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Plant roots are able to exude vast amounts of metabolites into the rhizosphere especially when subjected to phosphorus (P) deficiency to increase P solubility and thus its´ uptake. This causes noteworthy costs in terms of energy and carbon (C) for the plants. For this reason, we suggested that exudates reacquisition by roots could represent an energy saving strategy of plants. This study aimed at investigating the effect of P deficiency on the ability of hydroponically grown tomato plants to re-uptake specific metabolites generally present in root exudates by using 13C-labelled molecules. Hence, tomato plants have been grown for 21 days in full and P deficient nutrient solution. Exudates reuptake has been assessed by immersion of roots in a solution containing 13C labeled glycine, glucose, fructose, citrate, and malate. δ13C analysis was performed using a Continuous Flow Isotope Ratio Mass Spectrometer (CFIRMS). Results revealed that P deficient tomato plants were able to take up significantly more citrate (+37%) and malate (+37%), when compared to controls. While also glycine (+42%) and fructose (+49%) uptake was enhanced in P shortage, glucose acquisition was not affected by plants nutritional status. Unexpectedly, results also highlighted that P deficiency leads to a 13C enrichment in both tomato roots and shoots over time (shoots +2.66 ‰, roots +2.64 ‰, compared to control plants). This could be explained by stomata closure triggered by P deficiency resulting in an increased use of 13CO2 in respect to 12CO2, normally preferred by RuBisCO. Our findings highlight that tomato plants are able to take up a wide range of metabolites belonging to root exudates, thus optimizing C trade off. This trait is particularly evident when plants grew in P deficiency.

The determination of citrate exuded from soil-grown roots is very challenging due to its rapid microbial degradation and mineralization, sorption to the solid soil phase and ongoing release of organic molecules from organic matter breakdown. For this reason, our knowledge about citrate release is mainly based on experiments carried out in hydroponics. Results obtained in hydroponics cannot directly be transferred to soil-plant systems, as hydroponics represents an artificial environment. This study aimed to develop a localization and quantification technique for citrate exuded from soil-grown plant roots, based on diffusive gradients in thin film (DGT). Polyacrylamide gels containing precipitated zirconium hydroxide (ZrOH) were applied to the rhizosphere of soil grown plants, on which citrate is efficiently immobilized, thereby creating a zero sink to sample the citrate exuded from the roots. Citrate was eluted with 1 mL 0.5 mol L-1 NaOH from the ZrOH gel and quantified by ion chromatography. ZrOH gel discs were able to bind the citrate contained in 10 mL of 2.77 mg citrate L-1 solutions within a 4h uptake period. Elution efficiency was ~89%. ZrOH gel capacity at pH 8 was 200 µg per gel disc and 299 µg per gel disc at pH 4, which is sufficient to act as a zero sink for citrate released from plant roots. As a first exemplary method application, we grew white lupin plants in rhizotrons using a highly phosphorus deficient soil. ZrOH gel sheets were applied for 26 h onto cluster roots for citrate sampling following established DGT protocols. Gels were cut afterwards into 5×5 and 5×2 mm slices for obtaining a citrate exudation map. In both cases we were able to localize and quantify up to 7.89 µg citrate on individual gel slices, as well as to identify longitudinal and lateral citrate gradients around the cluster roots. Moreover, the characterization of ZrOH gels showed its suitability for citrate sampling in terms uptake kinetics and capacity. These results demonstrate that the developed method is suitable for citrate sampling and localization in a non-destructive way from soil-grown plant roots. As it is applicable to soil grown-roots and provides unprecedented spatial resolution, this sampling technique advances the experimental possibilities for researching root exudates considerably. Using suitable binding materials, this approach is also applicable to other carboxylates such as malate or oxalate and other compound classes such as phenolics, flavonolos etc. Furthermore, this technique can be combined with complementary imaging methods for mapping e.g. nutrients, contaminants, pH or enzyme activity distributions.