Seasonal Changes in the Composition and Content of Dehydrins in Winter Wheat Plants

Seasonal changes in the pattern and content of dehydrins in winter wheat (Triticum aestivum) plants grown under field and laboratory conditions were studied by one-dimensional PAGE and immunochemical methods. During hardening, plants accumulated dehydrin-like polypeptides with mol wts of 209, 196, 66, 50, and 41 kD. In winter, low-molecular-weight dehydrins with mol wts of 24, 22, 17, 15, and 12 kD were synthesized and accumulated as well. Their content dropped sharply in spring when plants became unhardened. Accumulation/disappearance of these proteins corresponded to the fluctuations in wintering plant frost tolerance before winter and in spring. It is assumed that both high- and medium-molecular-weight dehydrins are involved in plant stress responses and adaptation, whereas low-molecular-weight dehydrins are evidently involved only in the process of low-temperature adaptation.     

Accumulation of Dehydrins and ABA-Inducible Proteins in Wheat Seedlings during Low-Temperature Acclimation

Using one-dimensional SDS-PAGE and immunochemical methods, we detected the presence and estimated the content of dehydrins and ABA-inducible (RAB) polypeptides in etiolated seedlings of four spring and three winter wheat (Triticum aestivum L.) cultivars differing in frost hardiness. We hardened three-day-old seedlings at 4°C for nine days or grew them at 22°C for a day (control seedlings). We established that heat-stable cold-regulated (COR) polypeptides with mol wts of 209, 196, 169, 66, 50, and 41 kD, which are characteristic of hardened wheat seedlings, were homologous to polypeptides from a dehydrin family and polypeptides with mol wts of 209, 196, 66, 50, and 41 kD were immunologically related to RAB-proteins. We supposed that these COR polypeptides were involved in the prevention of local protein dehydration and denaturation during hypothermia. Analysis of the relative content of COR proteins revealed a close correlation between the cultivar frost hardiness and the concentration of these proteins. It seems evident that different accumulation of dehydrins and RAB polypeptides in different cultivars of a single species is one of the causes for different plant frost hardiness.     

Association of dehydrins with wheat mitochondria during low-temperature adaptation

In mitochondria from the crowns of field-grown winter wheat plants or their seedlings hardened in the laboratory, thermostable proteins immunologically related to dehydrins were detected. It was found that two dehydrins with mol wts of 63 and 52 kD bound with the outer mitochondrial membrane during autumnal hardening or during adaptation to low temperature in the laboratory. Dehydrins of similar mol wts were detected among proteins in the total membrane fraction from low-temperature-adapted wheat plants. In addition, dehydrins with mol wts of 209 and 196 kD were present in this fraction as well. Dehydrins of similar mol wts were bound with mitochondria from seedlings adapted to low temperature and those from the crowns of plants after autumnal hardening. In spring, the amount of dehydrins associated with mitochondria from the crowns declined to the level characteristic of early autumn. Dehydrin association with mitochondria is evidently an important defense mechanism of frost-resistant plants.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 221–226.Original Russian Text Copyright © 2005 by Borovskii, Stupnikova, Antipina, Anuchina, Voinikov.This revised version was published online in April 2005 with a corrected cover date.     

Polymorphism of Thermostable Proteins in Soft Wheat Seedlings during Low-Temperature Acclimation

The accumulation of thermostable stress proteins during hardening was studied in etiolated seedlings of spring (cvs. Rollo, Drott, Angara-86, and Tyumenskaya-80) and winter (moderately frost-resistant cv. Bezostaya-1 and highly frost-resistant cvs. Irkutskaya ozimaya and Zalarinka) wheat using one-dimensional SDS-PAGE. Hardening was performed at 4°C for nine days. Seedling tolerance to low subzero temperatures was estimated from electrolyte leakage and seedling survival after freezing. Hardening of all wheat genotypes tested resulted in the accumulation of thermostable cold-regulated (COR) polypeptides with mol wts of 209, 196, 169, 66, 50, and 41 kD. A densitometric analysis demonstrated a close correlation between the cultivar frost tolerance and the relative content of COR proteins, which evidently indicated the protective functions of the latter. These results led us to suggest that the level of specific protective agents, thermostable high-molecular-weight COR polypeptides in particular, determines the degree of plant frost resistance within a particular plant species.     

