A LEA 4 protein up-regulated by ABA is involved in drought response in maize roots

Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to high concentrations during the late stages of seeds development, which are integral to desiccation tolerance. LEA proteins also play a protective role under other abiotic stresses. We analyzed in silico a maize protein predicted to be highly hydrophilic and intrinsically disordered. This prediction was experimentally corroborated by solubility assays under denaturing conditions. Based on its amino acid sequence, we propose that this protein belongs to group four of the LEA proteins. The accumulation pattern of this protein was similar to that of dehydrins during the desiccation process that takes place during seed development. This protein was induced by exogenous abscisic acid in immature embryos, but during imbibition was down-regulated by gibberellins. It was also induced in maize roots under osmotic stress. So far, this is the first member of the LEA proteins belonging to group four to be characterized in maize, and it plays a role in the response to osmotic stress.     

Intracellular localization of group 3 LEA proteins in embryos of Artemia franciscana

Late embryogenesis abundant (LEA) proteins are accumulated by anhydrobiotic organisms in response to desiccation and improve survivorship during water stress. In this study we provide the first direct evidence for the subcellular localizations of AfrLEA2 and AfrLEA3m (and its subforms) in anhydrobiotic embryos of Artemia franciscana. Immunohistochemistry shows AfrLEA2 to reside in the cytoplasm and nucleus, and the four AfrLEA3m proteins to be localized to the mitochondrion. Cellular locations are supported by Western blots of mitochondrial, nuclear and cytoplasmic fractions. The presence of LEA proteins in multiple subcellular compartments of A. franciscana embryos suggests the need to protect biological structures in many areas of a cell in order for an organism to survive desiccation stress, and may explain in part why a multitude of different LEA proteins are expressed by a single organism.     

Temporal profiling of the heat-stable proteome during late maturation of Medicago truncatula seeds identifies a restricted subset of late embryogenesis abundant proteins associated with longevity

Developing seeds accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered and hydrophilic proteins that confer cellular protection upon stress. Many different LEA proteins exist in seeds, but their relative contribution to seed desiccation tolerance or longevity (duration of survival) is not yet investigated. To address this, a reference map of LEA proteins was established by proteomics on a hydrophilic protein fraction from mature Medicago truncatula seeds and identified 35 polypeptides encoded by 16 LEA genes. Spatial and temporal expression profiles of the LEA polypeptides were obtained during the long maturation phase during which desiccation tolerance and longevity are sequentially acquired until pod abscission and final maturation drying occurs. Five LEA polypeptides, representing 6% of the total LEA intensity, accumulated upon acquisition of desiccation tolerance. The gradual 30-fold increase in longevity correlated with the accumulation of four LEA polypeptides, representing 35% of LEA in mature seeds, and with two chaperone-related polypeptides. The majority of LEA polypeptides increased around pod abscission during final maturation drying. The differential accumulation profiles of the LEA polypeptides suggest different roles in seed physiology, with a small subset of LEA and other proteins with chaperone-like functions correlating with desiccation tolerance and longevity.     

Prediction of functions for two LEA proteins from mung bean

LEA (late embryogenesis abundant) proteins are associated with tolerance to water stress resulting from desiccation and cold shock. Although various functions have been proposed to LEA proteins, their precise role is not fully defined. In silico analysis of the amino acid sequence of two LEA proteins (early methionine-labeled Vigna, EMV) from the tropical legume crop, Vigna radiata identified a 20 residues motif 'GGQTRKQQLGSEGYHEMGRK' characteristic to group 1 LEA proteins. Structural analyses hypothesize these proteins to function like DNA/RNA binding proteins in protecting macromolecules/ membrane stabilization at the time of dehydration process.     

