Cytochrome c 6-like protein as a putative donor of electrons to photosystem I in the cyanobacterium Nostoc sp. PCC 7119

Most organisms performing oxygenic photosynthesis contain either cytochrome c 6 or plastocyanin, or both, to transfer electrons from cytochrome b 6-f to photosystem I. Even though plastocyanin has superseded cytochrome c 6 along evolution, plants contain a modified cytochrome c 6, the so called cytochrome c 6A, whose function still remains unknown. In this article, we describe a second cytochrome c 6 (the so called cytochrome c 6-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c 6A than to cyanobacterial cytochrome c 6. In this article, we conclude that the cytochrome c 6-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c 6 and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c 6 and plastocyanin. In any way, the Cyt c 6-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.Dirección General de Investigación Científica y Técnica BFU2006-01361/BMCJunta de Andalucía PAI BIO022 BIO19

Diversity of N2-fixing cyanobacteria from Andalusian paddy fields and analysis of their potential as bioinoculants

The marshes of the Guadalquivir River contain the largest area of rice cultivation in Spain, where more than 40,000 ha are used every year for rice production. These wetland areas provide a perfect place for rice cultivation, and represent a unique aquaticterrestrial habitat that hold more wintering waterfowl than any other European wetland. Paddies require large amounts nitrogen and phosphorus for their growth, development and production. Though, flooded conditions used for rice cultivation drastically diminish efficiency inorganic nitrogen fertilizers, being only 30–40% used by the plant, and in some cases even less. Large amounts of nitrogen fertilizers are dissolved in the surface water and lost, causing environmental pollution and health problems due to losses through N2O and NO volatilization, denitrification, and leaching (Ishii et al., 2011)

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

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

Cyt c6-3: A new isoform of photosynthetic Cyt c6 exclusive to heterocyst-forming cyanobacteria

All known cyanobacteria contain Cyt c6, a small soluble electron carrier protein whose main function is to transfer electrons from the Cyt b6 f complex to PSI, although it is also involved in respiration. We have previously described a second isoform of this protein, the Cyt c6-like, whose function remains unknown. Here we describe a third isoform of Cyt c6 (here called Cytc6-3), which is only found in heterocyst- forming filamentous cyanobacteria. Cyt c6-3 is expressed in vegetative cells but is specifically repressed in heterocysts cells under diazotrophic growth conditions. Although there is a close structural similarity between Cyt c6-3 and Cyt c6 related to the general protein folding, Cyt c6-3 presents differential electrostatic surface features as compared with Cyt c6, its expression is not copper dependent and has a low reactivity towards PSI. According to the different expression pattern, functional reactivity and structural properties, Cyt c6-3 has to play an as yet to be defined regulatory role related to heterocyst differentiation.Fundación de Investigación de la Universidad de Sevilla FIUS05710000Ministerio de Economía y Competitividad BIO2012-35271, BIO2015-64169-PJunta de Andalucía PAIDI BIO-02

Cytochrome c6 is the main respiratory and photosynthetic soluble electron donor in heterocysts of the cyanobacterium Anabaena sp. PCC 7120

Cytochrome c6 is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b6-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c6 and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c6 is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c6 has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c6 deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c6 plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin.Fundación de Investigación de la Universidad de Sevilla FIUS05710000Ministerio de Economía y Competitividad BIO2015-64169-PJunta de Andalucía PAIDI BIO-02

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

Site-directed Mutagenesis of Cytochromec 6 from Anabaena Species PCC 7119

A number of surface residues of cytochromec 6 from the cyanobacterium Anabaenasp. PCC 7119 have been modified by site-directed mutagenesis. Changes were made in six amino acids, two near the heme group (Val-25 and Lys-29) and four in the positively charged patch (Lys-62, Arg-64, Lys-66, and Asp-72). The reactivity of mutants toward the membrane-anchored complex photosystem I was analyzed by laser flash absorption spectroscopy. The experimental results indicate that cytochrome c 6 possesses two areas involved in the redox interaction with photosystem I: 1) a positively charged patch that may drive its electrostatic attractive movement toward photosystem I to form a transient complex and 2) a hydrophobic region at the edge of the heme pocket that may provide the contact surface for the transfer of electrons to P700. The isofunctionality of these two areas with those found in plastocyanin (which acts as an alternative electron carrier playing the same role as cytochromec 6) are evident

The Interactions of Cyanobacterial Cytochromec6 and Cytochrome f, Characterized by NMR

During oxygenic photosynthesis, cytochromec6 shuttles electrons between the membrane-bound complexes cytochrome bf and photosystem I. Complex formation between Phormidium laminosum cytochromef and cytochrome c6 from bothAnabaena sp. PCC 7119 and Synechococcus elongatus has been investigated by nuclear magnetic resonance spectroscopy. Chemical-shift perturbation analysis reveals a binding site on Anabaena cytochrome c6, which consists of a predominantly hydrophobic patch surrounding the heme substituent, methyl 5. This region of the protein was implicated previously in the formation of the reactive complex with photosytem I. In contrast to the results obtained for Anabaena cytochromec6, there is no evidence for specific complex formation with the acidic cytochrome c6 fromSynechococcus. This remarkable variability between analogous cytochromes c6 supports the idea that different organisms utilize distinct mechanisms of photosynthetic intermolecular electron transfer.European Commission HPRN-CT-1999-00095Spanish Ministry of Science and Technology BMC2000-0444Andalusian Government CVI-019

Hongos entomopatógenos: de la agricultura a la conservación del patrimonio histórico

Los métodos tradicionales utilizados en la conservación del patrimonio histórico incluyen principalmente el uso de insecticidas sintéticos, que presentan las desventajas de su toxicidad y de su falta de inocuidad sobre el sustrato a tratar. En este artículo se presenta una alternativa a estos métodos basada en el empleo de sustancias biocidas extraídas o provenientes de hongos entomopatógenos, como son Beauveria bassianay Metarhizium anisopliae. La presente propuesta incluye dos variantes no excluyentes como son el uso de conidios y la aplicación de geles con enzimas y toxinas provenientes de dichas especies. Esta metodología aporta ventajas con respecto a los métodos tradicionales, como la no toxicidad para el manipulador, la aplicabilidad a diferentes sustratos independientemente de su tamaño y localización, y su prácticamente nula reactividad frente al sustrato