METABOLIC ADAPTATIONS of the NON-MYCOTROPHIC PROTEACEAE to SOILS with LOW PHOSPHORUS AVAILABILITY Hans Lambers,1 Peta L

Annual Plant Reviews (2015) 48, 289–336 http://onlinelibrary.wiley.com doi: 10.1002/9781118958841.ch11

Chapter 11 METABOLIC ADAPTATIONS OF THE NON-MYCOTROPHIC PROTEACEAE TO SOILS WITH LOW PHOSPHORUS AVAILABILITY Hans Lambers,1 Peta L. Clode,2 Heidi-Jayne Hawkins,3 Etienne Laliberte,´ 1,4 Rafael S. Oliveira,1,5 Paul Reddell,6 Michael W. Shane,1 Mark Stitt7 and Peter Weston8 1School of Plant Biology, University of Western Australia, Crawley (Perth), Australia 2Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Crawley (Perth), Australia 3Department of Biology, University of Cape Town, South Africa; Conservation South Africa, Centre for Biodiversity Conservation, Kirstenbosch National Botanical Gardens, Claremont, South Africa 4Institut de Recherche en Biologie Veg´ etale,´ UniversitedeMontr´ eal,´ Montreal,´ Canada 5Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Sao˜ Paulo, Brazil 6EcoBiotics Ltd, Yungaburra, Queensland, Australia 7Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany 8Royal Botanic Gardens and Domain Trust, Sydney, New South Wales, Australia

Abstract: Proteaceae are almost all non-mycorrhizal and most species produce proteoid (= cluster) roots when grown in low-phosphorus (P) soils. In south-western Australia and the Cape Floristic Region of South Africa, Proteaceae have diversified more than anywhere else, and occur on the most severely P-impoverished soils in the landscape. Several traits related to their P nutrition account for the success of south-western Australian Proteaceae on P-impoverished soils: (i) a P-acquisition strategy based on carboxylate release from ephemeral cluster roots, which allows the species to ‘mine’ P that is ‘sorbed’ to soil particles; (ii) efficient use of P in

Annual Plant Reviews Volume 48: Phosphorus Metabolism in Plants, First Edition. Edited by William C. Plaxton and Hans Lambers. C⃝2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

289 290 Phosphorus Metabolism in Plants

photosynthesis, based on a very low investment in ribosomal RNA, extensive replacement of phospholipids by lipids that do not contain P, and allocation of P to photosynthetic cells and not epidermal cells; (iii) a very high P-remobilisation efficiency; and (iv) a high seed P content. Proteaceae in southern South Amer- ica do have a P-acquisition strategy based on carboxylate release, but lack the other P-efficiency traits. They occur on soils that contain vast amounts ofP,but with a very low P availability, and invest less biomass in cluster roots. However, these ephemeral structures live somewhat longer and release far more carboxylates when compared with Proteaceae from south-western Australia. The various aspects of P nutrition in Proteaceae across the world are discussed in a phylogenetic context.

Keywords: Cluster roots, delayed greening, phospholipids, phosphorus- acquisition efficiency, photosynthetic phosphorus-use efficiency, phosphorus toxicity, ribosomal RNA, seed phosphorus.

11.1 Introduction

The flowering plant family Proteaceae is considered of Gondwanan age,with a fossil record that dates back to the mid-Cretaceous, 120–90 million years ago (Johnson & Briggs, 1975; Dettmann & Jarzen, 1998; Doyle & Donoghue, 1992; Sauquet et al., 2009a), when the major Gondwanan continental blocks were still connected (Ali & Krause, 2011). Both, modern and fossil lineages are associated with all southern continents, but the present distribution of Proteaceae can only partly be explained by the break-up of Gondwana. Indeed, some sister groups post-date the break-up of Gondwana, indicating transoceanic dispersal (Barker et al., 2007). Proteaceae diversified faster in both the Cape Floristic Region of Southern Africa and in south-western Australia than in any other area (Sauquet et al., 2009a). These regions are severely nutrient- impoverished (Lambers et al., 2010), and Sauquet and coworkers (2009a) spec- ulated that higher soil fertility in other regions may have led to local extinctions of Proteaceae, partly due to strong competition for soil resources with species from other families. Shifts in climate are probably also involved. For example, large-scale extinctions of a hyperdiverse sclerophyll flora in eastern Australia occurred when the climate in this region markedly changed from a high-rainfall summer-wet climate in the Early Pleistocene to a much drier climate, whereas the climate was more stable in south-western Aus- tralia (Sniderman et al., 2013), at least along its coastal margin (Wyrwoll et al., 2014). The high diversity of Proteaceae in both south-western Aus- tralia (about 700 species) and the Cape Floristic Region in South Africa (>350 species) is likely the result of rapid speciation or slow rates of extinction, determined by low soil fertility, habitat fragmentation, and a relatively stable climate (Hopper, 2009; Sniderman et al., 2013). In the northern hemisphere, about 110 species of Proteaceae occur in southern India, Sri Lanka, Japan, south-east Asia, northern South America, northern Africa, and Central Metabolic adaptations of the non-mycotrophic Proteaceae 291

America (Venkata Rao, 1967; Pate et al., 2001; Weston, 2007). Since little physiological and biochemical research has been conducted on those taxa, this chapter focuses primarily on species of Proteaceae from the southern hemisphere, some of which have been studied in great detail. Proteaceae are basal eudicots, belonging to the Proteales, which also includes the sister group of the Proteaceae, the Platanaceae (including the London plane tree), and sister to those two families, the Nelumbonaceae (including the sacred lotus) (Gu et al., 2013). However, despite their phylogenetic proximity, the Platanaceae do not share many of the traits that are typical of the Proteaceae and which will be explored in this chapter. In particular, most species of Proteaceae are non-mycorrhizal (Brundrett, 2002), whereas Platanus forms symbiotic associations with arbuscular mycorrhizal fungi (Pope, 1980; Tisserant et al., 1996). Nelumbo is probably not mycorrhizal, despite records to the contrary (Koul et al., 2012), which appear to have relied on insufficiently rigorous criteria for recognising the presence of arbuscular mycorrhizas (see Brundrett, 2009). Given its aquatic habitat, Nelumbo is unlikely to be the host of mycorrhizal fungi, which typically require aerobic conditions (Smith & Read, 2008). Almost all species of Proteaceae produce specialised ‘proteoid’ or cluster roots when P is limiting (Figure 11.1; Purnell, 1960; Shane & Lambers, 2005a), but cluster roots have never been found in either Platanaceae or Nelumbonaceae. The functioning of these cluster roots is discussed next.

11.2 Phosphorus nutrition of Proteaceae, with a focus on south-western Australia

11.2.1 Phosphorus acquisition by non-mycorrhizal roots: cluster roots Cluster roots were first noted on a species of Proteaceae growing in the botanical gardens in Leipzig, Germany (cited in Purnell, 1960). The first detailed study on proteoid roots was by Purnell (1960), who coined the term ‘proteoid’ roots, being “ ... dense clusters of rootlets of limited growth along the lateral roots of many members of the family Proteaceae”. Since then, the term ‘cluster roots’ has been used for similar structures on root systems in several other families (Dinkelaker et al., 1995; Lamont, 1981; Shane & Lambers, 2005a). Phosphorus (P) deprivation generally favours lateral over primary root growth, especially in nutrient-rich soil patches, a response termed ‘topsoil foraging’ (Lynch & Brown, 2001). Likewise, cluster-root formation is most pronounced when plants are grown at a very low supply of P (Figure 11.1a, b), and the formation of cluster roots is suppressed when plants are grown at a higher P supply (Lamont, 1972; Shane et al., 2003; Zu´ niga-Feest˜ et al., 2010). Clusters may comprise a single ‘simple’ cluster root, as in Australian Hakea and the southern South American Embothrium species (Figures 11.1a, c, e, f), or 292 Phosphorus Metabolism in Plants

Figure 11.1 Cluster roots of Proteaceae species from plants grown at ≤1 μM phosphorus (P) in nutrient solution (a–c, f) or P-impoverished soils (d, e). (a) Compound cluster roots of the south-western Australian Banksia repens. (b) Simple cluster roots of south-western Australian Hakea prostrata. (c) Time course of development in H. prostrata from rootlet initiation at day 1 (far left) to senescence at day 20 (far right); that is, stages I and II are immature, stage III is mature, stage IV is senescing. (d) Claviform cluster root of the southern South American Gevuina avellana showing club-like tips of mature rootles. (e,f) Simple cluster roots of the southern South American Embothrium coccineum grown in porous pumice (e) or hydroponically (f). The cluster roots of only one Proteaceae species, Gevuina avellana, rootlets develop unusual club-shaped tips (d), referred to as claviform cluster roots (Ramirez et al., 2004). Photographs a–c, courtesy of Michael W. Shane; photographs d–f, courtesy of Michael W. Shane and A Zu´niga-Feest.˜ Metabolic adaptations of the non-mycotrophic Proteaceae 293

