Crop improvement through microbial biofertilisers and molecular markers of salt stress

The exponential growth of the population, the trend towards organic food consumption and the increasingly restrictive regulations imposed by the European Union are forcing the agricultural sector to evolve towards more sustainable practices that are less harmful to the environment. In addition to these challenges, farmers need to cope with other biotic and abiotic factors affecting plant productivity, such as drought, diseases and pests. To that end, biotechnological approaches to obtain food in a sustainable way are being explored. The lower Guadalquivir region, at the South of Spain, contains the largest area devoted to intensive agriculture practices in Andalusia. This region faces two main problems, which are high salinity in irrigation water and N contamination by synthetic fertilizers (Paredes et al., 2020). In this work we provide two different biotechnological approaches to address these problems: 1) We have characterized salt resistance in nine rice varieties that are being cultivated in the Guadalquivir paddies. This analysis has been complemented through morphological, physiological and biochemical approaches, using analytical methods such as HPLC and mass chromatography, among others. We have identified metabolites that are overproduced in salt stress conditions and might be used for the early detection of salt stress in the plant. A comparative analysis of the different rice varieties analysed provided valued information about the different tolerance to salt. 2) In order to reduce the use of synthetic nitrogen fertilizers, we tested three bioinoculants that had been previously isolated from cotton soils. First, we characterized them biochemically for the PGPR activities, comprising N2 fixation and production of plant phytohormones. Effectiveness of these bioinoculants was assayed in microcosms experiments. We found a significant plant growth stimulation in two of the three bioinoculants evaluated

Transcriptional regulation of genes involved in the symbiosis between Nostoc and Oryza

Motivation: Cyanobacteria of the genus Nostoc are capable of establishing symbiosis relationships with many different types of plants. In these mutualistic relationships the cyanobacterium provides the plant with fixed nitrogen, while the plant provides the cyanobacterium with protection from hostile environments and carbon compounds as energy for N2 fixation. It has recently been described that Nostoc punctiforme performs a stable symbiosis with Oryza sativa (Álvarez et al., 2020). In order to know the molecular mechanisms involved in the recognition between the plant and the cyanobacterium, a proteomic study was carried out in the early stages of co-culture of both organisms. In this study, proteins with homology to the Nod factors of Rhizobium sp. were identified in Nostoc, which could be related to signaling in the plant. The aim of this work is to study the regulation of the expression of the genes encoding these Nod proteins by means of RT-qPCR. Methods: The expression of Nostoc punctiforme Nod genes was studied in response to the presence of the plant at 1, 2, 3, 5 and 7 days of co-culture. On the one hand, a Nostoc punctiforme culture grown at 25°C in Roux flasks with 1% CO2, continuous illumination and at 30°C was prepared. On the other hand, Oryza sativa seedlings were obtained germination of seeds under axenic conditions. At one week of growth, the seedlings were transplanted into flasks with hydroponic medium. Co-culture was performed by adding a fixed amount of Nostoc to the Oryza culture medium, and incubating the mixture in thermostated chambers at 25°C, 12h light/dark cycles and 75% relative humidity. RNA was extracted from Nostoc samples that had been in contact with the plant. As a control, Nostoc incubated without the plant was used. After RNA retrotranscription, the resulting cDNA was used to evaluate the expression of the genes of interest. Results: It was observed that the expression of certain Nod genes is activated in the presence of Oryza, although there are other Nod genes whose expression remains unchanged in response to inoculation with the plant

Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana

In non-cyanogenic species, the main source of cyanide derives from ethylene and camalexin biosyntheses. In mitochondria, cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolised by the β-Cyanoalanine synthase CYS-C1, catalysing the conversion of cysteine and cyanide to hydrogen sulfide and β- cyanoalanine. The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine(thiol)lyase mitochondrial isoform, OAS-C, which catalyses the incorporation of sulfide to O-acetylserine to produce cysteine, thus generating a cyclic pathway in the mitochondria. The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme, showing defects in root hair formation. Genetic complementation with the OAS-C gene rescues the impairment of root hair elongation restoring the wild type phenotype. The mitochondria compromise their capacity to proper detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant. Consequently, we observe an accumulation of sulfide and cyanide and of the alternative oxidase, which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules. Our results allow us to suggest that the significance of OAS-C is related with its role in the proper sulfide and cyanide detoxification in mitochondria.Ministerio de Ciencia e Innovación BIO2010-15201Junta de Andalucía BIO–27

Cytochrome cM is probably a membrane protein similar to the C subunit of the bacterial nitric oxide reductase

