A barley (Hordeum vulgare L.) LEA3 protein, HVA1, is abundant in protein storage vacuoles

The HVA1 protein belongs to the LEA3 group, which is expressed during the late stage of seed maturation. It is also induced by exogenous abscisic acid (ABA) and a variety of environmental stresses in germinating barley (Hordeum vulgare L.). In the present work, the potential role of HVA1 was investigated by studying its tissue distribution and subcellular localization in mature and stressed seeds by immuno-microscopic methods. In the mature seed, HVA1 protein was detected in all tissues except the non-living starchy endosperm. During germination the amount of HVA1 protein decreased but did not totally disappear. Incubation with 100 μM ABA, cold treatment or drought stress dramatically increased HVA1 expression in the germinated seed. In this work, the distribution of a LEA3 group protein was studied in a cereal seed for the first time by immuno-electron microscopy. In the scutellum and aleurone layer, HVA1 was localized both in the cytoplasm and protein storage vacuoles (PSVs). HVA1 protein was found to be threefold more abundant in PSVs than in the cytoplasm of an unstressed seed tissue. The ratio increased with ABA or stress treatments to at least ninefold. The role of HVA1 in PSVs remains unclear: a previously suggested possibility is ion sequestration to prevent precipitation during stress. On the other hand, HVA1 protein could also be degraded in PSVs. HVA1 protein does not have the signal peptide typical of proteins which are glycosylated and targeted into the vacuole via the Golgi complex. Because HVA1 is not glycosylated, it may use an alternative, ER-independent vacuolar pathway, also found in yeast cells.     

A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state

Late embryogenesis abundant (LEA) proteins are a highly diverse group of polypeptides expected to play important roles in desiccation tolerance of plant seeds. They are also found in other plant tissues and in some anhydrobotic invertebrates, fungi, protists and prokaryotes. The LEA protein LEAM accumulates in the matrix space of pea (Pisum sativum) mitochondria during late seed maturation. LEAM is an intrinsically disordered protein folding into amphipathic α-helix upon desiccation. This suggests that it could interact with the inner mitochondrial membrane, providing structural protection in dry seeds. Here, we have used Fourier-transform infrared and fluorescence spectroscopy to gain insight into the molecular details of interactions of LEAM with phospholipid bilayers in the dry state and their effects on liposome stability. LEAM interacted specifically with negatively charged phosphate groups in dry phospholipids, increasing fatty acyl chain mobility. This led to an enhanced stability of liposomes during drying and rehydration, but also upon freezing. Protection depended on phospholipid composition and was strongly enhanced in membranes containing the mitochondrial phospholipid cardiolipin. Collectively, the results provide strong evidence for a function of LEAM as a mitochondrial membrane protectant during desiccation and highlight the role of lipid composition in the interactions between LEA proteins and membranes.     

Isolation and expression analysis of LEA genes in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.     

Pea seed mitochondria are endowed with a remarkable tolerance to extreme physiological temperatures

Most seeds are anhydrobiotes, relying on an array of protective and repair mechanisms, and seed mitochondria have previously been shown to harbor stress proteins probably involved in desiccation tolerance. Since temperature stress is a major issue for germinating seeds, the temperature response of pea (Pisum sativum) seed mitochondria was examined in comparison with that of mitochondria from etiolated epicotyl, a desiccation-sensitive tissue. The functional analysis illustrated the remarkable temperature tolerance of seed mitochondria in response to both cold and heat stress. The mitochondria maintained a well-coupled respiration between -3.5 degrees C and 40 degrees C, while epicotyl mitochondria were not efficient below 0 degrees C and collapsed above 30 degrees C. Both mitochondria exhibited a similar Arrhenius break temperature at 7 degrees C, although they differed in phospholipid composition. Seed mitochondria had a lower phosphatidylethanolamine-to-phosphatidylcholine ratio, fewer unsaturated fatty acids, and appeared less susceptible to lipid peroxidation. They also accumulated large amounts of heat shock protein HSP22 and late-embryogenesis abundant protein PsLEAm. The combination of membrane composition and stress protein accumulation required for desiccation tolerance is expected to lead to an unusually wide temperature tolerance, contributing to the fitness of germinating seeds in adverse conditions. The unique oxidation of external NADH at low temperatures found with several types of mitochondria may play a central role in maintaining energy homeostasis during cold shock, a situation often encountered by sessile and ectothermic higher plants.     

小麦TaLEA1基因的表达特征及抗逆功能验证

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁,因此耐盐基因挖掘对作物耐盐育种非常重要。许多研究表明胚胎发育晚期丰富蛋白(LEA)在植物应对非生物胁迫中发挥积极作用。本研究以小麦TaLEA1基因为研究对象,分析了其表达蛋白的理化性质及基因表达模式,并通过在拟南芥中过表达,分析Ta LEA1基因的抗逆功能。结果表明,TaLEA1基因的表达蛋白属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。Ta LEA1基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA1基因,显著提高了盐胁迫下转基因拟南芥的种子萌发率、根长及盐和旱胁迫下的叶绿素含量。本研究结果为LEA基因抗逆机理的研究和耐盐基因的挖掘提供了重要信息。     

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.     

