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South-western Australia is an ancient region known for its severely nutrient-impoverished soils (McArthur, 1991; Lambers et al., 2012) and exceptionally high plant biodiversity (Hopper, 2009). Among the macronutrients, phosphorus (P) is the least available nutrient in this region, as a consequence of prolonged soil weathering (Lambers et al., 2010; Laliberté et al., 2012). Sulfur is one of the few macronutrients that is found at concentrations similar to that considered adequate for growth of crop plants in Banksia (Proteaceae) species in this region (Denton et al., 2007). On the most severely P-impoverished soils, nonmycorrhizal Proteaceae are an important component of the vegetation (Pate & Bell, 1999). Under low-P conditions, plant species in this family typically form cluster roots that effectively ‘mine’ P by releasing large amounts of low-molecular-weight carboxylates (Lambers et al., 2008).
P-starved leaves tend to have low rates of photosynthesis per unit leaf area, at least in crop plants (Brooks et al., 1988; Rao & Terry, 1989; Fredeen et al., 1990). Leaves of Proteaceae species from south-western Australia, however, exhibit relatively fast rates of photosynthesis, despite having extremely low leaf P concentrations ([P]) (Denton et al., 2007). Consequently, some of these species exhibit a very high photosynthetic P-use-efficiency (PPUE) (Denton et al., 2007; Lambers et al., 2010). In view of dwindling phosphate rock reserves and increasing prices of P fertilizers (Gilbert, 2009), understanding the biochemical basis of this high PPUE would allow us to explore whether there are lessons for developing P-efficient crops (Lambers et al., 2011).
In barley (Hordeum vulgare) grown in nutrient solution at a growth-limiting P supply, the major P fractions in leaves are nucleic acids (30%), free orthophosphate (26%), P-containing metabolites (26%) and phospholipids (17%) (Chapin & Bieleski, 1982). Phospholipids are a component of the plasmalemma and of tonoplast, chloroplast, and mitochondrial membranes (Härtel et al., 2000; Andersson et al., 2003; Jouhet et al., 2004; Andersson et al., 2005). Phospholipids also play a role in signalling during plant development and in plant responses to stress (Cowan, 2006). Therefore, when considering changes in P distribution that could affect PPUE in mature leaves, changes in the concentrations of orthophosphate, P-containing metabolites and nucleic acids and membrane lipid composition are the most likely candidates (Veneklaas et al., 2012).
There is good evidence that rapid rates of photosynthesis require a fine balance between the concentrations of free phosphate and phosphorylated intermediates, and that photosynthesis is inhibited when free phosphate is depleted (Heldt et al., 1977; Stitt & Quick, 1989; see Stitt et al., 2010 for a recent review). The total concentration of P, adenine nucleotides and phosphorylated intermediates is constrained by the amount of phosphate in the cytoplasm. While there is evidence that shortage of phosphate in the cytosol and chloroplast can lead to remobilization of phosphate from the vacuole (Sharkey et al., 1986; Mimura, 1995), little is known about how this process is regulated. Eudicots tend to accumulate orthophosphate in epidermal cells (Conn & Gilliham, 2010); however, Hakea prostrata R. Br. (Proteaceae) accumulates P in its mesophyll cells (Shane et al., 2004). The accumulation of P in mesophyll cells may allow more efficient use of P for photosynthesis, which occurs in the mesophyll cells. Except for some studies indicating that enzyme concentrations of UDP-glucose pyrophosphorylase may increase in P-deficient plants (Ciereszko et al., 2001), little is known about how photosynthesis can be optimized to maintain flux when the total amount of P available for intermediary metabolism is decreased.
In a recent paper (Lambers et al., 2011), we hypothesized that a high PPUE might be partly attributable to a replacement of phospholipids by galactolipids or sulfolipids, which do not contain P. Upon P starvation of Arabidopsis thaliana plants, the phospholipid fraction in leaves declines from 36 to 19% (Dörmann & Benning, 2002) with a concomitant increase of galactolipids and sulfolipids. In P-replete plants, the thylakoid and the inner envelope membrane already contain quite high galactolipid concentrations, but other cellular membranes contain mainly phospholipids. During P-starvation, galactolipids are substituted for phospholipids in these extrachloroplastidic membranes (Härtel et al., 2000; Dörmann, 2007). The replacement of phospholipids by other lipids in several membranes in response to P starvation is a dynamic and reversible process (Andersson et al., 2003; Cruz-Ramírez et al., 2006; Gaude et al., 2008) and is seen in many plant species, including barley, oats (Avena sativa) and maize (Zea mays) (Tjellström et al., 2008). However, replacement of phospholipids by other lipids, while preventing leaf death under severe P limitation, might inexorably lead to a decline in the rate of photosynthesis (Brooks et al., 1988; Rao & Terry, 1989; Fredeen et al., 1990).
