**5. Heat shock proteins**

Another interesting family of stress-induced proteins is the heat shock family [54, 55].

It is known that heat stress may inhibit growth and development of plants. This may be due to heat stress itself or due to formation of ROS and other oxidants induced by heat stress. Heating also changes the structure of proteins, up to complete denaturation, and alters the activity of many enzymes. Disturbances in membranes structure, in their permeability and fluidity may cause partial or total disintegration of cells. Heat shock, that is, a short-term increase in temperature by 8–10 °C above the optimum one, is well known to induce rapid transient and reversible changes in gene expression in all living organisms. These changes result in the synthesis of specific group of polypeptides called heat shock proteins and suppression (complete or partial) of the synthesis of "normal" cell proteins synthesized by the cells before heat shock. This general biological phenomenon was qualified as a response to heat shock [55, 56]. The universality and conserved character of this response indicate its importance in cell physiology. Since heat shock proteins accumulation at heat shock in the cells of plant vegetative organs and seedlings was correlated with the development of plant tolerance to subsequent action of lethal temperatures, it was suggested that the response to heat shock is a manifestation of molecular mechanisms providing cell heat tolerance [57–59].

The induction of transcription of heat shock proteins is a common phenomenon in all living organisms. These proteins are grouped in plants into five classes according to their approxi‐ mate molecular weight: HSP100, HSP90, HSP70 (chaperones), HSP60 (chaperonins), small heat shock proteins (sHSPs), and ubiquitin (8.5 kD). Higher plants have at least 20 sHSPs and there might be 40 kinds of these sHSPs in one plant species. All of the major HSPs (that is, those expressed in very high amounts in response to heat and other stresses) have related functions: they ameliorate problems caused by protein misfolding and aggregation. However, each major HSP family has a unique mechanism of action. Some promote the degradation of misfolded proteins (ubiquitin, and various ubiquitin-conjugating enzymes); others bind to different types of folding intermediates and prevent them from aggregating (HSP70 and HSP60); and still another (HSP100) promotes the reactivation of proteins that have already aggregated [55].

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

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

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

Another interesting family of stress-induced proteins is the heat shock family [54, 55].

It is known that heat stress may inhibit growth and development of plants. This may be due to heat stress itself or due to formation of ROS and other oxidants induced by heat stress. Heating also changes the structure of proteins, up to complete denaturation, and alters the activity of many enzymes. Disturbances in membranes structure, in their permeability and fluidity may cause partial or total disintegration of cells. Heat shock, that is, a short-term increase in temperature by 8–10 °C above the optimum one, is well known to induce rapid transient and reversible changes in gene expression in all living organisms. These changes result in the synthesis of specific group of polypeptides called heat shock proteins and suppression (complete or partial) of the synthesis of "normal" cell proteins synthesized by the cells before heat shock. This general biological phenomenon was qualified as a response to heat shock [55, 56]. The universality and conserved character of this response indicate its importance in cell physiology. Since heat shock proteins accumulation at heat shock in the cells of plant vegetative organs and seedlings was correlated with the development of plant tolerance to subsequent action of lethal temperatures, it was suggested that the response to heat shock is a manifestation of molecular mechanisms providing cell heat tolerance [57–59].

The induction of transcription of heat shock proteins is a common phenomenon in all living organisms. These proteins are grouped in plants into five classes according to their approxi‐ mate molecular weight: HSP100, HSP90, HSP70 (chaperones), HSP60 (chaperonins), small heat shock proteins (sHSPs), and ubiquitin (8.5 kD). Higher plants have at least 20 sHSPs and there might be 40 kinds of these sHSPs in one plant species. All of the major HSPs (that is, those expressed in very high amounts in response to heat and other stresses) have related functions: they ameliorate problems caused by protein misfolding and aggregation. However, each major

seeds collected in 1993, and 23, 30, and 35–55 kD in seeds collected in 1994 [41, 42].

questions are of great interest and require further study.

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

seeds is of great interest.

**5. Heat shock proteins**

During two recent decades occurred after heat shock proteins discovery, the notions concern‐ ing their properties and role gradually widened and became much more complex. Now, we know that many heat shock proteins are molecular chaperons and facilitate protein–protein interactions in the cell, that heat shock proteins can be present in normal cells not subjected to stress, that they can be expressed at some developmental stages in the absence of heat shock, and that their synthesis can be induced by other stress types [57, 60–62]. Changes in gene expression resulting in heat shock proteins accumulation in the cells are evidently play an important physiological role and somehow protect cell structures and separate protein components against injuries induced by various stressors and increase cell tolerance and their adaptation to unfavorable environmental conditions [63]. Nevertheless, so far we did not decipher completely heat shock proteins functions and molecular mechanisms of their action. The possible heat shock effects on the synthesis of normal non-heat-shock proteins in various plant tissues are still less studied; it is not clear whether these effects are universal to the same degree as those of heat shock proteins gene expression. In many cases, heat shock suppressed the total protein synthesis and especially that of non-heat-shock proteins. This effect was evidently controlled on the level of translation because the normal pattern of protein synthe‐ sis was rapidly restored afterthe change in the temperature and transcription suppression with α-amanitin.Itis knownthatheat shock inhibitedtotalproteinsynthesis inthe vegetative organs of seedlings and plant cell cultures. This was related not to the inhibition of non-heat-shock mRNA synthesis but to incapability of these mRNAs to be translated under heat shock conditions. However, this specific response to heat shock was not evidently universal be‐ cause it was not observed in seed embryos during seed development and germination. Thus, it has been shown for soybean and common bean seeds that the synthesis of storage proteins and many other non-heat shock proteins and their mRNAs was not reduced and even was activated under heat shock and occurred along with the synthesis of heat shock proteins [64, 65]. In embryos of wheat [66], sorghum [67], maize [68], and pea [69], heat shock markedly activatedproteinsynthesisduringearlystagesofgermination,andheatshockproteinssynthesis was induced simultaneously with the synthesis of the bulk of proteins produced by embryo tissuesbeforeheatshock.Onthebasisofthesefacts,itwasconcludedthatsuchaspecificresponse of protein synthesis to heat shock observed in seed embryos of many grasses during seed development and germination could have a definite physiological significance; this could be a manifestation of additional molecular mechanisms improving embryo tolerance to unfavora‐ ble environmental conditions and, as a consequence, their viability [69].

Small heat shock proteins function as intracellular chaperones for other proteins. They play an important role in protein–protein interactions such as folding. They assist in the establish‐ ment of proper protein conformation and prevent unwanted protein aggregation. By helping to stabilize partially unfolded proteins, HSPs aid in transporting proteins across membranes within the cell [57]. HSPs recognize and bind to other proteins when these other proteins are in non-native conformations. The non-native conformations of these proteins could be due to protein-denaturing stress or due to immature peptides folded, assembled, or localized to an 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 enzymes against heat-induced inactivation.

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

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].