Cell-wall lectins during winter wheat cold hardening

The polypeptide composition and functional activity of cell-wall lectins from roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings during cold hardening were studied. Several phases of lectin activity changes were observed, which indicates their involvement in the development of general adaptation syndrome of the cell. After 0.5-h low-temperature treatment, marked alterations occurred in the profile of protein elution: lectins with mol wts of 78 and 42.5 kD disappeared and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It was established that 17.5-and 69-kD lectins and most lectins eluted with glucose were arabinogalactan proteins (AGP), which permitted a supposition that these lectins were involved in the interaction between the cell wall and cytoskeleton. After 7-day-long hardening, total protein content reduced and lectins with mol wts of 69 and 37 kD disappeared, which corresponded to reduced lectin activity by the end of hardening. A transient appearance of 37-and 69-kD lectins, which are AGP, might indicate their involvement in the triggering the development of plant-cell defense responses.     

Woody plants of Yakutia and low-temperature stress

Physiological and biochemical features of woody plants (Pinus sylvestris L. and Betula platyphylla Sukacz.) during transition from vegetative to frost-resistant state under conditions of extremely severe climate of Yakutia were studied. In P. sylvestris such transition was accompanied by a decrease in the content of chlorophylls long before first frosts and by an increase in the proportion of Xanth and simultaneous decrease in the content of β-carotene in needles during the first and second phases of hardening. In the period of preparation to dormancy, overwintering organs of both P. sylvestris (needles) and B. platyphylla (buds) accumulated the two groups of major dehydrins, with low mol wts of 15–21 kD and middle mol wts of 66–141 kD. Simultaneously, low temperature led to a great increase in the content of polyunsaturated fatty acids (FAs) in lipids of P. sylvestris needles and B. platyphylla buds, primarily linoleic acid and also eicosenoic FAs differing in the extent of desaturation. Observed qualitative and quantitative changes in pigments, total proteins, dehydrins, and FAs during autumn hardening of P. sylvestris and B. platyphylla plants presume their important role in the development of resistance of these tree species to low-temperature stress (down to −60°C) in the cryolithic zone of Yakutia.     

茶树叶片类脱水素蛋白的Western-blot分析

为了解茶树脱水素种类与功能,采用Western-blot技术,研究了不同季节及越冬过程中茶树叶片脱水素蛋白家族的表达模式。结果显示:(1)茶树叶片总蛋白提取采用酚-甲醇/醋酸铵沉淀法,用时短、蛋白浓度高、SDSPAGE电泳条带清晰,背景干净,满足茶树Western-blot技术要求。(2)在不同季节及越冬期中发现14~95kD共9种不同分子量的茶树类脱水素蛋白,其中95、65、48、37、34和14kD等6种蛋白表达量较为稳定,季节与越冬期变化不明显;58kD脱水素仅在冬季表达,越冬期不断上升,2月份增加到最高,表达丰度高;28kD脱水素蛋白在冬季表达量高,越冬期与茶树抗寒力变化规律一致;21kD脱水素在夏季和越冬期后期有较高的表达。研究表明,这3种脱水素可能在茶树抗逆中起着重要作用。     

The relationship between the differences in frost resistance of Arabidopsis and Thellungiella and heat shock proteins and dehydrins

Plants of extremophile Thellungiella (Thellungiella salsuginea (Pall.) OE Schulz) withstood freezing at ?15°C for 2 h without hardening, whereas plants of Arabidopsis (Arabidopsis thaliana (L.) Heinh.) were damaged at ?10°C and died at ?15°C under these conditions. The content of heat shock proteins (HSPs) HSP101, HSP60 and constitutive HSC70 was significantly higher in unhardened Thellungiella plants than in unhardened Arabidopsis plants. The spectrum of dehydrins (DHNs) in unhardened Thellungiella plants was more diverse and their total content was higher than in unhardened Arabidopsis plants. Frost resistance of Arabidopsis increased after hardening (4°C, 7 days), and there was an increase in the content of HSP101 and HSP60, as well as in the content of the DHN with a mol wt of 70 kD. Thellungiella plants survived after hardening at ?18°C, and the increase in the content of HSP101, HSP70, and HSP60 was significantly less pronounced than in Arabidopsis. At the same time, the content of DHNs in Thellungiella increased significantly during the hardening primarily because of the appearance of two DHNs (mol wts of 42 and 45 kD). It is assumed that an increased content of HSPs and DHNs and their greater diversity can be one of the factors of Thellungiella resistance to low temperatures as compared to Arabidopsis.     

Identification and characterization of dehydrins in horse chestnut recalcitrant seeds

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.     

