Purple bacteria for the biotechnological valorisation of agro-industrial streams

Waste management worldwide has received increasing attention from global policies in recent years. In particular, agro-industrial streams represent a global concern due to the huge volumes generated and a high number of residues, which produce an environmental and economic impact on the ecosystem. The use of biotechnological approaches to treat these streams could allow the production of desirable by-products to be reinjected into the production cycle through sustainable processes. Purple phototrophic bacteria (PPB) are targeted as microorganisms capable to reduce the pressure of agro-industrial streams on environmental issues, due to their metabolic versatility (autotrophic and/or heterotrophic growth under different conditions). This Ph.D. research project aims to assess the effectiveness of PPB cultivation for industrial streams valorisation in the applications of biogas desulfurization and microbial protein production. For these purposes, the first part of the present work is dedicated to the cultivation of purple sulfur bacteria (PSB) for biogas streams upgrading, cleaning biogas from sulfur compounds (H2S), and producing elemental sulfur (S0), potentially suitable as a slow-release fertilizer. The second part of the thesis, instead, sees the application of purple non-sulfur bacteria (PNSB) on streams rich in organics, such as molasses, generating biomass with high content of proteins and pigments, useful as supplements in animal feed. The assessment of the main metabolic mechanisms involved in the two processes is evaluated at a laboratory scale using flasks and a photobioreactor, to define the consumption of substrates and the accumulation of products both in the autotrophic (on biogas) and in heterotrophic grow (on molasses). In conclusion, the effectiveness of processes employing PPB for a sustainable valorisation of several agro-industrial streams has been proved promising, using actual residues, and coupling their treatments with the production of added-value by-products.

Technological processes study for high value health ingredients and foods production

This PhD thesis describes set up of technological models for obtaining high health value foods and ingredients that preserve the final product characteristics as well as enrich with nutritional components. In particular, the main object of my research has been Virgin Olive Oil (VOO) and its important antioxidant compounds which differentiate it from all other vegetables oils. It is well known how the qualitative and quantitative presence of phenolic molecules extracted from olives during oil production is fundamental for its oxidative and nutritional quality. For this purpose, agronomic and technological conditions of its production have been investigated. It has also been examined how this fraction can be better preserved during storage. Moreover, its relation with VOO sensorial characteristics and its interaction with a protein in emulsion foods have also been studied. Finally, an experimental work was carried out to determine the antioxidative and heat resistance properties of a new antioxidant (EVS-OL) when used for high temperature frying such as is typically employed for the preparation of french fries. Results of the scientific research have been submitted for a publication and some data has already been published in national and international scientific journals.

Physiology and Biotechnology of the Hydrogen Production with the Green Microalga Chlamydomonas reinhardtii

