Philosophiae Doctor - PhD / This study explores the roles of exogenously applied nitric oxide, exogenously applied caffeic acid and salt stress on the antioxidant system in cereal (exemplified by maize) and legume (using soybean as an example) plants together with their influence on membrane integrity and cell death.This study investigates changes in H2O2 content, root lipid peroxidation, root cell death and antioxidant enzymatic activity in maize roots in response to exogenously applied nitric oxide (NO) and salt stress. This part of the study is based on the partially understood interaction between NO and reactive oxygen species (ROS) such as H2O2 and the role of antioxidant enzymes in plant salt stress responses. The results show that application of salt (NaCl) results in elevated levels of H2O2 and an increase in lipid peroxidation, consequently leading to increased cell death. The study also shows that by regulating the production and detoxification of ROS through modulation of antioxidant enzymatic activities, NO plays a pivotal role in maize responses to salt stress. The study argues for NO as a regulator of redox homeostasis that prevents excessive ROS accumulation during exposure of maize to salinity stress that would otherwise be deleterious to maize. This study extends the role of exogenously applied NO to improve salt stress tolerance in cereals crops (maize) further to its role in enhancing salt stress tolerance in legumes. The effect of long-term exposure of soybean to NO and salt stress on root nodule antioxidant activity was investigated to demonstrate the role of NO in salt stress tolerance. The results show that ROS scavenging antioxidative enzymes like SOD, GPX and GR are differentially regulated in response to exogenous application of NO and salt stress. It remains to be determined if the NOinduced changes in antioxidant enzyme activity under salt stress are sufficient to efficiently reduce ROS accumulation in soybean root nodules to levels close to those of unstressed soybean root nodules. Furthermore, this study investigates the effect of long-term exposure of soybean to exogenous caffeic acid (CA) and salt stress, on the basis of the established role of CA as an antioxidant and the involvement of antioxidant enzymes in plant salt stress responses. The effect of CA on soybean nodule number, biomass (determined on the basis of nodule dry weight, root dry weight and shoot dry weight), nodule NO content, and nodule cyclic guanosine monophosphate (cGMP) content in response to salt stress was investigated. Additionally, CA-induced changes in nodule ROS content, cell viability, lipid peroxidation and antioxidant enzyme activity as well as some genes that encode antioxidant enzymes were investigated in the presence or absence of salt stress. The study shows that long-term exposure of soybean to salt stress results in reduced biomass associated with accumulation of ROS, elevated levels of lipid peroxidation and elevated levels of cell death. However, exogenously applied CA reversed the negative effects of salt stress on soybean biomass, lipid peroxidation and cell death. CA reduced the salt stress-induced accumulation of ROS by mediating changes in root nodule antioxidant enzyme activity and gene expression. These CA-responsive antioxidant enzymes were found to be superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR), which contributed to the scavenging of ROS in soybean nodules under salt stress. The work reported in Chapter 2 has been published in a peer-reviewed journal [Keyster M, Klein A, Ludidi N (2012) Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide. Plant Signaling and Behavior 7: 349-360]. My contribution to the published paper was all the work that is presented in Chapter 2,whereas the rest of the work in the paper (which is not included in Chapter 2) was contributed by Dr Marshall Keyster.

