We present a simple microfluidic device that uses an array of well-defined chambers to immobilize thousands of femtoliter- to picoliter-scale aqueous drops suspended in inert carrier oil. This device ...enables timelapse studies of large numbers of individual drops, while simultaneously enabling subsequent drop recovery.
• Pentatricopeptide repeat (PPR) proteins are modular RNA-binding proteins involved in different aspects of RNA metabolism in organelles. PPR proteins of the PLS subclass often contain C-terminal ...domains that are important for their function, but the role of one of these domains, the E domain, is far from resolved. Here, we elucidate the role of the E domain in CRR2 in plastids.
• We identified a surprisingly large number of small RNAs that represent in vivo footprints of the Arabidopsis PLS-class PPR protein CRR2. An unexpectedly strong base conservation was found in the nucleotides aligned to the E domain. We used both in vitro and in vivo experiments to reveal the role of the E domain of CRR2.
• The E domain of CRR2 can be predictably altered to prefer different nucleotides in its RNA ligand, and position 5 of the E1-motif is biologically important for the PPR–RNA interaction. The ‘code’ of the E domain PPR motifs is different from that of P- and S-motifs.
• The findings presented here show that the E domain of CRR2 is involved in sequence-specific interaction with its RNA ligand and have implications for our ability to predict RNA targets for PLS-PPRs and their use as biotechnological tools to manipulate specific RNAs in vivo.
Chitin is an abundant polysaccharide primarily produced as an industrial waste stream during the processing of crustaceans. Despite the limited applications of chitin, there is interest from the ...medical, agrochemical, food and cosmetic industries because it can be converted into chitosan and partially acetylated chitosan oligomers (COS). These molecules have various useful properties, including antimicrobial and anti-inflammatory activities. The chemical production of COS is environmentally hazardous and it is difficult to control the degree of polymerization and acetylation. These issues can be addressed by using specific enzymes, particularly chitinases, chitosanases and chitin deacetylases, which yield better-defined chitosan and COS mixtures. In this review, we summarize recent chemical and enzymatic approaches for the production of chitosan and COS. We also discuss a design-of-experiments approach for process optimization that could help to enhance enzymatic processes in terms of product yield and product characteristics. This may allow the production of novel COS structures with unique functional properties to further expand the applications of these diverse bioactive molecules.
Summary
The chloroplast is a prime target for genetic engineering in plants, offering various advantages over nuclear transformation. For example, chloroplasts allow the expression of polycistronic ...transcripts and thus to engineer complex metabolic pathways. Each cistron within such a longer transcript needs its own expression elements. Within the 5′‐UTR, such expression elements are needed for stabilizing mRNAs and for translation of the downstream reading frame. One of the few effective expression elements used so far in transplastomic approaches is the intercistronic expression element (IEE). The IEE is derived from the psbT‐psbH intergenic region and includes a target sequence of the RNA binding protein HCF107. We here show that excessive expression of the IEE can lead to specific defects of endogenous chloroplast mRNA stabilization, likely via depletion of HCF107. Key players in chloroplast transcript stabilization and translation are pentatricopeptide repeat (PPR) proteins, which are structurally related to HCF107. PPR proteins that stabilize mRNAs leave behind short RNA footprints that are indicators of their activity. We identified such sRNAs in tobacco, and demonstrate that they are sufficient to stabilize and stimulate translation of mRNAs from synthetic dicistronic transgenes in chloroplasts. Thus, minimal sequence elements are generally adequate to support key steps in chloroplast gene expression, i.e. RNA stability and translation. Furthermore, our analysis expands the repertoire of available expression elements to facilitate the assembly and expression of multi‐gene ensembles in the chloroplast.
Significance Statement
We demonstrate that binding sites for RNA‐binding proteins can be used to differentially activate translation and RNA stability of chloroplast transgenes, which is relevant for any biotechnological approach that utilizes the chloroplast as an expression platform. Intriguingly, overexpression of an sRNA sequence can lead to titration of cognate trans‐acting RNA binding proteins (RBPs) followed by defects in endogenous RNA processing events at least in the case of HCF107. This suggests that RBPs can act as limiting factors of chloroplast RNA metabolism.
Acclimation in plants – the Green Hub consortium Kleine, Tatjana; Nägele, Thomas; Neuhaus, H. Ekkehard ...
Plant journal,
April 2021, 2021-04-00, 20210401, Letnik:
106, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Summary
Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to ...which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to ‘smart breeding’ methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast‐related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.
Significance Statement
Acclimation allows plants to adapt at the physiological level to changes in their environment, and is pivotal to plant fitness, especially in the light of global warming. The Green Hub consortium aims to understand and enhance the protective effects of the cellular networks that mediate acclimation of plants including crops.
Cyanines derived from heptamethines were investigated in combination with iodonium salts as initiators of the radical polymerization of tripropylene glycol diacrylate and epoxides derived from ...bisphenol‐A‐diglycidylether. A new near‐infrared (NIR) LED prototype emitting at 805 nm with an exposure intensity of 1.2 W cm−2 facilitated initiation of both radical and cationic polymerization using sensitizers derived from cyanines. This new light‐emitting device has brought new insight into the photochemistry of cyanines with the general structure 1 because a combination of photonic and thermal processes strongly influences reaction pathways. In particular, cationic cyanines comprising a cyclopentene moiety and diphenylamino group in the center initiated the cationic polymerization of epoxides. Selective oxidation of this unit explains why specifically these derivatives may function as initiators for cationic polymerization. In contrast, when the diphenylamino group was replaced by a barbital group at the meso‐position cationic polymerization of epoxides was not initiated.
