Dehydrins is a complex family of hydrophilic heat-stable proteins. Their properties and functions are inadequately studied. They are accumulated in plant tissues in response to any external stimulus causing dehydration of the cells. These stimuli may result from drought, salt stress, cooling, treatments with hormones, and seed maturation. Dehydrins are identified in cyanobacteria, tissues of gymnospermous and angiospermous plants, herbaceous and woody species, vegetative organs, and various embryonic tissues of seeds. They are thought to be essential elements in plants' resistance or tolerance to dehydration. Plant seeds are of special interest for investigating this group of proteins. In the resistant to dehydration orthodox seeds, dehydrins are synthesized and accumulated at the final stages of maturation associated with seed desiccation. Recalcitrant seeds that are sensitive to dehydration do not dry out upon maturation and retain high moisture content and active metabolism. These seeds are capable of dehydrin production but remain sensitive to water loss and cannot cope with a profound desiccation, unlike orthodox-type seeds. The review considers main properties and functions of dehydrins, their structure, classification, spread, and intracellular localization. Roles of dehydrins in recalcitrant seeds are discussed.

Аннотация.Рекальцитрантные (неустойчивые к высыханию) семена древесных растений

In recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.), the bulk of protein in axial organs and cotyledons is accounted for by water-soluble proteins (albumins). In the cells of embryo, proteins are predominantly located in the cytosol, whereas the fraction of cell structures precipitate in the range from 1000 to 20000 g, accounting for only an insignificant part of total protein. Among the proteins of this fraction, there were no major components that could play a role of storage proteins. The aim of this work was to study deposition of protein in the vacuoles of cells of recalcitrant seeds of horse chestnut. Light microscopy and specific staining of protein and phytin did not detect protein bodies in the vacuoles of axial organs and cotyledons. Electron microscopy revealed traces of phytin in the vacuoles, but there were no formed globoids or considerable amount of protein therein. It is possible that precisely the absence of typical storage proteins and genetically determined desiccation in the course of maturation of recalcitrant seeds of horse chestnut stipulated preservation of the vacuoles that in mature recalcitrant seeds were not transformed into protein bodies.

The fraction of heat-stable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stress-induced dehydrin-like polypeptides. In our experiments, in tissues of dormant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Low-molecular-weight heat-stable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not cross-react with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissue-specificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heat-stable proteins during the entire period of stratification and also radicle emersion. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cycloheximide, or α-amanitin, dehydrins did not disappeared from the fraction of heat-stable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could cross-react with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50-kD heat-stable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heat-sensitive proteins and was named as a dehydrin-like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heat-stable cytosolic proteins. The role and function of major heat-stable proteins in horse chestnut seeds are yet to be studied.

Mechanisms of protection against photo-oxidation in selected desiccation-tolerant lichens and mosses have been investigated by measuring loss of light absorption during desiccation and chlorophyll fluorescence as indicators of photoprotection. Apparent absorption (1-T) spectra measured in the reflectance mode revealed stronger absorption of photosynthetic pigments in hydrated than in desiccated organisms, but differences were pronounced only in a cyanolichen, less so in some chlorolichens, and even less in mosses. Since the amplitude of chlorophyll fluorescence is a product of (1-T) light absorption by chlorophyll and quantum yield of fluorescence, and since fluorescence is inversely related to thermal energy dissipation, when chemical fluorescence quenching is negligible, fluorescence measurements were used to measure changes in energy dissipation. Preincubation of the hydrated organisms and desiccation in darkness excluded the contribution of mechanisms of energy dissipation to photoprotection which are dependent on the presence of zeaxanthin or on the light-dependent formation of a quencher of fluorescence within the reaction centre of photosystem II. Fast drying in darkness or in very low light was less effective in decreasing chlorophyll fluorescence than slow drying. Heating the desiccated organisms increased fluorescence by inactivating the mechanism responsible for fluorescence quenching. Glutaraldehyde inhibited fluorescence quenching during desiccation. Prolonged exposure of a desiccated moss or a desiccated lichen to very strong light caused more photo-induced damage after fast drying than after slow drying. The photo-oxidative nature of damage was emphasized by the observation that irreversible loss of fluorescence was larger in air than in a nitrogen atmosphere. It is concluded from these observations that desiccation-induced conformational changes of a chlorophyll protein complex result in the fast radiationless dissipation of absorbed light energy. This mechanism of photoprotection is more effective in preventing photo-oxidative damage than other mechanisms of energy dissipation which require light for activation such as zeaxanthin-dependent energy dissipation or quencher formation within the reaction centre of photosystem II.

