Phenoplate: An innovative method for assessing interacting effects of temperature and light on non-photochemical quenching in microalgae under chemical stress.

Rapid light curves are one of the most widely used methods for assessing the physiological state of photosynthetic organisms. While the method has been applied in a range of physiological studies over the last 20 years, little progress has been made in adapting it for the new age of multi-parametric phenotyping. In order to advance research that is aimed at evaluating the physiological impact of multiple factors, the Phenoplate was developed: a simultaneous assessment of temperature and light gradients. It was used to measure rapid light curves of three marine microalgae across a temperature gradient and altered phosphate availability. The results revealed that activation of photoprotective mechanisms occurred with high efficiency at lower temperatures, and relaxation of photoprotection was negatively impacted above a certain temperature threshold in Tetraselmis sp. It was observed that Thalassiosira pseudonana and Nannochloropsis oceanica exhibited two unique delayed non-photochemical quenching signatures: in combinations of low light with low temperature, and darkness with high temperature, respectively. These findings demonstrate that the Phenoplate approach can be used as a rapid and simple tool to gain insight into the photobiology of microalgae

The Arabidopsis Thylakoid Chloride Channel AtCLCe Functions in Chloride Homeostasis and Regulation of Photosynthetic Electron Transport.

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

Methyl Jasmonate and Methyl-β-Cyclodextrin Individually Boost Triterpenoid Biosynthesis in <i>Chlamydomonas Reinhardtii</i> UVM4.

The commercialisation of valuable plant triterpenoids faces major challenges, including low abundance in natural hosts and costly downstream purification procedures. Endeavours to produce these compounds at industrial scale using microbial systems are gaining attention. Here, we report on a strategy to enrich the biomass of the biotechnologically-relevant Chlamydomonas reinhardtii strain UVM4 with valuable triterpenes, such as squalene and (S)-2,3-epoxysqualene. C. reinhardtii UVM4 was subjected to the elicitor compounds methyl jasmonate (MeJA) and methyl-β-cyclodextrine (MβCD) to increase triterpene yields. MeJA treatment triggered oxidative stress, arrested growth, and altered the photosynthetic activity of the cells, while increasing squalene, (S)-2,3-epoxysqualene, and cycloartenol contents. Applying MβCD to cultures of C. reinhardtii lead to the sequestration of the two main sterols (ergosterol and 7-dehydroporiferasterol) into the growth medium and the intracellular accumulation of the intermediate cycloartenol, without compromising cell growth. When MβCD was applied in combination with MeJA, it counteracted the negative effects of MeJA on cell growth and physiology, but no synergistic effect on triterpene yield was observed. Together, our findings provide strategies for the triterpene enrichment of microalgal biomass and medium

A Cyanobacteria Enriched Layer of Shark Bay Stromatolites Reveals a New Acaryochloris Strain Living in Near Infrared Light.

The genus Acaryochloris is unique among phototrophic organisms due to the dominance of chlorophyll d in its photosynthetic reaction centres and light-harvesting proteins. This allows Acaryochloris to capture light energy for photosynthesis over an extended spectrum of up to ~760 nm in the near infra-red (NIR) spectrum. Acaryochloris sp. has been reported in a variety of ecological niches, ranging from polar to tropical shallow aquatic sites. Here, we report a new Acarychloris strain isolated from an NIR-enriched stratified microbial layer 4-6 mm under the surface of stromatolite mats located in the Hamelin Pool of Shark Bay, Western Australia. Pigment analysis by spectrometry/fluorometry, flow cytometry and spectral confocal microscopy identifies unique patterns in pigment content that likely reflect niche adaption. For example, unlike the original A. marina species (type strain MBIC11017), this new strain, Acarychloris LARK001, shows little change in the chlorophyll d/a ratio in response to changes in light wavelength, displays a different Fv/Fm response and lacks detectable levels of phycocyanin. Indeed, 16S rRNA analysis supports the identity of the A. marina LARK001 strain as close to but distinct from from the A. marina HICR111A strain first isolated from Heron Island and previously found on the Great Barrier Reef under coral rubble on the reef flat. Taken together, A. marina LARK001 is a new cyanobacterial strain adapted to the stromatolite mats in Shark Bay