500 4000 Total surface area of rootlets Number of rootlets

400 3000 root axis

−1 300

2000 root axis) −1 cm

200 2 Surface area (mm 1000

Number of rootlets cm 100

0 0 I II III IV Stage of cluster root development

Figure 11.2 Rootlet number and total surface area per cm of main root axis for simple cluster roots of Hakea prostrata. All data are mean ± standard error (n = 5). Each developmental stage is labelled according to the number of days following rootlet emergence observed at day 1 until senescence at day 20, as shown in Figure 11.1c. M.W. Shane, unpublished data. a ‘compound’ cluster root, as in Australian Banksia species (Figure 11.1a). The formation of clusters increases the root mass ratio (i.e. root mass as a fraction of total plant mass), while proliferation at a density of about 300 rootlets per cm main root axis in, for example, Hakea prostrata (harsh hakea) results in an approximately fivefold increase in root surface area (Figure 11.2). This would enhance P acquisition, as the root surface is the primary site of P uptake for non-mycorrhizal species (Chapter 5) and P has very limited mobility in soil (Chapter 1). However, the rootlets and the root hairs they produce are so close together that the zones from which they acquire P overlap (Jungk, 2001). The cluster-root structure is therefore more important to allow the build-up of exuded compounds than to enhance the uptake of nutrients from the rhizosphere. This is discussed in detail below. Cluster roots are ephemeral structures (Figure 11.1c) and only live for about three weeks in H. prostrata (Shane et al., 2004a). The rootlets are determi- nate and meristems are lost when rootlets mature and the vascular system becomes fully differentiated to the tip. Cluster roots of Proteaceae release large amounts of carboxylates (i.e. anions of organic acids) at relatively fast rates (Roelofs et al., 2001). The effect of carboxylates in the rhizosphere is to mobilise inorganic and organic phosphate (Pi and organic-P, respectively) 294 Phosphorus Metabolism in Plants that is sorbed onto soil particles (Lambers et al., 2006), replacing P via ligand exchange, thereby desorbing P, which thus enters into the soil solution (Geelhoed et al., 1998). In H. prostrata, this release of carboxylates takes place in an ‘exudative burst’ of mainly citrate (Shane et al., 2004a), as shown before for Lupinus albus (Fabaceae) (Watt & Evans, 1999). This developmentally regulated pattern of cluster-root exudation minimises the chance of the carboxylates being consumed by microorganisms before playing their role of mobilising sorbed P. In addition, cluster roots of Proteaceae may release a range of compounds that inhibit microbial activity, as shown for L. albus (Weisskopf et al., 2006a; Weisskopf et al., 2006b; Tomasi et al., 2008). In alkaline (calcareous) soil, the simultaneous release of protons during carboxylate exudation (to maintain charge balance) also enhances the availability of P, but in acid soil the release of protons actually decreases the availability of P (Lambers et al., 2008a). Therefore, Proteaceae from south-western Australia, most of which are found on acidic soils, release other cations (e.g. potassium) to maintain charge balance (Roelofs et al., 2001). ‘Plant-available’ soil [P] (e.g. resin- exchangeable P) in habitats in south-western Australia where species of Pro- teaceae are prominent is typically less than 1 mg kg−1 soil (Hayes et al., 2014; Lambers et al., 2013a), compared with, for example, 70–100 mg kg−1 soil in the 0- to 2-cm layer of well-fertilised agricultural soil, and 50–30 mg kg−1 at 8– 10 cm in no-till crop-production system (Guertal et al., 1991). In these nutrient- impoverished environments, non-mycorrhizal Proteaceae are notorious for their ability to thrive on some of the world’s most severely P-impoverished soils (Shane & Lambers, 2005a). This appears paradoxical, because mycorrhizas are well known to enhance plant P acquisition (Smith & Read, 2008), whereas Proteaceae is considered a non-mycorrhizal family (Brundrett, 2009; Shane & Lambers, 2005a). There are, however, some exceptions within the Proteaceae where species producing mycorrhizas are found, such as Hakea verrucosa, which is endemic on ultramafic soils in south-western Australia (Boulet & Lambers, 2005), and Placospermum coriaceum from tropical rain- forests in north-eastern Australia (Figure 11.3), both of which form arbuscular mycorrhizas. Why are non-mycorrhizal species of Proteaceae with cluster roots so suc- cessful on P-impoverished soils? This is accounted for by their remarkable capacity to mine P from soils, and thus access P that is not readily available for mycorrhizas. This strategy involves the developmentally pro- grammed release of carboxylates from their cluster roots, as opposed to the P-scavenging mycorrhizal strategy (Lambers et al., 2008b). Parfitt (1979) grew ryegrass plants that were either colonised by arbuscular mycorrhizal fungi or uncolonised in goethite, a major P-sorbing mineral in soil. He added P at a range of concentrations, showing that mycorrhizal fungi were only effective in enhancing plant P uptake over a very narrow range of soil P. Above that range, non-colonised plants grew as well as colonised plants, but below that range the mycorrhizas were ineffective. Consistent with these results, the mycorrhizal Placospermum coriaceum, which does not make cluster roots, Metabolic adaptations of the non-mycotrophic Proteaceae 295

Figure 11.3 Growth (a) and colonisation by mycorrhizal fungi (b) of seedlings of Placospermum coriaceum (a species which does not produce cluster roots) in response to increasing phosphorus supply with or without inoculation with spores of arbuscular mycorrhizal fungi. Data are mean ± standard error. P. Reddell, unpublished data. produces relatively less biomass at very low soil P than Darlingia darlingiana, Musgravia heterophylla and Carnavonia araliifolia var. montana, which are non- mycorrhizal Proteaceae species from similar rainforest habitats and soils that do form cluster roots (Figure 11.4). When soil P concentrations are increased

100

Carnarvonia araliifolia var. montana 25

% maximum dry% maximum weight Darligia darlingiana Musgravea heterophylla Plascospermum coriaceum 0

0 5 10 25 50 100 P supply (mg P kg−1 soil)

Figure 11.4 Comparative growth response (% of maximum shoot dry weight) to increasing phosphorus supply for seedlings of four species of rainforest Proteaceae from eastern Australia grown in a granitic soil. Three of the species (Darlingia darlingiana, Carnarvonia araliifolia var. montana and Musgravea heterophylla) form cluster roots, while Placospermum coriaceum forms arbuscular mycorrhizas and does not produce cluster roots. P. Reddell, unpublished data. 296 Phosphorus Metabolism in Plants further, all species produce more biomass, until soils contain very high levels of plant-available P, which are toxic for some species, as is common for various members of the Proteaceae (see below). The release of relatively large amounts of carboxylates, compared with what is commonly found in species without cluster roots, is associated with an increased production of carboxylates preceding and during the exudative burst (Shane et al., 2004a). Increased carbon use for the synthesis of carboxylates is associated with a decreased use of carbon in cluster-root growth and respiration. However, the abundance of the alternative oxidase increases at this developmental stage. The alternative cyanide-resistant respiratory pathway resides in the inner mitochondrial membrane, similar to the cytochrome pathway. The alternative oxidase allows the oxidation of NADH with O2 as the terminal electron acceptor, bypassing two sites of coupled ATP synthesis (Millenaar & Lambers, 2003). Its functioning is considered important to allow the oxidation of NADH that is produced during synthesis of citrate at a time when little ATP is required for biosynthetic reactions (Shane et al., 2004a). In cluster roots of L. albus, an enhanced expression of the gene encoding the alternative oxidase is associated with a higher in vivo activity of the alternative respiratory pathway (Florez-Sarasa et al., 2014). The carbon costs associated with the production and functioning of cluster roots in H. prostrata has been estimated to represent 52–100% of the daily produced photosyn- thates (Lambers et al., 2006). However, it must be borne in mind that cluster roots in south-western Australian Proteaceae are only produced in the wet season, that is two to three months per year, whereas photosynthesis is main- tained throughout much of the year (albeit at lower rates when water availability is restricted). Similarly high values have been found for L. albus,with exuded citrate estimated at 23% of the total plant dry weight (Dinkelaker et al., 1989). Phosphoenolpyruvate carboxylase (PEPC) plays a key role in plant accli- matisation to P deficiency (Chapters 3 and 4) (Plaxton & Tran, 2011; Vance et al., 2003). This includes controlling the production of carboxylates (citrate, malate) that are exuded by cluster roots. Phosphoenolpyruvate carboxylase catalyses the irreversible β-carboxylation of phosphoenolpyruvate (PEP), to form oxaloacetate and Pi; the oxaloacetate is then reduced to malate, which can be converted into citrate. Recently, Shane et al. (2013) discovered a novel post-translational mechanism for the reciprocal control of PEPC in cluster roots of H. prostrata. Immature cluster roots of H. prostrata contain an equiva- lent ratio of 110-kDa monoubiquitinated and 107-kDa phosphorylated PEPC polypeptides. Incubation with a de-ubiquitinating enzyme in vitro converted the PEPC heterotetramer of immature proteoid roots into a homotetramer, which results in a significant increase in the enzyme’s activity under sub- optimal, but physiologically relevant, assay conditions. Cluster-root maturation was paralleled by PEPC activation, via in-vivo de-ubiquitination and subsequent phosphorylation of the de-ubiquitinated subunits, but without any apparent change in PEPC protein abundance. This post-translational Metabolic adaptations of the non-mycotrophic Proteaceae 297 control was hypothesised to contribute to the massive synthesis and release of carboxylates from mature cluster roots, and also to promote metabolic P recy- cling, since Pi is a byproduct of the PEPC reaction (Chapter 4). In cluster roots of L. albus, carboxylates are released via a citrate-permeable channel (Zhang et al., 2004), and this is likely to be similar in Proteaceae. This contention is supported by an approximately 50% reduction in exudation when a plasmalemma anion channel blocker is applied to mature cluster roots of Leucadendron foedum, in which some exudation possibly also occurs via carboxylate:H+ symport. As expected, the vacuolar storage of major carboxylates occurs prior to exudation, since the inhibition of a tonoplast anion channel results in an increased exudation in the same species (H.-J. Hawkins, unpublished data). Protons are not always released as charge- balancing cations for the carboxylates. In some cases, as in L. albus (Zhu et al., 2005), other cations are quantitatively more important (Roelofs et al., 2001). Potassium citrate is more effective at mobilising P than citric acid is (Palomo et al., 2006). The fact that Proteaceae species are rather uncommon on calcareous soils (Hayes et al., 2014) is counterintuitive, as releasing large amounts of carboxylates and protons would appear to be an ideal strategy to cope with calcareous soils, where P strongly interacts with calcium (Ca) at high pH. It is possible that it is not the high pH but rather the high Ca concentration that excludes most Proteaceae from calcareous soils. Calcium sensitivity has been shown for other calcifuge species (Jefferies & Willis, 1964), but its physiological basis has yet to be explored. It is hypothesised that the Ca sensitivity of Proteaceae is a consequence of the localisation of P in their mesophyll cells, where Ca is generally also stored in most dicotyledons (see below). The upregulation of extracellular and intracellular acid phosphatase (APase) activity is recognised as a biochemical hallmark of plant P starvation (Chapter 10). Acid phosphatases catalyse the hydrolysis of phosphomonoesters and anhydrides within the acidic pH range. Secreted APases can hydrolyse organic-P (Tran et al., 2010), which can comprise a major fraction of total soil P, especially in older soils (Chapuis-Lardy et al., 2001; Turner et al., 2013). In addition, Bieleski and Johnson (1972) showed for the small aquatic plant Spirodela oligorrhiza that significant levels of phosphomonoesters can leak during growth under low-P conditions. Failure to recapture this released P could constitute a net loss of P form roots. It is not surprising, therefore, that cluster roots of H. undulata (Dinkelaker et al., 1997) and Banksia sessilis (Grier- son & Comerford, 2000) release APases. Measurements of extracellular APase activity of cluster roots of hydroponically grown, P-deprived H. prostrata (Figure 11.5) show that the majority of root extracellular APase activity is surface-bound with a smaller fraction accounted for by exuded APases. Although the method used to identify these differences has been available for many years (Barrett-Lennard et al., 1993), most current studies provide total root APase activity without differentiat- ing between cell wall-bound and exuded APases, which may be completely different proteins with a different substrate specificity. The acquisition of Pi 298 Phosphorus Metabolism in Plants

Root exudate Root surface

1.5 FW) −1 g −1 1 APase activity

(μmol pNP min 0.5

0 NCII I II III IVV Stage of cluster root development

Figure 11.5 The influence of cluster-root development on secretory and cell wall-bound APase activity of P-deficient Hakea prostrata grown with ≤1 μMP.Stages correspond to those given in Figure 11.1c (Shane et al., 2013); NC = non-cluster roots. Data are mean ± standard error (n = 5). Assay methods and conditions were as described in Barrett-Lennard et al. (1993) (M.W. Shane and W.C. Plaxton, unpublished data). from hydrolysed organic-P due to APase activity (exuded or cell wall-bound) is important and is likely enhanced by the release of carboxylates, because organic-P, like Pi, also tends to be sorbed onto soil particles (Anderson et al., 1974; Shang et al., 1992). In addition, the release of protons would favour the activity of APases, as it would bring the pH closer to their pH optimum. At present, no biochemical or functional studies have been reported for any P-starvation-induced extracellular APases that are associated with cluster roots of Proteaceae. However, there is a substantial amount of information on L. albus, which shows increased activities of both intracellular and extracellular acid phosphatases (Gilbert et al., 1999; Miller et al., 2001).

