Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH)

Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.     

Functional characterization of the thi1 promoter region from Arabidopsis thaliana

The Arabidopsis thaliana THI1 protein is involved in thiamine biosynthesis and is targeted to both chloroplasts and mitochondria by N-terminal control regions. To investigate thi1 expression, a series of thi1 promoter deletions were fused to the beta-glucuronidase (GUS) reporter gene. Transgenic plants were generated and expression patterns obtained under different environmental conditions. The results show that expression derived from the thi1 promoter is detected early on during development and continues throughout the plant's life cycle. High levels of GUS expression are observed in both shoots and roots during vegetative growth although, in roots, expression is restricted to the vascular system. Deletion analysis of the thi1 promoter region identified a region that is responsive to light. The smallest fragment (designated Pthi322) encompasses 306 bp and possesses all the essential signals for tissue specificity, as well as responsiveness to stress conditions such as sugar deprivation, high salinity, and hypoxia.     

Functional analysis of conserved motifs in the mechanosensitive channel homolog MscS-Like2 from Arabidopsis thaliana

The Mechanosensitive channel of Small conductance (MscS) of Escherichia coli has become an excellent model system for the structural, biophysical, and functional study of mechanosensitive ion channels. MscS, a complex channel with multiple states, contributes to protection against lysis upon osmotic downshock. MscS homologs are widely and abundantly dispersed among the bacterial and plant lineages, but are not found in animals. Investigation into the eukaryotic branch of the MscS family is in the beginning stages, and it remains unclear how much MscS homologs from eukaryotes resemble E. coli MscS with respect to structure, function, and regulation. Here we test the effect of mutating three conserved motifs on the function of MscS-Like (MSL)2, a MscS homolog localized to the plastids of Arabidopsis thaliana. We show that 1) a motif at the top of the cytoplasmic domain, referred to here as the PN(X)(9)N motif, is essential for MSL2 function and for its proper intraplastidic localization; 2) substituting polar residues for two large hydrophobic residues located in the predicted pore-lining transmembrane helix of MSL2 produces a likely gain-of-function allele, as previously shown for MscS; and 3) mis-expression of this allele causes severe defects in leaf growth, loss of chloroplast integrity, and abnormal starch accumulation. Thus, two of the three conserved motifs we analyzed are critical for MSL2 function, consistent with the conservation of structure and function between MscS family members in bacteria and plants. These results underscore the importance of plastidic mechanosensitive channels in the maintenance of normal plastid and leaf morphology.     

Biochemical and molecular characterization of a hydroxyjasmonate sulfotransferase from Arabidopsis thaliana

12-Hydroxyjasmonate, also known as tuberonic acid, was first isolated from Solanum tuberosum and was shown to have tuber-inducing properties. It is derived from the ubiquitously occurring jasmonic acid, an important signaling molecule mediating diverse developmental processes and plant defense responses. We report here that the gene AtST2a from Arabidopsis thaliana encodes a hydroxyjasmonate sulfotransferase. The recombinant AtST2a protein was found to exhibit strict specificity for 11- and 12-hydroxyjasmonate with K(m) values of 50 and 10 microm, respectively. Furthermore, 12-hydroxyjasmonate and its sulfonated derivative are shown to be naturally occurring in A. thaliana. The exogenous application of methyljasmonate to A. thaliana plants led to increased levels of both metabolites, whereas treatment with 12-hydroxyjasmonate led to increased level of 12-hydroxyjasmonate sulfate without affecting the endogenous level of jasmonic acid. AtST2a expression was found to be induced following treatment with methyljasmonate and 12-hydroxyjasmonate. In contrast, the expression of the methyljasmonate-responsive gene Thi2.1, a marker gene in plant defense responses, is not induced upon treatment with 12-hydroxyjasmonate indicating the existence of independent signaling pathways responding to jasmonic acid and 12-hydroxyjasmonic acid. Taken together, the results suggest that the hydroxylation and sulfonation reactions might be components of a pathway that inactivates excess jasmonic acid in plants. Alternatively, the function of AtST2a might be to control the biological activity of 12-hydroxyjasmonic acid.     

Genomic sequence of a calnexin homolog from Arabidopsis thaliana.

