Journal Article

Acclimation to Diverse Environmental Stresses Caused by a Suppression of Cytosolic Ascorbate Peroxidase in Tobacco BY-2 cells

Takahiro Ishikawa, Yukari Morimoto, Rapolu Madhusudhan, Yoshihiro Sawa, Hitoshi Shibata, Yukinori Yabuta, Ayako Nishizawa and Shigeru Shigeoka

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 8, pages 1264-1271
Published in print August 2005 | ISSN: 0032-0781
Published online August 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci135
Acclimation to Diverse Environmental Stresses Caused by a Suppression of Cytosolic Ascorbate Peroxidase in Tobacco BY-2 cells

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The active oxygen species (AOS) that arise from normal metabolic processes are kept under tight control by various antioxidant mechanisms. AOS are important signal molecules that regulate many physiological processes, including environmental stress responses. In this work, we have investigated the effect of lowered cytosolic ascorbate peroxidase (APX) activity in transgenic tobacco BY-2 cells, using two transformed BY-2 cell lines, cAPX-S2 and cAPX-S3, resulting from co-suppression by expression of Arabidopsis APX1 cDNA under the cauliflower mosaic virus (CaMV) 35S promoter. cAPX-S2 and cAPX-S3 possessed 50 and 75% lower cytosolic APX activity, respectively, compared with that in the untransformed cells. Chemical fluorescence analysis indicated that the AOS levels were markedly higher in the two APX-suppressed cell lines than in the wild-type cells. However, there were no substantial differences in the activity levels of the various other antioxidant enzymes. Interestingly, the APX-suppressed cells showed different responses and tolerances to environmental stresses, such as heat and salinity. Suppression subtractive hybridization revealed that several heat- and salt stress-inducible genes were up-regulated in cAPX-S3 cells. HSP70, DnaJ-like protein and purple acid phosphatase were among the constitutively induced genes. An in-gel kinase assay suggested that a mitogen-activated protein (MAP) kinase of approximately 46 kDa was predominantly active in the APX-suppressed cells, and transcript levels of both nicotiana protein kinase 1 (NPK1) and nucleoside diphosphate kinase 2 (NDPK2) were up-regulated. These data suggest the possibility that MAP kinase cascades are activated by subtle imbalances in the homeostasis of the cellular redox status caused by lowered cytosolic APX activity.

Keywords: Active oxygen species; Ascorbate peroxidase; Diverse stress tolerance; Redox; Tobacco BY-2; AOS, active oxygen species; APX, ascorbate peroxidase; AsA, ascorbate; CaMV, cauliflower mosaic virus; DHA, dehydroascorbate; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); H2DCF-DA, 2′,7′-dichlorofluorescein diacetate; HSP, heat shock protein; MAP, mitogen-activated protein; MBP, myelin basic protein; MDA, monodehydroascorbate; NDPK, nucleoside diphosphate kinase; NPK1, nicotiana protein kinase 1; SSH, suppression subtractive hybridization

Journal Article.  6279 words.  Illustrated.

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

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