Structural Model of the Cytosolic Domain of the Plant Ethylene Receptor 1 (ETR1)

Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.     

Functional characterization of domains in AtTRB1, a putative telomere-binding protein in Arabidopsis thaliana

Telomeres are nucleoprotein structures ensuring the stability of eukaryotic chromosome ends. Two protein families, TRFL (TFL-Like) and SMH (Single-Myb-Histone), containing a specific telobox motif in their Myb domain, have been identified as potential candidates involved in a functional nucleoprotein structure analogous to human "shelterin" at plant telomeres. We analyze the DNA-protein interaction of the full-length and truncated variants of AtTRB1, a SMH-family member with a typical structure: N-terminal Myb domain, central H1/5 domain and C-terminal coiled-coil. We show that preferential interaction of AtTRB1 with double-stranded telomeric DNA is mediated by the Myb domain, while the H1/5 domain is involved in non-specific DNA-protein interaction and in the multimerization of AtTRB1.     

Phosphorylation of Arabidopsis response regulator 7 (ARR7) at the putative phospho-accepting site is required for ARR7 to act as a negative regulator of cytokinin signaling

Cytokinins are plant hormones that regulate diverse aspects of plant growth and development. Arabidopsis cytokinin signal transduction utilizes a multi-step two-component signaling (TCS) system by histidyl–aspartidyl phosphorelays. We here show that phosphorylation of ARR7, an A-type response regulator that acts as a negative regulator of cytokinin signaling, is required for its function in plants. Phosphorylation of ARR7 is inhibited in vitro by mutation in a putative phospho-accepting Asp residue into an Asn residue (ARR7^D85N). While ectopic expression of ARR7 decreases root-growth inhibition, callus formation, and cytokinin-inducible gene expression, overexpression of ARR7 ^D85N at the similar level does not generate these phenotypes. ARR7^D85N is localized to the nucleus and the half-life of this mutant protein is similar to that of ARR7 in Arabidopsis mesophyll protoplasts. These results suggest that the phosphorylation of ARR7 is necessary for ARR7-mediated cytokinin response.     

Possible role of light in the maintenance of EIN3/EIL1 stability in Arabidopsis seedlings

To examine the mechanism of EIN3-mediated gene expression by ethylene, the expression patterns of ethylene-inducible genes by ethylene were monitored in Col-0 and ethylene signaling mutants. In Col-0, the inducibility of ACC oxidase by ethylene in light-grown seedlings was much higher than in dark-grown seedlings. While the expression of ACC oxidase was highly increased by ethylene not only in Col-0 but in ein3-1 under light treatment, this pattern was completely abrogated in etiolated ein3-1 seedlings, suggesting the expression of EIN3-mediated ACC oxidase genes could be affected by light. To check if the level of EIN3 and EIL1 was regulated by light, cell-free degradation assays were performed. This resulted in the rapid degradation of these proteins within 1h after adding dark-grown cell extracts and this degradation was retarded by light-grown extracts. Here, we propose that light may act as a negative regulator in the destabilization of EIN3/EIL1.     

Osmotic stress sensing in Populus: components identification of a phosphorelay system

To study the Populus response to an osmotic stress, we have isolated one cDNA encoding a histidine-aspartate kinase (HK1) and four cDNAs encoding histidine-containing phosphotransfer proteins (HPts), HPt1-4. The predicted HK1 protein shares a typical structure with ATHK1 and SLN1 osmosensors. The 4 HPTs are characterized by the histidine phosphotransfer domain. We have shown that HK1 is upregulated during an osmotic stress in hydroponic culture. We have detected an interaction between HK1 and HPt2, using the yeast two-hybrid system. These results suggest the existence of a multi-step phosphorelay pathway probably involved in osmotic stress sensing in Populus.     

CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana

The family of calcineurin B-like (CBL) proteins is a unique group of Ca²⁺ sensors in plants. CBLs relay the calcium signal by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). Extensive studies have demonstrated that the CBL-CIPK complexes mediate plant responses to a variety of external stresses. However, there are few reports on the CBL-CIPK involved in cold stress responses. In this study, we analyzed expression of CIPK7 and CBL1 in Arabidopsis during cold treatments. Expression of CIPK7 was induced by cold, and CIPK7 interacted with CBL1 in vitro. Moreover, affinity chromatography purification of CIPK7 from Arabidopsis plants using CBL1 suggested that CIPK7 may associate with CBL1 in vivo. Expression of CBL1 was cold inducible, and CBL1 had a role in regulating cold response. By comparing expression patterns of CIPK7 between wild-type and cbl1 mutant plants, we found the induction of CIPK7 by cold stress was influenced by CBL1. This is the first report to demonstrate that CIPK7 may play a role in cold response via its interaction with CBL1.     