The hydrogen production in the green microalga Chlamydomonas reinhardtii was evaluated by means of a detailed physiological and biotechnological study. First, a wide screening of the hydrogen productivity was done on 22 strains of C. reinhardtii, most of which mutated at the level of the D1 protein. The screening revealed for the first time that mutations upon the D1 protein may result on an increased hydrogen production. Indeed, productions ranged between 0 and more than 500 mL hydrogen per liter of culture (Torzillo, Scoma et al., 2007a), the highest producer (L159I-N230Y) being up to 5 times more performant than the strain cc124 widely adopted in literature (Torzillo, Scoma, et al., 2007b). Improved productivities by D1 protein mutants were generally a result of high photosynthetic capabilities counteracted by high respiration rates. Optimization of culture conditions were addressed according to the results of the physiological study of selected strains. In a first step, the photobioreactor (PBR) was provided with a multiple-impeller stirring system designed, developed and tested by us, using the strain cc124. It was found that the impeller system was effectively able to induce regular and turbulent mixing, which led to improved photosynthetic yields by means of light/dark cycles. Moreover, improved mixing regime sustained higher respiration rates, compared to what obtained with the commonly used stir bar mixing system. As far as the results of the initial screening phase are considered, both these factors are relevant to the hydrogen production. Indeed, very high energy conversion efficiencies (light to hydrogen) were obtained with the impeller device, prooving that our PBR was a good tool to both improve and study photosynthetic processes (Giannelli, Scoma et al., 2009). In the second part of the optimization, an accurate analysis of all the positive features of the high performance strain L159I-N230Y pointed out, respect to the WT, it has: (1) a larger chlorophyll optical cross-section; (2) a higher electron transfer rate by PSII; (3) a higher respiration rate; (4) a higher efficiency of utilization of the hydrogenase; (5) a higher starch synthesis capability; (6) a higher per cell D1 protein amount; (7) a higher zeaxanthin synthesis capability (Torzillo, Scoma et al., 2009). These information were gathered with those obtained with the impeller mixing device to find out the best culture conditions to optimize productivity with strain L159I-N230Y. The main aim was to sustain as long as possible the direct PSII contribution, which leads to hydrogen production without net CO2 release. Finally, an outstanding maximum rate of 11.1 ± 1.0 mL/L/h was reached and maintained for 21.8 ± 7.7 hours, when the effective photochemical efficiency of PSII (ΔF/F'm) underwent a last drop to zero. If expressed in terms of chl (24.0 ± 2.2 µmoles/mg chl/h), these rates of production are 4 times higher than what reported in literature to date (Scoma et al., 2010a submitted). DCMU addition experiments confirmed the key role played by PSII in sustaining such rates. On the other hand, experiments carried out in similar conditions with the control strain cc124 showed an improved final productivity, but no constant PSII direct contribution. These results showed that, aside from fermentation processes, if proper conditions are supplied to selected strains, hydrogen production can be substantially enhanced by means of biophotolysis. A last study on the physiology of the process was carried out with the mutant IL. Although able to express and very efficiently utilize the hydrogenase enzyme, this strain was unable to produce hydrogen when sulfur deprived. However, in a specific set of experiments this goal was finally reached, pointing out that other than (1) a state 1-2 transition of the photosynthetic apparatus, (2) starch storage and (3) anaerobiosis establishment, a timely transition to the hydrogen production is also needed in sulfur deprivation to induce the process before energy reserves are driven towards other processes necessary for the survival of the cell. This information turned out to be crucial when moving outdoor for the hydrogen production in a tubular horizontal 50-liter PBR under sunlight radiation. First attempts with laboratory grown cultures showed that no hydrogen production under sulfur starvation can be induced if a previous adaptation of the culture is not pursued outdoor. Indeed, in these conditions the hydrogen production under direct sunlight radiation with C. reinhardtii was finally achieved for the first time in literature (Scoma et al., 2010b submitted). Experiments were also made to optimize productivity in outdoor conditions, with respect to the light dilution within the culture layers. Finally, a brief study of the anaerobic metabolism of C. reinhardtii during hydrogen oxidation has been carried out. This study represents a good integration to the understanding of the complex interplay of pathways that operate concomitantly in this microalga.

A candidate gene approach to identify DNA markers associated with meat quality and production traits: investigation of several cathepsin genes in Italian heavy pigs.

