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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 reliable sampling of root exudates in soil grown plants is experimentally challenging. This study aimed at developing a citrate sampling and mapping technique with millimetre-resolution using DGT (diffusive gradients in thin films) ZrOH binding gels. Citrate adsorption kinetics, DGT capacity and stability of ZrOH gels were evaluated. ZrOH gels were applied to generate 2D maps of citrate exuded by white lupin roots grown in rhizotrosn in a phosphorus deficient soil. Citrate was adsorbed quantitatively and rapidly by the ZrOH gels, these gels can be stored after sampling for several weeks prior to analysis. The DGT capacity of the ZrOH gel for citrate depends on the ionic strength and the pH of the soil solution but was suitable for citrate sampling. 2D citrate maps of rhizotron grown plants have been generated for the first time at a millimetre resolution to measure an illustrated plant response to P fertilization. DGT-based citrate sampling is suitable for studying the root exudation in soil environments, at unprecedented spatial resolution. By changing binding material, the technique is also applicable to other exudate classes and might be used for the evaluation of whole root exudation crucial in specific cultivar breeding.HighlightWe present a novel, reliable, easy to use, non-destructive citrate sampling- and two-dimensional high-resolution imaging technique for soil grown plant roots.

Iron (Fe) bioavailability in soils is often limited and can be further exacerbated by a non- homogeneous distribution in the soil profile, which has been demonstrated to vary both in space and time. Consequently, plants respond with morphological and physiological modifications at the root level involving a complex local and systemic signaling machinery. The present work unravels the role of two phytohormones (i.e., ethylene and auxin) and their integrated signaling in plant response to Fe deficiency. Inhibitors of auxin polar transport and of ethylene biosynthesis (N-1-naphthylphthalamic acid - NPA and aminoethoxyvinylglycine - AVG, respectively) were applied on tomato (Solanum lycopersicum L.) plants grown by the split-root technique, which allows to simulate condition of Fe heterogeneous distribution. Results showed that plants, exposed to an uneven Fe supply, triggered a complex auxin-ethylene signaling. A systemic action of auxin on FERRIC REDUCTASE OXIDASE 1 (SlFRO1) expression was revealed, while ethylene signaling was effective both locally and systemically. In addition, the investigation of Fe concentration in tissues showed that when leaves overcame Fe deficiency a Fe &ldquo, steady state&rdquo, was maintained. Therefore, physiological adaptation to this heterogeneous Fe supply could be mediated by the integration of the complex signaling pathways prompted by both auxin and ethylene activities.

Due to the deliberate use of cupric fungicides in the last century for crop-defence programs, copper (Cu) has considerably accumulated in the soil. The concentrations of Cu often exceed the safety limits of risk assessment for Cu in soil and this may cause toxicity in plants. Copper toxicity induces nutritional imbalances in plants and constraints to plants growth. These aspects might be of paramount importance in the case of phosphorus (P), which is an essential plant macronutrient. In this work, hydroponically grown cucumber plants were used to investigate the influence of the exposure to different Cu concentrations (0.2, 5, 25 and 50 μM) on i) the phenotypic traits of plants, particularly at root level, ii) the nutrient content in both roots and shoots, and iii) the P uptake mechanisms, considering both the biochemical and molecular aspects. At high Cu concentrations (i.e. above 25 μM), the shoot and root growth resulted stunted and the P influx rate diminished. Furthermore, two P transporter genes (i.e. CsPT1.4 and CsPT1.9) were upregulated at the highest Cu concentration, albeit with different induction kinetics. Overall, these results confirm that high Cu concentrations can limit the root acquisition of P, most likely via a direct action on the uptake mechanisms (e.g. transporters). However, the alteration of root plasma membrane permeability induced by Cu toxicity might also play a pivotal role in the observed phenomenon.

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.

Among the different mineral nutrients, potassium (K) is recognized as the macronutrient that most strongly affects the quality parameters of crops. An adequate K nutrition is in fact associated with increased fruit size, enhanced nutraceutical properties (e.g., content of sugar, organic acids, antioxidants), improved color and increased shelf life for many agricultural products. Previous studies have shown that, in controlled conditions, the foliar application of K-based fertilizers during the fruit development period could improve fruit quality; however, it is not clear whether such positive results can also be obtained in field-cultivated plants. For these reasons, the aim of the present study was to assess if the repeated applications of K-based fertilizers during the production period (mid-June to late August) might affect the quality parameters of strawberry (Fragaria × ananassa 'Murano') fruits. Frigoplants, transplanted in April, were cultivated in grow bags on cocopeat (8 plants per linear meter) and treated foliarly spraying K(2)SO(4) (6 g L(-1)) either once or twice a week. Control plants were sprayed with distilled water. As expected, foliar K-fertilization increased the K concentration by 8-11% in fruits and by 7-5% in leaves according to the frequency of application (once and twice a week, respectively). In addition, strawberry fruits exhibited an enhanced total soluble solids content (approximately 12%) and sweetness index (approximately 13%). Conversely, other nutraceutical compounds (e.g., phenolics, flavonoids and flavonols) as well as the fruit yield were not affected by K treatments, regardless the fertilization levels. In conclusion, results here presented show that at the field scale the foliar K-fertilization during the development of strawberries can enhance the quality and the nutraceutical value of fruits.

Selenium (Se) is an essential nutrient for humans, due to its antioxidant properties, whereas, to date, its essentiality to plants still remains to be demonstrated. Nevertheless, if added to the cultivation substrate, plants growth resulted enhanced. However, the concentration of Se in agricultural soils is very variable, ranging from 0.01 mg kg-1 up to 10 mg kg-1 in seleniferous areas. Therefore several studies have been performed aimed at bio-fortifying crops with Se and the approaches exploited were mainly based on the application of Se fertilizers. The aim of the present research was to assess the biofortification potential of Se in hydroponically grown strawberry fruits and its effects on qualitative parameters and nutraceutical compounds. The supplementation with Se did not negatively affect the growth and the yield of strawberries, and induced an accumulation of Se in fruits. Furthermore, the metabolomic analyses highlighted an increase in flavonoid and polyphenol compounds, which contributes to the organoleptic features and antioxidant capacity of fruits; in addition, an increase in the fruits sweetness also was detected in biofortified strawberries. In conclusion, based on our observations, strawberry plants seem a good target for Se biofortification, thus allowing the increase in the human intake of this essential micronutrient.