Fe deficiency induces phosphorylation and translocation of Lhcb1 in barley thylakoid membranes

HvLhcb1 a major light-harvesting chlorophyll a/b-binding protein in barley, is a critical player in sustainable growth under Fe deficiency. Here, we demonstrate that Fe deficiency induces phosphorylation of HvLhcb1 proteins leading to their migration from grana stacks to stroma thylakoid membranes. HvLhcb1 remained phosphorylated even in the dark and apparently independently of state transition, which represents a mechanism for short-term acclimation. Our data suggest that the constitutive phosphorylation-triggered translocation of HvLhcb1 under Fe deficiency contributes to optimization of the excitation balance between photosystem II and photosystem I, the latter of which is a main target of Fe deficiency.     

Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.     

TWO SPECIFIC CAUSES OF CELL MORTALITY IN FREEZE‐THAW CYCLE OF YOUNG THALLI OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Porphyra yezoensis Ueda is an important marine aquaculture crop with single‐layered gametophytic thalli. In this work, the influences of thallus dehydration level, cold‐preservation (freezing) time, and thawing temperature on the photosynthetic recovery of young P. yezoensis thalli were investigated employing an imaging pulse‐amplitude‐modulation (PAM) fluorometer. The results showed that after 40 d of frozen storage when performing thallus thawing under 10°C, the water content of the thalli showed obvious effects on the photosynthetic recovery of the frozen thalli. The thalli with absolute water content (AWC) of 10%–40% manifested obvious superiority compared to the thalli with other AWCs, while the thalli thawed at 20°C showed very high survival rate (93.10%) and no obvious correlation between thallus AWCs and thallus viabilities. These results indicated that inappropriate thallus water content contributed to the cell damage during the freeze‐thaw cycle and that proper thawing temperature is very crucial. Therefore, AWC between 10% and 40% is the suitable thallus water content range for frozen storage, and the thawing process should be as short as possible. However, it is also shown that for short‐term cold storage the Porphyra thallus water content also showed no obvious effect on the photosynthetic recovery of the thalli, and the survival rate was extremely high (100%). These results indicated that freezing time is also a paramount contributor of the cell damage during the freeze‐thaw cycle. Therefore, the frozen nets should be used as soon as time permits.     

Effect of sediment type on microphytobenthos vertical distribution: Modelling the productive biomass and improving ground truth measurements

Intertidal flats are frequently colonised by microphytobenthos (MPB) assemblages that form transient biofilms at the sediment surface which are responsible for large fractions of estuarine primary production. The large spatio-temporal variability in MPB biomass distribution in concert with the fact that tidal flats can cover many km² makes the use of remote sensing particularly useful in assessing MPB distribution. Water content, sediment type and MPB vertical migration are variables that affect the relationship between ground truth measurements and remote sensing of benthic chlorophyll. The effect of chlorophyll depth distribution (top 2 mm) on the relationship between benthic chlorophyll and several remote sensing indices (NDVI, PI, R562/R647, derivative indices and PAM fluorescence) was investigated over a 2 year sampling period at 6 sites (Tagus estuary, Portugal). Additionally, the effect of the dark adaptation time required to measure the minimum fluorescence parameter (F₀) was also tested. Sediment type strongly affected MPB depth distribution with muddy sites showing a strong negative exponential decay in chlorophyll with distance from the surface while sandy sites had a homogenous distribution over the same scale (2 mm). Chlorophyll content (mass per unit mass, μg g^− 1) in the top 2 mm was better correlated with remote sensing indices than concentration (mass per unit volume, mg m^− 3), both for NDVI (0.72 vs. 0.45) and for PAM fluorescence (0.70 vs. 0.55). Separating the data by transect increased the correlation values in all situations. A fitted model of chlorophyll depth distribution showed that the effect of asymmetrical chlorophyll depth distribution was stronger on the correlations between chlorophyll concentration and NDVI than on chlorophyll content and NDVI (0.46-2 mm vs. 0.74-125 μm, muddy site) the same was valid for fluorescence (0.66-2 mm vs. 0.92-125 μm, muddy site). Dark adapting the samples for more than 5 min did not result in any significant difference in the relationship between F₀ and chlorophyll a. The residuals from the regression of chlorophyll content on NDVI were positively correlated (0.7) with the mass per unit of mass of sediment < 63 μm and negatively (− 0.6) with chlorophyll concentration, this indicates that if no correction is performed to account for chlorophyll depth distribution both units will be strongly affected by the mass of < 63 μm particles. The results demonstrate that although expressing chlorophyll a as concentration is generally a better option for ground truth measurements care should be taken to account for chlorophyll depth distribution since strong asymmetries within the sampling depth can introduce large errors.     

