In a previous study we found accumulation of two dehydrin-like proteins in the plant mitochondria under low temperature treatment [21]. Now five dlps have been found even at the control temperature due to a gel density decrease and an increased protein load (Fig. 1). We also re-calculated relative molecular masses of dlps. The bands corresponding to all these proteins were very weak or absolutely absent if we used antibodies blocked by dehydrin peptides (data not shown).
Two of these polypeptides had the same molecular masses in all the species (63 and 52 kD). Analysis of the thermostable fraction revealed that these dlps like most of dlps reported earlier [1, 8] were of heat-stable nature (Fig. 2). At the same time, the other three proteins did not appear to be thermostable: one heat-sensitive protein with the molecular mass 28 kD was registered in all three species and the other two had similar, but slightly different masses – 59 and 58 kD, 57 and 54 kD, 58 and 56 kD for maize, rye, wheat, respectively (Fig. 1). The finding that the proteins immunologically related to dehydrins was found to be constitutive and heat-sensitive was unusual. In the literature available we found only one reference concerned: Li with coworkers had described proteins related to dehydrins being constitutive and heat-sensitive, in fucoid algae [9]. It is interesting that all three heat-sensitive mitochondrial dlps from maize, wheat and rye were constitutive too. These proteins were not induced by all the treatments used (Fig. 1, 3, 4, 5). Based on this observation we concluded that these proteins were not involved in the stress reaction and adaptation.
As shown by us earlier [21] and as we show in the present work (Fig. 1,2) the two revealed thermostable dlps accumulated during cold treatment. Earlier we used data based on sonicated mitochondrial samples[21]. At present we study not only sonicated (Fig. 1,2) but also unsonicated ones (Fig. 3, 4, 5), which enables resolution of all mitochondrial dehydrins and not only primarily weakly bound with large fragments of mitochondria or membranes.
Analysis of the unsonicated mitochondrial fraction of total proteins (heat-sensitive and heat-stable) showed that freezing and drought stresses, and ABA treatment as well as cold caused strong accumulation of dlp63 in the wheat (Fig. 5) and rye (Fig. 4) mitochondria and had no effect in the mitochondria of maize (Fig. 3). The dlp52 was induced only by exogenous ABA and cold treatment in the mitochondria of rye and wheat, and in the rye ones also by drought (Figs. 1, 4, 5). At the same time in the thermostable fraction (Fig. 2) there was some increase of these proteins in maize mitochondria as well, although in less degree than in the rye and wheat ones. Apparently, the share of heat-stable protein in the total protein fraction of the same mol. wts was little. It made visualization of heat-stable polypeptides in the total fraction difficult.
Thus cold and ABA treatment resulted in significant accumulation of both heat-stable dlps in the rye and wheat mitochondria. The dlp 63 kD accumulated during all treatments used, but dlp 52 kD during cold and ABA only, but during drought also in the rye organelles. It suggests that dlp52 does not accumulate under freezing and drought conditions or its content is too low, in comparison with dlp63, to detect some changes. In any case it appears that both heat-stable proteins, particularly dlp63, may be associated with the protection of mitochondria against desiccation or other stresses caused by freezing.
The relative abundance of the thermostable dlps was higher in freezing tolerant species (rye or winter wheat) than in the maize as had been shown earlier [21]. At the same time the comparison of dehydrin contents among species based on immunochemical data should be accepted only with caution since we did not estimate binding affinity of antibodies in each species.
The treatment of exogenous ABA provided some confirmation of ABA participating in induction of heat-stable dlps 63 and 52 kD. It corroborates again an important role of ABA in plant responses to stresses related to dehydration not only at the whole-plant level but also at the subcellular level.
