Abstract
Two full-length cDNA clones, designated TrxhA and TrxhB, encoding different but very similar thioredoxin h polypeptides were isolated from wheat (Triticum aestivum cv. Chinese Spring) aleurone cells. The deduced proteins show a high similarity to each other and to thioredoxin h from other sources, in particular from T. aestivum and T. durum. One of them, TRXhA, was expressed in E. coli as a His-tagged polypeptide and used to raise polyclonal antibodies by immunization of rabbits. These antibodies identified a single band (ca. 13.5 kDa) in western blot analysis of protein extracts from all wheat organs analyzed. TRXhA and TRXhB when expressed in E. coli as intact polypetides showed indistinguishable electrophoretic mobility, which corresponded to the 13.5 kDa polypeptide detected in wheat protein extracts. The amount of thioredoxin h transcripts increased in scutellum and aleurone cells during germination but GA3 did not exert any stimulatory effect on thioredoxin h expression. Although northern blot analysis detected a single band, competitive RT-PCR showed that this band is due to the accumulation of both TrxhA and TrxhB mRNAs. These results suggest that the single band detected in western blots is due to the presence of at least two thioredoxin h polypeptides. Immunolocalization experiments confirmed the high content of thioredoxins h in scutellum and aleurone cells, and showed a low content in the starchy endosperm of germinating grains. Interestingly, though these proteins are evenly distributed in the cytosol, the highest levels of thioredoxins h were detected in the nucleus, both in aleurone and scutellum cells.
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Baulcombe, D.C. and Buffard, L.R. 1983. Gibberellic acid-regulated expression of α-amylase and six other genes in wheat aleurone layer. Planta 157: 493–501.
Besse, I., Wong, J.H., Kobrehel, K. and Buchanan, B.B. 1996. Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium. Proc. Natl. Acad. Sci. USA 93: 3169–3175.
Bethke, P.C., Lonsdale, J.E., Fath, A. and Jones, R.L. 1999. Hormonally regulated programmed cell death in barley aleurone cells. Plant Cell 11: 1033–1045.
Bodenstein-Lang, J., Buch, A. and Follman, H. 1989. Animal and plant mitochondria contain specific thioredoxins. FEBS Lett. 258: 22–26.
Brugidou, C., Marty, Y., Chartier, Y. and Meyer, Y. 1992. The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differentially expressed. Mol. Gen. Genet. 238: 285–293.
Buchanan, B.B. 1991. Regulation of CO 2 assimilation in oxygenic photosynthesis: the ferredoxin/thioredoxin system. Perspective on its discovery, present status and future development. Arch. Biochem. Biophys. 288: 1–9.
Buchanan, B.B., Adamidi, C., Lozano, R.M., Yee, B.C., Momma, M., Kobrehel, K., Eermel, K. and Frick, O.L. 1997. Thioredoxin-linked migration of allergic responses to wheat. Proc. Natl. Acad. Sci. USA 94: 5372–5377.
Cejudo, F.J., Murphy, G., Chinoy, C. and Baulcombe, D.C. 1992. A gibberellin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells. Plant J. 2: 937–948.
Dominguez, F. and Cejudo, F.J. 1995. Pattern of endoproteolysis during wheat seed germination. Physiol. Plant. 95: 253–259.
Dominguez, F. and Cejudo, F.J. 1999. Patterns of starchy endosperm acidification and protease gene expression in wheat grains following germination. Plant Physiol. 119: 81–88.
Fath, A., Bethke, P.C. and Jones, R.L. 1999. Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation. Plant J. 20: 305–315.
Florencio, F.J., Yee, B.C., Johnson, T.C. and Buchanan, B.B. 1988. An NADP/thioredoxin system in leaves: purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach. Arch. Biochem. Biophys. 266: 496–507.
Gautier, M.-F., Lullien-Pellerin, V., de Lamotte-Guéry, G., Guirao, A. and Joudrier, P. 1988. Characterization of wheat thioredoxin h cDNA and production of an active Triticum aestivum protein in Escherichia coli. Eur. J. Biochem. 252: 314–324.
González, M.C., Bevia, O., Echevarria, C., Vidal, J. and Cejudo, F.J. 1998. Expression and localization of phosphoenolpyruvate carboxylase in developing and germinating wheat grains. Plant Physiol. 116: 1249–1258.
