Cultivated Tomato (Solanum lycopersicum L.) Suffered a Severe Cytoplasmic Bottleneck during Domestication: Implications from Chloroplast Genomes

In various crops, genetic bottlenecks occurring through domestication can limit crop resilience to biotic and abiotic stresses. In the present study, we investigated nucleotide diversity in tomato chloroplast genome through sequencing seven plastomes of cultivated accessions from the Campania region (Southern Italy) and two wild species among the closest (Solanum pimpinellifolium) and most distantly related (S. neorickii) species to cultivated tomatoes. Comparative analyses among the chloroplast genomes sequenced in this work and those available in GenBank allowed evaluating the variability of plastomes and defining phylogenetic relationships. A dramatic reduction in genetic diversity was detected in cultivated tomatoes, nonetheless, a few de novo mutations, which still differentiated the cultivated tomatoes from the closest wild relative S. pimpinellifolium, were detected and are potentially utilizable as diagnostic markers. Phylogenetic analyses confirmed that S. pimpinellifolium is the closest ancestor of all cultivated tomatoes. Local accessions all clustered together and were strictly related with other cultivated tomatoes (S. lycopersicum group). Noteworthy, S. lycopersicum var. cerasiforme resulted in a mixture of both cultivated and wild tomato genotypes since one of the two analyzed accessions clustered with cultivated tomato, whereas the other with S. pimpinellifolium. Overall, our results revealed a very reduced cytoplasmic variability in cultivated tomatoes and suggest the occurrence of a cytoplasmic bottleneck during their domestication

The complete plastome sequences of eleven Capsicum genotypes: Insights into DNA variation and molecular evolution

Members of the genus Capsicum are of great economic importance, including both wild forms and cultivars of peppers and chilies. The high number of potentially informative characteristics that can be identified through next-generation sequencing technologies gave a huge boost to evolutionary and comparative genomic research in higher plants. Here, we determined the complete nucleotide sequences of the plastomes of eight Capsicum species (eleven genotypes), representing the three main taxonomic groups in the genus and estimated molecular diversity. Comparative analyses highlighted a wide spectrum of variation, ranging from point mutations to small/medium size insertions/deletions (InDels), with accD, ndhB, rpl20, ycf1, and ycf2 being the most variable genes. The global pattern of sequence variation is consistent with the phylogenetic signal. Maximum-likelihood tree estimation revealed that Capsicum chacoense is sister to the baccatum complex. Divergence and positive selection analyses unveiled that protein-coding genes were generally well conserved, but we identified 25 positive signatures distributed in six genes involved in different essential plastid functions, suggesting positive selection during evolution of Capsicum plastomes. Finally, the identified sequence variation allowed us to develop simple PCR-based markers useful in future work to discriminate species belonging to different Capsicum complexes

