In Vivo Metabolic Regulation of Alternative Oxidase under Nutrient Deficiency—Interaction with Arbuscular Mycorrhizal Fungi and Rhizobium Bacteria

The interaction of the alternative oxidase (AOX) pathway with nutrient metabolism is important for understanding how respiration modulates ATP synthesis and carbon economy in plants under nutrient deficiency. Although AOX activity reduces the energy yield of respiration, this enzymatic activity is upregulated under stress conditions to maintain the functioning of primary metabolism. The in vivo metabolic regulation of AOX activity by phosphorus (P) and nitrogen (N) and during plant symbioses with Arbuscular mycorrhizal fungi (AMF) and Rhizobium bacteria is still not fully understood. We highlight several findings and open questions concerning the in vivo regulation of AOX activity and its impact on plant metabolism during P deficiency and symbiosis with AMF. We also highlight the need for the identification of which metabolic regulatory factors of AOX activity are related to N availability and nitrogen-fixing legume-rhizobia symbiosis in order to improve our understanding of N assimilation and biological nitrogen fixation.FONDECYT from National Agency for Research and Development (ANID) 1191118Chilean Scholarship Program/Becas de doctorado nacional/2017 21180329European Union (EU) 75330

Ecophysiological effects of arbuscular mycorrhizal inoculation on arundo donax under mediterranean conditions

