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A new tool to study in-situ micronutrient uptake and translocation in plants

Final Report Summary - STABLE PLANT (A new tool to study in-situ micronutrient uptake and translocation in plants)

1. Background of the STABLE PLANT project
Zinc (Zn) is an essential micronutrient for humans, livestock, and plants. Agricultural soils in large parts of the world are deficient in Zn and crops growing on them have problems with taking the Zn up. This leads to widespread Zn (and other micronutrient) deficiency in the crops growing on the soils and consequently in the humans eating these crops. This is in particular true for regions where economies are entirely depending on crops as the major source of micronutrients.
While our understanding of micronutrient uptake in temperate soils and ‘western’ crops such as wheat, corn, or potatoes is well advanced, this is not true for rice. Our knowledge about the mechanisms of Zn uptake, translocation, and efficiency in rice or about the soil chemistry of Zn in submerged soils is very limited, and this is hindering the efficient breeding of Zn efficient rice traits. In grass species like rice, it has been proposed that the acquisition of micronutrient involves the excretion of phytosiderophores (PS) from the plant into the soil. These are non-protein amino acids of low molecular weight, able to form soluble complexes with metals. The roots then take up the complexes. While this mechanism is well known to operate for iron (Fe), it has only recently been suggested that phytosiderophores are also involved in the uptake of Zn and possibly other metals by rice and other plants. Recent research suggested that phytosiderophores secretion is sufficient to increase Zn uptake by more than five-fold in rice growing on a submerged soil with low Zn content. But this is yet to be established unequivocally.
Elemental uptake from the soil into the plant is associated with a discrimination of the stable isotopes. This is because the reactivity and structures of isotopes with different masses differs for each isotope. We know that environmental conditions (climate, soil) and physiological status of the plant play a major role in the control of isotopic variations found in plants. Stable isotopes have been widely applied to investigate the plant physiology and responses to the environment of light elements such as carbon (C), sulphur (S) or oxygen (O). Only the most recent development of the multicollector-inductively coupled plasma mass spectrometry technology (MC-ICP-MS), however, has enabled us to use stable isotopes of heavier metals such as Zn or Fe to study plant soil interactions. This technique has a great potential to help unravel the mechanisms of uptake of essential nutrients by plants, and make a great impact on developing plant nutrition science.

2. Objectives of STABLE PLANT
The objective of STABLE PLANT was (i) to test the role of phytosiderophores during the previously observed Zn isotope fractionation during uptake into rice and (ii) to improve our understanding of the mechanism of Zn efficiency in rice. The work should furthermore play a key role in establishing stable isotopes as novel technique for the study of metal uptake and translocation in plants and of trace metal cycling in the environment, and deliver new insights into the isotope fractionation associated with biological processes. We used Zn uptake by rice as our model system, but our results are applicable to other metals and plants.