The mediterranean sea we want

open58siThis paper presents major gaps and challenges for implementing the UN Decade of Ocean Science for Sustainable Development (2021-2030) in the Mediterranean region. The authors make recommendations on the scientific knowledge needs and co-design actions identified during two consultations, part of the Decade preparatory-phase, framing them in the Mediterranean Sea’s unique environmental and socio-economic perspectives. According to the ‘Mediterranean State of the Environment and Development Report 2020’ by the United Nations Environment Programme Mediterranean Action Plan and despite notable progress, the Mediterranean region is not on track to achieve and fully implement the Sustainable Development Goals of Agenda 2030. Key factors are the cumulative effect of multiple human-induced pressures that threaten the ecosystem resources and services in the global change scenario. The basin, identified as a climate change vulnerability hotspot, is exposed to pollution and rising impacts of climate change. This affects mainly the coastal zones, at increasing risk of extreme events and their negative effects of unsustainable management of key economic assets. Transitioning to a sustainable blue economy is the key for the marine environment’s health and the nourishment of future generations. This challenging context, offering the opportunity of enhancing the knowledge to define science-based measures as well as narrowing the gaps between the Northen and Southern shores, calls for a joint (re)action. The paper reviews the state of the art of Mediterranean Sea science knowledge, sets of trends, capacity development needs, specific challenges, and recommendations for each Decade’s societal outcome. In the conclusions, the proposal for a Mediterranean regional programme in the framework of the Ocean Decade is addressed. The core objective relies on integrating and improving the existing ocean-knowledge, Ocean Literacy, and ocean observing capacities building on international cooperation to reach the “Mediterranean Sea that we want”.openCappelletto M.; Santoleri R.; Evangelista L.; Galgani F.; Garces E.; Giorgetti A.; Fava F.; Herut B.; Hilmi K.; Kholeif S.; Lorito S.; Sammari C.; Lianos M.C.; Celussi M.; D'alelio D.; Francocci F.; Giorgi G.; Canu D.M.; Organelli E.; Pomaro A.; Sannino G.; Segou M.; Simoncelli S.; Babeyko A.; Barbanti A.; Chang-Seng D.; Cardin V.; Casotti R.; Drago A.; Asmi S.E.; Eparkhina D.; Fichaut M.; Hema T.; Procaccini G.; Santoro F.; Scoullos M.; Solidoro C.; Trincardi F.; Tunesi L.; Umgiesser G.; Zingone A.; Ballerini T.; Chaffai A.; Coppini G.; Gruber S.; Knezevic J.; Leone G.; Penca J.; Pinardi N.; Petihakis G.; Rio M.-H.; Said M.; Siokouros Z.; Srour A.; Snoussi M.; Tintore J.; Vassilopoulou V.; Zavatarelli M.Cappelletto M.; Santoleri R.; Evangelista L.; Galgani F.; Garces E.; Giorgetti A.; Fava F.; Herut B.; Hilmi K.; Kholeif S.; Lorito S.; Sammari C.; Lianos M.C.; Celussi M.; D'alelio D.; Francocci F.; Giorgi G.; Canu D.M.; Organelli E.; Pomaro A.; Sannino G.; Segou M.; Simoncelli S.; Babeyko A.; Barbanti A.; Chang-Seng D.; Cardin V.; Casotti R.; Drago A.; Asmi S.E.; Eparkhina D.; Fichaut M.; Hema T.; Procaccini G.; Santoro F.; Scoullos M.; Solidoro C.; Trincardi F.; Tunesi L.; Umgiesser G.; Zingone A.; Ballerini T.; Chaffai A.; Coppini G.; Gruber S.; Knezevic J.; Leone G.; Penca J.; Pinardi N.; Petihakis G.; Rio M.-H.; Said M.; Siokouros Z.; Srour A.; Snoussi M.; Tintore J.; Vassilopoulou V.; Zavatarelli M

Plastid Transformation: New Challenges in the Circular Economy Era

In a circular economy era the transition towards renewable and sustainable materials is very urgent. The development of bio-based solutions, that can ensure technological circularity in many priority areas (e.g., agriculture, biotechnology, ecology, green industry, etc.), is very strategic. The agricultural and fishing industry wastes represent important feedstocks that require the development of sustainable and environmentally-friendly industrial processes to produce and recover biofuels, chemicals and bioactive molecules. In this context, the replacement, in industrial processes, of chemicals with enzyme-based catalysts assures great benefits to humans and the environment. In this review, we describe the potentiality of the plastid transformation technology as a sustainable and cheap platform for the production of recombinant industrial enzymes, summarize the current knowledge on the technology, and display examples of cellulolytic enzymes already produced. Further, we illustrate several types of bacterial auxiliary and chitinases/chitin deacetylases enzymes with high biotechnological value that could be manufactured by plastid transformation

Tobacco Plastid Transformation as Production Platform of Lytic Polysaccharide MonoOxygenase Auxiliary Enzymes

Plant biomass is the most abundant renewable resource in nature. In a circular economy perspective, the implementation of its bioconversion into fermentable sugars is of great relevance. Lytic Polysaccharide MonoOxygenases (LPMOs) are accessory enzymes able to break recalcitrant polysaccharides, boosting biomass conversion and subsequently reducing costs. Among them, auxiliary activity of family 9 (AA9) acts on cellulose in synergism with traditional cellulolytic enzymes. Here, we report for the first time, the production of the AA9 LPMOs from the mesophilic Trichoderma reesei (TrAA9B) and the thermophilic Thermoascus aurantiacus (TaAA9B) microorganisms in tobacco by plastid transformation with the aim to test this technology as cheap and sustainable manufacture platform. In order to optimize recombinant protein accumulation, two different N-terminal regulatory sequences were used: 5′ untranslated region (5′-UTR) from T7g10 gene (DC41 and DC51 plants), and 5′ translation control region (5′-TCR), containing the 5′-UTR and the first 14 amino acids (Downstream Box, DB) of the plastid atpB gene (DC40 and DC50 plants). Protein yields ranged between 0.5 and 5% of total soluble proteins (TSP). The phenotype was unaltered in all transplastomic plants, except for the DC50 line accumulating AA9 LPMO at the highest level, that showed retarded growth and a mild pale green phenotype. Oxidase activity was spectrophotometrically assayed and resulted higher for the recombinant proteins without the N-terminal fusion (DC41 and DC51), with a 3.9- and 3.4-fold increase compared to the fused proteins

