Plant dehydrins: shedding light on structure and expression patterns of dehydrin gene family in barley

Dehydrins, an important group of late embryogenesis abundant proteins, accumulate in response to dehydration stresses and play protective roles under stress conditions. Herein, phylogenetic analysis of the dehydrin family was performed using the protein sequences of 108 dehydrins obtained from 14 plant species based on plant taxonomy and protein subclasses. Sub-cellular localization and phosphorylation sites of these proteins were also predicted. The protein features distinguishing these dehydrins categories were identified using various attribute weighting and decision tree analyses. The results revealed that the presence of the S motif preceding the K motif (Y_nSK_n, SK_n, and S_nKS) was more evident and the Y_nSK_n subclass was more frequent in monocots. In barley, as one of the most drought-tolerant crops, there are ten members of Y_nSK_n out of 13 HvDhns. In promoter regions, six types of abiotic stress-responsive elements were identified. Regulatory elements in UTR sequences of HvDhns were infrequent while only four miRNA targets were found. Furthermore, physiological parameters and gene expression levels of HvDhns were studied in tolerant (HV1) and susceptible (HV2) cultivars, and in an Iranian tolerant wild barley genotype (Spontaneum; HS) subjected to gradual water stress and after recovery duration at the vegetative stage. The results showed the significant impact of dehydration on dry matter, relative leaf water, chlorophyll contents, and oxidative damages in HV2 compared with the other studied genotypes, suggesting a poor dehydration tolerance, and incapability of recovering after re-watering in HV2. Under severe drought stress, among the 13 HvDhns genes, 5 and 10 were exclusively induced in HV1 and HS, respectively. The gene and protein structures and the expression patterns of HvDhns as well as the physiological data consistently support the role of dehydrins in survival and recovery of barley plants from drought particularly in HS. Overall, this information would be helpful for functional characterization of the Dhn family in plants.     

Expression analysis of dehydrin multigene family across tolerant and susceptible barley (Hordeum vulgare L.) genotypes in response to terminal drought stress

Dehydrins are one of the characteristic families of plant proteins that usually accumulate in response to drought. In the present study, gene expressions of dehydrin multigene family (13 genes) were examined in flag leaves of tolerant (Yousef) and susceptible (Moroco) barley varieties under terminal drought to characterize the involvement of dehydrins in the adaptive processes. The stomatal conductance, RWC, and Chl a, b contents had more reduction in Moroco than the Yousef which has more elevated osmotic adjustment. Drought stress increased significantly MDA and electrolyte leakage levels, but greater in Moroco, indicating a poor protection of cell and cytoplasmic membrane in this variety. Yousef variety had no reduction in grain yield under drought condition. Five genes (Dhn1, Dhn3, Dhn5, Dhn7 and Dhn9) were exclusively induced in Yousef under drought stress. In the stress condition, relative gene expression of Dhn3, Dhn9 had the direct correlations (P < 0.05) with Chl a, b contents, osmotic adjustment, stomatal conductance, plant biomass and grain yield, and the negative correlations (P < 0.05) with MDA and electrolyte leakage levels. The results supported the impending functional roles of dehydrin K_n and particularly Y_nSK_n types in dehydration tolerance of barley during the reproductive stage.     

Identification and transcriptional analysis of dehydrin gene family in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Dehydrins (DHNs) are a group II late embryogenesis abundant (LEA) proteins that play essential roles in plant growth, development and responses to diverse environmental stimuli. Here, four DHNs in cucumber genome were identified using bioinformatics-based methods according to the highly conserved K-, Y- and S-segments, including 1 Y_nK_n-type, 2 Y_nSK_n-type, and 1 SK_n-type DHNs. All of them are intrinsically disordered proteins (IDPs) and possess a large number of disorder-promoting amino acids. Secondary structure prediction revealed that each of them is composed of high proportion of alpha helix and random coil. Gene structure and phylogenetic analyses with DHNs from cucumber and several other species revealed that some closely related DHN genes had similar gene structures. A number of cis-elements involved in stress responses and phytohormones were found in each CsDHN promoter. The tissue expression profiles suggested that the CsDHN genes have overlapping, but different expression patterns. qRT-PCR results showed that three selected CsDHN genes could respond to heat, cold, osmotic and salt stresses, as well as to signaling molecules such as H₂O₂ and ABA. These results lay a solid foundation for future functional investigation of the cucumber dehydrin gene family in tissue development and stress responses in plants.     

