Periodic Reporting for period 1 - ExuNutriStress (Poplar root exudates response to multi-nutrients stress)
Reporting period: 2021-10-01 to 2023-09-30
According to the United Nations report, the world’s population will increase by 2 billion persons in 2050 and so, the challenge for the next thirty years is to double the current rate of food production. In the context of global changes, it is essential to maintain the productivity of agroecosystems to mitigate the increase in atmospheric carbon oxide (CO2) concentrations since photosynthesis is the main entry point for carbon (C) in ecosystems and the availability of mineral nutrients often limits it. Therefore, plant mineral nutrition is a key issue for atmospheric CO2 fixation. On the other hand, the arable land availability decreases due to climate change, and nutrient availability is one of the most serious limitations to crop yields. Unfortunately, nutrient deficiency in food crops often translates into a nutrient deficiency in humans as iron (Fe) deficiency anemia that affects around two billion people globally, according to the World Health Organization. In contrast, the nitrogen (N) pollution level expected by 2050 is projected to be 150% higher than in 2010, with the agricultural sector accounting for 60% of this increase. So, it is therefore urgent to reduce this pollution by finding strategies to increase nitrogen use efficiency. Moreover, it is estimated that around 27% of C allocated to roots is exuded to the rhizosphere through roots. However, this percentage can vary depending on plant species, age, and nutritional status. Thus, root exudates are also important for C sequestration in soils and an important resource for studying how plant metabolism is affected by nutrient deficiencies. In this way, we will gain a better understanding of how to correct these deficiencies with less impact on the environment and in a healthier way for humankind.
Then, ExuNutriStress is an interdisciplinary (plant physiology, analytical chemistry, and bioinformatics) approach to the effects of nutritional deficiencies (N and Fe), individually and combined, on root exudates profiles of poplar trees. The poplar tree (Populus spp) was chosen for this project because it is a fast-growth tree that combines rapid maturation and wide geographic distribution with economic relevance for wood and biomass production and has seldom been studied in terms of nutrition. The main nutrient deficiencies chosen were N and Fe because N is a fundamental macronutrient that takes part in proteins, chlorophyll, nucleic acids, nucleotides, and nucleosides, and its availability is a major determinant of yield. At the same time, iron, among other processes, is involved in photosynthesis, mitochondrial respiration, N assimilation, and hormone biosynthesis, and other plants are already known to use exudates to improve iron availability in soils. Nitrogen and iron deficiencies were tested individually and in combination since plant responses to multiple nutrient stresses are usually not the sum of the plant responses to each stress.
Then, ExuNutriStress is an interdisciplinary (plant physiology, analytical chemistry, and bioinformatics) approach to the effects of nutritional deficiencies (N and Fe), individually and combined, on root exudates profiles of poplar trees. The poplar tree (Populus spp) was chosen for this project because it is a fast-growth tree that combines rapid maturation and wide geographic distribution with economic relevance for wood and biomass production and has seldom been studied in terms of nutrition. The main nutrient deficiencies chosen were N and Fe because N is a fundamental macronutrient that takes part in proteins, chlorophyll, nucleic acids, nucleotides, and nucleosides, and its availability is a major determinant of yield. At the same time, iron, among other processes, is involved in photosynthesis, mitochondrial respiration, N assimilation, and hormone biosynthesis, and other plants are already known to use exudates to improve iron availability in soils. Nitrogen and iron deficiencies were tested individually and in combination since plant responses to multiple nutrient stresses are usually not the sum of the plant responses to each stress.
In ExuNutriStress, during the two years of the project, seven hydroponic experiments were carried out in growth chambers under controlled conditions. Two experiments for N deficiency, three for Fe deficiency, and two for the combined (N + Fe) deficiency. Each experiment was monitored periodically by assessing plant chlorosis status, through chlorophyll measurement by a SPAD meter, in leaves and pH in the nutrient solution. At the end of the experiment, the nutrient solution and the exudates in water were collected, and plants were separated into leaves, stems, and roots, weighted and their micro and macronutrients were analyzed by microwave plasma atomic emission spectroscopy (MP AES). Nitrogen and C content as well as jasmonic and salicylic acid concentrations were measured in roots. Due to the complexity of the sample matrix, it is important to note that we had to develop specific protocols to extract, prepare, and analyze metabolites in roots, nutrient solution, and exudation in pure water. The occurrence of mucilage in the exudation in water was one of the challenges in purifying these samples. In order to study the coverage of the entire diversity of compounds released by roots, we have carried out untargeted metabolomic analyses to obtain the root exudate profiles by applying liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) since it is compatible with aqueous samples and combines high-resolution and sensitivity. However, for data analysis, we have focused on three families of specialized metabolites: flavonoids, coumarins, and salicylates. The reasons for this choice are: (i) under N starvation the plants suffer an imbalance in N and C metabolisms that alters flavonoid production, (ii) poplar trees belong to the Salicaceae family and then salicylates are likely to be affected, and (iii) under Fe starvation, other plant species are known to excrete coumarins and/or flavins. Moreover, bioinformatic tools such as MZmine3, MetGem, Sirius, Cytoscape, and MetaboAnalyst were applied for molecular networking and statistical analysis.
As preliminary results, poplar roots accumulated specific flavonoids, salicylates, stilbenes, and coumarins under N, Fe, and N+Fe starvation as salicin, tremulacin, salicortin, and proanthocyanins. In the case of N deficiency, the Shikimate and phenylpropanoids pathway as well as the carbohydrates pathway were affected while for Fe deficiency, in addition to the pathways mentioned, fatty acids, amino acids, and alkaloids were also disturbed. These results were presented at the 15th Journées du Réseau Francophone de Métabolomique et Fluxomique (poster-Perpignan- May 2023) and the Plant Biology European meeting (oral communication- Marseille-July 2023). Moreover, the project ExuNutriStress has its website (https://exunutristress.com) and its evolution has been published periodically on the social media Instagram (https://www.instagram.com/exunutristress). The project was presented at Researcher’s Night in Paris (September 2022) and a scientific publication is being prepared.
As preliminary results, poplar roots accumulated specific flavonoids, salicylates, stilbenes, and coumarins under N, Fe, and N+Fe starvation as salicin, tremulacin, salicortin, and proanthocyanins. In the case of N deficiency, the Shikimate and phenylpropanoids pathway as well as the carbohydrates pathway were affected while for Fe deficiency, in addition to the pathways mentioned, fatty acids, amino acids, and alkaloids were also disturbed. These results were presented at the 15th Journées du Réseau Francophone de Métabolomique et Fluxomique (poster-Perpignan- May 2023) and the Plant Biology European meeting (oral communication- Marseille-July 2023). Moreover, the project ExuNutriStress has its website (https://exunutristress.com) and its evolution has been published periodically on the social media Instagram (https://www.instagram.com/exunutristress). The project was presented at Researcher’s Night in Paris (September 2022) and a scientific publication is being prepared.
The results obtained in ExuNutriStress will contribute to the study of the N and Fe cycle in poplar trees, unveiling possible strategies relying on root exudation of metabolites involved in N fixation as well as in solubilizing and complexing iron from iron hydroxides in calcareous soils. Then, it will highlight the effect of a highly relevant combined deficiency in N & Fe and the importance of the N/Fe balance in plant nutrition. We anticipate that ExuNutriStress results will be used in future research to formulate sustainable fertilizers, more efficient in correcting mineral deficiencies, and will reduce the use of synthetic fertilizers like iron chelates in vineyards, orchards, and agricultural forestry.