Here we test the hypothesis that mature leaves of Proteaceae that occur naturally on severely P-impoverished soils and exhibit a very high PPUE (Denton et al., 2007) invest relatively little P in phospholipids and predominantly use galactolipids and sulfolipids instead. We chose to test this hypothesis in a location that is well known for its high plant biodiversity (particularly Proteaceae) and its ancient, nutrient-impoverished soils, Lesueur National Park in south-western Australia (Hopper & Gioia, 2004) (Fig. 1). We compare the results on relative lipid composition in six Proteaceae species with those obtained on the model plant Arabidopsis thaliana, grown under both P-sufficient and P-starved conditions. This allows a comparison of the response of Proteaceae species from severely P-impoverished soils with that of a species commonly found in a relatively nutrient-rich habitat.
Figure 1. Location of (a) Lesueur National Park in Western Australia and (b) the sites in Lesueur National Park where three Banksia and three Hakea species were sampled.
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Soils in the natural habitat of six Proteaceae were severely P-impoverished, mature leaf [P] was very low, and PPUE was high, as previous studies had shown (Denton et al., 2007; Lambers et al., 2010). Most importantly, our results show, for the first time, that these Proteaceae species extensively replace phospholipids in mature leaves with lipids that do not contain P (i.e. galactolipids and sulfolipids), thus demonstrating that savings can be made in this P pool to a previously unknown extent, and offering a partial molecular explanation for their extremely high PPUE.
Total leaf [P]
As observed for south-western Australian Banksia and Hakea species (Wright et al., 2004; Shane & Lambers, 2005; Denton et al., 2007), mature leaf [P] was very low, without any visual signs of P deficiency. Interestingly, [P] of expanding leaves was about twice as high as that in mature leaves. This is partly accounted for by an increase in sclerenchymatic tissue that is associated with increasing leaf toughness during leaf development; that is, by ‘dilution’. However, there was probably also a change in chemical composition during leaf development, when the prominently present brownish or reddish pigments in soft expanding leaves disappeared. These pigments may have been metabolized or they could be masked by the increase in chlorophyll concentration. This change in colour and the gradual build-up of photosynthetic capacity are similar to the phenomenon of ‘delayed greening’ in rainforest plants (Kursar & Coley, 1992).
The decline in leaf [P] following full leaf expansion agrees with the ‘growth rate hypothesis’ (Elser et al., 2003). The demand for ribosomal RNA, and thus for P, would be relatively high during leaf expansion when rates of protein synthesis are higher. In contrast, in mature leaves, ribosomal RNA is required only to sustain protein turnover and hence investment in this fraction can be less (Lambers et al., 2010; Veneklaas et al., 2012). This aspect clearly requires further investigation.
Rates of photosynthesis were relatively high, with values for Banksia species being typically higher than those for Hakea species. As shown in Fig. 6, B. menziesii has sunken stomata as typically found for thick-leaved Banksia species; the depth of the stomatal crypt is closely correlated with the thickness of the leaf (Hassiotou et al., 2009). Conversely, the thick leaves of H. prostrata do not have stomatal crypts (Fig. 6). Unlike Banksia species, which commonly have several stomata located in crypts, Hakea species only occasionally have sunken stomata in shallow individual crypts (Groom et al., 1997; Jordan et al., 2008). The absence of deep stomatal crypts with multiple stomata leads to a longer path for CO2 diffusion from the air to the chloroplasts (Roth-Nebelsick et al., 2009). The lack of deep stomatal crypts possibly accounts for the lower rates of photosynthesis in Hakea species compared with those in Banksia (Fig. 5).
Given the relatively high rates of photosynthesis and very low mature leaf [P], PPUE values would be high (Lambers et al., 2010, 2011). We estimated PPUE by combining data on photosynthesis per unit leaf area (Fig. 5), data on leaf [P] per unit leaf dry weight, obtained for similar leaves of nearby plants (Fig. 4), and values for leaf area per unit dry weight, also for similar leaves of nearby plants. Although PPUE should ideally be estimated for the same leaves, the average estimated PPUE value for the six Proteaceae species was 305 μmol CO2 g−1 P s−1, with values as high as 488 μmol CO2 g−1 P s−1 for B. attenuata, the lowest being 169 μmol CO2 g−1 P s−1 for H. prostrata. These rates of photosynthesis expressed per unit leaf P are remarkably high, as found before for Proteaceae from south-western Australia (Lambers et al., 2010). Global average values for PPUE, as determined under field conditions, are 103 μmol CO2 g−1 P s−1 (Wright et al., 2004). Values for PPUE vary by an order of magnitude at any value for leaf mass per unit leaf area (LMA), and this correlated with variation in leaf N concentration (Reich et al., 2009). Mean values for PPUE are 59 mol CO2 g−1 s−1 for leaves with N : P < 15, whereas PPUE is 129 mol CO2 g−1 s−1 for leaves with N : P > 15 (Wright et al., 2004), but the biochemical basis of the N-linked difference in PPUE remains unclear.
