Functional dissection of hydrophilins during in vitro freeze protection

In plants, Late Embryogenesis Abundant (LEA) proteins typically accumulate in response to low water availability conditions imposed during development or by the environment. Analogous proteins in other organisms are induced when exposed to stress conditions. Most of this diverse set of proteins can be grouped according to properties such as high hydrophilicity and high content of glycine or other small amino acids in what we have termed hydrophilins. Previously, we showed that hydrophilins protect enzyme activities in vitro from low water availability effects. Here, we demonstrate that hydrophilins can also protect enzyme activities from the adverse effects induced by freeze-thaw cycles in vitro. We monitored conformational changes induced by freeze-thaw on the enzyme lactate dehydrogenase (LDH) using the fluorophore 1-anilinonaphthalene-8-sulfonate (ANS). Hydrophilin addition prevents enzyme inactivation and this effect is reflected in changes in the ANS-fluorescence levels determined for LDH. We further show that for selected plant hydrophilins, removal of certain conserved domains affects their protecting capabilities. Thus, we propose that hydrophilins, and in particular specific protein domains, have a role in protecting cell components from the adverse effects caused by low water availability such as those present during freezing conditions by preventing deleterious changes in protein secondary and tertiary structure.     

A general method of protein purification for recombinant unstructured non-acidic proteins

Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.     

A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene.

We recently reported that a Tetrahymena thermophila 58-kilodalton (kDa) mitochondrial protein (hsp58) was selectively synthesized during heat shock. In this study, we show that hsp58 displayed antigenic similarity with mitochondrially associated proteins from Saccharomyces cerevisiae (64 kDa), Xenopus laevis (60 kDa), Zea mays (62 kDa), and human cells (59 kDa). Furthermore, a 58-kDa protein from Escherichia coli also exhibited antigenic cross-reactivity to an antiserum directed against the T. thermophila mitochondrial protein. The proteins from S. cerevisiae and E. coli antigenically related to hsp58 were studied in detail and found to share several other characteristics with hsp58, including heat inducibility and the property of associating into distinct oligomeric complexes. The T. thermophila, S. cerevisiae, and E. coli macromolecular complexes containing these related proteins had similar sedimentation characteristics and virtually identical morphologies as seen with the electron microscope. The distinctive properties of the E. coli homolog to T. thermophila hsp58 indicate that it is most likely the product of the groEL gene.     

Two different late embryogenesis abundant proteins from Arabidopsis thaliana contain specific domains that inhibit Escherichia coli growth

Late embryogenesis abundant (LEA) proteins constitute a set of proteins widespread in the plant kingdom that show common physicochemical properties such as high hydrophilicity and high content of small amino acid residues such as glycine, alanine, and serine. Typically, these proteins accumulate in response to water deficit conditions imposed by the environment or during plant normal development. In this work, we show that the over-expression in Escherichia coli of proteins of the LEA 2 and the LEA 4 families from Arabidopsis thaliana leads to inhibition of bacterial growth and that this effect is dependent on discrete regions of the proteins. Our data indicate that their antimicrobial effect is achieved through their interaction with intracellular targets. The relevance of the cationic nature and the predicted structural organization of particular protein domains in this detrimental effect on the bacteria growth process is discussed.     

Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha

Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5 M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation–rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a “molecular shield”. Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins.     

Temperature-induced extended helix/random coil transitions in a group 1 late embryogenesis-abundant protein from soybean