CoordinateCleaner: Standardized cleaning of occurrence records from biological collection databases

© 2019 The Authors. Methods in Ecology and Evolution published by John Wiley & Sons Ltd on behalf of British Ecological Society. 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. 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. 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 datasets (18.5%) might be biased by rasterized coordinates. In PBDB, 1205 records (6.3%) are potentially problematic. All cleaning functions and the biodiversity institution database are open-source and available within the CoordinateCleaner r-package

Characterisation and Bioactivity Analysis of Peridinin-Chlorophyll a-Protein (PCP) Isolated from Symbiodinium tridacnidorum CS-73

Peridinin-Chlorophyll a-Proteins (PCP) are the major light harvesting proteins in photosynthetic dinoflagellates. PCP shows great variation in protein length, pigment ratio, sequence, and spectroscopic properties. PCP conjugates (PerCP) are widely used as fluorescent probes for cellular and tissue analysis in the biomedical field. PCP consists of a peridinin carotenoid; thereby, it can potentially be used as a bioactive compound in pharmaceutical applications. However, the biological activities of PCP are yet to be explored. In this study, we extracted, purified, and partially characterised the PCP from Symbiodinium tridacnidorum (CS-73) and explored its antioxidant, anti-cancer and anti-inflammation bioactivities. The PCP was purified using an ÄKTA™ PURE system and predicted to be of 17.3 kDa molecular weight (confirmed as a single band on SDS-PAGE) with an isoelectric point (pI) 5.6. LC-MS/MS and bioinformatic analysis of purified PCP digested with trypsin indicated it was 164 amino acids long with >90% sequence similarity to PCP of SymA3.s6014_g3 (belonging to clade A of Symbiodinium sp.) confirmed with 59 peptide combinations matched across its protein sequence. The spectroscopic properties of purified PCP showed a slight shift in absorption and emission spectra to previously documented analysis in Symbiodinium species possibly due to variation in amino acid sequences that interact with chl a and peridinin. Purified PCP consisted of a 19-amino-acid-long signal peptide at its N terminal and nine helixes in its secondary structure, with several protein binding sites and no DNA/RNA binding site. Furthermore, purified PCP exhibited antioxidant and in vitro anti-inflammation bioactivities, and anti-cancer activities against human metastatic breast adenocarcinoma (MDA-MB-231) and human colorectal (HTC-15) cancer cell lines. Together, all these findings present PCP as a promising candidate for continued investigations for pharmaceutical applications to cure chronic diseases, apart from its existing application as a fluorescent-probe.</jats:p

Multicenter Evaluation of Independent High-Throughput and RT-qPCR Technologies for the Development of Analytical Workflows for Circulating miRNA Analysis.

BACKGROUND:Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. METHODS:Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. RESULTS:We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. CONCLUSIONS:Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology

K+ and Cl- channels/transporters independently fine-tune photosynthesis in plants.

In variable light environments, plants adjust light use in photosynthetic electron transport and photoprotective dissipation in the thylakoid membrane. In this respect, roles of the K+/H+ antiporter KEA3, the Cl- channel/transporter CLCe and the voltage-dependent Cl- channel VCCN1 have been unraveled in Arabidopsis thaliana. Here we report that they independently adjust photosynthesis on the basis of analyses using single and higher order loss-of-function mutants. In short experiments of photosynthetic response on transition from dark to low light, we reveal a sequential functioning of VCCN1 and CLCe in the activation of photoprotection and of KEA3 in its downregulation to a low steady state while adjusting the electron transport. On transition from low to high light, VCCN1 accelerates the activation of photoprotection, whereas KEA3 slows it down on transition from high to low light. Based on parallel electrochromic band shift measurements, the mechanism behind is that VCCN1 builds up a pH gradient across the thylakoid membrane, whereas KEA3 dissipates this gradient, which affects photoprotection. CLCe regulates photosynthesis by a pH-independent mechanism likely involving Cl- homeostasis. Nevertheless, all genotypes grow well in alternating high and low light. Taken together, the three studied ion channels/transporters function independently in adjusting photosynthesis to the light environment

