Podcasts about rbcl

It's time!!Happy Dirty Thursday (AKA Dirty Alchemy: Black Friday Edition)For the next 48 hours we have 3 special specials for you:

This is TRAINING 3 of the free 4-Part Training "Sacred Growth: Nurturing Your Online Business like a Food Forest" - Get the Full Training here: https://www.thedirtyalchemy.com/forestAnd, the Regenerative Business Creation Lab is open now!! Click here to get all the details: https://regenerativebusinesscreationlab.com/When you look at a forest it's easy to see a wall of green. But as you spend more time with it your eyes start to see and recognize the different layers, different species, different shades.Whether you decided on an Oak Savannah, Recovering Forest, or Mature Forest from the last video, each of these have 7 layers in them that work together and play their part in the fertility of the overall system.Fertility here I'm using as the aliveness and regeneration of the whole. Your creativity, abundance, impact.If you read my book ‘Regenerative Business' then you may remember that one of Nature's principles is biodiversity. Biodiversity creates a strong, resilient system, and homogeneity causes weakness.A forest isn't just a mono crop of 1 type of tree and nothing else. It is a diverse network of many plants, trees, species, that are collaborating.In a traditional food forest, there are 7 layers that all work together. And by layers, I'm talking actual physical layers that are taking up 3-D space. So instead of acres and acres of corn taking up only 1 grassy layer, there are 7 stacking layers that are cohabitating 1 space.1. First we have the Tall Tree or Canopy LayerThis layer provides large quantities of food + shade.These are your large fruit and nut trees, as well as support trees that help improve your soil such as nitrogen fixers like alders.2. The second layer is the Sub-Canopy or Large Shrub LayerThis is commonly fruit trees like apples.3. The third layer is the Shrub Layer: Here we have shrubs, like raspberries, blueberries, and also medicinal plants. This layer provides a lot of nutrition and healing. It also provides nesting space for wildlife and increases biodiversity.4. Next we have our Herbaceous Layer: This is when we start adding in more traditional food crops like vegetables and flowers for beneficial insects. This layer Dies back in winter - if you have a cold winter - and brings a ton of variety, and where you can lay on things you love and novelty.If you remember from the last video, our Oak Savannah is for those who want way more of this layer. This playful novelty that requires a repeated energy investment. For example, doing a one-off mini workshop just because you want to.5. The 5th layer is the Groundcover Layer: The purpose here is to cover the ground and hold the soil in place so it doesn't wash or blow away in a storm. It also holds moisture in the ground so that you don't have to water as much. Think, strawberries for eating, clover for teas and also being able to be stepped on and acting like a living mulch to protect the soil below.6. The Root Crops Layer gives us grounding staples. Potatoes, garlic, bulbs, onions, sun chokes.7. And finally we have the vining Layer: Vines climb the woody plants and trees — You can think of this layer as a ladder that connects all of the other layers. Vining plants can grow all the way from the ground layer to the tops of tree canopies.For each of these layers we have the operational and marketing side. Kind of like plant growth versus plant pollination and fruiting.For our tall trees - which are the piece we design around, we have our core offers, funnels and launches.I know that the words “funnel” and “launch” can give people hives but it's the most straightforward way to describe them since people know what I'm talking about when I say them. Please sub in Evergreen Ecosystem for funnel and Sacred Attraction Formula for launch if that feels better for you.There's a concept in regenerative agriculture called “Companion Planting” where one plant takes certain nutrients from the soil while the other provides shade or a thick trunk for a vining plant to grow up. You can think of your Food Forest funnel like this - where you're intentionally planting offers and systems to work together harmoniously without your human intervention.Our tall trees give us a clear way to deliver epic transformation and to deepen the relationship with our core people.In a Mature Forest that means your entire business is focused on this.Our sub-canopy adds in another layer of promotions and offers that reenforces our main canopy. Maybe that looks like doing a flash sale on your birthday.The shrub layer is the all-important Community element of business. Communities come in so many shapes and sizes. You can create community through a blog, or on your social media, in a free Facebook group, a paid membership, or your podcast. This is a place where people want to visit and return to to be seen.With the herbaceous layer we have all the novelty that my Oak Savannah people crave. You do all the things - courses, workshops, physical products, more courses, webinars, whatever. And you have to tend to it and replant it and feed it. You work the land for your harvest. With our ground cover we are holding moisture and fertility in the soil. On a personal level that is our magical practices and mindset. On a business systems level that is client attracting things like ads and SEO.Our