternative explanations, in addition to what we have shown for the role of soil P, in the selective colonization potential of species of Proteaceae to edaphically different habitats.
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Concluding remarks |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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The present results help to explain why species of closely related genera of the Proteaceae fill marginally different niches. They also provide the start of an ecophysiological framework for understanding the enormous plant species richness in the south-west of Australia, a global biodiversity hotspot (Myers et al., 2000
; Hopper and Gioia, 2004), as related to edaphic factors. In G. crithmifolia, the net P-uptake rates by roots were down-regulated strongly and systemically, in contrast to H. prostrata. Moreover, better growth and P allocation to roots of G. crithmifolia helped to prevent toxic quantities of P accumulating in the leaves. These results on two closely related taxa endemic on different soil types indicate that a larger and more comprehensive survey of native species should be carried out. Species that differ in their susceptibility to developing P-toxicity symptoms in the species-rich flora of south-western Australia will provide valuable information on the significance of root adaptation and root physiology in the process of species diversification. |
Acknowledgements |
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We are grateful to Professor Kingsley Dixon at Kings Park and Botanic Garden for providing the Grevillea crithmifolia R.Br. seedlings for our experiments. We are also grateful to Dr Patrick Finnegan and Professor Stephen Hopper for providing valuable comments during the preparation of this manuscript. We thank Stuart Pearse for help with growing the plants and Paul Gioia for permission to reproduce the species distribution maps. We also thank the Journal of Experimental Botany, which supported the SEB Symposium Plant Frontiers Meeting 2005, at which the data included here were presented first, and two anonymous reviewers for helpful comments. This work was supported by the Australian Research Council.
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