Dehydrin-like Proteins Research Articles

Plant dehydrins and dehydrin-like proteins: characterization and participation in abiotic stress response.

Abiotic stress has a significant impact on plant growth and development. It causes changes in the subcellular organelles, which, due to their stress sensitivity, can be affected. Cellular components involved in the abiotic stress response include dehydrins, widely distributed proteins forming a class II of late embryogenesis abundant protein family with characteristic properties including the presence of evolutionarily conserved sequence motifs (including lysine-rich K-segment, N-terminal Y-segment, and often phosphorylated S motif) and high hydrophilicity and disordered structure in the unbound state. Selected dehydrins and few poorly characterized dehydrin-like proteins participate in cellular stress acclimation and are also shown to interact with organelles. Through their functioning in stabilizing biological membranes and binding reactive oxygen species, dehydrins and dehydrin-like proteins contribute to the protection of fragile organellar structures under adverse conditions. Our review characterizes the participation of plant dehydrins and dehydrin-like proteins (including some organellar proteins) in plant acclimation to diverse abiotic stress conditions and summarizes recent updates on their structure (the identification of dehydrin less conserved motifs), classification (new proposed subclasses), tissue- and developmentally specific accumulation, and key cellular activities (including organellar protection under stress acclimation). Recent findings on the subcellular localization (with emphasis on the mitochondria and plastids) and prospective applications of dehydrins and dehydrin-like proteins in functional studies to alleviate the harmful stress consequences by means of plant genetic engineering and a genome editing strategy are also discussed.

Correlation between dehydrin-like proteins and cold hardiness of grapevine

Winter survival of Vitis vinifera Linnaeus in cool climate viticultural areas can be jeopardized due to inadequate cold hardiness. Dehydrins are a family of proteins commonly found in plant tissue in response to dehydration stress and cold exposure. To determine their presence and relationship to cold hardiness in overwintering grapevines, compound buds of V. vinifera cv. Sauvignon blanc were sampled from a commercial vineyard every 2–3 weeks throughout the 2016–2017 winter. Proteins were extracted and separated by SDS–PAGE, and potential dehydrins were immunoblotted with a commercial antibody raised against the dehydrin K-segment consensus sequence. Six protein bands were identified in four Sauvignon blanc clones at 23, 26, 35, 41, 48, and 90 kDa, showing a serological relation to dehydrins due to their reaction with the K-segment antibody. The bands at 23, 41, 48, and 90 kDa were confirmed as dehydrins following trypsin digestion and liquid chromatography with tandem mass spectrometry with Mascot analysis. Their fluctuations throughout the dormant season were quantified by immunoblotting, and three patterns emerged, the 23, 26, and 35 kDa proteins peaked immediately prior to deacclimation; the 41 and 48 kDa proteins peaked during maximum hardiness and decreased towards deacclimation, while the 90 kDa plateaued during the same period. Maximum hardiness and relative dehydrin band intensity were positively correlated ( p < 0.050) for all but the 23 kDa protein. The variation in accumulation patterns and relationships to cold hardiness indicates that these dehydrin proteins are likely regulated by different molecular processes and could play different roles in cryoprotection throughout dormancy.

Dehydration Sensitivity at the Early Seedling Establishment Stages of the European Beech (Fagus sylvatica L.)

Shortage of water is a limiting factor for the growth and development of plants, particularly at early developmental stages. We focused on the European beech (Fagus sylvatica L.), which produces seeds and further seedlings in large intervals of up to ten years. To explore the beech seedling establishment process, six stages referring to embryo expansion were studied to determine sensitivity to dehydration. The characterization of the response of elongating embryonic axes and cotyledons included a viability test before and after dehydration and measurement of the amounts of electrolyte leakage, concentration, and arrangement of storage materials, changes in chaperone proteins related to water deficit, and accumulation of hydrogen peroxide and superoxide anion radicals. Elongating embryonic axes and cotyledons differed in water content, dehydration rates, membrane permeability before and after dehydration, protein, and lipid decomposition pattern, and amount of 44-kDa dehydrin and 22-kDa small heat shock protein (sHSP). Protruding embryonic axes were more sensitive to dehydration than cotyledons, although dehydration caused transient reinduction of three dehydrin-like proteins and sHSP synthesis, which accompany desiccation tolerance. Extended deterioration, including overproduction of hydrogen peroxide and depletion of superoxide anion radicals, was reported in dehydrated embryonic axes longer than 10 mm characterized by highly elevated cellular leakage. The apical part elongating embryonic axes consisting of the radicles was the most sensitive part of the seed to dehydration, and the root apical meristem area was the first to become inviable. The effects of severe dehydration involving ROS imbalance and reduced viability in beech seedlings with embryonic axes longer than 10 mm might help to explain the difficulties in beech seedling establishment observed in drought-affected environments. The conversion of environmental drought into climate-originated oxidative stress affecting beech seedling performance is discussed in this report.

Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins.

Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation.

Salinity–induced modulations in the protective defense system and programmed cell death in Nostoc muscorum

To study the biochemical adaptive responses of the blue green algae Nostoc muscorum to the salinity- induced stress they were exposed to various concentrations (5, 10, 15, 20 or 200 mM) of sodium chloride (NaCl). A dose-dependent inhibition of total protein content showed an adverse effect of NaCl on the growth of N. muscorum. Four-day treatment of NaCl (5–20 mM) progressively increased the content of the total peroxide with subsequent increase of the superoxide dismutase (SOD) activity, proline and total phenol content only up to 10 mM NaCl. Higher concentrations of NaCl caused significant decrease in both the enzymatic and non-enzymatic antioxidants. Induction of two polypeptides of ~29.10 and 40.15 kD as well as upregulation of many polypeptides as compared to control indicates the induction of SOD and dehydrin-like proteins, which supports the theory of adaptation against the salt stress. Furthermore, adaptation of N. muscorum to lower concentrations (5–20 mM) of NaCl was also confirmed by no fragmentation of DNA while DNA fragmentation indicating programmed cell death (PCD) could only be seen at 200 mM NaCl for 12 hours. We hypothesized that proline may confer a positive role to combat salinity stress and the same was confirmed by treatment of the test blue green algae with exogenous proline (1 and 10 μM). The results exhibited 16% reduction in the level of total peroxides, which is a well known oxidative stress marker in the 10 μM proline-treated NaCl group as compared to direct exposure to NaCl.

Identification and evolutionary relationships of partial gene sequences from dehydrin group in three species of cacti

Dehydrins or Group 2 Late Embryogenesis Abundant(LEA) proteins play an important role in the response and adaptationto different types of abiotic stresses such as droughts, high salinityand low temperatures. Using PCR techniques, we identified threegene fragments that encoded dehydrin-like proteins in three cactispecies Opuntia ficus-indica (OpfiDHN-like), Leuchtenbergia principis(LepDHN-like) and Mammillaria bombycina (MabDHN-like).Bioinformatic sequence analysis showed an identity between 96 and97% with the Opuntia streptacantha dehydrin 1 (OpsDHN1) gene,demonstrating that the amplified fragments corresponded to dehydrin-like gene sequences, and that the designed oligonucleotideswere effective for similar gene amplification in different cacti genera.Multiple OpfiDHN-like, LepDHN-like and MabDHN-like alignmentsshowed that they possessed three repetitions of the conservedK segment. Also, a histidine rich motif was found, which is believedto facilitate the binding of these proteins with metal ions that probablyevolved differently in the Opuntioidea and Cactoidea subfamiliesof the Cactaceae family. Bioinformatic tools demonstratedthat each of the three partial amino acid sequences corresponded toacidic, highly hydrophilic, and disordered protein fragments, whichare characteristics of dehydrin proteins. Phylogenetic analysis usingmaximum parsimony, indicated that cacti dehydrins-like proteinswere monophyletic, as well as those of other families.

Insights on germinability and desiccation tolerance in developing neem seeds (Azadirachta indica): Role of AOS, antioxidative enzymes and dehydrin-like protein

