Bioinformatics and protein expression analyses implicate LEA proteins in the drought response of Collembola

Humidity has a large impact on the distribution and abundance of terrestrial invertebrates, but the molecular mechanisms governing drought resistance are not fully understood. Some attention has been given to the role of the heat shock response as a component of desiccation tolerance, but recent focus has been on the chaperone-like LEA (late embryogenesis abundant) proteins in anhydrobiotic animals. This study investigates the expression of putative LEA proteins as well as the heat shock protein Hsp70 during drought stress in soil and surface dwelling species of Collembola (springtails). In silico analysis of four EST candidates from two species of Collembola showed the presence of a Group 3 LEA protein in Megaphorura arctica. In common with other Group 3 LEA proteins, the new sequence is predicted to be 100% natively unfolded, with a strong degree of lysine and alanine periodicity and with a negative average hydrophobicity of −1.273. The sequence clusters with members of the Group 3 LEA in plants. Furthermore, cross-species Western blotting showed drought-induced expression of putative LEA proteins in six species of Collembola. In the surface dwelling species, Orchesella cincta, degree of dehydration and length of exposure correlated with level of putative LEA protein. Hsp70 was also found to increase in individuals of O. cincta and Folsomia candida that had been exposed to drought conditions for 6 days. These results show the presence of a LEA protein-coding region in Collembola, but also indicate that several proteins are involved in response to dehydration stress, including Hsp70.     

Differential organ-specific response to salt stress and water deficit in nodulated bean (Phaseolus vulgaris)

Nodulated bean plants were exposed to mild salt stress or water deficit in such a way that the nodule's nitrogen‐fixing activity was reduced to about 25–30% that of controls. Water‐deprived plants showed a slight decrease in the weight of the aerial part, whereas the photosynthetic parameters were not significantly affected. In contrast, salt‐stressed plants displayed a reversible decrease in the quantum yield of photosystem II photochemistry. Five water‐deficit responsive cDNA clones encoding one lipid transfer protein, two late‐embryogenesis abundant (LEA) proteins and two proline‐rich proteins (PRPs) showed different organ‐specific expression patterns depending on the kind of stress applied. PRPs and one LEA protein, PvLEA‐18, exhibited the highest expression in nodules. Anti‐PvLEA‐18 antibodies were used to immunolocalize the protein in the nodule. PvLEA‐18 was localized in the cytoplasm and nucleus of nodule cortex cells, and preferentially in cells of the vascular bundles, showing enhanced accumulation under water deficit. To our knowledge, this is the first time that a LEA protein has been identified in legume nodules.     

Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to mammalian somatic cells

Late Embryogenesis Abundant (LEA) proteins are commonly found in plants and other organisms capable of undergoing severe and reversible dehydration, a phenomenon termed “anhydrobiosis”. Here, we have produced a tagged version for three different LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, binds to cellular membranes, and pTag-LEA-BFP, Artemia franciscana LEA protein group 3 that targets the mitochondria. Sheep fibroblasts transfected with single or all three LEA proteins were subjected to air drying under controlled conditions. After rehydration, cell viability and functionality of the membrane/mitochondria were assessed. After 4 h of air drying, cells from the un-transfected control group were almost completely nonviable (1% cell alive), while cells expressing LEA proteins showed high viability (more than 30%), with the highest viability (58%) observed in fibroblasts expressing all three LEA proteins. Growth rate was markedly compromised in control cells, while LEA-expressing cells proliferated at a rate comparable to non-air-dried cells. Plasmalemma, cytoskeleton and mitochondria appeared unaffected in LEA-expressing cells, confirming the protection conferred by LEA proteins on these organelles during dehydration stress. This is likely to be an effective strategy when aiming to confer desiccation tolerance to mammalian cells.     

Dehydration-specific induction of hydrophilic protein genes in the anhydrobiotic nematode Aphelenchus avenae

Some organisms can survive exposure to extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living nematode Aphelenchus avenae can be induced to enter the anhydrobiotic state by exposure to a moderate reduction in relative humidity. During this preconditioning period, the nematode accumulates large amounts of the disaccharide trehalose, which is thought to be necessary, but not sufficient, for successful anhydrobiosis. To identify other adaptations that are required for anhydrobiosis, we developed a novel SL1-based mRNA differential display technique to clone genes that are upregulated by dehydration in A. avenae. Three such genes, Aav-lea-1, Aav-ahn-1, and Aav-glx-1, encode, respectively, a late embryogenesis abundant (LEA) group 3 protein, a novel protein that we named anhydrin, and the antioxidant enzyme glutaredoxin. Strikingly, the predicted LEA and anhydrin proteins are highly hydrophilic and lack significant secondary structure in the hydrated state. The dehydration-induced upregulation of Aav-lea-1 and Aav-ahn-1 was confirmed by Northern hybridization and quantitative PCR experiments. Both genes were also upregulated by an osmotic upshift, but not by cold, heat, or oxidative stress. Experiments to investigate the relationship between mRNA levels and protein expression for these genes are in progress. LEA proteins occur commonly in plants, accumulating during seed maturation and desiccation stress; the presence of a gene encoding an LEA protein in an anhydrobiotic nematode suggests that some mechanisms of coping with water loss are conserved between plants and animals.     

