A novel late embryogenesis abundant protein and peroxidase associated with black point in barley grains

Black point of barley grain is a disorder characterised by a brown-black discolouration at the embryo end of the grain. Black point is undesirable to the malting industry and results in significant economic loss annually. To identify proteins associated with barley black point we utilised a proteomic approach with 2-DE to compare proteins from whole grain samples of black pointed and healthy grain. From this comparison two condition-specific proteins were identified: a novel 75 kDa late embryogenesis abundant (LEA) protein and a barley grain peroxidase 1 (BP1) that were specifically more abundant in healthy grain and black pointed grain, respectively. Although LEA protein was less abundant in black pointed grain, LEA gene expression was greater suggesting protein degradation had possibly occurred in black pointed grain. Similarly, the increase in BP1 in black pointed grain could not be explained by gene expression. Western blot analysis also revealed that the identified LEA protein is biotinylated in vivo. The role that each of these proteins might have in black point development is discussed.     

Dehydration-regulated processing of late embryogenesis abundant protein in a desiccation-tolerant nematode

Late embryogenesis abundant (LEA) proteins occur in desiccation-tolerant organisms, including the nematode Aphelenchus avenae, and are thought to protect other proteins from aggregation. Surprisingly, expression of the LEA protein AavLEA1 in A. avenae is partially discordant with that of its gene: protein is present in hydrated animals despite low cognate mRNA levels. Moreover, on desiccation, when its gene is upregulated, AavLEA1 is specifically cleaved to discrete, smaller polypeptides. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes, and main cleavage sites were mapped to 11-mer repeated motifs in the AavLEA1 sequence. Processed polypeptides retain function as protein anti-aggregants and we hypothesise that the expression pattern and cleavage of LEA protein allow rapid, maximal availability of active molecules to the dehydrating animal.     

Evolution of the Group 1 late embryogenesis abundant (Lea) genes: analysis of the Lea B19 gene family in barley

The highly conserved Group 1 late embryogenesis abundant (Lea) genes are present in the genome of most plants as a gene family. Family members are conserved along the entire coding region, especially within the extremely hydrophilic internal 20 amino acid motif, which may be repeated. Cloning of Lea Group 1 genes from barley resulted in the characterization of four family members named B19.1, B19.1b, B19.3 and B19.4 after the presence of this motif 1, 1, 3 and 4 times in each gene, respectively. We present here the results of comparative and evolutionary analyses of the barley Group 1 Lea gene family (B19). The most important findings resulting from this work are (1) the tandem clustering of B19.3 and B19.4, (2) the spatial conservation of putative regulatory elements between the four B19 gene promoters, (3) the determination of the relative age of the gene family members and (4) the chimeric nature of B19.3 and B19.4, reflecting a cross-over or gene-conversion event in their common ancestor. We also show evidence for the presence of one or two additional expressed B19 genes in the barley genome. Based on our results, we present a model for the evolution of the family in barley, including the 20 amino acid motif. Comparisons of the relatedness between the barley family and all other known Group 1 Lea genes using maximum parsimony (PAUP) analysis provide evidence for the time of divergence between the barley genes containing the internal motif as a single copy and as a repeat. The PAUP analyses also provide evidence for independent duplications of Group 1 genes containing the internal motif as a repeat in both monocots and dicots.     

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.     

Genome-wide identification and analysis of late embryogenesis abundant (LEA) genes in Prunus mume

Late embryogenesis abundant (LEA) proteins play important roles in plant desiccation tolerance. In this study, 30 LEA genes were identified from Chinese plum (Prunus mume) through genome-wide analysis. The PmLEA genes are distributed on all Chinese plum chromosomes except chromosome 3. Twelve (40 %) and five PmLEA genes are arranged in tandem and segmental duplications, respectively. The PmLEA genes could be divided into eight groups (LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, PvLEA18, dehydrin and seed maturation protein). Ten gene conversion events were observed and most of them (70 %) were identified in dehydrin group. Most PmLEA genes were highly expressed in flower (22/30) and up-regulated by ABA treatment (19/30).     

