Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress

Background  

Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress.     

Mitochondrial Low-Molecular-Weight Heat-Shock Proteins and the Tolerance of Cereal Mitochondria to Hyperthermia

Relationships between the appearance of low-molecular-weight heat-shock proteins (LMW HSPs) in maize, winter wheat, and winter rye mitochondria and the tolerance of the mitochondria to hyperthermia (42°C, 3 h) were studied using one-dimensional SDS-PAGE, immunochemical methods, and polarography. Heat shock inhibited respiration to a greater extent in the wheat and rye than in the maize mitochondria. A single 20-kD LMW HSP was found both inside and on the surface of mitochondria isolated from heat-treated wheat and rye seedlings. After heating maize seedlings, two LMW HSPs (28 and 23 kD) appeared inside the mitochondria, and three proteins (22, 20, and 19 kD) appeared on their surface. We suppose that the latter three proteins play an essential role in the protection of mitochondria from hyperthermic damage. It seems likely that the diversity of the hyperthermia-induced LMW HSPs in plant mitochondria affects their thermal stability.     

Photoperiodic induction of dormancy and freezing tolerance in Betula pubescens. Involvement of ABA and dehydrins

In many woody plants photoperiod signals the initiation of dormancy and cold acclimation. The photoperiod-specific physiological and molecular mechanisms have remained uncharacterised. The role of abscisic acid (ABA) and dehydrins in photope-riod-induced dormancy and freezing tolerance was investigated in birch, Betula pubescens Ehrh. The experiments were designed to investigate if development of dormancy and freezing tolerance under long-day (LD) and short-day (SD) conditions could be affected by manipulation of the endogenous ABA content, and if accumulation of dehydrin-like proteins was correlated with SD and/or the water content of the buds. Experimentally, the internal ABA content was increased by ABA application and by water stress treatment under LD, and decreased by blocking the synthesis of ABA with fluridone under SD. Additionally, high humidity (95% RH) was applied to establish if accidental water stress was involved in SD. ABA content was monitored by gas chromatography-mass spectrometry with selective ion monitoring (SIM). Short days induced a transient increase in ABA content, which was absent in 95% RH, whereas fluridone treatment decreased ABA. Short days induced a typical pattern of bud desiccation and growth cessation regardless of the treatment, and improved freezing tolerance except in the fluridone treatment. ABA content of the buds was significantly increased after spraying ABA on leaves and after water stress, treatments that did not induce cessation of growth and dormancy, but improved freezing tolerance. In addition to several constitutively produced dehydrins, two SD-specific proteins of molecular masses 34 and 36 kDa were found. Photoperiod- and experimentally-induced alterations in ABA contents affected freezing tolerance but not cessation of growth and dormancy. Therefore, involvement of ABA in the photoperiodic control of cold acclimation is more direct than in growth cessation and dormancy. As the typical desiccation pattern of the buds was found in all SD plants, and was not directly related to ABA content or to freezing tolerance, this pattern characterises the onset of photo-period-induced growth cessation and dormancy. The results provide evidence for the existence of various constitutively and two photoperiod-induced dehydrins in buds of birch, and reveal characteristics of dormancy and freezing tolerance that may facilitate further investigations of photoperiodic control of growth in trees.     

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.     

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.     

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.     

Differential accumulation of dehydrin-like proteins by abiotic stresses in Deschampsia antarctica Desv.

Dehydrins are proteins that accumulate during environmental stresses leading to cell dehydration. Deschampsia antarctica is one of the two vascular plants that have colonized the Maritime Antarctic. This plant is usually exposed to cold, salt and desiccating winds in the field. We proposed that among the factors that allow D. antarctica to survive the harsh environmental conditions is the presence of dehydrins. We studied the accumulation of dehydrins by abscisic acid (ABA), dehydration, NaCl and low osmotic potential. Western blots using an anti-dehydrin antibody revealed a complex pattern of dehydrin-like proteins (DLPs) accumulation in the different treatments. DLPs with apparent molecular weight of 58, 57, 55, 53, 48, 42, 32, 30, 28 and 25 kDa were detected in the different treatments. DLPs accumulation was associated with a decrease in the relative water content (RWC) of the plants. These results suggest that DLPs accumulation could contribute to explain how D. antarctica can survive under adverse Antarctic conditions.     

Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus).

The level of three major polypeptides of 65, 60, and 14 kD increased in response to chilling unit accumulation in floral buds of a woody perennial, blueberry (Vaccinium, section Cynaococcus). The level of the polypeptides increased most dramatically within 300 h of chilling and decreased to the prechilling level with the initiation of budbreak. Cold-hardiness levels were assessed for dormant buds of Vaccinium corymbosum and Vaccinium ashei after different chilling treatments until the resumption of growth. These levels coincided with the level of the chilling-responsive polypeptides. Like some other previously described cold-induced proteins in annual plants, the level of the chilling-induced polypeptides also increased in leaves in response to cold treatment; the chilling-induced polypeptides were heat stable, resisting aggregation after incubation at 95 degrees C for 15 min. By fractionating bud proteins first by isoelectric point (pI) and then by molecular mass, the pI values of the 65- and 60-kD polypeptides were found to be 7.5 to 8.0 and the pI value of the 14-kD polypeptide was judged to be 8.5. Purification of the 65- and 60-kD polypeptides, followed by digestion with endoproteinase Lys-C and sequencing of selected fragments, revealed similarities in amino acid composition between the 65- and 60-kD polypeptides and dehydrins. Indeed, antiserum to the lysine-rich consensus sequence EKKGIMDKIKEKLPG of dehydrin proteins cross-reacted to all three of the major chilling-responsive polypeptides of blueberry, identifying these as dehydrins or dehydrin-like proteins.     

Structure of the <Emphasis Type="Italic">TADHN</Emphasis> gene for dehydrin-like protein of soft wheat and activation of its expression by ABA and 24-epibrassinolide

The structure of the cloned fragment of wheat (Triticum aestivum L.) TADHN gene encoding dehydrin-like protein was examined. A comparative analysis of nucleotide and deduced amino acid sequences revealed a high homology of this fragment with sequences of the barley dhn8 gene and wheat wcor gene family. In deduced amino acid sequence of the TADHN fragment, a 15-residue region EKKGFLEKIKEKLPG was found, which corresponded to a highly conserved K-segment of dehydrins. Wheat seedling treatment with 3.7 μM ABA and 0.4 μM 24-epibrassinolide exerted similar stimulatory effects on expression of the TADHN gene, which indicates the involvement of dehydrins in the protective action of these phytohormones in wheat plants.     

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。     

Antifreeze protein accumulation in freezing-tolerant cereals

Freezing-tolerant plants withstand extracellular ice formation at subzero temperatures. Previous studies have shown that winter rye ( Secale cereale L.) accumulates proteins in the leaf apoplast during cold acclimation that have antifreeze properties and are similar to pathogenesis-related proteins. To determine whether the accumulation of these antifreeze proteins is common among herbaceous plants, we assayed antifreeze activity and total protein content in leaf apoplastic extracts from a number of species grown at low temperature, including both monocotyledons (winter and spring rye, winter and spring wheat, winter barley, spring oats, maize) and dicotyledons (spinach, winter and spring oilseed rape [canola], kale, tobacco). Apoplastic polypeptides were also separated by SDS-PAGE and immunoblotted to determine whether plants generally respond to low temperature by accumulating pathogenesis-related proteins. Our results showed that significant levels of antifreeze activity were present only in the apoplast of freezing-tolerant monocotyledons after cold acclimation at 5/2⁰C. Moreover, only a closely related group of plants, rye, wheat and barley, accumulated antifreeze proteins similar to pathogenesis-related proteins during cold acclimation. The results indicate that the accumulation of antifreeze proteins is a specific response that may be important in the freezing tolerance of some plants, rather than a general response of all plants to low temperature stress.     