Group 1 late embryogenesis-abundant (LEA) proteins are a subset of hydrophilins that are postulated to play important roles in protecting plant macromolecules from damage during freezing, desiccation, or osmotic stress. To better understand the putative functional roles of group 1 LEA proteins, we analyzed the structure of a group 1 LEA protein from soybean (Glycine max). Differential scanning calorimetry of the purified, recombinant protein demonstrated that the protein assumed a largely unstructured state in solution. In the presence of trifluoroethanol (50% [w/v]), the protein acquired a 30% alpha-helical content, indicating that the polypeptide is highly restricted to adopt alpha-helical structures. In the presence of sodium dodecyl sulfate (1% [w/v]), 8% of the polypeptide chain adopted an alpha-helical structure. However, incubation with phospholipids showed no effect on the protein structure. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein existed in equilibrium between two conformational states. Ultraviolet absorption spectroscopy studies also showed that the protein became more hydrated upon heating. Furthermore, circular dichroism spectral measurements indicated that a minimum of 14% of amino acid residues existed in a solvent-exposed, left-handed extended helical or poly (L-proline)-type (PII) conformation at 20 degrees C with the remainder of the protein being unstructured. The content of PII-like structure increased as temperature was lowered. We hypothesize that by favoring the adoption of PII structure, instead of the formation of alpha-helical or beta-sheet structures, group 1 LEA proteins retain a high content of surface area available for interaction with the solvent. This feature could constitute the basis of a potential role of LEA proteins in preventing freezing, desiccation, or osmotic stress damage.     

Prevalent structural disorder in E. coli and S. cerevisiae proteomes

Intrinsically unstructured proteins, which exist without a well-defined 3D structure, carry out essential functions and occur with high frequency, as predicted for genomes. The generality of this phenomenon, however, is questioned by the uncertainty of what fraction of genomes actually encodes for expressed proteins. Here, we used two independent bioinformatic predictors, PONDR VSL1, and IUPred, to demonstrate that disorder prevails in the recently characterized proteomes and essential proteins of E. coli and S. cerevisiae, at levels exceeding that estimated from the genomes. The S. cerevisiae proteome contains three times as much disorder as that of E. coli, with 50-60% of proteins containing at least one long (>30 residues) disordered segment. This evolutionary advance can be explained by the observation that disorder is much higher in Gene Ontology categories related to regulatory, as opposed to metabolic, functions, and also in categories unique to yeast. Thus, protein disorder is a widespread and functionally important phenomenon, which needs to be characterized in full detail for understanding complex organisms at the molecular level.     

Identification of two hydrophilins that contribute to the desiccation and freezing tolerance of yeast (Saccharomyces cerevisiae) cells

Hydrophilins are a group of proteins that are present in all organisms and that have been defined as being highly hydrophilic and rich in glycine. They are assumed to play important roles in cellular dehydration tolerance. There are 12 genes in the yeast Saccharomyces cerevisiae that encode hydrophilins and most of these genes are stress responsive. However, the functional role of yeast hydrophilins, especially in desiccation and freezing tolerance, is largely unknown. Here, we selected six candidate hydrophilins for further analysis. All six proteins were predicted to be intrinsically disordered, i.e. to have no stable structure in solution. The contribution of these proteins to the desiccation and freezing tolerance of yeast was investigated in the respective knock-out strains. Only the disruption of the genes YJL144W and YMR175W (SIP18) resulted in significantly reduced desiccation tolerance, while none of the strains was affected in its freezing tolerance under our experimental conditions. Complementation experiments showed that yeast cells overexpressing these two genes were both more desiccation and freezing tolerant, confirming the role of these two hydrophilins in yeast dehydration stress tolerance.     

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.     

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 Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties

Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.     

Suppression of Escherichia coli alkB mutants by Saccharomyces cerevisiae genes.

The alkB gene is one of a group of alkylation-inducible genes in Escherichia coli, and its product protects cells from SN2-type alkylating agents such as methyl methanesulfonate (MMS). However, the precise biochemical function of the AlkB protein remains unknown. Here, we describe the cloning, sequencing, and characterization of three Saccharomyces cerevisiae genes (YFW1, YFW12, and YFW16) that functionally complement E. coli alkB mutant cells. DNA sequence analysis showed that none of the three gene products have any amino acid sequence homology with the AlkB protein. The YFW1 and YFW12 proteins are highly serine and threonine rich, and YFW1 contains a stretch of 28 hydrophobic residues, indicating that it may be a membrane protein. The YFW16 gene turned out to be allelic with the S. cerevisiae STE11 gene. STE11 is a protein kinase known to be involved in pheromone signal transduction in S. cerevisiae; however, the kinase activity is not required for MMS resistance because mutant STE11 proteins lacking kinase activity could still complement E. coli alkB mutants. Despite the fact that YFW1, YFW12, and YFW16/STE11 each confer substantial MMS resistance upon E. coli alkB cells, S. cerevisiae null mutants for each gene were not MMS sensitive. Whether these three genes provide alkylation resistance in E. coli via an alkB-like mechanism remains to be determined, but protection appears to be specific for AlkB-deficient E. coli because none of the genes protect other alkylation-sensitive E. coli strains from killing by MMS.     