Dehydrin genes and their expression in recalcitrant oak (<Emphasis Type="Italic">Quercus robur</Emphasis>) embryos

In this work, three dehydrin genes, QrDhn1, QrDhn2, QrDhn3, were isolated from recalcitrant oak (Quercus robur). Their expression pattern was analyzed in both zygotic and somatic embryos as well as in vegetative tissues exposed to different kinds of abiotic stresses including desiccation, osmotic stress, and chilling. The QrDhn1 gene encoding for Y_nSK_n type dehydrin was expressed during later stages of zygotic embryo development but in somatic embryos only when exposed to osmotic or desiccation stress. In contrast, the other two oak dehydrin genes encoding for putative K_n type dehydrins were expressed only in somatic embryos (both not-treated and osmotically stressed) and leaves of oak seedlings exposed to desiccation. Behavior of these genes suggests that different dehydrins are involved in processes of seed maturation and response to altered osmotic (water status) conditions in somatic embryos. Revealing further members of dehydrin gene family in recalcitrant oak might contribute to clarify non-orthodox seed behavior as well as identify mechanisms contributing to desiccation tolerance in plants.     

Analysis of Brassica napus dehydrins and their Co-Expression regulatory networks in relation to cold stress

Dehydrins (DHNs) are plant specific cold and drought stress-responsive proteins that belong to late embryogenesis abundant (LEA) protein families. B. napus DHNs (BnDHNs) were computationally analyzed to establish gene regulatory- and protein-protein interaction networks. Promoter analyses suggested functionality of phytohormones in BnDHNs gene network. The relative expressions of some BnDHNs were analyzed using qRT-PCR in seedling leaves of both cold-tolerant (Zarfam) and -sensitive (Sari Gul) canola treated/untreated by cold. Our expression data were indicative of the importance of BnDHNs in cold tolerance in Zarfam. BnDHNs were classified into three classes according to the expression pattern. Moreover, expression of three BnDHN types, SK_n (BnLEA10 and BnLEA18), Y_nK_n (BnLEA90) and Y_nSK_n (BnLEA104) were significantly high in the tolerant cultivar at 12 h of cold treatment. Our findings put forward the possibility of considering these genes as screening biomarker to determine cold-tolerant breeding lines; something that needs to be further corroborated. Furthermore, these genes may have some implications in developing such tolerant lines via transgenesis.     

Plant dehydrins — Tissue location, structure and function

Dehydrins (DHNs) are part of a large group of highly hydrophilic proteins known as LEA (Late Embryogenesis Abundant). They were originally identified as group II of the LEA proteins. The distinctive feature of all DHNs is a conserved, lysine-rich 15-amino acid domain, EKKGIMDKIKEKLPG, named the K-segment. It is usually present near the C-terminus. Other typical dehydrin features are: a track of Ser residues (the S-segment); a consensus motif, T/VDEYGNP (the Y-segment), located near the N-terminus; and less conserved regions, usually rich in polar amino acids (the Φ-segments). They do not display a well-defined secondary structure. The number and order of the Y-, S-and K-segments define different DHN sub-classes: Y_nSK_n, Y_nKn, SK_n, K_n and K_nS. Dehydrins are distributed in a wide range of organisms including the higher plants, algae, yeast and cyanobacteria. They accumulate late in embryogenesis, and in nearly all the vegetative tissues during normal growth conditions and in response to stress leading to cellular dehydration (e.g. drought, low temperature and salinity). DHNs are localized in different cell compartments, such as the cytosol, nucleus, mitochondria, vacuole, and the vicinity of the plasma membrane; however, they are primarily localized to the cytoplasm and nucleus. The precise function of dehydrins has not been established yet, but in vitro experiments revealed that some DHNs (YSK_n-type) bind to lipid vesicles that contain acidic phospholipids, and others (K_nS) were shown to bind metals and have the ability to scavenge hydroxyl radicals [Asghar, R. et al. Protoplasma 177 (1994) 87–94], protect lipid membranes against peroxidation or display cryoprotective activity towards freezing-sensitive enzymes. The SK_n-and K-type seem to be directly involved in cold acclimation processes. The main question arising from the in vitro findings is whether each DHN structural type could possess a specific function and tissue distribution. Much recent in vitro data clearly indicates that dehydrins belonging to different subclasses exhibit distinct functions. An erratum to this article is available at .     

Expression of SK3-type dehydrin in transporting organs is associated with cold acclimation in Solanum species

The expression of a gene, encoding a dehydrin protein designated as DHN24 was analyzed at the protein level in two groups of Solanum species differing in cold acclimation ability. The DHN24 protein displays consensus amino acid sequences of dehydrins, termed K- and S-segments. The S-segment precedes three K-segments, classifying the protein into SK₃-type dehydrins. A group of Solanum species able to cold acclimation constituted by S. sogarandinum and S. tuberosum, cv. Aster, and a second one composed of a S. sogarandinum line, that lost ability to cold acclimation, and of S. tuberosum, cv. Irga, displaying low ability to cold acclimation were studied. Under control conditions, noticeable levels of the DHN24 protein was observed in stems, tubers, and roots of Solanum species. No protein was detected in leaves. During low temperature treatment the DHN24 protein level substantially increased in tubers, in transporting organs and in apical parts, and only a small increase was observed in leaves. The increase in protein abundance was only observed in the plants able to cold acclimate and was found to parallel the acclimation capacity. Upon drought stress, the DHN24 level decreased in stems and in leaves, but increased in apical parts. These results suggest that Dhn24 expression is regulated by organ specific factors in the absence of stress and by factors related to cold acclimation processes during low temperature treatment in collaboration with organ-specific factors. A putative function of the SK₃-type dehydrin proteins during plant growth and in the tolerance to low temperature is discussed.     

Osmotic solute responses of mycorrhizal citrus (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis>) seedlings to drought stress

This study investigated the accumulation of osmotic solutes in citrus (Poncirus trifoliata) seedlings colonized by Glomus versiforme subjected to drought stress or kept well watered. Development of mycorrhizae was higher under well watered than under drought-stressed conditions. Arbuscular mycorrhizal (AM) seedlings accumulated more soluble sugars, soluble starch and total non-structural carbohydrates in leaves and roots than corresponding non-AM seedlings regardless of soil-water status. Glucose and sucrose contents of well-watered and drought-stressed roots, fructose contents of well-watered roots and sucrose contents of drought-stressed leaves were notably higher in AM than in non-AM seedlings. K⁺ and Ca²⁺ levels in AM leaves and roots were greater than those in non-AM leaves and roots, while AM symbiosis did not affect the Mg²⁺ level. AM seedlings accumulated less proline than non-AM seedlings. AM symbiosis altered both the allocation of carbohydrate to roots and the net osmotic solute accumulations in response to drought stress. It is concluded that AM colonization enhances osmotic solute accumulation of trifoliate orange seedlings, thus providing better osmotic adjustment in AM seedlings, which did not correlate with proline but with K⁺, Ca²⁺, Mg²⁺, glucose, fructose and sucrose accumulation.     

Responses of the antioxidant defense system to drought stress in the leaves of Fargesia denudata seedlings,the staple food of the giant panda