[spa] El interés por los biocombustibles como alternativa viable a las fuentes no renovables de energía ha ido en aumento en la última década. Sin embargo, los biocombustibles no han estado exentos de polémica generando controversia a nivel social, económico y ambiental, como la competencia por la tierra con los cultivos básicos para alimentación humana y animal. Los biocombustibles de segunda generación se basan en gramíneas perennes como Arundo donax, plantas que no forman parte de la base alimentaria, capaces de producir grandes cantidades de biomasa con pocos aportes externos y con capacidad para adaptarse a condiciones de tierras marginales, en las que no se producen cultivos para alimentación humana o animal. A. donax es capaz de adaptarse a diferentes condiciones del suelo y presenta una alta producción de biomasa en condiciones de baja fertilidad en las zonas mediterráneas. Sin embargo, una de las desventajas de esta especie es su sensibilidad a la falta de agua y nutrientes durante las primeras etapas de crecimiento. Se ha demostrado que la simbiosis entre plantas y hongos formadores de micorrizas arbusculares (HMA) aumenta la resistencia de las plantas al estrés biótico y abiótico. En este escenario, el objetivo general de esta tesis doctoral es analizar la ecofisiología de A. donax en simbiosis con HMA, y su comportamiento tanto en condiciones control como bajo estrés salino. Las respuestas de las plantas en simbiosis y sin ella se evaluaron mediante el estudio de la fotosíntesis, la respiración in vivo y el metabolismo primario, el estado nutricional y la acumulación de biomasa. Los efectos fisiológicos y nutricionales de la simbiosis de A. donax con HMA también fueron estudiados bajo estrés salino y escasez de nutrientes. Además, se analizó el uso de HMA, entre otras técnicas, durante la producción de planta, con la intención de mejorar la calidad del plantel de A. donax para asegurar la supervivencia y mejorar la producción de biomasa en el primer y segundo año de cultivo. Los resultados de esta tesis doctoral muestran que A. donax presentó tolerancia a la sequía (a través de cambios morfo-anatómicos y de ajuste osmótico, y alta eficiencia en el uso del agua), a pesar de que la producción de biomasa se redujo significativamente. Además, presentó tolerancia a la salinidad moderada y una alta eficiencia en el uso del fósforo. Los cambios observados a nivel fisiológico en el simbionte generado por la simbiosis con Rizophagus irregularis y Funneliformis mosseae, permitieron una mayor producción de biomasa en comparación con las plantas no simbiontes tanto en condiciones control como bajo condiciones de estrés salino. El uso de HMA durante el periodo de aclimatación mejoró la producción de plantel, con tasas de supervivencia del 100% en campo y un aumento de la producción durante los primeros años. Sin embargo, otras técnicas como el uso de alveolos de gran tamaño y sustratos de alta calidad también pueden asegurar la supervivencia y producción del cultivo. Considerando todos los resultados en conjunto, se puede concluir que si bien A. donax por si sólo presenta características de alta productividad: tasas fotosintéticas elevadas, alta eficiencia en el uso de recursos (tanto agua como nutrientes) y moderada capacidad de resistencia al estrés, manteniendo producciones considerables que le hacen ser una especie adecuada para la producción de biomasa en tierras marginales, la simbiosis con HMA representa una herramienta interesante para mejorar todas estas características y asegurar el éxito de su cultivo bajo las condiciones impuestas por este tipo de tierras.[cat] L'interès pels biocombustibles com a alternativa viable a les fonts no renovables d'energia ha anat en augment en l'última dècada. No obstant això, els biocombustibles no han estat exempts de polèmica generant controvèrsia a nivell social, econòmic i ambiental, com la competència per les terres de cultiu amb els cultius bàsics per a alimentació humana i animal. Els biocombustibles de segona generació es basen en gramínies perennes (com ara Arundo donax), plantes que no formen part de la base alimentària, amb capacitat de produir grans quantitats de biomassa, amb pocs aports externs i amb capacitat per adaptar-se a condicions de terres marginals. Arundo donax és capaç d'adaptar-se a diferents condicions del sòl i presenta una alta producció de biomassa en condicions de baixa fertilitat en les zones mediterrànies. No obstant això, una de les desavantatges d'aquesta espècie és la seva sensibilitat a la falta d'aigua i nutrients durant les primeres etapes de creixement. S'ha demostrat que la simbiosi entre plantes i fongs formadors de micorizes arbusculars (FMA) augmenta la resistència a l'estrès biòtic i abiòtic del sòl. En aquest escenari, l'objectiu general d'aquesta tesi doctoral va ser estudiar els efectes ecofisiològics de la simbiosi micorrícica en A. donax, així com el seu comportament tant en condicions control com d’estrès salí. Les respostes de les plantes en simbiosi i sense es van avaluar mitjançant l'estudi de la fotosíntesi, la respiració in vivo i els canvis en el metabolisme primari, així com l'estat nutricional i l'acumulació de biomassa. Els efectes fisiològics i nutricionals de la simbiosi d'A. donax amb FMA també van ser analitzats sota salinitat i carència de fósfor. A més, es van investigar diferents possibilitats per millorar la qualitat del planter d'A. donax per assegurar la supervivència i la producció de biomassa en el primer i segon any de cultiu. Els resultats d'aquesta tesi doctoral mostren que A. donax presenta tolerància a la sequera (a través de canvis morfo-anatòmics i d'ajust osmòtic, i alta eficiència en l'ús de l'aigua), malgrat la biomassa produïda es veu reduïda. A més, A. donax va presentar tolerància a la salinitat moderada i alta eficiència en l'ús del fòsfor. No obstant això, els canvis observats a nivell fisiològic en el simbiont generats per la simbiosi amb R. irregularis i F. mosseae, permeten una major producció de biomassa en comparació de les plàntules no simbionts. La simbiosi amb FMA representa una valuosa eina per augmentar l’èxit de la implementació a camp i augmentar la biomassa de cultiu d'A. donax en terres marginals. L’ús de FMA durant el període d’aclimatació va millorar la producció del plantel, amb taxes de supervivència del 100% en camp i un augment de la producció durant els dos primers anys. No obstant, altres tècniques com l’ús d’alvèols de gran mida i de substrats d’alta qualitat també poden assegurar la supervivència i la producció del cultiu. Considerant tots els resultats en el seu conjunt, es pot concloure que si bé A. donax presenta característiques pròpies d’una espècie d’elevada productivitat: elevades taxes de fotosíntesi, elevada eficiència en l’ús dels recursos (aigua i nutrients, principalment), així com una moderada capacitat de resistència a l’estrès, mantenint produccions considerables que la fan ser una espècie adequada per a la producció de biomassa en terres marginals, la simbiosi amb FMA representa una eina interesant per millorar aquestes característiques i assegurar l’èxit del cultiu en les condicions imposades per aquest tipus de terres.[eng] The interest of biofuels has increased in the last decade as an alternative to non-renewable energy sources. However, biofuels presented several social, economic and ecological costs, like competitiveness with food crops and high water and fertilizer needs. The second-generation biofuels based on perennial grasses such as Arundo donax (giant reed) are non-food plant species that should be able to produce large amounts of biomass with low inputs and presented high adaptability to marginal lands. Giant reed is able to adapt to different soil conditions and presents high biomass production under low fertility conditions in Mediterranean areas. However, one of the handicaps of giant reed may be its sensitivity to lack of water and nutrients during the early stages of growth. Arbuscular mycorrhizal fungi (AM) symbiosis has been demonstrated to increase plant resistance to biotic and abiotic soil stresses in a variety of host plants. In this scenario, the aim of this study was to study the ecophysiological responses of A. donax to AM inoculation under control and salt stress conditions. Plant responses were evaluated through the study of the photosynthesis and respiration in vivo and on the levels of primary metabolites, nutritional status and biomass accumulation. Physiological and nutritional AM effects were also tested under salinity stress and lower nutrient inputs. Furthermore, we investigated different possiblities to improve A. donax plantets quality to ensure survival and biomass production in the first and second year of crop. The results of this PhD thesis show that A. donax presents tolerance to drought (through morpho-anatomical and osmotic adjustment, and high water use efficiency). In addition, it presents tolerance to moderate salinity and high phosphorus use efficiency. However, changes observed at the physiological level in the symbiont generated by the symbiosis with R. irregularis and F. mosseae, allowed a higher production of biomass compared to non-symbiotic plantlets. AM inoculation represented a valuable tool to increase the successful implementation and the increase of crop biomass of giant reed on marginal lands. The inoculation of micropropagated A. donax plantlets with AM at the acclimation stage increased the quality of the plantlets, what produced an increment in biomass production the first and the second year in the field. However, the use of bigger cells and better substrates would also ensure the plant survival and the biomass production. I can conclude that despite A. donax is a high productivity species, showing high photosynthetic rates and resources (water and nutrients) use efficiency, as well as a moderate stress resistance even under marginal land conditions, the inoculation with AM may be helpful to enhance all these traits in order to ensure the success of A. donax crop in such stressful areas

