Inflorescence-specific genes from Arabidopsis thaliana encoding glycine-rich proteins

Genomic and cDNA clones for three inflorescence-specific genes from Arabidopsis thaliana were isolated and characterized. The genes are tandemly organized in the genome on a 10 kb fragment. The expression of these genes is coordinately regulated in a developmental and organ-specific pattern. They are expressed predominantly in anthers at the later stage of flower development. The primary structure of the encoded gene products exhibits comparable features consisting of a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Little homology is observed either between the glycine-rich domain of the three genes or with previously described glycine-rich proteins from other plant species.     

Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen

Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturation labeling techniques. One hundred and three proteins differentially expressed (p value≤0.01) in pollen germinated for 6h compared with un-germination mature pollen, and 98 spots, which represented 71 proteins, were identified by LC-MS/MS. By bioinformatics analysis, 50 proteins were identified as secreted proteins. These proteins were mainly involved in cell wall modification and remodeling, protein metabolism and signal transduction. Three of the differentially expressed proteins were randomly selected to determine their subcellular localizations by transiently expressing YFP fusion proteins. The results of subcellular localization were identical with the bioinformatics prediction. Based on these data, we proposed a model for apoplastic proteins functioning in pollen germination and pollen tube growth. These results will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth.     

Gene atlas of iron-containing proteins in Arabidopsis thaliana

Iron (Fe) is an essential element for the development and physiology of plants, owing to its presence in numerous proteins involved in central biological processes. Here, we established an exhaustive, manually curated inventory of genes encoding Fe-containing proteins in Arabidopsis thaliana, and summarized their subcellular localization, spatiotemporal expression and evolutionary age. We have currently identified 1068 genes encoding potential Fe-containing proteins, including 204 iron-sulfur (Fe-S) proteins, 446 haem proteins and 330 non-Fe-S/non-haem Fe proteins (updates of this atlas are available at https://conf.arabidopsis.org/display/COM/Atlas+of+Fe+containing+proteins ). A fourth class, containing 88 genes for which iron binding is uncertain, is indexed as ‘unclear’. The proteins are distributed in diverse subcellular compartments with strong differences per category. Interestingly, analysis of the gene age index showed that most genes were acquired early in plant evolutionary history and have progressively gained regulatory elements, to support the complex organ-specific and development-specific functions necessitated by the emergence of terrestrial plants. With this gene atlas, we provide a valuable and updateable tool for the research community that supports the characterization of the molecular actors and mechanisms important for Fe metabolism in plants. This will also help in selecting relevant targets for breeding or biotechnological approaches aiming at Fe biofortification in crops.     

The Hsp90 family of proteins in Arabidopsis thaliana

The 90-kDa heat shock protein (Hsp90) is an essential molecular chaperone in eukaryotic cells, with key roles in the folding and activation of proteins involved in signal transduction and control of the cell cycle. A search for Hsp90 sequences in the Arabidopsis thaliana genome revealed that this family includes 7 members. The AtHsp90-1 through AtHsp90-4 proteins constitute the cytoplasmic subfamily, whereas the AtHsp90-5, AtHsp90-6, and AtHsp90-7 proteins are predicted to be within the plastidial, mitochondrial, and endoplasmic reticulum compartments, respectively. The deduced amino acid sequences of each of the cytoplasmic proteins contains the highly conserved C-terminal pentapeptide MEEVD. All of the AtHsp90 sequences include a conserved adenosine triphosphate-binding domain, whereas only the cytoplasmic and endoplasmic reticulum-resident sequences include an adjacent charged linker domain that is common in mammalian and yeast sequences. The occurrence of multiple AtHsp90 proteins in the cytoplasm and of family members in other subcellular compartments suggests a range of specific functions and target polypeptides.     

