**2. Dehydrins—stress-induced proteins**

The accumulation of dehydrin transcripts and proteins during dehydration and a correlation between the level of drought tolerance and the amount of dehydrin present strongly suggest that they are involved in protecting the plant from the negative effects of dehydration [9–11]. In recent years, dehydrins are studied very intensively, which resulted in the appearance of numerous reviews and research papers [12–18].

adapt to overcome them, to retain a capability of germination and to fulfill their physiological destination, that is, species preservation and distribution. Furthermore, in seeds the develop‐ mental program of the individual plant is switched over from embryogenesis to germination; in the periods of seed development and germination, seed embryos, being subjected to the action of unfavorable conditions, must change cell activity on the level of gene expression and induce the synthesis of anti-stress proteins to protect themselves and overcome stress effects. On the other hand, embryos have to provide expression of genes for proteins required for

Different environmental stresses to a plant may result in similar responses at the cellular and molecular level. This is due to the fact that the impacts of the stressors trigger similar strains and downstream signal transduction chains. Stressors such as drought (lack of environmental water), salinity (high osmolarity), and cold, especially frost (lack of liquid water), induce water deficiency [1]. All three forms of abiotic stress affect the water relations of a plant on the cellular as well as whole plant level, causing specific as well as unspecific reactions, damages and adaptation reactions. The stabilizing effect of liquid water on the membrane can be supported by compatible solutes and special proteins. At the metabolic level, osmotic adjustment by synthesis of low-molecular osmolytes (carbohydrates, betains, proline) can counteract cellular

During the periods of dehydration, a wide variety of plants can express dehydration proteins (dehydrins), which are also members of the plant late embryogenesis abundant (LEA) protein family. For the first time, LEA proteins were characterized in ripening seeds [2]. But they are widely investigated now [3–5]. However, their precise role has not been clear yet. These proteins are supposed to protect cells from water loss; they can behave as molecular chaper‐ ones [2]. Now the expression of LEA proteins has been shown not only in ripen seeds but also in other plant (or animal) tissues. LEA proteins are induced by osmotic or cold stress, by exogenous ABA (abscisic acid) [6]. It is believed that LEA proteins are non-catalytic proteins. Practically, all LEA proteins are water-soluble hydrophilic heat-stable and unfolded proteins. LEA proteins are synthesized in orthodox seeds, and the development of which at the final stage is associated with genetically determined drying. Their amount can reach 4% of total cell proteins [7]. At the same time, LEA proteins are found in desiccation-sensitive recalcitrant seeds. That is why the study of recalcitrant seeds is of great interest. On the one hand, these seeds produce LEA proteins, and on the other hand, they are sensitive to desiccation. Therefore, a characterization of LEA proteins in recalcitrant seeds can help understanding their role, for

example when plant cells are challenged by abiotic stress as cold or salt stress.

**2. Dehydrins—stress-induced proteins**

LEA proteins are classified into more than seven distinct groups [8]. Among the induced LEA proteins, dehydrins (group II of late embryogenesis abundant proteins) have been most commonly studied, but our knowledge of their fundamental role in the cell is incomplete.

The accumulation of dehydrin transcripts and proteins during dehydration and a correlation between the level of drought tolerance and the amount of dehydrin present strongly suggest

further development, that is, germination *per se*.

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

dehydration and turgor loss [1].

The dehydrins are very hydrophilic proteins and exhibit an unusually low level of recognizable structure [10, 19]. The main characteristic of the dehydrins is the presence of one or more lysinerich stretches of 15 amino acids (the sequence EKKGIMKIKEKLPG), called the K motifs (or Ksegments), that are predicted to form class A amphipathic alpha-helices [20, 21]. All dehydrins investigated have K-segment; it is usually used for preparing anti-dehydrin antibodies [12, 13]. The K-segment occurs 1–12 times, with 1 or 2 repeats being the most common. Since the K-segments can form amphipathic alfa-helices, they may stabilize membranes against dehydration. Dehydrins can also contain two other motifs: an N-terminal Y segment (consen‐ sus V/TDE/QYGNP) and a serine-rich S segment [11]. The Y-segment is similar to the nucleo‐ tide-binding domain found in bacteria. Typically, 1–3 Y-segments are present at the Nterminus of a dehydrin [11]. The S-segment contains a tract of Ser residues and is present in one or no copies in a dehydrin. Dehydrins extracted from drought-stressed plants are phos‐ phorylated on these serines [22]. The role of phosphorylation is not clear but may be correlated with translocation of dehydrins to the nucleus [23] or the increased negative charge could enhance the ability of the protein to bind divalent cations such as zinc. It has been proposed that the short amphipathic K segments of dehydrin polypeptides interact with solvent-exposed hydrophobic patches on proteins undergoing partial denaturation and thereby interfere with protein aggregate formation [10]. Amphipathic K helixes could also be involved in binding membrane lipids and thus could play a more specific role in protecting lipoproteins, proteins located in membranes, and/or the membrane structure itself [10, 24].

