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.     

Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs

Summary Earlier studies found that cotton (Gossypium hirsutum L.) cotyledons contain several mRNAs which are more abundant during late embryogenesis than in mid-embryogenesis or early germination. They are here termed Late embryogenesis-abundant mRNAs, encoded by Lea loci. Complementary DNA clones for 18 such mRNA sequences, defined at a hybridization criterion of T_m-15°C, were identified in a mature embryo cDNA library by differential cDNA hybridization. At a lower hybridization criterion, some sequence homology was found within several of these cloned Lea mRNA sequences. Each Lea mRNA sequence comprises 0.04–1.3% of mature embryo poly(A)⁺ mRNA, a level ten-fold to several hundred-fold higher than in young embryo or 24 h seedling poly(A)⁺ mRNA. Of 18 Lea mRNA sequences examined in cultured young embryos, the level of at least 13 are specifically increased by exogenous abscisic acid (ABA), several to a level near that in normal mature embryos. However, the abundance of several of the sequences does not appear to be significantly modulated by ABA. The LEA polypeptides encoded by 10 Lea mRNA sequences were identified by hybrid-arrested translation. They include most of the late embryogenesis-abundant, ABA-inducible, polypeptides previously identified. Preliminary results suggest that many of the individual Lea mRNA sequences are transcribed from 1–3 genes in each of cotton's two subgenomes.     

植物胚胎发育晚期丰富蛋白1组的结构与功能

植物胚胎发育晚期丰富蛋白（late embryogenesis abundant proteins,LEA）是植物胚胎发生后期种子中大量积累的一类蛋白质。根据蛋白质的氨基酸基序和保守结构特点,LEA蛋白一般分为6组,其中第1组LEA蛋白（LEA1）含有高度保守的20氨基酸基序。LEA1蛋白在水溶液中主要呈无规则结构,具高亲水性和热稳定性,与植物抗逆功能密切相关。本文就LEA1蛋白的功能和结构等方面的研究做一综述。     

LEAping to conclusions: A computational reanalysis of late embryogenesis abundant proteins and their possible roles

Background  

The late embryogenesis abundant (LEA) proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties.     

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.     

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).     

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。     

Analysis of the promoter activity of late embryogenesis abundant protein genes in barley seedlings under conditions of water deficit

Promoters of the late embryogenesis abundant protein genes, HVA1s, Dhn8s and Dhn4s from barley and wsi18j and rab16Bj from rice, were analysed in barley seedlings to assess their strength and timing of induction under water deficit conditions using a transient expression system. Of the drought-inducible promoters, Dhn4s exhibited the highest activity, followed by HVA1s, wsi18j and rab16Bj. Dehydration-induced #-glucuronidase expression levels driven by the Dhn4s, HVA1s and wsi18j promoters were higher than or comparable to that induced by a strong constitutive rice Act1 promoter. The expression patterns of wsi18j and Dhn8s differed from those of their homologous genes (wsi18 and Dhn8): the wsi18j promoter was strongly induced by abscisic acid, while the Dhn8s promoter was highly active in hydrated seedlings. HVA1s- and wsi18j-driven green fluorescent protein expression was induced within 1 h of drying under water deficit stress, while Dhn4s promoter activity was not detectable until 3 h, thus showing a difference in the timing of induction.     

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.     

Diverse LEA (late embryogenesis abundant) and LEA-like genes and their responses to hypersaline stress in post-diapause embryonic development of Artemia franciscana

From post-diapause cysts of Artemia franciscana, we defined fourteen LEA (late embryogenesis abundant) and LEA-like genes, including four novel members (Afrlea1-5, Afrlea3-5, Afrlea3-like1 and Afrlea3-like2), which were classified into four groups: G1, G3, G3-like (LEA group3-like), and SMP-like (seed-maturation-protein-like), based on their conserved and diversified sequence motifs and amino acid compositions among bacteria, plants, and animals. We also validated six representative genes based on quantitative RT-PCR, including three LEA and two LEA3-like genes that are down-regulated when dehydrated cysts hatch to desiccation-intolerant larvae as well as one SMP-like gene that is slightly up-regulated. We further tested their responses to hypersaline stress for four representatives-one from each group-and found that the expression of Afrlea1-5 and Afrlea3-2 were inducible but not Afrlea3-like1 and Afrsmp-like. This result suggested that the LEA and LEA-like genes may play different roles in resistance to hypersaline stress.     

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.     

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.     

Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves -- the link between jasmonate and abscisic acid

In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.