Fruit ripening-related expression of a gene encoding group 5 late embryogenesis abundant protein inCitrus

A cDNA and genomic clone (CuLEA5) encoding a group 5 late embryogenesis abundant protein (Lea5) was isolated from citrus fruit cDNA and genomic libraries. Sequence analysis indicated that the clone contains an open reading frame of 97 amino acids, and that the genomic structure is composed of two exons and one intron. A comparison of its amino sequence with other plant proteins showed that Lea5 proteins can be classified into two types - gymnosperm and angiosperm — based on a P-segment sequence designated by this study. Examination of its expression patterns indicated thatCuLEA5 has important roles during the development or ripening of seedless fruits and leaves inCitrus. The 5′-flanking region of the genomic DNA contains a number of putative hormonal- and stress-responsive elements. This is the first report that describes the expression ofLea5 during fruit ripening, as well as the sequence characteristics of its promoter region.     

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.     

Characterization of Phaseolus vulgaris cDNA clones responsive to water deficit: identification of a novel late embryogenesis abundant-like protein

Plant Molecular Biology - Six cDNA clones from Phaseolus vulgaris, whose expression is induced by water deficit and ABA treatment (rsP cDNAs) were identified and characterized. The sequence...     

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.     

A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L

The transport of metal micronutrients to developing organs in a plant is mediated primarily by the sieve elements. Ligands are thought to form complexes with the free ions in order to prevent cellular damage, but no binding partners have been unequivocally identified from plants so far. This study has used the phloem-mediated transport of micronutrients during the germination of the castor bean seedling to identify an iron transport protein (ITP). It is demonstrated that essentially all (55)Fe fed to seedlings is associated with the protein fraction of phloem exudate. It is shown that ITP carries iron in vivo and binds additional iron in vitro. ITP was purified to homogeneity from minute amounts of phloem exudate using immobilized metal ion affinity chromatography. It preferentially binds to Fe(3+) but not to Fe(2+) and also complexes Cu(2+), Zn(2+), and Mn(2+) in vitro. The corresponding cDNA of ITP was cloned using internal peptide fragments. The deduced protein of 96 amino acids shows high similarity to the stress-related family of late embryogenesis abundant proteins. Its predicted characteristics and its RNA expression pattern are consistent with a function in metal ion binding. The ITP from Ricinus provides the first identified micronutrient binding partner for phloem-mediated long distance transport in plants and is the first member of the late embryogenesis abundant protein family shown to have such a function.     

Purification of the major late Mr 89 000 heat-shock protein from spring barley seedlings

A fast and efficient method for the isolation of major late heat-shock protein (HSP89) from the seedlings of spring barley (Hordeum vulgare L. cv. Fatran) incubated at 40°C for 15 h was described.     

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。     

Implication of ABA and proline on cell membrane injury of water deficit stressed barley seedlings

The aim of this work was to examine the ability of ABA and proline to counteract the deleterious effect of water deficit stress on cell membrane injuries. Six-day-old seedlings of two barley genotypes (cv. Aramir, line R567) were treated with ABA (2·10⁻⁴ M) or proline (0.1 M) for 24 h, and then subjected to osmotic stress for 24h, by immersing their roots in polyethylene glycol (PEG 6000) solution of osmotic potential of −1.0 MPa and −1.5 MPa or by submerging the leaf pieces in PEG solution of osmotic potential of −1.6 MPa. Pretreatment of plants with ABA and proline caused an increase of free proline level in the leaves. Plants treated with ABA exhibited a lower membrane injury index under water stress conditions than those untreated even when no effect of this hormone on RWC in the leaves of stressed plants was observed. Pretreatment of plants with proline prevented to some extent membrane damage in leaves of the stressed seedlings, but only in the case when stress was imposed to roots. Improvement in water status of leaves was also observed in seedlings pretreatment with proline. The protective effect of both ABA and proline was more pronounced in line R567 that exhibited higher membrane injury under water deficit stress conditions.