Experiment: Dissecting the impact of CO2 and pH on the mechanisms of photosynthesis and calcification in the coccolithophore Emiliania huxleyi

Coccolithophores are important calcifying phytoplankton predicted to be impacted by changes in ocean carbonate chemistry caused by the absorption of anthropogenic CO2. However, it is difficult to disentangle the effects of the simultaneously changing carbonate system parameters (CO2, bicarbonate, carbonate and protons) on the physiological responses to elevated CO2. Here, we adopted a multifactorial approach at constant pH or CO2 whilst varying dissolved inorganic carbon (DIC) to determine physiological and transcriptional responses to individual carbonate system parameters. We show that Emiliania huxleyi is sensitive to low CO2 (growth and photosynthesis) and low bicarbonate (calcification) as well as low pH beyond a limited tolerance range, but is much less sensitive to elevated CO2 and bicarbonate. Multiple up-regulated genes at low DIC bear the hallmarks of a carbon-concentrating mechanism (CCM) that is responsive to CO2 and bicarbonate but not to pH. Emiliania huxleyi appears to have evolved mechanisms to respond to limiting rather than elevated CO2. Calcification does not function as a CCM, but is inhibited at low DIC to allow the redistribution of DIC from calcification to photosynthesis. The presented data provides a significant step in understanding how E. huxleyi will respond to changing carbonate chemistry at a cellular level

Ocean acidification increases the accumulation of toxic phenolic compounds across trophic levels

Increasing atmospheric CO2 concentrations are causing ocean acidification (OA), altering carbonate chemistry with consequences for marine organisms. Here we show that OA increases by 46-212% the production of phenolic compounds in phytoplankton grown under the elevated CO2 concentrations projected for the end of this century, compared with the ambient CO2 level. At the same time, mitochondrial respiration rate is enhanced under elevated CO2 concentrations by 130-160% in a single species or mixed phytoplankton assemblage. When fed with phytoplankton cells grown under OA, zooplankton assemblages have significantly higher phenolic compound content, by about 28-48%. The functional consequences of the increased accumulation of toxic phenolic compounds in primary and secondary producers have the potential to have profound consequences for marine ecosystem and seafood quality, with the possibility that fishery industries could be influenced as a result of progressive ocean changes.

Experiment: Adaptation of a globally important coccolithophore to ocean warming and acidification

Although oceanwarming and acidification are recognized as two major anthropogenic perturbations of today's oceanswe know very little about how marine phytoplankton may respond via evolutionary change.We tested for adaptation to ocean warming in combination with ocean acidification in the globally important phytoplankton species Emiliania huxleyi. Temperature adaptation occurred independently of ocean acidifcation levels. Exponential growth rates were were up to 16% higher in populations adapted for one year to warming when assayed at their upper thermal tolerance limit. Particulate inorganic (PIC) and organic (POC) carbon production was restored to values under present-day ocean conditions, owing to adaptive evolution, and were 101% and 55% higher under combined warming and acidification, respectively, than in non-adapted controls. Cells also evolved to a smaller size while they recovered their initial PIC:POC ratio even under elevated CO2. The observed changes in coccolithophore growth, calcite and biomass production, cell size and elemental composition demonstrate the importance of evolutionary processes for phytoplankton performance in a future ocean. At the end of a 1-yr temperature selection phase, we conducted a reciprocal assay experiment in which temperature-adapted asexual populations were compared to the respective non-adapted control populations under high temperature, and vice versa (1. Assay Data, Dataset #835336). Mean exponential growth rates ? in treatments subjected to high temperature increased rapidly under all high temperature-CO2 treatment combinations during the temperature selection phase (2. time series, Dataset #835339).

Decline in growth of foraminifer Marginopora rossi under eutrophication and ocean acidification scenarios

The combination of global and local stressors is leading to a decline in coral reef health globally. In the case of eutrophication, increased concentrations of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) are largely attributed to local land use changes. From the global perspective, increased atmospheric CO2 levels are not only contributing to global warming but also ocean acidification (OA). Both eutrophication and OA have serious implications for calcium carbonate production and dissolution among calcifying organisms. In particular, benthic foraminifera precipitate the most soluble form of mineral calcium carbonate (high-Mg calcite), potentially making them more sensitive to dissolution. In this study, a manipulative orthogonal two-factor experiment was conducted to test the effects of dissolved inorganic nutrients and OA on the growth, respiration and photophysiology of the large photosymbiont-bearing benthic foraminifer, Marginopora rossi. This study found the growth rate of M. rossi was inhibited by the interaction of eutrophication and acidification. The relationship between M. rossi and its photosymbionts became destabilized due to the photosymbiont's release from nutrient limitation in the nitrate-enriched treatment, as shown by an increase in zooxanthellae cells per host surface area. Foraminifers from the OA treatments had an increased amount of Chl a per cell, suggesting a greater potential to harvest light energy, however, there was no net benefit to the foraminifer growth. Overall, this study demonstrates that the impacts of OA and eutrophication are dose dependent and interactive. This research indicates an OA threshold at pH 7.6, alone or in combination with eutrophication, will lead to a decline in M. rossi calcification. The decline in foraminifera calcification associated with pollution and OA will have broad ecological implications across their ubiquitous range and suggests that without mitigation it could have serious implications for the future of coral reefs.