Expression in Escherichia coli of Three Different Soybean Late Embryogenesis Abundant (LEA) Genes to Investigate Enhanced Stress Tolerance

In order to identify the function of late embryogenesis abundant (LEA) genes, in vitro functional analyses were perfo rmed using an Escherichia coli heterologous expression system. Three soybean late embryogenesis abundant (LEA) genes, PM11 (GenBank accession No. AF004805; group 1), PM30 (AF1 17884; group 3), and ZLDE-2 (AY351918; group 2), were cloned and expressed in a pET-28a system.The gene products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. E. coli cells containing the recombinant plasmids or empty vector as controls were treated by salt and low temperature stress. Compared with control cells, the E. coli cells expressing either PM11 or PM30 showed a shorter lag period and improved growth when transferred to LB (Luria-Bertani) liquid media containing 800 mmol/L NaCl or 700 mmol/L KCl or after 4 ℃ treatment. E. coli cells expressing ZLDE-2 did not show obvious growth improvement both in either high KCl medium or after 4 ℃ treatment. The results indicate that the E. coli expression system is a simple, useful method to identify the functions of some stress-tolerant genes from plants.     

Functional Analysis of the Group 4 Late Embryogenesis Abundant Proteins Reveals Their Relevance in the Adaptive Response during Water Deficit in Arabidopsis