The germinability and desiccation tolerance (DT) in developing seed are regulated by cellular metabolism involving active oxygen species (AOS) and protective proteins during maturation drying. The aim of the present investigation was to unravel the functions of AOS (superoxide, H2O2 and OH-radical), antioxidative enzymes (SOD, CAT and APX) and dehydrin-like proteins in regulating the germinability and DT in undried and artificially desiccated developing neem seeds. Germination was first observed in seeds of 8 weeks after anthesis (waa) whereas DT was noticed from 9 waa. High levels of superoxide in undried and artificially desiccated seeds of 9 waa were rapidly declined up to 15 waa with simultaneous increase in levels of SOD (quantitative and isoenzymes) that dismutates superoxide with corresponding formation and accumulation of H2O2. Activities and isoenzymes of APX and CAT were promoted in seeds from 9 to 12 waa. Intensity of dehydrin-like proteins increased as development progressed in seeds with higher intensities in slow dried (SD) seeds. Desiccation modulated the metabolism for the acquisition of germinability and DT in the developing neem seeds from 8 to 15 waa by altering the levels of superoxide, H2O2 and OH-radical those possibly act as signalling molecules for reprogramming protective proteins. Desiccation mediated the expression of new bands of SOD and APX in undried as well as SD seeds during 9–12 waa but the bands were more intense in SD seeds. The superoxide and H2O2-regulated intensity of dehydrin-like protein in SD seeds further validated our conclusion.

Functional characterization of a dehydrin protein from Fagus sylvatica seeds using experimental and in silico approaches

A strong increase in the level of dehydrin/response ABA transcripts expression reported from the 14th week after flowering coincident with the accumulation of 26 and 44 kDa dehydrins in the embryonic axes of developing beech (Fagus sylvatica L.) seeds. Both transcript and protein levels were strongly correlated with maturation drying. These results suggest that the 44-kDa dehydrin protein is a putative dimer of dehydrin/response ABA protein migrating as a 26-kDa protein. Dehydrins and dehydrin-like proteins form large oligomeric complexes under native conditions and are shown as several spots differing in pI through isoelectrofocusing analyses. Detailed prediction of specific sites accessible for various post-translational modifications (PTMs) in the dehydrin/response ABA protein sequence revealed sites specific to acetylation, amidation, glycosylation, methylation, myristoylation, nitrosylation, O-linked β-N-acetylglucosamination and Yin-O-Yang modification, palmitoylation, phosphorylation, sumoylation, sulfation, and ubiquitination. Thus, these results suggest that specific PTMs might play a role in switching dehydrin function or activity, water binding ability, protein-membrane interactions, transport and subcellular localization, interactions with targeted molecules, and protein stability. Despite the ability of two Cys residues to form a disulfide bond, –SH groups are likely not involved in dimer arrangement. His-rich regions and/or polyQ-tracts are potential candidates as spatial organization modulators. Dehydrin/response ABA protein is an intrinsically disordered protein containing low complexity regions. The lack of a fixed structure and exposition of amino acids on the surface of the protein structure enhances the accessibility to 40 predicted PTM sites, thereby facilitating dehydrin multifunctionality, which is discussed in the present study.

Multiple Subcellular Localizations of Dehydrin-like Proteins in the Embryonic Axes of Common Beech (Fagus sylvatica L.) Seeds During Maturation and Dry Storage

The accumulation and localization of dehydrin-like proteins were analyzed in the embryonic axes of beech (Fagus sylvatica L.) seeds during maturation between the 12th and 19th week after flowering and in the dry state after 2 years of storage. Three dehydrin-like proteins were reported using an antibody specific to the K-segment, and their subcellular localization was investigated using an immunocytochemistry approach. All dehydrin-like proteins were phosphorylated, and two, with molecular masses of 26 and 44 kDa, accumulated in mature seeds. During seed maturation, dehydrin-like proteins were frequently detected inside the round or segmented nuclei, along the cell plasma membrane and associated with small vesicles. Moreover, they were found to be attached to the mitochondrial and amyloplast envelopes. These proteins were also located in the cytosol and were associated with membrane structures throughout the cytoplasm. Conversely, in the embryonic axes of stored seeds, fewer specific locations were found; dehydrin-like proteins were associated mostly with amyloplasts, and were detected to a lesser extent in the nuclei, vacuoles, and other cytoplasmic structures. Bioinformatic tools were used to predict the putative beech dehydrin sites that interact with DNA, proteins, and lipid membranes to highlight the reported subcellular locations. Here, we present and discuss the possible roles of dehydrin-like proteins in seeds with respect to their subcellular localizations.