root layer works below the surface - it's the systems and operations we have in place that when done right - we plant once and it continues to feed us for years.And finally our vines. Touching every layer of our business is the people that support the business' mission - the team. Our vines, our team maximize production within the area of planting. They help us to make the most of space and time within the system we have created. If you've been on the RBCL sales page then you know that there are more things at play than just the food forest - other species and elements.For example we have to acknowledge the flowers that each of these layers put out — this echoes how our branding and messaging is woven into each layer of our business. With a Mature Forest we have super consistent branding and marketing since we're centralized around 1 or a few core offers. Versus our Oak Savannah that has all sorts of crops and plants and flowers so we get to play and develop different messaging and branding across the business.There are also important elements like sun and water that your forest needs. These are the people or the visibility needed for your ecosystem to thrive. Because if you don't have people interacting with your business, it shrivels up and dies, as you as the gardener keep pouring your energy into it.Head on over to the RBCL info page if you want to play with our interactive forest image with hotspots explaining the different pieces at play in your business ecosystem.So now you have your quiz results, your chosen food forest type, and the 7 layers to fill. Take out your journal, or download this video's workbook to fill in your 7 layers + identify what may be lacking in your business now.After that - head to the next video to address arguably the most important part of your ecosystem - the climate and soil quality - AKA what energy and programming you're seeding your forest with.

This episode we're join by RBCL founder Moonie Spot & his partner Basil about the league & the positive effect its had on the community.

In the present study, the structure and mechanism of two assembly chaperones of Rubisco, Raf1 and RbcX, were investigated. The role of Raf1 in Rubisco assembly was elucidated by analyzing cyanobacterial and plant Raf1 with a vast array of biochemical and biophysical techniques. Raf1 is a dimeric protein. The subunits have a two-domain structure. The crystal structures of two separate domains of Arabidopsis thaliana (At) Raf1 were solved at resolutions of 1.95 Å and 2.6–2.8 Å, respectively. The oligomeric state of Raf1 proteins was investigated by size exclusion chromatography connected to multi angle light scattering (SEC-MALS) and native mass spectrometry (MS). Both cyanobacterial and plant Raf1 are dimeric with an N-terminal domain that is connected via a flexible linker to the C-terminal dimerization domain. Both Raf1 poteins were able to promote assembly of cyanobacterial Rubisco in an in vitro reconstitution system. The homologous cyanobacterial system resulted in very high yields of active Rubisco (>90%), showing the great efficiency of Raf1 mediated Rubisco assembly. Two distinct oligomeric complex assemblies in the assembly reaction could be identified via native PAGE immunoblot analyses as well as SEC-MALS and native MS. Furthermore, a structure-guided mutational analysis of Raf1 conserved residues in both domains was performed and residues crucial for Raf1 function were identified. A new model of Raf1 mediated Rubisco-assembly could be proposed by analyzing the Raf1-Rubisco oligomeric complex with negative stain electron microscopy. The final model was validated by determining Raf1-Rubisco interaction sites using chemical crosslinking in combination with mass spectrometry. Taken together, Raf1 acts downstream of chaperonin-assisted Rubisco large subunit (RbcL) folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by Rubisco small subunit (RbcS) results in holoenzyme formation. In the second part of this thesis, the role of eukaryotic RbcX proteins in Rubisco assembly was investigated. Eukaryots have two distinct homologs of RbcX, RbcX-I and RbcX-II. Both, plant and algal RbcX proteins were found to promote cyanobacterial Rubisco assembly in an in vitro reconstitution system. Mutation of a conserved residue important for Rubisco assembly in cyanobacterial RbcX also abolished assembly by eukaryotic RbcX, underlining functional similarities among RbcX proteins from different species. The crystal structure of Chlamydomonas reinhardtii (Cr) RbcX was solved at a resolution of 2.0 Å. RbcX forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Structural analysis of a fusion protein of CrRbcX and the C-terminal peptide of RbcL suggests that the peptide binding mode of CrRbcX may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution. Preliminary analysis of RbcX in Chlamydomonas indicated that the protein functions as a Rubisco assembly chaperone in vivo. Therefore, RbcX was silenced using RNAi in Chlamydomonas which resulted in a photosynthetic growth defect in several transformants when grown under light. RbcX mRNA levels were highly decreased in these transformants which resulted in a concomitant decrease of Rubisco large subunit levels. Biochemical and structural analysis from both independent studies in this thesis show that Raf1 and RbcX fulfill similar roles in Rubisco assembly, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.