Functional assessment of hydrophilic domains of late embryogenesis abundant proteins from distant organisms

Late embryogenesis abundant (LEA) proteins play a protective role during desiccation and oxidation stresses. LEA3 proteins are a major group characterized by a hydrophilic domain (HD) with a highly conserved repeating 11-amino acid motif. We compared four different HD orthologs from distant organisms: (i) DrHD from the extremophilic bacterium Deinococcus radiodurans; (ii) CeHD from the nematode Caenorhabditis elegans; (iii) YlHD from the yeast Yarrowia lipolytica; and (iv) BnHD from the plant Brassica napus. Circular dichroism spectroscopy showed that all four HDs were intrinsically disordered in phosphate buffer and then folded into α-helical structures with the addition of glycerol or trifluoroethanol. Heterologous HD expression conferred enhanced desiccation and oxidation tolerance to Escherichia coli. These four HDs protected the enzymatic activities of lactate dehydrogenase (LDH) by preventing its aggregation under desiccation stress. The HDs also interacted with LDH, which was intensified by the addition of hydrogen peroxide (H₂O₂), suggesting a protective role in a chaperone-like manner. Based on these results, the HDs of LEA3 proteins show promise as protectants for desiccation and oxidation stresses, especially DrHD, which is a potential ideal stress-response element that can be applied in synthetic biology due to its extraordinary protection and stress resistance ability.     

Common amino acid sequence domains among the LEA proteins of higher plants

LEA proteins are late embryogenesis abundant in the seeds of many higher plants and are probably universal in occurrence in plant seeds. LEA mRNAs and proteins can be induced to appear at other stages in the plant's life by desiccation stress and/or treatment with the plant hormone abscisic acid (ABA). A role in protecting plant structures during water loss is likely for these proteins, with ABA functioning in the stress transduction process. Presented here are conserved tracts of amino acid sequence among LEA proteins from several species that may represent domains functionally important in desiccation protection. Curiously, an 11 amino acid sequence motif is found tandemly repeated in a group of LEA proteins of vastly different sizes. Analysis of this motif suggests that it exists as an amphiphilic helix which may serve as the basis for higher order structure.     

Molecular anhydrobiology: identifying molecules implicated in invertebrate anhydrobiosis

Studies in anhydrobiotic plants have defined many genes whichare upregulated during desiccation, but comparable studies ininvertebrates are at an early stage. To develop a better understandingof invertebrate anhydrobiosis, we have begun to characterisedehydration-inducible genes and their proteins in anhydrobioticnematodes and bdelloid rotifers; this review emphasises recentfindings with a hydrophilic nematode protein. Initial work withthe fungivorous nematode Aphelenchus avenae led to the identificationof two genes, both of which were markedly induced on slow drying(90–98% relative humidity, 24 hr) and also by osmoticstress, but not by heat or cold or oxidative stresses. The firstof these genes encodes a novel protein we have named anhydrin;it is a small, basic polypeptide, with no counterparts in sequencedatabases, which is predicted to be natively unstructured andhighly hydrophilic. The second is a member of the Group 3 LEAprotein family; this and other families of LEA proteins arewidely described in plants, where they are most commonly associatedwith the acquisition of desiccation tolerance in maturing seeds.Like anhydrin, the nematode LEA protein, Aav-LEA-1, is highlyhydrophilic and a recombinant form has been shown to be unstructuredin solution. In vitro functional studies suggest that Aav-LEA-1is able to stabilise other proteins against desiccation-inducedaggregation, which is in keeping with a role of LEA proteinsin anhydrobiosis. In vivo, however, Aav-LEA-1 is apparentlyprocessed into smaller forms during desiccation. A processingactivity was found in protein extracts of dehydrated, but nothydrated, nematodes; these shorter polypeptides are also activeanti-aggregants and we hypothesise that processing LEA proteinserves to increase the number of active molecules availableto the dehydrating animal. Other LEA-like proteins are beingidentified in nematodes and it seems likely therefore that theywill play a major role in the molecular anhydrobiology of invertebrates,as they are thought to do in plants.     

Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation

Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.     

Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause

Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis—life without water—during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H₂O₂) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.     

