Higher Plant Arabidopsis Thaliana Research Articles

Phylogenetic and Structural Analyses of VPS13 Proteins in Archaeplastida Reveal Their Complex Evolutionary History in Viridiplantae.

VPS13 is a lipid transfer protein family conserved among Eukaryotes and playing roles in fundamental processes involving vesicular transport and membrane expansion including autophagy and organelle biogenesis. VPS13 folds into a long hydrophobic tunnel, allowing lipid transport, decorated by distinct domains involved in protein localization and regulation. Whereas VPS13 organization and function have been extensively studied in yeast and mammals, information in organisms originating from primary endosymbiosis is scarce. In the higher plant Arabidopsis thaliana, four paralogs, AtVPS13S, X, M1, and M2, were identified, AtVPS13S playing a role in the regulation of root growth, cell patterning, and reproduction. In this work, we performed phylogenetic, as well as domain and structural modeling of VPS13 proteins in Archaeplastida in order to understand their general organization and evolutionary history. We confirmed the presence of human VPS13B orthologues in some phyla and described two new VPS13 families presenting a particular domain arrangement: VPS13R in Rhodophytes and VPS13Y in Chlorophytes and Streptophytes. By focusing on Viridiplantae, we were able to draw the evolutionary history of these proteins made by multiple gene gains and duplications as well as domain rearrangements. We showed that some Chlorophytes have only three (AtVPS13M, S, Y) whereas some Charophytes have up to six VPS13 paralogs (AtVPS13M1, M2, S, Y, X, B). We also highlighted specific structural features of VPS13M and X paralogs. This study reveals the complex evolution of VPS13 family and opens important perspectives for their functional characterization in photosynthetic organisms.

Production of 5-aminolevulinic acid from glutamate by overexpressing HemA1 and pgr7 from Arabidopsis thaliana in Escherichia coli.

The important metabolic intermediate 5-aminolevulinic acid (ALA) is useful for cancer treatment or plant growth regulation and has consequently received much attention. In this study, we introduced the HemA1 and pgr7 genes from the higher plant Arabidopsis thaliana into recombinant Escherichia coli to overproduce extracellular 5-aminolevulinic acid via the C5 pathway. In the E. coli BL21 (DE3) strain background, the ALA concentration of the strain expressing both HemA1 and pgr7 was the highest and reached 3080.62mg/L. Among the 7 tested hosts, ALA production was the highest in E. coli Transetta (DE3). In E. coli Transetta GTR/GBP, the expression levels of zwf, gnd, pgl and RhtA were upregulated. Glutamate induced the expression of the GltJ, GltK, GltL and GltS genes that are in involved in glutamate uptake. The recombinant E. coli Transetta GTR/GBP was able to produce 7642mg/L ALA in modified minimal medium supplemented with 10g/L glutamate and 15g/L glucose after 48h of fermentation at 22°C. The results provide persuading evidence for the efficient production of ALA from glucose and glutamate in E. coli expressing A. thaliana HemA1 and pgr7. Further optimization of the fermentation process should be done to improve the ALA production to an industrially relevant level.

Sigma factor 1 in chloroplast gene transcription and photosynthetic light acclimation.

Sigma factors are dissociable subunits of bacterial RNA polymerase that ensure efficient transcription initiation from gene promoters. Owing to their prokaryotic origin, chloroplasts possess a typical bacterial RNA polymerase together with its sigma factor subunit. The higher plant Arabidopsis thaliana contain as many as six sigma factors for the hundred or so of its chloroplast genes. The role of this relatively large number of transcription initiation factors for the miniature chloroplast genome, however, is not fully understood. Using two Arabidopsis T-DNA insertion mutants, we show that sigma factor 1 (SIG1) initiates transcription of a specific subset of chloroplast genes. We further show that the photosynthetic control of PSI reaction center gene transcription requires complementary regulation of the nuclear SIG1 gene at the transcriptional level. This SIG1 gene regulation is dependent on both a plastid redox signal and a light signal transduced by the phytochrome photoreceptor.

Singlet oxygen imaging using fluorescent probe Singlet Oxygen Sensor Green in photosynthetic organisms

Formation of singlet oxygen (1O2) was reported to accompany light stress in plants, contributing to cell signaling or oxidative damage. So far, Singlet Oxygen Sensor Green (SOSG) has been the only commercialized fluorescent probe for 1O2 imaging though it suffers from several limitations (unequal penetration and photosensitization) that need to be carefully considered to avoid misinterpretation of the analysed data. Herein, we present results of a comprehensive study focused on the appropriateness of SOSG for 1O2 imaging in three model photosynthetic organisms, unicellular cyanobacteria Synechocystis sp. PCC 6803, unicellular green alga Chlamydomonas reinhardtii and higher plant Arabidopsis thaliana. Penetration of SOSG differs in both unicellular organisms; while it is rather convenient for Chlamydomonas it is restricted by the presence of mucoid sheath of Synechocystis, which penetrability might be improved by mild heating. In Arabidopsis, SOSG penetration is limited due to tissue complexity which can be increased by pressure infiltration using a shut syringe. Photosensitization of SOSG and SOSG endoperoxide formed by its interaction with 1O2 might be prevented by illumination of samples by a red light. When measured under controlled conditions given above, SOSG might serve as specific probe for detection of intracellular 1O2 formation in photosynthetic organisms.

Zeaxanthin-dependent nonphotochemical quenching does not occur in photosystem I in the higher plant Arabidopsis thaliana

Nonphotochemical quenching (NPQ) is the process that protects the photosynthetic apparatus of plants and algae from photodamage by dissipating as heat the energy absorbed in excess. Studies on NPQ have almost exclusively focused on photosystem II (PSII), as it was believed that NPQ does not occur in photosystem I (PSI). Recently, Ballottari et al. [Ballottari M, et al. (2014) Proc Natl Acad Sci USA 111:E2431-E2438], analyzing PSI particles isolated from an Arabidopsis thaliana mutant that accumulates zeaxanthin constitutively, have reported that this xanthophyll can efficiently induce chlorophyll fluorescence quenching in PSI. In this work, we have checked the biological relevance of this finding by analyzing WT plants under high-light stress conditions. By performing time-resolved fluorescence measurements on PSI isolated from Arabidopsis thaliana WT in dark-adapted and high-light-stressed (NPQ) states, we find that the fluorescence kinetics of both PSI are nearly identical. To validate this result in vivo, we have measured the kinetics of PSI directly on leaves in unquenched and NPQ states; again, no differences were observed. It is concluded that PSI does not undergo NPQ in biologically relevant conditions in Arabidopsis thaliana The possible role of zeaxanthin in PSI photoprotection is discussed.

A model for the ergosterol biosynthetic pathway in Chlamydomonas reinhardtii

ABSTRACTErgosterol and 7-dehydroporiferasterol are the predominant sterols in the membranes of the alga Chlamydomonas reinhardtii Dangeard. Ergosterol is primarily found in most fungi, which produce their end-product sterols by way of the intermediate lanosterol. In contrast, plants rarely make ergosterol and plant sterols are made via the intermediate cycloartenol. The cycloartenol-lanosterol bifurcation has been used as a means to evolutionarily categorize species based on their sterol production. Use of bioinformatics has revealed that the green alga, C. reinhardtii is probably producing sterols using a pathway very similar to that of higher plants. The Chlamydomonas genome was searched for genes that encoded proteins exhibiting high similarity to sterol biosynthetic proteins in the higher plant Arabidopsis thaliana or the yeast Saccharomyces cerevisiae. Genes with the greatest similarity were chosen and annotated. To establish whether these genes were expressed, the presence of their transcripts was determined and quantitative RT-PCR was performed. It was observed that many of the transcripts coordinately increased in abundance after deflagellation, which induces an increase in membrane biosynthesis. This work demonstrates that C. reinhardtii has all of the genes necessary for the biosynthesis of ergosterol and 7-dehydroporiferasterol and that this alga uses the higher plant pathway to make a sterol normally associated with fungi. Evidence is also presented that other organisms commonly known as algae, including chlorophytes, haptophytes, rhodophytes, bacillariophytes, a phaeophyte and a cryptomonad, are likely to utilize the same biosynthetic pathway, making sterols via a cycloartenol intermediate.