Cytochrome cM was first described in 1994 and its sequence has been found in the genome of manifold cyanobacterial species ever since. Numerous studies have been carried out with the purpose of determining its function, but none of them has given place to conclusive results so far. Many of these studies are based on the assumption that cytochrome cM is a soluble protein located in the thylakoid lumen of cyanobacteria. In this work, we have reevaluated the sequence of cyto-chrome cM, with our results showing that its most probable 3D structure is strongly similar to that of the C subunit of the bacterial nitric oxide reductase. The potential presence of an α-helix tail, which could locate this protein in the thylakoid membrane, further supports this hypothesis, thus providing a new, unexpected role for this redox protein.Fundación Investigación Universidad de Sevilla FIUS0571000

The singular properties of photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum suggest new alternative functions

Cytochrome c 550 is an extrinsic component in the luminal side of photosystem II (PSII) in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c 550 from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C-terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c 550 show distinctive electrostatic features as compared with cyanobacterial cytochrome c 550 . In addition, co-immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c 550 from P. tricornutum is mainly located in the thylakoid domain of the chloroplast – where it interacts with PSII –, it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO 2 concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes.Ministerio de Economía, Industria y Competitividad BIO2015-64169-PJunta de Andalucía PAIDI BIO-02

Endophytic colonization of rice (Oryza sativa L.) by the symbiotic strain nostoc punctiforme PCC 73102

Cyanobacteria are phototrophic microorganisms able to establish nitrogen-fixing symbiotic associations with representatives of all four of the major phylogenetic divisions of terrestrial plants. Despite increasing knowledge on the beneficial effects of cyanobacteria in rice fields, the information about the interaction between these microorganisms and rice at the molecular and structural levels is still limited.We have used the model nitrogen-fixing cyanobacterium Nostoc punctiforme to promote a long-term stable endophytic association with rice. Inoculation with this strain of hydroponic cultures of rice produces a fast adherence of the cyanobacterium to rice roots. At longer times, cyanobacterial growth in the proximity of the roots increased until reaching a plateau. This latter phase coincides with the intracellular colonization of the root epidermis and exodermis. Structural analysis of the roots revealed that the cyanobacterium use an apoplastic route to colonize the plant cells. Moreover, plant roots inoculated with N. punctiforme show both the presence of heterocysts and nitrogenase activity, resulting in the promotion of plant growth under nitrogen deficiency, thus providing benefits for the plant.Fundación de Investigación de la Universidad de Sevilla FIUS5710000Ministerio de Economía y Competitividad BIO2015-64169-PCorporación Tecnológica de Andalucía BFE1430

Quantitative Proteomics at Early Stages of the Symbiotic Interaction Between Oryza sativa and Nostoc punctiforme Reveals Novel Proteins Involved in the Symbiotic Crosstalk

Symbiosis between cyanobacteria and plants is considered pivotal for biological nitrogen deposition in terrestrial ecosystems. Despite extensive knowledge of the ecology of plant–cyanobacterium symbioses, little is known about the molecular mechanisms involved in recognition between partners. Here, we conducted a quantitative sequential window acquisition of all theoretical fragment ion spectra mass spectrometry pipeline to analyze protein changes in Oryza sativa and Nostoc punctiforme during early events of symbiosis. We found differentially expressed proteins in both organisms linked to several biological functions, including signal transduction, adhesion, defense-related proteins and cell wall modification. In N. punctiforme we found increased expression of 62 proteins that have been previously described in other Nostoc–plant symbioses, reinforcing the robustness of our study. Our findings reveal new proteins activated in the early stages of the Nostoc–Oryza symbiosis that might be important for the recognition between the plant and the host. Oryza mutants in genes in the common symbiosis signaling pathway (CSSP) show reduced colonization efficiency, providing first insights on the involvement of the CSSP for the accommodation of N. punctiforme inside the plant cells. This information may have long-term implications for a greater understanding of the symbiotic interaction between Nostoc and land plants.Fundación General Consejo Superior de Investigaciones Científicas (Programa ComFuturo)Universidad de Sevilla, Junta de Andalucía y fondos FEDER de la Unión Europea UE)-US–138074

Changes in rice rhizosphere and bulk soil bacterial communities in the Doñana wetlands at different growth stages

