Mitochondrial low-molecular-weight heat shock proteins and tolerance of crop plantís mitochondria to hyperthermia --Korotaeva, NE, Antipina, AI, Grabelnych, OI, Varakina, NN, Borovskii, GB, Voinikov, VK Plants are known to synthesize, under heat shock, a large diversity of low-molecular-weight heat shock proteins (LMW HSPs) that function as protectors on the biochemical level. The a-crystallin-related, low-molecular-weight heat shock proteins range in size from approx 17 to 30 kDa and share a conserved C-terminal domain common to all eukaryotic LMW HSPs and to the a-crystallin proteins of the vertebrate eye lens. LMW HSPs act in vivo as molecular chaperones to bind partially denatured proteins, preventing irreversible protein inactivation and aggregation (Waters, E.R. et al., J. Exp. Bot. 47:325-338 1996).

It is known that LMW HSPs play an important role in protection of the organelles from hyperthermia damage. Chaperone activity of organelle LMW HSPs contributes to the development of thermotolerance. For example, appearance of LMW HSPs in wheat mitochondria correlates with thermotolerance emergence, according to investigation of thermotolerant and non-tolerant varieties of wheat (Joshi, C.P. et al., TAG 95:834-841 1997). We suppose that there may be a correlation between thermostability of the species and expression of LMW HSPs. The aim of our investigation was to determine whether a correlation exists between thermotolerance among species and mitochondria LMW HSPs (mit LMW HSPs) accumulation, including their number and polymorphism. We chose for our investigation maize as a thermotolerant species, and wheat and rye as less tolerant species.

Three-day-old etiolated seedlings of maize, wheat, and rye were grown at 23 C (wheat and rye) and 27 C (maize). Some of the cut seedlings were placed in water for 3 hours at 42 C, thus being subjected to heat shock. Untreated seedlings were "control". Mitochondria were extracted from the control and shocked seedlings by the method of differential centrifugation with further purification by discontinuous Percoll gradient as described elsewhere (Borovskii, G.B. et al., J. Plant Physiol. 156:797-800 2000). Isolated mitochondria were used for the extraction of the proteins and the measuring of the energetic activity. Proteins were subjected to SDS-PAGE (14% of acrylamide) using a mini-Protean II cell (Bio-Rad, USA) according to the manufacturerís instruction. Western blotting and immunodetection were carried out, as described previously (Timmons, T.M. and Dunbar, B.S., Meth. Ensimol. 182:679-688, 1990) using anti-a-crystallin primary antibodies, kindly provided by Dr. Craig A. Downs (Heckathorn, S.A. et al., Plant Physiol. 116:439-444 1998).

Western blot showed the appearance of LMW HSPs immunochemically related to a-crystallin in all three species after heat shock (Fig. 1). Five mit LMW HSPs, 28, 23, 22, 20 and 19 kD, were found in maize, and only one mit LMW HSP 20 kD was found in wheat and rye. It should be noted that LMW HSPs were detected only for "shock" samples. Perhaps the differences in number of LMW HSPs in maize on the one hand, and in wheat and rye on the other hand, are related to differences in stability of the species to heat shock.

Other authors have discovered LMW HSPs 22 and 30 kD in maize mitochondria under heat shock (42 C, 3 h.) (Lund, A.A. et al., Plant Physiol. 116:1097-1110 1998). Based on the similarity of molecular weights, the LMW HSPs 23 and 29 kD which we detected, are likely the proteins 22 and 30 kD mentioned above. However, other mit LMW HSPs were not detected by these authors, while we identified an additional three LMW HSPs 21, 20 and 19 kD (Fig. 1). According to our account, this does not contradict the results of Lund A. et. al., inasmuch as they used maize grown at 29 C, when LMW HSPs immunochemically related to a-crystallin appear in total maize protein fraction at 27 C (see the article "Appearance of HSPs immunochemically related to alpha-crystallin at the temperature close to optimum in the absence of dehydration in crops" in this MNL). In this case only part of the proteins seem newly synthesized, i.e. HSPs, when comparing "control" and "shock" samples.