Polymerization in a new light: New LED prototypes are used to initiate radical and cationic photopolymerization using cationic polymethine initiators derived from heptamethines. A combination of photonic and thermal processes strongly influences the reaction pathways.
Chloroplast RNA metabolism is controlled and excecuted by hundreds of nuclear-encoded, chloroplast-localized RNA binding proteins. Contrary to the nucleo-cytosolic compartment or bacteria, there is ...little evidence for non-coding RNAs that play a role as riboregulators of chloroplasts. We mined deep-sequencing datasets to identify short (16-28 nt) RNAs in the chloroplast genome and found 50 abundant small RNAs (sRNAs) represented by multiple, in some cases, thousands of sequencing reads, whereas reads are in general absent from the surrounding sequence space. Other than sRNAs representing the most highly abundant mRNAs, tRNAs and rRNAs, most sRNAs are located in non-coding regions and many are found a short distance upstream of start codons. By transcript end mapping we show that the 5' and 3' termini of chloroplast RNAs coincide with the ends of sRNAs. Sequences of sRNAs identified in Arabidopsis are conserved between different angiosperm species and in several cases, we identified putative orthologs in rice deep sequencing datasets. Recently, it was suggested that small chloroplast RNA fragments could result from the protective action of pentatricopeptide repeat (PPR) proteins against exonucleases, i.e. footprints of RNA binding proteins. Our data support this scenario on a transcriptome-wide level and suggest that a large number of sRNAs are in fact remnants of PPR protein targets.
Hybrid bidentate phosphine-phosphorodiamidite ligands based on a chiral Betti base backbone and diphenylphosphinoaniline derivatives have been prepared (BettiPhos). The ligands possess a stereogenic ...P atom at the phosphorodiamidite moiety, whose configuration can be largely controlled by the synthetic route and the choice of base and solvent. The new ligands were applied in the rhodium-catalyzed asymmetric hydroformylation (AHF) of vinyl esters and vinyl amides. Very high enantioselectivities of up to 97% ee accompanied by excellent regioselectivities (up to b/l > 1000) were obtained using the BettiPhos ligand (S C,S C,R P,S C)-4b bearing an additional chiral group at the aniline nitrogen. The catalyst resting state RhH(CO)2{(S C,S C,R P,S C)-4b} was investigated by high pressure-NMR studies, revealing an equatorial–apical coordination of the bidentate ligand where the two phosphorus donors rapidly exchange their positions through an intermediate with the ligand bound via the phosphine group only.
Bacterial group II introns encode maturase proteins required for splicing. In organelles of photosynthetic land plants, most of the group II introns have lost the reading frames for maturases. Here, ...we show that the plastidial maturase MatK not only interacts with its encoding intron within trnk-UUU, but also with six additional group II introns, all belonging to intron subclass llA. Mapping analyses of RNA binding sites revealed MatK to recognize multiple regions within the trnK intron. Organellar group ll introns are considered to be the ancestors of nuclear spliceosomal introns. That MatK associates with multiple intron ligands makes it an attractive model for an early trans-acting nuclear splicing activity.
Summary
Synthesis of the D1 reaction center protein of Photosystem II is dynamically regulated in response to environmental and developmental cues. In chloroplasts, much of this regulation occurs at ...the post‐transcriptional level, but the proteins responsible are largely unknown. To discover proteins that impact psbA expression, we identified proteins that associate with maize psbA mRNA by: (i) formaldehyde cross‐linking of leaf tissue followed by antisense oligonucleotide affinity capture of psbA mRNA; and (ii) co‐immunoprecipitation with HCF173, a psbA translational activator that is known to bind psbA mRNA. The S1 domain protein SRRP1 and two RNA Recognition Motif (RRM) domain proteins, CP33C and CP33B, were enriched with both approaches. Orthologous proteins were also among the enriched protein set in a previous study in Arabidopsis that employed a designer RNA‐binding protein as a psbA RNA affinity tag. We show here that CP33B is bound to psbA mRNA in vivo, as was shown previously for CP33C and SRRP1. Immunoblot, pulse labeling, and ribosome profiling analyses of mutants lacking CP33B and/or CP33C detected some decreases in D1 protein levels under some conditions, but no change in psbA RNA abundance or translation. However, analogous experiments showed that SRRP1 represses psbA ribosome association in the dark, represses ycf1 ribosome association, and promotes accumulation of ndhC mRNA. As SRRP1 is known to harbor RNA chaperone activity, we postulate that SRRP1 mediates these effects by modulating RNA structures. The uncharacterized proteins that emerged from our analyses provide a resource for the discovery of proteins that impact the expression of psbA and other chloroplast genes.
Significance Statement
To discover proteins involved in the post‐transcriptional regulation of the chloroplast psbA gene, we employed several methods to comprehensively analyze proteins that associate with maize psbA mRNA in vivo. Functional analyses of three top candidates revealed a putative RNA chaperone that impacts the expression of the chloroplast psbA, ndhC, and ycf1 genes, and two RNA Recognition Motif (RRM) domain proteins that bind psbA mRNA but have little effect on psbA expression under the growth conditions sampled.
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