Acute stress enhances the expression of neuroprotection- and neurogenesis-associated genes in the hippocampus of a mouse restraint model

Stress arises from an external demand placed on an organism that triggers physiological, cognitive and behavioural responses in order to cope with that request. It is thus an adaptive response useful for the survival of an organism. The objective of this study was to identify and characterize global changes in gene expression in the hippocampus in response to acute stress stimuli, by employing a mouse model of short-term restraint stress. In our experimental design mice were subjected to a one time exposure of restraint stress and the regulation of gene expression in the hippocampus was examined 3, 12 and 24 hours thereafter. Microarray analysis revealed that mice which had undergone acute restraint stress differed from non-stressed controls in global hippocampal transcriptional responses. An up-regulation of transcripts contributing directly or indirectly to neurogenesis and neuronal protection including, Ttr, Rab6, Gh, Prl, Ndufb9 and Ndufa6, was observed. Systems level analyses revealed a significant enrichment for neurogenesis, neuron morphogenesis- and cognitive functions-related biological process terms and pathways. This work further supports the hypothesis that acute stress mediates a positive action on the hippocampus favouring the formation and the preservation of neurons, which will be discussed in the context of current data from the literature

High-level production of single chain monellin mutants with enhanced sweetness and stability in tobacco chloroplasts

Plastid-based MNEI protein mutants retain the structure, stability and sweetness of their bacterial counterparts, confirming the attractiveness of the plastid transformation technology for high-yield production of recombinant proteins. The prevalence of obesity and diabetes has dramatically increased the industrial demand for the development and use of alternatives to sugar and traditional sweeteners. Sweet proteins, such as MNEI, a single chain derivative of monellin, are the most promising candidates for industrial applications. In this work, we describe the use of tobacco chloroplasts as a stable plant expression platform to produce three MNEI protein mutants with improved taste profile and stability. All plant-based proteins were correctly expressed in tobacco chloroplasts, purified and subjected to in-depth chemical and sensory analyses. Recombinant MNEI mutants showed a protein yield ranging from 5% to more than 50% of total soluble proteins, which, to date, represents the highest accumulation level of MNEI mutants in plants. Comparative analyses demonstrated the high similarity, in terms of structure, stability and function, of the proteins produced in plant chloroplasts and bacteria. The high yield and the extreme sweetness perceived for the plant-derived proteins prove that plastid transformation technology is a safe, stable and cost-effective production platform for low-calorie sweeteners, with an estimated production of up to 25–30 mg of pure protein/plant

High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass

Biofuels production from plant biomasses is a complex multi‐step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from hyperthermophilic Bacteria and Archaea and test their application in the bioconversion of an important industrially pretreated biomass feedstock for production of second‐generation biofuels. The selected enzymes, endoglucanase, endoxylanase and β‐glucosidase, were expressed in tobacco plastome with a protein yield upto 75 % of total soluble proteins. The accumulation of endoglucanase gave altered plant phenotypes whose severity was directly linked to the enzyme yield and that was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endoxylanase and β‐glucosidase, produced at very high level without detrimental effects on plant development, were enriched by heat treatment. Both biocatalysts retained the main features of the native or recombinantly expressed enzymes, but resulted more thermophilic than the E. coli recombinant counterparst. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid‐derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid‐derived xylanase, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β‐glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher. The very high production level of hyperthermophilic enzymes, their stability and bioconversion effciencies described in this study demonstrate that plastid transformation represents a real cost‐effective production platform for cellulolytic enzymes

Developmental and extracellular matrix-remodeling processes in rosiglitazone-exposed neonatal rat cardiomyocytes

Objective: The objective of this study was to investigate the effects of rosiglitazone (Avandia®) on gene expression in neonatal rat ventricular myocytes. Materials &amp; methods: Myocytes were exposed to rosiglitazone ex vivo. The two factors examined in the experiment were drug exposure (rosiglitazone and dimethyl sulfoxide vs dimethyl sulfoxide), and length of exposure to drug (½ h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h, 36 h and 48 h). Results: Transcripts that were consistently expressed in response to the drug were identified. Cardiovascular system development, extracellular matrix and immune response are represented prominently among the significantly modified gene ontology terms. Conclusion: Hmgcs2, Angptl4, Cpt1a, Cyp1b1, Ech1 and Nqo1 mRNAs were strongly upregulated in cells exposed to rosiglitazone. Enrichment of transcripts involved in cardiac muscle cell differentiation and the extracellular matrix provides a panel of biomarkers for further analysis in the context of adverse cardiac outcomes in humans. Original submitted 15 November 2013; Revision submitted 14 February 2014 </jats:p