The Donana ˜ wetlands comprise an emblematic Mediterranean landscape protected as a UNESCO World Heritage Site. Some parts of these wetlands have been transformed into intensive rice cultivation areas, which are currently the most productive rice-growing areas in Europe. We examined the bacterial communities in these domesticated soils as they are key for plant health and productivity and have a strong influence on biochemical cycles. To identify the bacteria, we used metabarcoding analysis coupled with metabolic predictions and cooccurrence networks. This analysis was performed in the bulk and rhizosphere soils during different stages in the growing season. These soil compartments had a greater effect on the bacterial communities than the plant phenological stages. The diversity and richness of the bacterial population inhabiting the rhizosphere was much lower than that in the bulk soil, comprising taxa that were significantly more represented in this soil compartment, such as bacteria from the genus Hydrogenophaga, three genera from the order Rhizobiales, and unclassified genera from the families Desulfocapsaceae and Actinobacteria. Rhizosphere co-occurrence networks revealed a high number of negative connections, indicating unstable bacterial communities that may be highly influenced by biotic and abiotic factors. Rhizosphere networks mostly rely on two taxa belonging to the phyla Proteobacteria and Cyanobacteria, which are the predicted network hubs in this soil compartment. The bulk soil conserved high bacterial diversity and richness that was stable throughout the growth period of rice. Anaerobic bacteria from genera Marmoricola, the uncultured Gemmatimonadota bacteria SDR1034 terrestrial group, Anaerolinea, and the sulphur oxidizer, Thiobacillus were highly represented. This analysis provides valuable information for understanding bacterial diversity in the rhizosphere of rice cultivated in this region, which is critical for enhancing plant growth and productivity.Corporación Tecnológica de Andalucía (CTA) - BFE14300Universidad de Sevilla - VI PPIT-U

Phylogenetic and functional analysis of cyanobacterial Cytochrome c6-like proteins

All known photosynthetic cyanobacteria carry a cytochrome c6 protein that acts transferring electrons from cytochrome b6f complex to photosystem I, in photosynthesis, or cytochrome c oxidase, in respiration. In most of the cyanobacteria, at least one homologue to cytochrome c6 is found, the so-called cytochrome c6B or cytochrome c6C. However, the function of these cytochrome c6-like proteins is still unknown. Recently, it has been proposed a common origin of these proteins as well as the reclassification of the cytochrome c6C group as c6B, renaming the new joint group as cytochrome c6BC. Another homologue to cytochrome c6 has not been classified yet, the formerly called cytochrome c6-3, which is present in the heterocyst-forming filamentous cyanobacteria Nostoc sp. PCC 7119. In this work, we propose the inclusion of this group as an independent group in the genealogy of cytochrome c6-like proteins with significant differences from cytochrome c6 and cytochrome c6BC, with the proposed name cytochrome c6D. To support this proposal, new data about phylogeny, genome localisation and functional properties of cytochrome c6-like proteins is provided. Also, we have analysed the interaction of cytochrome c6-like proteins with cytochrome f by isothermal titration calorimetry and by molecular docking, concluding that c6-like proteins could interact with cytochrome b6f complex in a similar fashion as cytochrome c6. Finally, we have analysed the reactivity of cytochrome c6-like proteins with membranes enriched in terminal oxidases of cyanobacteria by oxygen uptake experiments, concluding that cytochrome c6D is able to react with the specific copper-oxidase of the heterocysts, the cytochrome c oxidase 2.Fundación de Investigación de la Universidad de Sevilla FIUS05710000Junta de Andalucía PAIDI AGR-288Consejo Superior de Investigaciones Científicas 2022527

Cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the transcriptional profile in arabidopsis

In Arabidopsis thaliana, DES1 is the only identified l-Cysteine desulfhydrase located in the cytosol, and it is involved in the degradation of cysteine and the concomitant production of H2S in this cell compartment. Detailed characterization of the T-DNA insertion mutants des1-1 and des1-2 has provided insight into the role of sulfide metabolically generated in the cytosol as a signaling molecule. Mutations of L-CYS DESULFHYDRASE 1 (DES1) impede H2S generation in the Arabidopsis cytosol and strongly affect plant metabolism. Senescence-associated vacuoles are detected in mesophyll protoplasts of des1 mutants. Additionally, DES1 deficiency promotes the accumulation and lipidation of the ATG8 protein, which is associated with the process of autophagy. The transcriptional profile of the des1-1 mutant corresponds to its premature senescence and autophagy-induction phenotypes, and restoring H2S generation has been shown to eliminate the phenotypic defects of des1 mutants. Moreover, sulfide is able to reverse ATG8 accumulation and lipidation, even in wild-type plants when autophagy is induced by carbon starvation, suggesting a general effect of sulfide on autophagy regulation that is unrelated to sulfur or nitrogen limitation stress. Our results suggest that cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the transcriptional profile of Arabidopsis.European Regional Development Fund BIO2010-15201 BFU2009-07368Junta de Andalucía BIO-27