Phosphorus (P) is essential for plant growth and function and is often present in soils at concentrations that are deficient for plant growth. Not only is P important for driving plant productivity and plant diversity in natural systems (Lambers et al. 2015; Turner et al. 2018; Wassen et al. 2005; Zemunik et al, 2015), it is often a limiting nutrient for productivity in many managed agricultural systems. This is particularly true for the ancient and highly weathered soils in Australia, which are among the most naturally P-deficient in the world. The adaptation of plants to soils with low levels of plant-available P has been studied extensively across a wide range of ecosystems. Indeed, research conducted in the Lambers’ laboratory at the University of Western Australia has been instrumental in helping to understand how native plants and crops adapt and respond to P-limited environments, and especially the role of root exudates in mobilizing soil P (Lambers et al. 2002; Lambers et al. 2008; Lambers et al. 2011; Lambers et al. 2015). The pivotal articles and reviews published in Plant and Soil, including many by Lambers and his team, have revealed much about the chemistry of soil P (McLaughlin et al. 2011; Weaver and Wong 2011) and led to potential strategies for managing fertilizer use in agriculture (Richardson et al. 2011a; Simpson et al. 2011; McIvor et al. 2011).
In addition to the physiological and morphological changes to roots (Lynch and Brown 2001; Richardson et al. 2009), P deficiency is often associated with an increased release of organic anions (also referred to as carboxylates) from roots into the rhizosphere. Organic anions increase the availability of P in soil for plant uptake, especially under conditions of P deficiency. The increased rates of organic anion release in response to P deficiency have been described in many native Australian plant species that develop cluster roots (e.g., the Proteacea: Roelofs et al. 2001; Lambers et al. 2002; Lambers et al. 2015) as well as in a wide range of agricultural species with and without cluster roots, including pasture legumes and grasses, grain legumes and cereals (Nuruzzaman et al. 2006; Pearse et al. 2006; Wang et al. 2013; Wouterlood et al. 2004; Kidd et al. 2018). The release of citrate and malate from the specialized cluster roots of white lupin (Lupinus albus L.) has been studied in greatest detail (e.g., Veneklass et al. 2003; Shane et al. 2008; Wang et al, 2013). In this species, citrate concentrations of 5 to 50 µmol g−1 soil (corresponding to expected soil solution concentrations of 1 to 10 mM) have been reported around the cluster roots (Dinkelaker et al 1989; Gerke et al. 1994).
Various studies using soil-grown plants have shown that organic anions released from roots can increase the concentration of the plant-available P (as orthophosphate) in the soil solution and many examples (e.g., as outlined in references above) have been reported. The effectiveness of different organic anions in mobilizing P, however, is highly dependent on soil type and the form of P either present in soil or provided to the plants. Plant species and genotype is also important because it determines the type and amount of each organic anion released and the dynamics of that release (Wang and Lambers 2020). More direct examples of P mobilization by organic anions have extracted soils in vitro with different organic anion solutions (Khademi et al. 2009; Ryan et al. 2014). For example, using seven contrasting Australian soils differing in pH and P content, Ryan et al. (2014) showed that both 1 mM and 10 mM citrate increased the concentration of orthophosphate, with greater mobilization occurring in acidic soils than for alkaline-calcareous soils. Citrate was able to mobilize P from both the inorganic and organic P fractions and this varied markedly between soils. Organic anions are proposed to mobilize P from sparingly-available pools of soil P through a number of processes including: (i) competition for sorption sites (i.e., desorption of orthophosphate), (ii) ligand-promoted mineral dissolution or exchange reactions with cations of iron (Fe 2+/3+), aluminum (Al3+) or calcium (Ca2+), or (iii) interactions with soil microorganisms. For the latter process, microorganisms may directly release organic anions themselves, or mobilize P by promoting root growth or through biomass turnover (Richardson et al. 2011a; Richardson et al. 20011b; Wang and Lambers, 2020). Organic anion release is also commonly associated with release of protons (H+) which can induce local regions of acidification that further influence the desorption and diffusion of P in soil (Barrow et al. 2017). Local acidification may also promote the solubilization of precipitated pools of Ca-P prevalent in alkaline soils (Jones 1998).
