ISO-TRACER: Stable isotope ratio analysis of oxygen in phosphate for phosphorus tracing in soil-plant systems

Phosphorous (P) is one of the major limiting nutrients in plant production and is a finite resource. Standard soil analyses aimed at quantifying the plant available P fraction, like Olsen-P extraction or Hedley sequential extractions, are insufficient for providing information on P availability during a growth period. Furthermore, determining the phosphate content of plant tissue does not generate knowledge about P uptake efficiency from different fertilizers or internal P utilization. Phosphorous only has one stable isotope so researchers have applied P radioisotopes to elucidate P cycling processes in the soil-plant system. However, this approach is hampered by short half-lives, requires licensing and is not applicable at a natural abundance level. Therefore, research using a compoundspecific isotope analysis (CSIA) approach by analyzing the stable isotope ratio of oxygen (O) in phosphate has gained much attention during the past two decades. Oxygen has three stable isotopes (16O, 17O and 18O), and applying this approach has enhanced the comprehension of soil phosphorus dynamics and biogeochemical cycling but remains underexplored in plants. This PhD project aimed to apply the CSIA approach using oxygen isotopes of inorganic phosphate as a proxy tracer of P to study P utilization by plants.

An optimized and more uniform protocol for extraction of inorganic P using 0.3 M trichloroacetid acid (TCA-P) and subsequent purification as silver phosphate for oxygen isotope ratio analysis from plant material was developed, which has the potential to be used for a large variety of plant species (paper I). Drying of the plant material before TCA-P extraction enhanced the applicability of the method. A thorough clean-up before the first purification step and a final vacuum roasting step to eliminate coprecipitated oxygen-bearing species before analysis was implemented. The optimized method was pivotal for conducting the studies carried out in this project and was used in manuscript I and in two case studies (case study I and II) using barley as a model plant. In manuscript I, plants were grown in a greenhouse using three different fertilizers and three different water isotope signatures. The study showed that for all treatments, the original oxygen isotope signature (δ18O) from the fertilizer was lost in plant leaves and was mainly influenced by the oxygen isotope composition of the plant water. The majority of the δ18OTCA-P values deviated from calculated equilibrium values, thus no clear indication of full equilibrium or kinetically controlled fractionation was observed for the oxygen isotope exchange between oxygen atoms of ambient water and inorganic P. Generally the δ18OTCA-P value of plant leaves was similar between fertilizer treatments, however, treatments receiving the most enriched irrigation water did not show the same trend.

Case study I investigated whether the oxygen isotope composition of the P source was preserved in the inorganic P in barley xylem sap (δ18Oxylem-P). Xylem sap was collected from plants grown hydroponically with mineral P sources of different oxygen isotope signatures and water having two different isotope signatures. Here we observed that the original P source oxygen isotope composition was lost in the phosphate collected from xylem sap and the δ18Oxylem-P value increased with increasing water isotope signature. Case study II explored the impact of nutrient deficiencies on the δ18OTCA-P value of barley leaves, where barley was grown without nitrogen (N), potassium (K) or P. Significantly different δ18OTCA-P values was observed for plants grown without P and N, whereas control plants and plants grown without K had similar values.

In conclusion, the PhD project provides a uniform and optimized method for plant inorganic phosphate extraction and purification for oxygen isotope ratio analysis. It was observed that the original oxygen isotope composition of the P source was lost inside the xylem sap and leaves, suggesting a rapid turnover of P inside the plant imprinting oxygen from ambient water into the inorganic phosphate. Thus, there are obvious limitations for using the CSIA approach of studying oxygen isotope ratios of phosphate in plants as a proxy tracer of P during a growth period, and gaining knowledge about P uptake efficiency from different fertilizers. Yet, clear deviations from theoretically calculated equilibrium values were observed and nutrient deficiencies have an impact on the δ18OTCAP value. Although δ18O values are difficult to interpret, the CSIA approach proves to be a complementary tool in soil-plant P research but further work is required to understand what controls the δ18O values of inorganic P in plant tissue. Accordingly, by analyzing the δ18OP value from different plant compartments and different P compounds (not just inorganic P), such information is likely to uncover knowledge gaps in long-term plant metabolic strategies when grown under different conditions like nutrient deficiencies and broadening our understanding of internal plant P use efficiency.