Small proteins in cyanobacteria provide a paradigm for the functional analysis of the bacterial micro-proteome.

BackgroundDespite their versatile functions in multimeric protein complexes, in the modification of enzymatic activities, intercellular communication or regulatory processes, proteins shorter than 80 amino acids (μ-proteins) are a systematically underestimated class of gene products in bacteria. Photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of 19 small proteins of less than 50 amino acids. In analogy, previously unstudied small ORFs with similar degrees of conservation might encode small proteins of high relevance also in other functional contexts.ResultsHere we used comparative transcriptomic information available for two model cyanobacteria, Synechocystis sp. PCC 6803 and Synechocystis sp. PCC 6714 for the prediction of small ORFs. We found 293 transcriptional units containing candidate small ORFs ≤80 codons in Synechocystis sp. PCC 6803, also including the known mRNAs encoding small proteins of the photosynthetic apparatus. From these transcriptional units, 146 are shared between the two strains, 42 are shared with the higher plant Arabidopsis thaliana and 25 with E. coli. To verify the existence of the respective μ-proteins in vivo, we selected five genes as examples to which a FLAG tag sequence was added and re-introduced them into Synechocystis sp. PCC 6803. These were the previously annotated gene ssr1169, two newly defined genes norf1 and norf4, as well as nsiR6 (nitrogen stress-induced RNA 6) and hliR1(high light-inducible RNA 1) , which originally were considered non-coding. Upon activation of expression via the Cu2+.responsive petE promoter or from the native promoters, all five proteins were detected in Western blot experiments.ConclusionsThe distribution and conservation of these five genes as well as their regulation of expression and the physico-chemical properties of the encoded proteins underline the likely great bandwidth of small protein functions in bacteria and makes them attractive candidates for functional studies.

Studies of the PfkB Family of Proteins in Arabidopsis thaliana

The phosphofructokinase B (PkfB) family of carbohydrate kinases is a large group that includes members with specificity for a diverse array of carbohydrate substrates, including fructose, ribose, fructose‐6‐phosphate, tagatose‐6‐phosphate, adenosine, myo‐inositol, and others. Although there are 21 genes encoding PfkB family members in the higher plant Arabidopsis thaliana, only a few have been studied in detail. Two Arabidopsis proteins with fructokinase activity were identified and characterized via native PAGE and in‐gel assays, though the genes were not identified. Another class of PfkB proteins identified in Arabidopsis lacks demonstrated enzymatic activity, but is necessary for the accumulation of photosynthetic transcripts in chloroplasts. The fructokinase‐like proteins 1 and 2 (FLN1 and FLN2, respectively) and NARA5 all belong to this class, although the mechanism of how NARA5 exerts it's phenotype remains elusive. FLN1 and 2 have been identified in proteomics studies as members of a large protein complex that interacts with, and likely regulates, the prokaryotic‐type plastid RNA polymerase which specifically transcribes photosynthetic genes in the plastid.We have found that FLN1 and FLN2 interact with each other in an ATP‐dependent manner both in vitro and in vivo. Phenotypic analysis suggests that the presence of both proteins is necessary for proper levels of transcription. Our data on the interaction between FLN1 and FLN2 suggests a mechanism where the FLN proteins act as a biochemical switch to activate transcription under conditions that favor their binding to each other.We have also begun characterization of the remainder of the Arabidopsis PfkB family and have identified a group of seven highly similar Arabidopsis PfkB proteins. These seven proteins were expressed in E. coli and enzymatic assays performed. All are active as fructokinases with high specificity for fructose. T‐DNA mediated inactivation of five of the seven genes resulted in no overt phenotype which hints at possible redundancy between some of these gene products. Using GFP fusions, we have determined that all localize to the cytosol except one, which localizes to plastids. Higher order mutants and biochemical analysis, along with expression analysis will allow us to tease apart the differences and the importance of each of these family members.We have characterized another PfkB protein that appears to be the sole Arabidopsis ribokinase (AtRBSK). Its biochemical properties and cofactor requirements are similar to that of known mammalian and prokaryotic RBSKs. T‐DNA insertion into AtRBSK results in the hyper‐accumulation of ribose in both the roots and leaves of 10 day‐old seedlings compared to wild type. Arabidopsis lacking AtRBSK grow poorly on media supplemented with ribose as the only sugar. We are currently dissecting the pathway that leads to the production of ribose in plants to put AtRBSK into a biological context.Our studies of the PfkB family in Arabidopsis are yielding promising results and will continue to further our understanding of many fundamental processes in plant biology ranging from central metabolism to the regulation of transcription in chloroplasts.Support or Funding InformationThe authors gratefully acknowledge support from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US. Department of Energy (contracts DE‐FG02–09ER16077 and DE‐SC0002175) to JC

