Solution structure of a late embryogenesis abundant protein (LEA14) from Arabidopsis thaliana, a cellular stress-related protein

We report the three-dimensional structure of a late embryogenesis abundant (LEA) protein from Arabidopsis thaliana gene At1g01470.1. This protein is a member of Pfam cluster PF03168, and has been classified as a LEA14 protein. LEA proteins are expressed under conditions of cellular stress, such as desiccation, cold, osmotic stress, and heat. The structure, which was determined by NMR spectroscopy, revealed that the At1g01470.1 protein has an alphabeta-fold consisting of one alpha-helix and seven beta-strands that form two antiparallel beta-sheets. The closest structural homologs were discovered to be fibronectin Type III domains, which have <7% sequence identity. Because fibronectins from animal cells have been shown to be involved in cell adhesion, cell motility, wound healing, and maintenance of cell shape, it is interesting to note that in plants wounding or stress results in the overexpression of a protein with fibronectin Type III structural features.     

胚胎发育晚期丰富蛋白(LEA蛋白)与植物抗逆性研究进展

胚胎发育晚期丰富蛋白(LEA蛋白)在自然条件下主要在种子发育晚期大量积累,植物LEA基因也在多种非生物胁迫下诱导表达。植物LEA蛋白是植物应对失水胁迫(包括干旱、盐碱、冷冻等)逆境的一种广泛存在的亲水性应答蛋白,具有很强的热稳定性。本论文就LEA蛋白的结构、分类、功能及抗逆性分子机制进行了概述与总结,为分离新的LEA蛋白成员,进行功能分析以及进一步发掘其潜在应用价值提供参考。     

Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana

We analyzed the Arabidopsis thaliana genome sequence to detect Late Embryogenesis Abundant (LEA) protein genes, using as reference sequences proteins related to LEAs previously described in cotton or which present similar characteristics. We selected 50 genes representing nine groups. Most of the encoded predicted proteins are small and contain repeated domains that are often specific to a unique LEA group. Comparison of these domains indicates that proteins with classical group 5 motifs are related to group 3 proteins and also gives information on the possible history of these repetitions. Chromosomal gene locations reveal that several LEA genes result from whole genome duplications (WGD) and that 14 are organized in direct tandem repeats. Expression of 45 of these genes was tested in different plant organs, as well as in response to ABA and in mutants (such as abi3, abi5, lec2 and fus3) altered in their response to ABA or in seed maturation. The results demonstrate that several so-called LEA genes are expressed in vegetative tissues in the absence of any abiotic stress, that LEA genes from the same group do not present identical expression profile and, finally, that regulation of LEA genes with apparently similar expression patterns does not systematically involve the same regulatory pathway.     

Distinct functions of evolutionary conserved MSF1 and late embryogenesis abundant (LEA)-like domains in mitochondria

PRELID1, the only late embryogenesis abundant (LEA) domain-containing protein in humans, exerts cytoprotective effects through its LEA domain within the mitochondria. Although PRELID1 homologs in vertebrates contain the LEA domain, homologs in lower eukaryotes are thought to lack this domain. In this study, we identify a novel LEA-like domain in a yeast PRELID1 homolog, Ups2p, which contains sequence conservation with the LEA domain of human PRELID1. PRELID1 homologs, including Ups2p, are known to contain the PRELI/MSF1 domain. Our study reveals that the MSF1 domain of Ups2p maintains proper mitochondrial electron transport chain function, respiratory competency, and mitochondrial phosphatidylethanolamine metabolism. The Ups2p MSF1 domain mediates cardiolipin depletion in the absence of Ups1p. However, the Ups2p LEA-like domain is responsible for cardiolipin depletion resulting from UPS2 overexpression. The regulation of phosphatidylethanolamine levels by the MSF1 domain is antagonized by the Ups2p LEA-like domain. We demonstrate that the yeast LEA-like domain protects cells from oxidative stress and can be functionally replaced by the human LEA domain. Together our studies suggest distinct roles of MSF1 and LEA-like domains in mitochondrial function and resistance to oxidative stress.     

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.     

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.     

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.     

A late embryogenesis abundant protein HVA1 regulated by an inducible promoter enhances root growth and abiotic stress tolerance in rice without yield penalty

Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress‐inducible promoter was designed to control the expression of a late embryogenesis abundant protein (HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water‐use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin‐dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress‐suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty.     

Evaluation of the protective activities of a late embryogenesis abundant (LEA) related protein, Cor15am, during various stresses in vitro

Arabidopsis Cor15am is a late embryogenesis abundant (LEA) related protein that has been shown to exhibit cryoprotective activity in vitro. In this study, we further investigated the mechanisms by which Cor15am protects substrates from inactivation. Although Cor15am did not exhibit refolding activity, it showed protective activity against various stresses in vitro. This might be attributable to the activity of Cor15am in attenuating the aggregation of the substrates. Our data indicate that Cor15am functions as a protectant against various stresses by preventing protein aggregation.     

A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.     

