The Availability of Organically Reared Livestock in the European Union

The first regulation on organic farming (Council Regulation EEC No 2092/91) was drawn up in 1991, laying down the rules for farmers wishing to claim official recognition of their organic status. Since 1991, this Regulation has been amended on numerous occasions, in particular in August 1999 by Council Regulation (EC) No 1804/1999, which extended its scope to cover organic livestock production. According to this Regulation organic livestock production should take place in organic conditions; namely that livestock must come from production units in the organic production system and throughout their life, this system of production must be applied. However, at the time of implementing these harmonised rules for organic livestock production, the current development of the sector was such that there was not a sufficient range of organically reared livestock species (including both livestock species for production and livestock species for breeding) and breeds available on the market. Section 3 of Part B of Technical Annex I therefore provides a number of derogations to the general principle of organic production, including a derogation that livestock must come from production units in the organic production system (hereafter referred to as ¿the derogation¿). These derogations have been extended and slightly amended on a number of occasions in recent years. However, it is acknowledged that these derogations cannot be extended indefinitely without justification. Moreover, the European Action Plan for Organic Food and Farming clearly states that end dates of the transitional periods for the derogations should be respected to ensure the integrity of organic agriculture. The aim of this Study was to carry out an economic analysis to assess the availability of organically reared livestock in the EU-25 and to evaluate the impact of the removal of the derogation that livestock must come from production units in the organic production system on the economic sustainability of the organic livestock sector in selected EU Member States. A case study methodology was used to carry out this economic analysis, focusing on pig, egg and broiler production systems in selected EU Member States. The Study found that most countries make full use of the derogation on sourcing non-organic livestock. However, the extent to which this derogation is used was found to vary considerably by livestock species and Member State. The notable exceptions to this general rule were: Organic broiler production. In Austria (virtually) all organic broiler production takes place without using the derogation and the UK a significant proportion of organic broiler production takes place without using the derogation. Organic egg production. In the UK a significant proportion of organic egg production takes place without using the derogation. Organic pig production. In Germany, the Netherlands and the UK, a significant proportion of organic pig production takes place without using the derogation. In Portugal, organic pig production was also found to take place without using the derogation, but significantly the organic replacements used were found to originate from units within the organic production system itself and throughout its life this system of production is applied.JRC.DG.J.5-Agriculture and Life Sciences in the Econom

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency

The conserved Fanconi anemia nuclease Fan1 and the SUMO E3 ligase Pli1 act in two novel Pso2-independent pathways of DNA interstrand crosslink repair in yeast

DNA interstrand cross-links (ICLs) represent a physical barrier to the progression of cellular machinery involved in DNA metabolism. Thus, this type of adduct represents a serious threat to genomic stability and as such, several DNA repair pathways have evolved in both higher and lower eukaryotes to identify this type of damage and restore the integrity of the genetic material. Human cells possess a specialized ICL-repair system, the Fanconi anemia (FA) pathway. Conversely yeasts rely on the concerted action of several DNA repair systems. Recent work in higher eukaryotes identified and characterized a novel conserved FA component, FAN1 (Fanconi anemia-associated nuclease 1, or FANCD2/FANCI-associated nuclease 1). In this study, we characterize Fan1 in the yeast Schizosaccharomyces pombe. Using standard genetics, we demonstrate that Fan1 is a key component of a previously unidentified ICL-resolution pathway. Using high-throughput synthetic genetic arrays, we also demonstrate the existence of a third pathway of ICL repair, dependent on the SUMO E3 ligase Pli1. Finally, using sequence-threaded homology models, we predict and validate key residues essential for Fan1 activity in ICL repair

Introduction of a leaky stop codon as molecular tool in Chlamydomonas reinhardtii.

Expression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct

Molecular Physiology of the Chlamydomonas Pyrenoid

Operation of the CO2-concentrating mechanism (CCM) in the model green alga *Chlamydomonas reinhardtii* relies on confining Rubisco in a proteinaceous microcompartment, the chloroplastic pyrenoid. Despite a long history of research, this enigmatic structure lacks a full definition both in terms of physiological function as well as molecular composition. The work presented here used a unique set of pyrenoid-less mutants (which differ from wild-type only with respect to the genes coding for the small subunit of Rubisco) to address two key questions: (i) the function of the pyrenoid in photosynthesis in enabling the CCM to supply Rubisco with inorganic carbon under CO2 limiting conditions; (ii) the mechanism of Rubisco aggregation underlying pyrenoid formation. Firstly, cells that are unable to aggregate Rubisco were found severely limited by access to CO2, yet fully able to compensate any structural changes within the chloroplast. Secondly, *in silico* and *in vitro* analyses of Rubisco protein interactions established that a linking agent is required for Rubisco aggregation. A Rubisco interactome was characterised using native gel electrophoresis and co-IP assays followed by mass spectrometry. In a complementary approach, a forward genetic screen based on high-throughput immunofluorescence localization of Rubisco aimed to identify key pyrenoid assembly factors. The present study combined and developed a wide range of molecular, physiological and computational techniques, to show that Rubisco aggregation forming the pyrenoid is achieved through a complex network of protein interactions in order to effectively supply CO2 via the CCM.BBSRC Studienstiftun

Supplementary Code from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

SI figure 1 from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

SI figure 2 from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

SI figure 4 from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

SI figure 3 from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality