**4. Dehydrins in horse chestnut seeds during dormancy and germination**

The mature horse chestnut (*Aesculus hippocastanum* L.) seeds are not tolerant to dehydration (i.e., belong to the recalcitrant seed type), but they are resistant to long cold stress, for instance, during winter in the central Russia when seeds are under the snow cover. The second biological peculiarity of horse chestnut seeds is that they are in the state of deep dormancy, that is, they are incapable of germinating under favorable conditions without prior prolonged incubation at low above-zero temperatures and high water content, known as stratification.

The analysis of horse chestnut seed proteins made it possible to reveal a number of unique characteristics of their proteome, which distinguish recalcitrant horse chestnut seeds from the majority of orthodox seeds and is evidently related to the specific features of their physiological behavior. These characteristics include extremely low content of globulins, predominance of water-soluble proteins located in the cytosol, and the high level of non-compartmentalized heat-stable proteins [50, 52].

The presence of heat-stable proteins (i.e., proteins resistant to high-temperature denaturation) in horse chestnut seeds is the most interesting feature of the object of our study. The results obtained demonstrated that heat-stable proteins accumulate during maturation of horse chestnut seeds and are present in freshly picked seeds in considerable amounts. These proteins account for more than 30% of soluble cytosolic proteins of the axial organs and most of soluble proteins of cotyledons (more than 80%). It is possible that in some seeds certain heat-stable proteins may function as storage deposits, being a source of nitrogen for the seedling. The involvement of heat-stable proteins in some other specific functions related to their extreme temperature resistance, characteristic amino acid composition, and high cellular content cannot be ruled out.

In order to identify and characterize stress-induced dehydrin-like polypeptides in mature recalcitrant horse chestnut seeds, we analyzed the fraction of cytosolic heat-stable proteins isolated in the period of seed dormancy and germination [52]. In our experiments, in tissues of dormant seeds, dehydrin was identified by immunoblotting as a single bright band of the polypeptide with a mol wt of about 50 kD. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. Apparently, recalcitrant seed germination is accompanied by dehydrin disappearance, like this occurs during orthodox seed germination.

are synthesized before maturation drying [6]. And maturation drying is genetically deter‐ mined in orthodox seeds. During embryogenesis and maturation drying of orthodox seeds, dehydrins are synthesized and seeds may survive dry storage. "Typical" recalcitrant seeds [48, 49] such as *Avicennia* and *Brunguiera* have no dehydrins and high water content and they do not survive water loss during storage. Intermediate seeds (*Coffea, Barringtinia*) have no dehydrins during embryogenesis but have genes of dehydrins and may synthesize dehydrins after partial water loss in storage, so these seeds may survive [48, 49]. And "atypical" recalci‐ trant seeds (*Camellia, Castanea, Euterpe, Quercus*) accumulate some dehydrins at the end of embryogenesis and undergo a weak maturation drying and can be stored for a limited time [48, 49]. So, the role of dehydrins in recalcitrant seeds is not clear, and further comprehensive

322 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

**4. Dehydrins in horse chestnut seeds during dormancy and germination**

The mature horse chestnut (*Aesculus hippocastanum* L.) seeds are not tolerant to dehydration (i.e., belong to the recalcitrant seed type), but they are resistant to long cold stress, for instance, during winter in the central Russia when seeds are under the snow cover. The second biological peculiarity of horse chestnut seeds is that they are in the state of deep dormancy, that is, they are incapable of germinating under favorable conditions without prior prolonged incubation

The analysis of horse chestnut seed proteins made it possible to reveal a number of unique characteristics of their proteome, which distinguish recalcitrant horse chestnut seeds from the majority of orthodox seeds and is evidently related to the specific features of their physiological behavior. These characteristics include extremely low content of globulins, predominance of water-soluble proteins located in the cytosol, and the high level of non-compartmentalized

The presence of heat-stable proteins (i.e., proteins resistant to high-temperature denaturation) in horse chestnut seeds is the most interesting feature of the object of our study. The results obtained demonstrated that heat-stable proteins accumulate during maturation of horse chestnut seeds and are present in freshly picked seeds in considerable amounts. These proteins account for more than 30% of soluble cytosolic proteins of the axial organs and most of soluble proteins of cotyledons (more than 80%). It is possible that in some seeds certain heat-stable proteins may function as storage deposits, being a source of nitrogen for the seedling. The involvement of heat-stable proteins in some other specific functions related to their extreme temperature resistance, characteristic amino acid composition, and high cellular content

In order to identify and characterize stress-induced dehydrin-like polypeptides in mature recalcitrant horse chestnut seeds, we analyzed the fraction of cytosolic heat-stable proteins isolated in the period of seed dormancy and germination [52]. In our experiments, in tissues of dormant seeds, dehydrin was identified by immunoblotting as a single bright band of the polypeptide with a mol wt of about 50 kD. During radicle emergence, not only the fraction of

at low above-zero temperatures and high water content, known as stratification.

studies are required.

heat-stable proteins [50, 52].

cannot be ruled out.

