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.     

GTP Promotes the Formation of Early-Import Intermediates But Is Not Required during the Translocation Step of Protein Import into Chloroplasts

Dehydrins are a family of proteins (LEA [late-embryogenesis abundant] D11) commonly induced by environmental stresses associated with low temperature or dehydration and during seed maturation drying. Our previous genetic studies suggested an association of an approximately 35-kD protein (by immunological evidence a dehydrin) with chilling tolerance during emergence of seedlings of cowpea (Vigna unguiculata) line 1393-2-11. In the present study we found that the accumulation of this protein in developing cowpea seeds is coordinated with the start of the dehydration phase of embryo development. We purified this protein from dry seeds of cowpea line 1393-2-11 by using the characteristic high-temperature solubility of dehydrins as an initial enrichment step, which was followed by three chromatography steps involving cation exchange, hydrophobic interaction, and anion exchange. Various characteristics of this protein confirmed that indeed it is a dehydrin, including total amino acid composition, partial amino acid sequencing, and the adoption of alpha-helical structure in the presence of sodium dodecyl sulfate. The propensity of dehydrins to adopt alpha-helical structure in the presence of sodium dodecyl sulfate, together with the apparent polypeptide adhesion property of this cowpea dehydrin, suggests a role in stabilizing other proteins or membranes. Taken together, the genetic, physiological, and physicochemical data are at this stage consistent with a cause-and-effect relationship between the presence in mature seeds of the approximately 35-kD dehydrin, which is the product of a single member of a multigene family, and an increment of chilling tolerance during emergence of cowpea seedlings.     

A predicted N-terminal helical domain of a Group 1 LEA protein is required for protection of enzyme activity from drying.

Late embryogenesis abundant (LEA) proteins have been repeatedly implicated in the acquisition of desiccation tolerance in angiosperm seed embryos. However, the mechanism(s) by which protection occurs is not well understood. While the Group 1 LEA proteins are predicted to be largely unordered in solution, there is strong evidence that upon drying these proteins undergo a structural transition that leads to an increase in alpha-helical content. Several studies also suggest there is a direct interaction between Group 1 LEA proteins and other molecules in the cytoplasm that may be critical for the establishment of desiccation tolerance during embryo maturation. We have produced a recombinant Group 1 LEA protein and show that it is capable of protecting the enzyme lactate dehydrogenase from the deleterious effects of drying. We have also evaluated the ability of various altered recombinant Group 1 LEA proteins to protect in the same assay. Our results suggest that the highly conserved 20 amino acid Group 1 LEA signature motif is not required for protection in our in vitro assay. However, introduction of two juxtaposed proline residues into an N-terminal helical domain predicted to exist in the hydrated structure significantly compromises the ability of the recombinant protein to provide protection from drying. These results suggest that the N-terminal domain of Group 1 LEA proteins may be important for proper folding during dehydration.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

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.     

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.     

第三组胚胎晚期丰富蛋白lea3基因研究概述

晚期胚胎富集蛋白(late embryogenesis abundant protein,LEA蛋白)是在高等植物胚胎发育晚期大量积累的一类蛋白,根据其结构特点LEA蛋白一般分为6组,其中第3组LEA蛋白(LEA3)含有11个氨基酸串联重复的基元序列,可以形成α-螺旋结构,能在干旱胁迫的环境中保护生物大分子,减轻水份胁迫对植物造成的伤害,与植物抗逆性密切相关。该文就lea3基因及其蛋白的结构、功能、基因表达和应用等进行简要的综述,并对lea3基因及其蛋白今后的研究方向和应用前景进行了展望。     

Dehydration-specific induction of hydrophilic protein genes in the anhydrobiotic nematode Aphelenchus avenae

