Differential regulation of five Pht1 phosphate transporters from maize (Zea mays L.).

Maize is one of the most important crops in the developing world, where adverse soil conditions and low fertilizer input are the two main constraints for stable food supply. Understanding the molecular and biochemical mechanisms involved in nutrient uptake is expected to support the development of future breeding strategies aimed at improving maize productivity on infertile soils. Phosphorus is the least mobile macronutrient in the soils and it is often limiting plant growth. In this work, five genes encoding Pht1 phosphate transporters which contribute to phosphate uptake and allocation in maize were identified. In phosphate-starved plants, transcripts of most of the five transporters were present in roots and leaves. Independent of the phosphate supply, expression of two genes was predominant in pollen or in roots colonized by symbiotic mycorrhizal fungi, respectively. Interestingly, high transcript levels of the mycorrhiza-inducible gene were also detectable in leaves of phosphate-starved plants. Thus, differential expression of Pht1 phosphate transporters in maize suggests involvement of the encoded proteins in diverse processes, including phosphate uptake from soil and transport at the symbiotic interface in mycorrhizas, phosphate (re)translocation in the shoot, and phosphate uptake during pollen tube growth.

Crystal structure of UDP-N-acetylglucosamine enolpyruvyltransferase, the target of the antibiotic fosfomycin.

BACKGROUND: The ever increasing number of antibiotic resistant bacteria has fuelled interest in the development of new antibiotics and other antibacterial agents. The major structural element of the bacterial cell wall is the heteropolymer peptidoglycan and the enzymes of peptidoglycan biosynthesis are potential targets for antibacterial agents. One such enzyme is UDP-N-acetylglucosamine enolpyruvyltransferase (EPT) which catalyzes the first committed step in peptidoglycan biosynthesis: the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 3-hydroxyl of UDP-N-acetylglucosamine (UDPGlcNAc). EPT is of potential pharmaceutical interest because it is inhibited by the broad spectrum antibiotic fosfomycin. RESULTS: The crystal structure of substrate-free EPT has been determined at 2.0 A resolution. The structure reveals a two-domain protein with an unusual fold (inside out alpha/beta barrel) which is built up from the sixfold repetition of one folding unit. The only repetitive element in the amino acid sequence is a short motif, Leu-X3-Gly(Ala), which is responsible for the formation of hydrogen-bond interactions between the folding units. An enzyme which catalyzes a similar reaction to EPT, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), has a very similar structure despite an amino acid sequence identity of only 25%. To date, only these two enzymes appear to display this characteristic fold. CONCLUSIONS: The present structure reflects the open conformation of the enzyme which is probably stabilized through two residues, a lysine and an arginine, located in the cleft between the domains. Binding of the negatively charged UDPGlcNAc to these residues could neutralize the repulsive force between the two domains, thereby allowing the movement of a catalytically active cysteine residue towards the cleft.

Superinduction of phenylalanine ammonia-lyase in gherkin hypocotyls caused by the inhibitor, L-alpha-aminooxy-beta-phenylpropionic acid.

The extractable activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5) and the concentration of sugar esters of p-coumaric and ferulic acids in the hypocotyls of etiolated gherkin seedlings increase upon irradiation with white light. Treatment of intact seedlings with the phenylalanine ammonia-lyase inhibitors alpha-aminooxyacetic acid and L-alpha-aminooxy-beta-phenylpropionic acid during illumination causes enhanced formation of the lyase and reduces the accumulation of hydroxycinnamic acids. Enzyme activity in excised hypocotyl segments floating on buffer increases in the dark as well as in the light, while hydroxycinnamic acids accumulate only in the light. Phenylalanine ammonia-lyase formation in the segments is inhibited by cinnamic acid and, to a lesser extent, p-coumaric acid, while it is slightly enhanced by caffeic acid and is not affected by ferulic acid. Aminooxyphenylpropionate dramatically promotes phenylalanine ammonia-lyase formation in the segments in darkness and light prevents the accumulation of hydroxycinnamic acids in the light. Aminooxyphenylpropionate does not, however, affect the time course of apparent lyase formation and decay. Cinnamic acid, the product of the lyase reaction, antagonizes the effect of aminooxyphenylpropionate. It is proposed that the reaction product(s) are involved to some extent in the regulation of the pool of active lyase in the hypocotyl tissue.

Functional reconstitution of the malate carrier of barley mesophyll vacuoles in liposomes.