A new temperature-insensitive allele of the Arabidopsis AXR6/CUL1 locus derived from a missense mutation in the C-terminal RBX1 binding region

We isolated a new recessive allele at the AUXIN RESISTANT6/CULLIN1 (AXR6/CUL1) locus, axr6–101, from an EMS-mutagenized population of Arabidopsis thaliana, the Landsberg erecta ecotype. axr6–101 is auxin resistant and semi-dwarf similar to the other recessive axr6 mutants. The axr6–101 phenotype is caused by the E716K substitution of the CUL1 protein, which is likely to affect its ability to bind to the C-terminal RING domain of RING-box 1 (RBX1). The previously reported allele of AXR6, cul1–7, is caused by a substitution at T510 that binds to the N-terminal β-strand of RBX1. Although cul1–7 shows temperature-sensitive phenotype, the axr6–101 phenotype is largely unaffected by temperature. axr6–101 may provide an important genetic resource for study of the structure−function relationship of the CUL1 protein.     

The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions

The development of mitochondria and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting membranes. The aim of the present work was: (1) to enhance our understanding of the biochemical machinery of mitochondrial membranes and (2) to test the versatility of the procedure developed for the identification of the hydrophobic proteome of the chloroplast envelope [Molecular and Cellular Proteomics 2 (2003) 325-345]. A proteomic analysis was performed, to provide the most exhaustive view of the protein repertoire of these membranes. For this purpose, highly purified mitochondria were prepared from Arabidopsis cultured cells and membrane proteins were extracted. To get a more exhaustive array of membrane proteins from Arabidopsis mitochondria, from the most to the less hydrophobic ones, various extraction procedures (chloroform/methanol extraction, alkaline or saline treatments) were applied. LC-MS/MS analyses were then performed on each membrane subfraction, leading to the identification of more than 110 proteins. The identification of these proteins is discussed with respect to their mitochondrial localization, their physicochemical properties and their implications in the metabolism of mitochondria. In order to provide a new overview of the biochemical machinery of the plant mitochondria, proteins identified during this work were compared to the lists of proteins identified during previous proteomic analyses performed on plant and algae mitochondria (Arabidopsis, pea, Chlamydomonas, rice, etc.). A total of 502 proteins are listed. About 40% of the 114 proteins identified during this work were not identified during previous proteomic studies performed on mitochondria.     

Quantitative analysis of herbivore-induced cytosolic calcium by using a Cameleon (YC 3.6) calcium sensor in Arabidopsis thaliana

Ca²⁺ is a key player in plant cell responses to biotic and abiotic stress. Owing to the central role of cytosolic Ca²⁺ ([Ca²⁺]_cyt) during early signaling and the need for precise determination of [Ca²⁺]_cyt variations, we used a Cameleon YC 3.6 reporter protein expressed in Arabidopsis thaliana to quantify [Ca²⁺]_cyt variations upon leaf mechanical damage (MD), herbivory by 3rd and 5th instar larvae of Spodoptera littoralis and S. littoralis oral secretions (OS) applied to MD. YC 3.6 allowed a clear distinction between MD and herbivory and discriminated between the two larvae instars. To our knowledge this is the first report of quantitative [Ca²⁺]_cyt determination upon herbivory using a Cameleon calcium sensor.     

Desaturase mutants reveal that membrane rigidification acts as a cold perception mechanism upstream of the diacylglycerol kinase pathway in Arabidopsis cells

Membrane rigidification could be the first step of cold perception in poikilotherms. We have investigated its implication in diacylglycerol kinase (DAGK) activation by cold stress in suspension cells from Arabidopsis mutants altered in desaturase activities. By lateral diffusion assay, we showed that plasma membrane rigidification with temperature decrease was steeper in cells deficient in oleate desaturase than in wild type cells and in cells overexpressing linoleate desaturase. The threshold for the activation of the DAGK pathway in each type of cells correlated with this order of rigidification rate, suggesting that cold induced-membrane rigidification is upstream of DAGK pathway activation.     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

TE-Tracker: systematic identification of transposition events through whole-genome resequencing

Background

Transposable elements (TEs) are DNA sequences that are able to move from their location in the genome by cutting or copying themselves to another locus. As such, they are increasingly recognized as impacting all aspects of genome function. With the dramatic reduction in cost of DNA sequencing, it is now possible to resequence whole genomes in order to systematically characterize novel TE mobilization in a particular individual. However, this task is made difficult by the inherently repetitive nature of TE sequences, which in some eukaryotes compose over half of the genome sequence. Currently, only a few software tools dedicated to the detection of TE mobilization using next-generation-sequencing are described in the literature. They often target specific TEs for which annotation is available, and are only able to identify families of closely related TEs, rather than individual elements.