Identification of novel families of membrane proteins from the model plant Arabidopsis thaliana

MOTIVATION: The completion of the Arabidopsis genome offers the first opportunity to analyze all of the membrane protein sequences of a plant. The majority of integral membrane proteins including transporters, channels, and pumps contain hydrophobic alpha-helices and can be selected based on TransMembrane Spanning (TMS) domain prediction. By clustering the predicted membrane proteins based on sequence, it is possible to sort the membrane proteins into families of known function, based on experimental evidence or homology, or unknown function. This provides a way to identify target sequences for future functional analysis. RESULTS: An automated approach was used to select potential membrane protein sequences from the set of all predicted proteins and cluster the sequences into related families. The recently completed sequence of Arabidopsis thaliana, a model plant, was analyzed. Of the 25,470 predicted protein sequences 4589 (18%) were identified as containing two or more membrane spanning domains. The membrane protein sequences clustered into 628 distinct families containing 3208 sequences. Of these, 211 families (1764 sequences) either contained proteins of known function or showed homology to proteins of known function in other species. However, 417 families (1444 sequences) contained only sequences with no known function and no homology to proteins of known function. In addition, 1381 sequences did not cluster with any family and no function could be assigned to 1337 of these.     

Mitochondrial localization of Arabidopsis thaliana Isu Fe-S scaffold proteins

Isu are scaffold proteins involved in iron-sulfur cluster biogenesis and playing a key role in yeast mitochondria and Escherichia coli. In this work, we have characterized the Arabidopsis thaliana Isu gene family. AtIsu1,2,3 genes encode polypeptides closely related to their bacterial and eukaryotic counterparts. AtIsu expression in a Saccharomyces cerevisiae Deltaisu1Deltanfu1 thermosensitive mutant led to the growth restoration of this strain at 37 degrees C. Using Isu-GFP fusions expressed in leaf protoplasts and immunodetection in organelle extracts, we have shown that Arabidopsis Isu proteins are located only into mitochondria, supporting the existence of an Isu-independent Fe-S assembly machinery in plant plastids.     

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.     

An Arabidopsis thaliana protein homologous to cyanobacterial high-light-inducible proteins

An Arabidopsis thaliana cDNA clone encoding a novel 110 amino acid thylakoid protein has been sequenced. The in vitro synthesized protein is taken up by intact chloroplasts, inserted into the thylakoid membrane and the transit peptide is cleaved off during this process. The mature protein is predicted to contain 69 amino acids, to form one membrane-spanning -helix and to have its N-terminus at the stromal side of the thylakoid membrane. The protein showed similarity to the LHC, ELIP and PsbS proteins of higher plants, but more pronounced to the high-light-inducible proteins (HLIPs) of cyanobacteria and red algae, to which no homologue previously has been detected in higher plants. As for HLIP and ELIP, high light increases the mRNA levels of the corresponding gene. Sequence comparisons indicate that the protein may bind chlorophyll and form dimers in the thylakoid membrane. The level of expression of the protein seems to be far lower than that of normal PSI and PSII subunits.     

Functional characterization of selected LEA proteins from Arabidopsis thaliana in yeast and in vitro

Main conclusion

Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying. To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are “atypical”, since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.     

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.     

Arabidopsis thaliana hairy roots for the production of heterologous proteins

To evaluate the ability of Arabidopsis thaliana hairy roots to produce heterologous proteins, hypocotyls were transformed with Rhizobium rhizogenes harbouring a green fluorescent protein gene (gfp) fused to a plant signal peptide sequence. Hairy root transgenic lines were generated from wild-type or mutant genotypes. A line secreted GFP at 130 mg/l of culture medium. Unlike as was previously found with turnip hairy roots, a His-tag was still attached to approximately 50?% of the protein. Control of the pH and addition of a protease inhibitor to the culture medium resulted in up to 87?% of the GFP retaining the His-tag. A. thaliana hairy roots expressing the human serpina1 (α-1-antitrypsin) gene secreted the protein, which was visible on a PAGE gel. Protein activity in the culture medium was demonstrated using an elastase inhibition assay. A. thaliana hairy roots can now be considered for the production of heterologous proteins, making it possible to mine the numerous genetic resources for enhancing protein production and quality.     