Multiple genome alignment for identifying the core structure among moderately related microbial genomes

Background

LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown.

Results

We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE) and/or low temperature response (LTRE) elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded.

Conclusion

The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for future efforts to elucidate the functional role of these enigmatic proteins.     

过表达TaLEA1和TaLEA2基因提高转基因拟南芥的耐盐性

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁。耐盐基因挖掘对作物耐盐育种非常重要。LEA蛋白家族是一个多基因家族,在植物应对非生物胁迫中发挥重要作用。本课题组前期研究阐明小麦TaLEA1基因在拟南芥中过表达可以提高转基因植物的耐盐性和抗旱性。本研究系统分析了小麦TaLEA2基因表达蛋白的理化性质、基因表达模式及启动子功能区域,并在拟南芥中过表达TaLEA2基因及共表达TaLEA1和TaLEA2基因,分析TaLEA2基因的抗逆功能及2个LEA基因的抗逆效果。结果表明,TaLEA2基因的表达产物属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。TaLEA2基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA2基因,或过表达TaLEA1和TaLEA2基因都能够提高转基因拟南芥的耐盐性和抗旱性,转基因株系的种子萌发率、根长及叶绿素含量显著高于野生型,且双基因过表达的转基因植物的抗逆能力高于单个基因过表达株系。本研究结果为LEA基因抗逆机理的研究和多基因共转提高植物抗逆性提供了重要信息。     

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

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

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.     

Disordered plant LEA proteins as molecular chaperones

Plants often respond to abiotic stresses by the increased expression of LEA (late embryogenesis abundant) proteins, so called because they also accompany seed formation. Whereas the cellular function of LEA proteins in mitigating the damage caused by stress is clear, the molecular mechanisms of their action are rather enigmatic. Several models have been developed, based on their putative activities as ion sinks, stabilizers of membrane structure, buffers of hydrate water, antioxidants and/or chaperones. Due to their known structural flexibility, this latter idea has received little experimental attention thus far. Recently, however, it has been suggested that intrinsically disordered proteins (IDPs) may exert chaperone activity by an “entropy transfer” mechanism. In our subsequent study published in the May issue of Plant Physiology, we provided evidence that two group 2 LEA proteins, ERD (early response to dehydration) 10 and 14, are potent molecular chaperones. This observation may have far-reaching implications, as it may explain how LEA proteins of ill-defined structures protect plant cells during dehydration, and it may also lead to the general experimental validation of the entropy transfer model of disordered chaperones.Key words: abiotic stress, dehydration stress, stress tolerance, late embryogenesis abundant protein, chaperone, disordered protein, unstructured protein     

Function and stress tolerance of seed mitochondria

Seeds of higher plant are desiccation tolerant, which suggests that their mitochondria exhibit particular properties. Insight into the function of seed mitochondria, especially in legume and model plants, has been fostered by the development of proteomics. Seed mitochondria are functional at the onset of imbibition, and their integrity and performance systematically improves during germination. This suggests that repair and biogenesis mechanisms exist, and this is supported by morphological and biochemical evidence. Seed mitochondria generate and operate in a hypoxic environment. They accumulate stress proteins, such as a small heat-shock protein and a late embryogenesis abundant protein. The mitochondria of pea ( Pisum sativum L.) seed also display a biased phospholipid composition likely to favour desiccation tolerance. These specific biochemical properties surely contribute to the remarkable tolerance of seed mitochondria to extreme temperatures. Recent progress towards the resolution of the seed mitochondrial proteome is discussed in light of the growing body of genomic data.     

Group 1 and 2 LEA protein expression correlates with a decrease in water stress induced protein aggregation in horsegram during germination and seedling growth

Plants produce an array of proteins as a part of a global response to protect the cell metabolism when they grow under environmental conditions such as drought and salinity that generate reduced water potential. The synthesis of hydrophilic proteins is a major part of the response to water deficit conditions. An increased expression of LEA proteins is thought to be one of the primary lines of defense to prevent the loss of intercellular water during adverse conditions. These LEA proteins are known to prevent aggregation of a wide range of other proteins. In this study we report the water stress induced protein aggregation and its abrogation followed by expression of group 1 and group 2 LEA proteins of water soluble proteomes in horsegram. Water stress caused an increased protein aggregation with magnitude and duration of stress in horsegram seedlings. Tissue-specific expression of LEA 1 protein decreased in the embryonic axis when compared to cotyledons in 24 h stressed seedlings. We found no cross reaction of LEA 1 with proteome of 48 h stressed embryonic axis and 72 h stressed root and shoot samples. However, LEA 2 antibodies were cross reacted with four polypeptides with different molecular mass in shoot tissue samples and found no reaction with root proteome as evidenced from immuno-blot analysis. The role of LEA proteins in relation to protein aggregation during water stressed conditions was discussed.     