The malate carrier of barley (Hordeum vulgare L.) mesophyll vacuoles was highly purified by chromatography on hydroxyapatite followed by affinity-chromatography using 5-amino-1,2,3-benzenetricarboxylic acid as ligand. The carrier, reconstituted in asolectin liposomes, had properties similar to those described previously for the carrier in intact vacuoles (Martinoia, E., Flügge, U.I., Kaiser, G., Heber, U. and Heldt, H.W. (1985) Biochim. Biophys. Acta 806, 311-319). The apparent Km for malate uptake was 2-3 mM, and the uptake was inhibited by other carboxylic acids (preferentially tricarboxylic). The sulfhydryl reagent, p-chloromercuribenzenesulfonate, as well as the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, also inhibited malate uptake. The transport was dependent on the membrane potential with an optimum at about 35 mV.

Only the Mature Form of the Plastidic Chorismate Synthase Is Enzymatically Active.

Coding regions of a cDNA for precursor and mature chorismate synthase (CS), a plastidic enzyme, from Corydalis sempervirens were expressed in Escherichia coli as translational fusions to glutathione-S-transferase. Fusion proteins were purified, and precursor and mature forms of CS were then released by proteolytic cleavage with factor Xa. Although mature CS was enzymatically active after release, activity could be detected neither for the precursor CS nor for corresponding glutathione-S-transferase fusion proteins. In contrast, two other shikimate pathway enzymes (shikimate kinase and 5-enol-pyruvylshikimate-3-phosphate synthase) have previously been shown to be as enzymatically active as their respective higher molecular weight precursors. By expression of unfused, mature CS from C. sempervirens in E. coli, it was possible to obtain large quantities of enzymatically active CS protein compared to yields from plant cell cultures. Expression levels in E. coli approached 1% of total soluble protein. No differences were found between authentic CS isolated from cell cultures and CS expressed in and purified from E. coli, which made possible a more detailed biochemical characterization of CS. Quaternary structure analysis of the purified mature CS indicated that the enzyme exists as a dimer, in contrast to the active tetrameric structures determined for E. coli and Neurospora crassa enzymes.

Inhibition of lignin formation by L-alpha-aminooxy-beta-phenylpropionic acid, an inhibitor of phenylalanine ammonia-lyase.

Mungbean (Vigna radiata (L.) Wilczek) seedlings grown for 9 days on filter paper soaked with 0.3 to 1 mM L-alpha-aminooxy-beta-phenylpropionic acid (AOPP), a potent inhibitor of L-phenylalanine ammonia-lyase, had a greatly reduced anthocyanin content, and the cell walls of the xylem vessels did not stain with the phloroglucinol/HCl or safranine/astrablue reagents indicating the absence of lignin-like material. Furthermore, vanillin was detectable in nitro-benzene-oxidized lignin preparations only from control seedlings, but not from AOPP-treated seedlings. Scanning electron microscopy of hypocotyl cross sections revealed collapsed xylem vessels in seedlings grown in the presence of AOPP indicating that lignin is required for resistance against the tensile forces in the conducting cells of the xylem. AOPP enhanced the growth of cultured cells of Lonicera prolifera Rehd. while it inhibited the production of extracellular material that gave a positive reaction with phloroglucinol/HCl.

The UDP-N-acetylglucosamine 1-carboxyvinyl-transferase of Enterobacter cloacae. Molecular cloning, sequencing of the gene and overexpression of the enzyme.

The UDP-N-acetylglucosamine 1-carboxyvinyltransferase (enolpyruvyltransferase, EC 2.5.1.7) which catalyses the first committed step in the biosynthesis of the bacterial cell-wall peptidoglycan was purified to near homogeneity from Enterobacter cloacae and the NH2-terminal amino-acid sequence determined. Using the polymerase chain reaction a 53-bp DNA fragment was synthesized; this fragment encodes the NH2-terminal sequence of the enzyme. A clone was then isolated which contained an open reading frame of 1257 bp coding for a protein of 419 amino acids. This protein was overexpressed 100-fold in transformed Escherichia coli cells and shown to possess the enolpyruvyltransferase activity. The overall amino-acid sequence of the enolpyruvyltransferase is significantly similar to that of the 5-enolpyruvylshikimate 3-phosphate synthase, the only other enzyme known to catalyse the transfer of the enolpyruvate moiety of phosphoenolpyruvate to a substrate.

ABI1 of Arabidopsis is a protein serine/threonine phosphatase highly regulated by the proton and magnesium ion concentration.

The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, maintenance of seed dormancy, and inhibition of plant growth. All three responses are regulated by the ABI1 gene product. The ABI1 protein (ABI1p) has been characterized as a protein serine/threonine phosphatase of type 2C that is highly affected in its activity by changes in the proton and magnesium ion concentrations. In the ABA-insensitive mutant abi1 of Arabidopsis thaliana a single amino acid exchange in the primary structure results in both a dominant insensitive phenotype and a strongly reduced protein phosphatase activity in vitro by possibly impairing metal ion coordination.

Cloning and expression in yeast of a higher plant chorismate mutase. Molecular cloning, sequencing of the cDNA and characterization of the Arabidopsis thaliana enzyme expressed in yeast.