The responses of the antioxidant defense system in plant species to drought stress are still relatively unknown. In order to further understand how the system responds to drought stress, the leaves of Fargesia denudata seedlings were investigated. Antioxidant enzyme activities, antioxidant contents, hydrogen peroxide (H₂O₂), superoxide anion (O ₂ ^·? ) and MDA contents in the seedling leaves were measured under well-watered (WW), moderate drought-stressed (MD), and severe drought-stressed (SD) treatments. Although drought stress significantly increased H₂O₂ and O ₂ ^·? levels in F. denudata leaves, only weak lipid peroxidation was observed. This is attributed to the higher superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) activities in F. denudata leaves during the entire drought period. Reduced and oxidized ascorbate (AsA and DHA) contents were almost not affected by drought except that DHA under SD showed an obvious increase on day 30. Furthermore, reduced glutathione (GSH) content under drought stress significantly decreased, while oxidized glutathione (GSSG) markedly increased under SD on days 30 and 45 as well as under MD on day 30; as a result, the ratio GSH/GSSG declined considerably. These results indicated that GSH was involved in scavenging H₂O₂ and O ₂ ^·? under drought stress and it was more sensitive to drought stress in scavenging H₂O₂ and O ₂ ^·? than AsA. As a result, a highly efficient antioxidant defense system in drought-stressed F. denudate leaves operated mainly through the synergistic functioning of SOD, CAT, APX, MDHAR, DHAR, GR, and GSH against oxidative damage.     

Soil Drench Treatment with ?-Aminobutyric Acid Increases Drought Tolerance of Potato

The non-protein amino acid β-aminobutyric acid (BABA) is known to be a priming agent for a more efficient activation of cellular defence responses and a potent inducer of resistance against biotic and abiotic stresses in plants. Nevertheless, most of the studies on priming have been carried out in Arabidopsis. In potato, the effect of BABA was demonstrated only on biotic stress tolerance. We investigated the effect of BABA on the drought tolerance of potato and found that soil drenched with BABA at a final concentration of 0.3 mM improves the drought tolerance of potato. Water loss from the leaves of the primed plants is attenuated and the yield is increased compared to the unprimed drought-stressed plants. The metabolite composition of the tubers of the BABA-treated plants is less affected by drought than the tuber composition of the non-treated plants. Nitric oxide and ROS (reactive oxygen species) production is increased in the BABA-treated roots but not in the leaves. In the leaves of the BABA-treated plants, the expression of the drought-inducible gene StDS2 is delayed, but the expression of ETR1, encoding an ethylene receptor, is maintained for a longer period under the drought conditions than in the leaves of the non-treated, drought-stressed control plants. This result suggests that the ethylene-inducible gene expression remains suppressed in primed plants leading to a longer leaf life and increased tuber yield compared to the non-treated, drought-stressed plants. The priming effect of BABA in potato, however, is transient and reverts to an unprimed state within a few weeks.     

Identification of Reference Genes for Quantitative Real-Time PCR in Date Palm (Phoenix dactylifera L.) Subjected to Drought and Salinity

Date palm is an important crop plant in the arid and semi-arid regions supporting human population in the Middle East and North Africa. These areas have been largely affected by drought and salinity due to insufficient rainfall and improper irrigation practices. Date palm is a relatively salt- and drought-tolerant plant and more recently efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Quantitative real-time PCR (qPCR) is a promising technique for the analysis of stress-induced differential gene expression, which involves the use of stable reference genes for normalizing gene expression. In an attempt to find the best reference genes for date palm’s drought and salinity research, we evaluated the stability of 12 most commonly used reference genes using the geNorm, NormFinder, BestKeeper statistical algorithms and the comparative ΔC_T method. The comprehensive results revealed that HEAT SHOCK PROTEIN (HSP), UBIQUITIN (UBQ) and YTH domain-containing family protein (YT521) were stable in drought-stressed leaves whereas GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH), ACTIN and TUBULIN were stable in drought-stressed roots. On the other hand, SMALL SUBUNIT RIBOSOMAL RNA (25S), YT521 and 18S ribosomal RNA (18S); and UBQ, ACTIN and ELONGATION FACTOR 1-ALPHA (eEF1a) were stable in leaves and roots, respectively, under salt stress. The stability of these reference genes was verified by using the abiotic stress-responsive CYTOSOLIC Cu/Zn SUPEROXIDE DISMUTASE (Cyt-Cu/Zn SOD), an ABA RECEPTOR, and a PROLINE TRANSPORTER 2 (PRO) genes. A combination of top 2 or 3 stable reference genes were found to be suitable for normalization of the target gene expression and will facilitate gene expression analysis studies aimed at identifying functional genes associated with drought and salinity tolerance in date palm.