Winter rye antifreeze activity increases in response to cold and drought, but not abscisic acid

Antifreeze activity increases in winter rye ( Secale cereale L.) during cold acclimation as the plants accumulate antifreeze proteins (AFPs) that are similar to glucanases, chitinases and thaumatin-like proteins (TLPs) in the leaf apoplast. In the present work, experiments were conducted to assess the role of drought and abscisic acid (ABA) in the regulation of antifreeze activity and accumulation of AFPs. Antifreeze activity was detected as early as 24 h of drought treatment at 20°C and increased as the level of apoplastic proteins increased. Apoplastic proteins accumulated rapidly under water stress and reached a level within 8 days that was equivalent to the level of apoplastic proteins accumulated when plants were acclimated to cold temperature for 7 weeks. These drought-induced apoplastic proteins had molecular masses ranging from 11 to 35 kDa and were identified as two glucanases, two chitinases, and two TLPs, by using antisera raised against cold-induced rye glucanase, chitinase, and TLP, respectively. Apoplastic extracts obtained from plants treated with ABA lacked the ability to modify the growth of ice crystals, even though ABA induced the accumulation of apoplastic proteins within 4 days to a level similar to that obtained when plants were either drought-stressed for 8 days or cold-acclimated for 7 weeks. These ABA-induced apoplastic proteins were identified immunologically as two glucanases and two TLPs. Moreover, the ABA biosynthesis inhibitor fluridone did not prevent the accumulation of AFPs in the leaves of cold-acclimated rye plants. Our results show that cold acclimation and drought both induce antifreeze activity in winter rye plants and that the pathway regulating AFP production is independent of ABA.     

Dehydrins associated with the development of frost resistance of Asian white birch

Seasonal changes in the content of dehydrins in Asian white birch (Betula platyphylla Sukacz.) growing under extreme cold conditions of Eastern Siberia (Central Yakutia) were studied for the first time by SDS-PAGE and immunoblotting. Several polypeptides, including putative storage proteins, which content was higher in winter than in other periods, were observed. Intraspecies polymorphism of dehydrins was detected during plant dormancy. The two groups of dehydrins were found: dehydrins with mol wts of 56-73 kD, which were present year-round, and dehydrins with mol wts of 15–21 kD, evidently related to the development of frost resistance because they were absent in summer but present in large amounts in winter. Under low winter temperatures, the highest level of dehydrins coincided with the lowest content of water in buds, which was accompanied by increased plant frost resistance to the highest values.     

The comparison of proteins with immunochemical affinity to stress protein 310 kD in cytoplasmatic proteins of winter rye,winter wheat,Elymus and maize

The search for proteins, immunochemically related to winter rye CSP 310 among the native cytoplasmatic proteins of a number of cultivated cereals with different tolerance to low temperatures—maize, winter wheat and winter rye and the very low temperature tolerant wild grass—Elymus sibiricus was carried out. Western blotting showed that among the native cytoplasmatic proteins of all species investigated there are proteins immunochemically related to CSP 310 protein with molecular weights about 230 and about 140–110 kD. Proteins with molecular weights about 480 and 310 kD were found in significant amounts only in winter rye. In E. sibiricus proteins with molecular weights 380–320 kD were present but these were not present among the cytoplasmatic protein spectra of the other species. In each case the proteins immunochemically related to CSP 310 consisted of different combinations of two types of subunits.     

Accumulation of soluble sugars, heat-stable proteins and dehydrins in cryopreservation of protocorm-like bodies ofDendrobium candidumby the air-drying method

Protocorm-like bodies (PLBs) of Dendrobium candidum were successfully cryopreserved by the air-drying method. The optimal water content before freezing seemed to be at the range of 0.1 g H₂O/g DW (11 % on fresh weight basis) to 0.5 g H₂O/g DW (33 % on fresh weight basis). Changes in soluble sugars, heat-stable proteins and dehydrins during desiccation of PLBs were analyzed. Extensive accumulation of soluble sugars was observed at water content of about 7.2 g H₂O/g DW (after 24 h desiccation), and the sugars content did not increase further during the following desiccation. The amount of heat-stable protein increased significantly when water content decreased to 1.0 g H₂O/g DW (after approximately 66 h desiccation). Results from immunological detection showed that two bands of the heat-stable proteins with respective molecular masses of 28.7 and 34.3 kDa were dehydrins which appeared when water content dropped to 1.0 g H₂O/g DW. Therefore, it seemed that accumulation of dehydrins happened later than that of soluble sugars. Interestingly, exogenous ABA treatment of PLBs before desiccation could also induce the accumulation of soluble sugars, heat-stable proteins and dehydrins. The possible roles of these substances in the acquisition of dehydration and freezing tolerance were discussed.     