Some organisms can survive exposure to extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living nematode Aphelenchus avenae can be induced to enter the anhydrobiotic state by exposure to a moderate reduction in relative humidity. During this preconditioning period, the nematode accumulates large amounts of the disaccharide trehalose, which is thought to be necessary, but not sufficient, for successful anhydrobiosis. To identify other adaptations that are required for anhydrobiosis, we developed a novel SL1-based mRNA differential display technique to clone genes that are upregulated by dehydration in A. avenae. Three such genes, Aav-lea-1, Aav-ahn-1, and Aav-glx-1, encode, respectively, a late embryogenesis abundant (LEA) group 3 protein, a novel protein that we named anhydrin, and the antioxidant enzyme glutaredoxin. Strikingly, the predicted LEA and anhydrin proteins are highly hydrophilic and lack significant secondary structure in the hydrated state. The dehydration-induced upregulation of Aav-lea-1 and Aav-ahn-1 was confirmed by Northern hybridization and quantitative PCR experiments. Both genes were also upregulated by an osmotic upshift, but not by cold, heat, or oxidative stress. Experiments to investigate the relationship between mRNA levels and protein expression for these genes are in progress. LEA proteins occur commonly in plants, accumulating during seed maturation and desiccation stress; the presence of a gene encoding an LEA protein in an anhydrobiotic nematode suggests that some mechanisms of coping with water loss are conserved between plants and animals.     

Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress

Hsp101 is a molecular chaperone that is required for the development of thermotolerance in plants and other organisms. We report that Arabidopsis thaliana Hsp101 is also regulated during seed development in the absence of stress, in a pattern similar to that seen for LEA proteins and small Hsps; protein accumulates during mid-maturation and is stored in the dry seed. Two new alleles of the locus encoding Hsp101 (HOT1) were isolated from Arabidopsis T-DNA mutant populations. One allele, hot1-3, contains an insertion within the second exon and is null for Hsp101 protein expression. Despite the complete absence of Hsp101 protein, plant growth and development, as well as seed germination, are normal, demonstrating that Hsp101 chaperone activity is not essential in the absence of stress. In thermotolerance assays hot1-3 shows a similar, though somewhat more severe, phenotype to the previously described missense allele hot1-1, revealing that the hot1-1 mutation is also close to null for protein activity. The second new mutant allele, hot1-2, has an insertion in the promoter 101 bp 5' to the putative TATA element. During heat stress the hot1-2 mutant produces normal levels of protein in hypocotyls and 10-day-old seedlings, and it is wild type for thermotolerance at these stages. Thus this mutation has not disrupted the minimal promoter sequence required for heat regulation of Hsp101. The hot1-2 mutant also expresses Hsp101 in seeds, but at a tenfold reduced level, resulting in reduced thermotolerance of germinating seeds and underscoring the importance of Hsp101 to seed stress tolerance.     

Study of AtSUS2 localization in seeds reveals a strong association with plastids

Sucrose synthase (SUS) is a key enzyme in sucrose metabolism. This enzyme catalyzes the reversible conversion of sucrose and UDP to UDP-glucose and fructose. In the Arabidopsis SUS gene family (six members), SUS2 is strongly and specifically expressed in Arabidopsis seeds during the maturation phase. Using specific antibodies, we have shown that SUS2 is localized in the embryo, endosperm and seed coat with differential patterns. During the maturation phase, the SUS2 protein seems to be mainly co-localized with plastids in the embryo. This novel finding is discussed in relation to the role of this enzyme in storage organs.     

A RING-H2 zinc-finger protein gene RIE1 is essential for seed development in Arabidopsis

RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. A novel gene, RIE1, encoding a RING-H2 zinc-finger protein was identified in Arabidopsis thaliana and is characterized in this paper. RIE1 encodes a predicted protein product of 359 amino acids residues with a molecular mass of 40 kDa, with a RING-H2 zinc-finger motif located at the extreme end of the C-terminus. Characterization of a Dissociation (Ds) insertion line (SGT4559) and a T-DNA insertion line (SRIE1) demonstrated that disruption of RIE1 is embryo-lethal. SGT4559 heterozygous plants produced seeds with embryo development arrested from globular to torpedo stages. Some mutant seeds were rescued by embryo culture, and the mutant (rie1) plants seemed to grow normally compared to wild-type plants, except that the mutants produced only abnormal seeds. However, RIE1 was expressed in different tissues throughout the whole plant as revealed by northern blot analysis and gene fusion assay of RIE1 promoter with the beta-glucuronidase (GUS) gene. Our results indicated that RIE1 plays an essential role in seed development.     