Chorismate mutase (EC 5.4.99.5) catalyzes the first step in the branch of the shikimate pathway which leads to the aromatic amino acids, phenylalanine and tyrosine. We have isolated a cDNA for this enzyme from the higher plant, Arabidopsis thaliana, by complementing a yeast strain (aro7) with a cDNA library from A. thaliana. This is the first chorismate mutase cDNA isolated from a plant. It encodes a protein of 334 amino acids. The identity of the deduced amino acid sequence is 41% to the chorismate mutase sequence from Saccharomyces cerevisiae. The N-terminal portion of the deduced amino acid sequence has no homology to the S. cerevisiae sequence but resembles known plastid-specific transit peptides. The A. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids, as previously described for plastidic chorismate mutase isozymes.

Differential expression of genes coding for ABC transporters after treatment of Arabidopsis thaliana with xenobiotics.

ATP-binding cassette (ABC) transporters are thought to be involved in many cellular detoxification mechanisms. Performing a BLAST search, we found four distinct expressed sequence tags (EST) of Arabidopsis thaliana highly similar to the human and fungal glutathione-conjugate ABC transporters. We studied the expression of the corresponding genes in response to various xenobiotics in an effort to gain information on their function. The abundance of transcripts corresponding to one of the genes (EST1) was not affected by the various compounds tested, whereas the abundance of transcripts corresponding to the other three genes (EST2, EST3, EST4) was increased by 1-chloro-2,4-dinitrobenzene, primisulfuron and IRL 1803. Treatment of Arabidopsis with either primisufuron or IRL 1803 resulted in a more than 40-fold increase in EST2-specific transcripts.

Time-resolved solid-state REDOR NMR studies of UDP N-acetylglucosamine enolpyruvyl transferase.

The new method of time-resolved solid-state rotational echo double resonance (REDOR) NMR spectroscopy introduced recently by this laboratory has been applied to the enzyme uridine N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase (EPT), with the goal of probing the interactions between reactive species and their enzyme active site. The approach has been used in a qualitative fashion with the enzyme-inhibitor and enzyme-intermediate complexes of uniformly 15N-labeled UDP-NAG EPT, trapped under steady-state and pre-steady-state conditions. A different set of intermolecular interactions between the substrates UDP-NAG, UDP-NAG plus 3-Z-fluorophosphoenolpyruvate, covalent O-phosphothioketal, and UDP-NAG plus phosphoenolpyruvate trapped under time-resolved conditions (after 50 ms reaction time), and the EPT enzyme active site were observed, and this is contrasted to a similar study of the interactions in a related enzyme, 5-enolpyruvyl-shikimate-3-phosphate synthase.

A herbicide antidote (safener) induces the activity of both the herbicide detoxifying enzyme and of a vacuolar transporter for the detoxified herbicide.

In plants potentially toxic compounds are ultimately deposited in the large central vacuole. In this report we show that isolated barley mesophyll vacuoles take up the glucoside conjugate of the herbicide derivate [5-hydroxyphenyl]primisulfuron. Transport is stimulated by Mg-ATP and is distinct from that previously described for glutathione conjugates. Treatment of barley with different herbicide antidotes (safeners) revealed that the safener cloquintocet-mexyl doubles the vacuolar transport activities for both the glutathione and glucoside conjugates. Stimulation of the uptake of the metolachlor-glutathione conjugate was the result of an increased uptake velocity whereas the Km remained unaltered, suggesting that the higher activity was due to a higher expression of the transporter. These results indicate that modulation of vacuolar transport activities are an integral part of the detoxification mechanism of plants.

Mechanism of Action of the Herbicide 2-Chloro-3(4-chlorophenyl) Propionate and its Methyl Ester: Interaction with Cell Responses Mediated by Auxin.

Chlorfenprop-methyl (the herbicidal component of BIDISIN®), and to a lesser extent the free acid, chlorfenprop, inhibit auxin mediated cell responses in coleoptiles of Avena sativa L. and Zea mays L., such as cell elongation, auxin-uptake, -transport and -metabolism, acidification of growth media, and binding of naphthyl-I-acetic acid to auxin-specific binding sites in homo-genates of corn coleoptiles. Within a very narrow concentration range (1 to 2µM) chlorfenprop-methyl arrests growth from 0 to 100% in sensitive cultivars. The compound displays neither auxin-nor anti-auxin-activity, and only the l(-)-enantiomer is active. The interaction of the herbicide with auxin at the level of membranes is proposed.

Differential expression of three purple acid phosphatases from potato.