11.2.2 Proteaceae species that do not produce cluster roots Cluster roots are absent in Persoonia (Purnell, 1960) and other members of the Proteaceae tribe Persoonieae (Garnieria, Toronia and Acidonia), but their feeder roots form a dense cover of persistent root hairs up to 6 mm long (Lamont, 1982). Cluster roots are also reported to be absent in Agastachys odorata and Symphionema montanum (Lee, 1978), and in the mycorrhizal species referred to above, Placospermum coriaceum (P. Reddell, unpublished data). However, one Metabolic adaptations of the non-mycotrophic Proteaceae 299 of the present authors (H.L.) has recently observed cluster roots in A. odorata in Tasmania (unpublished data) which suggests that Symphionema ought to be re-examined for the presence of cluster roots as well. Weston (2007) spec- ulated that cluster roots probably evolved in the family’s stem lineage and were secondarily lost in both Persoonioideae and Symphionematoideae. The Proteaceae species that do not form cluster roots form two distinct clades, both of which are nested within the clade of cluster-root-forming Proteaceae (Weston & Barker, 2006). Mycorrhizal Proteaceae species, that is H. verrucosa and P. coriaceum, are not closely related. It is envisaged that ancestors of all Proteaceae were mycorrhizal, and that their non-mycorrhizal status evolved in founder members of the Proteaceae (Brundrett, 2002). The mycorrhizal status of some Proteaceae species would then be a more recent and secondary trait. For example, in H. verrucosa, mycorrhizas most likely evolved under the selection pressure of soils that are rich in nickel (Ni), where the fungi can help to prevent Ni toxicity, which might result from the release of carboxylic acids, in the host plant. A similar situation has arisen in other typical non-mycorrhizal families (Brassicaceae and Caryophyllaceae) with species on soils that are rich in Ni (Lambers et al., 2009 and references therein). The P-mobilising carboxylates that are exuded by cluster roots of Pro- teaceae also mobilise micronutrients in the rhizosphere, such as manganese (Mn). This explains why Proteaceae species tend to exhibit high leaf [Mn] (Child & Smith, 1960; Jaffre,´ 1979; Rabier et al., 2007). The suppression of cluster roots in H. prostrata during growth at a high P supply decreases Mn accumulation in mature leaves (Shane & Lambers, 2005b). Therefore, leaf Mn concentration might be used as an easily measured above-ground trait to act as a proxy for carboxylate release. If this is the case, the finding that the leaves of Persoonia longifolia, which does not produce cluster roots, contain high concentrations of Mn (P.E. Hayes, unpublished data) suggests that this species also depends on a carboxylate-releasing strategy to acquire P from the nutrient-poor soils in its natural habitat. Further research is required to evaluate this possibility.

11.2.3 Phosphorus toxicity Phosphorus toxicity is a common, though not universal, phenomenon for south-western Australian Proteaceae species (Handreck, 1991; Parks et al., 2000; Shane et al., 2004b; Shane et al., 2004c; de Campos et al., 2013). An exces- sive accumulation of P leads to toxicity also in other plant species, though this is not normally seen until genetic lesions in signalling pathways lead to such an accumulation of P. One example outside the Proteaceae is the pho2 mutant in the model plant Arabidopsis thaliana, in which a feedback mechanism that regulates Pi transfer to the shoot is incapacitated (Chapters 2 and 5) (Delhaize & Randall, 1995; Bari et al., 2006). The P sensitivity of Proteaceae is accounted for by a very low capacity to down-regulate Pi-uptake, such that 300 Phosphorus Metabolism in Plants leaves accumulate Pi to toxic levels (de Campos et al., 2013; Shane et al., 2004b; Shane et al., 2004c). Species that strongly down-regulate their Pi-uptake, such as Grevillea crithmifolia, are not P-sensitive (Shane & Lambers, 2006). Interest- ingly, the remobilisation of Pi from senescing leaves is decreased in mir156- overexpressing Arabidopsis lines, in which the E2 ligase PHO2 is supressed (Chiou et al., 2006). Plants exposed to high levels of Pi often show symptoms of micronutrient deficiency, even though they show normal leaf micronutrient concentrations (Lambers et al., 2002). This occurs because micronutrients in leaves or soil can be precipitated by excess Pi, rendering them unavailable. In Leucadendron ‘Safari Sunset’, excess Pi has also been shown to precipitate foliar Mn, but not foliar Fe or Zn (Hawkins et al., 2008). The extent to which species down-regulate their Pi-uptake capacity at a high Pi supply is inversely correlated with their capacity to remobilise Pi from senescing leaves (de Campos et al., 2013). It is surmised that this reflects a common control of Pi transport in both roots, which absorb Pi from the rhizosphere, and senescing leaves, which export Pi to the phloem. This point remains to be further explored, especially in comparison with Proteaceae such as G. crithmifolia that do down-regulate their Pi-uptake capacity and are not P-sensitive (Shane & Lambers, 2006).

11.2.4 High rates of photosynthesis despite low leaf P concentrations Many species of Proteaceae in south-western Australia show very low leaf P concentrations ([P]) (approximately 0.2 mg g−1 dry weight (DW) (Denton et al., 2007a; Lambers et al., 2012b). By contrast, the optimal leaf [P] for most crops is around 4 mg g−1 (McLachlan et al., 1987; Fohse¨ et al., 1988; Rodr´ıguez et al., 2000). This very low leaf [P] in south-western Australian species of Pro- teaceae is partly accounted for by the scleromorphic nature of the leaves, essentially ‘diluting’ the concentration of nutrients (Figure 11.6). Using the average leaf dry matter content of approximately 0.55 g DW g−1 fresh weight (FW), Sulpice et al. (2014), calculated that the very low leaf [P] on a dry weight basis, 10-fold lower than in plants showing an ‘adequate’ leaf [P] (Epstein & Bloom, 2005), is reduced to a 1.8-fold difference when expressed on a FW basis. As discussed below, the very low leaf [P] is also associated with several biochemical adaptations. In crop plants and Arabidopsis, P-starved leaves tend to have low rates of photosynthesis per unit leaf area (Brooks et al., 1988; Rao & Terry, 1989; Fredeen et al., 1990; Jacob & Lawlor, 1991; Ghannoum & Conroy, 2007). In contrast, and despite their low leaf [P], species of Proteaceae from P-impoverished soils in south-western Australia exhibit relatively rapid rates of photosynthesis per unit leaf area (approximately 20 μmol m−2 s−1 in Banksia species and 15 μmol m−2 s−1 in Hakea species (Denton et al., 2007a; Lambers et al., 2012b), when measured under ambient conditions in their Metabolic adaptations of the non-mycotrophic Proteaceae 301

(a)

(b) (c)

Figure 11.6 Hand-cut transverse sections of mature scleromorphic leaf leaves of (a) Banksia victoriae, and (b and c) Hakea baxteri. The lower leaf surface is at the bottom in each micrograph. UV-induced autofluorescence is shown in panels a and b).(a) Stomatal crypts lined with stomata (white arrows). Thick cell walls of epidermis, fibres and vascular tissues fluoresce blue. Chlorophyll in palisade parenchyma and guard cells fluoresces red. (b) Stomata occur on upper and lower leaf surface (short white arrows) directly above a single layer of palisade parenchyma (fluoresces red). Thick cell walls fluoresce blue, particularly, relatively long cells interspersed within each layer of palisade parenchyma. These cells may act as ‘struts’ to brace and strengthen leaves (long white arrows). (c) Same section as in panel (b), but stained with phloroglucinol/HCl (pink-red colour specific for hydroxycinnamyl aldehyde structures in lignin). Apparent cell-wall lignification of ‘struts’ and central tissue layer comprising one vein in cross-section visible with bundle cap of fibres on either side of vascular tissues of xylem (x)and phloem (∗). Scale bars: (a) 240 μm; (b) 180 μm. 302 Phosphorus Metabolism in Plants natural habitat. As a result, the photosynthetic P-use efficiency (PPUE) is −1 −1 extremely high in these Proteaceae species: 0.2 to 0.5 μmol CO2 [g leaf P] s −1 −1 (Denton et al., 2007a), compared with 0.059 μmol CO2 g s for species with < −1 −1 leaves with N:P ratios 15, and 0.129 μmol CO2 g s for species with leaves with N:P >15 (Wright et al., 2004). The higher rate of photosynthesis in Banksia leaves compared with Hakea leaves is associated with the presence of sunken stomata in Banksia species with thick leaves (Hassiotou et al., 2009; Lambers et al., 2012b) (Figure 11.6a,c). Sunken stomata increase photosynthetic rates, because they reduce the diffusion pathway of CO2 to mesophyll cells. Sunken stomata are absent in Banksia species (e.g. B. littoralis) with thin leaves, and in all Hakea species (Lambers et al., 2012b; Lambers et al., 2014). To discover the underlying mechanism of a high PPUE in south-western Australian Proteaceae species, four major P-containing fractions in leaves need to be analysed in detail: Pi, phospholipids, nucleic acids (predominantly ribosomal RNA, rRNA), and low-molecular-weight phosphorylated metabolites (Lambers et al., 2011). Averaged for all cases where total [P] <4mgg−1 DW, small P-containing metabolites represent 17%, phospholipid P = 23%, Pi = 25%, nucleic acid P (mainly RNA) = 35% (Veneklaas et al., 2012). There- fore, nucleic acid P would appear to be the most important P pool where gains in P-use efficiency (PUE) could be made, although other pools still represent significant proportions of total leaf P. Foliar Pi in dicotyledons is mostly stored in the vacuoles of epidermal cells, as opposed to mesophyll cells in monocotyledons (Conn & Gilliham, 2010). Interestingly, in H. prostrata (Proteaceae) (Shane et al., 2004b) and B. attenuata (Figure 11.7a), [Pi] in epidermal cells is always very low. Instead, accumulation of Pi at higher P supply takes place in mesophyll cells (Shane et al., 2004b). Inorganic phosphate plays an essential role during photosynthesis. Follow- ing its incorporation into ATP by the thylakoid ATP synthase, Pi is transferred to the phosphorylated intermediates of the Calvin–Benson cycle, and released again during the synthesis of end-products such as starch, sucrose, and amino acids. With the exception of starch, most of these end-products are synthesised in the cytosol. Photosynthate is exported as triose-P from chloro- plasts, Pi is released in the cytosol, and returns to the chloroplast in a strict counterexchange with triose-P (Stitt et al., 2010). There is good evidence that rapid rates of photosynthesis require a fine balance between the levels of free Pi and phosphorylated intermediates, and that photosynthesis is inhibited when free Pi is depleted (see Stitt et al., 2010 for a recent review). As photosynthesis occurs in mesophyll cells and not in epidermal cells, it is envisaged that the accumulation of Pi in the mesophyll may allow a more efficient use of Pi. The total level of Pi, adenine nucleotides and other phosphorylated metabolites is constrained by the amount of Pi in the cytoplasm. While the depletion of Pi in the cytosol and chloroplast leads to a remobilisation of Pi from the vacuole (Sharkey et al., 1986; Mimura, 1995), little is known about how this process is regulated. There is no information on other Proteales Metabolic adaptations of the non-mycotrophic Proteaceae 303

