Use of nitrate as a metabolic modulator in ammonium-fed leafy vegetable production
Global agriculture's dependence on nitrogen-based fertilizers and high soil nitrification rates causes excessive nitrate accumulation in soil and plants, leading to environmental and health problems. Nitrate accumulation in plans is especially notable in nitratepreferring leafy vegetables,...
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| Formato: | tesis doctoral |
| Fecha de publicación: | 2026 |
| País: | España |
| Recursos: | Universidad Pública de Navarra |
| Repositorio: | Academica-e. Repositorio Institucional de la Universidad Pública de Navarra |
| OAI Identifier: | oai:dnet:academicae__::0703be1f07d3e6f51e1215ac3918daca |
| Acesso em linha: | https://hdl.handle.net/2454/56793 |
| Access Level: | acceso embargado |
| Palavra-chave: | Nitrate accumulation Leafy vegetables Ammonium-based fertilization Alleviation Carbon metabolism Methylglyoxal Nitrate-ammonium interaction |
| Resumo: | Global agriculture's dependence on nitrogen-based fertilizers and high soil nitrification rates causes excessive nitrate accumulation in soil and plants, leading to environmental and health problems. Nitrate accumulation in plans is especially notable in nitratepreferring leafy vegetables, frequently exceeding European Union (EU) food safety limits. Ammonium-based fertilization is a promising alternative to lower nitrate content in edible tissues. However, ammonium as the sole nitrogen source is toxic to most crops, particularly to nitrate-preferring species like spinach (Spinacia oleracea L.) and other leafy vegetables. This toxicity, among other, is linked to disruptions in central carbon metabolism, promoting the synthesis of methylglyoxal (MG), a toxic compound. It is known that millimolar levels of nitrate can alleviate ammonium toxicity. However, the mechanisms by which alleviation also could occur at non-nutritive doses (i.e., micromolar range) remain poorly explored. Furthermore, the specific function of micromolar nitrate in regulating ammonium-induced carbon overflow in primary metabolism is not yet fully elucidated. This thesis hypothesized that the co-supply of small, micromolar concentrations of nitrate could alleviate ammonium toxicity while maintaining the Benefit of low nitrate content in edible tissues. We further proposed that this alleviation is driven by a specific regulatory action on carbon metabolism, likely mediated by nitric oxide (NO) derivatives (like GSNO) through redox-based post-translational modifications (PTMs), preventing MG synthesis. The general aim of this thesis was to bridge the gap between fundamental knowledge of the nitrate-ammonium interaction and its practical application in agriculture. We sought to gain mechanistic insights into how nitrate regulates carbon metabolism to inform the formulation of improved, ammonium-dominant nutrient solutions for the comercial production of leafy vegetables with reduced nitrate content. Our findings confirm that at micromolar concentrations, nitrate is rapidly translocated to the leaves where it exerts a regulatory function. This micromolar supply triggers a reorganization of carbon flow, effectively redirect carbon to different metabolic pathways, avoiding the spontaneous synthesis of toxic MG. A key target of this regulatory action was identified as the photosynthetic enzyme ABtype glyceraldehyde-3-phosphate dehydrogenase (AB-GAPDH), a crucial component of central carbon metabolism. Our data established that this regulatory mechanism is posttranslational, as no changes in AB-GAPDH transcript or protein abundance were detected. Instead, the enzyme’s activity is modulated through redox-dependent PTMs. Consistent with our initial hypothesis, we observed that the levels of GSNO were significantly higher in ammonium-fed plants supplemented with micromolar nitrate compared to those without, strongly suggesting that S-nitrosation is the likely (though yet to be definitively confirmed) mechanism of this redox-based regulation. While the micromolar nitrate supply provided an important mechanistic insight, we found it was insufficient to sustain agronomically acceptable yields in advanced-stage leafy vegetables on its own. This demonstrated that a higher, albeit still minimized, nitrate content is required for practical cultivation. We therefore explored different nitrate-to-ammonium ratios to balance yield, stress tolerance, and nitrate accumulation. For some leafy vegetables like rocket (Eruca sativa Mill.) and borage (Borago officinalis L.), a 3.33:6.66 (mM) nitrate-to-ammonium ratio emerged as the optimal solution. For the highly ammonium-sensitive spinach, however, none of the tested ratios simultaneously achieved the dual objective of lowering nitrate accumulation while maintaining acceptable yields. This emphasises the importance of tailoring fertilisation strategies to the specific physiology of each crop. This thesis provides a novel knowledge on nitrate’s function as a post-translational regulator of central carbon metabolism in the context of ammonium nutrition. This discovery opens new perspectives for applied agriculture. It provides a fundamental scientific basis for the formulation of ammonium-dominant fertilizers that incorporate low levels of nitrate not just as a nutrient, but as a critical regulator. This approach paves the way for the commercial production of leafy vegetables that are high-yield, high-quality and compliant with EU food safety standard for nitrate content. |
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