The extensive replacement of phospholipids by galactolipids and sulfolipids offers a partial explanation for the high PPUE values of the present Proteaceae. However, as phospholipids represent c. 20% of all P in leaves of plants grown at a limiting P supply (Chapin & Bieleski, 1982; Poirier et al., 1991), additional factors must play an important role as well (Veneklaas et al., 2012). Preferential allocation of orthophosphate to mesophyll cells in H. prostrata (Proteaceae) (Shane et al., 2004), close to where photosynthesis occurs, may also contribute to a high PPUE. This allocation pattern differs from what is generally found in eudicots, which tend to accumulate orthophosphate in epidermal cells (Conn & Gilliham, 2010). The significance of both the phosphorylated intermediates and the ribosomal RNA fraction (Lambers et al., 2010; Veneklaas et al., 2012) is currently being studied.
During leaf development, the fraction of phospholipids declined three- to five-fold (dependent on the Proteaceae species), and phospholipids were replaced to a major extent by galactolipids and to a lesser extent by sulfolipids. Replacement of phospholipids by galactolipids or sulfolipids is also considered a hallmark of P starvation (Tjellström et al., 2008) and has been described for a range of species upon P starvation, including A. thaliana, barley, oats and maize (Dörmann & Benning, 2002; Tjellström et al., 2008). The transcription of genes involved in the synthesis of galactolipids and sulfolipids is up-regulated rapidly under P starvation in leaves of A. thaliana (e.g. Hammond et al., 2003; Morcuende et al., 2007) and other plant species (e.g. Hammond et al., 2011). The three- to four-fold decline of the phospholipid fraction during leaf development, for example, 56.2–9.6% in B. attenuata (Fig. 6), is much greater than what is observed in the comparison of young and mature leaves of P-stressed A. thaliana, where the phospholipid fraction declined by less than two-fold, from c. 59 to 41% (Fig. 9). Moreover, the decline of phospholipids observed in the comparison of mature leaves from P-sufficient and P-starved A. thaliana plants is much smaller: c. 63 to 41% (Fig. 9) or 36–19% (Dörmann & Benning, 2002). Also remarkable is that the replacement in the present six Proteaceae species occurred without any signs of P deficiency of the leaves and while maintaining high photosynthetic activities (Fig. 5), whereas rates of photosynthesis decrease dramatically in barley leaves when plants are grown with a limiting P supply (Foyer & Spencer, 1986). In fact, increasing the P supply to H. prostrata grown in a natural soil collected from its native habitat to that commonly used for crop plants only marginally increases rates of photosynthesis of glasshouse-grown plants, and markedly reduces it when the P supply is increased further, when P-toxicity symptoms develop (Shane & Lambers, 2005).
Why would phospholipids be a major component in expanding leaves and then be replaced or diluted by other lipids at a later developmental stage? Phosphorus deficiency causes phospholipid replacement in membranes in a range of species (Härtel et al., 2000; Dörmann & Benning, 2002; Dörmann, 2007; Tjellström et al., 2008). Considering the very low rates of photosynthesis in young, expanding leaves compared with mature ones, this shift may reflect increased investment in chloroplast membranes (Forde & Steer, 1976). Galactolipids are a major and phospholipids only a minor component of chloroplast membranes (Bahl et al., 1976; Dörmann, 2007). Increased investment of galactolipids and sulfolipids in chloroplast membranes of fully expanded leaves cannot entirely explain the shift we observed. During development, organelles other than chloroplasts are actively built up, and in their membranes phospholipid must have been replaced by other lipids as a result of P shortage. It is likely that Proteaceae have adapted to this situation, and that membrane perturbation deriving from phospholipid replacement is minimized in a manner that deserves further investigation. In addition, phospholipids play a role in signalling during plant development and this may require greater investment in phospholipids during leaf expansion (Cowan, 2006); however, it is not clear if that signalling component is quantitatively important. The plasma membrane leaflet facing the apoplast (probably the major water permeability barrier) contains only trace amounts of galactolipids (Tjellström et al., 2010). Phospholipids possibly play a vital role in the plasma membrane and tonoplast when they require a high degree of lipid order, during leaf expansion. This aspect deserves further study, if we wish to exploit this trait linked to a high PPUE in P-efficient crop plants.
In south-western Australia, Proteaceae are very successful at growing on the world's most P-impoverished soils. They exhibit very low mature leaf [P] and very high PPUE. While the lipid fraction of young, expanding leaves of the studied species, on average, contains 46.0% phospholipids, mature leaves show as little as 9.6% phospholipids. This shift is much greater than what is known for other species and we clearly showed that it is not simply attributable to dilution by other lipids during normal leaf development. The reduction in the phospholipid fraction from young to mature leaves indicates that these Proteaceae species extensively replace phospholipids with nonphospholipids during leaf development. This coincides with relatively high rates of photosynthesis and no signs of P deficiency of mature leaves. This P investment pattern offers a partial explanation for the high PPUE of the investigated species. Further research is warranted to explore whether this mechanism to increase PPUE is worth applying in future crop plants, in view of dwindling rock phosphate reserves and increasing P-fertilizer prices (Gilbert, 2009).