Figure 11.7 Phosphorus (P) and calcium (Ca) concentrations in the South African Leucadendron ‘Safari Sunset’ (L. laureolum × L. salignum) species (a, b) and the south-western Australian Banksia attenuata (c, d). Note that for both B. attenuata and Leucadendron ‘Safari Sunset’, besides xylem and phloem, P is mainly located in the mesophyll, with relatively little in the epidermis (a, c). This elemental location is opposite to what occurs for calcium (b, d) in these plants. (a) An example of a leaf scan of Leucadendron ‘Safari Sunset’ using particle-induced X-ray emission spectrometry (micro-PIXE) with the graph of P concentration per tissue type shown alongside. Microanalysis was performed on cryo-fixed, 250- to 500-mm hand-cut, transverse leaf sections revealing distinct tissue layers including epidermis (e), mesophyll (m) and one vascular bundle where the bundle sheath (b), sclerenchyma (s), xylem (x) and phloem (p) are shown; see labels in panel (b). The lower epidermis is at the bottom of the images. Concentration data were obtained by drawing regions of interest, with the spectrum from each pixel within the region of interest summed and quantified. (b) An example of a leaf scan of Leucadendron ‘Safari Sunset’ using micro-PIXE with the graph of Ca concentration per tissue type for four leaves shown alongside. Methodological details in panels (a) and (b) are the same. (c) Phosphorus concentration in B. attenuata leaves scanned using energy-dispersive spectroscopy (EDS). Microanalysis was performed on cryopreserved, freeze-substituted, resin-embedded material. The X-ray element maps were obtained from transverse leaf sections revealing distinct tissue layers, including the adaxial epidermis, hypodermis, mesophyll, and sclerenchyma. Concentration data were extracted in a way similar to above. (d) Calcium concentrations in B. attenuata leaves scanned using EDS. Methodological details are as for panel (c). Data are mean ± standard error. Panels (a) and (b) reproduced from Hawkins et al. (2008); panels (c) and (d) from P. Clode (unpublished data). 304 Phosphorus Metabolism in Plants with respect to allocation of P in leaf cells. The preferential allocation of P to mesophyll cells, rather than to epidermal cells, in south-western Australian Proteaceae species, offers a partial explanation for the high PPUE of these species. In crop plants and Arabidopsis, the concentration of phospholipids markedly declines in plants during P starvation, and this is compensated by an increase in the level of galactolipids and sulfolipids (Chapter 9) (Calderon- Vazquez et al., 2008; Gaude et al., 2008; Yamaryo et al., 2008). In six Proteaceae species growing in their natural P-impoverished habitat, phospholipid concentrations are high in young leaves, but markedly decline during normal leaf development, whereas those of galactolipids and sulfolipids strongly increase (Figure 11.8). Whilst the contribution of phospholipids decreases by less than twofold in Arabidopsis and crop species such as maize and bar- ley, it decreases by over fourfold in the Proteaceae species. However, photosynthetic rates increase sharply from young to mature leaves (Lambers et al., 2012b). Therefore, these Proteaceae species extensively replace phospholipids with lipids that do not contain P, without compromising photosynthesis. The low investment in phospholipids, relative to other lipids, offers a partial explanation for a high photosynthetic rate per unit leaf P in Proteaceae adapted to P-impoverished soils (Lambers et al., 2012b). It is currently not known why the Proteaceae can continue to photosynthesise at normal rates despite low phospholipid levels, and why they replace their phospholipids during leaf development, rather than make galactolipids and sulfolipids during leaf expansion. Perhaps this is because membranes with galactolipids and sulfolipids are leakier than those with phospholipids, making them maladap- tive for expanding leaves. However, this warrants further investigation. In Arabidopsis and crop species, ribosome abundance is high in the growing cells of young leaves, and much lower in mature leaves (Detchon & Possing- ham, 1972; Dean & Leech, 1982; Baerenfaller et al., 2012; Sulpice et al., 2014). In

Figure 11.8 Average lipid profiles of young expanding and mature fully expanded leaves of three Banksia and three Hakea species, growing in their natural habitat, in comparison with Arabidopsis thaliana, grown at either high or low supply of phosphorus (Lambers et al., 2012b). Metabolic adaptations of the non-mycotrophic Proteaceae 305

(a) (b)

(e) (f)

(g)

Figure 11.9 Average biochemical characteristics of young expanding and mature fully expanded leaves of three Banksia and three Hakea species in comparison with Arabidopsis thaliana (Sulpice et al., 2014). (a) Plastidic ribosomal RNA expressed on a fresh weight basis. (b) Cytosolic ribosomal RNA expressed on a fresh weight basis. (c) Plastidic ribosomal RNA expressed on a protein basis. (d) Cytosolic ribosomal RNA expressed on a protein basis. (e) Protein. (f) Rubisco activity expressed on a protein basis. (g) Glucose-6-phosphate concentration (Glc6P) on a protein basis. Data are mean ± standard error.

Arabidopsis, rRNA levels are high in young leaves, but in mature leaves they are much lower than in P-replete plants (Sulpice et al., 2014). Compared with young leaves of Arabidopsis, immature leaves of six Proteaceae species growing in their natural P-impoverished habitat contain very low levels of rRNA (expressed on a FW basis), especially plastidic rRNA (Figure 11.9a, b) (Sulpice et al., 2014). Proteaceae also show a slow development of their photosynthetic apparatus (‘delayed greening’); this can be seen by visual inspection as well as by biochemical analyses, which reveal that young leaves have very low levels of chlorophyll and Calvin–Benson cycle enzymes (Sulpice et al., 2014). Cru- cially, ‘delayed greening’ is associated with extremely low levels of plastidic rRNA (Figure 11.9a, b). In mature leaves of these six species of Proteaceae, rRNA, soluble protein and Calvin–Benson cycle enzyme activities are very low on a FW basis (Figure 11.9e, f) (Sulpice et al., 2014). Mature leaves of the 306 Phosphorus Metabolism in Plants

Proteaceae also show very low levels of rRNA (expressed on a FW basis), but cytosolic rRNA levels are particularly low (Figure 11.9a, b). Expressed per unit protein, however, rRNA levels of Proteaceae are quite similar or somewhat higher than those in Arabidopsis. The low ribosome abundance in the young leaves of these Proteaceae con- tributes in a major way to their high PPUE. This is envisaged to act in three ways: (i) less P is invested in ribosomes; (ii) the rate of growth and, hence, the demand for P is low; and (iii) the especially low plastidic ribosome abundance in young leaves delays formation of the photosynthetic machinery, spreading investment of P in rRNA in time. Arabidopsis and crop plants show a large decrease in the levels of phosphorylated intermediates and free nucleotides when they are grown under P- limiting conditions (Brooks et al., 1988; Hurry et al., 2000; Zrenner et al., 2006; Morcuende et al., 2007; Veneklaas et al., 2012). For technical reasons, it was not possible to reliably measure the concentrations of most phosphorylated intermediates and adenine nucleotides in leaf material from the six Proteaceae studied. However, it was possible to measure glucose-6-phosphate, which is a major P-containing metabolite (Gibon et al., 2009; Sulpice et al., 2009), which occupies a central position in metabolism, being involved in the pathways of sucrose and starch synthesis, sucrose and starch degradation, and glycolysis (Stitt et al., 2010). In the following, it is assumed that the response of glucose-6-phosphate can be used as a proxy for the response of other phosphorylated intermediates, as well as free adenine nucleotides. Although soluble protein and Calvin–Benson cycle enzyme activities are low in Pro- teaceae species growing in their natural P-impoverished habitat (as discussed above), they maintain normal levels of glucose-6-phosphate (expressed per unit leaf soluble protein) (Figure 11.9g) (Sulpice et al., 2014). Thus, unlike other species, which show a decrease in the concentration of phosphorylated metabolites (Veneklaas et al., 2012), the investigated south-western Australian Proteaceae do not show low levels of glucose-6-phosphate when growing in their P-impoverished natural habitat (Sulpice et al., 2014). Decreasing the concentration of phosphorylated metabolites is therefore not part of the strategy used by these species to economise P; it would require higher levels of enzymes, and thus rRNA, a much larger pool of P in leaves. From a systems perspective, the strategy of Proteaceae to function at low levels of rRNA but ‘normal’ levels of phosphorylated intermediates rep- resents a very effective strategy to maximise PPUE. The low P allocation for rRNA will lead to a low rate of protein synthesis, while maintenance of the concentration of P-containing intermediates of carbon metabolism will allow enzymes – and hence the rRNA that is required for their synthesis – to operate effectively. Interestingly, this adaptive metabolic strategy of Proteaceae from P-impoverished habitats is the exact opposite of that found in plants that have adapted to cold environments, in which increased enzyme activities at low temperatures partially compensate for the corre- sponding decrease in their catalytic activity (Stitt & Hurry, 2002; Usadel Metabolic adaptations of the non-mycotrophic Proteaceae 307 et al., 2008). The low enzyme levels may explain why these Proteaceae typically do not show any leaf expansion during winter, which is very mild in the Mediterranean climate in which they occur. Leaf growth tends to occur in late spring and early summer, when temperatures have increased (Veneklaas & Poot, 2003). The low investment of P in rRNA in young leaves may pre- clude their expansion at low temperatures, but low rRNA and protein levels do not prevent high rates of photosynthesis in winter (Veneklaas & Poot, 2003). These results underline the point that Proteaceae which, are adapted to P-impoverished soils in south-western Australia respond to low P in a very different manner to Arabidopsis and crop plants. The latter have been bred to maximise growth and seed yield with large inputs of P-containing fer- tilisers, and their response to low P indicates that they continue to priori- tise rapid growth rates, even in the presence of low P. Whilst such a strategy allows faster growth in the short term, it also means that mature leaves are acutely limited by P, causing greater losses of P associated with leaf turnover, as shown for a faster-growing grass, Molinia caerulea, in comparison with the slower-growing evergreen shrub, Calluna vulgaris (Aerts, 1990). In contrast, the Proteaceae appear to pace their growth closely to the P supply, and to avoid or minimise acute P limitation of metabolism in mature leaves. This finding opens up important questions about how ribosome abundance isreg- ulated in young leaves of Proteaceae, to what extent this is genetically ‘hard- wired’, and to what extent ribosome biogenesis might be regulated by the momentary P supply or by signals that integrate information about the P supply in the preceding season or seasons.