Results

We present TE-Tracker, a general and accurate computational method for the de-novo detection of germ line TE mobilization from re-sequenced genomes, as well as the identification of both their source and destination sequences. We compare our method with the two classes of existing software: specialized TE-detection tools and generic structural variant (SV) detection tools. We show that TE-Tracker, while working independently of any prior annotation, bridges the gap between these two approaches in terms of detection power. Indeed, its positive predictive value (PPV) is comparable to that of dedicated TE software while its sensitivity is typical of a generic SV detection tool. TE-Tracker demonstrates the benefit of adopting an annotation-independent, de novo approach for the detection of TE mobilization events. We use TE-Tracker to provide a comprehensive view of transposition events induced by loss of DNA methylation in Arabidopsis. TE-Tracker is freely available at http://www.genoscope.cns.fr/TE-Tracker.

Conclusions

We show that TE-Tracker accurately detects both the source and destination of novel transposition events in re-sequenced genomes. Moreover, TE-Tracker is able to detect all potential donor sequences for a given insertion, and can identify the correct one among them. Furthermore, TE-Tracker produces significantly fewer false positives than common SV detection programs, thus greatly facilitating the detection and analysis of TE mobilization events.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0377-z) contains supplementary material, which is available to authorized users.     

Assessment of 1H NMR spectroscopy and multivariate analysis as a technique for metabolite fingerprinting of Arabidopsis thaliana

An approach to metabolite fingerprinting of crude plant extracts that utilizes 1H nuclear magnetic resonance (NMR) spectroscopy and multivariate statistics has been tested. Using ecotypes of Arabidopsis thaliana as experimental material, a method has been developed for the rapid analysis of unfractionated polar plant extracts, enabling the creation of reproducible metabolite fingerprints. These fingerprints could be readily stored and compared by a variety of chemometric methods. Comparison by principal component analysis using SIMCA-P allowed the generation of residual NMR spectra of the compounds that contributed significantly to the differences between samples. From these plots, conclusions were drawn with respect to the identity and relative levels of metabolites differing between samples.     

Characterization of a heat-stable protein with antimicrobial activity from Arabidopsis thaliana

A heat-stable protein with antimicrobial activity was isolated from Arabidopsis thaliana plants by buffer-soluble extraction and two chromatographic procedures. The results of MALDI-TOF analysis revealed that the isolated protein shares high sequence identity with aspen SP1. To determine the exact antimicrobial properties of this protein, a cDNA encoding the protein was isolated from an A. thaliana leaf cDNA library and named AtHS1. AtHS1 mRNA was induced by exposure to external stresses, such as salicylic acid and jasmonic acid. We also analyzed the antimicrobial activity of recombinant AtHS1 expressed in Escherichia coli. This protein inhibited pathogenic fungal strains, except for Phytophthora infestans and Phytophthora nicotianae, and it exhibited antibacterial activity against E. coli and Staphylococcus aureus. These results suggest that AtHS1 shows good potential for use as a natural material in the study of antimicrobial agents.     

The structure of "defective in induced resistance" protein of Arabidopsis thaliana, DIR1, reveals a new type of lipid transfer protein

Screening of transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants defective in systemic acquired resistance led to the characterization of dir1-1 (defective in induced resistance [systemic acquired resistance, SAR]) mutant. It has been suggested that the protein encoded by the dir1 gene, i.e., DIR1, is involved in the long distance signaling associated with SAR. DIR1 displays the cysteine signature of lipid transfer proteins, suggesting that the systemic signal could be lipid molecules. However, previous studies have shown that this signature is not sufficient to define a lipid transfer protein, i.e., a protein capable of binding lipids. In this context, the lipid binding properties and the structure of a DIR1-lipid complex were both determined by fluorescence and X-ray diffraction. DIR1 is able to bind with high affinity two monoacylated phospholipids (dissociation constant in the nanomolar range), mainly lysophosphatidyl cholines, side-by-side in a large internal tunnel. Although DIR1 shares some structural and lipid binding properties with plant LTP2, it displays some specific features that define DIR1 as a new type of plant lipid transfer protein. The signaling function associated with DIR1 may be related to a specific lipid transport that needs to be characterized and to an additional mechanism of recognition by a putative receptor, as the structure displays on the surface the characteristic PxxP structural motif reminiscent of SH3 domain signaling pathways.