Calnexin is a membrane-bound protein of the ER in animal cells (Wada et al., 1991). It shows considerable similarity to the major calcium-sequestering protein of the ER lumen, calreticulin, with two calcium-binding regions--a high-affinity, low-capacity region in the ER lumen and a low-affinity, high-capacity region in the cytoplasm. The protein is postulated to act as a calcium-regulated chaperone during protein maturation (Ou et al., 1993). We have isolated a genomic sequence showing significant homology to the animal gene over the predicted coding sequence (Table I). A partial cDNA from Zea mays was isolated from an expression library made from 6-d coleoptiles (Clontech, Palo Alto, CA). The library was screened using a monoclonal antibody raised against a small number of microsomal proteins resulting from a partial purification of plasma membrane Ca2+ ATPase (Briars et al., 1988). The partial cDNA showed sequence homology to the calcium-binding region common to calreticulin and calnexin. The fragment was used to screen a genomic library constructed from Arabidopsis thaliana (cv Larasbonerecta), and a 15-kb fragment was isolated and subcloned and the relevant subfragments were sequenced. The coding region contains five introns, two in the N-terminal region and three in the C-terminal region. The predicted amino acid sequence shows a high level of homology with the animal calnexin, although the terminal highly acidic calcium-binding region is shorter. A cDNA for a putative homolog of calnexin was isolated from A. thaliana (cv Columbia) by Huang et al.(1993); our coding sequence shows 85% identity and 92% similarity determined by FASTA (Wisconsin Genetics Computer Group package); however, the differences are greater than would be expected between cultivars of the same species. A Southern blot probed with DNA from the central calcium-binding region shows multiple bands. This, combined with the sequence heterogeneity, suggests that calnexin belongs to a family of related genes.     

Functional and structural characterization of the catalytic domain of the starch synthase III from Arabidopsis thaliana

Glycogen and starch are the major energy storage compounds in most living organisms. The metabolic pathways leading to their synthesis involve the action of several enzymes, among which glycogen synthase (GS) or starch synthase (SS) catalyze the elongation of the alpha-1,4-glucan backbone. At least five SS isoforms were described in Arabidopsis thaliana; it has been reported that the isoform III (SSIII) has a regulatory function on the synthesis of transient plant starch. The catalytic C-terminal domain of A. thaliana SSIII (SSIII-CD) was cloned and expressed. SSIII-CD fully complements the production of glycogen by an Agrobacterium tumefaciens glycogen synthase null mutant, suggesting that this truncated isoform restores in vivo the novo synthesis of bacterial glycogen. In vitro studies revealed that recombinant SSIII-CD uses with more efficiency rabbit muscle glycogen than amylopectin as primer and display a high apparent affinity for ADP-Glc. Fold class assignment methods followed by homology modeling predict a high global similarity to A. tumefaciens GS showing a fully conservation of the ADP-binding residues. On the other hand, this comparison revealed important divergences of the polysaccharide binding domain between AtGS and SSIII-CD.     

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.     

Biochemical and molecular characterization of ACH2, an acyl-CoA thioesterase from Arabidopsis thaliana

By using computer-based homology searches of the Arabidopsis genome, we identified the gene for ACH2, a putative acyl-CoA thioesterase. With the exception of a unique 129-amino acid N-terminal extension, the ACH2 protein is 17-36% identical to members of a family of acyl-CoA thioesterases that are found in both prokaryotes and eukaryotes. The eukaryotic homologs of ACH2 are peroxisomal acyl-CoA thioesterases that are up-regulated during times of increased fatty acid oxidation, suggesting potential roles in peroxisomal beta-oxidation. We investigated ACH2 to determine whether it has a similar role in the plant cell. Like its eukaryotic homologs, ACH2 carries a putative type 1 peroxisomal targeting sequence (-SKL(COOH)), and maintains all the catalytic residues typical of this family of acyl-CoA thioesterases. Analytical ultracentrifugation of recombinant ACH2-6His shows that it associates as a 196-kDa homotetramer in vitro, a result that is significant in light of the cooperative kinetics demonstrated by ACH2-6His in vitro. The cooperative effects are most pronounced with medium chain acyl-CoAs, where the Hill coefficient is 3.8 for lauroyl-CoA, but decrease for long chain acyl-CoAs, where the Hill coefficient is only 1.9 for oleoyl-CoA. ACH2-6His hydrolyzes both medium and long chain fatty acyl-CoAs but has highest activity toward the long chain unsaturated fatty acyl-CoAs. Maximum rates were found with palmitoleoyl-CoA, which is hydrolyzed at 21 micromol/min/mg protein. Additionally, ACH2-6His is insensitive to feedback inhibition by free CoASH levels as high as 100 microm. ACH2 is most highly expressed in mature tissues such as young leaves and flowers rather than in germinating seedlings where beta-oxidation is rapidly proceeding. Taken together, these results suggest that ACH2 activity is not linked to fatty acid oxidation as has been suggested for its eukaryotic homologs, but rather has a unique role in the plant cell.     

Characterization of a CENP-C homolog in Arabidopsis thaliana

Centromere protein C (CENP-C) is a component of the kinetochore essential for correct segregation of sister chromatids in mammals. In Arabidopsis thaliana, a single-copy gene encoding a protein homologous to CENP-C has been found by homology in the whole-genome sequence. To investigate the CENP-C homolog (AtCENP-C), we cloned cDNAs by RT-PCR and determined its full-length coding sequence. Antibodies against the synthetic peptide for the C-terminal residues of AtCENP-C detected a polypeptide in Arabidopsis cell extracts on western blots. Immunofluorescence labeling with the antibodies and fluorescence in situ hybridization demonstrated clearly that AtCENP-C is present at the centromeric regions throughout the cell cycle.     