Arid and semi-arid regions, mostly found in developing countries with exponentially increasing populations, are in chronic lack of water thereby severely limiting agricultural production. Irrigation with saline water, which is available in large quantities, could be an obvious solution, but current crops are all salt sensitive. Although major efforts are underway to breed salt tolerant crops, no breakthrough results have yet been obtained. One alternative could rely on plant-interacting microbiota communities. Indeed, rhizophere and endosphere microbial communities are distinct from those of the surrounding soils, and these specific communities contribute to plant growth and health by increasing nutrient availability or plant resistance towards abiotic and biotic stresses. Here we show that plant microbe interactions induce plant tolerance to multiple stresses. From a collection of strains isolated from the desert plant Indigofera argentea, we could identify at least four different strategies to induce salt stress tolerance in Arabidopsis thaliana. A deep analysis of Enterobacter sp. SA187 showed that it induces Arabidopsis tolerance to salinity through activation of the ethylene signaling pathway. Interestingly, although SA187 does not produce ethylene as such, the association of SA187 with plants induces the expression of the methionine salvage pathway in SA187 resulting in the conversion of bacterially produced 2-keto-4-methylthiobutyric acid (KMBA) to ethylene. In addition, a metabolic network characterization of both SA187 and Arabidopsis in their free-living and endophytic state revealed that the sulfur metabolic pathways are strongly upregulated in both organisms. Furthermore, plant genetic experiments verified the essential role of the sulfur metabolism and ethylene signaling in plant salt stress tolerance. Our findings demonstrate how successful plant microbes of a given community can help other plants to enhance tolerance to abiotic stress, and reveal a part of the complex molecular communication process during beneficial plant-microbe interaction.

Philosophiae Doctor - PhD / This study explores the roles of exogenously applied nitric oxide, exogenously applied caffeic acid and salt stress on the ontioxidant system in cereal (exemplified by maize) and legume (using soybean as an example) plants together with their influence on membrane integrity and cell death. This study investigates changes in H₂O₂ content, root lipid peroxidation, root cell death and antioxidant enzymatic activity in maize roots in response to exogenously applied nitric oxide (NO) and salt stress. This part of the study is based on the partially understood interaction between NO and reactive oxygen species (ROS) such as H₂O₂ and the role of antioxidant enzymes in plant salt stress responses. The results show that application of salt (NaCl) results in elevated levels of H₂O₂ and an increase in lipid peroxidation, consequently leading to increased cell death. The study also shows that by regulating the production and detoxification of ROS through modulation of antioxidant enzymatic activities, NO plays a pivotal role in maize responses to salt stress. The study argues for NO as a regulator of redox homeostasis that prevents excessive ROS accumulation during exposure of maize to salinity stress that would otherwise be deleterious to maize. This study extends the role of exogenously applied NO to improve salt stress tolerance in cereals crops (maize) further to its role in enhancing salt stress tolerance in legumes. The effect of long-term exposure of soybean to NO and salt stress on root nodule antioxidant activity was investigated to demonstrate the role of NO in salt stress tolerance. The results show that ROS scavenging antioxidative enzymes like SOD, GPX and GR are differentially regulated in response to exogenous application of NO and salt stress. It remains to be determined if the NO induced changes in antioxidant enzyme activity under salt stress are sufficient to efficiently reduce ROS accumulation in soybean root nodules to levels close to those of unstressed soybean root nodules. Furthermore, this study investigates the effect of long-term exposure of soybean to exogenous caffeic acid (CA) and salt stress, on the basis of the established role of CA as an antioxidant and the involvement of antioxidant enzymes in plant salt stress responses. The effect of CA on soybean nodule number, biomass (determined on the basis of nodule dry weight, root dry weight and shoot dry weight), nodule NO content, and nodule cyclic guanosine monophosphate (cGMP) content in response to salt stress was investigated. Additionally, CA-induced changes in nodule ROS content, cell viability, lipid peroxidation and antioxidant enzyme activity as well as some genes that encode antioxidant enzymes were investigated in the presence or absence of salt stress. The study shows that long-term exposure of soybean to salt stress results in reduced biomass associated with accumulation of ROS, elevated levels of lipid peroxidation and elevated levels of cell death. However, exogenously applied CA reversed the negative effects of salt stress on soybean biomass, lipid peroxidation and cell death. CA reduced the salt stress-induced accumulation of ROS by mediating changes in root nodule antioxidant enzyme activity and gene expression. These CA-responsive antioxidant enzymes were found to be superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR), which contributed to the scavenging of ROS in soybean nodules under salt stress. The work reported in Chapter 2 has been published in a peer-reviewed journal [Keyster M, Klein A, Ludidi N (2012) Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide. Plant Signaling and Behavior 7: 349-360]. My contribution to the published paper was all the work that is presented in Chapter 2, whereas the rest of the work in the paper (which is not included in Chapter 2) was contributed by Dr Marshall Keyster.

