Markus Piotrowski

Ruhr-Universität Bochum | RUB · Pflanzenphysiologie

Nicotianamine Synthase (NAS) catalyzes the biosynthesis of the low-molecular-mass metal chelator nicotianamine (NA) from the 2-aminobutyrate moieties of three S-adenosylmethionine molecules. NA has central roles in metal nutrition and metal homeostasis of flowering plants. The enzymatic function of NAS remains poorly understood. Crystal structures...

Nicotianamine Synthase (NAS) catalyzes the biosynthesis of nicotianamine (NA) from the 2-aminobutyrate moieties of three S -adenosylmethionine molecules. NA has central roles in metal nutrition and metal homeostasis of flowering plants. Despite the availability of crystal structures of archaeal and bacterial NAS-like proteins that carry out simpler...

IRON‐REGULATED TRANSPORTER1 (IRT1) is the root high‐affinity ferrous iron uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous iron (Fe) supplementation. Here we provide evidence supporting a second role of IRT1 in root‐to‐shoot partitioning of Fe. We show that irt1 mutants over‐accumulate Fe...

IRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous iron (Fe) supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot mobilization of Fe. We show that the irt1-2 ( pam42 ) mutant ove...

The beneficial root-colonizing fungus Colletotrichum tofieldiae mediates plant growth promotion (PGP) upon phosphate (Pi) starvation in Arabidopsis thaliana. This activity is dependent on the Trp metabolism of the host, including indole glucosinolate (IG) hydrolysis. Here, we show that C. tofieldiae resolves several Pi starvation-induced molecular...

Nitrilases are commercially important biocatalysts found in diverse taxa that catalyse the hydrolysis of nitriles to amides and acids. In vitro they form rings, terminating spirals or long filaments that seem to be necessary for activity. In vivo , NIT1 assemblies have been observed in Arabidopsis . Substrate size is correlated with the helical twi...

Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues...

Plant terpenoids are a large and highly diverse class of metabolites with an important role in the immune defense. They find wide industrial application as active pharmaceutical ingredients, aroma and fragrance compounds. Several Eremophila sp. derived terpenoids have been documented. To elucidate the terpenoid metabolism, the transcriptome of juve...

Nitrilases consist of a group of enzymes that catalyze the hydrolysis of organic cyanides. They are found ubiquitously distributed in the plant kingdom. Plant nitrilases are mainly involved in the detoxification of ß-cyanoalanine, a side-product of ethylene biosynthesis. In the model plant Arabidopsis thaliana a second group of Brassicaceae-specifi...

BiNGO analysis of selected up- and down-regulated genes in cyp79b2/cyp79b3. After filtering the microarray data according to the arbitrary set values of a p ≤ 0.05 and a log2 ratio > 1.2, gene enrichment and clustering in functional groups was analyzed using the BiNGO software package in Cytoscape 3.4.0. Over- and under-represented GO groups are gi...

Functional expression of C-terminally c-myc-tagged NITRILASE 1 from Arabidopsis thaliana. The NIT1:c-myc fusion construct as well as an empty vector control (pEXP1) were expressed in Escherichia coli as outlined in Material and methods. Aliquots of 12.5 μg of protein were subjected to SDS-gel electrophoresis (12.5%) and immunoblotting. The top pane...

Microarray analysis of the cyp79b2/cyp79b3 double mutant in comparison to wild-type Arabidopsis. RNA from 2 week-old cyp79b2/cyp79b3 and wild-type Arabidopsis seedlings was prepared and subjected to microarray analysis using affimetrix Ath1 genome chips. For each genotype three chips were hybridized and the data statistically analyzed employing the...

BiNGO analysis of selected up- and down-regulated genes in cyp79b2/cyp79b3. Graphical representations of over- and under-represented GO groups are provided in two separate figures.

Primary root elongation of wild type, NIT1ox, nit1-3, and NIT2RNAi mutants grown on media containing either IAN or IAA. Seedlings were grown on media containing risings amounts of IAN and IAA, respectively. Since the obtained transgenic lines for NIT1ox and NIT2RNAi showed no phenotypic differences, only data obtained for NIT1ox-J5 and NIT2RNAi-8 a...

The Biopython Project is a long-running distributed collaborative effort, supported by the Open Bioinformatics Foundation, which develops a freely available Python library for biological computation.We present here details of the latest Biopython release - version 1.66. New features include: extended Bio.KEGG and Bio.Graphics modules to support dra...

The Australian plant Eremophila belongs to the Myoporaceae tribe and includes over 200 different species. Some Eremophila species have been used in the Aboriginal medicine to alleviate symptoms like skin lesions and sore throat [1]. Various studies showed antibacterial effects of different Eremophila species against a wide range of Gram positive ba...