Comparative assessment of metabolic responses to cold acclimation and deacclimation in annual bluegrass and creeping bentgrass

The level of freezing tolerance achieved by perennial grasses and their capacity to remain cold acclimated throughout winter is critical for their winter survival. Research is necessary to better understand the metabolic processes associated with cold acclimation and deacclimation. This study was conducted to assess the response of creeping bentgrass (Agrostis stolonifera L.) (CB) and two ecotypes of annual bluegrass (Poa annua L.), one freezing-tolerant (AB-T) and one freezing-sensitive (AB-S), to cold acclimation and deacclimation. Following cold acclimation, plants were exposed to 8°C for 0.5, 1, 3, and 5 d to induce deacclimation. Plants were assessed for their freezing tolerance (lethal temperature resulting in 50% mortality, LT50), their concentrations in soluble sugars and amino acids, and for changes in dehydrin-like proteins. Fully acclimated CB achieved higher level of LT50 (−21.5°C) than AB-T (−19.8°C), followed by AB-S (−15.3°C). Total soluble sugars, mainly high molecular weight (HMW) fructans, accumulated in each species/ecotypes during cold acclimation with higher levels measured in CB. Dehydrin-like proteins were present in each species but they were mostly constitutive in AB while they were cold-inducible and linked with the LT50 in CB. Freezing tolerance decreased during deacclimation in each species. However, at each step of deacclimation, CB maintained higher freezing tolerance than AB in which the LT50 reached the low level of non-acclimated plants after five days at 8°C. The depletion of HMW fructans observed during deacclimation followed a similar trend, with higher levels remaining at the end in CB, followed by AB-T and then by AB-S. The higher susceptibility of AB to winter injury is associated with both its lower cold acclimation capacity and deacclimation sensitivity.

Dehydrin-like Proteins in the Necrotrophic Fungus Alternaria brassicicola Have a Role in Plant Pathogenesis and Stress Response

In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease.

A novel dehydrin-like protein from Aspergillus fumigatus regulates freezing tolerance

We recently reported on DprA and DprB, dehydrin-like proteins involved in the protection against oxidative, osmotic and pH stress in Aspergillus fumigatus. A third dehydrin-like gene, DprC, was detected in the genome of the filamentous fungus and repressed during conidial germination. The deletion mutant was impaired in freezing tolerance. The DprC-mediated response was dependent on the SakA MAP kinase pathway, and accordingly, SakA phosphorylation was induced by cold shock. When fused to eGFP, the protein was associated with the vacuoles.

Dehydrin metabolism is altered during seed osmopriming and subsequent germination under chilling and desiccation in Spinacia oleracea L. cv. Bloomsdale: Possible role in stress tolerance

Osmopriming improves seed germination performance as well as stress tolerance. To understand the biochemistry of osmopriming-induced seed stress tolerance, we investigated dehydrin (DHN) accumulation patterns at protein and transcript level (determined by immunoblotting and qPCR) during priming, and subsequent germination under optimal and stress conditions (i.e. chilling and desiccation) in spinach ( Spinacia oleracea L. cv. Bloomsdale) seeds. Our data indicate enhanced germination performance of primed seeds is accompanied by increased accumulation of three dehydrin-like proteins (DLPs): 30, 26, and 19-kD. Moreover, 30, 26 and 19-kD DLPs that first only transiently accumulated during priming re-accumulated in response to stresses, suggesting an evidence for ‘cross-tolerance’, which is initially induced by priming and later recruited during post-priming germination under stresses. Study with CAP85, a spinach DHN, corroborates above observations at the gene-expression and protein accumulation level. Additionally, our results suggest that during seed germination and seedling establishment, CAP85 expression may be regulated by the interplay of two factors: seedling development and stress responses. In conclusion, our data suggest that 30, 26, and 19-kD dehydrin-like proteins and CAP85 may be used as potential biochemical/molecular markers for priming-induced stress tolerance in ‘Bloomsdale’ spinach.

A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogenAspergillus fumigatus

During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.

Levels of ABA, its precursors and dehydrin-like proteins during mangrove leaf development and senescence

—Abscisic acid (ABA) and dehydrin proteins are thought to confer tolerance to plant tissue under physiological stress and drought. Rhizophora mucronata, a true mangrove species, is subjected to physiological drought from fluctuating high saline conditions where leaf loss or senescence is considered a possible regulation mechanism to combat stress. Levels of ABA and proteins that cross reacted with an anti – dehydrin antibody were assessed through development with the aim of correlating these factors to physiological water stress or salinity stress in R. mucronata leaves. Younger leaves showed lower levels of ABA than mature and senescing leaves. In situ production and translocation from mature to younger leaves may contribute to these observations. The presence of ABA in senescing leaves is thought to be due to the presence of low levels of physiological activity. Proteins detected by anti–dehydrin antibody require cDNA confirmation, but the visibly increasing intensity of a band at ~64kDa through development suggests potential correlation to drought or salinity stress which is expected to be maximal in maturing leaves. The absence of the dehydrin–like proteins in senescing leaves is postulated to be due to the lack of energy investment to synthesise these proteins in dying leaves.Keywords: mangroves, Rhizophora mucronata, ABA, leaves, senescence, dehydrins.

