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First published online April 2, 2004; 10.1104/pp.103.035857

Plant Physiology 134:1708-1717 (2004)
© 2004 American Society of Plant Biologists

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ENVIRONMENTAL STRESS AND ADAPTATION

Protein Cryoprotective Activity of a Cytosolic Small Heat Shock Protein That Accumulates Constitutively in Chestnut Stems and Is Up-Regulated by Low and High Temperatures1

Maria-Angeles Lopez-Matas2, Paulina Nuñez3, Alvaro Soto4, Isabel Allona, Rosa Casado, Carmen Collada, Maria-Angeles Guevara4, Cipriano Aragoncillo and Luis Gomez*

Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros de Montes, Universidad Politecnica de Madrid, E–28040 Madrid, Spain

Heat shock, and other stresses that cause protein misfolding and aggregation, trigger the accumulation of heat shock proteins (HSPs) in virtually all organisms. Among the HSPs of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. We analyzed the occurrence of sHSPs in vegetative organs of Castanea sativa (sweet chestnut), a temperate woody species that exhibits remarkable freezing tolerance. A constitutive sHSP subject to seasonal periodic changes of abundance was immunodetected in stems. This protein was identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry and internal peptide sequencing as CsHSP17.5, a cytosolic class I sHSP previously described in cotyledons. Expression of the corresponding gene in stems was confirmed through cDNA cloning and reverse transcription-PCR. Stem protein and mRNA profiles indicated that CsHSP17.5 is significantly up-regulated in spring and fall, reaching maximal levels in late summer and, especially, in winter. In addition, cold exposure was found to quickly activate shsp gene expression in both stems and roots of chestnut seedlings kept in growth chambers. Our main finding is that purified CsHSP17.5 is very effective in protecting the cold-labile enzyme lactate dehydrogenase from freeze-induced inactivation (on a molar basis, CsHSP17.5 is about 400 times more effective as cryoprotectant than hen egg-white lysozyme). Consistent with these observations, repeated freezing/thawing did not affect appreciably the chaperone activity of diluted CsHSP17.5 nor its ability to form dodecameric complexes in vitro. Taken together, these results substantiate the hypothesis that sHSPs can play relevant roles in the acquisition of freezing tolerance.


1 This work was supported by Plan Nacional de Biotecnología, Ministerio de Ciencia y Tecnología, Spain (grant no. BIO99–0931) and by Comunidad Autónoma de Madrid (grant no. 07M/0047/2000).

2 Present address: Facultad de Ciencias, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, E–28049 Madrid, Spain.

3 Present address: Departamento de Silvicultura, Facultad de Ciencias Forestales, Universidad de Chile, Santiago, Chile.

4 Present address: Centro de Investigación Forestal-CIFOR, Instituto Nacional de Investigaciones Agrarias, Ministerio de Agricultura, Pesca y Alimentación, E–28040 Madrid, Spain.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.035857.

* Corresponding author; e-mail lgomez{at}montes.upm.es; fax 34–91–3366387.

Received November 7, 2003; returned for revision January 13, 2004; accepted January 13, 2004.




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