The fungus Venturia inaequalis clone No. 36 isolated from Malus domestica cv. Gloster excretes a melanoprotein of 36 kDa in relatively high amounts during growth in liquid culture. The protein was isolated from the culture medium and purified to homogeneity. It was shown to contain melanin. After raising an antiserum against the isolated protein, t...

The intercellular washing fluid (IWF) of Malus domestica cv. Holsteiner Cox before and after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the leaves was investigated in a comparative manner. SDS-PAGE in combination with ESI Q-ToF mass spectrometry, and homology search in relevant data bases revealed the highly up-regul...

Size exclusion analysis of stromal DLA2 complexes. Stromal proteins (treated with RNase or not) from wild-type CC-406 grown under indicated conditions were separated by SEC (A–C). Fractions 1 to 14 were subjected to protein gel blot analysis using the DLA2 antibody. Molecular masses were calculated by parallel analysis of high molecular mass calibr...

cpPDC activity of SEC fractions. PDC activity was measured from SEC-separated crude thylakoids (A) and stroma (B) fractions 1–6 of mixotrophically grown wild-type cells. Prior to SEC, proteins were treated with RNase or not as indicated. The highest cpPDC activity measured in fraction 1 was set to 100%. Mean values and standard deviations of three...

DLA2 protein accumulation under different growth conditions. A total of 30 µg of whole cell proteins from a cell-wall-deficient wild-type strain grown under mixotrophic (M), heterotrophic (H), or photoautotrophic (P) conditions were separated by SDS-PAGE and subjected to immunoblot analysis. The blot was probed with antibodies against DLA2 and the...

Determination of the copy number of the DLA2 gene in C. reinhardtii by genomic Southern blotting. (A) Schematic representation of the genomic region containing the DLA2 gene (shown: chromosome 3, 1415567–1424567 in JGI v4). Enzyme restriction sites leading to respective restriction fragments shown under (B) are indicated. Grey bar represents the po...

Immunoprecipitation of DLA2 protein and bound RNA. (A) Immunoprecipitation of DLA2 protein. Proteins from crude thylakoids (cTs) of 5 L mixotrophically grown C. reinhardtii cell-wall-deficient wild-type cells were solubilized in lysis buffer (10 mM tricine/KOH, pH 7.8, 10 mM EDTA, 1% Triton X-100, and Roche Complete mini protease inhibitor cocktail...

Alignment of predicted Rossmann folds within the amino acid sequence of PDC-E2 subunits and human GAPDH. For accession numbers, see Figure 2. H. sapiens, HsGAPDH (accession no: NP_002037). Highly conserved residues are written in bold. The Rossmann fold consensus sequence is shown below the alignment as given on the NIH MBI laboratory prediction se...

psbA mRNA accumulation in iDLA2 lines under different growth conditions. For Northern blot analyses (upper panel) of indicated RNAi lines (iDLA2-1, -2, -3), and the control strain (WT-NE) total RNA was extracted by using TRI Reagent (Sigma-Aldrich) according to the manufacturer's instructions. A total of 3 µg were separated electrophoretically, blo...

Lipid accumulation in iDLA2 lines. Lipids from whole cells were isolated according to Rengstl et al. [83] from 50 mL photoautotrophically grown wild-type transformed with the empty vector NE 537 (WT-NE) or DLA2-RNAi lines (iDLA2-1, -2, -3). After extraction, lipids were separated by thin-layer chromatography visualized by incineration. The mayor ch...

Quantification of D1 and D2 protein synthesis rates in DLA2–RNAi lines under various growth conditions. D1 and D2 protein synthesis rates were determined as described in Figure 7. Quantification of signals was performed by using AlphaEaseFC software (Alpha Innotech Corp.) and calculating the ratio of D1 or D2, respectively, over AtpA/B. Values obta...

Prediction of RNA binding residues in E2 subunits of various organisms. Possible RNA binding residues within the amino acid sequence of E2 subunits from C. reinhardtii (A), Synechocystis sp. PCC 6803 (B), S. cerevisiae (C), and H. sapiens (D) were predicted by using the RNAbindR software with set “optimal prediction” (http://bindr.gdcb.iastate.edu/...

Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals aff...

Plants have evolved a variety of mechanisms for dealing with insect herbivory among which chemical defense through secondary metabolites plays a prominent role. Physiological, behavioural and sensorical adaptations to these chemicals provide herbivores with selective advantages allowing them to diversify within the newly occupied ecological niche....

