Elzbieta Krol

Perception of the Arabidopsis danger signal peptide 1 involves the pattern recognition receptor AtPEPR1 and its close homologue AtPEPR2

Plasma membrane-borne pattern recognition receptors, which recognize microbe-associated molecular patterns and endogenous damage-associated molecular patterns, provide the first line of defense in innate immunity. In plants, leucine-rich repeat receptor kinases fulfill this role, as exemplified by FLS2 and EFR, the receptors for the microbe-associated molecular patterns flagellin and elongation factor Tu. Here we examined the perception of the damage-associated molecular pattern peptide 1 (AtPep1), an endogenous peptide of Arabidopsis identified earlier and shown to be perceived by the leucine-rich repeat protein kinase PEPR1. Using seedling growth inhibition, elicitation of an oxidative burst and induction of ethylene biosynthesis, we show that wild type plants and the pepr1 and pepr2 mutants, affected in PEPR1 and in its homologue PEPR2, are sensitive to AtPep1, but that the double mutant pepr1/pepr2 is completely insensitive. As a central body of our study, we provide electrophysiological evidence that at the level of the plasma membrane, AtPep1 triggers a receptor-dependent transient depolarization through activation of plasma membrane anion channels, and that this effect is absent in the double mutant pepr1/pepr2. The double mutant also fails to respond to AtPep2 and AtPep3, two distant homologues of AtPep1 on the basis of homology screening, implying that the PEPR1 and PEPR2 are responsible for their perception too. Our findings provide a basic framework to study the biological role of AtPep1-related danger signals and their cognate receptors.

The Functional Significance of Individual Variation in Basal Metabolic Rate

Basal metabolic rate (BMR) was established as a common reference point allowing comparable measures across different individuals and species. BMR is often regarded as a minimal rate of metabolism compatible with basic processes necessary to sustain life. One confusing aspect, however, is that BMR is highly variable, both within and between species. A potential explanation for this variability is that while individuals with high BMRs may suffer the disadvantage of having to feed for longer to cover the extra energy demands, this may be offset by advantages that accrue because of the high metabolic rate. One suggested advantage is that high levels of BMR are a consequence of maintaining a morphology that permits high rates of the maximal sustained rate of metabolism (SusMR)—the rate of metabolism that can be sustained for days or weeks. We have been studying the energetics of MF1 laboratory mice during peak lactation to investigate this idea. In this article, we review some of our work in connection with three particular predictions that derive from the hypothesised links among morphology, basal metabolism, and sustained metabolic rate. By comparing groups of individuals, for example, lactating and nonlactating individuals, the patterns that emerge are broadly consistent with the hypothesis that BMR and SusMR are linked by morphology. Lactating mice have bigger organs connected with energy acquisition and utilisation, greater resting metabolic rates in the thermoneutral zone, called RMRt (approximately equivalent to BMR), and high sustainable rates of maximal energy intake. However, when attempts are made to establish these relationships across individuals within lactating mice, the associations that are anticipated are either absent or very weak and depend on shared variation due to body mass. At this level there is very little support for the suggestion that variation in RMRt (and thus BMR) is sustained by associations with SusMR.

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe-associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode-impaled mesophyll cells and root hairs of Col-0 Arabidopsis plants revealed rapid, dose-dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion-selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species-independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2-associated receptor-like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1-dependent, calcium-associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.

Limits to sustained energy intake IX: a review of hypotheses

Several lines of evidence indicate that animals in the wild may be limited in their maximal rates of energy intake by their intrinsic physiology rather than food availability. Understanding the limits to sustained energy intake is important because this defines an envelope within which animals must trade-off competing activities. In the first part of this review, we consider the initial ideas that propelled this area and experimental evidence connected with them. An early conceptual advance in this field was the idea that energy intake could be centrally limited by aspects of the digestive process, or peripherally limited at the sites of energy utilisation. A model system that has been widely employed to explore these ideas is lactation in small rodents. Initial studies in the late 1980s indicated that energy intake might be centrally limited, but work by Hammond and colleagues in the 1990s suggested that it was more likely that the limits were imposed by capacity of the mammary glands, and other works tended to support this view. This consensus, however, was undermined by studies that showed milk production was higher in mice at low temperatures, suggesting that the capacity of the mammary gland is not a limiting factor. In the second part of the review we consider some additional hypotheses that might explain these conflicting data. These include the heat dissipation limits hypothesis, the seasonal investment hypothesis and the saturated neural control hypothesis. Current evidence with respect to these hypotheses is also reviewed. The limited evidence presently available does not unambiguously support any one of them.