Philosophiae Doctor - PhD

La transition des activités humaines vers des modèles de développement durable pourrait contribuer à mieux s’adapter aux enjeux mondiaux interconnectés comme les changements climatiques, l’insécurité alimentaire, la santé globale, et l’épuisement des ressources. Cette thèse représente l’un des premiers travaux de recherche visant à combiner l’utilisation des saules à croissance rapide (Salix sp.) pour la phytoremédiation avec leur potentiel pour les bioraffineries. La métabolomique non-ciblée utilisée avec des saules dans deux études complémentaires a permis d’acquérir à la fois des connaissances appliquées sur la valorisation des composés extractibles et une compréhension fondamentale des mécanismes de tolérance au sel. Des saules matures, utilisés pour traiter des eaux usées, ont été évalués à travers un processus de transformation allant de la biomasse brute aux rendements finaux (bioéthanol, lignines, composés bioactifs). L’irrigation avec des eaux usées a triplé les rendements en biomasse, avec une augmentation significative du glucane au détriment d’une réduction des extractibles. Ces variations suggèrent l’allocation des ressources pour soutenir la croissance plutôt que les molécules de défense. Un prétraitement par liquide ionique a fractionné la biomasse, une méthode compatible avec des biomasses contaminées et permettant d’obtenir des composantes séparées de grande qualité. La saccharification enzymatique a livré les rendements en bioéthanol tandis qu’un contre-solvant a précipité la lignine brute, avec une efficacité similaire aux saules témoins. Malgré une teneur réduite en extractibles, l’irrigation par les eaux usées a enrichi le profil phytochimique en flavonoïdes et lignans, potentiellement associés aux perturbations causées par l’inondation et la salinité, et ayant de possibles propriétés antimicrobiennes et antioxydantes. En intégrant deux approches vertes, cette étude démontre une augmentation significative des rendements annuels de bioproduits par unité de surface cultivée, renforçant la viabilité économique et la durabilité environnementale de chacune des deux approches prises individuellement. Une seconde étude s’intéresse plus profondément aux mécanismes de tolérance d’un saule face à deux sels abondants, le NaCl et le Na2SO4, la salinisation étant généralement associée à l’utilisation d’eaux usées et fréquente sur de nombreuses terres marginales. Malgré une conductance stomatique réduite, Salix miyabeana a maintenu sa capacité photosynthétique et sa production de biomasse, même à des niveaux élevés de sel. Les cations et anions salins sont gérés différemment par la plante, qui restreint largement l’absorption du Na+ aux racines, alors que le Cl- et le SO42- sont transportés jusque dans les feuilles. Les analyses révèlent une tolérance résultant à la fois de la plasticité des métabolites constitutifs et des métabolites spécialisés associés aux organes. Les réponses nuancées en fonction des organes et des ions indiquent des adaptations spécifiques dans l’allocation du carbone, la production d’antioxydants ou le métabolisme du soufre par exemple. Seulement 3% des métabolites constituaient une réponse généralisée aux deux types de sels, incluant une réduction de saccharides et un enrichissement de composés phénoliques simples, comparativement aux 28% impliqués au total. Le NaCl induit une réduction du métabolisme énergétique à l’échelle de toute la plante, potentiellement combinée au renforcement racinaire par l’augmentation de précurseurs de la lignine et d’antioxydants. À niveaux équivalents d’électroconductivité et de sodium, Na2SO4 induit une perturbation métabolique plus limitée. Même à concentration double de Na2SO4, l’activité métabolique se caractérise par un enrichissement préférentiel, probablement facilité par l’assimilation de l’excès de SO42- en composés organiques plutôt que leur séquestration. Notre étude des ajustements métaboliques complexes des saules, une espèce non-modèle mais d’importance biologique, environnementale et économique, a contribué à approfondir notre compréhension de leur résilience face aux stress abiotiques, informant ainsi les approches de gestion de l'eau et des terres ainsi que les pratiques de production durables. / Transitioning towards sustainable