To become biologically active, a protein must fold into a distinct three-dimensional structure. Many non-native proteins require molecular chaperones to support folding and assembly. These molecular chaperones are important for de novo protein folding as well as refolding of denatured proteins under stress conditions. A certain subset of chaperones, the chaperonins, are required for the folding of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco); furthermore, correct folding of Rubisco is also aided by the Hsp70 chaperone system. Rubisco catalyzes the initial step of CO2 assimilation in the Calvin-Benson-Bassham (CBB) cycle. Unfortunately, this enzyme is extremely inefficient, not only does it exhibit a slow catalytic rate (three CO2 molecules fixed per second per Rubisco) but it also discriminates poorly between the assimilation of CO2 and O2 to its sugar-phosphate substrate ribulose-1,5-bisphosphate (RuBP), the latter resulting in loss of photosynthetic efficiency. Due to these inefficiencies, carbon fixation by Rubisco is the rate limiting step of the CBB cycle. Photosynthetic organisms must produce tremendous amounts of Rubisco to alleviate these shortcomings; therefore significant quantities of nitrogen stores are invested in the production of Rubisco making Rubisco the most abundant protein on earth. These drawbacks of Rubisco have important implications in increasing CO2 concentrations and temperatures in the context of global warming. The ability to engineer a more efficient Rubisco could potentially reduce photosynthetic water usage, increase plant growth yield, and reduce nitrogen usage is plants. However, eukaryotic Rubisco cannot fold and assemble outside of the chloroplast, hindering advancements in creating a more efficient Rubisco. Form I Rubisco, found in higher plants, algae, and cyanobacteria, is a hexadecameric complex consisting of a core of eight ~50 kDa large subunits (RbcL), which is capped by four ~15 kDa small subunits (RbcS) on each end. The discovery of a Rubisco-specific assembly chaperone, RbcX, has lead to a better understanding of the components necessary for the form I Rubisco assembly process. RbcX is a homodimer of ~15 kDa subunits consisting of four α- helices aligned in an anti-parallel fashion along the α4 helix. RbcX2 functions as a stabilizer of folded RbcL by recognizing a highly conserved C-terminal sequence of RbcL: EIKFEFD, termed the C-terminal recognition motif. As has been demonstrated by studies of cyanobacterial Rubisco, de novo synthesized RbcL is folded by the chaperonins, whereupon RbcX2 stabilizes the folded RbcL monomer upon release from the folding cavity and then assists in the formation of the RbcL8 core. RbcX2 forms a dynamic complex with RbcL8 and as a result, RbcX2 is readily displaced by RbcS docking in an ATP-independent manner, thereby creating the functional holoenzyme. However, the exact mechanism by which RbcS binding displaces RbcX2 from the RbcL8 core is still unknown. Furthermore, though much advancement has been made in the understanding of form I Rubisco folding and assembly, an exact and detailed mechanism of form I Rubisco assembly is still lacking. The highly dynamic complex of RbcL/RbcX is critical for the formation of the holoenzyme; however it has hindered attempts to characterize critical regions of RbcL that interact with the peripheral regions of RbcX2. An important observation arose when heterologous RbcL and RbcX2 components interacted; a stable complex could form enabling in depth characterization of the RbcL/RbcX2 interaction. In the present study, the detailed structural mechanism of RbcX2-mediated cyanobacterial form I Rubisco assembly is elucidated. To obtain molecular insight into the RbcX2-mediated assembly process of cyanobacterial form I Rubisco, cryo-EM and crystallographic studies in concert with mutational analysis were employed by taking advantage of the high affinity interaction between RbcL and RbcX2 in the heterologous system (Synechococcus sp. PCC6301 RbcL and Anabaena sp. CA RbcX2). Structure guided mutational analysis based on the 3.2 Å crystal structure of the RbcL8/(RbcX2)8 assembly intermediate were utilized to determine the precise interaction site between the body of RbcL and the peripheral region of RbcX2. From these studies a critical salt bridge could be identified that