Limits to sustained energy intake VI. Energetics of lactation in laboratory mice at thermoneutrality

SUMMARY The limits to sustained energy intake are important because of their implications for reproductive output, foraging behaviour and thermoregulatory capabilities. Recent attempts to elucidate the nature of the limits to sustained energy intake have focused on peak lactation, which is the most energetically demanding period for female mammals. The hypothesis that performance of lactating animals is limited peripherally by the capacity of mammary glands to produce milk has received the most attention. However, some empirical data cannot be explained by the peripheral limitation hypothesis. Here, we present a novel hypothesis that the limits to sustained energy intake at peak lactation are imposed by the capacity of the animal to dissipate body heat generated as a by-product of processing food and producing milk. To test the heat dissipation limit hypothesis we challenged reproducing MF1 laboratory mice (N=67) with a reduced potential heat flow between the animal and the environment by exposing them to 30°C (thermoneutral zone). We compared their food intake and reproductive output at peak lactation with animals studied previously at 21°C (N=71) and 8°C (N=15). Mice lactating at 30°C had a significantly lower mean asymptotic food intake (12.4 g day-1) than those at 21°C (23.5 g day-1) and 8°C (28.6 g day-1). On average, mice at 30°C raised significantly fewer (9.8) and smaller (6.1 g) pups than those at 21°C (11.3 pups; 7.0 g per pup) and smaller pups than those at 8°C (9.6 pups; 7.3 g per pup). Consequently, mean litter mass at 30°C (56.0 g) was significantly lower than at 21°C (77.1 g) and at 8°C (68.7 g). The mean rate of litter mass increase at 30°C (2.1 g day-1) was also lower than at 21°C (3.1 g day-1). The reduced food intake and low reproductive output in mice lactating at 30°C are consistent with the heat dissipation limit hypothesis.

Animal models of obesity

Background Obesity stems from a prolonged imbalance between the levels of energy intake and expenditure, with the resultant surplus being stored as body lipids. Our understanding of the regulation of food intake and the physiological basis of differences in energy expenditure is owed, in large part, to studies made on animals. Moreover, animal models have been a cornerstone of studies of environmental effects, such as epigenetics, responses to high-fat and low-calorie diets and the identification and development of pharmaceuticals for obesity treatment. This review provides some examples of the animal work that has been performed, and focuses on the variation in approaches that have been taken and their potential, rather than aiming to be a comprehensive summary.

Limits to sustained energy intake. X. Effects of fur removal on reproductive performance in laboratory mice.

SUMMARY The maximum rate of sustained energy intake (SusEI) may limit reproductive effort and other aspects of animal performance. We have previously suggested that lactating mice are not limited centrally by the alimentary tract or peripherally by the mammary glands, but that the limits to SusEI are imposed by the capacity of the animal to dissipate body heat generated as a by-product of processing food and producing milk. To explore the nature of the limits to SusEI, we bred MF1 laboratory mice at 21°C and then dorsally shaved lactating females to reduce their external insulation and thereby elevate their capacity to dissipate body heat. These mice increased their food intake by 12.0% and assimilated on average 30.9 kJ day–1 more energy than unshaved animals. With nearly identical mean litter sizes (11.4 pups for shaved and 11.3 pups for unshaved mice), shaved mothers exported 15.2% (22.0 kJ day–1) more energy as milk than control individuals. The elevated milk production of shaved mice enabled them to wean litters that were 15.4% (12.2 g) heavier than offspring produced by unshaved mice. Our results argue against central, peripheral or extrinsic limits to SusEI at peak lactation and provide strong support for the heat dissipation limit hypothesis. More generally, we see many situations where heat dissipation may be a previously unrecognised factor constraining the evolution of endothermic animals – for example, the latitudinal and altitudinal trends in clutch and litter sizes and the migration patterns of birds.

The contribution of animal models to the study of obesity

Summary Obesity results from prolonged imbalance of energy intake and energy expenditure. Animal models have provided a fundamental contribution to the historical development of understanding the basic parameters that regulate the components of our energy balance. Five different types of animal model have been employed in the study of the physiological and genetic basis of obesity. The first models reflect single gene mutations that have arisen spontaneously in rodent colonies and have subsequently been characterized. The second approach is to speed up the random mutation rate artificially by treating rodents with mutagens or exposing them to radiation. The third type of models are mice and rats where a specific gene has been disrupted or overexpressed as a deliberate act. Such genetically-engineered disruptions may be generated through the entire body for the entire life (global transgenic manipulations) or restricted in both time and to certain tissue or cell types. In all these genetically-engineered scenarios, there are two types of situation that lead to insights: where a specific gene hypothesized to play a role in the regulation of energy balance is targeted, and where a gene is disrupted for a different purpose, but the consequence is an unexpected obese or lean phenotype. A fourth group of animal models concern experiments where selective breeding has been utilized to derive strains of rodents that differ in their degree of fatness. Finally, studies have been made of other species including non-human primates and dogs. In addition to studies of the physiological and genetic basis of obesity, studies of animal models have also informed us about the environmental aspects of the condition. Studies in this context include exploring the responses of animals to high fat or high fat/high sugar (Cafeteria) diets, investigations of the effects of dietary restriction on body mass and fat loss, and studies of the impact of candidate pharmaceuticals on components of energy balance. Despite all this work, there are many gaps in our understanding of how body composition and energy storage are regulated, and a continuing need for the development of pharmaceuticals to treat obesity. Accordingly, reductions in the use of animal models, while ethically desirable, will not be feasible in the short to medium term, and indeed an expansion in activity using animal models is anticipated as the epidemic continues and spreads geographically.

