Progress 09/01/03 to 08/31/08
Outputs
Vitamin C is an essential redox molecule in eukaryotic cells. We have continued the study of cytochromes b561, a class of membrane proteins, ubiquitously present in plants and animals. These proteins use ascorbate as a substrate and play a poorly understood role in vitamin C metabolism. Using Arabidopsis thaliana as a model system, we have generated a knock-out plant, not expressing one of the cytochromes. Our recent results demonstrate that these proteins provide a new link between iron and vitamin C metabolism.
Impacts
The discovery of a new link between iron and ascorbate metabolism is a significant contribution to our understanding of the role of vitamin C. Since Cytochromes b occur in plants and animals, our results are also highly relevant to understanding mammalian vitamin C metabolism.
Publications
- Berczi A, Su D, Lakshminarasimhan M, Vargas A, Asard H (2005) Heterologous expression and site-directed mutagenesis of an ascorbate-reducible cytochrome b561. Arch. Biochem. Biophys. 443: 82-92
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Progress 10/01/03 to 09/30/04
Outputs
The major objective of the project is the characterization of the role in ascorbate metabolism of a newly described class of membrane proteins, cytochromes b561 (Cyts b561). The following progress has been made: 1) We have successfully generated recombinant Cyt b561 proteins in yeast. The recombinant proteins are ascorbate reducible and have characteristics similar to that of the native proteins. We are currently characterizing the substrate specificity of this protein, which will be a major contribution to understanding its physiological function. 2) A recently identified knock-out mutant plant in which one of the Cyt b561 genes has been knocked out, has been partially characterized. Of major interest is the observation that these plants seem to have an impaired iron metabolism, supporting a new physiological role for these proteins.
Impacts
This project contributes to our understanding of ascorbate (vitamin C) metabolism in plants, which is potentially of agricultural importance.
Publications
- Griesen D, Su D, Berczi A, Asard H (2004) Localization of an ascorbate-reducible cytochrome b561 in the plant tonoplast. Plant Physiology 134: 726-734
- Potters G, Horemans N, Bellone S, Caubergs R, Trost P, Guisez Y, Asard H (2004) Dehydroascorbate influences the plant cell cycle through a glutathione-independent reduction mechanism. Plant Physiology 134: 1479-1487
- Verelst W, Kapila J, Engler JD, De Almeida J, Stone J, Caubergs R, Asard H (2004) Tissue-specific and developmental regulation of Arabidopsis thaliana cytochrome b561 genes. Physiol Plant 120: 312-318 (Agricultural Research Division no. 13822)
- Chen S, Vaghchhipawala Z, Li W, Asard H, Dickman MB (2004) Tomato phospholipids hydroperoxide glutathione peroxidase inhibits cell death induced by Bax and oxidative stress in yeast and plants. Plant Physiology 135: 1630-1641
- Szarka A, Horemans, Banhegyi G, Asard H (2004) Facilitated glucose and dehydroascorbate transport in plant mitochondria. Arch Biochem Biophys 428: 73-80
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Progress 10/01/02 to 09/30/03
Outputs
The major objective of the project is the characterization of the role in ascorbate metabolism of a newly described class of membrane proteins, cytochromes b561 (Cyt b561). Major progress has been made on 1) the heterologous expression of the Arabidopsis Cyt b561 homologues in yeast; 2) the tissue and subcellular localization of the plant Cyt b561 isoforms, and 3) the identification of a knock-out mutant in one of the Cyt b561 isoforms. 1 - Heterlogous expression in yeast. We have successfully generated recombinant Cyt b561 proteins in yeast. The recombinant proteins are ascorbate reducible and characteristics similar to the native proteins. This result is major significance as it allows us to study the protein catalytic properties in great detail, and allows the generation of site-directed mutants to study the Cyt b561 structure-function relationship. 2 - Tissue and subcellular localization. Using the reporter gene GUS, fused to the Cyt b561 promoters we have been
able to determine the organ distribution for three of the four Cyt b561 isoforms from Arabidopsis. All proteins are predominantly expressed in the plant root system. One of the isoforms is localized at the vacuolar membrane. 3 - Knock-out mutant. Recently we have identified Arabidopsis lines in which the expression of one of the Cyt b561 isoforms is repressed. These plants are of major interest as they are essential tools in the characterization of the physiological role of these proteins.
Impacts
This project contributes to our understanding of ascorbate (vitamin C) metabolism in plants, which is potentially of agricultural importance.
Publications
- Berczi A, Asard H (2003) Soluble proteins, an often overlooked contaminant in plasma membrane preparations. Trends Plant Sci 8: 250-251 (Agricultural Research Division no. 14078)
- Potters G, De Gara L, Asard H, Horemans N (2002) Ascorbate and glutathione: guardians of the cell cycle, partners in crime ? Plant Physiol Biochem 40: 537-548 (Agricultural Research Division no. 13692)
- Pasternak T, Prinsen E, Ayaydin F, Miskolczi P, Potters G, Asard H, Van Onckelen H, Dudits D, Feher A (2002) The role of auxin, pH and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa. Plant Physiol 129: 1807-1819 (Agricultural Research Division no. 13694)
- Berczi A, Caubergs RJ, Asard H (2003) Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves. Protoplasma 221: 47-56 (Agricultural Research Division no. 13693)
- Bashtovyy D, Berczi A, Asard H, Pali T (2003) Structure prediction for the di-heme cytochrome b-561 protein family. Protoplasma 221: 31-40 (Agricultural Research Division no. 13700)
- Raeymaekers T, Asard H, Potters G, Guisez Y, Horemans N (2003) The copper-mediated oxidative busrt in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures. Protoplasma 221: 93-100
- Verelst W, Asard H (2003) A phylogenetic study of cytochrome b561 proteins. Genome Biology 4:R38 (Agricultural Research Division no. 14061)
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