Identification and Roles of Photosystem II Assembly, Stability, and Repair Factors in Arabidopsis.

Photosystem II (PSII) is a multi-component pigment-protein complex that is responsible for water splitting, oxygen evolution, and plastoquinone reduction. Components of PSII can be classified into core proteins, low-molecular-mass proteins, extrinsic oxygen-evolving complex (OEC) proteins, and light-harvesting complex II proteins. In addition to these PSII subunits, more than 60 auxiliary proteins, enzymes, or components of thylakoid protein trafficking/targeting systems have been discovered to be directly or indirectly involved in de novo assembly and/or the repair and reassembly cycle of PSII. For example, components of thylakoid-protein-targeting complexes and the chloroplast-vesicle-transport system were found to deliver PSII subunits to thylakoid membranes. Various auxiliary proteins, such as PsbP-like (Psb stands for PSII) and light-harvesting complex-like proteins, atypical short-chain dehydrogenase/reductase family proteins, and tetratricopeptide repeat proteins, were discovered to assist the de novo assembly and stability of PSII and the repair and reassembly cycle of PSII. Furthermore, a series of enzymes were discovered to catalyze important enzymatic steps, such as C-terminal processing of the D1 protein, thiol/disulfide-modulation, peptidylprolyl isomerization, phosphorylation and dephosphorylation of PSII core and antenna proteins, and degradation of photodamaged PSII proteins. This review focuses on the current knowledge of the identities and molecular functions of different types of proteins that influence the assembly, stability, and repair of PSII in the higher plant Arabidopsis thaliana.

Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms.

BackgroundStarch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C.ResultsIn this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII-A, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding.ConclusionsThe results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1598-6) contains supplementary material, which is available to authorized users.

Radiation-induced epigenetic bystander effects demonstrated in Arabidopsis thaliana.