Semi-viviparous embryo development and dehydrin expression in the mangrove Rhizophora mucronata Lam.

Rhizophora mucronata Lam. is a tropical mangrove with semi-viviparous (cotyledon body protrusion before shedding), non-quiescent and non-desiccating (recalcitrant) seeds. As recalcitrance has been thought to relate to the absence of desiccation-related proteins such as dehydrins, we for the first time systematically described and classified embryogenesis in R. mucronata and assessed the presence of dehydrin-like proteins. Embryogenesis largely follows the classic pattern till stage eight, the torpedo stage, with the formation of a cotyledonary body. Ovule and embryo express radical adaptations to semi-vivipary in the saline environment: (1) A large, highly vacuolated and persistent endosperm without noticeable food reserves that envelopes the developing embryo. (2) Absence of vascular tissue connections between embryo and maternal tissue, but, instead, transfer layers in between endosperm and integument and endosperm and embryo. Dehydrin-like proteins (55–65 kDa) were detected by the Western analysis, in the ovules till stage 10 when the integuments are dehisced. An additional 50 kDa band was detected at stages 6–8. Together these results suggest a continuous flow of water with nutrients from the integument via the endosperm to the embryo, circumventing the vascular route and probably suppressing the initially induced dehydrin expression.

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.

BackgroundDehydrins 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.ResultsAll the analyzed plant species showed constitutive accumulation of thermostable mitochondrial putative dehydrins ranging from 50 to 70 kDa. The mitochondrial dehydrin-like proteins observed in cauliflower and Arabidopsis ranged from 10 to 100 kDa and in lupin imbibed seeds and hypocotyls - from 20 to 90 kDa. Cold treatment increased mainly the accumulation of 10-100 kDa cauliflower and Arabidopsis dehydrin-like proteins, in the patterns different in cauliflower leaf and inflorescence mitochondria. However, in lupin mitochondria, cold affected mainly 25-50 kDa proteins and seemed to induce the appearance of some novel dehydrin-like proteins. The influence of frost stress on cauliflower leaf mitochondrial dehydrin- like proteins was less significant. The impact of heat stress was less significant in lupin and Arabidopsis than in cauliflower inflorescence mitochondria. Cauliflower mitochondrial dehydrin-like proteins are localized mostly in the mitochondrial matrix; it seems that some of them may interact with mitochondrial membranes.ConclusionsAll the results reveal an unexpectedly broad spectrum of dehydrin-like proteins accumulated during some abiotic stress in the mitochondria of the plant species analyzed. They display only limited similarity in size to those reported previously in maize, wheat and rye mitochondria. Some small thermolabile dehydrin-like proteins were induced under stress conditions applied and therefore they are likely to be involved in stress response.

Cold-hardiness of olive (Olea europaeaL.) cultivars in cold-acclimated and non-acclimated stages: seasonal alteration of antioxidative enzymes and dehydrin-like proteins

