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Storage triacylglycerols in castor bean seeds are enriched in the hydroxylated fatty acid ricinoleate. Extensive tissue-specific RNA-Seq transcriptome and lipid analysis will help identify components important for its biosynthesis. Storage triacylglycerols (TAGs) in the endosperm of developing castor (Ricinus communis) seeds are highly enriched in ricinoleic acid (18:1-OH). We have analysed neutral lipid fractions from other castor tissues using TLC, GLC and mass spectrometry. Cotyledons, like the endosperm, contain high levels of 18:1-OH in TAG. Pollen and male developing flowers accumulate TAG but do not contain 18:1-OH and leaves do not contain TAG or 18:1-OH. Analysis of acyl-CoAs in developing endosperm shows that ricinoleoyl-CoA is not the dominant acyl-CoA, indicating that either metabolic channelling or enzyme substrate selectivity are important in the synthesis of tri-ricinolein in this tissue. RNA-Seq transcriptomic analysis, using Illumina sequencing by synthesis technology, has been performed on mRNA isolated from two stages of developing seeds, germinating seeds, leaf and pollen-producing male flowers in order to identify differences in lipid-metabolic pathways and enzyme isoforms which could be important in the biosynthesis of TAG enriched in 18:1-OH. This study gives comprehensive coverage of gene expression in a variety of different castor tissues. The potential role of differentially expressed genes is discussed against a background of proteins identified in the endoplasmic reticulum, which is the site of TAG biosynthesis, and transgenic studies aimed at increasing the ricinoleic acid content of TAG. Several of the genes identified in this tissue-specific whole transcriptome study have been used in transgenic plant research aimed at increasing the level of ricinoleic acid in TAG. New candidate genes have been identified which might further improve the level of ricinoleic acid in transgenic crops.

Background New bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it’s insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin ( Galanthus nivalis agglutinin; GNA) . Results We report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris . PAF was orally toxic to pea ( Acyrthosiphon pisum ) and peach potato ( Myzus persicae ) aphids with respective, Day 5 LC 50 values of 54 µM and 105 µM derived from dose–response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC 50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees ( Bombus terristrus ) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins. Conclusions Our results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies.

Carbon dioxide is vital to the chemistry of life processes including metabolism, cellular homoeostasis, and pathogenesis. CO 2 is generally unreactive but can combine with neutral amines to form carbamates on proteins under physiological conditions. The most widely known examples of this are CO 2 regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase and haemoglobin. However, the systematic identification of CO 2 -binding sites on proteins formed through carbamylation has not been possible due to the ready reversibility of carbamate formation. Here we demonstrate a methodology to identify protein carbamates using triethyloxonium tetrafluoroborate to covalently trap CO 2 , allowing for downstream proteomic analysis. This report describes the systematic identification of carbamates in a physiologically relevant environment. We demonstrate the identification of carbamylated proteins and the general principle that CO 2 can impact protein biochemistry through carbamate formation. The ability to identify protein carbamates will significantly advance our understanding of cellular CO 2 interactions. Carbon dioxide can interact with proteins to form carbamate post-translational modifications. Here, the authors developed a strategy to identify carbamate post-translational modifications by trapping carbon dioxide and subsequently identifying the carbamylated proteins.

Background Integrating medical data using databases from different sources by record linkage is a powerful technique increasingly used in medical research. Under many jurisdictions, unique personal identifiers needed for linking the records are unavailable. Since sensitive attributes, such as names, have to be used instead, privacy regulations usually demand encrypting these identifiers. The corresponding set of techniques for privacy-preserving record linkage (PPRL) has received widespread attention. One recent method is based on Bloom filters. Due to superior resilience against cryptographic attacks, composite Bloom filters (cryptographic long-term keys, CLKs) are considered best practice for privacy in PPRL. Real-world performance of these techniques using large-scale data is unknown up to now. Methods Using a large subset of Australian hospital admission data, we tested the performance of an innovative PPRL technique (CLKs using multibit trees) against a gold-standard derived from clear-text probabilistic record linkage. Linkage time and linkage quality (recall, precision and F-measure) were evaluated. Results Clear text probabilistic linkage resulted in marginally higher precision and recall than CLKs. PPRL required more computing time but 5 million records could still be de-duplicated within one day. However, the PPRL approach required fine tuning of parameters. Conclusions We argue that increased privacy of PPRL comes with the price of small losses in precision and recall and a large increase in computational burden and setup time. These costs seem to be acceptable in most applied settings, but they have to be considered in the decision to apply PPRL. Further research on the optimal automatic choice of parameters is needed.