Growth of Pieris rapae and S. littoralis on wildtype and transgenic dhurrin-producing A. thaliana plants. First instar larvae of P. rapae (experiments 1–3) or S. littoralis (experiments 4–6) were allowed to feed on plants of one of the two genotypes of A. thaliana ad libitum. After 10 d, surviving larvae were counted and weighted. The number of ind...

Qualitative analysis of aromatic glucosinolate metabolites in P. rapae feces extracts. Feces samples were from larvae that had fed Col-0 leaves to which 2-phenylethylglucosinolate 2 (A), or benzylglucosinolate 1 (B) had been applied, or from larvae that had ingested leaves with no added glucosinolate (C). Glycine conjugates 5–7 (Fig. 1C) were detec...

The A. thaliana 35S:CYP79A2×Nit2-RNAi double mutant produces benzylglucosinolate, but lacks nitrilase activity. A. Freeze-dried leaves of F1 plants from a cross of A. thaliana 35S:CYP79A2 and A. thaliana Nit2-RNAi were extracted in methanol. Shown are representative LC-MS MRM traces obtained with these extracts in comparison to traces obtained from...

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling,...

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling,...

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All members of the YidC/Oxa1/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that Alb4 is required in assembly and/or stability of the CF1...

Nitrilases, enzymes that catalyze the hydrolysis of organic cyanides, are ubiquitous in the plant kingdom. The typical plant nitrilase is a nitrilase 4 homolog which is involved in the cyanide detoxification pathway. In this pathway, nitrilase 4 converts beta-cyanoalanine, the intermediate product of cyanide detoxification, into asparagine, asparti...

In previous reports we have reported that theobroxide induces characteristic accumulation of allene oxide cyclase (AOC; EC 5.3.99.6) protein and jasmonic acid (JA) in Pharbitis nil. In the present study, PnAOC, an AOC gene from Pharbitis nil was cloned. Immunofluorescence assays indicated that the AOC protein is located in the chloroplast of vascul...

The present study investigated the inhibitory effect on the conidial germination of Venturia inaequalis by using living whole cells, extracellular protein fraction and individual proteins from the non-pathogenic antagonistic bacterium Pseudomonas fluorescens Bk3.The bacterial suspension of P. fluorescens Bk3 from growth in minimal medium showed up...

The potential of plant nitrilases to convert indole-3-acetonitrile into the plant growth hormone indole-3-acetic acid has earned them the interim title of "key enzyme in auxin biosynthesis". Although not widely recognized, this view has changed considerably in the last few years. Recent work on plant nitrilases has shown them to be involved in the...

Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in...

Lotus japonicus accumulates the hydroxynitrile glucosides lotaustralin, linamarin, and rhodiocyanosides A and D. Upon tissue disruption, the hydroxynitrile glucosides are bioactivated by hydrolysis by specific beta-glucosidases. A mixture of two hydroxynitrile glucoside-cleaving beta-glucosidases was isolated from L. japonicus leaves and identified...

Gene expression in chloroplasts is regulated mainly at the posttranscriptional level. In the green alga Chlamydomonas reinhardtii, synthesis of the D2 protein (PsbD), which is the rate-determining subunit for the assembly of photosystem II, depends on the RNA stability factor Nac2. In addition, the RNA binding protein RBP40 has been implicated in t...

Members of the nitrilase 4 (NIT4) family of higher plants catalyze the conversion of β-cyanoalanine to aspartic acid and asparagine, a key step in cyanide detoxification. Grasses (Poaceae) possess two different NIT4 homologs (NIT4A and NIT4B), but none of the recombinant Poaceae enzymes analyzed showed activity with β-cyanoalanine, whereas protein...

During fungal fruiting body development, specialized cell types differentiate from vegetative mycelium. We have isolated a protein from the ascomycete Sordaria macrospora that is not present during vegetative growth but accumulates in perithecia. The protein was sequenced by mass spectrometry and the corresponding gene was termed app (abundant peri...

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+-binding affinity...

The auxin indole-3-acetic acid (IAA), which is essential for plant growth and development, is suggested to be synthesized via several redundant pathways. In maize (Zea mays), the nitrilase ZmNIT2 is expressed in auxin-synthesizing tissues and efficiently hydrolyses indole-3-acetonitrile to IAA. Zmnit2 transposon insertion mutants were compromised i...

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RN...

Cyanoalanine hydratase (E.C. 4.2.1.65) is an enzyme involved in the cyanide detoxification pathway of higher plants and catalyzes the hydrolysis of beta-cyano-L-alanine to asparagine. We have isolated the enzyme from seedlings of blue lupine (Lupinus angustifolius) to obtain protein sequence information for molecular cloning. In contrast to earlier...