functions as a guidepoint for correct dimer formation, and it was observed that RbcX2 exclusively mediates Rubisco dimer assembly. Furthermore, the mechanism of RbcX2 displacement from the RbcL8 core by RbcS binding was elucidated as well as indications of how RbcS docking on the RbcL8 core is imperative for full form I Rubisco catalytic function by stabilizing the enzymatically competent conformation of an N-terminal loop of Rubisco termed the ‘60ies loop’. Finally, initial attempts in in vitro reconstitution of eukaryotic Rubisco are reported along with the characterization of Arabidopsis thaliana RbcX2 binding to the C-terminal recognition motif of the Rubisco large subunit from various species.

Genetic crosses between the dioecious Bryonia dioica Jacq. (Cucurbitaceae) and the monoecious B. alba L. in 1903 provided the first clear evidence for Mendelian inheritance of dioecy and made B. dioica the classic case of XY sex determination in plants. We use chloroplast (cp) and nuclear (nr) DNA sequences from 129 individuals representing all morphological species to study species relationships and distribution, sexual system evolution, and association of ploidy-level with dioecy in Bryonia. Chloroplast and nuclear trees mostly fit morphological species concepts; there are seven dioecious and three monoecious species, together ranging from the Canary Islands to Central Asia. Bryonia verrucosa, the morphologically most differing species from the Canary Islands is sister to all other species. Our data argue for the inclusion of the narrowly endemic Central Asian species B. lappifolia and B. melanocarpa in B. monoica. Conflicts between cp and nr topologies imply that the dioecious hexaploid B. cretica arose from hybridization(s) involving the diploid species B. dioica, B. syriaca, and/or B. multiflora. The tetraploid B. marmorata likely originated via autopolyploidy. The nr phylogeny implies at least two transitions between dioecy and monoecy, but no correlation between change in sexual system and ploidy level. Fossil-calibrated molecular clocks using family-wide rbcL data with a Bryonia-centered sampling suggest that the deepest divergence in Bryonia occurred ca. ten million years ago and that monoecious and dioecious species crossed in the classic studies are separated by several million years of evolution. Traits, such as annual regrowth from a tuberous rootstock and other adaptations to a seasonal climate, as well as species and haplotype abundance, point to an origin of Bryonia in the Middle East. Species and haplotype poverty north of the Alps together suggest recolonization there after the last glacial maximum. Most species of Bryonia have 10 chromosomes (as confirmed by my own counts), and there appears to be no morphologically distinct pair that would represent the sex chromosomes. However, we know from the crossings carried out by Correns and others that in B. dioica, sex shows monofactorial dominant inheritance, setting up the hypothesis that B. dioica may have a pair of chromosomes on which key sex-determining gene(s) and sexlinked genes have accumulated. To gain insight into the possible presence of such a pair of sex chromosomes in B. dioica, it is necessary to sequence a fairly long sex-linked region to study its substitution behavior and to eventually visualize its physical placement using FISH. As a first step towards this goal, I developed a sex-linked SCAR marker for B. dioica from AFLP bands and sequenced it for individuals representing the full distribution range of the species from Scotland to North Africa. The region north of the Alps harbours distinct Y and X alleles that differ in a 197-bp indel, with the Y allele being perfectly linked to the male sex. In southern Europe, however, the XY system appears to break down (to an extent that is not clear), and there are signs of recombination between the Y and X homologues. Population genetic analyses suggest that the sex-linked region I amplified (i.e., the SCAR marker) experienced different evolutionary pressures in northern and southern Europe. These findings fit the evidence from my phylogenetic and phylogeographic analyses that the XY system in Bryonia is evolutionarily labile. Overall, my work suggests that Bryonia may be a good, but very complex, system in which to study the early steps of plant sex chromosome evolution.