Hirota, K., Matsui, M., Iwata, S., Nishiyama, A., Mori, K. and Yodoi, J. 1997. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc. Natl. Acad. Sci. USA 94: 3633–3638.
Holmgren, A. 1979. Reduction of disulfides by thioredoxin. J. Biol. Chem. 254: 9113–9119.
Izawa, S., Maeda, K., Sugiyama, K., Mano, J., Inoue, Y. and Kimura, A. 1999. Thioredoxin deficiency causes the constitutive activation of Yap-1, an AP-1-like transcription factor in Saccharomyces cerevisiae. J. Biol. Chem. 274: 28459–28464.
Jacquot, J.-P., Lancelin, J.-M. and Meyer, Y. 1997. Thioredoxins: structure and function in plant cells. New Phytol. 136: 543–570.
Johnson, T.C., Wada, K., Buchanan, B.B. and Holmgren, A. 1987. Purothionin: reduction by the wheat seed thioredoxin system and potential function as a secondary thiol messenger in redox control. Plant Physiol. 85: 446–451.
Kobrehel, K., Wong, J.H., Balogh, A., Kiss, F., Yee, B.C. and Buchanan, B.B. 1992. Specific reduction of wheat storage proteins by thioredoxin ℏ. Plant Physiol. 99: 919–924.
Kuo, A., Cappelluti, S., Cervantes-Cervantes, M., Rodriguez, M., and Bush, D.S. 1996. Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells. Plant Cell 8: 259–269.
Laughner, B.J., Sehnke, P.C. and Ferl, R.J. 1998. A novel member of the thioredoxin superfamily. Plant Physiol. 118: 987–996.
Lozano, R.M., Wong, J.H., Yee, B.C., Peters, A., Kobrehel, K. and Buchanan, B.B. 1996. New evidence for a role for thioredoxin h in germination and seedling development. Planta 200: 100–106.
Marcus, F., Chamberlain, S.H., Chu, C., Masiarz, F.R., Shin, S., Ye, B.C. and Buchanan, B.B. 1991. Plant thioredoxin ℏ:ananimal-like thioredoxin occurring in multiple cell compartments. Arch. Biochem. Biophys. 287: 195–198.
Mouaheb, N., Thomas, D., Verdoucq L., Monfort, P. and Meyer, Y. 1998. In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 95: 3312–3317.
Pedrajas, J.R., Kosmidou, E., Miranda-Vizuete, A., Gustafsson, J.-A., Wright, A.P.H. and Spyrou, G. 1999. Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae. J. Biol. Chem. 274: 6366–6373.
Rivera-Madrid, R., Mestres, D., Marinho, P., Jacquot, J.P., Decottignies, P., Miginiac-Maslow, M. and Meyer, Y. 1995. Evidence for five divergent thioredoxin h sequences in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 92: 5620–5624.
Rozell, B., Holmgren, A. and Hansson, H.A. 1988. Ultrastructural demonstration of thioredoxin and thioredoxin reductase in rat hepatocytes. Eur. J. Cell Biol. 46: 470–477.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, NY.
Schagger, H. and von Jagow, G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368–379.
Spyrou, G., Enmark, E., Miranda-Vizuete, A. and Gustafsson, J.-A. 1997. Cloning and expression of novel mammalian thioredoxin. J. Biol. Chem. 272: 2936–2941.
Watson, M.E.E. 1984. Compilation of published signal sequences. Nucl. Acids Res. 12: 5145–5164.
Wang, M., Oppedijk, B.J., Lu, X., van Duijn, B. and Schilperoort, R.A. 1996. Apoptosis in barley aleurone during germination and its inhibition by abscisic acid. Plant Mol. Biol. 32: 1125–1134.
Wong, J.H., Kobrehel, K. and Buchanan, B.B. 1995. Thioredoxin and seed proteins. Meth. Enzymol. 252: 228–240.
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Serrato, A.J., Crespo, J.L., Florencio, F.J. et al. Characterization of two thioredoxins h with predominant localization in the nucleus of aleurone and scutellum cells of germinating wheat seeds. Plant Mol Biol 46, 361–371 (2001). https://doi.org/10.1023/A:1010697331184
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DOI: https://doi.org/10.1023/A:1010697331184