The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress

Late embryogenesis abundant (LEA) proteins are hydrophilic, mostly intrinsically disordered proteins, which play major roles in desiccation tolerance. In Arabidopsis thaliana, 51 genes encoding LEA proteins clustered into nine families have been inventoried. To increase our understanding of the yet enigmatic functions of these gene families, we report the subcellular location of each protein. Experimental data highlight the limits of in silico predictions for analysis of subcellular localization. Thirty-six LEA proteins localized to the cytosol, with most being able to diffuse into the nucleus. Three proteins were exclusively localized in plastids or mitochondria, while two others were found dually targeted to these organelles. Targeting cleavage sites could be determined for five of these proteins. Three proteins were found to be endoplasmic reticulum (ER) residents, two were vacuolar, and two were secreted. A single protein was identified in pexophagosomes. While most LEA protein families have a unique subcellular localization, members of the LEA_4 family are widely distributed (cytosol, mitochondria, plastid, ER, and pexophagosome) but share the presence of the class A α-helix motif. They are thus expected to establish interactions with various cellular membranes under stress conditions. The broad subcellular distribution of LEA proteins highlights the requirement for each cellular compartment to be provided with protective mechanisms to cope with desiccation or cold stress.     

Long-term droughts change the hatching patterns of zooplankton resting eggs from permanent and temporary lakes

相似文献   

Functional characterization of selected LEA proteins from Arabidopsis thaliana in yeast and in vitro

Main conclusion

Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying. To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are “atypical”, since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.     

MtPM25 is an atypical hydrophobic late embryogenesis‐abundant protein that dissociates cold and desiccation‐aggregated proteins

Late embryogenesis‐abundant (LEA) proteins are one of the components involved in desiccation tolerance (DT) by maintaining cellular structures in the dry state. Among them, MtPM25, a member of the group 5 is specifically associated with DT in Medicago truncatula seeds. Its function is unknown and its classification as a LEA protein remains elusive. Here, evidence is provided that MtPM25 is a hydrophobic, intrinsically disordered protein that shares the characteristics of canonical LEA proteins. Screening protective activities by testing various substrates against freezing, heating and drying indicates that MtPM25 is unable to protect membranes but able to prevent aggregation of proteins during stress. Prevention of aggregation was also found for the water soluble proteome of desiccation‐sensitive radicles. This inhibition was significantly higher than that of MtEM6, one of the most hydrophilic LEA protein associated with DT. Moreover, when added after the stress treatment, MtPM25 is able to rapidly dissolve aggregates in a non‐specific manner. Sorption isotherms show that when it is unstructured, MtPM25 absorbs up to threefold more water than MtEM6. MtPM25 is likely to act as a protective molecule during drying and plays an additional role as a repair mechanism compared with other LEA proteins.     

A predicted N-terminal helical domain of a Group 1 LEA protein is required for protection of enzyme activity from drying.

Late embryogenesis abundant (LEA) proteins have been repeatedly implicated in the acquisition of desiccation tolerance in angiosperm seed embryos. However, the mechanism(s) by which protection occurs is not well understood. While the Group 1 LEA proteins are predicted to be largely unordered in solution, there is strong evidence that upon drying these proteins undergo a structural transition that leads to an increase in alpha-helical content. Several studies also suggest there is a direct interaction between Group 1 LEA proteins and other molecules in the cytoplasm that may be critical for the establishment of desiccation tolerance during embryo maturation. We have produced a recombinant Group 1 LEA protein and show that it is capable of protecting the enzyme lactate dehydrogenase from the deleterious effects of drying. We have also evaluated the ability of various altered recombinant Group 1 LEA proteins to protect in the same assay. Our results suggest that the highly conserved 20 amino acid Group 1 LEA signature motif is not required for protection in our in vitro assay. However, introduction of two juxtaposed proline residues into an N-terminal helical domain predicted to exist in the hydrated structure significantly compromises the ability of the recombinant protein to provide protection from drying. These results suggest that the N-terminal domain of Group 1 LEA proteins may be important for proper folding during dehydration.     

Identification of Anhydrobiosis-related Genes from an Expressed Sequence Tag Database in the Cryptobiotic Midge Polypedilum vanderplanki (Diptera; Chironomidae)

Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.