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Multicellular development requires the careful orchestration of gene expression to correctly create and position specialized cells. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are differentiated from vegetative cells in a reproducibly periodic and physiologically relevant pattern. While many genetic factors required for heterocyst development have been identified, the role of HetZ has remained unclear. Here, we present evidence to clarify the requirement of hetZ for heterocyst production and support a model where HetZ functions in the patterning stage of differentiation. We show that a clean, nonpolar deletion of hetZ fails to express the developmental genes hetR, patS, hetP and hetZ correctly and fails to produce heterocysts. Complementation and overexpression of hetZ in a hetP mutant revealed that hetZ was incapable of bypassing hetP, suggesting that it acts upstream of hetP. Complementation and overexpression of hetZ in a hetR mutant, however, demonstrated bypass of hetR, suggesting that it acts downstream of hetR and is capable of bypassing the need for hetR for differentiation irrespective of nitrogen status. Finally, protein-protein interactions were observed between HetZ and HetR, Alr2902 and HetZ itself. Collectively, this work suggests a regulatory role for HetZ in the patterning phase of cellular differentiation in Anabaena.

We conducted soil warming experiment in a cool-temperate forest with peat soil in northern Japan, during the snowless seasons of 2007–2009. Our objective was to determine whether or not the heterotrophic respiration rate and the temperature sensitivity would change by soil warming. We elevated the soil temperature by 3 °C at 5 cm depth by means of overhead infrared heaters and continuously measured soil CO2 fluxes by using a fifteen-channel automated chamber system. Trenching treatment was also carried out to separate heterotrophic respiration and root respiration from the total soil respiration. The fifteen chambers were divided into three groups each with five replications for the control, unwarmed-trenched, and warmed-trenched treatments. We found that heterotrophic respiration contributed 71 % of the total soil respiration with the remaining 29 % accounted to autotrophic respiration. Soil warming enhanced heterotrophic respiration by 74 % (mean 6.11 ± 3.07 S.D. μmol m−2 s–1) as compared to the unwarmed-trenched treatment (mean 3.52 ± 1.74 μmol m−2 s–1). Soil CO2 efflux, however, was weakly correlated with soil moisture, probably because the volumetric soil moisture (33–46 %) was within a plateau region for root and microbial activities. The enhancement in heterotrophic respiration with soil warming in our study suggests that global warming will accelerate the loss of carbon from forested peatlands more seriously than other upland forest soils. On the other hand, soil warming did not cause significant change in the temperature sensitivity, Q10, (2.79 and 2.74 determined using hourly efflux data for unwarmed- and warmed-trenched, respectively), but increased their basal respiration rate at 0 °C (0.93 and 1.21 μmol m−2 s−1, respectively). Results suggest that if we predict the soil heterotrophic respiration rate in future warmer environment using the current relationship between soil temperature and heterotrophic respiration, the rate can be underestimated.

Multicellular development requires the careful orchestration of gene expression to correctly create and position specialized cells. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are differentiated from vegetative cells in a reproducibly periodic and physiologically relevant pattern. While many genetic factors required for heterocyst development have been identified, the role of HetZ has remained unclear. Here, we present evidence to clarify the requirement of hetZ for heterocyst production and support a model where HetZ functions in the patterning stage of differentiation. We show that a clean, nonpolar deletion of hetZ fails to express the developmental genes hetR, patS, hetP and hetZ correctly and fails to produce heterocysts. Complementation and overexpression of hetZ in a hetP mutant revealed that hetZ was incapable of bypassing hetP, suggesting that it acts upstream of hetP. Complementation and overexpression of hetZ in a hetR mutant, however, demonstrated bypass of hetR, suggesting that it acts downstream of hetR and is capable of bypassing the need for hetR for differentiation irrespective of nitrogen status. Finally, protein-protein interactions were observed between HetZ and HetR, Alr2902 and HetZ itself. Collectively, this work suggests a regulatory role for HetZ in the patterning phase of cellular differentiation in Anabaena.