Proteomics of desiccation tolerance during development and germination of maize embryos

Maize seeds were used to identify the key embryo proteins involved in desiccation tolerance during development and germination. Immature maize embryos (28N) during development and mature embryos imbibed for 72 h (72HN) are desiccation sensitive. Mature maize embryos (52N) during development are desiccation tolerant. Thiobarbituric acid reactive substance and hydrogen peroxide contents decreased and increased with acquisition and loss of desiccation tolerance, respectively. A total of 111 protein spots changed significantly (1.5 fold increase/decrease) in desiccation-tolerant and -sensitive embryos before (28N, 52N and 72HN) and after (28D, 52D and 72HD) dehydration. Nine pre-dominantly proteins, 17.4 kDa Class I heat shock protein 3, late embryogenesis abundant protein EMB564, outer membrane protein, globulin 2, TPA:putative cystatin, NBS-LRR resistance-like protein RGC456, stress responsive protein, major allergen Bet v 1.01C and proteasome subunit alpha type 1, accumulated during embryo maturation, decreased during germination and increased in desiccation-tolerant embryos during desiccation. Two proteins, Rhd6-like 2 and low-molecular-weight heat shock protein precursor, showed the inverse pattern. We infer that these eleven proteins are involved in seed desiccation tolerance. We conclude that desiccation-tolerant embryos make more economical use of their resources to accumulate protective molecules and antioxidant systems to deal with maturation drying and desiccation treatment.     

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。     

Expression of three ABA-regulated clones and their relationship to maturation processes during the embryogenesis of chick-pea seeds

Water stress inhibits germination of chick-pea seeds and produces specific changes in gene expression. some of which are coincident with those induced by the exogenous application of abscisic acid (ABA). Three cDNA clones, GAB-8, GAB-9 and GAB-11, were previously identified as under the regulation of ABA and osmotic stress in embryonic axes of germinating chick-pea. Here we try to establish a relationship between the changes in gene expression induced by ABA and stress conditions during germination and those naturally occurring during the desiccation process that leads to seed maturation. Our results show that the germinative capacity of chick-pea is related to the water content of the organ. In vitro translation of the mRNAs from developing seed reveals that in the later stages of seed maturation some polypeptides appear that previously were found to be regulated by ABA and by water deficit in germinating seeds. Hybridization by northern blot of embryogenic mRNAs with GAB-8. GAB-9 and GAB-11 clones shows that the mRNAs corresponding to such clones only appear in the later phases of seed formation, coinciding with seed dehydration, and persisting until seeds became fully mature. The results suggest that these mRNAs are probably related to the response to dehydration that occurs during seed maturation, and that the pattern of expression of these ABA-regulated clones coincides with that of the established late embryogenesis-abundant (LEA) genes.     

The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis

We investigated the role of the oilbody proteins in developing and germinating Arabidopsis thaliana seeds. Seed oilbodies are simple organelles comprising a matrix of triacylglycerol surrounded by a phospholipid monolayer embedded and covered with unique proteins called oleosins. Indirect observations have suggested that oleosins maintain oilbodies as small single units preventing their coalescence during seed desiccation. To understand the role of oleosins during seed development or germination, we created lines of Arabidopsis in which a major oleosin is ablated or severely attenuated. This was achieved using RNA interference techniques and through the use of a T-DNA insertional event, which appears to interrupt the major (18 kD) seed oleosin gene of Arabidopsis and results in ablation of expression. Oleosin suppression resulted in an aberrant phenotype of embryo cells that contain unusually large oilbodies that are not normally observed in seeds. Changes in the size of oilbodies caused disruption of storage organelles, altering accumulation of lipids and proteins and causing delay in germination. The aberrant phenotypes were reversed by reintroducing a recombinant oleosin. Based on this direct evidence, we have shown that oleosins are important proteins in seed tissue for controlling oilbody structure and lipid accumulation.