A non-K+-solubilizing PGPB (Bacillus megaterium) increased K+ deprivation tolerance in Oryza sativa seedlings by up-regulating root K+ transporters

Potassium is one of the principal macronutrients required by all plants, but its mobility is restricted between soil compartments. Numerous studies have shown that Plant Growth Promoting Bacteria (PGPB) can facilitate nutrient uptake. The present work examined the effects of the PGPB (Bacillus megaterium) on rice plants subjected to potassium deprivation. To study only direct effects of B. megaterium, we first checked its lack of capacity to solubilize soil K. Rice plants were provided with 1.5 mM K (100%) or 0.015 mM K (1%) and growth related parameters, nutrient concentrations and gene expression of K transporters were determined. After two weeks, the 1% K treatment reduced growth of non-inoculated plants by about 50% compared with the 100% K treatment. However, there was no effect of reduced K nutrition on growth of inoculated plants. The reduction in growth in non-inoculated plants was accompanied by a similar reduction in K concentration in both roots and leaves and an overall 80% reduction of the plant potassium concentrations. In inoculated plants a 50% reduction occurred only in leaves. The expression of the K transporters HKT1;1, 1;2, 1;5, 2;2, 2;3 and 2;4 was up-regulated by the inoculation of B. megaterium under K deprivation conditions, explaining their higher K tissue concentrations and growth. Thus, the bacterial strain improved plant potassium nutrition without affecting K availability in the soil. The results demonstrate the potential of this bacteria for using as a biofertilizer to reduce the amount of potassium fertilizers to be applied in the field.A. Romero-Munar is funded by a fellowship from the Government of the Andalusia and the European Social Fund

Dual inoculation with Rhizophagus irregularis and Bacillus megaterium improves maize tolerance to combined drought and high temperature stress by enhancing root hydraulics, photosynthesis and hormonal responses