Lectin activity and composition in the wheat cell walls under the effects of low temperature and inhibitors of calcium signaling pathway

In the roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings, the effects of neomycin (100 μM), an inhibitor of phospholipase C, and dilthiazem (250 μM), a blocker of calcium channels on lectin activity and composition at low-temperature treatment (2–3°C) were studied. Hypothermia induced the appearance of two peaks of cell wall-bound lectin activity, e.g., in 0.5 and 6 h. Under these conditions, the inhibitors suppressed lectin activity. In 0.5 h of hypothermia, substantial changes in total profile of proteins were observed: lectins with mol wts of 85, 78, and 54 kD disappeared, and novel lectins with mol wts of 110, 105, 70, 50, and 34.5 kD appeared. In the presence of dilthiazem, the set of lectin proteins remained similar to that in unhardened plants, and the increase in the lectin content and activity was observed only after 3-h exposure to low temperature. This indicates that blocking dilthiazem-sensitive calcium channels slowed plant response to stress and did not permitt the cell to start rapidly the development of defense mechanisms. The important role of lectins with mol wts of 110 and 60 kD in the formation of freeze tolerance is supposed because these lectins did not appear in the presence of dilthiazem.     

Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the lambda phage DNA with N⁶-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca²⁺/Mg²⁺- and Zn²⁺-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16–80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.     

DNA-Binding Proteins and Structural Characteristics of Chloroplast Nucleoids

A comparative analysis of proteins from chloroplast nucleoids was performed in two higher-plant species (Pisum sativumL. andArabidopsis thalianaL.) and a green alga Chlamydomonas reinhardtiiDang. In the nucleoids of the higher plants and the alga, 26–27 proteins were detected with their mol wts ranging from 10 to more than 94 kD. In all the species tested, the distribution of nucleoid proteins by their mol wts was similar, especially between the predominant proteins with mol wts of 10 to 40 kD. Six DNA-binding proteins (12–18 kD) were detected in nucleoids fromCh. reinhardtiichloroplasts after in vivocovalent cross-linking between chloroplast DNA and proteins. Under an electron miscroscope, some regular structures resembling nucleosome-like particles of bacterial cells were revealed.     

Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment

Background

Dehydrins are known as Group II late embryogenesis abundant proteins. Their high hydrophilicity and thermostability suggest that they may be structure stabilizers with detergent and chaperone-like properties. They are localised in the nucleus, cytoplasm, and plasma membrane. We have recently found putative dehydrins in the mitochondria of some cereals in response to cold. It is not known whether dehydrin-like proteins accumulate in plant mitochondria in response to stimuli other than cold stress.

Results

We have found five putative dehydrins in the mitochondria of winter wheat, rye and maize seedlings. Two of these polypeptides had the same molecular masses in all three species (63 and 52 kD) and were thermostable. Drought, freezing, cold, and exogenous ABA treatment led to higher accumulation of dehydrin-like protein (dlp) 63 kD in the rye and wheat mitochondria. Protein 52 kD was induced by cold adaptation and ABA. Some accumulation of these proteins in the maize mitochondria was found after cold exposition only. The other three proteins appeared to be heat-sensitive and were either slightly induced or not induced at all by all treatments used.

Conclusions

We have found that, not only cold, but also drought, freezing and exogenous ABA treatment result in accumulation of the thermostable dehydrins in plant mitochondria. Most cryotolerant species such as wheat and rye accumulate more heat-stable dehydrins than cryosensitive species such as maize. It has been supposed that their function is to stabilize proteins in the membrane or in the matrix. Heat-sensitive putative dehydrins probably are not involved in the stress reaction and adaptation of plants.     

Proteins of cotyledons of mature horse chestnut seeds

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.     

Dehydrin stress proteins in <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. needles under conditions of extreme climate of Yakutia

This is the first study to investigate stress proteins dehydrins with the use of specific antibodies in the Scots pine (Pinus sylvestris L.) needles and their changes in the annual cycle under extreme climate of Yakutia. No pronounced polymorphism of major dehydrins (14–15 and 66 kDa) has been found during the winter dormancy period of P. sylvestris. A clear correlation between the seasonal variations in dehydrins and changes in the water content in needles was revealed. Consistently high levels of dehydrins was retained throughout the period of low negative temperatures. It is assumed that dehydrins can participate in the formation of P. sylvestris L. resistance to the permafrost conditions.     

Photosynthetic and biochemical characterization of in vitro-derived African violet (Saintpaulia ionantha H. Wendl) plants to ex vitro conditions