Photosystem II function and dynamics in three widely used Arabidopsis thaliana accessions.

Columbia-0 (Col-0), Wassilewskija-4 (Ws-4), and Landsberg erecta-0 (Ler-0) are used as background lines for many public Arabidopsis mutant collections, and for investigation in laboratory conditions of plant processes, including photosynthesis and response to high-intensity light (HL). The photosystem II (PSII) complex is sensitive to HL and requires repair to sustain its function. PSII repair is a multistep process controlled by numerous factors, including protein phosphorylation and thylakoid membrane stacking. Here we have characterized the function and dynamics of PSII complex under growth-light and HL conditions. Ws-4 displayed 30% more thylakoid lipids per chlorophyll and 40% less chlorophyll per carotenoid than Col-0 and Ler-0. There were no large differences in thylakoid stacking, photoprotection and relative levels of photosynthetic complexes among the three accessions. An increased efficiency of PSII closure was found in Ws-4 following illumination with saturation flashes or continuous light. Phosphorylation of the PSII D1/D2 proteins was reduced by 50% in Ws-4 as compared to Col-0 and Ler-0. An increase in abundance of the responsible STN8 kinase in response to HL treatment was found in all three accessions, but Ws-4 displayed 50% lower levels than Col-0 and Ler-0. Despite this, the HL treatment caused in Ws-4 the lagest extent of PSII inactivation, disassembly, D1 protein degradation, and the largest decrease in the size of stacked thylakoids. The dilution of chlorophyll-protein complexes with additional lipids and carotenoids in Ws-4 may represent a mechanism to facilitate lateral protein traffic in the membrane, thus compensating for the lack of a full complement of STN8 kinase. Nevertheless, additional PSII damage occurs in Ws-4, which exceeds the D1 protein synthesis capacity, thus leading to enhanced photoinhibition. Our findings are valuable for selection of appropriate background line for PSII characterization in Arabidopsis mutants, and also provide the first insights into natural variation of PSII protein phosphorylation

The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain.

Phosphorus (P) is an essential macronutrient, and P deficiency limits plant productivity. Recent work showed that P deficiency affects electron transport to photosystem I (PSI), but the underlying mechanisms are unknown. Here, we present a comprehensive biological model describing how P deficiency disrupts the photosynthetic machinery and the electron transport chain through a series of sequential events in barley (Hordeum vulgare). P deficiency reduces the orthophosphate concentration in the chloroplast stroma to levels that inhibit ATP synthase activity. Consequently, protons accumulate in the thylakoids and cause lumen acidification, which inhibits linear electron flow. Limited plastoquinol oxidation retards electron transport to the cytochrome b6f complex, yet the electron transfer rate of PSI is increased under steady-state growth light and is limited under high-light conditions. Under P deficiency, the enhanced electron flow through PSI increases the levels of NADPH, whereas ATP production remains restricted and, hence, reduces CO2 fixation. In parallel, lumen acidification activates the energy-dependent quenching component of the nonphotochemical quenching mechanism and prevents the overexcitation of photosystem II and damage to the leaf tissue. Consequently, plants can be severely affected by P deficiency for weeks without displaying any visual leaf symptoms. All of the processes in the photosynthetic machinery influenced by P deficiency appear to be fully reversible and can be restored in less than 60 min after resupply of orthophosphate to the leaf tissue