Abstract
World population will exceed 11 billion people by the end of the century leading to an increasing global demand for food, which is becoming more and more challenging to satisfy. In order to achieve the 60-110% enhancement in crop yields necessary, scientist have to develop new more sustainable strategies. The rhizosphere, commonly known as the soil influenced by the activity of roots is of crucial relevance to increase crop performance. A main driving factor in the soil-root interface are root exudates shaping the biological, chemical and physical properties of the rhizosphere. Among essential plant nutrients, phosphorus (P) is one of the most important macronutrients and its deficiency represents one of the main constraints for crop production; P deficient plants adopt several mechanisms to cope with the nutrient shortage (e.g. enhancement of plant root surface, root exudation, etc.).
The reliable quantification of root exudation and nutrient uptake is a very challenging task, especially when considering single root segments. Most approaches to this purpose necessitate root handling as for instance root dissecting and/or cutting. However, there is a knowledge gap on how much these techniques affect root physiology. Thus, the first study presented in this thesis aimed at assessing the effect of different root handling techniques on the phosphate (Pi) uptake and carboxylate exudation of white lupin roots. White lupin plants were grown hydroponically in a full and Pi-deficient nutrient solution for 60 days. Phosphate uptake and carboxylate exudation of cluster and non-cluster roots were measured using custom made cells 1, 4 and 8 hours after the onset of light. Three different experimental set-ups were used: i) without cutting the root apparatus from the shoots, nor dissecting the root into smaller root sections - named intact plant (IP); ii) separating the roots from the shoots, without dissecting the root into smaller sections– named intact root (IR); iii) separating the roots form the shoots and dissecting the roots in different sections – named dissected roots (DR). The sampling at 8 hours after the onset of light led to the most significant alterations of the root Pi uptake induced by the sampling method. Generally, roots were mainly affected by the DR sampling method, indicating that results of studies in which roots are cut/dissected require a carefully interpretation. Additionally, the study revealed that the root tip showed a very high Pi uptake rate, suggesting that the tip could act as a Pi sensor. Citrate, malate and lactate could be detected in juvenile, mature and senescent cluster root exudation. We observed a significant effect of the handling method on carboxylate exudation only at sampling hour 1 and 8 after the onset of light, although no clear and distinctive trend could be observed. Results revealed that the root handling as well as the sampling time point greatly influence root physiology and therefore should not be neglected when interpreting rhizosphere dynamics.
Plant roots are able to exude vast amounts of metabolites into the rhizosphere in response to P deficiency causing noteworthy costs in terms of energy and carbon (C) for the plants. Therefore, exudates’ reacquisition by roots could represent an energy saving strategy of plants. The second study presented in this thesis aimed at investigating the effect of P deficiency on the ability of hydroponically grown tomato plants to re-acquire specific compounds generally present in root exudates by using 13C-labelled molecules. Results showed that P deficient tomato plants were able to take up citrate (+37%) and malate (+37%), particularly when compared to controls. While glycine (+42%) and fructose (+49%) uptake was enhanced in P shortage, glucose acquisition was not affected by the nutritional status. Unexpectedly, results also showed that P deficiency led to a 13C enrichment in both tomato roots and shoots over time (shoots - +2.66 ‰, roots - +2.64 ‰, compared to control plants), probably due to stomata closure triggered by P deficiency. These findings highlight that tomato plants are able to take up a wide range of metabolites belonging to root exudates, thus maximizing C trade off. This trait was particularly evident when plants grew in P deficiency.
The reliable sampling of root exudates in soil-grown plants is experimentally challenging. Thus, the third study presented in this thesis 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 rhizotrones in a P deficient soil. Citrate was adsorbed quantitatively and rapidly by the ZrOH gels. Furthermore, these gels can be stored after sampling for several weeks prior to analysis without being microbially degraded. Even though the DGT capacity of the ZrOH gel for citrate depends on the ionic strength and the pH of the soil solution, the gels resulted suitable for citrate sampling. Two-D citrate maps of rhizotron-grown plants have been generated for the first time at a millimetre resolution to measure a 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 could also be applicable to other exudate classes and might be used for the evaluation of whole root exudation, being this latter crucial in specific cultivar breeding.
Overall, the understanding of the rhizosphere dynamics triggering root exudation and nutrient mobilization, uptake, translocation and allocation could be of great importance for rhizosphere management practices leading towards a more sustainable agricultural production. Such knowledge could be fundamental to develop breeding programs to increase the nutrient use efficiency of crops to better exploit resources already present in the soil and better adapt to low quality and low fertility soils, limiting the addition of external chemical inputs.