Radiation-induced bystander effects (RIBE) in vivo in the higher plant Arabidopsis thaliana ( A. thaliana ) have been well demonstrated in terms of effects on development and genetics. However, there is not yet robust evidence regarding RIBE-mediated epigenetic changes in plants. To address this, in the current work the roots of A. thaliana seedlings were locally irradiated with 10 Gy of α particles, after which DNA methylation in bystander aerial plants were detected using the methylation-sensitive amplification polymorphism (MSAP) and bisulfite sequencing PCR (BSP). Results showed that irradiation of the roots led to long-distance changes in DNA methylation patterns at some CCGG sites over the whole genome, specifically from hemi-methylation to non-methylation, and the methylation ratios, mainly at CG sites, strongly indicating the existence of RIBE-mediated epigenetic changes in higher plants. Root irradiation also influenced expressions of DNA methylation-related MET1, DRM2 and SUVH4 genes and demethylation-related DML3 gene in bystander aerial plants, suggesting a modulation of RIBE to the methylation machinery in plants. In addition, the multicopy P35S:GUS in A. thaliana line L5-1, which is silenced epigenetically by DNA methylation and histone modification, was transcriptionally activated through the RIBE. The transcriptional activation could be significantly inhibited by the treatment with reactive oxygen species (ROS) scavenger dimethyl sulfoxide (DMSO), indicative of a pivotal role of ROS in RIBE-mediated epigenetic changes. Time course analyses showed that the bystander signaling molecule(s) for transcriptional activation of multicopy P35S:GUS, although of unknown chemical nature, were generated in the root cells within 24 h postirradiation.

Duplication and divergence of the Psb27 subunit of Photosystem II in the green algal lineage

Photosystem II (PSII) is subject to light-induced damage and continuously undergoes repair to restore the damaged photosystems. During repair of PSII, the Psb27 protein interacts with the CP43 subunit of PSII. Two Psb27-like proteins (Psb27-H1 and Psb27-H2) are involved in the repair of PSII in the higher plant Arabidopsis thaliana. Here, we present evidence that duplication and divergence of Psb27 occurred in the green algal lineage and that the Psb27-H2 protein shares c. 30% sequence identity with Psb27-H1. Structural modelling of Arabidopsis thaliana Psb27-H1 and Psb27-H2 indicate that the two proteins have different distributions of surface charge. We suggest that the green algal lineage Psb27-H2 protein occupies a different binding niche than does Psb27-H1.

Localization and Phylogenetic Analysis of Enzymes Related to Organellar Genome Replication in the Unicellular Rhodophyte Cyanidioschyzon merolae

Plants and algae possess plastids and mitochondria harboring their own genomes, which are replicated by the apparatus consisting of DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, and primer removal enzyme. In the higher plant Arabidopsis thaliana, organellar replication-related enzymes (OREs) are similar in plastids and mitochondria because many of them are dually targeted to plastids and mitochondria. In the red algae, there is a report about a DNA replicase, plant/protist organellar DNA polymerase, which is localized to both plastids and mitochondria. However, other OREs remain unclear in algae. Here, we identified OREs possibly localized to organelles in the unicellular rhodophyte Cyanidioschyzon merolae. We then examined intracellular localization of green fluorescent protein-fusion proteins of these enzymes in C. merolae, whose cell has a single plastid and a single mitochondrion and is suitable for localization analysis, demonstrating that the plastid and the mitochondrion contain markedly different components of replication machinery. Phylogenetic analyses revealed that the organelle replication apparatus was composed of enzymes of various different origins, such as proteobacterial, cyanobacterial, and eukaryotic, in both red algae and green plants. Especially in the red alga, many enzymes of cyanobacterial origin remained. Finally, on the basis of the results of localization and phylogenetic analyses, we propose a model on the succession of OREs in eukaryotes.

The Fission Yeast FANCM Ortholog Directs Non-Crossover Recombination During Meiosis

The formation of healthy gametes depends on programmed DNA double-strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway. We also define the Rad51/Dmc1-mediator Swi5-Sfr1 as a major determinant in biasing the recombination process in favor of Mus81, to ensure the appropriate amount of COs to guide meiotic chromosome segregation. The conservation of these proteins from yeast to humans suggests that this interplay may be a general feature of meiotic recombination.

