Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L.
Introduction
Haploid pollen embryogenesis is considered an important tool for basic research as well as for plant breeding, allowing the generation of isogenic lines and new varieties by means of double-haploid plants (Clement et al., 2001). In Brassica napus it is well established that with a treatment of 32 °C for at least 8 h, isolated microspores in the culture can be deviated from their normal gametophytic developmental pathway toward the haploid embryogenic development (Pechan et al., 1991). In parallel, when microspores are cultured at a constant 18 °C temperature, gametophytic-like pollen development can be reproduced in vitro (Custers et al., 1994). Therefore, the study of heat stress effects on B. napus microspores can be useful to understand this switch. Previous studies have been focused on the molecular responses to this stress (Cordewener et al., 1995, Cordewener et al., 2000; Custers et al., 1994; Pechan et al., 1991), but cellular studies trying to localize stress-related molecules in situ during B. napus microspore embryogenesis induction are few (Coronado et al., 2002; Testillano et al., 2000).
In virtually all prokaryotes and eukaryotes studied, the most important response to heat stress is the synthesis of a set of protective proteins, known as heat-shock proteins (Hsps; reviewed in Lindquist, 1986; Morimoto et al., 1997; Vierling, 1991). Among these proteins, the most abundant and evolutionarily conserved is the Hsp70 family (Boorstein et al., 1994; Lindquist, 1986). In this family, the Hsp70 isoform acts as a chaperone for protein folding in eukaryotes (Sung et al., 2001), also being inducible by exposure to sublethal temperatures. In B. napus microspore embryogenesis, its expression is increased after exposure to the induction treatment (Cordewener et al., 1995; Testillano et al., 2000).
Hsp90 is the second most conserved Hsp (Lindquist, 1986) and the most constitutively expressed Hsp in eukaryotes (Jakob and Buchner, 1994), also being strongly induced upon exposure to sublethal temperatures (Lindquist, 1986; Vierling, 1991). Unlike Hsp70, Hsp90 seems to have a more specific role in heat stress, rather than under other types of stress, and specifically complexes with certain proteins (Rutherford and Lindquist, 1998). Despite its widely reported involvement in heat-shock response in many eukaryotes, up to now there are few data on Hsp90 in B. napus (Krishna et al., 1997; Park et al., 1998) and none about its putative involvement in heat-induced haploid embryogenesis.
In this work, the Hsp70 and Hsp90 ultrastructural localization, distribution patterns, and association in cultured microspores, induced (embryogenic) and noninduced (gametophytic), during the first stages of embryogenesis induction, and the initiation of haploid embryogenic development were studied.
Section snippets
Plant material
B. napus L. Cv. Topas donor plants were grown in Sanyo growth cabinets (, 18 °C, 80% humidity, and a photoperiod of 16/8).
For microspore culture, 3.3- to 3.4-mm flower buds were dissected and squashed in NLN-13 medium (Custers et al., 1994). The isolated microspores were induced to embryogenesis by a 32 °C heat treatment and cultured in darkness at 25 °C. Some of these cultures were kept under noninductive conditions, 18 °C, as a control for embryogenesis induction.
Antibodies
An anti-Hsp70
Immunoblotting
Immunoblotting assays were performed on total protein extracts from freshly isolated microspores and 32 °C-treated cultures (composed of induced and noninduced microspores). Anti-Hsp70 in freshly isolated microspores showed no signal under the experimental conditions used (Fig. 1), suggesting a very low level of constitutive Hsp70, probably below the sensitivity of the method. Under the same conditions anti-Hsp90 showed a band, approximately 90 kDa. In 32 °C-treated cultures, anti-Hsp70 and 90
Microscopical and immunocytochemical approaches allow specific features in embryogenesis-induced microspores to be distinguished
Several reports have been published concerning biochemical and molecular aspects of microspore embryogenesis in B. napus (Cordewener et al., 1995, Cordewener et al., 2000; Custers et al., 1994; Pechan et al., 1991) as well as in other species (Chupeau et al., 1998; Garrido et al., 1993; Touraev et al., 1997; Zarsky et al., 1995). However, since inductive efficiency can still be largely improved, embryogenic cultures also include a significant amount of microspores exposed to induction treatment
Acknowledgements
The collaboration of Drs. J.H.G. Cordewener, J.B.M. Custers, and M.M. van Lookeren-Campagne during the stay of J.M.S.S. in their lab in CPRO-DLO (Wageningen, The Netherlands) is especially acknowledged. Thanks are also due to the Electron Microscopy Service of Institute Cajal, CSIC, Madrid; to Ms. M.A. Ollacarizqueta (Confocal and CCD Camera Service of C.I.B.) for her excellent technical support; and to Dr. Jotham Austin and Ms. Beryl Ligus-Walker for checking the Englishstyle. J.M.S.S. was a
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Present address: MCD Biology, University of Colorado, UCB 347, Boulder, CO 80309, USA