3. Main Results of STABLE PLANT
3.1. Confirmation of the role of PS in Zn uptake in rice
Six rice genotypes were grown in Zn deficient soil from Tiaong (Philippines) during field trials in 2012. We analysed soils and plants (stems) for various major and trace element concentrations, for Zn isotope ratios, plant mass and plant length. We found that the Zn isotope signature in the stems of all the genotypes were significantly enriched in the heavier isotope compared to the free Zn2+ leachable from the soil using a dilute HCl solution (so called leachates). This suggests that an important contribution of the Zn taken up is in the form of a Zn-PS complex. This hints again to the crucial role of the phytosiderophores during the Zn uptake in rice grown on Zn deficient submerged soils and supports strongly our hypothesis.
3.2 Determination of the isotopic composition of plant-available Zn
A key question to date has been if the isotopic pool the phytosiderophores assesses in soils is different to the Zn readily bioavailable or in solution and if this signature changes during the dissolution process. To this end, we determined Zn availability and isotopic fractionation during phytosiderophore promoted dissolution. Zinc deficient soils were collected from our field station in the Philippines dissolved in a buffered EDTA solution and analysed after different time intervals. EDTA (Ethylenediaminetetraacetic acid) was chosen as model ligand as it has a similar affinity for Zn2+ like phytosiderophores. Up to 18 µg Zn/g soil were dissolved in solutions containing EDTA, whereas no measurable Zn was dissolved in absence of EDTA. This confirmed the important role of phytosiderophores in mobilising Zn from soil. The isotope data showed that the Zn in solution is enriched in the heavier Zn isotopes with respect to the soil, confirming that there during the formation of Zn complexes with EDTA leads to significant isotopic fractionation with the heavy Zn preferentially incorporated in the complex. The dissolution of Zn is best modelled using a logarithmic function (y = 1.453 ln(x) + 9.705; R2=0.9435) and showed three distinct phases: i) An initial phase of rapid release and the highest δ66Zn signature, ii) A transition phase from 24 h with isotope signatures similar to those of rice shoots, and iii) A slow-release phase from 48h, reaching a plateau after around 96h with a isotopically heavy Zn signatures around δ66Zn=1.39.
3.3 The effect of pH and PS concentration on the extend of isotope fractionation
To constrain the isotopic composition of the plant available Zn extracted from the Zn deficient soils, it is crucial to know the effect of phytosiderophore concentration and of soil pH on Zn availability and isotopic fractionation. To this end, zinc deficient soil was dissolved in solutions with different EDTA concentration (0.15 to 3 mM) and different pH (6 to 8). The Zn contents in the leachates were independent of the solution pH but dependent on the concentration of EDTA, implying that the EDTA (representing phytosiderophores) is the main factor controlling the dissolution and uptake of Zn. The isotope signature of the leachates extracted at pH 6 and 7 were in a similar range (δ66ZnLyon ranging between 0.6-1.0 ‰) and in line with the that of the soil (δ66ZnLyon = 0.8‰). The leachates extracted at pH 8, however, were isotopically heavier in the leachates with the lowest EDTA concentrations, i.e. a δ66ZnLyon value of 1.6 ‰ in solutions with 0.150 mM EDTA. Our results suggest that a higher concentration of phytosiderophores in the soil is likely to result in higher amounts of dissolved Zn, in agreement with the main hypothesis of STABLE PLANT. The experimental data suggests further that the isotopic signature of the PS-Zn complexes changes with pH, suggesting that a different Zn pool is assessed in the soils. This is supported by other elements.

4. Conclusions
The project STABLE PLANT has allowed to obtain a clearer view with respect to the isotope systematics in the rice-soil environment and the role of PS in Zn uptake in rice grown in Zn deficient soils. Our experiments confirmed that Zn in rice plants grown in soils with low Zn availability is isotopically heavier than the plant available Zn. This supports the initial hypothesis that the involvement of PS is an important Zn uptake mechanism present in all varieties of rice. Our results show that EDTA, used here as a PS proxy, is able to mobilise Zn from soils where from very little would be dissolved without it. This capacity is present in the pH range typical for the soil and rhizosphere environment. Phytosiderophores therefore play a crucial role of Zn acquisition in deficient soils. A greater concentration of EDTA resulted in a higher amount of Zn available in the soil solution, which reinforces the view that PS are the mechanism behind the improved performance of rice varieties tolerant to Zn deficiency in soils. Several parameters (time, pH, and PS concentration) determine the Zn isotopic composition of soil leachates. Our findings have important implications for the future interpretation of isotope data from the soil solutions, and for the use of the isotope technique in plant-soil interaction studies.

5. Wider impact in the society
The impact of STABLE PLANT extends to various areas. In the scientific sphere, it has permitted to develop a novel technique for the study of plant-soil interactions, and to obtain new basic knowledge in the area of plant nutrition, a field of great socioeconomic importance. It has also permitted the collaboration of various research institutions, by which a multidisciplinary research team was put together to face the challenge posed by Zn deficiency in soils from the most comprehensive and multidisciplinary approach possible. This network will continue to work beyond this project, and further benefits will be collected from the expertise and results generated during the duration of STABLE PLANT. The development of the project has also resulted in the training of young researchers in isotope techniques and others, which will improve their skills and employability.
The most direct impact of STABLE PLANT is for the work of rice breeders, who lack sufficient information about relevant traits to use in their selection programs. This bottleneck is currently the main hurdle in the development of Zn-efficient varieties of rice. We hope that the results provided by this project will help to relieve this circumstance and modestly contribute to tackle Zn malnutrition.

6. Contact details
Dominik Weiss, Imperial College of London,
Cristina Caldelas, +34 (0)657345950, @crcaldelas