**6. Heat shock proteins in recalcitrant horse chestnut seeds**

In the work [72], first data are obtained about functioning of molecular mechanisms providing for perception and transduction of heat signal and inducing heat shock proteins synthesis in the cells of embryo axes of dormant recalcitrant seeds, which are in metabolically active state but could not germinate. Embryo axes, cotyledon pieces, and cotyledon petioles were excised from recalcitrant horse chestnut (*Aesculus hippocastanum* L.) seeds in different times after the start of cold stratification and incubated at 28 or 40 °С on the medium containing <sup>35</sup>S-methio‐ nine and 50 μg/ml chloramphenicol for 4 h. The total rate of protein synthesis, the pattern of synthesized polypeptides, their distribution between subcellular fractions, and their relation to heat denaturing (5°min at 75 °C) were assessed. It was established that in all embryo parts, especially in axes, heat shock markedly activated protein synthesis in the beginning of stratification and to a lesser degree after ten weeks of stratification; heat shock suppressed protein synthesis at radicle emersion and especially during axial organ growth. Independently of the duration of stratification, which gradually released seed deep dormancy, isolated axes, cotyledons, and cotyledon petioles synthesized *in vivo* numerous diverse polypeptides at both 28 and 40°С. Newly synthesized polypeptides were present in the fractions of cell structures and cytosol; they differed in molecular weights, the intensity of labeling, and tolerance to heat denaturing. None of the dominating polypeptides present initially in all embryo parts and belonging mainly to heat-stable proteins was synthesized either at 28°С or 40°С. Some proteins synthesized at 40°С could be considered heat shock proteins because they were not synthesized at 28°С or their synthesis was markedly activated by heat shock. No less than 10 proteins behaved as obvious heat shock proteins; they were predominantly heat-sensitive soluble cytosolic proteins. All heat shock proteins, except those with mol wts of 220 and 34 kD, were highly labeled proteins. Some of them were characteristic of cell structures (220, 90, 20, and 18 kD); others were detected only in the cytosol (100, 80, and 34 kD). The synthesis of heat shock proteins did not depend on transcription and occurred on pre-existing mRNAs. An embryo capability of responding to heat shock did not depend on the seed physiological state and their germinability; it was similarly manifested in stratified and non-stratified seeds [72].

appropriate cellular compartment. In the presence of ATP at normal physiological concentra‐ tions, sHSPs change their conformation and releases denatured protein, allowing other molecular chaperones such as HSP70 to renature the protein and renew its biological activity. In the absence of ATP, sHSPs such as α-crystallin are more efficient than HSP70 in preventing stress-induced protein aggregation. *In vitro*, sHSPs selectively bind and stabilize proteins and prevent their aggregation at elevated temperatures in an ATP-independent way and protect

In recalcitrant of chestnut (*Castanea sativa*) seeds, a 20-kD protein has been purified from cotyledons, where it accumulates at levels comparable to those of major seed storage proteins [58]. This protein, termed Cs sHSP 1, forms homododecameric complexes under non-dena‐ turing conditions and appears to be homologous to cytosolic class I small heat shock proteins (sHSPs) from plant sources. *In vitro* evidence has been obtained that the isolated protein can function as a molecular chaperone; it not only increases, at stoichiometric levels, the renatu‐ ration yields of chemically denatured citrate synthase but also prevents the irreversible thermal inactivation of this enzyme. Although a role in desiccation tolerance has been hypothesized for seed sHSPs, this does not seem to be the case for Cs sHSP 1. The presence of immunolog‐ ically related proteins in orthodox and recalcitrant seeds of 13 woody species has been investigated [58]. The results indicate that the presence of Cs sHSP 1- like proteins, even at high levels, is not enough to confer desiccation tolerance and that the amount of these proteins does not furnish a reliable criterion to identify desiccation-sensitive seeds. Additional proteins or mechanisms appear necessary to keep the viability of orthodox seeds upon shedding. The synthesis of small HSPs may be an important part of seed developmental program [61]. As detected in pea [70], and in other species, the class I sHSPs accumulate in Arabidopsis seeds at midmaturation and decline during germination [61]. The similarity of sHSP regulation in such diverse species supports the conclusion that there is a selective advantage to this pattern of sHSP accumulation. The correlation of sHSP expression with the development of desiccation tolerance and dormancy suggests a possible role for the sHSPs in either or both of these

It was found that one major small heat shock protein existed with a molecular mass of 22 kDa and was detectable at high concentrations in seeds of three Acer species (orthodox and recalcitrant). After the seeds were dried, the content of this protein significantly increased. The largest content of this protein was observed in the oldest seeds, especially in embryonic axes. The proteins identified may play a protective role during water deficit and storage [47].