SUMMARYSeasonal patterns of antioxidative enzymes and proteins and their relations to cold-hardiness of nine olive (Olea europaeaL.) cultivars (Ascolona, Domat, Gemlik, Hojoblanca, Lecquest, Manzanilla, Meski, Samanli and Uslu) are documented in the current study. Fully expanded, uniformly sized leaves from 2-year-old shoots of the cultivars were collected from 20-year-old trees in cold-acclimated (CA, in January) and non-acclimated (NA, in July) stages. Leaf samples were exposed to low temperature at 4, −5, −10 and −20°C for 12 h to determine their cold-hardiness (LT50; assessed by electrolyte leakage). Cold-acclimation produced an increase in freezing tolerance of all cultivars (by lowering LT50). Domat and Lecquest were found to have the highest cold-hardiness among the nine cultivars investigated. Ascolona, Gemlik, Hojoblanca had moderate cold-hardiness, while Samanli, Meski, Uslu and Manzanilla were more sensitive. Activities of catalase (CAT: EC 1·11·1·6), ascorbate peroxidase (APX: EC 1·11·1·11) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase significantly varied depending on the cold-acclimation stage and the cold-hardiness level of the cultivars. Activities of the three antioxidative enzymes and total soluble proteins (TSP) were higher in the CA stage than in the NA stage. Although no accumulation of major polypeptides, except a 23 kDa protein, was detected either in CA samples or NA samples by SDS-PAGE, anti-dehydrin immunoblots revealed that the 43 and 23 kDa polypeptides were detectable during cold-acclimation of olive cultivars. Accumulation of both 43 and 23 kDa dehydrin was significantly higher in the CA stage than in the NA stage in all cultivars. Accumulation of 43 kDa dehydrin was correlated with cold-hardiness of the cultivars, while 23 kDa dehydrin was considered as cultivar-dependent since its accumulation was not parallel to LT50values of the cultivars. Indeed, the tissues of cvs Domat, Lecquest, Ascolona, Hojoblanca and Gemlik were found to enhance the structural stability of cellular membranes in the CA stage by increasing both the activity of such enzymes as CAT, APX and NADPH oxidase to activate the antioxidative systems and the expression of 43 kDa dehydrins.

Detection and subcellular localization of dehydrin-like proteins in quinoa (Chenopodium quinoa Willd.) embryos

The aim of this study was to characterize the dehydrin content in mature embryos of two quinoa cultivars, Sajama and Baer La Unión. Cultivar Sajama grows at 3600-4000 m altitude and is adapted to the very arid conditions characteristic of the salty soils of the Bolivian Altiplano, with less than 250 mm of annual rain and a minimum temperature of -1 degrees C. Cultivar Baer La Unión grows at sea-level regions of central Chile and is adapted to more humid conditions (800 to 1500 mm of annual rain), fertile soils, and temperatures above 5 degrees C. Western blot analysis of embryo tissues from plants growing under controlled greenhouse conditions clearly revealed the presence of several dehydrin bands (at molecular masses of approximately 30, 32, 50, and 55 kDa), which were common to both cultivars, although the amount of the 30 and 32 kDa bands differed. Nevertheless, when grains originated from their respective natural environments, three extra bands (at molecular masses of approximately 34, 38, and 40 kDa), which were hardly visible in Sajama, and another weak band (at a molecular mass of approximately 28 kDa) were evident in Baer La Unión. In situ immunolocalization microscopy detected dehydrin-like proteins in all axis and cotyledon tissues. At the subcellular level, dehydrins were detected in the plasma membrane, cytoplasm and nucleus. In the cytoplasm, dehydrins were found associated with mitochondria, rough endoplasmic reticulum cisternae, and proplastid membranes. The presence of dehydrins was also recognized in the matrix of protein bodies. In the nucleus, dehydrins were associated with the euchromatin. Upon examining dehydrin composition and subcellular localization in two quinoa cultivars belonging to highly contrasting environments, we conclude that most dehydrins detected here were constitutive components of the quinoa seed developmental program, but some of them (specially the 34, 38, and 40 kDa bands) may reflect quantitative molecular differences associated with the adaptation of both cultivars to contrasting environmental conditions.

Changes in late embryogenesis abundant proteins and a small heat shock protein during storage of beech ( Fagus sylvatica L.) seeds

Beech seed physiology, including the effect of stress proteins like late embryogenesis abundant (LEA) and small heat shock proteins (sHSP) on viability during storage, is not fully understood. Four lots of beech ( Fagus sylvatica L.) seeds have been stored for 1, 4, 6 and 8 years at −10 °C and 8–9% moisture content (MC). Under these conditions, the germination capacity ranges from 81.5% to 100% in the youngest seeds. However, the seeds decrease in vigour with prolonged time of storage. Dehydrins and dehydrin-like proteins were identified both in cotyledons and embryonic axes of the dry stored seeds. In general, decreased contents of LEA proteins as well as reduced content of total soluble protein were detected during prolonged storage. The contents of soluble proteins in embryonic axes and nearly all detected dehydrins and dehydrin-like proteins were correlated with germination capacity. Moreover a sHSP with molecular mass of approximately 22 kDa was identified. 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.