Alleviation of solar ultraviolet radiation (UVR)-induced photoinhibition in diatom Chaetoceros curvisetus by ocean acidification

The study aimed to unravel the interaction between ocean acidification and solar ultraviolet radiation (UVR) in Chaetoceros curvisetus. Chaetoceros curvisetus cells were acclimated to high CO2 (HC, 1000 ppmv) and low CO2 concentration (control, LC, 380 ppmv) for 14 days. Cell density, specific growth rate and chlorophyll were measured. The acclimated cells were then exposed to PAB (photosynthetically active radiation (PAR) + UV-A + UV-B), PA (PAR + UV-A) or P (PAR) for 60 min. Photochemical efficiency (phi PSII), relative electron transport rate (rETR) and the recovery of ?PSII were determined. HC induced higher cell density and specific growth rate compared with LC. However, no difference was found in chlorophyll between HC and LC. Moreover, phi PSII and rETRs were higher under HC than LC in response to solar UVR. P exposure led to faster recovery of phi PSII, both under HC and LC, than PA and PAB exposure. It appeared that harmful effects of UVR on C. curvisetus could be counteracted by ocean acidification simulated by high CO2 when the effect of climate change is not beyond the tolerance of cells.

Spontaneous mutations recovered as mosaics in the mouse specific-locus test

The specific-locus test (SLT) detects new mutants among mice heterozygous for seven recessive visible markers. Spontaneous mutations can be manifested not only as singleton whole-body mutants in controls (for which we report new data), but as mosaics—either visible (manifesting mottled coat color) in the scored generation (G2) or masked, among the wild-type parental generation (G1). Masked G1 mosaics reveal themselves by producing clusters of whole-body mutants in G2. We provide evidence that most, if not all, mosaics detected in the SLT (both radiation and control progenies) result from a single-strand spontaneous mutation subsequent to the last premeiotic mitosis and before the first postmeiotic one of a parental genome—the “perigametic interval.” Such events in the genomes of the G1 and G0 result, respectively, in visible and masked 50:50 mosaics. Per cell cycle, the spontaneous mutation rate in the perigametic interval is much higher than that in pregamete mitotic divisions. A clearly different locus spectrum further supports the hypothesis of different origin, and casts further doubt on the validity of the doubling-dose risk-estimation method. Because mosaics cannot have arisen in mitotic germ cells, and are not induced by radiation exposure in the perigametic interval, they should not be included in calculations of radiation-induced germ-line mutation rates. For per-generation calculations, inclusion of mosaics yields a spontaneous frequency 1.7 times that calculated from singletons alone for mutations contributed by males; including both sexes, the multiple is 2.2.

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Multicopy plasmids are clustered and localized in Escherichia coli

We localized the multicopy plasmid RK2 in Escherichia coli and found that the number of fluorescent foci observed in each cell was substantially less than the copy number of the plasmid, suggesting that many copies of RK2 are grouped into a few multiplasmid clusters. In minimal glucose media, the majority of cells had one or two foci, with a single focus localized near midcell, and two foci near the 1/4 and 3/4 cell positions. The number of foci per cell increased with cell length and with growth rate, and decreased upon entering stationary phase, suggesting a coordination of RK2 replication or segregation with the bacterial cell cycle. Time-lapse microscopy demonstrated that partitioning of RK2 foci is achieved by the splitting of a single focus into two or three smaller foci, which are capable of separating with rapid kinetics. A derivative of the high-copy-number plasmid pUC19 containing the lacO array was also localized by tagging with GFP-LacI. Whereas many of the cells contained numerous, randomly diffusing foci, most cells exhibited one or two plasmid clusters located at midcell or the cell quarter positions. Our results suggest a model in which multicopy plasmids are not always randomly diffusing throughout the cell as previously thought, but can be replicated and partitioned in clusters targeted to specific locations.

The Regulation of Photosynthetic Electron Transport during Nutrient Deprivation in Chlamydomonas reinhardtii1

The light-saturated rate of photosynthetic O2 evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O2 evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150–2159).