Three cDNAs encoding purple acid phosphatase (PAP) were cloned from potato (Solanum tuberosum L. cv. Désirée) and expression of the corresponding genes was characterised. StPAP1 encodes a low-molecular weight PAP clustering with mammalian, cyanobacterial, and other plant PAPs. It was highly expressed in stem and root and its expression did not change in response to phosphorus (P) deprivation. StPAP2 and StPAP3 code for high-molecular weight PAPs typical for plants. Corresponding gene expression was shown to be responsive to the level of P supply, with transcripts of StPAP2 and StPAP3 being most abundant in P-deprived roots or both stem and roots, respectively. Root colonisation by arbuscular mycorrhizal fungi had no effect on the expression of any of the three PAP genes. StPAP1 mRNA is easily detectable along the root axis, including root hairs, but is barely detectable in root tips. In contrast, both StPAP2 and StPAP3 transcripts are abundant along the root axis, but absent in root hairs, and are most abundant in the root tip. All three PAPs described contain a predicted N-terminal secretion signal and could play a role in extracellular P scavenging, P mobilisation from the rhizosphere, or cell wall regeneration.

Biomass allocation is an important determinant of the tannin concentration in growing plants.

BACKGROUND AND AIMS: Condensed tannins (CTs) in the diet affect consumers in a concentration-dependent manner. Because of their importance in plant defence against herbivores and pathogens as well as their potential application against gastrointestinal parasites of ruminants in agronomy, an understanding of the seasonal dynamics of CT concentrations during plant growth is essential. METHODS: Over a vegetation period, CT concentrations in leaves, stems and roots and the biomass proportions between these organs were investigated in Onobrychis viciifolia, Lotus corniculatus and Cichorium intybus. Based on the experimental data, a model has been suggested to predict CT concentrations in harvestable biomass of these species. KEY RESULTS: During the experiment, leaf mass fractions of plants decreased from 85, 64, 85 to 30, 18, 39 % d. wt in Onobrychis, Lotus and Cichorium, respectively, and proportions of stems and roots increased accordingly. While CT concentrations almost doubled in leaves in Onobrychis (from 52 to 86 mg g(-1) d. wt, P<0.001) and Lotus (from 25 to 54 mg g(-1) d. wt, P<0.001), they were stable at low levels in expanding leaves of Cichorium (5 mg g(-1) d. wt) and in stems and roots of all investigated species. Due to an inverse effect of the increasing CT concentrations in leaves and simultaneous dilution from increasing proportions of 'CT-poor' stems, CT concentrations in harvestable biomass were stable over time in all investigated species: 62, 26 and 5 mg g(-1) d. wt for Onobrychis, Lotus and Cichorium, respectively. CONCLUSIONS: As a consequence of the unequal distribution of tannins in different plant parts and due to the changing biomass proportions between them, various herbivores (e.g. a leaf-eating insect and a grazing ruminant) may find not only different concentrations of CT in their diets but also different CT dynamics during the season. For the prediction of seasonal variations of CT concentrations, biomass allocation and accumulation of none-CT plant material are likely to be as important predictors as the knowledge of CT synthesis and its regulation.

Evidence against the occurrence of adenosine-3':5'-cyclic monophosphate in higher plants.

Previous reports on the incorporation of [(14)C]adenine into adenosine-3':5'-cyclic monophosphate (cyclic AMP) in oat (Avena sativa L.) and maize (Zea mays L.) coleoptile sections, chick-pea (Cicer arietinum L.) embryos and barley (Hordeum vulgare L.) aleurone layers were reexamined. Separation of labelled nucleotides on DEAE-Sephadex A 25 showed that a peak of (14)C activity, previously considered to be cyclic AMP, is not identical with this compound. Attempts to detect the cyclic nucleotide by means of a highly specific protein-kinase assay in various plant tissues (Nicotiana tabacum L., tissue culture; Catharanthus roseus Don., tissue culture; Lycopersicon esculentum Mill, seedlings; Nicotiana tabacum, pith parenchyma; Avena sativa, coleoptiles) failed even though up to 100 g of plant material was extracted and a number of control experiments were carried out to insure that cyclic AMP, if present in the extracts, could be measured.

Metabolism of 1-aminoethylphosphinate generates acetylphosphinate, a potent inhibitor of pyruvate dehydrogenase.

The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate.

Degradation of the Phosphonate Herbicide Glyphosate by Arthrobacter atrocyaneus ATCC 13752.

Of nine authentic Arthrobacter strains tested, only A. atrocyaneus ATCC 13752 was capable of using the herbicide glyphosate [N-(phosphonomethyl)glycine] as its sole source of phosphorus. Contrary to the previously isolated Arthrobacter sp. strain GLP-1, which degrades glyphosate via sarcosine, A. atrocyaneus metabolized glyphosate to aminomethylphosphonic acid. The carbon of aminomethylphosphonic acid was entirely converted to CO(2). This is the first report on glyphosate degradation by a bacterial strain without previous selection for glyphosate utilization as a source of phosphorus.