FANCM Limits Meiotic Crossovers

The number of meiotic crossovers (COs) is tightly regulated within a narrow range, despite a large excess of molecular precursors. The factors that limit COs remain largely unknown. Here, using a genetic screen in Arabidopsis thaliana, we identified the highly conserved FANCM helicase, which is required for genome stability in humans and yeasts, as a major factor limiting meiotic CO formation. The fancm mutant has a threefold-increased CO frequency as compared to the wild type. These extra COs arise not from the pathway that accounts for most of the COs in wild type, but from an alternate, normally minor pathway. Thus, FANCM is a key factor imposing an upper limit on the number of meiotic COs, and its manipulation holds much promise for plant breeding.

Molecular characterization and expression analysis of two Na+/H+ antiporter genes in the marine red alga Porphyra yezoensis

Na+/H+ antiporters are known to play a crucial role in pH and Na+ homeostasis. In the present study, we characterized the molecular structures and expression patterns of two Na+/H+ antiporters from the marine red alga Porphyra yezoensis (designated PySOS1 and PyNhaD). The full-length cDNAs of PySOS1 and PyNhaD were 5122 and 1804 bp, and contained open reading frames (ORFs) of 4773 and 1275 bp, respectively. The deduced amino acid sequences showed high similarity to SOS1 and NhaD from the higher plant Arabidopsis thaliana. PySOS1 and PyNhaD contained conserved sequences found in the cation–proton antiporter. Quantitative real-time PCR analysis revealed that both antiporter genes were expressed in both the gametophyte and sporophyte of P. yezoensis. In addition, mRNA expression of PySOS1 and PyNhaD was simultaneously upregulated by light irradiation, suggesting that coordinated activity between the two is important in pH and Na+ homeostasis under light conditions. Moreover, the expression levels of both genes were partially reduced by the photosynthetic inhibitors DCMU and DBMIB, suggesting that upregulation is linked to photosynthesis-related metabolism. These findings provide an initial step towards understanding Na+/H+ antiporters in marine red algae.

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.

Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes

Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic α-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% α-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% α-helical in the dry state. However, the dry protein contained 15% β-sheets. FTIR spectroscopy revealed the β-sheets to be largely due to aggregation. β-Sheet content was reduced and α-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates.

The intrinsically disordered late embryogenesis abundant protein LEA18 from Arabidopsis thaliana modulates membrane stability through binding and folding

Intrinsically disordered proteins (IDPs) constitute a substantial part of cellular proteomes. Late embryogenesis abundant (LEA) proteins are mostly predicted to be IDPs associated with dehydration tolerance in many plant, animal and bacterial species. Their functions, however, are largely unexplored and also their structure and interactions with potential target molecules have only recently been experimentally investigated in a small number of proteins. Here, we report on the structure and interactions with membranes of the Pfam LEA_1 protein LEA18 from the higher plant Arabidopsis thaliana. This functionally uncharacterized positively charged protein specifically aggregated and destabilized negatively charged liposomes. Isothermal titration calorimetry showed binding of the protein to both charged and uncharged membranes. LEA18 alone was largely unstructured in solution. While uncharged membranes had no influence on the secondary structure of LEA18, the protein partially folded into β-sheet structure in the presence of negatively charged liposomes. These data suggest that LEA18 does not function as a membrane stabilizing protein, as suggested for other LEA proteins. Instead, a possible function of LEA18 could be the composition-dependent modulation of membrane stability, e.g., during signaling or vesicle-mediated transport.

Recombinant formate dehydrogenase from Arabidopsis thaliana: Preparation, crystal growth in microgravity, and preliminary X-ray diffraction study

Crystals of high-purity recombinant NAD+-dependent formate dehydrogenase from the higher plant Arabidopsis thaliana (AraFDH) were grown in microgravity in the Modul’-1 protein crystallization apparatus on the International Space Station. The space-grown crystals have larger sizes than those grown on Earth. X-ray diffraction data suitable for determining the three-dimensional structure were collected from the space-grown crystals to a resolution of 1.22 A using an X-ray synchrotron source. The crystals belong to sp. gr. P43212; the unit-cell parameters are a = b = 107.865 A, c = 71.180 A, α = β = γ = 90°.