To become biologically active, proteins have to acquire their correct three-dimensional structure by folding, which is frequently followed by assembly into oligomeric complexes. Although all structure relevant information is contained in the amino acid sequence of a polypeptide, numerous proteins require the assistance of molecular chaperones which prevent the aggregation and promote the efficient folding and/or assembly of newly-synthesized proteins. The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which catalyzes carbon fixation in the Calvin-Benson-Bassham cycle, requires chaperones in order to acquire its active structure. In plants and cyanobacteria, RuBisCO (type I) is a complex of approximately 550 kDa composed of eight large (RbcL) and eight small (RbcS) subunits. Remarkably, despite the high abundance and importance of this enzyme, the characteristics and requirements for its folding and assembly pathway are only partly understood. It is known that folding of RbcL is accomplished by chaperonin and most likely supported by the Hsp70 system, whereas recent findings indicate the additional need of specific chaperones for assembly. Nevertheless, this knowledge is incomplete, reflected by the fact that in vitro reconstitution of hexadecameric RuBisCO or synthesis of functional plant RuBisCO in E. coli has not been accomplished thus far. In this thesis, attempts to reconstitute type I RuBisCO in vitro did not result in production of active enzyme although a variety of reaction conditions and additives as well as chaperones of different kind, origin and combination were applied. The major obstacle for reconstitution was found to be the incapability to produce RbcL8 cores competent to form RbcL8S8 holoenzyme. It could be shown that the RbcL subunits interact properly with the chaperonin GroEL in terms of binding, encapsulation and cycling. However, they are not released from GroEL in an assembly-competent state, leading to the conclusion that a yet undefined condition or (assembly) factor is required to shift the reaction equilibrium from GroEL-bound RbcL to properly folded and released RbcL assembling to RbcL8 and RbcL8S8, respectively. Cyanobacterial RbcX was found to promote the production of cynanobacterial RbcL8 core complexes downstream of chaperonin-assisted RbcL folding, both in E. coli and in an in vitro translation system. Structural and functional analysis defined RbcX as a homodimeric, arc-shaped complex of approximately 30 kDa, which interacts with RbcL via two distinct but cooperating binding regions. A central hydrophobic groove recognizes and binds a specific motif in the exposed C-terminus of unassembled RbcL, thereby preventing the latter from uncontrolled misassembly and establishing further contacts with the polar peripheral surface of RbcX. These interactions allow optimal positioning and interconnection of the RbcL subunits, resulting in efficient assembly of RbcL8 core complexes. As a result of the highly dynamic RbcL-RbcX interaction, RbcS can displace RbcX from the core-complexes to produce active RbcL8S8 holoenzyme. Species-specific co-evolution of RbcX with RbcL and RbcS accounts for limited interspecies exchangeability of RbcX and for RbcX-supported or -dependent assembly modes, respectively. In summary, this study helped to specify the problem causing prevention of proper in vitro reconstitution of type I RuBisCO. Moreover, the structural and mechanistic properties of RbcX were analyzed, demonstrating its function as specific assembly chaperone for cyanobacterial RuBisCO. Since the latter is very similar to RuBisCO of higher plants, this work may not only augment the general understanding of type I RuBisCO synthesis, but it might also contribute to advancing the engineering of catalytically more efficient crop plant RuBisCO both in heterologous systems and in planta.

