1. Species occurrence records from online databases are an indispensable resource in ecological, biogeographical and palaeontological research. However, issues with data quality, especially incorrect geo-referencing or dating, can diminish their usefulness. Manual cleaning is time-consuming, error prone, difficult to reproduce and limited to known geographical areas and taxonomic groups, making it impractical for datasets with thousands or millions of records.2. Here, we present CoordinateCleaner, an r-package to scan datasets of species occurrence records for geo-referencing and dating imprecisions and data entry errors in a standardized and reproducible way. CoordinateCleaner is tailored to problems common in biological and palaeontological databases and can handle datasets with millions of records. The software includes (a) functions to flag potentially problematic coordinate records based on geographical gazetteers, (b) a global database of 9,691 geo-referenced biodiversity institutions to identify records that are likely from horticulture or captivity, (c) novel algorithms to identify datasets with rasterized data, conversion errors and strong decimal rounding and (d) spatio-temporal tests for fossils.3. We describe the individual functions available in CoordinateCleaner and demonstrate them on more than 90 million occurrences of flowering plants from the Global Biodiversity Information Facility (GBIF) and 19,000 fossil occurrences from the Palaeobiology Database (PBDB). We find that in GBIF more than 3.4 million records (3.7%) are potentially problematic and that 179 of the tested contributing This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Multiple K+ transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K + transporters is limited. Potassium transport and a well-balanced K + homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K + transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higherorder loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na + /H + antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis.

No abstract

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn 4 CaO 5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn 2+ and Ca 2+ homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn 2+ and Ca 2+ ions were differently sequestered in pam71, with Ca 2+ enriched in pam71 thylakoids relative to the wild type. The changes in Ca 2+ homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn 2+ , but not Ca 2+ . Furthermore, PAM71 suppressed the Mn 2+ -sensitive phenotype of the yeast mutant Dpmr1. Therefore, PAM71 presumably functions in Mn 2+ uptake into thylakoids to ensure optimal PSII performance.

In natural habitats, plants frequently experience rapid changes in the intensity of sunlight. To cope with these changes and maximize growth, plants adjust photosynthetic light utilization in electron transport and photoprotective mechanisms. This involves a proton motive force (PMF) across the thylakoid membrane, postulated to be affected by unknown anion (Cl−) channels. Here we report that a bestrophin-like protein from Arabidopsis thaliana functions as a voltage-dependent Cl− channel in electrophysiological experiments. AtVCCN1 localizes to the thylakoid membrane, and fine-tunes PMF by anion influx into the lumen during illumination, adjusting electron transport and the photoprotective mechanisms. The activity of AtVCCN1 accelerates the activation of photoprotective mechanisms on sudden shifts to high light. Our results reveal that AtVCCN1, a member of a conserved anion channel family, acts as an early component in the rapid adjustment of photosynthesis in variable light environments.

BACKGROUND In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

Chloride ions can be translocated across cell membranes through Cl− channels or Cl−/H+ exchangers. The thylakoid-located member of the Cl− channel CLC family in Arabidopsis thaliana (AtCLCe) was hypothesized to play a role in photosynthetic regulation based on the initial photosynthetic characterization of clce mutant lines. The reduced nitrate content of Arabidopsis clce mutants suggested a role in regulation of plant nitrate homeostasis. In this study, we aimed to further investigate the role of AtCLCe in the regulation of ion homeostasis and photosynthetic processes in the thylakoid membrane. We report that the size and composition of proton motive force were mildly altered in two independent Arabidopsis clce mutant lines. Most pronounced effects in the clce mutants were observed on the photosynthetic electron transport of dark-adapted plants, based on the altered shape and associated parameters of the polyphasic OJIP kinetics of chlorophyll a fluorescence induction. Other alterations were found in the kinetics of state transition and in the macro-organization of photosystem II supercomplexes, as indicated by circular dichroism measurements. Pre-treatment with KCl but not with KNO3 restored the wild-type photosynthetic phenotype. Analyses by transmission electron microscopy revealed a bow-like arrangement of the thylakoid network and a large thylakoid-free stromal region in chloroplast sections from the dark-adapted clce plants. Based on these data, we propose that AtCLCe functions in Cl− homeostasis after transition from light to dark, which affects chloroplast ultrastructure and regulation of photosynthetic electron transport.

SUMMARYThe Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high-phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO 2 fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non-photochemical quenching than the wild type, in line with the increased contribution of DpH to the proton-motive force across thylakoids. Small-angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long-range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild-type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro-organization of complexes and induction of photoprotective mechanisms.