Late-Embryogenesis Abundant (LEA) proteins accumulate to high levels during the last stages of seed development, when desiccation tolerance is acquired, and in vegetative and reproductive tissues under water deficit, leading to the hypothesis that these proteins play a role in the adaptation of plants to this stress condition. In this work, we obtained the accumulation patterns of the Arabidopsis (Arabidopsis thaliana) group 4 LEA proteins during different developmental stages and plant organs in response to water deficit. We demonstrate that overexpression of a representative member of this group of proteins confers tolerance to severe drought in Arabidopsis plants. Moreover, we show that deficiency of LEA proteins in this group leads to susceptible phenotypes upon water limitation, during germination, or in mature plants after recovery from severe dehydration. Upon recovery from this stress condition, mutant plants showed a reduced number of floral and axillary buds when compared with wild-type plants. The lack of these proteins also correlates with a reduced seed production under optimal irrigation, supporting a role in fruit and/or seed development. A bioinformatic analysis of group 4 LEA proteins from many plant genera showed that there are two subgroups, originated through ancient gene duplication and a subsequent functional specialization. This study represents, to our knowledge, the first genetic evidence showing that one of the LEA protein groups is directly involved in the adaptive response of higher plants to water deficit, and it provides data indicating that the function of these proteins is not redundant to that of the other LEA proteins.Water deficit is a common environmental condition that leads to various responses that help in the adaptation or adjustment of an organism to this stress, considered one of the most important environmental stresses influencing plant productivity (Bray, 1997; Morison et al., 2008). The adverse effects of this environmental stress need to be counteracted, mainly because of the increasing soil desertification in cultivated and uncultivated regions. This fact demands that plants tolerate drying periods and elevated salt concentrations in the soil, which may be accompanied by extreme temperatures. Also, an interest in understanding the mechanisms by which plants sense and respond to these environmental cues accounts for the most important reasons to study in detail the responses that have been selected in plants to cope with water deficit.The acquisition of desiccation tolerance during late stages of seed development is correlated with the induction of a set of small, highly hydrophilic proteins called Late-Embryogenesis Abundant (LEA) proteins (Dure et al., 1989). These proteins are ubiquitous in plants, and although there are several classifications, we will follow that of Battaglia et al. (2008), where they are classified into seven groups on the basis of sequence similarity. Analysis of the protein sequences in these groups from different plant species defined distinctive motifs within groups (Dure, 1993; Battaglia et al., 2008). The number of members is different for each LEA protein group and varies according to the plant species. Most LEA proteins are hydrophilins, a set of proteins characterized by their biased amino acid composition, richness in Gly and other small and/or charged residues, and high hydrophilicity index (Garay-Arroyo et al., 2000). This amino acid composition promotes their flexible structure in solution, existing mainly as random coils, with the exception of the hydrophobic or atypical LEA proteins (Singh et al., 2005). Moreover, hydrophilic LEA proteins from groups 2, 3, and 4 show a prevalence of typical spectroscopic patterns of intrinsically unstructured proteins, with the occurrence of transitions from intrinsically unstructured proteins to ordered conformations in the presence of helix-promoting solvents or air drying (McCubbin et al., 1985; Russouw et al., 1995; Eom et al., 1996; Lisse et al., 1996; Ismail et al., 1999; Wolkers et al., 2001; Soulages et al., 2002, 2003; Goyal et al., 2003; Shih et al., 2004; Tolleter et al., 2007). Their high content of water-interacting residues facilitates the scavenging of water molecules, which is of special importance during developmental stages where a programmed desiccation of tissues takes place, as in the dry seed (Dure et al., 1989), or when cells experience changes in their water status (Colmenero-Flores et al., 1999). Remarkably, there is also an elevated induction in the expression of these proteins in vegetative tissues after exposure to water deficit in basically all plants that have been analyzed. In recent years, proteins with similar characteristics and expression patterns have also been detected to be induced in response to osmotic stress in bacteria and yeast (Stacy and Aalen, 1998; Garay-Arroyo et al., 2000), algae (Honjoh et al., 1995, 2000; Tanaka et al., 2004), nematodes (Solomon et al., 2000; Browne et al., 2004), rotifers (Tunnacliffe et al., 2005), and arthropods (Menze et al., 2009).One of the hypotheses regarding their function is that these proteins may act as protectors of macromolecules and/or some cellular structures during water deficit, by preferentially interacting with the available water molecules and providing a hydration shell to protect “target” integrity and function (Bray, 1997; Garay-Arroyo et al., 2000; Hoekstra et al., 2001). The use of an in vitro dehydration assay, in which the activity of malate dehydrogenase and lactate dehydrogenase was measured in the presence or absence of a hydrophilic protein, showed that plant hydrophilins (LEA proteins from groups 2, 3, and 4) and hydrophilins from Saccharomyces cerevisiae and Escherichia coli were able to protect these enzymatic activities under low water availability conditions (Reyes et al., 2005). Similarly, in vitro assays using the same or other enzymes have been used to assess the protective capacities of LEA proteins under dehydration and cold (Honjoh et al., 2000; Hara et al., 2001; Bravo et al., 2003; Goyal et al., 2005; Grelet et al., 2005; Nakayama et al., 2007; Reyes et al., 2008). In some of these assays, the ratio of LEA protein to enzyme was 1:1, suggesting that the LEA protein protective activity is not only due to the formation of a preferential hydration shell but also to an additional effect probably related to a direct interaction with their targets (Reyes et al., 2005, 2008).There are many reports of LEA proteins expressed in transgenic plants under the control of regulated or constitutive promoters, showing tolerant phenotypes under drought, high salinity, or freezing stress (Xu et al., 1996; Sivamani et al., 2000; NDong et al., 2002; Chandra Babu et al., 2004; Puhakainen et al., 2004; Fu et al., 2007; Lal et al., 2007; Xiao et al., 2007; Dalal et al., 2009). Also, the heterologous expression in bacteria and yeast of some LEA proteins confers salt and freezing tolerance (Imai et al., 1996; Zhang et al., 2000; Liu and Zheng, 2005). However, this “gain-of-function” approach does not necessarily reflect their direct participation in the plant adjustment or adaptation to these stress conditions but rather their potential to confer tolerance when ectopically expressed. In contrast, the results of a “loss-of-function” approach will lead to a direct indication of the participation of a particular gene within this process. Even though there is a large extent of information regarding the different properties of LEA proteins, our knowledge concerning their role in plant adaptation to water-limiting conditions is insufficient.In this work, we focus on the study of the group 4 LEA proteins of Arabidopsis (Arabidopsis thaliana). With only three genes in the genome (AtLEA4-1, AtLEA4-2, and AtLEA4-5), the AtLEA4 group is one of the smallest groups in Arabidopsis (Battaglia et al., 2008; Hundertmark and Hincha, 2008), which makes it accessible for a loss-of-function analysis. The LEA4 proteins are characterized by a high content of A, T, and G amino acid residues, the latter highly represented in unstructured proteins. They have a conserved N-terminal domain of 70 to 80 residues, predicted to form amphipathic α-helices, and a less conserved C-terminal region with variable size and random coil structure (Dure, 1993). Like other LEA proteins, the LEA4 group is highly accumulated in all embryo tissues of dry seeds (Roberts et al., 1993). Recently, Wise (2002) performed a bioinformatics analysis and questioned the existence of a group 4 of LEA proteins as a distinct group of LEA proteins from group 3. The algorithm used the overrepresentation/underrepresentation of particular amino acids within small motifs in the protein, giving rise to a different classification for these proteins (Wise, 2003). In support of the original classification proposed by Dure et al. (1989) and because of the high sequence conservation within this group in plants, in this work, we present genetic and functional evidence that group 4 of LEA proteins is indeed a distinct group conserved in the plant kingdom. The results reported here show that overexpression of one of the AtLEA4 proteins in Arabidopsis leads to a tolerant phenotype compared with their wild-type counterparts in their capability to endure severe water deficit and that the reduction in the accumulation levels of these proteins leads to plants more sensitive to water-limiting conditions than their wild-type genotypes. Altogether, these data constitute, to our knowledge, the first direct evidence indicating that LEA4 proteins are involved in the adaptive response of vascular plants to withstand water deficit.     