Climate change is leading to combined drought and high temperature stress in many areas, drastically reducing crop production, especially for high-water-consuming crops such as maize. This study aimed to determine how the co-inoculation of an arbuscular mycorrhizal (AM) fungus (Rhizophagus irregularis) and the PGPR Bacillus megaterium (Bm) alters the radial water movement and physiology in maize plants in order to cope with combined drought and high temperature stress. Thus, maize plants were kept uninoculated or inoculated with R. irregularis (AM), with B. megaterium (Bm) or with both microorganisms (AM + Bm) and subjected or not to combined drought and high temperature stress (D + T). We measured plant physiological responses, root hydraulic parameters, aquaporin gene expression and protein abundances and sap hormonal content. The results showed that dual AM + Bm inoculation was more effective against combined D + T stress than single inoculation. This was related to a synergistic enhancement of efficiency of the phytosystem II, stomatal conductance and photosynthetic activity. Moreover, dually inoculated plants maintained higher root hydraulic conductivity, which was related to regulation of the aquaporins ZmPIP1;3, ZmTIP1.1, ZmPIP2;2 and GintAQPF1 and levels of plant sap hormones. This study demonstrates the usefulness of combining beneficial soil microorganisms to improve crop productivity under the current climate-change scenario.This work is a part of the I+D+i Project PID2020-112816RB-I00, financed by Ministerio de Ciencia e Innovación MCIN/AEI/10.13039/501100011033 and FEDER “A way to make Europe”

Biological nitrogen fixation improves production of ATP and nitrogen status in Lotus japonicus

1 página - Poster presentado en Iberian Plant Biology 2023. XVIII Portuguese-Spanish Congress on Plant Biology and the XXV Meeting of the Spanish Society of Plant Biology. 9-12 Julio 2023, Braga, Portugal.There is a lack of studies unravelling the effect of legume‐rhizobia interactions on plant respiratory energy efficiency. In plants, the existence of the alternative oxidase pathway (AOP) in the mETC confers metabolic flexibility by regulating the dissipation of reducing equivalents from TCA cycle, helping to maintain redox status and nutrient balance but decreasing yield of respiration. It is thought that carbon requirements of the symbiont and nitrogen transfer to the plant from nodules may affect the activities of both cytochrome oxidase pathway (COP) and AOP in plant organs for the benefit of plant yield in N poor soils. The main objective of this research was to create different plant N status by growing plants of WT Lotus japonicus at 5 mM and 10 mM KNO3, and in symbiosis with Mesorhizobium meliloti (0 mM KNO3). Besides, plants displaying spontaneous nodule formation (snf) mutations were grown at 1 mM KNO3. By isotope‐ratio mass spectrometry, we evaluated discrimination against 18O during respiration, and δ13C and δ14N in plant organs to determine ATP synthesis, changes in plant C economy, and N transfer from nodules. By high‐performance liquid chromatography (HPLC) and inductively coupled plasma (ICP) spectrometry, we also determined the content of NADH and nutrients as proxies of changes in TCA cycle activity and in plant nutrient economy. Our results indicated that nitrogen is vital in the modulation of respiratory metabolism, and that symbiosis improves production of ATP via COP, probably due to an incremented photosynthetic demand of symbiont for inorganic carbon and improved N status. Overall, our results shed some light into the complexity of legume‐rhizobia interactions involving plant respiration and essential plant nutrients

Effect of nitrogen supply and Rhizobia symbiosis in the isotopic composition of essential plant elements, nutrient content, TCA cycle activity and respiratory energy balance of Lotus japonicus

Resumen de la comunicación oral presentada en: I Spanish-Portuguese Congress on Beneficial Plant-Microbe Interactions (BeMiPlant) and XVIII National Meeting of the Spanish Society of Nitrogen Fixation (XVIII SEFIN). Oeiras, Portugal, 17-19 octubre (2022)This work was supported by FONDECYT No. 1191118 from National Agency for Research and Development (ANID) and the Chilean Scholarship Program/Becas de doctorado nacional/2017–21180329

In vivo metabolic regulation of alternative oxidase under nutrient deficiency-interaction with arbuscular mycorrhizal fungi and Rhizobium bacteria

The interaction of the alternative oxidase (AOX) pathway with nutrient metabolism is important for understanding how respiration modulates ATP synthesis and carbon economy in plants under nutrient deficiency. Although AOX activity reduces the energy yield of respiration, this enzymatic activity is upregulated under stress conditions to maintain the functioning of primary metabolism. The in vivo metabolic regulation of AOX activity by phosphorus (P) and nitrogen (N) and during plant symbioses with Arbuscular mycorrhizal fungi (AMF) and Rhizobium bacteria is still not fully understood. We highlight several findings and open questions concerning the in vivo regulation of AOX activity and its impact on plant metabolism during P deficiency and symbiosis with AMF. We also highlight the need for the identification of which metabolic regulatory factors of AOX activity are related to N availability and nitrogen-fixing legume-rhizobia symbiosis in order to improve our understanding of N assimilation and biological nitrogen fixation