Mapping of interaction domains of putative telomere-binding proteins AtTRB1 and AtPOT1b from Arabidopsis thaliana

We previously searched for interactions between plant telomere-binding proteins and found that AtTRB1, from the single-myb-histone (Smh) family, interacts with the Arabidopsis POT1-like-protein, AtPOT1b, involved in telomere capping. Here we identify domains responsible for that interaction. We also map domains in AtTRB1 responsible for interactions with other Smh-family-members. Our results show that the N-terminal OB-fold-domain of AtPOT1b mediates the interaction with AtTRB1. This domain is characteristic for POT1- proteins and is involved with binding the G-rich-strand of telomeric DNA. AtPOT1b also interacts with AtTRB2 and AtTRB3. The central histone-globular-domain of AtTRB1 is involved with binding to AtTRB2 and 3, as well as to AtPOT1b. AtTRB1-heterodimers with other Smh-family-members are more stable than AtTRB1-homodimers. Our results reveal interaction networks of plant telomeres.     

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.     

Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana

Epithiospecifier protein (ESP) is a protein that catalyses formation of epithionitriles during glucosinolate hydrolysis. In vitro assays with a recombinant ESP showed that the formation of epithionitriles from alkenylglucosinolates is ESP and ferrous ion dependent. Nitrile formation in vitro however does not require ESP but only the presence of Fe(II) and myrosinase. Ectopic expression of ESP in Arabidopsis thaliana Col-5 under control of the strong viral CaMV 35S promoter altered the glucosinolate product profile from isothiocyanates towards the corresponding nitriles.     

Expression and characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana

The present study reports the recombinant expression, purification, and partial characterization of a typical aspartic proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris. The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant aspartic proteinases with activity in acid pH range, maximal activity at pH 4.0, K_m of 44 μM, and k_cat of 55 s⁻¹ using a synthetic substrate. The enzyme was inhibited by pepstatin A.     

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2_1–64) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2_1–64 and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.     

The pleiotropic mutation dar1 affects plant architecture in Arabidopsis thaliana

Shoot architecture is shaped upon the organogenic activity of the shoot apical meristem (SAM). Such an activity relies on the balance between the maintenance of a population of undifferentiated cells in the centre of the SAM and the recruitment of organ founder cells at the periphery. A novel mutation in Arabidopsis thaliana, distorted architecture1 (dar1), is characterised by disturbed phyllotaxy of the inflorescence and consumption of the apical meristem late in development. SEM and light microscopy analyses of the dar1 SAM reveal an abnormal partitioning of meristematic domains, and mutations known to affect the SAM structure and function were found to interact with dar1. Moreover, the mutant shows an alteration of the root apical meristem (RAM) structure. Those observations support the hypothesis that DAR1 has a role in meristem maintenance and it is required for the normal development of Arabidopsis inflorescence during plant life.     

Peroxidase activity of annexin 1 from Arabidopsis thaliana

On the basis of earlier reports suggesting that annexin A1 from Arabidopsis thaliana (AnnAt1) participates in limiting the excessive levels of reactive oxygen species during oxidative burst in plants, we examined the sensitivity of recombinant AnnAt1 to hydrogen peroxide and its peroxidase activity. Purified recombinant protein remains mostly alpha-helical and binds to lipids in a calcium-dependent manner. Upon oxidation recombinant AnnAt1 exhibits a tendency to form dimers in vitro. AnnAt1 is also sensitive to the presence of reducing agents, suggesting that AnnAt1 is a redox sensor in plant cells. Moreover, using two independent methods we found that AnnAt1 displayed peroxidase activity which is probably related to the presence of a heme-binding domain within AnnAt1, as present in other peroxidases. Indeed, site-directed mutagenesis within this domain resulted in a complete abrogation of the activity of AnnAt1. Furthermore, this activity was found to be sensitive to the phosphorylation state of the protein.     

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.     

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.     