11.2.5 Leaf longevity

Whilst a high PPUE is important to use P efficiently in CO2 assimilation in the short term, a high leaf longevity allows the efficient use of P for carbon gain in the long term (Lambers et al., 2008a). The longevity of leaves of six Proteaceae in a Banksia woodland in south-western Australia ranged from 29 months for Stirlingia latifolia to 40 months for Adenanthos cygnorum (average 34.8 months) (Veneklaas & Poot, 2003). Higher values have been found for B. baueri, a shrub with horizontal leaves (60 months) and B. repens, a ground creeper with large vertical leaves (156 months) (Witkowski et al., 1992). With the exception of the longevity of B. repens leaves, these values are by no means exceptional (Reich et al., 1992). This is most likely accounted for by the fact that temperature, rather than drought or nutrient availability, is the primary driver of contrasting responses of leaf longevity (van Ommen Kloeke et al., 2012). It might also be noted that the maintenance of high levels of phosphorylated metabolites in mature leaves of the Proteaceae will allow an efficient use of the enzyme complement, and thus decrease high-light stress, which commonly occurs when the activity of the Calvin cycle is curtailed (Lambers et al., 2008a). The relatively high ribosome abundance in mature leaves will 308 Phosphorus Metabolism in Plants

(a) (b)

Figure 11.10 Delayed greening in young developing leaves of Proteaceae species from south-western Australia (a–c) and eastern Australia (d). (a) Hakea amplexicaulis. (b) Banksia grandis.(c)Banksia attenuata.(d)Lomatia silaifolia. Photographs (a) and (b) from Rafael S. Oliveira; photograph (c) from Owen K. Atkin; photograph (d) from Peter Weston. facilitate protein turnover and repair. It will be interesting in the future to measure the rates of protein turnover and repair in these plants growing in the field on P-impoverished soils, and to compare these with the ratesin Ara- bidopsis and crop plants growing in P-replete and P-limiting conditions.

11.2.6 Delayed greening The Proteaceae discussed above, and many others in the same P- impoverished environment, typically show a slow development of the photosynthetic apparatus (‘delayed greening’) (Figure 11.10). Their leaves have very low levels of chlorophyll, soluble protein, Rubisco and other Calvin– Benson cycle enzymes (Figure 11.9) (Sulpice et al., 2014). Young expanding leaves have a yellow or reddish colour in some Hakea and Banksia species (Figure 11.10), and a yellowish-brown colour in other Banksia species (Lambers et al., 2012b). Delayed greening has mainly been studied for Metabolic adaptations of the non-mycotrophic Proteaceae 309 tropical species (Kursar & Coley, 1992a; Close & Beadle, 2003; Cai et al., 2005), but is also reported for temperate species (Hughes et al., 2007). Delayed greening is often considered a defence against herbivory (Kursar & Coley, 1992b; Numata et al., 2004) as well as offering protection against high light intensities (Hughes et al., 2007). Delayed greening may result in a low protein level in younger leaves which would decrease their nutritional value, while the red and yellow pigments may be phenolic defence compounds (Kursar & Coley, 1992b) and provide protection against high levels of radi- ation (Hughes et al., 2007). Phenolics occur in the cotyledons of Proteaceae, where higher concentrations are associated with a better defence against herbivores (Hanley & Lamont, 2001). In the Proteaceae referred to here, soluble protein levels are, on average 1.5-fold higher in immature soft than in mature tough leaves (Sulpice et al., 2014). However, if the young expanding leaves had developed their photosynthetic machinery, as occurs in plants that do not exhibit delayed greening, the difference in total soluble protein would have been considerably greater, making the soft expanding leaves a more attrac- tive potential target for herbivores. Whilst the activities of most enzymes were low in young expanding leaves of these Proteaceae, their leaves showed relatively high in-vitro activities of shikimate dehydrogenase, a key enzyme of the phenyl propanoid pathway involved in the synthesis of red and yellow pigments, assuming that these are phenolics (D´ıaz et al., 1997; Peek et al., 2013). This comparison indicates that delayed greening is not necessarily associated with a decreased nutritional value per se, and emphasises the potential importance of high levels of defence metabolites in the young leaves. In temperate dicotyledonous species, leaf expansion and chloroplast biogenesis typically occur simultaneously (Detchon & Possingham, 1972; Dean & Leech, 1982; Baerenfaller et al., 2012). In Quercus glauca, delayed greening is considered important in the context of partitioning of N used for leaf expansion and N used for chloroplast development, which are two major sinks for N (Miyazawa et al., 2003). During leaf growth, both cell expansion and biogenesis of the photosynthetic apparatus require the synthesis of large amounts of protein (Miyazawa et al., 2003); in particular, Rubisco accounts for 30–40% of the total leaf protein in C3 plants, and light-harvesting complexes are also quite abundant. Similar arguments apply to the partitioning of P, and possibly with even more force. Ribosomes represent a large proportion of the total RNA and protein in growing cells (Warner, 1999), including those in young leaves (Detchon & Possingham, 1972; Dean & Leech, 1982; Baerenfaller et al., 2012; Sulpice et al., 2014), whilst of the rRNA in young leaves almost half is accounted for by plastid rRNA (Sulpice et al., 2014). Delayed greening may increase PUE, and this might be the primary reason for the synthesis of non- plastidic pigments, which then can protect the leaves against high light in the absence of a mature photosynthetic machinery.Indeed, it is possible that prior construction of the leaf, with phenolic pigments, a thick epidermis and scleromorphic structure may aid assembly of the photosynthetic machinery in the harsh light conditions experienced by these Proteaceae in south-western 310 Phosphorus Metabolism in Plants

Australia. A temporal separation of leaf expansion and establishment of the photosynthetic machinery lengthens the time until a leaf transitions from being a net importer to a net exporter of carbon. However, this is unlikely to be a major disadvantage in plants such as the Proteaceae, which produce leaves that have an average lifetime of two to three years. Their mature leaves accumulate large amounts of starch (Sulpice et al., 2014), which can be mobilised as sucrose to support growth of young leaves until they develop a strong photosynthetic capacity and become self-sufficient for carbon. Despite the importance of delayed greening to P economy in the species of Proteaceae studied by Sulpice et al. (2014), this phenomenon is not universal in south-western Australian Proteaceae, not even within a single genus, Hakea (H. Lambers, pers. obs.). Further integrative studies of the ecology, physiology, biochemistry and molecular biology of delayed greening might provide further insight into its ecophysiological significance. It will also be of interest to learn how the initiation of chloroplast biogenesis is uncoupled from light signalling, and what signals trigger this process at a later stage in leaf development in these Proteaceae species. This might in turn throw light on the reason for the very low plastidic ribosome abundance in their young leaves.

11.2.7 Efficient and proficient P remobilisation from senescing organs Although the leaves of Banksia function at very low [P], a major fraction is remobilised during leaf senescence, so that senesced leaves of some species contain as little as 19 mg P g−1 DW (Denton et al., 2007a; Hayes et al., 2014). As a result, leaf litter decomposition is slow, and most of the nutrients will be returned to the soil during fires. It would be interesting to assess if highly efficient remobilisation is due, in part, to very little P being present in the epidermal cells and most in the mesophyll cells. If that allocation pattern is important, monocotyledons would be expected to be more efficient at P remobilisation than dicotyledons. In a global comparison, graminoids, indeed, show a greater P-remobilisation efficiency (82%) than global average values (50%) (Vergutz et al., 2012). Little is known about P-remobilisation from senescing roots in general (but see Freschet et al., 2010); however, highly efficient P-remobilisation (80–90%) from senescing cluster roots and leaves of H. prostrata (Shane et al., 2004a; Shane et al., 2014) appears to match what is known for leaves of other Pro- teaceae from south-western Australia (Denton et al., 2007a). The up-regulation of intracellular APase activity is a ubiquitous P- starvation response, which allows scavenging of Pi from P-esters (Chapter 10) (Tran et al., 2010; Plaxton & Tran, 2011). Understanding the metabolism that drives the efficient remobilisation of P from senescing organs to younger growing regions and developing seeds in these Australian extremophile species will significantly enhance the present understanding of their high P-resorption efficiency and proficiency (Denton et al., 2007a). The metabolic Metabolic adaptations of the non-mycotrophic Proteaceae 311 networks that mediate P remobilisation from senescing leaves are poorly understood, but evidence from Arabidopsis has demonstrated a key role for the vacuolar and cell wall-targeted purple APase (PAP) isozyme AtPAP26, which is one of 29 PAP isozymes encoded by the Arabidopsis genome (Robin- son et al., 2012; Shane et al., 2014). The P-remobilisation efficiency of senescing roots and leaves of H. prostrata has also been linked with a striking up-regulation of cell wall-localised and intracellular PAPs, and ribonucle- ase (RNase) activity (Shane et al., 2014). The up-regulation and dual target- ing of PAPs and RNases to the cell wall and vacuolar compartments likely make a crucial contribution to highly efficient P remobilisation during senescence (Shane et al., 2014). Further, as already mentioned, the mir399/PHO2 signalling network that senses Pi levels in the Arabidopsis shoots and regulates Pi allocation between the roots and shoots (Bari et al., 2006; Chiou et al., 2006; Pant et al., 2008) may also influence the remobilisation of P from senescing leaves (Chiou et al., 2006).