The review considers and sums up the results of studies of physiological and biochemical characteristics of the dormant and germinating recalcitrant seed (the object of the study, the seed of horse chestnut, Aesculus hippocastanum L., is viewed as an exemplary case). The results of analysis of the proteomes of the axes and cotyledons have been studied and the effects of the stratification have been assessed. Gene expression has been studied at the level of protein synthesis: the protein-synthesizing capacity of the cells of the embryonic axis and cotyledon storage parenchyma of mature seed and seed undergoing stratification. The extent to which the functionally active translation machinery of ripe seed depends on transcription has been assessed, and the ability to synthesize protein under the conditions of stratification has been established. It is concluded that the embryonic axis of dormant seed lacks innate dormancy and that the isolated axis exhibits diverse sensitivity to exogenous abscisic acid and other physiologically active compounds.

This is the first characterization of proteins from storage parenchyma of cotyledons of mature dormant recalcitrant horse chestnut (Aesculus hippocastanum L.) seeds and evaluation the cell protein-synthesizing capacity. It was established that the content of protein in cotyledons did not exceed 0.5% of tissue fresh weight. Soluble proteins (the proteins of the postmitochondrial supernatant or cytosol) comprised the bulk (up to 90%) of total proteins. Protein of subcellular structures (20000 g-pellet) comprised 5–7% of total protein. Cotyledon proteins were heterogenous in their charges and molecular weights of subunits. Cotyledon protein was easily extracted with a salt (1 M NaCl); they comprised 90% of water-soluble albumin-like proteins. The proportion of globulins was insignificant; it did not exceed 5%. Most water-soluble proteins (more than 80%) were tolerant to heat denaturing. Among these heat-stable proteins, two major groups of polypeptides dominated: an electrophoretically homogeneous component with a mol wt of 24–25 kD and a complex group from three to five polypeptides with mol wts in the range between 6 and 12 kD. Native heat-stable proteins had disulfide bonds. Four fractions of heat-stable proteins were obtained by ammonium sulfate fractionation; three of them were alike in their polypeptide composition and contained major components with mol wts of 24–25 and 5–12 kD. It was established that the active translational machinery functioned in the cells of storage parenchyma in cotyledons of mature dormant horse chestnut seeds. During each stage of stratification, cotyledon fragments incorporated 35S-methionine into TCA-insoluble material more actively than axial organs. We discuss cotyledon protein composition, their function as a storage organ, and a possible role of heat-stable proteins.

This is the first characterization of proteins from axial organs of recalcitrant horse chestnut seeds during deep dormancy, dormancy release, and germination. We demonstrated that, during the entire period of cold stratification, axial organs were enriched in easily soluble albumin-like proteins and almost devoid of globulins. About 80% of the total protein was found in the cytosol. Approximately one third of cytosolic proteins were heat-stable polypeptides, which were major components of total proteins. Heat-stable proteins comprised three groups of polypeptides with mol wts of 52–54, 24–25, and 6–12 kD with a predominance of low-molecular-weight proteins. The polypeptide patterns of heat-stable and thermolabile proteins differed strikingly. Heat-stable proteins accumulated in axes during the late seed maturation, comprising more than 30% of the total protein in axes of mature seeds. The polypeptide patterns of the total protein of axial organs and its particular fractions did not change in the course of seed dormancy and release. At early germination, the content of heat-stable proteins in axes decreased and their polypeptide pattern changed both in the cytosol and cell structures. We believe that at least some heat-stable proteins can function as storage proteins in the axes. Localization of storage proteins in the cells of axial organs and the role of heat-stable proteins in recalcitrant seeds are discussed.