The cathepsin enzymes represent an important family of lysosomal proteinases with a broad spectrum of functions in many, if not in all, tissues and cell types. In addition to their primary role during the normal protein turnover, they possess highly specific proteolytic activities, including antigen processing in the immune response and a direct role in the development of obesity and tumours. In pigs, the involvement of cathepsin enzymes in proteolytic processes have important effects during the conversion of muscle to meat, due to their influence on meat texture and sensory characteristics, mainly in seasoned products. Their contribution is fundamental in flavour development of dry-curing hams. However, several authors have demonstrated that high cathepsin activity, in particular of cathepsin B, is correlated to defects of these products, such as an excessive meat softness together with abnormal free tyrosine content, astringent or metallic aftertastes and formation of a white film on the cut surface. Thus, investigation of their genetic variability could be useful to identify DNA markers associated with these dry cured hams parameters, but also with meat quality, production and carcass traits in Italian heavy pigs. Unfortunately, no association has been found between cathepsin markers and meat quality traits so far, in particular with cathepsin B activity, suggesting that other genes, besides these, affect meat quality parameters. Nevertheless, significant associations were observed with several carcass and production traits in pigs. A recent study has demonstrated that different single nucleotide polymorphisms (SNPs) localized in cathepsin D (CTSD), F (CTSF), H and Z genes were highly associated with growth, fat deposition and production traits in an Italian Large White pig population. The aim of this thesis was to confirm some of these results in other pig populations and identify new cathepsin markers in order to evaluate their effects on cathepsin activity and other production traits. Furthermore, starting from the data obtained in previous studies on CTSD gene, we also analyzed the known polymorphism located in the insulin-like growth factor 2 gene (IGF2 intron3-g.3072G>A). This marker is considered the causative mutation for the quantitative trait loci (QTL) affecting muscle mass and fat deposition in pigs. Since IGF2 maps very close to CTSD on porcine chromosome (SSC) 2, we wanted to clarify if the effects of the CTSD marker were due to linkage disequilibrium with the IGF2 intron3-g.3072G>A mutation or not. In the first chapter, we reported the results from these two SSC2 gene markers. First of all, we evaluated the effects of the IGF2 intron3-g.3072G>A polymorphism in the Italian Large White breed, for which no previous studies have analysed this marker. Highly significant associations were identified with all estimated breeding values for production and carcass traits (P<0.00001), while no effects were observed for meat quality traits. Instead, the IGF2 intron3-g.3072G>A mutation did not show any associations with the analyzed traits in the Italian Duroc pigs, probably due to the low level of variability at this polymorphic site for this breed. In the same Duroc pig population, significant associations were obtained for the CTSD marker for all production and carcass traits (P < 0.001), after excluding possible confounding effects of the IGF2 mutation. The effects of the CTSD g.70G>A polymorphism were also confirmed in a group of Italian Large White pigs homozygous for the IGF2 intron3-g.3072G allele G (IGF2 intron3-g.3072GG) and by haplotype analysis between the markers of the two considered genes. Taken together, all these data indicated that the IGF2 intron3-g.3072G>A mutation is not the only polymorphism affecting fatness and muscle deposition in pigs. In the second chapter, we reported the analysis of two new SNPs identified in cathepsin L (CTSL) and cathepsin S (CTSS) genes and the association results with meat quality parameters (including cathepsin B activity) and several production traits in an Italian Large White pig population. Allele frequencies of these two markers were evaluated in 7 different pig breeds. Furthermore, we mapped using a radiation hybrid panel the CTSS gene on SSC4. Association studies with several production traits, carried out in 268 Italian Large White pigs, indicated positive effects of the CTSL polymorphism on average daily gain, weight of lean cuts and backfat thickness (P<0.05). The results for these latter traits were also confirmed using a selective genotype approach in other Italian Large White pigs (P<0.01). In the 268 pig group, the CTSS polymorphism was associated with feed:gain ratio and average daily gain (P<0.05). Instead, no association was observed between the analysed markers and meat quality parameters. Finally, we wanted to verify if the positive results obtained for the cathepsin L and S markers and for other previous identified SNPs (cathepsin F, cathepsin Z and their inhibitor cystatin B) were confirmed in the Italian Duroc pig breed (third chapter). We analysed them in two groups of Duroc pigs: the first group was made of 218 performance-tested pigs not selected by any phenotypic criteria, the second group was made of 100 Italian Duroc pigs extreme and divergent for visible intermuscular fat trait. In the first group, the CTSL polymorphism was associated with weight of lean cuts (P<0.05), while suggestive associations were obtained for average daily gain and backfat thickness (P<0.10). Allele frequencies of the CTSL gene marker also differed positively among the visible intermuscular extreme tails. Instead, no positive effects were observed for the other DNA markers on the analysed traits. In conclusion, in agreement with the present data and for the biological role of these enzymes, the porcine CTSD and CTSL markers: a) may have a direct effect in the biological mechanisms involved in determining fat and lean meat content in pigs, or b) these markers could be very close to the putative functional mutation(s) present in other genes. These findings have important practical applications, in particular the CTSD and CTSL mutations could be applied in a marker assisted selection (MAS) both in the Italian Large White and Italian Duroc breeds. Marker assisted selection could also increase in efficiency by adding information from the cathepsin S genotype, but only in the Italian Large White breed.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from Complex I

Evidence accumulated in the last ten years has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes or respirasomes. Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. Following this line of thought we have decided to directly investigate ROS production by Complex I under conditions in which the complex is arranged as a component of the supercomplex I1III2 or it is dissociated as an individual enzyme. The study has been addressed both in bovine heart mitochondrial membranes and in reconstituted proteoliposomes composed of complexes I and III in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. The results of this investigation provide experimental evidence that the production of ROS is strongly increased in either model; supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I . This is the first demonstration that dissociation of the supercomplex I1III2 in the mitochondrial membrane is a cause of oxidative stress from Complex I. Previous work in our laboratory demonstrated that lipid peroxidation can dissociate the supramolecular assemblies; thus, here we confirm that preliminary conclusion that primary causes of oxidative stress may perpetuate reactive oxygen species (ROS) generation by a vicious circle involving supercomplex dissociation as a major determinant.