Light harvesting is fundamental for production of ATP and reducing equivalents for CO 2 fixation during photosynthesis. However, electronic energy transfer (EET) through a photosystem can harm the photosynthetic apparatus when not balanced with CO 2 . Here, we show that CO 2 binding to the light-harvesting complex modulates EET in photosynthetic cyanobacteria. More specifically, CO 2 binding to the allophycocyanin alpha subunit of the light-harvesting complex regulates EET and its fluorescence quantum yield in the cyanobacterium Synechocystis sp. PCC 6803. CO 2 binding decreases the inter-chromophore distance in the allophycocyanin trimer. The result is enhanced EET in vitro and in live cells. Our work identifies a direct target for CO 2 in the cyanobacterial light-harvesting apparatus and provides insights into photosynthesis regulation. The transfer of electronic energy through a photosystem can harm the photosynthetic apparatus when not balanced with CO 2 fixation. Here, the authors show that CO 2 modulates electronic energy transfer in cyanobacteria by binding to and enhancing the activity of the light-harvesting complex.

Plants reprogramme their proteome to alter cellular metabolism for effective stress adaptation. Intracellular proteomic responses have been extensively studied, and the extracellular matrix stands as a key hub where peptide signals are generated/processed to trigger critical adaptive signal transduction cascades inaugurated at the cell surface. Therefore, it is important to study the plant extracellular proteome to understand its role in plant development and stress response. This study examined changes in the soluble extracellular sub-proteome of sorghum cell cultures exposed to a combination of sorbitol-induced osmotic stress and heat at 40 °C. The combined stress significantly reduced metabolic activity and altered protein secretion. While cells treated with osmotic stress alone had elevated proline content, the osmoprotectant in the combined treatment remained unchanged, confirming that sorghum cells exposed to combined stress utilise adaptive processes distinct from those invoked by the single stresses applied separately. Reactive oxygen species (ROS)-metabolising proteins and proteases dominated differentially expressed proteins identified in cells subjected to combined stress. ROS-generating peroxidases were suppressed, while ROS-degrading proteins were upregulated for protection from oxidative damage. Overall, our study provides protein candidates that could be used to develop crops better suited for an increasingly hot and dry climate.

Plants reprogram gene expression as an adaptive response to survive high temperatures. While the identity and functions of intracellular heat stress-responsive proteins have been extensively studied, the heat response of proteins secreted to the extracellular matrix is unknown. Here, we used Sorghum bicolor, a species adapted for growth in hot climates, to investigate the extracellular heat-induced responses. When exposed to 40 °C for 72 h, heat-sensitive Arabidopsis cell suspension cultures died, while ICSB338 sorghum cell cultures survived by activation of a transcriptional response characterized by the induction of HSP70 and HSP90 genes. Quantitative proteomic analysis of proteins recovered from cell culture medium revealed specific heat stress-induced protein accumulation within the sorghum secretome. Of the 265 secreted proteins identified, 31 responded to heat (≥2-fold change), with 84% possessing a predicted signal peptide for targeting to the classical secretory pathway. The differentially accumulated proteins have putative functions in metabolism, detoxification, and protein modifications. A germin (SORBI_3003G427700) was highly heat-inducible at both protein and gene level. Overall, our study reveals new insights into sorghum responses to heat and provides a useful resource of extracellular proteins that could serve as targets for developing thermotolerant crops. Data are available via ProteomeXchange with identifier PXD021536.

Drought stress severely affects crop productivity and threatens food security. As current trends of global warming are predicted to exacerbate droughts, developing drought-resilient crops becomes urgent. Here, we used the drought-tolerant (BW35695) and drought-sensitive (BW4074) wheat varieties to investigate the physiological, biochemical, and leaf proteome responses underpinning drought tolerance. In response to drought, the tolerant variety had higher osmolyte accumulation and maintained higher leaf water content than the sensitive variety. BW35695 also had an enhanced antioxidant enzyme capacity and reduced reactive oxygen species (ROS), resulting in diminished membrane lipid damage, as reflected by malondialdehyde content. Proteomic analysis revealed that drought-induced differential expression of proteins involved in diverse biological processes in both wheat varieties, including primary and secondary metabolism, protein synthesis/folding/degradation, defense/ROS detoxification, energy, transcription, and cell structure. Notably, photosynthesis emerged as the most enriched biochemical process targeted for suppression in the drought-tolerant BW35695 wheat, but not in drought-sensitive BW4074, possibly as a survival strategy for averting cell damage inflicted by photosynthesis-derived ROS. Additionally, protein synthesis-related proteins were highly upregulated in BW35695, presumably to drive cell-wide stress-adaptive responses. The protein network identified here will be useful in further studies to understand the molecular basis for divergent drought response phenotypes in crops.

When exposed to drought stress many plants reprogram their gene expression to activate adaptive biochemical and physiological responses for survival. However, most of the well-studied adaptive responses are common between drought-sensitive and drought-tolerant species, making it difficult to identify the key mechanisms underpinning successful drought tolerance in crops. We developed a sorghum experimental system that compares between drought-sensitive (ICSB338) and enhanced drought-tolerant (SA1441) varieties. We show that sorghum activates a swift and robust stomatal shutdown to preserve leaf water content when water stress has been sensed. Water uptake is enhanced via increasing root cell water potential through the rapid biosynthesis of predominantly glycine betaine and an increased root-to-shoot ratio to explore more soil volume for water. In addition to stomatal responses, there is a prompt accumulation of proline in leaves and effective protection of chlorophyll during periods of water limitation. Root and stomatal functions rapidly recover from water limitation (within 24 h of re-watering) in the drought-tolerant variety, but recovery is impaired in the drought-sensitive sorghum variety. Analysis of the root proteome revealed complex protein networks that possibly underpin sorghum responses to water limitation. Common and unique protein changes between the two sorghum varieties provide new targets for future use in investigating sorghum drought tolerance.

Background Probabilistic record linkage is a process used to bring together person-based records from within the same dataset (de-duplication) or from disparate datasets using pairwise comparisons and matching probabilities. The linkage strategy and associated match probabilities are often estimated through investigations into data quality and manual inspection. However, as privacy-preserved datasets comprise encrypted data, such methods are not possible. In this paper, we present a method for estimating the probabilities and threshold values for probabilistic privacy-preserved record linkage using Bloom filters. Methods Our method was tested through a simulation study using synthetic data, followed by an application using real-world administrative data. Synthetic datasets were generated with error rates from zero to 20% error. Our method was used to estimate parameters (probabilities and thresholds) for de-duplication linkages. Linkage quality was determined by F-measure. Each dataset was privacy-preserved using separate Bloom filters for each field. Match probabilities were estimated using the expectation-maximisation (EM) algorithm on the privacy-preserved data. Threshold cut-off values were determined by an extension to the EM algorithm allowing linkage quality to be estimated for each possible threshold. De-duplication linkages of each privacy-preserved dataset were performed using both estimated and calculated probabilities. Linkage quality using the F-measure at the estimated threshold values was also compared to the highest F-measure. Three large administrative datasets were used to demonstrate the applicability of the probability and threshold estimation technique on real-world data. Results Linkage of the synthetic datasets using the estimated probabilities produced an F-measure that was comparable to the F-measure using calculated probabilities, even with up to 20% error. Linkage of the administrative datasets using estimated probabilities produced an F-measure that was higher than the F-measure using calculated probabilities. Further, the threshold estimation yielded results for F-measure that were only slightly below the highest possible for those probabilities. Conclusions The method appears highly accurate across a spectrum of datasets with varying degrees of error. As there are few alternatives for parameter estimation, the approach is a major step towards providing a complete operational approach for probabilistic linkage of privacy-preserved datasets.