In embryo axes excised from mature horse chestnut (Aesculus hippocastanum L.) seeds, both freshly-fallen and subjected to cold stratification, the ability for growth was studied. While excised axes were kept on water at 28°C for 3 days, their fresh weight and length increased, the polypeptide composition of soluble proteins changed, the content of some heat-stable polypeptides decreased, and the capacity for protein synthesis in vivo retained. All these processes were similar to those in the axes of intact seeds during stratification until radicle protrusion. Growth of excised axes accelerated with the increasing duration of stratification. Cycloheximide (50 mg/l) and α-amanitin (7 mg/l) inhibited axis growth, but an inhibitor of ABA synthesis fluridone (5 mg/l) and a natural cytokinin dihydrozeatin (10–5 M) did not influence the growth rate. The growth capacity of axes excised from dormant and germinating horse chestnut seeds indicates the absence of dormancy in the axes of mature seeds. ABA (10–5 M) suppressed completely the growth of axes detached from seeds experiencing cold stratification but still not germinating, although protein synthesis was not inhibited. The axes excised from the seeds after radicle emergence were insensitive to ABA and grew actively in its presence. ABA-induced growth inhibition might be related to the suppressed synthesis of minor polypeptides required for growth or to the activated synthesis of some growth-retarding proteins. The conclusion was drawn that the excised axes could be used as a model for studying the processes preceding visible germination of recalcitrant seeds.

This is the first characterization of the protein-synthesizing capacity of axial organs of recalcitrant horse chestnut seeds, which were subjected to cold wet stratification, during deep dormancy, dormancy release, and germination. The embryo axis length, fresh weight, and protein content remained almost unchanged during the entire period of stratification but increased after radicle protrusion. Isolated embryo axes were capable of radioactive amino acid uptake and incorporation, starting from the first hours of their incubation at 28°C. The capacity for protein synthesis was easily detected since the first days of stratification; its rate did not change until germination and increased after radicle protrusion. Cycloheximide suppressed protein synthesis by 95%; during stratification, protein synthesis was insensitive to α-amanitin (7 μg/ml) but was inhibited by 40% during germination. This indicates the presence of preformed translatable mRNAs in stratifying recalcitrant seeds, whereas in germinating seeds protein synthesis depends on transcription to a greater extent. Proteins synthesized by the axes were located in various subcellular compartments but mostly in the cytosol; among cytosolic proteins, thermolabile proteins dominated. The number and set of synthesized polypeptides remained almost unchanged during stratification. Most pronounced changes in protein composition occurred during germination and concerned mainly minor components of cytosolic heat-stable proteins. It seems evident that, under dormancy and during dormancy release, a constant set of genes was expressed due to preformed mRNA activity. Noticeable changes in gene expression occurred at the level of protein synthesis only during seed germination. Thus, the competent translational machinery is present in axis cells of dormant mature recalcitrant seeds capable of performing protein synthesis under favorable conditions. It seems probable that inability of these seeds to germinate is due to the absence of special gene expression, but not to disfunctioning of protein-synthesizing machinery.

The mobilization of protein and phytin was studied in aleurone grains of the endosperm of germinating castor beans (Ricinus communis L.). Differential staining of protein and phytin zones of aleurone grains indicated that mobilization of phytin preceded that of proteins. Biochemical assays demonstrated that, after 64 h of seed imbibition, phytin content decreased by 14%, and, after six days, phytin was digested by 87%. This process was correlated with increasing phytase activity. At the same time, phytin content increased in cotyledons and axial organs. The breakdown of storage proteins began after radicle emergence. The activities of two proteolytic enzymes accelerated after three and five days of germination, respectively, in correlation with the degradation of storage proteins and conversion of aleurone grains into aleurone vacuoles by the third day. We concluded that the mobilization of phytin and protein in the endosperm preceded the mobilization of storage lipids, and this determined the composition of metabolites flowing from the endosperm to the seedling.