Functional expression of phosphagen kinase systems confers resistance to transient stresses in Saccharomyces cerevisiae by buffering the ATP pool

Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.     

Involvement and relative importance of at least two distinct mechanisms in the effects of 2-mercaptoethanol on murine lymphocytes in culture

2-Mercaptoethanol (2-ME) exerts several effects on murine lymphocytes in culture that might explain its ability to enhance survival and growth of these cells. The uptake of the essential amino acid cystine and consequently the maintenance of intracellular glutathione levels are enhanced by 2-ME. Furthermore, 2-ME (even in the disulfide form) causes lymphocytes to release thiols into the culture medium. These effects might protect the cells from oxidative damage. The additional cystine provided by treatment of lymphocyte cultures with 2-ME might also allow adequate protein synthesis to support survival and/or growth. This study was conducted to assess the relative importance of the antioxidant and protein synthesis effects of 2-ME. As expected, 2-ME increased cystine uptake at all concentrations that enhanced growth and survival, but four nonthiol antioxidants that enhanced growth and/or survival either did not substantially affect cystine uptake or decreased it and did not affect the release of cystine or its products. The results presented here demonstrate that antioxidant protection is necessary and sufficient for lymphocyte survival and that cystine uptake in untreated lymphocytes is sufficient to support the protein synthesis needed for survival and limited growth. However, we also noted that concentrations of 2-ME that stimulated maximal growth more than doubled protein synthesis as measured at 8 hr. Thus the portion of the effects of 2-ME not accounted for by antioxidant action could be accounted for by enhanced protein synthesis.     

Interactive Effects of Fire, Soil Climate, and Moss on CO2 Fluxes in Black Spruce Ecosystems of Interior Alaska

Fire is an important control on the carbon (C) balance of the boreal forest region. Here, we present findings from two complementary studies that examine how fire modifies soil organic matter properties, and how these modifications influence rates of decomposition and C exchange in black spruce (Picea mariana) ecosystems of interior Alaska. First, we used laboratory incubations to explore soil temperature, moisture, and vegetation effects on CO₂ and DOC production rates in burned and unburned soils from three study regions in interior Alaska. Second, at one of the study regions used in the incubation experiments, we conducted intensive field measurements of net ecosystem exchange (NEE) and ecosystem respiration (ER) across an unreplicated factorial design of burning (2 year post-fire versus unburned sites) and drainage class (upland forest versus peatland sites). Our laboratory study showed that burning reduced the sensitivity of decomposition to increased temperature, most likely by inducing moisture or substrate quality limitations on decomposition rates. Burning also reduced the decomposability of Sphagnum-derived organic matter, increased the hydrophobicity of feather moss-derived organic matter, and increased the ratio of dissolved organic carbon (DOC) to total dissolved nitrogen (TDN) in both the upland and peatland sites. At the ecosystem scale, our field measurements indicate that the surface organic soil was generally wetter in burned than in unburned sites, whereas soil temperature was not different between the burned and unburned sites. Analysis of variance results showed that ER varied with soil drainage class but not by burn status, averaging 0.9 ± 0.1 and 1.4 ± 0.1 g C m⁻² d⁻¹ in the upland and peatland sites, respectively. However, a more complex general linear model showed that ER was controlled by an interaction between soil temperature, moisture, and burn status, and in general was less variable over time in the burned than in the unburned sites. Together, findings from these studies across different spatial scales suggest that although fire can create some soil climate conditions more conducive to rapid decomposition, rates of C release from soils may be constrained following fire by changes in moisture and/or substrate quality that impede rates of decomposition. Author contributions: JAO: performed research, analyzed data, contributed new methods, wrote the paper; MRT: designed laboratory study, performed research, analyzed data; JWH: designed field study, performed research; KLM: performed research; LEP: performed research, contributed new method; GS: performed research; JCN: performed research.     

THE TIME-COURSE OF POLAR MOVEMENT OF GIBBERELLIN THROUGH ZEA ROOTS

The polarity of movement of gibberellin through sections cut from near the root tips of Zea mays L. was studied, using methods like those we previously used in roots for auxin and in petioles for auxins, cytokinins, and gibberellic acid (GA-3). One μg GA-3 was added in a donor agar block and gibberellin activity in the receiver agar at the opposite end of the section was measured directly with a modified barley endosperm bioassay. The movement of gibberellin was away from the root tip (basipetal) and thus opposite in direction to the polarity of auxin through such root sections. The time-course of basipetal movement was dissimilar to that for gibberellin or auxin movement through petiole sections. It took 14-18 hr for gibberellin activity equivalent to 6 ng GA-3 to collect in the basal receivers on roots. Apical receivers showed activity equivalent to 1.6 ng GA-3 at 14-18 hr. Less than 0.01 ng equivalent GA-3 was collected from sections to which GA-3 was not added, so the 6 and 1.6 ng were almost entirely due to the added GA-3. These general conclusions were confirmed with an experiment using ¹⁴C-GA-3. A decline in activity in receivers was found in some experiments at 18 hr, paralleling earlier results with GA-3, IAA, and adenine in petioles and IAA in roots.     

Communal egg-laying behaviour and the consequences of egg aggregation in the brown anole (Anolis sagrei)

Communal nesting is a behaviour that involves multiple females laying eggs in the same nest or nesting site. This behaviour may be a consequence of a shortage of preferable nest sites (constraint hypothesis) or an adaptation generated by fitness benefits associated with egg aggregation (adaptive hypothesis). Experimental tests of these hypotheses require information about maternal nest site choice and its fitness consequences. To address these, we studied a lizard (brown anole; Anolis sagrei) that produces single-egg clutches, but often aggregates eggs in nest sites. In a lab study, females were given the option of nesting in (a) soil previously used as nest substrate vs. fresh soil and (b) soil with eggshells vs. without eggshells. We also experimentally examined the effects of egg aggregation by incubating eggs singly, in groups of four, and in groups of nine. We recorded egg surface temperature, water uptake, and hatchling morphology. Females were more likely to nest in pots with used soil and with eggshells than in pots with fresh soil or without eggshells. We observed no effects of egg aggregation on egg survival, egg temperature, or most measures of hatchling morphology. However, singly-incubated eggs absorbed more water than eggs incubated in the four and nine egg aggregations and this resulted in offspring with greater body condition (i.e. heavier for their length) at hatching. The behavioural experiment demonstrates that females actively choose nest sites that have been used previously (as expected under the adaptive hypothesis), but the egg-aggregation experiment shows no benefits to offspring based on the variables measured. Thus, results of the behaviour study support the adaptive hypothesis; however, results from our egg-incubation study do not. Likely, the adaptive and constraint hypotheses are not mutually exclusive, and a diversity of factors influence the evolution of communal nesting behaviour.