Abstract
The rhizosphere, i.e., the zone of soil influenced by root activity, plays a pivotal role in enhancing crop performance and yield. A key factor in the soil-root interface is the release of root exudates, which shape the biological, chemical, and physical properties of the rhizosphere. Among essential nutrients, phosphorus (P) and iron (Fe) are two of the most important for plant growth, and their deficiency poses one of the biggest challenges to crop production. P- and Fe-deficient plants deploy several strategies to manage nutrient shortages, such as increasing root surface area and enhancing root exudation. Root exudation provides undiscussed benefits to the plants, increasing, among other things, nutrients availability and uptake. However, this process comes with substantial energy and carbon (C) costs. Consequently, the reacquisition of these exudates by roots could offer a potential energy-saving strategy for plants and represent an unexplored adaptation to nutrient deficiencies. The first two studies of the present PhD thesis focused on the optimisation and development of the methodological approaches needed to fill this knowledge gap. Accordingly, the first study investigated the unknown relationship between the natural abundance of stable carbon isotope ratios (δ13C), and consequently 13C discrimination (Δ), and nutrient deficiencies in different plant species. The results aimed at advancing the knowledge about the 13C “baseline” in plants experiencing nutrient deficiencies. This will in turn improve the reliability and the interpretation of the results derived by future studies exploiting isotope labelling and tracing as methodological approach. The δ13C of barley (Hordeum vulgare L.), cucumber (Cucumis sativus L.), maize (Zea mays L.), and tomato (Solanum lycopersicum L.) was analysed using an isotope-ratio mass spectrometer (IRMS) after exposing the hydroponically grown plants to P, Fe and combined P/Fe deficiencies over a two week period. Simultaneously, plant physiological status was measured using an Infrared Gas Analyzer (IRGA). The results revealed distinct variations based on time, treatment, species, and tissue type. Moreover, the physiological parameters showed limited correlation with δ 13C changes, indicating that δ 13C is not solely determined by photosynthetic Δ. Besides providing this information about δ13C dynamics, the results allowed us to conclude that the use of δ 13C as a predictor for nutritional deficiencies, as hypothesised in literature, is not recommended due to its inability to accurately differentiate between nutrient stresses, especially when multiple stresses occur simultaneously. The second study focused more strictly on the optimization of the experimental plan, namely sampling technique and duration. Indeed, these are critical parameters that must be carefully considered prior to conducting any full-scale experiment. Thus, for the second study of this PhD thesis hydroponically grown tomato plants were exposed to P, Fe and P/Fe shortages for one week, after which root exudates were collected for 2, 4 and 6 hours. The results demonstrate that the duration of exudate collection significantly influences the exudation dynamics of tomato plants under nutrient deficiencies. An incorrect selection of collection duration may hinder the accurate detection of differences in root exudate patterns across various nutrient deficiencies. Therefore, the determination of the optimal exudate collection duration for each specific experimental setup, 4 h in this case, is essential to ensure reliable and representative data on root exudation. With all the required knowledge and optimized protocols derived by the first and second studies of the PhD, it was possible to investigate the reacquisition of root exudates. Hence, for the third study of this PhD tomato seedlings were grown hydroponically for 3 weeks under Control conditions, Fe and P deficiencies, with sampling twice per week. IRMS was used to measure δ 13C in roots and shoots following a 2 hour exposure to 13C-labeled glycine at concentrations of 0, 50, or 500 μmol L−1 . Glycine was used since it is one of the most abundant amino acids (AA) released by roots and plays a crucial role in various rhizosphere processes, such as nutrient mobilization and microorganisms recruitment. Plant physiological status was evaluated using an IRGA, while ionome analysis was performed using Inductively Coupled Plasma Mass Spectrometry. Glycine uptake varied by concentration, indicating the involvement of root uptake transporters with different substrate affinities. Uptake decreased over time, with significantly higher rates in Fe and P deficiencies compared to the Control, underscoring the importance of glycine acquisition during early growth, i.e. germination, and in nutrient-deficient plants. Translocation of glycine to shoots declined over time in P starved and Control plants but increased in Fe deficient plants, suggesting a potential role of glycine in Fe transport via the xylem. These findings demonstrate that glycine acquisition and its translocation to shoots are influenced by Fe and P deficiencies. This adaptive response to nutrient limitations could enhance plant fitness, making it a valuable trait for selecting cultivars that are better suited to withstand abiotic stress. To push the boundaries of research forward, for the fourth study of this PhD thesis the same experimental setup used for the third study was applied to citric acid. Citric acid was selected to explore a different class of compounds: organic acids (OA) rather than AA. Citric acid is recognized as a key component of root exudates of many plant species and plays a vital role in nutrient mobilization and overall nutrient stress responses. In addition to the analyses performed in the third study, root morphology was also investigated and a flux analysis was performed at 14 days after treatment (DAT). More precisely at 14 DAT, tomato plants were exposed to the 13C labelled citric acid for 2h as for the other sampling days and also for 15, 30 and 60 min. The samples were analysed both with an IRMS for the Bulk Stable Isotope Analysis (BSIA) as well as with Liquid Chromatography Time Of Flights Mass Spectrometry for a Compound Specific Isotope Analysis (CSIA). The results of this study show how citric acid uptake increased in tomato plants exposed to P deficiency. These outcomes are particularly interesting considering the fact that root acquisition of OA seems to offer limited benefits for photosynthetic organisms, as stated in the literature, mainly due to low concentrations in soil compared to other compounds and the lack of essential nutrients like N in their backbone. Hence, these results are hinting at potential biological functions beyond known nutrient uptake roles. Despite similar root morphologies, differences in citric acid uptake between P and Fe starved plants were evident, suggesting additional physiological adaptations beyond morphological ones. Fe deficiency reduced citric acid uptake, supporting the limited benefits hypothesis, whereas roots of P starved plants exhibited increased uptake, especially under severe deficiency. The differential citric acid uptake and metabolization in roots P and Fe deficient plants suggests specific adaptations to nutrient deficiencies. We observed a particularly significant enrichment of GABA in roots of P starved plants, potentially enhancing stress responses and the chances of survival under P deficiency conditions. These findings challenge the notion of limited benefits of OA uptake in Fe deficiency and suggest a crucial role for citric acid uptake in plant adaptation to P deficiency. In conclusion, understanding the dynamics of root exudation and reacquisition that drive nutrient mobilization, uptake, translocation, and allocation is crucial for effective rhizosphere management practices aimed at promoting sustainable agricultural production. This knowledge could serve as a foundation for developing breeding programs focused on enhancing nutrient use efficiency in crops, enabling them to better utilize existing soil resources and adapt to low-quality and low-fertility soils, thereby reducing the reliance on external chemical inputs.