Journal Article

Molecular Cloning and Characterization of a Senescence-induced Tau-class Glutathione <i>S</i>-transferase from Barley Leaves

Tadashi Kunieda, Taketomo Fujiwara, Toyoki Amano and Yuzo Shioi

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 9, pages 1540-1548
Published in print September 2005 | ISSN: 0032-0781
Published online September 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci167
Molecular Cloning and Characterization of a Senescence-induced Tau-class Glutathione S-transferase from Barley Leaves

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Glutathione S-transferases (GSTs) (EC 2.5.1.18) are multifunctional proteins involved in such diverse intracellular events as primary and secondary metabolism, signaling and stress metabolism. In this study, we found a senescence-induced tau-class GST (SIGST) in senescent leaves of barley (Hordeum vulgare L.). The SIGST was purified 19-fold to homogeneity from initial crude extracts by three steps of chromatography with a yield of 5%. The purified SIGST had a GSH-conjugating activity and peroxidase (POD) activity at the same level of 1.7 µmol min–1 mg protein–1, although restricted substrate selectivity could be seen in POD activity. Barley SIGST is a slightly acidic protein with a molecular weight of 49 k and is composed of two subunits. The enzyme exhibited a single pH optimum at pH 8.3. The K m values were 0.285 mM for GSH and 0.293 mM for 1-chloro-2,4-dinitrobenzene. In most respects, the barley enzyme resembles those that have been reported from other higher plants. The SIGST gene was cloned from cDNA of senescent barley leaves. DNA sequence analysis shows that the cloned SIGST had only one base different from the barley embryo GST, ECGST. The obtained sequence indicates that SIGST is classified into the plant-specific tau class. mRNA expression analysis showed that in addition to senescence, SIGST was strongly induced by treatment with a herbicide and low temperature. The responses to these stresses suggest that SIGST may be involved at least partly in the secondary metabolism as an antioxidant and enhancement of enzymatic activity during senescence.

Keywords: Barley (Hordeum vulgare L.); Cloning; Glutathione S-transferase; Senescence; Stress response; AOS, active oxygen species; CDNB, 1-chloro-2,4-dinitrobenzene; GA3, gibberellic acid; GST, glutathione S-transferase; HRP, horseradish peroxidase; JA, jasmonic acid; POD, peroxidase; RT–PCR, reverse transcription–PCR; SIGST, senescence-induced GST

Journal Article.  6576 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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