Limits to sustained energy intake VII. Milk energy output in laboratory mice at thermoneutrality

SUMMARY The limits to sustained energy intake at peak lactation could be imposed peripherally, by the capacity of the mammary glands, or centrally, by the capacity of the animal to dissipate body heat generated as a by-product of processing food and producing milk. To distinguish between the two hypotheses, we examined milk energy output at peak lactation in MF1 laboratory mice exposed to 30°C (N=12), 21°C (N=10; published data) and 8°C (N=10; published data). The peripheral limitation hypothesis predicts that milk energy output will remain constant at different temperatures, while the heat dissipation limit hypothesis predicts a decline in milk energy output as temperature increases. Since estimates of milk energy output in small mammals can vary depending on the calculation method used, we evaluated the milk energy output of mice (N=24) using four different methods: (1) as the difference between metabolizable energy intake and daily energy expenditure of the female, (2) from female water turnover, (3) from pup water turnover and (4) from the energy budget of the litter. We assessed these four methods by comparing their accuracy, precision and sensitivity to changes in parameters involved in the calculations. Methods 1, 3 and 4 produced similar estimates of milk energy output, while those derived from female water turnover were significantly lower and more variable. On average, mice at 30°C exported significantly less energy as milk (87.7 kJ day–1) than mice at 21°C (166.7 kJ day–1) and 8°C (288.0 kJ day–1). This reduction in milk energy output at 30°C was caused by a significant decline in both milk flow (20.0 g day–1, 12.9 g day–1 and 8.5 g day–1 at 8°C, 21°C and 30°C, respectively) and gross energy content of milk (14.6 kJ g–1, 13.1 kJ g–1 and 10.5 kJ g–1 at 8°C, 21°C and 30°C, respectively). Milk produced at 30°C contained significantly less total solids (34.4%) than milk at 21°C (40.9%) and 8°C (41.5%) and significantly less fat (20.0%) than milk at 21°C (26.4%) and 8°C (30.3%). The reduced milk energy output in mice exposed to 30°C, paralleled by their reduced food intake and low reproductive output, argues against the peripheral limitation hypothesis and provides strong support for the heat dissipation limit hypothesis.

Limits to sustained energy intake VIII. Resting metabolic rate and organ morphology of laboratory mice lactating at thermoneutrality

SUMMARY We have previously shown that the food intake and milk production of MF1 laboratory mice lactating at 30°C, 21°C and 8°C increase as temperature declines. These data suggest that mice are not limited peripherally by the capacity of the mammary glands to produce milk but are limited by the capacity of the animal to dissipate body heat generated as a by-product of food processing and milk production. Here, we measure resting metabolic rate (RMR; prior to breeding and at peak lactation) and organ morphology (at peak lactation) in MF1 mice exposed to 30°C (thermoneutrality) and compare these traits with the same parameters measured previously in mice at 21°C and 8°C. The masses of visceral organs primarily responsible for energy flux (heart, lungs, stomach, small intestine, large intestine, liver, pancreas, spleen and kidneys) increased as temperature declined. The masses of all these organs differed between mice exposed to 8°C and 21°C, whereas only the masses of heart, liver and kidneys differed between mice at 21°C and 30°C. The increases in organ masses were paralleled by increases in RMR at peak lactation above the levels measured prior to breeding, with mice at 8°C and 21°C having significantly higher increases in RMR than mice at 30°C (29.6 kJ day–1, 25.5 kJ day–1 and 8.1 kJ day–1, respectively). The observed changes in visceral organs and RMR are consistent with both the heat dissipation and peripheral limit hypotheses. However, mice exposed to 8°C had substantially larger mammary glands than mice at 21°C or 30°C (2.450 g, 1.115 g and 0.956 g dry mass, respectively), which argues against the peripheral limitation hypothesis and is consistent with the heat dissipation limit hypothesis. In addition, cold exposure resulted in greater masses of brown adipose tissue, white adipose tissue, pelage and tail. We discuss these changes in the context of the potential thermoregulatory benefits from use of the heat generated as a by-product of milk synthesis.