Applying Pulse Amplitude Modulation (PAM) fluorometry to microalgae suspensions: stirring potentially impacts fluorescence

The use of microalgae suspensions in PAM-fluorometers such as the Water-PAM (Walz GmbH, Germany) presents the problem of maintaining a homogeneous sample. The Water-PAM is marketed with an optional accessory for stirring the sample within the cuvette while in the emitter–detector (ED) unit. This stirring device can help to prevent cells from settling out of suspension over the time-course of chlorophyll-a fluorescence measurements. The ED unit was found to provide a vertically heterogeneous light environment and, therefore, cells within a single sample can exist in different quenched states. Enhancing cell movement by stirring was found to substantially influence measured fluorescence yield while performing induction curve and rapid light curve analyses. This is likely to result from relatively unquenched cells outside the main light-path moving into a higher light region and thus emitting disproportionately more fluorescence than quenched cells. Samples containing cells with high sinking rates or motile species may encounter similar (but reduced) problems. This effect can be mitigated by: (a) reducing analysis time to minimise the distance cells can sink/swim during the measurement procedure and avoiding the necessity of stirring; (b) limiting the proportion of sample outside the light path by minimising sample volume or; (c) by activating the stirrer only for short periods between saturation pulses and allowing enough time after stirring for quenching to stabilise before activation of the saturation pulse. Alternatively, modifications to the instrument providing a vertical dimension to the LED-array could resolve the issue by providing a more homogeneous light environment for the sample.     

An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.     

Coumarin-3-carboxylic acid derivatives as potentiators and inhibitors of recombinant and native N-methyl-d-aspartate receptors

N-Methyl-d-aspartate receptors (NMDARs) are known to be involved in a range of neurological and neurodegenerative disorders and consequently the development of compounds that modulate the function of these receptors has been the subject of intense interest. We have recently reported that 6-bromocoumarin-3-carboxylic acid (UBP608) is a negative allosteric modulator with weak selectivity for GluN2A-containing NMDARs. In the present study, a series of commercially available and newly synthesized coumarin derivatives have been evaluated in a structure-activity relationship (SAR) study as modulators of recombinant NMDAR activity. The main conclusions from this SAR study were that substituents as large as iodo were accommodated at the 6-position and that 6,8-dibromo or 6,8-diiodo substitution of the coumarin ring enhanced the inhibitory activity at NMDARs. These coumarin derivatives are therefore excellent starting points for the development of more potent and GluN2 subunit selective inhibitors, which may have application in the treatment of a range of neurological disorders such as neuropathic pain, epilepsy and depression. Surprisingly, 4-methyl substitution of UBP608 to give UBP714, led to conversion of the inhibitory activity of UBP608 into potentiating activity at recombinant GluN1/GluN2 receptors. UBP714 also enhanced NMDAR mediated field EPSPs in the CA1 region of the hippocampus. UBP714 is therefore a novel template for the development of potent and subunit selective NMDAR potentiators that may have therapeutic applicability in the treatment of patients with cognitive deficits or schizophrenia.     

Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1–Ccr4 and Pan2–Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2–PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3′ end of poly(A).