Organic P in soil typically accounts for at least 50% of the total soil P and forms of organic P can be identified and quantified using solution 31P NMR spectroscopy (Turner et al. 2002; McLaren et al. 2019a). Most of the organic P in soil occurs as phosphomonoesters (e.g., inositol hexakisphosphates, lower order inositol phosphates and other sugar-phosphates), phosphodiesters (e.g., phospholipids and nucleic acids, primarily as DNA) and a large pool of poorly characterized monoester compounds (McLaren et al. 2019a). Of these, the myo and scyllo stereoisomers of inositol hexakisphosphate are the most prevalent in many soils (Turner 2007). Similar to orthophosphate anions, inositol hexakisphosphates are readily adsorbed in soils and can also be precipitated with Al3+, Fe2+/3+ and Ca2+ (Celi and Barberis 2005; Jackman and Black 1951; Tang et al. 2006). Inositol phosphates are furthermore complexed within high molecular weight soil organic matter as structurally complex, supra- and macro-molecular monoester material that otherwise remains poorly characterized (Hong and Yamane 1981; McLaren et al. 2019b). Whilst various studies have demonstrated that organic anion extractions can increase the concentration of organic P (Otani and Ae 1999; Hayes et al. 2000; Wei et al. 2010, Ryan et al. 2014), the identity of the organic P compounds released and their contribution to plant nutrition remains to be further investigated (Richardson et al., 2005; George et al. 2018).
The nature of organic P in soil has been characterized by the lability of extracted P to dephosphorylation by various phosphatase enzymes, which show differing specificity toward mono-ester and di-ester forms of organic P (Hayes et al. 2000; Bünemann 2008; Darch et al. 2016; Jarosch et al. 2019). A meta-analysis by Bünemann (2008) showed that up to 60% of organic P was typically amendable to dephosphorylation across a wide range of soil extracts and water samples, with phytases (i.e., inositol hexakisphosphate phosphohydrolyases) generally showing the greatest release of orthophosphate. Significantly, using two Australian pasture soils, Hayes et al. (2000) showed that up 40% of the total P extracted by 50 mM citric acid was hydrolyzed by a highly purified phytase, whereas up to 79% was hydrolyzed by a commercially available phytase preparation that exhibited a wider substrate specificity. By contrast, lesser quantities of organic P (<17% and <9%) were dephosphorylated by the commercial phytase in water and 0.5 M sodium bicarbonate extracts, respectively, even though bicarbonate itself extracted three to four times more organic P (Hayes et al. 2000). This indicates a potentially strong interaction between organic anions in the mobilization of organic P substrates and their subsequent lability to phosphatases to release bioavailable P as orthophosphate.
In this study we sought to further investigate the interaction between organic anions and phosphatases in the mobilization of soil organic P and lability of the extracted P to dephosphorylation. We hypothesized that different combinations of organic anions and phosphatases would differentially influence P mobilization across a range of contrasting soil types, and that that greater release of P would occur when specific combinations of organic anion and phosphatase were present as potential functional components of root exudates. To test these hypotheses, we used a range of citrate, malate and oxalate concentrations in combination with commercially available preparations of acid-phosphomonoesterase (PME), phosphodiesterase (PDE) and phytase (PHY) to examine P release from six contrasting agricultural soils. Solution 31P NMR spectroscopy was used to identify and quantify the presence of phosphomonoester and phosphodiester forms of organic P directly in the extracts. Our findings demonstrate that organic anions both mobilize organic P from soil and render it more labile to dephosphorylation by phosphatases. These results have important implications for the P nutrition of plants and the dynamics of P in soils.