Expression of a dehydrin-like protein in maize seedlings germinated from seed exposed to freezing

Dehydrins are a family of heat-soluble, hydrophilic proteins that share a considerable degree of sequence homology. Their expression has been reported in numerous plant species in response to a multitude of environmental stresses including low temperature, freezing, and desiccation. It has also been established that exposing plant tissues to freezing temperatures generates desiccation stress. We observed differential accumulation of a dehydrin-like protein and corresponding mRNA in three-day-old maize (Zea mays L) seedlings germinated under favorable environmental conditions from seed that had been exposed to freezing temperatures during maturation. This represents the first documented situation in which a dehydrin-like protein differentially accumulates under favorable environmental conditions. We believe that the dehydrin-like protein and corresponding mRNA are synthesized de novo in seedlings that are germinated from seed that have been exposed to freezing in response to desiccation-like stress that persists under favorable environmental conditions resulting from freeze-induced damage sustained by the ungerminated embryo.     

Characterization of an 80-kDa dehydrin-like protein in barley responsive to cold acclimation

Barley ( Hordeum vulgare L.) exposed to low temperature increases its freezing tolerance. This increase has been associated with several metabolic changes caused by low temperature, including expression of dehydrins (DHN), a family of proteins induced by dehydration and cold acclimation. DHNs play an undetermined role in dehydration responses during freezing. We have studied the accumulation of an 80-kDa DHN-like protein (P-80) in barley under cold acclimation 6/4°C (day/night), postulating that it is localized in tissues where primary ice nucleation occurs. P-80 was absent in nonacclimated plants and was detectable after 48 h of cold acclimation, reaching a stable level after 6 days. P-80 decreased when plants were returned to 20–25°C. Drought, ABA and high temperature did not increase the levels of P-80, suggesting that its expression could be specifically regulated by cold. Immunolocalization by tissue printing and fresh cross sections of leaves showed the protein to be associated with vascular tissues and epidermis. The localization of P-80 is consistent with our hypothesis because vascular tissue and the epidermis are preferential ice nucleation zones during the onset of freezing. The differential accumulation of P-80 may have an adaptive value by participating in tolerance mechanisms during freeze-induced dehydration.     

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.     

Synthesis of Freezing Tolerance Proteins in Leaves, Crown, and Roots during Cold Acclimation of Wheat

Protein synthesis was studied in leaves, crown, and roots during cold hardening of freezing tolerant winter wheat (Triticum aestivum L. cv Fredrick and cv Norstar) and freezing sensitive spring wheat (T. aestivum L. cv Glenlea). The steady state and newly synthesized proteins, labeled with [³⁵S]methionine, were resolved by one- and two-dimensional polyacrylamide gels. The results showed that cold hardening induced important changes in the soluble protein patterns depending upon the tissue and cultivar freezing tolerance. At least eight new proteins were induced in hardened tissues. A 200 kilodalton (kD) (isoelectric point [pl] 6.85) protein was induced concomitantly in the leaves, crown, and roots. Two proteins were specifically induced in the leaves (both 36 kD, pl 5.55 and 5.70); three in the crown with M_r 150 (pl 5.30), 45 (pl 5.75), and 44 kD (pl > 6.80); and two others in the roots with M_r 64 (pl 6.20) and 52 kD (pl 5.55). In addition, 19 other proteins were synthesized at a modified rate (increased or decreased) in the leaves, 18 in the crown and 23 in the roots. Among the proteins induced or increased in hardened tissues, some were expressed at a higher level in the freezing tolerant cultivars than in the sensitive one, indicating a correlation between the synthesis and accumulation of these proteins and the degree of freezing tolerance. These proteins, suggested to be freezing tolerance proteins, may have an important role in the cellular adaptation to freezing.