The results of this study indicate the presence of specific mitochondrial dlps in three non-cereal plant species: cauliflower, Arabidopsis thaliana
and yellow lupin, which considerably extends the results obtained by Borovskii et al.
]. The detection and partial characterisation of those proteins were performed with the help of different antibodies; two of them were directed against the K-segment of dehydrins and one - against the Solanum sogarandinum
DHN24 dehydrin (for SK3
-type of dehydrins). This approach minimized the possibility of detection of artefacts resulting from non-specific cross-immunoreactions. In all the analyses only abundant dlps were taken into account. The two antibodies specific to the dehydrin K-segment gave similar results on all Western blots; however, limited similarity of the dlps pattern was obtained when antibodies against K-segment and against potato DHN24 dehydrin were used. This may be due to the relatively different amounts of K-segments or K-segment- related sequence motifs in different dlps, or to the non-equal number of accessible epitops for dlps recognized by those antibodies. The last possibility is more probable, as in this study the spectrum of dlps that crossreacted with the DHN24 antibody was always smaller. Additionally, stress conditions could affect not only the relative level of dlps, but also the number of immunoreactive epitops of particular proteins. Notably, Rorat et al.
] speculated that the Brassicaceae
family may be characterized by the presence of two types of cytoplasmic dehydrins: SK3
(including DHN24) and SK2
. Therefore, it cannot be excluded that at least part of the mitochondrial dlps of cauliflower and Arabidopsis
reported in the present study may contain aminoacid motifs similar to different dehydrins. However, only limited similarity in size of the presently reported cauliflower and Arabidopsis
mitochondrial dlps with known dehydrins of related plant species was detected, as most of the described Brassica
dehydrins range from 19 to 22 kDa in size; an exception is the 31 kDa ERD10 dehydrin of Brassica napus
]). The size of ten known Arabidopsis
dehydrins also varies from 10 to 29 kDa [2
]. However, RAB18 [Gen Bank:NP_201441
, ERD14 [Gen Bank:NP_177745
] and LTI30 [Gen Bank:NP_190666
dehydrins as well as 3 other proteins [Gen Bank:NP_195554
] resemble in size the dlps detected in Arabidopsis
mitochondria in the present study. It should be added that despite the fact that all dehydrin subclasses contain K-segment, it may be less conservative in sequence among different plants, as suggested for gymnosperms (reviewed by Close et al.
]). This fact may also indicate that some truly diverse dlps were identified in lupin, cauliflower and Arabidopsis
Notably, in cauliflower, Arabidopsis
and lupin mitochondria two distinct groups of dlps seem to be present: one containing higher-molecular-weight proteins (>50 kDa) and the other composed of small dlps which are more variable in size than in cereal mitochondria (Figures , , , ; [16
]). Previous studies reported tissue specific localisation of plant dehydrins under stress conditions [13
]. Here the expression of dlps under the abiotic stress conditions analyzed was also monitored in the mitochondria prepared from different plant organs. This approach made it possible to determine novel dlps in plant species and organs, which extended the existing data. The level of dlps was studied in the mitochondria isolated both from imbibed seeds and hypocotyls of yellow lupin and from cauliflower and Arabidopsis
leaves as well as from cauliflower early inflorescences. The influence of cold and heat stress on the accumulation of various dlps in the lupin mitochondria from imbibed seeds and hypocotyls was different. Generally, in the lupin imbibed seed mitochondria, contrary to the lupin hypocotyls, the quantitative changes were rather limited to higher-molecular weight dlps; however, the level of some small thermolabile dlps in lupin hypocotyl mitochondria was clearly affected by stress conditions, which was not the case for cereal mitochondria dehydrin-related proteins [16
]. In fact, the dlps detected in lupin imbibed seed mitochondria may represent a distinct set of proteins active under dessication, imbibition and germination. However, comparison of dlps of the dried and imbibed seed mitochondria was impossible due to the low quality of the dried seed mitochondria. In addition, it seems that some proteins (ca. 25, 35, 45 kDa) may be good candidates for cold-induced dlps in yellow lupin mitochondria. Moreover, protein 25 kDa, accumulating in yellow lupin hypocotyl mitochondria under heat stress, displayed similarity in size to SK3
-type RAB16-like Lupinus albus
]); however, yellow lupin 25 kDa protein could not be detected by dehydrin SK3
-type specific antibodies. This suggests that some lupin mitochondrial dlps may not necessarily be similar to the known dehydrins in the species analyzed up till now.