Characterization of Cadmium Binding, Uptake, and Translocation in Intact Seedlings of Bread and Durum Wheat Cultivars

High Cd content in durum wheat (Triticum turgidum L. var durum) grain grown in the United States and Canada presents potential health and economic problems for consumers and growers. In an effort to understand the biological processes that result in excess Cd accumulation, root Cd uptake and xylem translocation to shoots in seedlings of bread wheat (Triticum aestivum L.) and durum wheat cultivars were studied. Whole-plant Cd accumulation was somewhat greater in the bread wheat cultivar, but this was probably because of increased apoplastic Cd binding. Concentration-dependent 109Cd2+-influx kinetics in both cultivars were characterized by smooth, nonsaturating curves that could be dissected into linear and saturable components. The saturable component likely represented carrier-mediated Cd influx across root-cell plasma membranes (Michaelis constant, 20–40 nm; maximum initial velocity, 26–29 nmol g−1 fresh weight h−1), whereas linear Cd uptake represented cell wall binding of 109Cd. Cd translocation to shoots was greater in the bread wheat cultivar than in the durum cultivar because a larger proportion of root-absorbed Cd moved to shoots. Our results indicate that excess Cd accumulation in durum wheat grain is not correlated with seedling-root influx rates or root-to-shoot translocation, but may be related to phloem-mediated Cd transport to the grain.

Coordinating DNA replication initiation with cell growth: differential roles for DnaA and SeqA proteins.

We describe here the development of a new approach to the analysis of Escherichia coli replication control. Cells were grown at low growth rates, in which case the bacterial cell cycle approximates that of eukaryotic cells with G1, S, and G2 phases: cell division is followed sequentially by a gap period without DNA replication, replication of the single chromosome, another gap period, and finally the next cell division. Flow cytometry of such slowly growing cells reveals the timing of replication initiation as a function of cell mass. The data show that initiation is normally coupled to cell physiology extremely tightly: the distribution of individual cell masses at the time of initiation in wild-type cells is very narrow, with a coefficient of variation of less than 9%. Furthermore, a comparison between wild-type and seqA mutant cells shows that initiation occurs at a 10-20% lower mass in the seqA mutant, providing direct evidence that SeqA is a bona fide negative regulator of replication initiation. In dnaA (Ts) mutants the opposite is found: the mass at initiation is dramatically increased and the variability in cell mass at initiation is much higher than that for wild-type cells. In contrast to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go through two initiation events per cell division cycle, and all the origins present in each cell are not initiated in synchrony. The implications for the complex interplay amongst growth, cell division, and DNA replication are discussed.

Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae.

We quantitate the absolute levels of individual mRNAs per yeast cell by hybridizing total yeast RNA with an excess of gene-specific 32P-oligonucleotides, and digesting the resulting RNA-DNA hybrids with S1 nuclease. By comparing the his3 hybridization signal from a known amount of yeast cells to the signal generated by a known amount of his3 RNA synthesized in vitro, we determine that yeast strain KY114 growing in yeast extract/peptone/glucose medium at 30 degrees C contains seven molecules of his3 mRNA per cell. Using a galactose shut-off procedure, we determined that the half-life of his3 mRNA is approximately 11 min under these conditions. From these observations, we calculate that one his3 mRNA molecule is synthesized every 140 s. Analysis of other his3 promoter derivatives suggests that the maximal transcriptional initiation rate in yeast cells is one mRNA molecule every 6-8 s. Using his3 as an internal standard, the number of mRNA molecules per cell have been determined for ded1, trp3, rps4, and gall under a variety of growth conditions. From these results, the absolute mRNA level of any yeast gene can be determined in a single hybridization experiment. Moreover, the rate of transcriptional initiation can be determined for mRNAs whose decay rates are known.

cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe the cdc18'+gene is required both for initiation of DNA replication and for coupling mitosis to the completion of S phase. Cells lacking Cdc18 fail to enter S phase but still undergo nuclear division. Expression of cdc18+ is sufficient to drive a G1-arrested cdc10ts mutant into the S phase of the cell cycle, indicating that cdc18+ represents a critical link between passage through START and the initiation of DNA replication. Here we show that Cdcl8 is a highly unstable protein that is expressed only once per cell cycle at the boundary between GI and S phase. De novo synthesis of Cdc18 is required before, but not after, the initiation of DNA replication, indicating that Cdc18 function is not necessary once the initiation event has occurred. Overproduction of the protein results in an accumulation of cells with DNA content of greater than 2C and delays mitosis, suggesting that Cdc18 is sufficient to cause reinitiation of DNA replication within a given cell cycle. Our data indicate that the synthesis of Cdc18 protein is a critical rate-limiting step in the initiation of DNA replication during each cell cycle. The extreme lability of the protein may contribute to the prevention of reinitiation.