In relation to wheat mit LMW HSPs our results were in accordance with the data of other authors (Joshi, C.P. et al., TAG 95:834-841 1997). As far as we know, our data about rye mit LMW HSP is the first such reported.

For determining the thermotolerance of the mitochondria, the activity of the mitochondria respiration after heat shock was measured. The respiration of the mitochondria was recorded polarographically at 27 C using a platinum electrode of a close type in a 1.4 ml volume cell. 10mM malate in the presence of 10 mM glutamate was used as an oxidation substrate. Polarograms were used to calculate the rates of the oxygen uptake in state 3 (phosphorylate respiration) and in state 4 (nonphosphorylate respiration) (Estabrook, R.W., Methods Enzymology 10:41-47, 1967).

The rate of the oxidative activity declined after heat shock (42 C, 3 h.) to a great extent in all three species (Table 1). However, in maize the decrease of the oxidative activity of the mitochondria after stress was less than in wheat and rye. Indeed the rate of phosphorylative and nonphosphorylative respiration in maize mitochondria after heat shock decreased 38.3 % and 30.4 %, while in the wheat and rye mitochondria that were 63% and 59.5 % (wheat), and 65% and 60.6 % (rye) accordingly. Thus, although the mitochondria of all species were damaged under heat shock, the thermotolerance of maize mitochondria was superior to that of mitochondria of wheat and rye.

The thermotolerance of the maize mitochondria concurs with the accumulation of a number of LMW HSPs immunochemically related to a-crystallin. Our data permit us to suppose that the diversity of LMW HSPs plays an important role in the protection of respiration processes of mitochondria from heat shock damage. It is known that the majority of LMW HSPs are chaperones, i.e. they stabilize protein structure and prevent damage and resultant turnover. For example mit LMW HSP of tomato is a chaperone (Liu, J.A. et al., Plant Cell Physiol. 40:1297-1304 1999). The mit LMW HSP protects NADH:ubiquinone oxidoreductase of the electron transport chain during heat stress in mitochondria of apples (Downs, C.A. et al., FEBS Letters. 430:246-250 1998). Based on information from the literature and our research we expect that various LMW HSPs influence different proteins or recognize various transitional states of partly denatured proteins. In this case maize mitochondria have a more abundant composition of chaperones than wheat and rye, and enhanced capabilities for preventing damage to the enzymes of the electron transport chain.

The activity of maize mitochondria is more thermotolerant than that of organelles of wheat and rye. The number of LMW HSPs, immunochemically related to a-crystallin, appearing under heat stress in mitochondria correlates with thermotolerance of the organelles, and correspondingly with thermotolerance of the species.

This work was supported by the Russian Fund of Basic Research (project 99-04-48121).

Figure 1. Immunodetection of LMW HSPs, related to a-crystallin, among the mitochondrial proteins of maize (1, 2), wheat (3, 4) and rye (5, 6). Three-day-old seedlings were shocked at 42 C for 3 h. (2, 4, 6) or left for 3 h. at the growing temperature (1, 3, 5) before the isolation of mitochondria. Extracted mitochondrial proteins were divided by SDS-PAGE. Molecular weight standards are on the right.

Table 1. The influence of heat shock (42 C. 3 h.) on the oxidative activity of the mitochondria of maize, wheat and rye. All experiments were made in three biological replications. The data obtained were analysed statistically, means and S.D. (P>0.95) are presented.
Variants  The rate of oxygen uptake (nmol O2 / min mg protein)
    State 3 State 4
maize  control 86.6±3.9  29.3±1.4
  shock 53.5±1.5 20.4±1.2
wheat control 81.1±2.3 35.4±1.8
  shock 29.9±2.0 14.3±0.8
rye control 82.9±1.1 37.8±1.1
  shock 29.2±3.2 14.9±1.1


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