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Occurrence of mitochondria-targeted Late Embryogenesis Abundant (LEA) gene in animals increases organelle resistance to water stress.

ABSTRACT: Anhydrobiotic animals survive virtually complete loss of cellular water. The mechanisms that explain this phenomenon are not fully understood but often include the accumulation of low molecular weight solutes such as trehalose and macromolecules like Late Embryogenesis Abundant (LEA) proteins. Here we report for the first time the occurrence of a mitochondria-targeted LEA gene (Afrlea3m) product in an animal species. The deduced molecular mass of the 307-amino acid polypeptide from the brine shrimp Artemia franciscana is 34 kDa. Bioinformatic analyses reveal features typical of a Group 3 LEA protein, and subcellular localization programs predict targeting of the mature peptide to the mitochondrial matrix, based on an N-terminal, amphipathic presequence. Real-time quantitative PCR shows that Afralea3m mRNA is expressed manyfold higher in desiccation-tolerant embryonic stages when compared with intolerant nauplius larvae. Mitochondrial localization of the protein was confirmed by transfection of human hepatoma cells (HepG2/C3A) with a nucleotide construct encoding the first 70 N-terminal amino acids of AfrLEA3m in-frame with the nucleotide sequence for green fluorescence protein. The chimeric protein was readily incorporated into mitochondria of these cells. Successful targeting of a protein to human mitochondria by use of an arthropod signaling sequence clearly reveals the highly conserved nature of such presequences, as well as of the import machinery. Finally, mitochondria isolated from A. franciscana embryos, which naturally contain AfrLEA3m and trehalose, exhibit resistance to water stress (freezing) as evidenced by an unchanged capacity for oxidative phosphorylation on succinate + rotenone, a resistance that is absent in mammalian mitochondria lacking AfrLEA3m.

SUBMITTER: Menze MA

PROVIDER: S-EPMC2667758 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Occurrence of mitochondria-targeted Late Embryogenesis Abundant (LEA) gene in animals increases organelle resistance to water stress.

Menze Michael A MA Boswell Leaf L Toner Mehmet M Hand Steven C SC

The Journal of biological chemistry 20090219 16

Anhydrobiotic animals survive virtually complete loss of cellular water. The mechanisms that explain this phenomenon are not fully understood but often include the accumulation of low molecular weight solutes such as trehalose and macromolecules like Late Embryogenesis Abundant (LEA) proteins. Here we report for the first time the occurrence of a mitochondria-targeted LEA gene (Afrlea3m) product in an animal species. The deduced molecular mass of the 307-amino acid polypeptide from the brine shr ...[more]

PMID: 19228698

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Project description:BackgroundLEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown.ResultsWe present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE) and/or low temperature response (LTRE) elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded.ConclusionThe analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for future efforts to elucidate the functional role of these enigmatic proteins.

Project description:Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-?-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions.

Dataset Information

Occurrence of mitochondria-targeted Late Embryogenesis Abundant (LEA) gene in animals increases organelle resistance to water stress.

Publications

Occurrence of mitochondria-targeted Late Embryogenesis Abundant (LEA) gene in animals increases organelle resistance to water stress.

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