However, there are data that LMW HSPs imunochemically related to a-crystallin appear in plants at normal temperature, for example during embryogenesis (Carranco, R. et al., 272:27470-27475, 1997). Here the expression of LMW HSPs plays a general protective role in desiccation tolerance (Wehmeyer, N. et al., Plant Phys. 122:1099-1108, 2000). Thus LMW HSPs are the part of the underlying mechanisms of cell protection against dehydration damage. The aim of our work is testing whether the expression of LMW HSPs occurs in the absence of dehydration at normal temperature conditions. For comparison, we chose maize as a thermotolerant species, and wheat and rye as less not tolerant species.
We used three-day-old etiolated seedlings of maize, wheat, and rye, which were grown at 23 C (wheat and rye) and 27 C (maize). The cut seedlings were placed in water for 3 hours at 42 C, thus being subjected to heat shock. Total proteins were extracted from control and shocked seedlings as described elsewhere (Borovskii, G.B. et al., J. Plant Physiol. 156:797-800 2000). Proteins were subjected to SDS-PAGE (14 % of acrylamide) using a mini-Protean II cell (Bio-Rad, USA) according to the manufacturer’s instructions. Western blot and immunodetecton were carried out as was described previously (Timmons, B.R. and Dunbar, B.S., Methods Enzymol. 182:679-688, 1991). Antibodies to a—crystallin sequence, kindly provided by Dr. Craig A. Downs, were used for detection of LMW HSPs (Heckathorn, S.A. et al., Plant Physiol. 116:439-444, 1998).
Electrophoresis of total proteins did not demonstrate a distinct quantitative or qualitative difference between "control" and "shock" in LMW HSPs of all the species (data not shown). Immunoblotting showed that protein samples from all three species contained LMW HSPs as were shown immunologically (Fig. 1). Maize samples included the group of HSPs 22-18 kD, and wheat and rye included one HSP 20 kD. Percentage of the maize LMW HSPs related to a-crystallin is higher than in the wheat and rye. This may be due to higher thermotolerance of maize.
LMW HSPs are clearly apparent in the "control" samples (Fig. 1). It is best expressed in wheat, slightly less in maize, and weak in rye. This suggests that either LMW HSPs, as well as high-molecular HSPs, may be synthesized constitutively, or alternatively seedling germination temperature (23 C for wheat and rye and 27 C for maize) may lead to their expression. To check this we chose the temperature of 20 C for germination of the seeds of all the species. Immunoreaction demonstrated a practically complete absence of LMW HSPs in "controls" of maize, wheat and rye grown at 20 C, which is in agreement with our supposition (Fig. 2). Appearance of LMW HSPs in all the species at the temperature close to optimum (23 C) supports the possibility of constitutive synthesis of these proteins in the seedlings. On the other hand, appearance of the LMW HSPs within the frameworks of optimal temperature may prove a high level of thermosensitivity of the LMW HSPs synthesis reaction for all three species.
This work was supported by the Russian Fund of Basic Research (project 99-04-48121).
Figure 1. Immunodetection of the proteins immunochemically related to a-crystallin. The proteins were extracted from three-day-old seedlings of maize (1, 2), wheat (3, 4) and rye (5, 6), grown at 27 C (maize) or at 23 C (wheat and rye). Before the extraction of the proteins, seedlings were shocked at 42 C for 3h. (2, 4, 6) or left for 3 h. at the growing temperature (1, 3, 5). Molecular weight markers are indicated on the right.
2. Immunodetection of the proteins, immunochemically related to a-crystallin.
The proteins were extracted from three-day-old seedlings of maize (1, 2,
7, 8), wheat (3, 4, 9, 10) and rye (5, 6, 11, 12), grown at 20 C (lines
1 - 6), at 27 C (lines 7, 8) and at 23 C (lines 9-12). Before the extraction
of the proteins, seedlings were shocked at 42 C for 3h. (2, 4, 6, 8, 10,
12) or left for 3 h. at the growing temperature (1, 3, 5, 7, 9, 11). Molecular
weight markers are indicated on the right.
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