Chloroplast gene expression is predominantly regulated at the posttranscriptional levels of mRNA stability and translation efficiency. The expression of psbA, an important photosynthesis-related chloroplast gene, has been revealed to be regulated via its 5’- untranslated region (UTR). Some cis-acting elements within this 5’UTR and the correlated trans-acting factors have been defined in Chlamydomonas. However, no in vivo evidence with respect to the cis-acting elements of the psbA 5’UTR has been so far achieved in higher plants such as tobacco. To attempt this, we generated a series of mutants of the tobacco psbA 5’UTR by base alterations and sequence deletions, with special regard to the stem-loop structure and the putative target sites for ribosome association and binding of nuclear regulatory factors. In addition, a versatile plastid transformation vector pKCZ with an insertion site in the inverted repeat region of the plastid genome was constructed. In all constructs, the psbA 5’UTR (Wt or modified) was used as the 5’ leader of the reporter gene uidA under control of the same promoter, Prrn, the promoter of the rRNA operon. Through biolistic DNA delivery to tobacco chloroplasts, transplastomic plants were obtained. DNA and RNA analyses of these transplastomic plants demonstrated that the transgenes aadA and uidA had been correctly integrated into the plastome at the insertion site, and transcribed in discrete sizes. Quantitative assays were also done to determine the proportion of intact transplastome, the uidA mRNA level, Gus activity, and uidA translation efficiency. The main results are the following: 1) The insertion site at the unique MunI between two tRNA genes (trnR-ACG and trnNGUU) is functional. Vector pKCZ has a large flexibility for further DNA manipulations and hence is useful for future applications. 2) The stem-loop of the psbA 5’UTR is required for mRNA stabilisation and translation. All mutants related to this region showed a 2~3 fold decrease in mRNA stability and a 1.5~6 fold reduction in translation efficiency. The function of this stem-loop depends on its correct sequence and secondary conformation. 3) the AU-box of the psbA 5’UTR is a crucial translation determinant. Mutations of this element almost abolished translation efficacy (up to 175-fold decrease), but did not significantly affect mRNA accumulation. The regulatory role of the AU-Box is sequencedependent and might be affected by its inner secondary structure. 4) The internal AUG codon of the psbA 5’UTR is unable to initiate translation. An introduction of mRNA translatability from this codon failed to direct the translation of reporter uidA gene, overriding the mutation of the AU-Box. 5) The 5’end poly(A) sequence does not confer a distinct regulatory signal. The deletion of this element only insignificantly affected mRNA abundance and translation. However, this mutation might slightly disturb the conformation of the stem-loop, resulting in a moderate decrease in translation efficiency (~1.5 fold). 6) The SD(Shine-Dalgarno)-like RBS (ribosome binding site) of the psbA 5’UTR appears to be an indispensable element for translation initiation. Mutation of this element led to a dramatically low expression of the uidA gene as seen by Gus staining. 7) The 5’end structural sequence of the rbcL 5’UTR does not convey a high mRNA stabilising effect to the psbA 5’UTR in a cycling condition of the light and the dark. Their distinct roles appear to be involved in darkness adaptation. Furthermore, with respect to the overall regulatory function of the psbA 5’UTR, two models are proposed, i.e. dual RBS-mediated translation initiation, and cpRBPs-mediated mRNA stability and translation. The mechanisms for mRNA stabilisation entailed by the rbcL 5’UTR are also discussed. Direct repeat-mediated transgene loss after chloroplast transformation and other aspects related to the choice of insertion site and plastid promoter are also analysed.