11.2.8 Seed P reserves Seed P concentrations of south-western Australian Proteaceae species are remarkably high, up to 36 mg g−1 DM in H. pycnoneura (average 13.2 mg P g−1 DM; 1.4 mg per seed) (Kuo et al., 1982; Milberg & Lamont, 1997; Denton et al., 2007a; Groom & Lamont, 2010). For comparison, the average value for a wide range of crop species is 3.5 mg P g−1 DM (Marschner, 1995). Phosphorus is associated with globoid-rich tissue in seeds of several investigated Proteaceae species (Kuo et al., 1982). Seed P can contribute up to 48% of the total above- ground P, such as in B. hookeriana (Witkowski & Lamont, 1996). Seed set in Banksia species in south-western Australia tends to be very low; commonly only a few percent of all the flowers produce seeds (Fuss & Sedgley, 1991; Lamont & Wiens, 2003). Since seed set can be increased by addition of nutrients (Stock et al., 1989), a low seed set appears to be a mechanism allowing the seeds to accumulate large amounts of nutrients, in particular P. It will be interesting to learn if the low seed set is due to low fertilisation rates, or to seed abortion, possibly to coordinate the seed production with available P and ensure a high P content in each individual seed. The high [P] in seeds of Proteaceae from P-impoverished soils facilitates seedling establishment and early growth in soils with extremely low P availability; it also allows investment in deep roots that access the water table which is vitally important in a seasonally dry environment. More generally, it will make the initial growth of these species largely independent of a need to acquire P from the P-impoverished soils in which they live, providing a strong competitive advantage over other species that contain less P in their seeds and/or use this P less efficiently (Hocking, 1982; Milberg & Lamont, 1997). Denton et al. (2007b) calculated that for 35-week-old seedlings of nine Banksia species, the P content of the sown seeds could have contributed as much as 12–70% to the total seedling P; the potential contribution of seed P 312 Phosphorus Metabolism in Plants to seedling P was strongly correlated with seed size. However, at this stage, all nine Banksia species had already invested heavily in cluster root growth and carboxylate release (Denton et al., 2007b), indicating that while seedling establishment is supported by P reserves form the seed, further growth is dependent on acquired P from the soil.

11.3 Comparison of species of Proteaceae in south-western Australia with species elsewhere

11.3.1 The Cape Floristic Region in South Africa Proteaceae in the Cape Floristic Region in South Africa have been studied in some detail (Lamont, 1982), and appear to function in a similar manner as those in south-western Australia, but with subtle differences (as noted in this section). South African Proteaceae have long been known to produce cluster roots (Lamont, 1982). The similarities between Proteaceae from South Africa and south-western Australia may be partly accounted for by the close phylogenetic relationship between Adenanthos and Isopogon in south-western Australia, which form a paraphyletic doublet of outgroups to the ‘Cape Clade’ (subtribe Leucadendrinae) comprising a large number of South African Proteaceae, with Leucodendron being sister to the rest of this clade (Figure 11.11; Barker et al., 2002). Leaf [P] is, on average, somewhat higher in South African Proteaceae in the Cape Floristic Region than in south-western Australian Proteaceae (Rundel et al., 1988; Lambers et al., 2010), with values of about 0.4 mg P g−1 leaf DM (Wright et al., 2004) to 0.6 mg P g−1 leaf DM (Hawkins & Cramer, 2011). Pho- tosynthetic rates of Cape Proteaceae vary between low and moderately high; from 3 to 18 μmol m−2 s−1 (Mooney et al., 1983; Van der Heyden & Lewis, 1990; West et al., 2012). The average leaf mass per area (LMA) for 64 Pro- teaceae species is 252 g DM m−2 (Wright et al., 2004). Combining these values from different datasets gives a range for PPUE of 0.02 to 0.14 mmol CO2

Figure 11.11 (Continued) NZeal – New Zealand; SWAus – mesic to semi-arid south western Australia; SWPac – Vanuatu and Fiji; Tas – Tasmania; TropAf – sub-Saharan Africa except for the Cape Floristic Region; TSAm – tropical South America except Brazil. Where a coloured square is placed to the left of a generic name, this indicates that at least one species in that genus has been screened for the presence/absence of proteoid roots and arbuscular mycorrhizas. The colour of the square indicates the following: black – proteoid roots and arbuscular mycorrhizas both absent; red – proteoid roots absent but arbuscular mycorrhizas present; blue – proteoid roots present but arbuscular mycorrhizas absent; yellow – proteoid roots and arbuscular mycorrhizas both present. The character phylogenies of two characters were inferred by parsimony optimisation on this chronogram using the Mesquite software package (Maddison & Maddison, 2011). Inferred ancestral character-state combinations are colour-coded as for genera; grey-coloured lineages indicate equivocal ancestral character-state reconstructions. Metabolic adaptations of the non-mycotrophic Proteaceae 313

Figure 11.11 Chronogram for genera of the Proteaceae and outgroups, modified from Sauquet et al. (2009b) with Dryandra synonymised under Banksia (see Mast & Thiele, 2007) and the recently named genera Lasjia and Nothorites inserted at the positions and timing indicated by Mast et al. (2008). The number in parentheses to the right of each generic name indicates the number of species recognised by the National Herbarium of New South Wales (NSW) in that genus. The abbreviations to the right of the species numbers are codes summarising presence in geographic areas as follows: Asia – mainland South East Asia between Nepal, Sri Lanka, southern Japan and the western margin of New Guinea; Brazil – Brazil; CAm – Central America; CAus – arid Australia; CFRAf – Cape Floristic Region of Africa; SthSAm – mesic temperate South America; EAus – mesic eastern mainland Australia between Spencers Gulf and the southern limit of the Wet Tropics; Mad – Madagascar; MTAus – Australian monsoon tropics; NCal – New Caledonia; NEAus – Australian Wet Tropics; NGuin – New Guinea; 314 Phosphorus Metabolism in Plants

[g leaf P]−1 s−1; the highest value is about half of the lowest value given above for south-western Australia Banksia species. It would appear that, to date, only one report has been made on the localisation of Pi in leaves of Proteaceae species in South Africa, and this shows that Pi is allocated to mesophyll cells in Leucadendron ‘Safari Sunset’ (Figure 11.7b, d; Hawkins et al., 2008). This is very similar to the situation found for south-western Australia species (as discussed above), but different from the general pattern for dicotyledons (Conn & Gilliham, 2010). Furthermore, no reports appear to have been made on delayed greening in Proteaceae from the Cape Region, although anecdotal evidence is available of this phenomenon in some South African species in this region. The seed [P] and P content of the Proteaceae of the Cape Floristic Region are lower than for south-western Australian Proteaceae, at 5.8 versus 13.2 mg P g−1 DM and 0.3 versus 1.4 mg per seed, respectively (Groom & Lamont, 2010). These differences (and the others referred to above) reflect differences in selection pressure, because the soils in the Cape Floristic Region are marginally less infertile than those in south-western Australia (Witkowski & Mitchell, 1987; McArthur, 1991). Nevertheless, all leaf P concentrations of Proteaceae species from South Africa are much lower and the seed P concentrations much higher than global average values (Epstein & Bloom, 2005; Marschner, 1995). South African Proteaceae have long been known to be P-sensitive (Nichols & Beardsell, 1981), and this can pose a problem in floriculture (Hawkins et al., 2007; Hawkins et al., 2008). As with species of Proteaceae elsewhere, P-sensitivity is not a universal phenomenon, and some show a relatively tight control of their Pi-uptake capacity (Shane et al., 2008).

11.3.2 Eastern Australia For the sake of this chapter, the term “eastern Australia” is used for any part of Australia located east of the Nullarbor, including South Australia. Cluster- root functioning in eastern Australian Proteaceae species has been studied in Telopea speciosissima (Grose, 1989), B. integrifolia (Grierson & Attiwill, 1989; Grierson, 1992; Grierson & Comerford, 2000) and H. actites (Schmidt et al., 2003). As detailed comparisons, such as those carried out for a southern South American species (Delgado et al., 2014), have yet to be made, no knowledge is currently available regarding possible subtle differences with south-western Australian species. The average leaf [P] for 11 Proteaceae in eastern Australia is 692 mg P g−1 DW (Westman & Rogers, 1977; Bennett & Attiwill, 1996; Wright et al., 2004). This is two- to threefold higher than values for south-western Australian Pro- teaceae species, despite the fact that most species are from shared genera (Wright et al., 2004; Denton et al., 2007a; M.W. Shane, unpublished data). This may reflect phenotypic variations due to differences in soil P availability, or stronger selection pressures for higher PUE in south-western Australia. The Metabolic adaptations of the non-mycotrophic Proteaceae 315

Table 11.1 Data on nitrogen (N) and phosphorus (P) concentrations and on specific leaf area (SLA) for 395 individual trees at 16 primary rainforest sites intheWet Tropics of north Queensland, eastern Australia. Sites are classified according to soil parent material as either fertile (basalt) or infertile (granites and rhyolites). There were eight sites on the fertile soils and eight sites on the infertile soils. Annual rainfall at all sites exceeds 2500 mm. Twelve species of Proteaceae were present in the sampling plots, with a total of 32 individual trees sampled for these species (P. Reddell, unpublished data).

No. of Leaf N Leaf P SLA species (mg g−1 DW) (mg g−1 DW) (m2 kg−1) Other Prot. Other Prot. Other Prot. Other Prot.

All sites 363 32 17.7 12.1 1.081 0.640 8.59 7.53 Fertile sites 185 15 19.3 13.2 1.444 0.870 9.26 7.39 Infertile sites 178 17 16.0 11.2 0.710 0.431 7.92 7.73

Prot. = Proteaceae. data compiled in Table 11.1 reveal that it is likely a combination of both points. For one common Proteaceae (Cardwellia sublimis) that occurred on both fertile and infertile soils in this sampling, there was a significant difference in leaf P concentration between the soil types: 760 ± 140 μgPg−1 leaf DW (n = 6) on the fertile sites, and 550 ± 60 μgPg−1 leaf DW (n = 5) on the infertile sites, indicating phenotypic variation. However, even the lowest values in Table 11.1 are higher than typical values for south-western Australian Pro- teaceae, so there is also evidence for a difference in selection pressure. Pro- teaceae growing on infertile rainforest soils have significantly lower leaf [P] than those growing on more fertile rainforest soils: 430 versus 870 μgPg−1 leaf DW. This difference is not related to leaf scleromorphy (as measured by specific leaf area (SLA), leaf thickness and leaf toughness; data forSLAare included in Table 11.1). Across both soil types, rainforest Proteaceae consis- tently have much lower average leaf P concentrations (∼40% less) than the average found in non-Proteaceae species representing a wide range of plant families that were growing on these same sites. On fertile sites this lower leaf P concentration in Proteaceae compared with other species can only partly be accounted for by higher levels of scleromorphy in the leaves of Proteaceae, as assessed using SLA, but this is not the case when comparing species on infertile sites (Table 11.1). These data indicate that Proteaceae species from these sites are less efficient than their south-western Australian counterparts, but more efficient than co-occurring non-Proteaceae species from a range of different families. Some Proteaceae from eastern Australia, such as Lomatia silaifolia, Persoonia mollis,andMacadamia tetraphylla, show delayed greening (Figure 11.10d). At present, it appears that only one report exists of seed [P] in Banksia species from eastern Australia, showing an average of 11.8 mg P g−1 DW for two species (Grundon, 1972), which is very similar to the average 316 Phosphorus Metabolism in Plants for 11 Banksia species from south-western Australia (11.0 mg P g−1 DW; Groom & Lamont, 2010). For H. gibbosa, Grundon (1972) found a seed [P] of 15.2 mg P g−1 DW, while Groom & Lamont (2010) presented an average seed [P] for 13 Hakea species from south-western Australia of 16.7 mg P g−1 DW, again remarkably similar. However, this comparison was biased, as it compared highly species-rich genera in south-western Australia (Mast & Thiele, 2007; Speck, 1958) with the same genera in eastern Australia, rather than also including other genera that are more closely related to those in southern South America and Brazil. One such species is Macadamia integrifolia,sisterto Brabejum (Cape Region in South Africa) and Panopsis (which is widespread in tropical South and Central America) (Figure 11.11; Weston & Barker, 2006), which has a seed [P] of 1.9 mg g−1 (Thomas & Gordon, 1977, as cited in Groom & Lamont, 2010). However, as this species produces large edible nuts this value may also be biased, and further information on seed [P] is warranted. Phosphorus toxicity is quite common among eastern Australian Proteaceae species, such as Telopea speciosissima (Grose, 1989), B. serrata (Groves & Keraitis, 1976), B. ericifolia (Parks et al., 2007), but is by no means universal (Nichols & Beardsell, 1981; Handreck, 1991).

