Mitochondria were extracted from three-day-old maize shoots by differential centrifugation as described previously (Pobezhimova et al., J. Therm. Biol. 21:283-288, 1996). Cold stress treatment was performed at 4 C for 1 h. The electrophoresis of native proteins was performed as described previously (Kolesnichenko et al., J. Therm. Biol. 25:203-209, 2000). Protein relative molecular weights were determined using the HMW kit of markers (Pharmacia, Sweden). Western blotting and ethydium bromide staining of native gels were performed as described previously (Kolesnichenko et al., J. Therm. Biol. 25:203-209, 2000).
Ethydium bromide staining of native maize proteins showed the absence of nucleic acids (NA) binding to 300-310 kD cytoplasmic proteins (Fig. 1A). CSP 310 — like maize cytoplasmic proteins isolated by affinity chromatography as described previously (Kolesnichenko et al., J. Therm. Biol. 24:211-215, 1999) also showed the absence of NA (Fig. 1B). On the other hand, western blotting of the native maize mitochondrial proteins showed an increase of CSP 310 concentration during cold stress (Fig. 2). Therefore, we can suppose that the increased CSP 310 content in maize mitochondria depends on the synthesis of this protein de novo during cold shock or on possible structural changes in cytoplasmatic CSP 310-like proteins with different molecular mass but not on the presence of non-active CSP 310 "depot" in cytoplasm.
Based on the data obtained we can conclude that in maize the defense mechanisms against cold stress connected with uncoupling in mitochondria caused by cold stress protein CSP 310 differ from those in winter cereals.
Acknowledgements: The work has been performed with the support of the Russian Foundation of Basic Research (project 01-04-48953).
Figure 1. A. Ethydium bromide staining of native electrophoresis of control (1) and stressed (2) at 4 C, 1h maize cytoplasmic proteins; B. Ethydium bromide staining of cytoplasmic CSP-310-like proteins isolated by affinity chromatography from maize (1) and winter wheat (2).
2. Western blotting of control (1) and stressed (2) at 4 C, 1h maize
native mitochondrial proteins with antibodies toward CSP 310 from winter
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