Significant changes in the abundance of various dlps were also very evident in the mitochondria from Arabidopsis
leaves and cell cultures. Generally, in the latter case quite numerous dlps were detected (Figure ), which is consistent with the strong accumulation of SK2
-type dehydrins in poplar cell cultures in the stationary phase [31
]; interestingly, the level of those proteins increased constantly from the early exponential phase. Notably, the Arabidopsis
cell cultures used in the present study displayed a high number of mitochondrial dlps in the stationary phase of growth. Those dlps were immunorective both with the dehydrin K-segment and with dehydrin SK3
-type specific antibodies.
The mitochondrial dlps from unstressed cauliflower inflorescences were less abundant, but the level of some of them significantly increased after cessation of cold and heat stress and subsequent plant growth under control conditions. This result can indicate that the changes in dlps abundance in cauliflower mitochondria are time-dependent and associated with the possible acquiring of tolerance both to cold and to heat treatment. It is possible that cytoplasmic dehydrins and dehydrin-related proteins may interact with mitochondrial membranes and prevent them from destabilisation during stress and conditioning to stress. Curiously enough, plant recovery after heat stress, contrary to the direct heat and cold stress in mitochondria of cauliflower inflorescences, resulted in accumulation of dlps quite different in size, possibly because the induction of distinct dlps, specific to heat stress response and acting in protein chaperoning during local water fluctuations. These changes may be present after plant shifting from heat stress to control growth conditions.
Dlps from the mitochondria of cauliflower leaves displayed relatively lower changes in abundance after cold treatment than the Arabidopsis
ones (Figures , ). Interestingly, Soitamo et al.
] observed similarly sized dehydrins in whole leaf protein extracts from Arabidopsis
plants under cold/light treatment.
In the present study the most thermostable polypeptides were found among high-molecular weight dlps (Figure , Table , , ). Generally, the thermostability of mitochondrial dlps described in this study for all the analyzed plants seems not to correlate with their level of accumulation under stress conditions. Yellow lupin thermolabile mitochondrial dlps of ca. 25 kDa displayed differences in the level under cold and heat stress conditions, contrary to the thermolabile 28 kDa protein in cereal mitochondria, which showed constitutive expression [16
The study of subcellular localisation of cauliflower dlps led to the conclusion that most of those proteins are present in the mitochondrial matrix (Figure ). Previously Borovskii et al.
] had shown that some small cereal dlps are water-soluble proteins and suggested that other dlps could associate with cellular membranes. The application of pronase E for digestion of native cauliflower mitochondria made it possible to confirm that only some high-molecular weight dlps, like 65 kDa, are sensitive to proteolytic digestion (Figure ). This corresponds to the results of Borovskii et al.
] in the case of 63 kDa dehydrin-related protein of wheat. The interactions of cauliflower mitochondrial dlps with membranes were also checked during the salt extraction (Figure ). Generally, the results coming from those experiments were fully consistent with those obtained from pronase digestion. The cauliflower dlp of ca. 65 kDa (probably corresponding to wheat 63 kDa protein) was removed from the membranes, which suggests its rather peripheral localisation on the external side of the outer mitochondrial membrane (Figure ). Considering the high level of thermostability as well as the results coming from salt extraction and pronase E digestion, the protein of ca. 65 kDa seems to be a putative candidate for a true cauliflower cytoplasmic dehydrin interacting with mitochondria. The possibility of interactions of dehydrins and dehydrin-related proteins with higher plant mitochondria should be confirmed in future by functional targeting assays, unless the cloning of cDNAs for those proteins becomes possible.
The results of the current study made it possible to discuss again [16
] the putative roles of dehydrins and dehydrin-related proteins in plant mitochondria under abiotic stress treatment. It seems that some large thermostable putatively cytoplasmic dehydrins associate with the outer mitochondrial membrane, whereas smaller dehydrin-related proteins are actively imported into the mitochondrial matrix and therefore could also stabilize the inner mitochondrial membrane and/or protect matrix enzymes against damage. During acclimation to stress conditions, the interactions of cytoplasmic dehydrins with the outer mitochondrial membrane may get strengthened and in the mitochondrial matrix the pool of dehydrin-like proteins may be actively rearranged in number and size. This phenomenon indicates that some conserved mechanisms regarding the expression of genes encoding mitochondrial dehydrin-related proteins may be engaged in stress response. This observation should be additionally confirmed by functional tests in the future.