Determination of inorganic phosphorus in plants

The fraction of inorganic phosphorus (inorganic P, Pi) occurs in plants as approximately half of the total phosphorus (TP) and mainly as orthophosphates, which can be present as H₃PO₄, H₂PO₄⁻, HPO₄²⁻ and PO₄³⁻, with respective pKa values of 2.12, 7.21, and 12.67. Among those forms, H₂PO₄⁻ is the most efficiently absorbed by plants [32, 33]. The remaining forms of inorganic phosphorus present in plants are pyrophosphates [34] and polyphosphates [35] (Fig. 2).

Inorganic and organic forms of phosphorus present in plants forming the total phosphorous pool together with an indication of the methods allowing for determination of particular forms of P in plant material

Most methods for phosphate determination are based on the spectrophotometric detection of a coloured phosphomolybdate complex [36]. Two colourimetric methods, the molybdenum blue method [37] and the malachite green assay [38], are commonly used to quantify orthophosphate extracted from plants. The indication for molybdenum blue takes place in two stages. The first stage involves the reaction between orthophosphate ions and molybdate ions in an acidic solution and results in the formation of a yellow phosphomolybdate complex. In contrast, the second stage entails reducing molybdenum in the complex to form an intense blue-coloured product.

All molybdenum blue methods are normally applied in an aqueous solution and require a strong acid, a source of molybdate (Mo(VI)) and a reductant. Ascorbic acid is the most widely used reductant, and antimony is used to catalyse the reduction of molybdenum by ascorbic acid. The absorbance of the resulting complex was measured with a spectrophotometer at 880 nm [39].

One of the most sensitive methods for the determination of phosphate is the colourimetric method with malachite green, also called the micromethod, because it allows the determination of even nanomolar concentrations of phosphorus in samples. Aromatic amine called malachite green, in the presence of ammonium molybdate reacts stoichiometrically with Pi and forms a coloured complex characterized by a maximum absorption at 660 nm [40]. Any colourimetric method detects free phosphate, although it cannot distinguish between the different pools of P present in the sample [41, 42]. Both spectrophotometric methods, molybdenum blue and malachite green, have their pros and cons [42]. The molybdenum blue method can be automated by flow injection analysis [43] and has a broad calibration range of linearity at high and very low concentrations (0.004–1.2 mg Pi L⁻¹). The appearance of colour in this method is independent of temperature, but the method can be time-consuming, labour-intensive, and may generate significant chemical waste, as some reagents are briefly stable at room temperature; thus, they have to be replaced with newly prepared reagents [44]. For comparison, the method with malachite green is linear over a range between 0.007–0.6 mg Pi L^–1 [42] and is widely used in plant science research due to its simplicity and the high stability of the assay reagents [19]. In both procedures, the adaptation of the assay to small samples may allow accurate measurements of Pi in the range of nanograms (of 0,3 to 8 ng of Pi per sample). The calibration curve and the sample volume are crucial elements for the reproducibility and accuracy of results. The disadvantages of both mentioned methods are the destruction of plant tissues' integrity and the limitation of measurements to the total content of Pi in plant samples. Nevertheless, the sensitivity of those methods and their reliability make it feasible to analyse small samples at the tissue level [19].

Ion chromatography (IC), a subset of liquid chromatography, is the most frequently chosen chromatographic method for the determination of phosphates. This method was first elaborated to measure the concentrations of inorganic anions with a conductivity detector [45]. In comparison to colourimetric methods, IC allows the detection of orthophosphate ions in real-time and synchronous analysis of ortho- and pyrophosphate anions and other ionic species [46]. The analyses of environmental samples showed that the concentration of Pi determined by the IC method was usually less than that obtained by the molybdenum blue method [47]. Many IC methods for routine anion determination, including inorganic phosphate, have been well documented as standard methods [48]. When using the IC technique, the stationary phase and eluent should be matched to the type of samples. It should also be noted that especially a high ion content may influence the obtained results. The presence of high concentrations of anions (chloride, nitrate, and sulphate) may disrupt or even make the IC determination of phosphate impossible [49]. Depending on the type of cations, the peak areas of phosphate may fluctuate. It was proven that ferric iron(III) significantly decreases, whereas aluminium(III) ions slightly increase the peak areas during phosphate IC determination [50].

Despite various interferences in real samples, the analysis of phosphates by IC has been more accepted for complex environmental samples such as plant samples, especially when it is compared to the molybdenum blue method [51, 52].

Current scientific reports indicate that inorganic polyphosphates (polyP) may also exist in plants [53]. Polyphosphates are linear polymers of inorganic phosphate (Pi) units with sizes ranging from tripolyphosphate (three Pi units) to long-chain polyP (approximately 1000 Pi units) linked by phosphoanhydride bonds. The existence of polyP in plant tissues has been established using microscopy (TEM) [54, 55] and biochemical methods of extraction [56], which are now known to produce artefacts. Recently, polyP-specific dyes and polyP-binding domains were used to detect polyP in plant cells. Although the presence of significant polyP stores was not confirmed in plants, it is possible that higher plants accumulate polyP in specific organs or cells, only in certain developmental stages or in response to certain environmental stimuli [35].