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Tissue distribution of short-chain alcohol dehydrogenase (SAD) proteins in pea (Pisum sativum) in the absence and presence of UV-B stress, and heterologous stress-induced expression from the SAD promoter
Örebro University, Department of Natural Sciences.ORCID iD: 0000-0001-9713-2365
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Biological Sciences
Research subject
Biochemistry; Biology
Identifiers
URN: urn:nbn:se:oru:diva-2941OAI: oai:DiVA.org:oru-2941DiVA, id: diva2:135683
Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2017-10-18Bibliographically approved
In thesis
1. Characterization of stress-inducible short-chain dehydrogenases/reductases (SDR) in plants: study of a novel small protein family from Pisum sativum (pea)
Open this publication in new window or tab >>Characterization of stress-inducible short-chain dehydrogenases/reductases (SDR) in plants: study of a novel small protein family from Pisum sativum (pea)
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In pea (Pisum sativum), the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). The SAD genes are transiently expressed in plants after short exposures to ultraviolet-B radiation, which in turn leads to formation of SAD protein in leaf and stem tissue upon prolonged irradiation. SAD gene expression is also seen as a result of wounding stress.

The recombinant SAD-C protein (which was the most highly over-expressed isoform in Escherichia coli of the isoforms) was shown to be a tetramer that probably consists of a dimer of dimers and which possesses quinone-reducing capability. The enzyme shows approximately the same NADH- and NADPH-dependent activity with 2,5- and 2,6-dimethylbenzoquinone and menadione as substrates.

Western blotting and immunohistochemistry (IHC) shows that the SAD protein is present to a smaller or larger extent in all the different pea tissues examined. Environmental stress such as UV-B radiation clearly increases the content of SAD in leaf and stem tissue but not in roots. This indicates that increased expression of the SAD genes, as a result of UV-B exposure, is limited to the exposed tissue. This is substantiated by the heterologous GUS expression from the pea SAD-C promoter in Arabidopsis during wounding. Only the wound site and the vicinal tissue show transcription from this promoter. In non-stressed tissue (as well as in UV-B-stressed leaves and stem), SAD predominantly occurred in epidermal or sub-epidermal cells as judged by IHC. The protoderm of the pea seed cotyledonary axis contains the most heavily stained cells. This indicates a possible role for the SAD protein in development as well as in protection against environmental stress. Also, discrete staining was obtained in particular cell types of the ovary.

To be able to understand the biochemical and physiological role of the SAD enzyme, an in silico modeling study of the SAD protein structure was performed. The simulations of our SAD protein, as well as of related proteins with known crystal structure (3alfa,20beta-hydroxysteroid dehydrogenase and secoisolariciresinol dehydrogenase), allowed us to obtain an energy-minimized structure for the monomeric SAD as well as important data on the cofactor interaction in the active site.

Crystallization of recombinant SAD-C has been performed. The needle-like crystals, which diffract to 3.5Å, contain probably eight monomers in the asymmetric unit, presumably containing a pair of tetramers.

SAD enhances the reduction rates of quinones with NADH. However, NADH can also accomplish reduction of certain quinones non-enzymatically. Both theoretical calculations and experimental techniques were used to elucidate the structural and electronic pre-requisites for this non-enzymatic quinone reduction.

Place, publisher, year, edition, pages
Örebro: Örebro universitetsbibliotek, 2005. p. 43
Series
Örebro Studies in Life Science ; 2
Keyword
Cell biology
National Category
Biological Sciences
Research subject
Biology
Identifiers
urn:nbn:se:oru:diva-195 (URN)91-7668-458-x (ISBN)
Public defence
2005-12-02, Hörsal P2, Prismahuset, Örebro universitet, Örebro, 10:00 (English)
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Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2017-10-18Bibliographically approved

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Scherbak, NikolaiStrid, Åke

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