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.     

Expression in Escherichia coli of Three Different Soybean Late Embryogenesis Abundant （LEA） Genes to Investigate Enhanced Stress Tolerance

In order to identify the function of late embryogenesis abundant (LEA) genes, in vitro functional analyses were performed using an Escherichia coli heterologous expression system. Three soybean late embryogenesis abundant (LEA) genes, PMll (GenBank accession No. AF004805; group 1), PM30(AF117884; group 3), and ZLDE-2 (AY351918; group 2), were cloned and expressed in a pET-28a system.The gene products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. E. coli cells containing the recombinant plasmids or empty vector as controls were treated by salt and low temperature stress. Compared with control cells, the E. coli cells expressing either PMll or PM30 showed a shorter lag period and improved growth when transferred to LB (Luria-Bertani) liquid media containing 800 mmol/L NaC1 or 700 mmol/L KC1 or after 4℃ treatment. E. coli cells expressing ZLDE-2 did not show obvious growth improvement both in either high KC1 medium or after 4℃ treatment. The results indicate that the E. coli expression system is a simple, useful method to identify the functions of some stress-tolerant genes from plants.     

长春花Crlea基因的克隆及原核表达初步分析

晚期胚胎丰富（Late Embryogenesis Abundant, LEA）蛋白是植物在干旱胁迫下响应并被描述为具有潜在的抗旱功能的一类重要的抗旱蛋白。通过建立干旱胁迫下长春花（Catharanthus roseus）的cDNA文库并进行测序筛选分析,首次分离得到Crlea（Crlea for Catharanthus roseus late embryogenesis abundant）全长基因。该基因具有492 bp的开放读码框,编码163个氨基酸,其中偏性氨基酸含量占总蛋白的55.9%。同源性分析表明该假定蛋白与胡萝卜（Daucus carota）LEA DC3 的同源性达69%。亲水性分析表明具有极强的亲水性。为进一步验证CrLEA蛋白的功能,构建了Crlea基因的原核表达载体并在大肠杆菌中对其表达进行了分析。结果表明,原核载体成功的表达了CrLEA蛋白,亲水性实验及热稳定性实验表明CrLEA蛋白具有极强的亲水性和热稳定性。     

Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins

Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0647-3) contains supplementary material, which is available to authorized users.