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.     

The induction of anhydrobiosis in the sleeping chironomid: current status of our knowledge

An African chironomid, Polypedilum vanderplanki, is the only insect known to be capable of extreme desiccation tolerance, or anhydrobiosis. In the 1950s and 1960s, Hinton strenuously studied anhydrobiosis in this insect from a physiological standpoint; however, nobody has afterward investigated the phenomenon. In 2000, research on mechanisms underlying anhydrobiosis was resumed due to successful establishment of a rearing system for P. vanderplanki. This review is focused on the latest findings on the physiological and molecular mechanisms underlying the induction of anhydrobiosis in P. vanderplanki. Early experiments demonstrated that the induction of anhydrobiosis was possible in isolated tissues and independent from the control of central nervous system. However, to achieve successful anhydrobiosis, larvae need a slow regime of desiccation, allowing them to synthesize molecules, which will protect cells and tissues against the deleterious effects of dehydration. Trehalose, a nonreducing disaccharide, which accumulates in P. vanderplanki larvae up to 20% of the dry body mass, is thought to replace the water in its tissues. Similarly, highly hydrophilic proteins called the late embryogenesis abundant (LEA) proteins are expressed in huge quantities and act as a molecular shield to protect biological molecules against aggregation and denaturation. This function is shared by heat shock proteins, which are also upregulated during the desiccation process. At the same time, desiccating larvae express various antioxidant molecules and enzymes, to cope with the massive oxidative stress, which is responsible for general damage to membranes, proteins, and DNA in dehydrating cells. Finally, specific water channels, called aquaporins, accelerate dehydration, and trehalose together with LEA proteins forms a glassy matrix, which protects the biological molecules and the structural integrity of larvae in the anhydrobiotic state.     

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.     

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.     

Conformation of a group 2 late embryogenesis abundant protein from soybean. Evidence of poly (L-proline)-type II structure

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (L-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (L-proline)-type II-like helical conformation at 12 degrees C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80 degrees C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt alpha-helical structure and to interact with phospholipid bilayers through amphipathic alpha-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation, ionic, or osmotic stress-related damage to macromolecular structures.     

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.     

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.     

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.     

A LEA model peptide protects the function of a red fluorescent protein in the dry state

We tested whether a short model peptide derived from a group 3 late embryogenesis abundant (G3LEA) protein is able to maintain the fluorescence activity of a red fluorescent protein, mKate2, in the dry state. The fluorescence intensity of mKate2 alone decreased gradually through repeated dehydration-rehydration treatments. However, in the presence of the LEA model peptide, the peak intensity was maintained almost perfectly during such stress treatments, which implies that the three dimensional structure of the active site of mKate2 was protected even under severe desiccation conditions. For comparison, similar experiments were performed with other additives such as a native G3LEA protein, trehalose and BSA, all of whose protective abilities were lower than that of the LEA model peptide.     

Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation

Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.     

Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein

Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in resurrection plants and in plant seeds, and the recent discovery of a dehydration-induced Group 3 LEA-like gene in the nematode Aphelenchus avenae suggests a similar association in anhydrobiotic animals. Despite their importance, little is known about the structure of Group 3 LEA proteins, although computer modeling and secondary structure algorithms predict a largely alpha-helical monomer that forms coiled coil oligomers. We have therefore investigated the structure of the nematode protein, AavLEA1, in the first such analysis of a well characterized Group 3 LEA-like protein. Immunoblotting and subunit cross-linking experiments demonstrate limited oligomerization of AavLEA1, but analytical ultracentrifugation and gel filtration show that the vast majority of the protein is monomeric. Moreover, CD, fluorescence emission, and Fourier transform-infrared spectroscopy indicate an unstructured conformation for the nematode protein. Therefore, in solution, no evidence was found to support structure predictions; instead, AavLEA1 seems to be natively unfolded with a high degree of hydration and low compactness. Such proteins can, however, be induced to fold into more rigid structures by partner molecules or by altered physiological conditions. Because AavLEA1 is associated with desiccation stress, its Fourier transform-infrared spectrum in the dehydrated state was examined. A dramatic but reversible increase in alpha-helix and, possibly, coiled coil formation was observed on drying, indicating that computer predictions of secondary structure may be correct for the solid state. This unusual finding offers the possibility that structural shifts in Group 3 LEA proteins occur on dehydration, perhaps consistent with their role in anhydrobiosis.     

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.     

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.