Climatic variabilities on millennial and longer time scales with a bipolar seesaw pattern have been documented in paleoclimatic records, but their frequencies, relationships with mean climatic state, and mechanisms remain unclear. Understanding the processes and sensitivities that underlie these changes will underpin better understanding of the climate system and projections of its future change. We investigate the long-term characteristics of climatic variability using a new ice-core record from Dome Fuji, East Antarctica, combined with an existing long record from the Dome C ice core. Antarctic warming events over the past 720,000 years are most frequent when the Antarctic temperature is slightly below average on orbital time scales, equivalent to an intermediate climate during glacial periods, whereas interglacial and fully glaciated climates are unfavourable for a millennial-scale bipolar seesaw. Numerical experiments using a fully coupled atmosphere-ocean general circulation model with freshwater hosing in the northern North Atlantic showed that climate becomes most unstable in intermediate glacial conditions associated with large changes in sea ice and the Atlantic Meridional Overturning Circulation. Model sensitivity experiments suggest that the prerequisite for the most frequent climate instability with bipolar seesaw pattern during the late Pleistocene era is associated with reduced atmospheric CO2 concentration via global cooling and sea ice formation in the North Atlantic, in addition to extended Northern Hemisphere ice sheets.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocysts arranged in a periodic pattern when deprived of a fixed source of nitrogen. In a genetic screen for mutations that prevent diazotrophic growth, open reading frame all1758, which encodes a putative serine/threonine phosphatase, was identified. Mutation of all1758 resulted in a number of seemingly disparate phenotypes that included a delay in the morphological differentiation of heterocysts, reduced cell size, and lethality under certain conditions. The mutant was incapable of fixing nitrogen under either oxic or anoxic conditions, and lacked the minor heterocyst-specific glycolipid. Pattern formation, as indicated by the timing and pattern of expression from the promoters of hetR and patS fused transcriptionally to the gene for green fluorescent protein (GFP), was unaffected by mutation of all1758, suggesting that its role in the formation of heterocysts is limited to morphological differentiation. Transcription of all1758 was constitutive with respect to both cell type and conditions of growth, but required a functional copy of all1758. The reduced cell size of the all1758 mutant and the location of all1758 between the cell division genes ftsX and ftsY may be indicative of a role for all1758 in cell division. Taken together, these results suggest that the protein encoded by all1758 may represent a link between cell growth, division and regulation of the morphological differentiation of heterocysts.

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.

HetR, master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120, stimulates heterocyst differentiation via transcriptional autoregulation and is negatively regulated by PatS and HetN, both of which contain the active pentapeptide RGSGR. However, the direct targets of PatS and HetN remain uncertain. Here, we report experimental evidence for direct binding between HetR and the C-terminal RGSGR pentapeptide, PatS-5. Strains with a hetR allele coding for conservative substitutions at residues 250-256 had altered patterns of heterocysts and, in some cases, reduced sensitivity to PatS-5. Cysteine scanning mutagenesis coupled with electron paramagnetic resonance (EPR) spectroscopy showed quenching of spin label motion at HetR amino acid 252 upon titration with PatS-5, indicating direct binding of PatS-5 to HetR. Gel shift assays indicated that PatS-5 disrupted binding of HetR to a 29 base pair inverted-repeat-containing DNA sequence upstream from hetP. Double electron-electron resonance EPR experiments confirmed that HetR existed as a dimer in solution and indicated that PatS-5 bound to HetR without disrupting the dimer form of HetR. Isothermal titration calorimetry experiments corroborated direct binding of PatS-5 to HetR with a K(d) of 227 nM and a 1:1 stoichiometry. Taken together, these results indicated that PatS-5 disrupted HetR binding to DNA through a direct HetR/PatS interaction. PatS-5 appeared to either bind in the vicinity of HetR amino acid L252 or, alternately, to bind in a remote site that leads to constrained motion of this amino acid via an allosteric effect or change in tertiary structure.