Xylogalacturonan exists in cell walls from various tissues of Arabidopsis thaliana

Evidence is presented for the presence of xylogalacturonan (XGA) in Arabidopsis thaliana. This evidence was obtained by extraction of pectin from the seeds, root, stem, young leaves and mature leaves of A. thaliana, followed by treatment of these pectin extracts with xylogalacturonan hydrolase (XGH). Upon enzymatic treatment, XGA oligosaccharides were primarily produced from pectin extracts obtained from the young and mature leaves and to a lesser extent from those originating from the stem of A. thaliana. The oligosaccharide GalA(3)Xyl was predominantly formed from these pectin extracts. No XGA oligosaccharides were detected in digests of pectin extracts from the seeds and roots. A low number of XGA oligosaccharides was obtained from pectins of A. thaliana. This indicates a uniform distribution of xylose in XGA from A. thaliana. The predominant production of GalA(3)Xyl, as well as the release of linear GalA oligosaccharides pointed to a lower degree of xylose substitution in XGA from A. thaliana than in XGA from apple and potato. The estimated amount of XGA accounted for approximately 2.5%, 7% and 6% (w/w) of the total carbohydrate in the pectin fraction of the stem, young leaves and mature leaves, respectively.     

cDNA isolation, characterization, and protein intracellular localization of a katanin-like p60 subunit from Arabidopsis thaliana

Summary Katanin, a heterodimeric protein with ATP-dependent microtubule-severing activity, localizes to the centrosome in animal cells. Widespread occurrence is suspected as several species contain homologs to the katanin p60 subunit. Recently we isolated anArabidopsis thaliana cDNA with significant identity to the p60 subunit of sea urchin katanin. Like p60, the encoded protein is a member of the AAA superfamily of ATPases, containing the Walker ATP binding consensus and the signature AAA minimal consensus sequences within a single larger AAA/CAD amino acid motif. Phylogenetic analysis placed the encoded protein in the AAA subfamily of cytoskeleton-interactive proteins, where it formed a strongly supported clade with 4 other members identified as katanin p60 subunits. The clone was named AtKSSArabidopsis thaliana kataninlike protein small subunit). Western blots, performed using a polyclonal antibody raised against recombinant AtKSS, revealed AtKSS is present in protein extracts of all Arabidopsis organs examined. To evaluate potential interactions between AtKSS and the cytoskeleton, the intracellular localization of AtKSS was correlated with that of tubulin. AtKSS was found in perinuclear regions during interphase, surrounding the spindle poles during mitosis, but was absent from the preprophase band and phragmoplast microtubule arrays. These data support the thesis that AtKSS is an Arabidopsis homolog of the p60 subunit of katanin. Its cell cycle-dependent distribution is consistent with microtubule-severing activity, but additional studies will better define its role.     

Natural variation for seed oil composition in Arabidopsis thaliana

The biochemical pathways involved in the biosynthesis and accumulation of storage lipids in seeds have been extensively studied. However, the regulatory mechanisms of those pathways, their environmental interactions and the ecological implications of variation are poorly understood. We have initiated a new approach: the analysis of natural variation in Arabidopsis thaliana. Three hundred and sixty accessions were surveyed for content of oil, very long chain fatty acids (VLCFAs) and polyunsaturated fatty acids (PUFAs) in their seeds. The results revealed extensive natural variation. A core set of accessions, the seeds of which reproducibly contain extreme amounts of oil, VLCFAs and PUFAs have been identified. Reproducible oil content ranged from 34.6 to 46.0% of seed dry weight. VLCFA content ranged from 13.0 to 21.2% of total fatty acids. PUFA content, ranged from 53.3 to 66.1% of total fatty acids. Interactions were also identified for PUFA and VLCFA content of seeds with vernalisation of plants. Mapping of the regions of the genome involved in controlling the traits was conducted in an F(2) population and indicated that natural variation at the loci FAE1 and FAD3 might be involved in the regulation of VLCFA and PUFA content, respectively. A set of accessions, which capture a broad range of the natural variation for these traits available in A. thaliana, has been selected to form a core set which can be used to further dissect the genetics of the regulation of seed lipid traits and to identify the genes involved.     

Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha

CHL1 (AtNRT1.1) is a dual-affinity nitrate transporter of Arabidopsis thaliana, in which phosphorylation at Thr 101 switches CHL1 from low to high nitrate affinity. CHL1 expressed in a Hansenula polymorpha high-affinity nitrate-transporter deficient mutant (Deltaynt1) restores nitrate uptake and growth. These events take place at nitrate concentrations as low as 500 muM, suggesting that CHL1 has a high-affinity for nitrate in yeast. Accordingly, CHL1 expressed in H. polymorpha presents a K (m) for nitrate of about 125 muM. The absence of nitrate, the CHL1 gene inducer, showed the high turnover rate of CHL1 expressed in yeast, which is counteracted by nitrate CHL1 induction. Furthermore, H. polymorpha strains expressing CHL1 become sensitive to 250 muM chlorate, as expected for CHL1 high-affinity behaviour. Given that CHL1 presented high affinity by nitrate, we study the role of CHL1 Thr101 in yeast. Strains producing CHL1Thr101Ala, unable to undergo phosphorylation, and CHL1Thr101Asp, where CHL1 phosphorylation is constitutively mimicked, were used. Yeast strains expressing CHL1Thr101Ala, CHL1Thr101Asp and CHL1 at the same rate showed that Deltaynt1CHL1Thr101Ala is strikingly unable to transport nitrate and contains a very low amount of CHL1 protein; however, Deltaynt1CHL1Thr101Asp restores nitrate uptake and growth, although no significant changes in nitrate affinity were observed. Our results show that CHL1-Thr101 is involved in regulating the levels of CHL1 expressed in yeast and suggest that the phosphorylation of this residue could be involved in targeting this nitrate transporter to the plasma membrane. The functional expression of CHL1 in H. polymorpha reveals that this yeast is a suitable tool for evaluating the real nitrate transport capacity of plant putative nitrate transporters belonging to different families and study their regulation and structure function relationship.     

Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1

Phytochelatin (PC), a class of heavy metal-binding peptides, is synthesized from the tripeptide glutathione (GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS). In the present study, the PC production rate catalyzed by recombinant Arabidopsis PCS1 (rAtPCS1) in the presence of a constant free Cd(II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates. In contrast, the PC production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a maximum, which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased. These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd(II) binds to activate the enzyme. The affinity constant, optimized using a one-site mathematical model, successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or 10 μM) but not for the assay using higher concentrations (50 or 500 μM), where a sigmoidal increase in PCS activity was evident. Furthermore, the PCS activity determined at a constant GSH concentration as a function of Cd(II) concentration also reached a maximum. These findings demonstrate that rAtPCS1 also possesses a second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect. A two-site mathematical model was applied successfully to account for the observed phenomena, supporting the suggestion that rAtPCS1 possesses two Cd(II) binding sites.     

Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H₂-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H₂ production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H₂ with a H₂ production rate of 32.9 ml/h/l. The H₂ production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H₂ to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH₂ production with C. butyricum CGS2.     

The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression

Summary The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the Al gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 by DNA fragment extending from –982 to –634 by upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 F1 gene and of several highly expressed plant genes.     

Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana

The 119-amino acid residue prolyl cis/trans isomerase from Arabidopsis thaliana (PIN1At) is similar to the catalytic domain of the human hPIN1. However, PIN1At lacks the N-terminal WW domain that appears to be essential for the hPIN1 function. Here, the solution structure of PIN1At was determined by three-dimensional nuclear magnetic resonance spectroscopy. The PIN1At fold could be superimposed on that of the catalytic domain of hPIN1 and had a 19 residue flexible loop located between strand beta1 and helix alpha1. The dynamical features of this beta1/alpha1-loop, which are characteristic for a region involved in protein-protein interactions, led to exchange broadening in the NMR spectra. When sodium sulfate salt was added to the protein sample, the beta1/alpha1 loop was stabilized and, hence, a complete backbone resonance assignment was obtained. Previously, with a phospho-Cdc25 peptide as substrate, PIN1At had been shown to catalyze the phosphoserine/phosphothreonine prolyl cis/trans isomerization specifically. To map the catalytic site of PIN1At, the phospho-Cdc25 peptide or sodium sulfate salt was added in excess to the protein and chemical shift changes in the backbone amide protons were monitored in the (1)H(N)-(15)N heteronuclear single quantum coherence spectrum. The peptide caused perturbations in the loops between helix alpha4 and strand beta3, between strands beta3 and beta4, in the alpha3 helix, and in the beta1/alpha1 loop. The amide groups of the residues Arg21 and Arg22 showed large chemical shift perturbations upon phospho-Cdc25 peptide or sulfate addition. We conclude that this basic cluster formed by Arg21 and Arg22, both located in the beta1/alpha1 loop, is homologous to that found in the hPIN1 crystal structure (Arg68 and Arg69), which also is involved in sulfate ion binding. We showed that the sulfate group competed for the interaction between PIN1At and the phospho-Cdc25 peptide. In the absence of the WW domain, three hydrophobic residues (Ile33, Ile34, and Leu35) located in the long flexible loop and specific for the plant PIN-type peptidyl prolyl cis/trans isomerases (PPIases) could be an additional interaction site in PIN1At. However, phospho-peptide addition did not affect the resonances of these residues significantly. Electrostatic potential calculations revealed a negatively charged area not found in hPIN1 on the PIN1At molecular surface, which corresponds to the surface shielded by the WW domain in hPIN1. Based on our experimental results and the molecular specificities of the PIN1At enzyme, functional implications of the lack of WW domains in this plant PIN-type PPIase will be discussed.