models that harmoniously integrate human activities within natural cycles could address interconnected global challenges such as climate change, social and economic inequalities, food insecurity, global health, and resource depletion. Phytoremediation, leveraging plant-microorganism interactions to mitigate soil and water pollution, offers a potentially efficient and environment-friendly solution, while also being economically viable and socially acceptable. This thesis represents one of the initial efforts to integrate the application of fast-growing willows (Salix sp.) for phytoremediation, capitalizing on their stress tolerance, with its potential for widerange biorefineries, harnessing their high biomass productivity as a renewable resource for bioenergy and bio-based products. Two complementary studies employed untargeted metabolomics to acquire both applied knowledge on the extensive valorization potential of extractable phytochemicals, and fundamental understanding of their salt stress tolerance mechanisms. Mature field-grown willows used for phytofiltration of primary municipal wastewater effluent were comprehensively characterized to assess their biorefinery potential through a complete process, from raw biomass to final bioethanol, lignin, and extractives yields. Wastewater irrigation tripled biomass yield and structural biochemical compound quantification revealed a significant increase in constitutive glucan along with a significant decrease in extractives, suggesting resource investment into growth over specialized defensive compounds. Ionic liquid pretreatment enabled biomass fractionation, chosen for its versatility in recovering multiple highquality streams and its suitability for contaminated biomass. Subsequently, enzymatic saccharification determined the final bioethanol yields, while counter-solvent precipitation recovered crude lignins. Despite a generalized reduction of extractives, wastewater irrigation enriched the extractable phytochemical profile with flavonoids and lignans, potentially associated with flooding, salinity, antimicrobial and antioxidant activities. Wastewater irrigated willows maintained quality and conversion potential, enhancing the annual valorization yields per growing surface, and strengthening the economic viability and environmental sustainability. These results demonstrated mutual benefits of integrating wastewater treatment and biorefineries. A subsequent study investigated Salix miyabeana tolerance and metabolic response to two environmentally abundant salts, NaCl and Na2SO4, considering salinity as a potential consequence of wastewater irrigation. Despite reduced stomatal conductance, willows maintained photosynthetic capacity and biomass productivity even under high soil salinity levels, demonstrating their salt tolerance. Elemental analysis revealed differential fate of salt ions, restricted Na+ absorption in roots, while Cl- and SO4 2- accumulated in aerial parts. Untargeted metabolomics exposed willow’s efficient metabolic plasticity in response to salts, modulating both constitutive and organ-specialized metabolites. Differential organ- and anion-specific responses indicated nuanced adaptation mechanisms, involving adjustments in carbon allocation, antioxidant production, and sulfur metabolism. Only 3% of metabolites constituted a generalized salt stress response, including reduced saccharides alongside enriched simple phenolics, as compared to 28% involved in overall salt response. NaCl triggered plant-wide energy metabolism reduction, potentially coupled with reinforced root structure through increased lignin precursors and antioxidant compounds. At similar soil EC and sodium concentration, Na2SO4 caused lower metabolic disruption, and as Na2SO4 concentration increased, willow was able to support the metabolic enrichment instead of depletion, likely associated with the ability to integrate SO4 2- in sulfur metabolism instead of passive sequestration. Our investigation into the complex metabolic adjustments of willows, a non-model species but of biological, environmental and economic importance, contributed to deepen our understanding of its resilience towards abiotic stresses, further informing effective water and land management approaches as well as sustainable production practices.