In the work [72], first data are obtained about functioning of molecular mechanisms providing for perception and transduction of heat signal and inducing heat shock proteins synthesis in the cells of embryo axes of dormant recalcitrant seeds, which are in metabolically active state but could not germinate. Embryo axes, cotyledon pieces, and cotyledon petioles were excised from recalcitrant horse chestnut (*Aesculus hippocastanum* L.) seeds in different times after the

enzymes against heat-induced inactivation.

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

processes, as has been previously hypothesized [62, 71].

**6. Heat shock proteins in recalcitrant horse chestnut seeds**

The analysis of heat shock action on gene expression in embryo tissues in dormant horse chestnut seeds demonstrated translation activation leading to the induction of a wide set of heat shock protein synthesis at the maintenance of the bulk of normal (non-shock) cellular protein synthesis. Due to this specific embryo response to heat shock, at early stages of germination and even under unfavorable conditions, embryo cells retain a capability of continuation or supporting on the sufficient level of the synthesis of proteins required for cell activity switching over to new developmental program, from embryogenesis to germination, and thus increase the reliability of germination. We believe that the absence of discrimination of non-heat shock mRNA translation during heat shock is specific to embryo tissues and could be considered an additional mechanism facilitating seed adaptation to unfavorable environ‐ mental conditions and successful germination.

Horse chestnut seeds survive successfully (and even require for deep dormancy release) the period of long cooling (18–22 weeks), retaining the high water content in their cells, and thus they are well adapted to overcome or correct damages arising under these conditions. However, according to current knowledge, heat shock proteins just fulfill this protective function. Therefore, it might be that, in mature dormant horse chestnut seeds, some amounts of required heat shock proteins are already present. These heat shock proteins could be synthesized and accumulated under the influence of elevated temperatures in the embryo cells during seed development or after their falling, that is, in response to heat shock, and they were preserved in the cells after seed entry into deep dormancy in the metabolically active state; they could improve embryo tolerance to unfavorable environmental conditions during stratification and thus increase seed viability.

We believe that the absence of discrimination of non-heat shock mRNA translation during heat shock is specific to embryo tissues and could be considered an additional mechanism facili‐ tating seed adaptation to unfavorable environmental conditions and successful germination.

Induction of heat shock proteins synthesis is a universal feature of the response to heat shock. According to our data, all tissues isolated from the embryos of dormant recalcitrant horse chestnut seeds responded to heat shock not only by continuation of non-heat shock protein synthesis but also by induction of similar sets of heat shock proteins. Two observations are of interest. We did not observe any dramatic changes in the set of heat shock proteins synthesized by isolated axes in the response to heat shock in the course of stratification, which evidently facilitate seed deep dormancy release. Moreover, heat shock proteins synthesis was readily detected in axes excised from non-stratified seeds, that is, it did not depend on seed capability of germination. This indicates independence of heat shock proteins synthesis at heat shock in the course of stratification of the embryo physiological state and its capability of germination. At the same time, some of our data indicate that stratification still somehow affected embryo physiological state. Thus, in the course of stratification, sensitivity of isolated axis growth to abscisic acid and indol-3-acethyc acid decreased [53], some characteristics of the proteome changed [50], and sensitivity of isolated axis translation to heat shock changed as well. However, this did not affect embryo tissue capacity to respond to heat shock. It is likely that signals providing for dormancy state, its release, and seed germination do not interact with signals leading to heat shock proteins synthesis induction. Furthermore, heat shock proteins' gene expression in isolated axes of dormant recalcitrant horse chestnut seeds was not depend‐ ent on transcription and was controlled predominantly on the level of translation. This means that all components required for the complex molecular mechanism of heat shock proteins gene expression were present in axis cells of mature seeds and were evidently produced still during seed development, may be under the influence of elevated temperatures. After mature seed falling, this mechanism is retained in the cells in the functionally active state and is capable of a rapid initiation of heat shock proteins synthesis in response to heat shock or another stress. However, the realization of this mechanism of heat shock proteins accumulation under natural conditions of stratification seems not very probable because the rate of protein synthesis under low temperature is low and heat signal is absent. Nevertheless, horse chestnut seeds survive successfully the period of long cooling (18–22 weeks), retaining the high water content in their cells and thus they are well adapted to overcome or correct damages arising under these conditions. However, according to current knowledge, heat shock proteins just fulfill this protective function. Therefore, it might be that, in mature dormant horse chestnut seeds, some amounts of required heat shock proteins are already present. These heat shock proteins could be synthesized and accumulated under the influence of elevated temperatures in the embryo cells during seed development or after their falling, that is, in response to heat shock, and they were preserved in the cells after seed entry into deep dormancy in metabolically active state; they could improve embryo tolerance to unfavorable environmental conditions during stratification and thus increase seed viability. This suggestion is supported by our observation that one of heat shock proteins, ubiquitin, was present in dormant horse chestnut seeds in functionally active state (i.e., in association with dehydrins) [52].