Cold-induced accumulation of the thermostable mitochondrial dlps in all species studied (Fig. 1, 2) was accompanied by increasing of plant cryotolerance. This increase had been shown previously [22]. The quantity of survived seedlings under 24 hours at -8°C rose from zero to 61% after 7 days of acclimation. There are also a number of papers describing cold acclimation of rye at low positive temperatures from 2 to 5 [23]. Though maize is not a freezing-tolerant species, maize seedlings could acclimate at the temperature near low limit of growth [24, 25]. The connection between accumulation of the mitochondrial dlps and increase of plant cryotolerance suggests an important role of the dlps in the development of the tolerant state of cryotolerant cereals.
The freezing treatment that we used presumably led to damage of cells or even death due to severe dehydration. It has recently been shown that after freezing of even very sensitive species like bean, tobacco, tomato and cucumber, ice was extracellular. Only corn had combination of extracellular and intracellular damage [26]. The increase of dlp63 in rye and wheat mitochondria under freezing treatment perhaps resulted from very fast association of this protein with organelles under cellular dehydration. This suggests presence of this protein in the cytoplasm before freezing. The absence of dlp63 accumulation in the maize mitochondria may be explained by low contents of this protein in the cytoplasm of maize cells. Thus, the mitochondria of freezing-tolerant cultures such as winter wheat and rye have cryotolerant mechanisms that are different from those of freezing-sensitive species such as maize.
The accumulation of dehydrins during various treatments has been studied in different plant species [1]. The accumulations of dehydrins (WCS120) related to freezing tolerance were demonstrated also on cereal species [20]. In wheat and other cereals the wcs120 gene family was coordinately regulated by low temperatures and the resulting proteins accumulated in significant amounts in freezing-tolerant members of Poaceae. Immunogold labeling indicated that the WCS120 protein family was localized in both the nucleus and cytoplasm of cold acclimated tissues [27]. The presence of these proteins in high amounts in the nucleus indicates that they may protect or stabilize the transcriptional machinery from inactivation [20].
Another LT-responsive dehydrin referred to as WCOR410 was identified [19]. Due to its acidic nature and the absence of the glycine-rich repeat presented in the cold-regulated dehydrin WCS120 [20], WCOR410 is thought to belong to a different subtype of the D-11 protein family, the so-called acidic dehydrin [19]. The WCOR 410 protein family is preferentially associated with the plasma membrane of cells in the sensitive vascular transition area where freezing-induced dehydration is likely to be more severe. The authors [19] proposed that this dehydrin might play a role in preventing destabilization of the plasma membrane that occurs under dehydrative conditions. These results confirm, at least in part, the previous hypothesis of the authors that dehydrin subtypes can accomplish their function in different compartments [19].
Our data concerning presence and accumulation of dehydrins in the mitochondria of cereals, to our mind, also confirm this hypothesis. Mitochondrial membranes are very permeable for the water which moves out of the cells into the intercellular spaces concomitantly with freezing temperatures decreasing and drought stress increasing. Dehydration may lead to damage of the mitochondria. Apparently there are some tolerance mechanisms in the cell against the injuring effect of dehydration on these organelles. Our results concerning the accumulation of heat-stable dehydrin-like proteins in the mitochondria under the cold treatment, drought and freezing stress suppose that mitochondrial dlps should be involved in the freezing- and dehydrative-tolerance mechanisms. First, as was mentioned before, the revealed heat-stable dehydrins have the K segment. It has been proposed to form an amphipathic α-helix which is responsible for the lipid-associating properties [14]. Possibly, a role of the dehydrin K segment (and respectively of dlps containing this site) is hydrophobic interaction with partially denatured mitochondrial proteins or membranes which stabilizes them. Second, the hydrophilic nature of the dlps is well suited to replace water and stabilize mitochondrial membranes through polar interactions during dehydration.
The higher accumulation of the dlps in winter wheat and rye mitochondria probably reflects stability of the mitochondria, cells and the whole plant under dehydration and freezing. The target of mitochondrial dlps action is unclear. Precise localization of these proteins in the organelles and the finding of targets of it action would give more information about the function of mitochondrial dlps. Further experiments concerning dlp localization in mitochondria are in progress.