African violet (Saintpaulia ionantha H. Wendl) is one of the most easily and commonly tissue-cultured ornamental plants. Despite this, there are limited reports on photosynthetic capacity and its impact on the plant quality during acclimatization. Various growth, photosynthetic and biochemical parameters and activities of antioxidant enzymes and dehydrins of micropropagated plants were assessed under three light intensities (35, 70, and 100 µmol m^?2 s^?1 photosynthetic photon flux density – PPFD). Fresh and dry plant biomass, plant height, and leaf area were optimal with high irradiance (70–100 µmol m^?2 s^?1 PPFD). Chlorophyll and carotenoid contents and net photosynthesis were optimal in plants grown under 70 µmol m^?2 s^?1 PPFD. Stomatal resistance, malondialdehyde content, and F_v/F_m values were highest at low light irradiance (35 µmol m^?2 s^?1 PPFD). The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, increased as light irradiance increased, signaling that high light irradiance was an abiotic stress. The accumulation of 55, 33, and 25 kDa dehydrins was observed with all light treatments although the expression levels were highest at 35 µmol m^?2 s^?1 PPFD. Irradiance at 70 µmol m^?2 s^?1 PPFD was suitable for the acclimatization of African violet plants. Both low and high irradiance levels (35 and 100 µmol m^?2 s^?1 PPFD) induced the accumulation of antioxidants and dehydrins in plants which reveals enhanced stress levels and measures to counter it.     

Dehydrins in Buds of Main Birch Species under Conditions of Karelia

The composition and seasonal dynamics of stress proteins-dehydrins in the buds of the main birch species (downy birch (Betula pubescens Ehrh.), silver birch (B. pendula Roth)) and its varieties (Karelian birch (B. pendula var. carelica (Mercklin) Hämet-Ahti)), growing in northwest Russia (on the example of the Republic of Karelia) were investigated for the first time. It was shown that the level of low-molecular dehydrins, mainly with a molecular mass of 17 kD, is subjected to major seasonal changes, regardless of the specific features of the birch. The maximal level of 17 kD dehydrin was formed during the autumn preparation of plants to dormancy and was persistently preserved during the cold period of the year. The content of medium-molecular weight dehydrins of 66–69 kD was almost at the same level all year round. Significant inter-and intraspecific polymorphism of the major dehydrins of 17 and 66–69 kD in the buds of downy birch, silver birch, and Karelian birch during dormancy was not found. The significant similarity in the composition of total proteins and dehydrins, as well as the uniform nature of their seasonal changes, mainly 17 kD dehydrin, indicates the phylogenetic proximity and similar mechanisms of adaptation of the main species of the genus Betula L. to the temperate continental climate of Karelia.     

Dehydrin stress proteins in birch buds in regions with contrasting climate

Dehydrin stress proteins were identified in buds of silver birch (Betula pendula Roth) grown in regions with contrasting climate, Karelia and Central Yakutia, using specific antibodies. Two types of dehydrins present in the plant buds, proteins with average (56–73 kDa) and low (14–21 kDa) molecular weight, as well as 17-kDa dehydrin, were detected in all studied plants. The most sensitive to seasonal changes are 14- to 21-kDa dehydrins, the level of which, regardless of the region where the birch grows, significantly increased during the autumn–winter period. The intraspecific polymorphism of dehydrins was more pronounced in B. pendula grown under the sharply continental climate of Yakutia, which is probably due to the peculiarities of the adaptation of woody plants to the extremely low temperatures of the cryolitic zone.     

Dormancy development during cold hardening of in vitro cultured Malus domestica Borkh. plants in relation to their frost resistance and cryotolerance

The development of dormancy, frost resistance and cryotolerance of in vitro apple plants (Malus domestica Borkh.), cv. Greensleeves during their exposure to cold hardening was studied. In vitro cultures were cold hardened at 4°C under a short photoperiod up to 25?weeks. The dormancy status, non-structural saccharides, proline, water content and frost resistance were evaluated for optimization of cryopreservation. According to regrowth tests, in vitro cultures exhibited endogenous dormancy after the maximal frost resistance was reached. The highest regeneration ability of shoot tips after cryopreservation by encapsulation–dehydration method coincided with the period of the plant’s dormant state and maximum of frost resistance. All studied saccharides and proline exhibited the maximal values at the beginning of cold hardening and/or the dormancy phase. Contrary to the accumulation of saccharides and proline, water content showed the inverse time behaviour. According to these results, the cold hardening-induced endodormancy, high frost resistance and accumulation of saccharides and proline are the important prerequisites for the successful cryopreservation of shoot tips of in vitro grown apple plants.     

Survival types of high mountain plants under extreme temperatures

Extreme temperatures are a main factor limiting plant growth in high mountain habitats. During winter, the risk of frost damage is highest at windblown and often snow-free sites. During summer, actively growing plants are particularly endangered by episodic cold spells, but also by short-term overheating. The current review gives an overview of extreme temperatures in the European Alps and observations of temperature damage on plants in their natural habitats. Furthermore, seasonal time courses of frost and heat resistance derived from laboratory tests on different plant growth forms are presented. Study species were the cushion plants Silene acaulis, Minuartia sedoides, Saxifraga oppositifolia and Carex firma collected on wind-exposed ridges; the rosette plant Soldanella alpina collected on snow-protected sites, and three Sempervivum species collected in xerothermic habitats.