As a first step towards using world-wide a common 36Cl standard-type material for the normalization of 36Cl AMS results, we have initiated an interlaboratory comparison, but with differences in the execution of the project compared to other round-robin exercises: a) participants have given up anonymity to better optimize discussion and conclusions, b) preliminary results have been shown at various scientific meetings and workshops to discuss first trends and to increase the number of participants, c) participants had the possibility to repeat their AMS measurements and/or change measurement setups or data evaluation to improve individual data [e.g. 1]. We are aware and acknowledge that these later changes by some of the participants, which led to different overall data compared with the initial results, are not fully consistent with the idea of a normal proficiency test layout. However, these data changes were accepted to better meet the main objectives of the intercomparison: to identify and eliminate differences between AMS laboratories, thus, improving the 36Cl data quality world-wide as soon as possible. The evaluation of the final results of the eight participating AMS laboratories for three synthetic AgCl samples with 36Cl/Cl ratios at the 10-11 (SM-Cl-11), 10-12 (SM-Cl-12), and 10-13 (SM-Cl-13) level shows no difference in the sense of simple statistical significance [2]. However, more detailed statistical analyses indicate a certain interlaboratory bias and an under-estimation of uncertainties by some of the laboratories. The round-robin data demonstrate that 36Cl/Cl results from two individual AMS laboratories can differ by up to 17% (Fig. 1). Thus, the necessity for further work on harmonizing the 36Cl business on a world-wide scale and on improving the measurements is obvious. Fig. 1: Regression of normalized results for the three investigated samples in the order of ascending average. [1] V. Alfimov et al., Ion Beam Physics, ETH Zurich, Annual Report (2009) 13. [2] S. Merchel et al., Anal. Bioanal. Chem., submitted.

A first international 36Cl interlaboratory comparison has been initiated. Evaluation of the final results of the eight participating accelerator mass spectrometry (AMS) laboratories on three synthetic AgCl samples with 36Cl/Cl ratios at the 10-11, 10-12, and 10-13 level shows no difference in the sense of simple statistical significance. However, more detailed statistical analyses demonstrate certain interlaboratory bias and underestimation of uncertainties by some laboratories. Subsequent remeasurement and reanalysis of the data from some AMS facilities, the round-robin data indicates that 36Cl/Cl data from two individual AMS laboratories can differ by up to 17%. Thus, the demand for further work on harmonising the 36Cl-system on a world-wide scale and enlarging the improvement of measurements is obvious.

Driven by the progress in AMS and its spreading application within geosciences, measurements of increasing numbers of samples with low isotopic ratios will be required in the future. Therefore, we have examined the linearity of 10Be/9Be as a function of isotope ratio by distributing 3 secondary standards (dilutions of NIST4325: 10-12-10-14) to 9 AMS labs. The problem of low ratio samples is even more crucial for 36Cl mainly due to the high volatility of chlorine. Thus, we have prepared large quantities of 3 36Cl/Cl solutions from a certified 36Cl activity (NIST4943) by dilution with NaCl. AgCl precipitated from these solutions (10-11-10-13) has been distributed to 9 AMS labs. Some measurements are still ongoing. First results from 6 labs for each nuclide show that these interlaboratory exercises are very valuable.

Driven by the progress in accelerator mass spectrometry (AMS) and its spreading application within geosciences, the number of samples with low isotopic ratios will increase. Therefore, we have examined the linearity of 10Be/9Be as a function of isotope ratio by distributing three secondary standards (dilutions of NIST4325: 10-12-10-14) to nine AMS labs. The problem of low ratio samples is even more crucial for 36Cl. Thus, we have prepared large quantities of three 36Cl/Cl solutions from a certified 36Cl activity (NIST4943) by dilution with NaCl. AgCl precipitated from these solutions (10-11-10-13) has been distributed to nine AMS labs. We are still awaiting some final results, but first results from eight labs for each nuclide show that these interlaboratory exercises are very valuable, as they show maximum differences between individual AMS labs up to 35% for 10Be, and 25% for 36Cl, respectively. Our data indicate that actual 36Cl measurements at LLNL, PRIME Lab and ANU can differ by more than 10%. The implications of this for 36Cl in-situ production rates, determined earlier at these facilities, cannot be ignored. Some, although not all, of the current disagreement concerning 36Cl production rates may result from these discrepancies. However, we are assuming that our new data at the 10-12-10-13 level are still representative for earlier measurements of in-situ samples at that level. This work was partially funded by CRONUS-EU (Marie-Curie Action, 6th FP #511927).

Round-robin studies on relative sensitivity factors (RSFs) in secondary ion mass spectrometry (SIMS) were conducted using bulk GaAs samples uniformly doped with various impurity elements. A total of 31 laboratories participated in two round-robins. More than 30 sets of relative ion intensities were obtained for B, Si, Cr, Mn, Fe, Cu, Zn, In and Te in GaAs. The RSFs for both positive and negative ions were derived for several types of SIMS instruments. The effect of primary ion incident angle was examined using quadrupole-based instruments and found to be the determining factor of the instrumental dependence of RSF.