Water loss from Jackass Penguin Spheniscus demersus eggs during natural incubation

The water budget of incubating Jackass Penguin eggs was studied on Marcus Island, South Africa, and complementary measurements were made in the laboratory. The mean ambient temperature was 16-5 "C and the mean humidity was 12-4 Torr (89% relative humidity). The temperature of incubated live and water-filled eggs ranged between 14^oCand 37 ^oC. The mean calculated egg temperature was 34-9' C. The mean brood patch temperature was 37-1 ^oC, slightly lower than the cloacal temperature (37.8 ^oC). The mean brood patch area was about 38 cm². The rate of water loss was 411 mg day^-1. The total diffusive water loss during 37 days of incubation was, as predicted, 15-2% of the initial 100-3 g egg mass. The total pore number was 6245 per egg and the shell thickness was 577 fira. It is suggested that the eggshell parameters, incubation length and nesting behaviour are compensated in such a way that an egg-to-nest water vapour pressure difference lower than commonly found is sufficient to bring about the normal total water loss.     

The complex network of non-cellulosic carbohydrate metabolism

Partitioning of carbon dominates intracellular fluxes in both photosynthetic and heterotrophic plant tissues, and has vast influence on both plant growth and development. Recently, much progress has been made in elucidating the structures of the biosynthetic and degradative pathways that link the major and minor pools of soluble carbohydrates to cellular polymers such as starch, heteroglycans and fructans. In most cases, the regulatory properties of these pathways have been elucidated and the enzymes involved have been investigated using reverse genetics approaches. Although many of the results from these approaches were merely confirmatory, several of them were highly unexpected. The challenge ahead is to achieve better understanding of metabolic regulation at the network level in order to develop more rational strategies for metabolic engineering.     

Determination of metabolic fluxes in a non-steady-state system

Estimation of fluxes through metabolic networks from redistribution patterns of (13)C has become a well developed technique in recent years. However, the approach is currently limited to systems at metabolic steady-state; dynamic changes in metabolic fluxes cannot be assessed. This is a major impediment to understanding the behaviour of metabolic networks, because steady-state is not always experimentally achievable and a great deal of information about the control hierarchy of the network can be derived from the analysis of flux dynamics. To address this issue, we have developed a method for estimating non-steady-state fluxes based on the mass-balance of mass isotopomers. This approach allows multiple mass-balance equations to be written for the change in labelling of a given metabolite pool and thereby permits over-determination of fluxes. We demonstrate how linear regression methods can be used to estimate non-steady-state fluxes from these mass balance equations. The approach can be used to calculate fluxes from both mass isotopomer and positional isotopomer labelling information and thus has general applicability to data generated from common spectrometry- or NMR-based analytical platforms. The approach is applied to a GC-MS time-series dataset of (13)C-labelling of metabolites in a heterotrophic Arabidopsis cell suspension culture. Threonine biosynthesis is used to demonstrate that non-steady-state fluxes can be successfully estimated from such data while organic acid metabolism is used to highlight some common issues that can complicate flux estimation. These include multiple pools of the same metabolite that label at different rates and carbon skeleton rearrangements.     

Identification of enzyme activity quantitative trait loci in a Solanum lycopersicum x Solanum pennellii introgression line population

Activities of 28 enzymes from central carbon metabolism were measured in pericarp tissue of ripe tomato fruits from field trials with an introgression line (IL) population generated by introgressing segments of the genome of the wild relative Solanum pennellii (LA0716) into the modern tomato cultivar Solanum lycopersicum M82. Enzyme activities were determined using a robotized platform in optimized conditions, where the activities largely reflect the level of the corresponding proteins. Two experiments were analyzed from years with markedly different climate conditions. A total of 27 quantitative trait loci were shared in both experiments. Most resulted in increased enzyme activity when a portion of the S. lycopersicum genome was substituted with the corresponding portion of the genome of S. pennellii. This reflects the change in activity between the two parental genotypes. The mode of inheritance was studied in a heterozygote IL population. A similar proportion of quantitative trait loci (approximately 30%) showed additive, recessive, and dominant modes of inheritance, with only 5% showing overdominance. Comparison with the location of putative genes for the corresponding proteins indicates a large role of trans-regulatory mechanisms. These results point to the genetic control of individual enzyme activities being under the control of a complex program that is dominated by a network of trans-acting genes.     

Analysis of a range of catabolic mutants provides evidence that phytanoyl-coenzyme A does not act as a substrate of the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex in Arabidopsis during dark-induced senescence

The process of dark-induced senescence in plants is not fully understood, however, the functional involvement of an electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO), has been demonstrated. Recent studies have revealed that the enzymes isovaleryl-coenzyme A (CoA) dehydrogenase and 2-hydroxyglutarate dehydrogenase act as important electron donors to this complex. In addition both enzymes play a role in the breakdown of cellular carbon storage reserves with isovaleryl-CoA dehydrogenase being involved in degradation of the branched-chain amino acids, phytol, and lysine while 2-hydroxyglutarate dehydrogenase is exclusively involved in lysine degradation. Given that the chlorophyll breakdown intermediate phytanoyl-CoA accumulates dramatically both in knockout mutants of the ETF/ETFQO complex and of isovaleryl-CoA dehydrogenase following growth in extended dark periods we have investigated the direct importance of chlorophyll breakdown for the supply of carbon and electrons during this process. For this purpose we isolated three independent Arabidopsis (Arabidopsis thaliana) knockout mutants of phytanoyl-CoA 2-hydroxylase and grew them under the same extended darkness regime as previously used. Despite the fact that these mutants accumulated phytanoyl-CoA and also 2-hydroxyglutarate they exhibited no morphological changes in comparison to the other mutants previously characterized. These results are consistent with a single entry point of phytol breakdown into the ETF/ETFQO system and furthermore suggest that phytol is not primarily metabolized by this pathway. Furthermore analysis of isovaleryl-CoA dehydrogenase/2-hydroxyglutarate dehydrogenase double mutants generated here suggest that these two enzymes essentially account for the entire electron input via the ETF complex.