Emisión del gas invernadero óxido nitroso por la simbiosis Rhizobium etli-Phaseolus vulgaris

Abstract

Nitrous oxide (N2O) is a potent greenhouse gas (GHG) and ozone depleting agent (O3) due to its high radiative potential, which is about 310 times higher than that of CO2 (reviewed by Aryal et al., 2020), and its high stability in the atmosphere. With an estimated half-life in the atmosphere of about 116±9 years (Prather et al., 2015), this gas falls into the classification of long-lived greenhouse gases. Although the emission of N2O to the atmosphere is relatively low compared to other gases (0.03 %), due to the above-mentioned characteristics, it is estimated to contribute 6% to global warming, being the third most impactful gas after carbon dioxide (CO2) and methane (CH4) (IPCC, 2019). N2O is produced both in natural environments (mainly oceans, forests and savannahs) and by anthropogenic sources (agriculture, biomass burning, power plants, wastewater treatment plants, combustion engines and nitric acid production). Over the last four decades, N2O emission due to anthropogenic sources has increased substantially, rising from 5.6 Tg N/year in the 1980s to 7.3 Tg N/year in the decade 2007-2016. Up to 87% of this increase is due to direct emission from agriculture (71%) and indirect emission from anthropogenic additions of nitrogen to soils (16%) (Tian et al., 2020). Most natural sources, and those derived from agriculture, emit N2O due to microbial metabolism of nitrogen compounds and the increase in emissions is mainly attributed to the extensive use of nitrogen fertilisers. These compounds are metabolised by soil microorganisms, with nitrification and microbial denitrification being the main processes involved in the emission of this gas. While nitrification occurs under aerobic conditions, denitrification is a process that occurs in oxygenlimited environments. Denitrification consists of the sequential reduction of nitrate (NO3-) or nitrite (NO2-) to nitric oxide (NO), N2O and finally dinitrogen (N2). Four enzymes are involved in this process: respiratory nitrate reductases (Nar or Nap), nitrite reductases (NirS or NirK), nitric oxide reductases (cNor, qNor or CuANor) and nitrous oxide reductase (Nos). This process is a substantial source of their gaseous intermediates NO and N2O. Therefore, knowledge of the microorganisms, as well as the environmental and regulatory factors involved in this process is vital to establish and develop mitigation strategies. In this sense, the "Nitrogen Metabolism in Rhizospheric Bacteria" (NitroRhiz) group of the Department of Soil and Plant Microbiology of the “Estación Experimental of Zaidín” (CSIC) has developed several projects in this line of research. Rhizobia are soil diazotrophic organisms with a very special ability, restricted to a small group of microorganisms, that is to fix atmospheric N2 (inaccessible to most organisms) to a bioavailable form, ammonium (NH4+). In addition, another unique feature of this group is their dual life form, being able to live freely or in association with leguminous plants. This symbiotic association is a great advantage for legumes, as it allows them to thrive in environments with a shortage of nitrogen (N), an essential macronutrient for plants and life on this planet. For this reason, legumes have traditionally been used in crop rotation systems to increase soil fertility. Moreover, these plants are an important source of vegetable protein as well as other nutritionally valuable metabolites and are the basis of diets in some countries. For all these reasons, the rhizobia-legume symbiosis has enormous potential for the development of sustainable agriculture. Recently, however, the NitroRhiz group has demonstrated the ability of soybean (Glycine max) and alfalfa (Medicago truncatula) nodules, two of the most agro-economically important legumes, to produce NO and N2O through denitrification in bacteroids, the specialised forms of rhizobia in the nodules. Specifically, NO (Sánchez et al., 2010) and N2O (Tortosa et al., 2015; 2020) production has been demonstrated in soybean nodules inoculated with Bradyrhizobium diazoefficiens in response to NO3- and flooding. Similarly, N2O production by the Ensifer meliloti-M. truncatula symbiosis, has also been found to be induced in response to NO3- and flooding (Pacheco et al., 2023). Likewise, the importance of copper as a modulating factor of the denitrification process and, therefore, of N2O production has been established in both symbiotic systems (Tortosa et al., 2020; Pacheco et al., 2023). In this Thesis, the study of N2O emission by the Rhizobium etli-Phaseolus vulgaris symbiosis has been addressed. P. vulgaris (common bean) is a legume of great agro-economic importance worldwide. R. etli CE3, derived from R. etli CFN42, is an "incomplete" denitrifier, as it lacks respiratory nitrate reductases (Nar, Nap), which carry out the first step of denitrification (reduction of NO3- to NO2-), as well as nitrous oxide reductase (Nos), the enzyme that carries out the last step of denitrification (reduction of N2O to N2). However, this bacterium possesses the nirK and nor genes, which encode a nitrite reductase (NirK) and a nitric oxide reductase (cNor), respectively (Bueno et al., 2005; Gómez-Hernández et al., 2011). In this Thesis, it has been possible to identify and characterise an assimilatory nitrate reductase (NarB) that reduces NO3- to NO2- in the bacterial cytosol, as well as a NO3-/NO2- transporter (NarK) that allows the extrusion of NO2- from the cytoplasm to the periplasmic space, where, under oxygen (O2)-limited conditions, it will be reduced to NO and N2O by the NirK and cNor enzymes, respectively. Under free-living conditions, the narB (built in this work), nirK and norC (Gómez-Hernández et al, 2011) mutant strains did not emit N2O when grown with NO3- as the sole source of N unlike the WT strain. While strain narK (built in this work), produced less N2O than WT. In addition, it was found that NarB is necessary for NO3- dependent induction of nor genes expression, and that the signal molecule that induces the expression of these genes could be NO. All these results have demonstrated the ability of R. etli to produce N2O from NO3- thanks to the coupling of two fundamental pathways of the N cycle: NO3- assimilation and denitrification. Moreover, in this Thesis it has been demonstrated for the first time that the nodules of common bean plants inoculated with R. etli and irrigated with a solution containing NO3-, emit N2O, while flooding has been ruled out as a factor inducing this emission, due to the high sensitivity of the R. etli-common bean symbiosis to this stress. The involvement of R. etli NarB, NarK, NirK and cNor proteins in N2O release from bean nodules has also been confirmed and demonstrated by using mutant strains in the genes coding for these proteins. The involvement of NarB in N2O emission, both under free living conditions and in symbiosis, has also been demonstrated using a strain overexpressing NarB. Furthermore, the role of NarB and cNor in the modulation of NO levels, a central signalling molecule in plants, has been demonstrated by electron paramagnetic resonance (EPR) spectroscopy analysis of whole nodules. The knowledge generated in this Thesis on N2O emission by the R. etlicommon bean symbiosis increases the knowledge generated by the NitroRhiz group in recent years and has allowed the identification of new systems involved in N2O emission in legumes of great agronomic interest in our country, such as common beans. This knowledge will be very valuable for the design of appropriate N2O emission mitigation strategies for leguminous crops.