According to the presence of the K-, S-, and Y-segments, dehydrins can be divided into five structural subgroups: Kn, SKn, KnS, YnKn, and YnSKn [10, 11]. Although not specifically included in the YSK naming system, dehydrins also contain Φ-segments, which are rich in Gly, Thr, and many other polar amino acids. This poorly conserved segment tends to be located between the Y-, S-, and K-segments.

Dehydrins are evolutionarily conserved among photosynthetic organisms including angio‐ sperms, gymnosperms, ferns, mosses, liverworts, algae and cyanobacteria, as well as in some non-photosynthetic organisms such as yeast [17, 25]. Dehydrins seem to be very ancient proteins—a 40-kD protein was observed in Calothrix sp. strain PCC 7601, and in *Nostoc* sp. strain Mac-R2, an osmotic-induced doublet at 39 and 40 kD was observed. It appears that cyanobacteria produce a dehydrin-like protein under osmotic stress [25].

The expression of many dehydrins increases by the phytohormone abscisic acid (ABA), they are also referred as RAB proteins (Responsive to ABA) [26–28]. On the other hand, protein level of some dehydrins is regulated by low temperature only [22]. In particular, studies of stress-induced accumulation of five dehydrins in *Arabidopsis* revealed that two of them (LTI30 and COR47) accumulated primarily in response to low temperature. The level of another two proteins (ERD14 and LTI29) was upregulated by ABA and low temperatures, whereas RAB18 was only found in ABA-treated plants [22]. Borovsky and coworkers [15] have found that not only cold but also drought, freezing, and exogenous ABA treatment also result in accumulation of heat-stable dehydrin-like proteins in plant mitochondria. The most tolerant winter wheat and rye accumulate more of the heat-stable dehydrins than maize. Cold-induced accumulation of the heat-stable mitochondrial dehydrin-like proteins in all species studied was accompanied by increasing of plant cryotolerance [15].

Dehydrins have been most extensively studied in relation to drought and cold stresses [29– 35]. Some experimental studies provide evidence that dehydrins contribute to freezing stress tolerance in plants and suggest that this could be partly due to their protective effect on membranes [36]. Dehydrins stabilize plant plasma and organellar membranes in conditions of stress, and further zinc may be an important co-factor in stabilization [37, 38].

The hypothesis that dehydrins have detergent and chaperone-like properties and may interact with compatible solutes to serve as structural stabilizers of macromolecules under conditions of water deficit [10] is now experimentally evident [17, 39].

Numerous *in vitro* functions have been described and proposed for dehydrins, including cryoprotection of lactate dehydrogenase (LDH), cryoprotection of purified protoplasts and chloroplasts, prevention of water loss, binding of excess ions, binding of nucleic acids, prevention of protein aggregation at elevated temperatures, and prevention of ice crystal growth. RAB18 (Y2SK2) accumulates in response to the phytohormone abscisic acid (ABA), drought, and low temperature [22]. LTI29 and COR47 (SK3) accumulate primarily in response to low temperature but also to ABA and salt stress [22]. LTI30 (K6) accumulates mainly under cold stress [22]. ERD14 (SK2) is present in non-stressed plants although the protein level is upregulated by stress, particularly drought stress [22]. Dehydrin XERO (YSK2) mRNA has been found to be constitutively expressed [39].

Of these functions, the most extensively studied has been the cryoprotection of LDH, where it has been shown that dehydrins are more effective than small molecules such as sucrose at protecting LDH activity from freeze-thaw damage [39].

The following are the functions of dehydrins with experimental evidence: binding to water and ions, binding to phospholipids, radical scavenging, phosphorylation, binding to calcium, protection of enzymes, binding to cytoskeletons, and binding to nucleic acids [17].

DHN genes are also expressed significantly in seeds toward the end of maturation, a period when the seed undergoes a developmentally programmed reduction in water content [22, 40]. The LEA/dehydrin proteins have been estimated to comprise up to 4% of the total seed protein [7].