Auxin and its transport play a role in plant tolerance to arsenite‐induced oxidative stress in Arabidopsis thaliana

The role of auxin in plant development is well known; however, its possible function in root response to abiotic stress is poorly understood. In this study, we demonstrate a novel role of auxin transport in plant tolerance to oxidative stress caused by arsenite. Plant response to arsenite [As(III)] was evaluated by measuring root growth and markers for stress on seedlings treated with control or As(III)‐containing medium. Auxin transporter mutants aux1, pin1 and pin2 were significantly more sensitive to As(III) than the wild type (WT). Auxin transport inhibitors significantly reduced plant tolerance to As(III) in the WT, while exogenous supply of indole‐3‐acetic acid improved As(III) tolerance of aux1 and not that of WT. Uptake assays using H³‐IAA showed As(III) affected auxin transport in WT roots. As(III) increased the levels of H₂O₂ in WT but not in aux1, suggesting a positive role for auxin transport through AUX1 on plant tolerance to As(III) stress via reactive oxygen species (ROS)‐mediated signalling. Compared to the WT, the mutant aux1 was significantly more sensitive to high‐temperature stress and salinity, also suggesting auxin transport influences a common element shared by plant tolerance to arsenite, salinity and high‐temperature stress.     

Enhanced oxidative stress in the ethylene-insensitive (ein3-1) mutant of Arabidopsis thaliana exposed to salt stress

To better understand the role of ethylene signaling in plant stress tolerance, salt-induced changes in gene expression levels of ethylene biosynthesis, perception and signaling genes were measured in Arabidopsis thaliana plants exposed to 15 days of salinity. Among the genes analyzed, EIN3 showed the highest expression level increase under salt stress, suggesting a key role for this ethylene-signaling component in response to salt stress. Therefore, we analyzed the salt stress response over 15 days (by adding 100 mM NaCl to the nutrient solution) in the ein3-1 mutant compared to the wild-type (Col-0) in terms of growth, oxidative stress markers (lipid peroxidation, foliar pigments and low-molecular-weight antioxidants) and levels of growth- and stress-related phytohormones (including cytokinins, auxins, gibberellins, abscisic acid, jasmonic acid and salicylic acid). The ein3-1 mutant grew similarly to wild-type plants both under control and salt stress conditions, which was associated with a differential time course evolution in the levels of the cytokinins zeatin and zeatin riboside, and the auxin indole-3-acetic acid between the ein3-1 mutant and the wild-type. Despite showing no signs of physiological deterioration under salt stress (in terms of rosette biomass, leaf water and pigment contents, and PSII efficiency) the ein3-1 mutant showed enhanced lipid peroxidation under salt stress, as indicated by 2.4-fold increase in both malondialdehyde and jasmonic acid contents compared to the wild-type. We conclude that, at moderate doses of salinity, partial insensitivity to ethylene might be compensated by changes in endogenous levels of other phytohormones and lipid peroxidation-derived signals in the ein3-1 mutant exposed to salt stress, but at the same time, this mutant shows higher oxidative stress under salinity than the wild-type.     

Heterologous expression of two <Emphasis Type="Italic">Physcomitrella patens</Emphasis> group 3 late embryogenesis abundant protein (LEA3) genes confers salinity tolerance in arabidopsis

Salinity stress is a major limiting factor in agriculture and adversely affecting the whole plant. As a halophyte, the moss Physcomitrella patens, has been suggested to be an ideal model plant to study salinity tolerance and adaption. Two abiotic stress-responsive Group 3 Late Embryogenesis Abundant protein genes had been identified in P. patens and named as PpLEA3-1 and PpLEA3-2, respectively. Functions of these two genes were analyzed by heterologous expressions in Arabidopsis, driven either by their native P. patens promoters or by the 35S CaMV constitutive promoter. Phenotype analysis revealed that pLEA3::LEA3, pLEA3::LEA3::GFP and 35S::LEA3::GFP transgenic lines had stronger salinity resistance than that in the wild type and empty-vector control. Further analysis showed that the contents of proline and soluble sugar were increased and the malondialdehyde (MDA) were repressed in these transgenic plants after exposure to salinity stress. Our observations indicate that these two Group 3 PpLEA genes played a role in the adaption to salinity stress.     

Ectopic phytocystatin expression leads to enhanced drought stress tolerance in soybean (Glycine max) and Arabidopsis thaliana through effects on strigolactone pathways and can also result in improved seed traits

Ectopic cystatin expression has long been used in plant pest management, but the cysteine protease, targets of these inhibitors, might also have important functions in the control of plant lifespan and stress tolerance that remain poorly characterized. We therefore characterized the effects of expression of the rice cystatin, oryzacystatin‐I (OCI), on the growth, development and stress tolerance of crop (soybean) and model (Arabidopsis thaliana) plants. Ectopic OCI expression in soybean enhanced shoot branching and leaf chlorophyll accumulation at later stages of vegetative development and enhanced seed protein contents and decreased the abundance of mRNAs encoding strigolactone synthesis enzymes. The OCI‐expressing A. thaliana showed a slow‐growth phenotype, with increased leaf numbers and enhanced shoot branching at flowering. The OCI‐dependent inhibition of cysteine proteases enhanced drought tolerance in soybean and A. thaliana, photosynthetic CO₂ assimilation being much less sensitive to drought‐induced inhibition in the OCI‐expressing soybean lines. Ectopic OCI expression or treatment with the cysteine protease inhibitor E64 increased lateral root densities in A. thaliana. E64 treatment also increased lateral root densities in the max2‐1 mutants that are defective in strigolactone signalling, but not in the max3‐9 mutants that are defective in strigolactone synthesis. Taken together, these data provide evidence that OCI‐inhibited cysteine proteases participate in the control of growth and stress tolerance through effects on strigolactones. We conclude that cysteine proteases are important targets for manipulation of plant growth, development and stress tolerance, and also seed quality traits.