The parasitic flagellate Trichomonas vaginalis contains hydrogenosomes, anaerobic organelles related to mitochondria, that generate ATP from the fermentative conversion of pyruvate to acetate, CO2 and molecular hydrogen. Although an essentially anaerobic organism, Trichomonas encounters low oxygen concentrations in its natural habitat and has to pr...

Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catal...

The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in...

In order to understand better the survival and mutual interaction of epiphytic bacteria and fungi on apple plants, bacteria collected from these plants were cultivated on intact adaxial, stoma free cuticle membranes originally obtained from apple. The bacteria were labelled with luciferase genes from Vibrio harveyi in order to follow up their devel...

The bacteriophage T4 alpha- and beta-glucosyltransferases (AGT and BGT) catalyse the transfer of glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine of T4 DNA in an alpha- and beta-conformation, respectively. Following the 3D structure of BGT and a secondary structure alignment of AGT and BGT, we performed a site-directed mutagenesis...

Leaves of apple (Malus domestica cv. Elstar) were infected with a cloned isolate of the apple scab Venturia inaequalis. The intercellular washing fluid (IWF) of these plants was collected and the variation in the composition of proteins in the IWF was analysed by SDS-PAGE and two-dimensional gel electrophoresis during and after the infection with V...

A recent study demonstrated that gravi‐incompetent coleoptile‐less seedlings of rye exhibit gravi‐competence after exogenous application of ethylene. Treatments and conditions which induce and interfere with this phenomenon were analysed in more detail. Aminocyclopropane‐1‐carboxylic acid (ACC) as a precursor of ethylene has similar gravicompetence...

We isolated two nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward beta-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic aci...

The cloning, expression and characterization of plant agmatine iminohydrolase (AIH, also known as agmatine deiminase, EC 3.5.3.12) is described. Recombinant AIH of Arabidopsis thaliana forms dimers and catalyzes the specific conversion of agmatine to N-carbamoylputrescine and ammonia. Biochemical data suggested that cysteine side chains are involve...

A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His(6)-tagged protein and purified to apparent homogeneity by Ni(2+)-chelate affinity chromatography. The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria....

An HPLC/GC-MS/MS technique (high-pressure liquid chromatography in combination with gas chromatography-tandem mass spectrometry) has been worked out to analyze indole-3-acetamide (IAM) with very high sensitivity, using isotopically labelled IAM as an internal standard. Using this technique, the occurrence of IAM in sterile-grown Arabidopsis thalian...

The plastid transcription kinase (PTK), a component of the major RNA polymerase complex from mustard chloroplasts, has been implicated in redox-mediated regulation of plastid gene expression. A cloning strategy to define the PTK gene(s) resulted in the isolation of a full-length cDNA for a protein with overall high homology with the alpha subunit o...

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 microM). Autophosphorylation...

Arabidopsis thaliana expresses four nitrilases, three of which (NIT1, NIT2 and NIT3) are able to convert indole-3-acetonitrile to indole-3-acetic acid (IAA), the plant growth hormone, while the isozyme NIT4 is a beta-cyano-l-alanine hydratase/nitrilase. NIT3 promoter activity is marginal in leaves or roots of vegetative plants and undetectable in b...

Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5'-->3' direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-t...

Nitrilases (nitrile aminohydrolases, EC 3.5.5.1) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases ofArabidopsis thaliana, the isoform NIT4 is the most divergent one, and homologs of NIT4 are also known from species not belonging to the Brassicaceae like Nicotiana tabacum andOryz...

The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protei...

Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent...

About thirty years after the initial identification of 14-3-3 proteins in mammalian brain, they are now thought to be ubiquitous among eukaryotes. We identified five cDNAs encoding 14-3-3 proteins of Nicotiana tabacum L. using a polymerase chain reaction (PCR)-based screening strategy. A phylogenetic analysis was carried out with 14-3-3 amino-acid...

Upon binding to a high-affinity plasma membrane (PM) protein (a member of the 14-3-3 family of regulatory proteins), the fungal phytotoxin fusicoccin (FC) activates the H+- ATPase by hindering the inhibitory interaction of the enzyme’s C-terminus with its catalytic site. Protease protection experiments carried out with sealed PM vesicles of differe...

Protein phosphorylation was analyzed in Bryonia dioica tendrils using in vitro assays. The major microsomal phosphoproteins had apparent molecular masses of 120 (p120), 58 (p58), 45 (p45) and 36 (p36) kDa and contained phosphoserine; p36 was additionally phosphorylated at threonine residues, and p120 was found to be tendril-specific. Phosphorylatio...