11.3.3 Southern South America All six Proteaceae species from southern South America belong to clades that are thought to have arrived on the continent during the break-up of Gond- wana; Lomatia and Orites are genera that occur both in southern South Amer- ica and in eastern Australia, the southern South American Embothrium and Gevuina are closely related to the eastern Australian genera Telopea and Hicks- beachia/Bleasdalea, respectively (Figure 11.11) (Prance & Plana, 1998; Weston & Barker, 2006; Barker et al., 2007; Mast et al., 2008; Carpenter, 2012). Cluster roots have been found on all six southern South American Proteaceae species, but are produced lower in the soil profile than is common for south-western Australian species (Lambers et al., 2012a). Their functioning has been studied in detail for Embothrium coccineum (Zu´ niga-Feest˜ et al., 2010; Delgado et al., 2013). There are many similarities with what is known for south-western Australian species, but also a significant difference. Compared with H. prostrata, E. coccineum produces far fewer cluster roots, but these live longer and release more citrate per unit cluster-root weight (Delgado et al., 2014). This difference in cluster-root functioning is related to the concentration of total soil P, which is about 100-fold higher in the southern South American soils inhab- ited by Proteaceae. However, the concentration of plant-available P is very similar in both habitats, thus offering an explanation for why the cluster-root strategy is used in the southern South American soils (Lambers et al., 2012a). Releasing more carboxylates in a low-P sandy soil cannot further enhance P availability, whereas in a young volcanic soil with a high total P concentration, the release of more carboxylates can mobilise far more P (Delgado et al., 2014). Given that the southern South American species are phylogenetically Metabolic adaptations of the non-mycotrophic Proteaceae 317 very close to eastern Australian species, and since both function at higher leaf [P] than south-western Australian species, further studies of cluster-root functioning of some eastern Australian species (e.g., Lomatia, Orites, Telopea and Hicksbeachia) are warranted. Leaf [P] of the southern South American E. coccineum is about threefold higher than that of south-western Australian species (Lambers et al., 2012a). No data are currently available on PPUE for southern South American Pro- teaceae, but given the information on leaf [P] and gas exchange, PPUE is expected to be quite low, compared with values for south-western Australian species; this is confirmed by recent data on E. coccineum referred to by Lam- bers et al. (2012a). The P-remobilisation efficiency of this species is only about 14% (Lambers et al., 2012a) and Lomatia hirsuta also shows virtually the same [P] in green leaves as in senesced leaves (Diehl et al., 2008). Leaf longevity for four southern South American Proteaceae species ranges from 8.4 months for E. coccineum, grown at a high light intensity, to 65 months for Gevuina avellana, grown at a low light intensity (average 33.2 months) (Lusk & Corcuera, 2011). The leaves of E. coccineum live for a relatively short period, but the average leaf longevity of the southern South American species is remarkably similar to that of south-western Australian species. Delayed greening has not been reported for southern South American species, and has not been noted by the authors of the present chapter, who are familiar with the habit of most of the southern South American Proteaceae species. The average seed [P] of the six southern South American Proteaceae species is 3.2 mg P g−1 DM (Delgado et al., 2014), less than for the species in the Cape Region, and much less than for the south-western Australian species. This is in line with the higher soil P status of the southern South American soils. Southern South American Proteaceae species do not appear to be particularly sensitive to elevated Pi concentrations in their root environment, provided that the Pi supply is not enhanced too suddenly (Delgado et al., 2014; Zu´ niga-Feest˜ et al., 2010).

11.3.4 Brazil Large cluster roots are produced by Euplassa legalis (P. Costa, unpublished data), which occurs naturally in the Atlantic rainforest of Brazil and is taxonomically close to the southern South American Gevuina and the eastern Australian Hicksbeachia and Bleasdalea (Figure 11.11) (Weston & Barker, 2006). Cluster roots have also been found in Roupala rhombifolia and R. montana, albeit in very low numbers (A. Abrahao˜ & M. de Campos, pers. obs.); these species occur in the cerrado and its genus is sister to Neorites, a monospecific genus that occurs in the Wet Tropics of north-eastern Australia (Figure 11.11) (Weston & Barker, 2006). The leaf [Mn] of R. montana is high (156 μgMng−1 leaf DW), which suggests that this species uses a carboxylate-releasing P- mobilising strategy (de Campos, 2012). 318 Phosphorus Metabolism in Plants

The leaf [P] of the cerrado taxon R. montana var. montana,measuredin its natural habitat, is 0.43–0.51 mg g−1 DW in mature leaves (Amaury de Medeiros & Haridasan, 1985; Nardoto et al., 2006). This is about twice that in leaves of south-western Australian species, but less than in eastern Aus- tralian species. The light-saturated rate of photosynthesis of R. montana var. −2 −1 montana is 14 μmol CO2 m s , which is less than that of south-western Australian species that have lower leaf [P]; if a similar leaf mass per unit leaf area is assumed, the PPUE is about threefold less, similar to global average values (Wright et al., 2004; Lambers et al., 2010;). Phosphorus remobilisation from senescing leaves is about 60% (Nardoto et al., 2006), and similar to the global average value (Vergutz et al., 2012). To the present authors’ knowledge, no data are presently available for other Brazilian species. Mature individuals of both R. montana var. montana and R. montana var. brasiliensis do not exhibit delayed greening in their natural habitat, but seedlings and saplings of R. montana var. montana do show this phenomenon (H. Lambers & R.S. Oliveira, pers. obs.). Currently, no information is available on the delayed greening or seed [P] of other Proteaceae species in Brazil. Proteaceae are poorly represented in cerrado (Felfili & Da Silva, 1993), although the soil fertility in the landscape is relatively low: total P concentration in topsoils range from 301 to 456 mg kg−1 (Chapuis-Lardy et al., 2001) with plant-available soil P being about 4 mg kg−1 (Cruz Ruggiero et al., 2002; Amorim & Batalha, 2007). However, sites with very low soil P concentrations (<0.1 mg kg−1) have also been reported (Haridasan, 2008). Whilst cerrado soils have low P concentrations on a global scale (Marques et al., 2004), the values are considerably higher than those in soils in south-western Aus- tralia, where species of Proteaceae occur in large numbers (Lambers et al., 2006; Laliberte´ et al., 2012; Hayes et al., 2014). It is likely that the ancestral cerrado Proteaceae, which are taxonomically closer to eastern Australian taxa than to their south-western Australian counterparts, lacked some of the P- efficiency traits of the south-western Australian species, precluding them from diversification in cerrado vegetation. There is currently no evidence available of the P sensitivity of Brazilian Proteaceae.

11.4 Perspectives

The information compiled in this chapter shows that there are at least six key traits related to P economy that appear to mediate the remarkable ability of the south-western Australian Proteaceae to thrive on severely P-impoverished soils: (i) specialised, non-mycorrhizal cluster roots that ‘mine’ P; (ii) a reduction in P investment and slower protein synthesis and growth in young leaves due to very low ribosome abundance, and the maintenance of normal levels of phosphorylated metabolites and ribosomes for protein turnover in mature leaves, leading to a high PPUE; (iii) the Metabolic adaptations of the non-mycotrophic Proteaceae 319 replacement of phospholipids by galactolipids and sulfolipids, also leading to a high PPUE; (iv) a preferential allocation of Pi to mesophyll cells, rather than to epidermal cells, leading to a further increase in PPUE; (v) a high P- remobilisation efficiency of senescing leaves and roots; (vi) a high seed [P] that will support seedling establishment in P-impoverished soils. A delayed greening of leaves is exhibited by many south-western Australian species, but this is not a universal P-efficiency trait. Extended leaf longevity will contribute more significantly to long-term PUE than will a delayed greening strategy. Species from the Cape Floristic Region of South Africa, most of which are closely related to the predominantly south-western Australian genus Adenan- thos, share the P-efficient traits of the south-western Australian Proteaceae as far as they have been studied, but they have not evolved to quite the same extent. Species in southern South America which are more closely related to eastern Australian Proteaceae have effective cluster roots, which function in a different manner in E. coccineum; this reflects the higher soil P status but similar P availability in its natural habitat compared with that of south-western Australian Proteaceae (Delgado et al., 2014). The southern South American species lack at least some of the other P-efficiency traits of Proteaceae of south-western Australia and South Africa (Lambers et al., 2012a). Based on the currently limited information brought together in this chapter, it would appear that eastern Australian and Brazilian species function somewhere between the species from south-western Australia and those in southern South America, possibly reflecting the intermediate soil P concentration in Brazil and eastern Australia. Until now, studies of Proteaceae that are adapted to grow on P- impoverished soils have provided a good description of P-economy traits that allow them to acquire Pi from the soil and use it very efficiently in photosynthesis and growth. However, it remains unclear as to what genetic and molecular mechanisms underlie these adaptive traits, and how these traits evolved. With the advent of next-generation sequencing (NGS), new perspectives are opening up for a deeper analysis of non-model species. On the one hand, it will be possible to use NGS to obtain genome sequences for selected Proteaceae, including groups of phylogenetically related plants which contain species that are adapted, and species that are not adapted, to severely P-impoverished soils. This may reveal changes in genes or gene vari- ants that correlate with the development of traits that support survival on P- impoverished soils. Given that these traits are present in Banksia and Hakea species, it will be possible to obtain at least two – and probably more – lineages to search for conserved features. Geographical clines such as those in south-western Australia, between the coast where the soils are younger and contain more P, and those at least 10 km inland which are older and severely P-impoverished, may provide a good location to identify such sets of species. In parallel, it should be possible to use a wider spectrum of functional ‘omics’ tools (e.g. as discussed in Chapters 3 and 8) to probe the responses 320 Phosphorus Metabolism in Plants and underlying signalling networks more deeply. The ability to make stable transformants (genetically manipulated to knockout the expression of specific genes/enzymes, or to overexpress them) of plants suchas H. prostrata would be very valuable, and this has already been achieved with L. albus (Uhde-Stone et al., 2005). Whilst technical problems still need to be solved to allow the application of proteomics and metabolomics to the intransi- gent leaf extracts of these species, large-scale transcriptomics analyses using NGS should be possible. The interpretation of such studies will be supported by parallel genome sequencing, as well as emerging tools for the assembly and the establishment of a gene annotation and gene ontology for newly sequenced species (Lohse et al., 2012; Lohse et al., 2014). Questions that can be approached will include a more systematic cataloguing of the differing response of crop plants and these Proteaceae species to P supply. With regards to understanding the underlying signalling mechanisms, such data will provide insights into whether canonical P-signalling pathways, including the PHR1 signalling network and the mir399/PHO2 sub-network from Arabidop- sis (Chapter 2), are conserved or modified in Proteaceae. Focused studies of the regulation of ribosome biogenesis and chloroplast biogenesis by developmental programmes and the Pi supply, and of the mechanisms that determine to which cell types Pi is preferentially allocated, are also needed. It will be important to learn how the distribution of Pi is regulated between the maintenance of [Pi] in mature organs, in order to maximise their longevity, and allocation to growth of new leaves and roots. For all these studies, the inclusion of related Proteaceae species that are not adapted to severely P-impoverished soils might provide a better – and certainly complementary – control than Arabidopsis or crop species. One of the key characteristics of Proteaceae that are adapted to P- impoverished soils is that even moderate levels of readily available Pi in their growth media may be toxic. As discussed above, this is because these species do not have the ability to exert strong feedback inhibition on Pi uptake. But, is this due to negative mutations that have accumulated in the absence of any counterselection during millions of years of evolution in very-low P conditions? Or is it because this trait is closely linked with the molecular and genetic mechanisms that allow these Proteaceae species to maximise Pi uptake and Pi-resorption efficiency? Detailed temporal studies of changes in transcripts, proteins (including their post-translational mod- ifications), metabolic fluxes, metabolism, and growth after the addition of subtoxic and toxic levels of Pi in P-sensitive and closely related P-insensitive species could provide insights into differences in the metabolic, molecular and signalling response to P. Questions also arise with respect to the interaction between P-economy traits and other aspects of plant growth and metabolism. One set of questions revolves around the energetic costs of cluster roots. What are the construction costs and the running costs (i.e. the costs of synthesising and exuding citrate)? How much P is acquired by a ‘typical’ cluster root? How much carbon gain Metabolic adaptations of the non-mycotrophic Proteaceae 321 will be supported by this P when it is invested in photosynthesis and dark respiration, and hence plant growth? Knowledge about the P availability of soils in the field, published studies of the composition, exudation rates and respiration rates of cluster roots, photosynthesis rates and leaf longevity, together with the analysis of Pi allocation to different cellular components described in this chapter provide a starting point for quantitative models that describe the carbon cost of each molecule of Pi that is acquired by a plant, and the carbon gain that a molecule of Pi will support in the following season and years. However, important questions that need to be pursued to further parame- terise such models include learning how roots sense elevated soil Pi levels where they preferentially locate cluster roots, and learning more about the costs of maintenance and turnover, and the associated respiratory costs. Another set of questions revolves around the interaction between Pi uptake and metabolism and N and S uptake and metabolism. One of the key adaptations in the low-P adapted Proteaceae is the low ribosome abundance in young leaves which will lead to slow rates of protein synthesis and a low demand for N and S. Soil N is also low in the areas where these plants are grown (Lambers et al., 2010). However, whilst both young and mature leaves of investigated south-western Australian Proteaceae are characterised by low protein concentrations on a fresh weight basis, the concentrations of total free amino acids were only marginally lower (Sulpice et al., 2014). Questions arise as to whether there is a tight control of N and S uptake and metabolism when P is limiting, as is found for other species (Rufty et al., 1993; de Magalhaes˜ et al., 1998; Gniazdowska & Rychter, 2000), and also how the allocation of N and S is regulated between the maintenance of protein levels in mature leaves, allocation to growth in young leaves, and the production of protein- rich cluster roots. Another general question is how carbon, N, S and P interact to regulate ribosome biogenesis and ribosome degradation and, thus, ribosome abundance. A further set of questions revolves around an emerging interaction between sucrose transport and P signalling in Arabidopsis, and whether a similar interplay occurs in Proteaceae that are adapted to severely P- impoverished soils. Briefly, there is growing evidence that long-distance sucrose transport plays an important role in P responses (Hammond & White, 2008; Hammond & White, 2011). In L. albus, sugars play a role in cluster-root formation (Cheng et al., 2011). The Arabidopsis pho3 mutation, which was identified in a screen based on reduced acid phosphatase activity during Plimita- tion, was ultimately identified as a mutant of the phloem sucrose/protein cotransporter AtSUC2 (Lloyd & Zakhleniuk, 2004). Further, P-deficiency responses in roots can be decreased by either a reduction in photosynthesis or by stem girdling, which blocks phloem transport (Liu et al., 2005; Karthikeyan et al., 2007). In addition, it was shown recently that ubiquitous sucrose transporter overexpression (Lei et al., 2011) and the directed overexpression of three phloem sucrose transporters (AtSUC2, AtSUC1, ZmSUT1)areunderthe control of a phloem companion cell-specific promoter (Dasgupta et al., 2014), 322 Phosphorus Metabolism in Plants and led to the induction of P-deficiency responses in the roots as well asan inhibition of growth that can be reversed by increasing the Pi supply to the root. Unlike nitrate, ammonium and sulfate, Pi is not assimilated via a dedi- cated and highly regulated assimilation pathway. Instead, the vast bulk of the Pi that enters metabolism does so via the mitochondrial ATP synthase (oxida- tive phosphorylation) or, when leaves are exposed to light, via thylakoid ATP synthase (photophosphorylation). The Pi moiety is transferred from ATP to phosphorylated intermediates of central metabolism which in turn are used to synthesise nucleotides, phospholipids and other P-containing metabolites. As the ATP synthases are central pathways for cellular energy metabolism, they can hardly be down-regulated to control the flux of Pi into metabolism. It is possible that the use of Pi is instead regulated via changes in the availability of other resources, for example via the transport of sucrose. Interesting analo- gies have been noted to the way in which a low ribosome abundance regulates and restricts protein synthesis and, thence, the downstream demand for Pi in the south-western Australian Proteaceae that are adapted to grow on severely P-impoverished soils. It will be interesting to learn if Pi availability, use and signalling interact with the transport of sucrose and hence carbon allocation in Proteaceae species, and if this interaction underlies some of the P-economy traits or the sensitivity of these species to P toxicity. Seedling establishment plays a key role in the success of a species. In the fire-prone environment of south-western Australia, seedling recruitment is largely restricted to the winter following a fire (Miller & Dixon, 2014). As noted above, the seeds of Proteaceae contain a high P content, and interesting questions have arisen with respect to the use of this P during germination: whether it is all immediately used for seedling growth, especially root growth to access water; or whether part of it is actually stored for use at a later stage in the life history. Which of the six P-efficiency traits identified in Proteaceae would be desirable in crops plants? These might include understanding how these Pro- teaceae achieve an extensive substitution of phospholipids by galactolipids and sulfolipids, and how surplus Pi can be directed to the vacuole of mesophyll cells, rather than epidermal cells. On the other hand, the decrease in growth rate that is an unavoidable consequence of a low rRNA abundance in young leaves may be problematic, unless it is possible to tightly couple rRNA abundance with P availability. If low ribosome abundance were restricted to fully expanded leaves, and not expressed in expanding leaves, it might actually be a highly desirable trait in some crop species. Delayed greening likely carries a negative trade-off in annual crop plants, because it will decrease carbon-assimilation rates in expanding leaves. Likewise, a high seed [P] will obviously decrease yield in low-P cultivation regimes and, more importantly, render iron and zinc less available for human and animal con- sumption (Welch & Graham, 2004). The value of cluster roots in an agricultural context depends heavily on soil conditions, as they would presumably only be more effective than mycorrhizas in severely P-impoverished soils and Metabolic adaptations of the non-mycotrophic Proteaceae 323 in soils with high amounts of total P, but with low availability, due to the low soil pH and higher levels of Fe and Al (Lambers et al., 2012a; Lambers et al., 2013b; Delgado et al., 2014). Which traits should be taken into account in the context of ecological restoration? It has been shown that the P-sensitivity of many Proteaceae deserves attention, but even when Pi is supplied at non-toxic levels, the slow growth and low competitive ability of many Proteaceae may lead to their exclusion (Heddle & Specht, 1975; Lambers et al., 2013a). Sources of Pi include not only the common causes of eutrophication, but also the spraying of phosphite (Chapters 1 and 2) to slow down the spread of the plant pathogen Phy- tophthora cinnamomi (Lambers et al., 2013a). It is therefore pivotal to discover less-harmful alternatives for phosphite. The non-mycorrhizal carboxylate-releasing P-acquisition traits of Pro- teaceae are likely also to be important in biodiversity hotspots for promoting coexistence through facilitation (Muler et al., 2014). Likewise, this trait could be exploited to achieve overyielding in the context of intercropping in agriculture (Li et al., 2014). In summary, the studies of highly P-efficient Proteaceae have led to new discoveries of P-efficiency traits that would never have been revealed if plant scientists had persisted with the usual crop or model species. Yet, these studies have allowed the exploration of similar efficiency traits in crop species, such as Triticum aestivum (wheat) (Aziz et al., 2014). The six P-efficiency traits of Proteaceae have shown what some plant species have evolved, and it is now the task of research groups worldwide to explore which of these traits are undesirable in crops, and which are likely to lead to more P-efficient crop cultivars that can be used in an era when P will becomea much more expensive and less-available resource.

Acknowledgements

These studies were supported by the Australian Research Council (ARC), with Discovery Projects (DP0209245, DP0985685, DP110101120, DP130100005) to H.L. (and P.L.C. for DP130100005), an ARC Australian Research Fellowship to M.W.S. (DP1092856), to E.L. via an ARC DECRA (DE120100352), and to M.S. by the Max Planck Society and the European Union (collaborative project TiMet under contract no. 245143).

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