Characterization of Arabidopsis thaliana methyl-CpG-binding domain (MBD) proteins

Cytosine methylation at symmetrical CpG and CpNpG sequences plays a key role in the epigenetic control of plant growth and development; yet, the way by which the methylation signal is interpreted into a functional state has not been elucidated. In animals, the methylation signal is recognized by methyl-CpG-binding domain (MBD) proteins that specifically bind methylated CpG dinucleotides. In Arabidopsis thaliana, 12 putative MBD proteins were identified and classified into seven subclasses. Here, we characterized six MBD proteins representing four subclasses (II, III, IV, and VI) of the Arabidopsis MBD family. We found that AtMBD7 (subclass VI), a unique protein containing a double MBD motif, as well as AtMBD5 and AtMBD6 (subclass IV), bind specifically symmetrically methylated CpG sites. The MBD motif derived from AtMBD6, but not from AtMBD2, was sufficient for binding methylated CpG dinucleotides. AtMBD6 precipitated histone deacetylase (HDAC) activity from the leaf nuclear extract. The examined AtMBD proteins neither bound methylated CpNpG sequences nor did they display DNA demethylase activity. Our results suggest that AtMBD5, AtMBD6, and AtMBD7 are likely to function in Arabidopsis plants as mediators of the CpG methylation, linking DNA methylation-induced gene silencing with histone deacetylation.     

Detection of membrane-bound cytokinin-binding proteins in Arabidopsis thaliana cells.

In order to isolate cytokinin-binding proteins (CBPs), we have developed new affinity probes constituted of a cytokinin such as zeatin riboside ([9R]Z) conjugated to a carrier protein. These probes were used for detecting CBPs in an ELISA procedure. The efficiency of the cytokinin conjugate in detecting CBPs was controlled with protein model: proteins having an affinity for cytokinin such as the monoclonal anti-[9R]Z antibodies did bind the cytokinin conjugate whereas proteins unable to bind cytokinin such as bovine serum albumin did not. Using these new affinity probes, we showed that CBPs are present in the membrane fraction of in vitro cultured Arabidopsis thaliana cells. The nature of the protein at the detected binding sites was demonstrated by submitting the microsomal proteins to a proteolytic treatment, which was found to eradicate the binding. Free biologically active cytokinins or monoclonal anti-[9R]Z antibodies inhibited the binding, thus showing the specificity of the interaction. The detected CBPs were partially solubilized from the membranes with potassium chloride, indicating their peripheral membrane location. The separation by anion exchange chromatography of solubilized microsomal proteins revealed the existence of two different CBPs. They were present at higher levels in cells during the exponential growth phase.     

Systematic prediction of autophagy-related proteins using Arabidopsis thaliana interactome data

Autophagy is a self-degradative process that is crucial for maintaining cellular homeostasis by removing damaged cytoplasmic components and recycling nutrients. Such an evolutionary conserved proteolysis process is regulated by the autophagy-related (Atg) proteins. The incomplete understanding of plant autophagy proteome and the importance of a proteome-wide understanding of the autophagy pathway prompted us to predict Atg proteins and regulators in Arabidopsis. Here, we developed a systems-level algorithm to identify autophagy-related modules (ARMs) based on protein subcellular localization, protein–protein interactions, and known Atg proteins. This generates a detailed landscape of the autophagic modules in Arabidopsis. We found that the newly identified genes in each ARM tend to be upregulated and coexpressed during the senescence stage of Arabidopsis. We also demonstrated that the Golgi apparatus ARM, ARM13, functions in the autophagy process by module clustering and functional analysis. To verify the in silico analysis, the Atg candidates in ARM13 that are functionally similar to the core Atg proteins were selected for experimental validation. Interestingly, two of the previously uncharacterized proteins identified from the ARM analysis, AGD1 and Sec14, exhibited bona fide association with the autophagy protein complex in plant cells, which provides evidence for a cross-talk between intracellular pathways and autophagy. Thus, the computational framework has facilitated the identification and characterization of plant-specific autophagy-related proteins and novel autophagy proteins/regulators in higher eukaryotes.