Proteins arising during the late stages of embryogenesis in Pisum sativum L.

Two abscisic acid (ABA)-responsive seed proteins, ABR17 and ABR18 (ABA-responsive 17000-M_r and 18000-M_r, respectively), previously found to be induced in cultured embryos of pea (Pisum sativum L.) are major components synthesised during normal seed desiccation. The ABR17 and ABR18 proteins showed different patterns of accumulation. The ABR18 protein was abundant in the testa during early seed development but in desiccating seed it was synthesised in the embryo, indicating spacial as well as temporal regulation of expression. The ABR18 protein was undetectable soon after germination but reappeared after adding ABA. The ABR17 protein was not detected in the testa but appeared in the embryo just prior to maximum fresh weight. The ABR17 protein continued to be synthesised during germination and was also present in non-stressed leaves. A high level of endogenous ABA or added ABA increased levels of translatable ABR17 mRNA. The ABR17 and ABR18 proteins were further characterised so as to help determine their structure and function. Neither protein appeared to contain a signal peptide but both proteins appeared to be glycosylated. The proteins had similar amino-acid compositions and limited Nterminal analysis showed 56% sequence identity. Neither protein had any significant N-terminal sequence homology to any of the late embryogenesis-abundant (LEA) proteins or dehydrins. Both proteins, however, show striking homology with a pea disease-resistance-response protein and the major birch pollen allergen, indicating that the ABR17 and ABR18 proteins may be members of a distinct group of stress-induced proteins.Abbreviations ABA (±) cis,trans-abscisic acid - ABR17 M_r-17200 ABA-responsive protein - ABR18 M_r-18 100 ABA-responsive protein - FW fresh weight - I_gG immunoglobulin G - LEA late embryogenesis-abundant - M_r apparent molecularmass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station.     

Towards the identification of late-embryogenic-abundant phosphoproteome in Arabidopsis by 2-DE and MS

Late-embryogenesis-abundant (LEA) proteins accumulate as plant seeds desiccate and also in vegetative organs during periods of stress. They are predicted to play a role in plant stress tolerance. In the present study, we have initiated the characterization of phosphorylated LEA proteins present in the Arabidopsis seed, using a strategy that combines the thermostability (solubility upon heating) of many LEA-type proteins with the use of phosphoaffinity chromatography to obtain an enriched subpopulation of phosphoproteins. The specificity and efficiency of the procedure was assessed by alkaline phosphatase treatment and by a specific stain for phosphoproteins, in addition to the immunodetection of AtRab18, a phosphorylated LEA protein present in the mature dry seed. The phosphoproteins were identified by MS either by PMF using MALDI-TOF MS after 2-DE separation, or by peptide sequencing using both capillary LC MS/MS (LC muESI-ITMS/MS) and nanoLC coupled to nanoESI-MS/MS (LC-nanoESI-Q-TOF-MS/MS). Several LEA-type and storage-like proteins were identified as components of the phosphoproteome of the Arabidopsis seed.     

Overexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli and provides enhanced osmotic stress tolerance and ABA sensitivity in Arabidopsis thaliana

Previous studies have shown that the late embryogenesis abundant (LEA) group 3 proteins significantly respond to changes in environmental conditions. However, reports that demonstrate their biological role, especially in Arabidopsis, are notably limited. This study examines the functional roles of the Arabidopsis LEA group 3 proteins AtLEA3-3 and AtLEA3-4 in abiotic stress and ABA treatments. Expression of AtLEA3-3 and AtLEA3-4 is upregulated by ABA, high salinity, and osmotic stress. Results on the ectopic expression of AtLEA3-3 and AtLEA3-4 in E. coli suggest that both proteins play important roles in resistance to cold stress. Overexpression of AtLEA3-3 in Arabidopsis (AtLEA3-3-OE) confers salt and osmotic stress tolerance that is characterized during germination and early seedling establishment. However, AtLEA3-3-OE lines show sensitivity to ABA treatment during early seedling development. These results suggest that accumulation of AtLEA3-3 mRNA and/or proteins may help heterologous ABA re-initiate second dormancy during seedling establishment. Analysis of yellow fluorescent fusion proteins localization shows that AtLEA3-3 and AtLEA3-4 are mainly distributed in the ER and that AtLEA3-3 also localizes in the nucleus, and in response to salt, mannitol, cold, or BFA treatments, the localization of AtLEA3-3 and AtLEA3-4 is altered and becomes more condensed. Protein translocalization may be a positive and effective strategy for responding to abiotic stresses. Taken together, these results suggest that AtLEA3-3 has an important function during seed germination and seedling development of Arabidopsis under abiotic stress conditions.