Since horse chestnut seeds contain along with heat-stable proteins numerous heat-sensitive proteins, it was of importance to elucidate whether dehydrin-like proteins are present among the latter. Nobody analyzed this protein fraction for the presence of dehydrin-like proteins. In the fraction of heat-sensitive proteins of horse chestnut seeds, we detected a component with a mol wt of 80 kD, which cross-reacted with anti-dehydrin antibody, that is, it was immuno‐ logically revealed as dehydrin-like protein. The analysis of different subcellular protein fractions of axial organs, cotyledons, and cotyledon petioles showed that both 50-kD dehydrin and 80-kD dehydrin-like protein could be detected in the total homogenate protein. It seems important that one of the axis heat-sensitive polypeptides cross-reacted with the anti-dehydrin antibody but differed from heat-stable dehydrin by a higher molecular weight (about 80 kD). This is the first indication on the possible presence of dehydrin-like proteins among heatsensitive polypeptides of horse chestnut seeds [52].

During stratification, there were no substantial changes in the content of 50-kD dehydrin. However, this protein disappeared rapidly during seed germination. Since a small heat shock protein ubiquitin plays an important role in cell protein degradation, it was of interest to elucidate whether there is any connection between dehydrin, which should disappear during seed germination, and ubiquitin, which marks specifically proteins destined for degradation. It turned out that 50-kD dehydrin cross-reacted with anti-ubiquitin antibody. This means that dehydrin ubiquitination might provide for dehydrin rapid disappearance after radicle protrusion. This fact seems very interesting because just 50-kD dehydrin disappeared firstly during horse chestnut seed germination.

Earlier we have established that embryo axes of dormant seeds are not in the dormant state and could germinate *in vitro* (72 h on water at 28 °С) in each period of stratification of dormant seeds [53]. Like during seed germination *in vivo*, 50-kD dehydrin was not detected in such axes germinated *in vitro*. As we have demonstrated [53], treatment of excised axes with ABA (10–5 M), cycloheximide, or α-amanitin suppressed their germination. Under these conditions of suppressed growth, 50-kD dehydrin remained in the axes and was easily detected by immu‐ noblotting. The mechanism of ABA inhibitory action on excised axis germination remains unknown. It is also unknown whether ABA induces 50-kD dehydrin synthesis in axes or simply prevents its degradation, resulting in its presence in axes on the level detected before germination. However, at the comparison of ABA-treated axes with those treated with cycloheximide, or α-amanitin, the substantially stronger signal may be noted in the case of ABA treatment. This may indicate indirectly on the induction of dehydrin synthesis at germination inhibition by ABA [52].

The molecular weight of the single dehydrin we detected in the horse chestnut seeds was slightly above 50 kD. At the same time, other researchers reported other values [41, 42], for example, 12, 14, and 18 kD or 30–55 kD. So far it is difficult to explain such differences in dehydrin sizes. It is not excluded that this is related to some specific feature of horse chestnut plants or their populations in different countries. It seems more likely that these differences are related to the influence of different growth conditions. Thus, seeds collected in different years differed in the size of dehydrins: 12, 14, and 18 kD in seeds collected in 1992; 14 kD in seeds collected in 1993, and 23, 30, and 35–55 kD in seeds collected in 1994 [41, 42].

However, dehydrins comprise only a small part of heat-stable proteins of recalcitrant seeds. The functions of other heat-stable proteins accumulating in horse chestnut seeds in great amounts during dormancy and germination, the reasons for their extreme heat resistance, and their relation to the low-temperature action during stratification remain unclear. These questions are of great interest and require further study.

We believe that the presence of the proteome of horse chestnut seeds of hydrophilic proteins capable of holding moisture may be related to the recalcitrant character of these seeds. Heatstable proteins also promote the resistance of highly watered seed to cold stress under the conditions of stratification, thereby keeping the embryo viable. From our point of view, the mere presence of a large amount of heat-stable proteins in dormant recalcitrant horse chestnut seeds is of great interest.