Functional characterization of pathogen-responsive protein AtDabb1 with an antifungal activity from Arabidopsis thaliana

A plant antifungal protein was purified from Arabidopsis thaliana leaves by using a typical procedure consisting of anion exchange chromatography and high-performance liquid chromatography. We determined the amino acid sequence of the purified protein using MALDI-TOF/MS analysis, and found that the sequence matched that of a hypothetical Arabidopsis protein in GenBank (accession number NP_175547). We designated the protein as AtDabb1. After the cDNA encoding the AtDabb1 gene was cloned from an Arabidopsis leaf cDNA library, the recombinant protein was expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the AtDabb1 gene was induced by pathogen-related signaling molecules including salicylic acid and jasmonic acid. These results suggest that AtDabb1 may contribute to the induced plant defense mechanism against diverse pathogenic fungi.     

Cytosolic and plastidic chorismate mutase isozymes from Arabidopsis thaliana: molecular characterization and enzymatic properties

The three aromatic amino acids phenylalanine, tyrosine, and tryptophan are synthesized in the plastids of higher plants. There is, however, biochemical evidence that a cytosolic isoform exists of the enzyme catalysing the first step of that branch of the pathway which is specific for the synthesis of phenylalanine and tyrosine, i.e. chorismate mutase (CM). We now report on the isolation of a cDNA clone encoding a cytosolic CM isozyme from Arabidopsis thaliana that was identified by complementing a CM-deficient Escherichia coli strain. The deduced amino acid sequence of this isozyme was 50% identical to that of a previously isolated plastidic CM, and 41% identical to that of yeast CM. The organ-specific expression patterns of the two CM genes were rather similar, but only the gene encoding the plastidic isozyme was elicitor- and pathogen-inducible. The plastidic CM expressed in E. coli was activated by tryptophan and inhibited by phenylalanine and tyrosine, whereas the cytosolic isozyme was insensitive. The existence of a cytosolic CM isozyme implies that either a cytosolic pathway (partial or complete) for the biosynthesis of phenylalanine and tyrosine exists, or that prephenate, originating from chorismate in the cytosol, is utilized for the synthesis of metabolites other than these two aromatic amino acids.     

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

Preparation and functional characterization of thylakoids from Arabidopsis thaliana

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O₂ and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana

The Rho family of GTPases are in higher eukaryotes divided into 3 major subfamilies; the Rho, Rac and Cdc42 proteins. In plants, however, the Rho family is restricted to one large family of Rac-like proteins. From work with mammalian phagocytes the Rac proteins are known to activate a multicomponent NADPH-dependent oxidase which results in accumulation of H₂O₂, a process termed oxidative burst. In plants a similar oxidative burst is observed and plays an important role in its defence against pathogen infections, suggesting a similar role for the plant Rac-like proteins. The Rho family of GTPases proteins are also involved in control of cell morphology, and are also thought to mediate signals from cell membrane receptors.In a broad search for members of the Ras superfamily in plants, several new small GTP-binding proteins were found. We report here the identification and molecular cloning of 5 rac-like cDNAs from Arabidopsis thaliana, Arac1–5. The Rac-like proteins deduced from the cDNA sequences all share 80–95% homology, but show considerably more diversity on the nucleotide level, indicating that this is an ancient gene family. Four of the rac genes were found to be expressed in all tissues examined, but one gene, Arac2, was expressed exclusively in the root, hypocotyl and stem. Our results show that the rac gene family in A. thaliana consists of at least 10 different genes.     

Functional bioinformatics for Arabidopsis thaliana

MOTIVATION: The genome of Arabidopsis thaliana, which has the best understood plant genome, still has approximately one-third of its genes with no functional annotation at all from either MIPS or TAIR. We have applied our Data Mining Prediction (DMP) method to the problem of predicting the functional classes of these protein sequences. This method is based on using a hybrid machine-learning/data-mining method to identify patterns in the bioinformatic data about sequences that are predictive of function. We use data about sequence, predicted secondary structure, predicted structural domain, InterPro patterns, sequence similarity profile and expressions data. RESULTS: We predicted the functional class of a high percentage of the Arabidopsis genes with currently unknown function. These predictions are interpretable and have good test accuracies. We describe in detail seven of the rules produced.     

Functional bioinformatics for Arabidopsis thaliana

The authors would     

Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana

The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana. Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L. Sullivan and R.D. Vierstra, Proc. Natl. Acad. Sci. USA 86 (1989) 9861-9865). The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa. The protein encoded by AtUBC2-1 is shown to accept ¹²⁵I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions. It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme.