Putrescine N ‐methyltransferase (PMT) catalyses S ‐adenosylmethionine (SAM)‐dependent methylation of putrescine in tropane alkaloid biosynthesis. PMT presumably evolved from the ubiquitous spermidine synthase (SPDS). SPDS protein structure suggested that only few amino acid exchanges in the active site were necessary to achieve PMT activity. Protein modelling, mutagenesis, and chimeric protein construction were applied to trace back evolution of PMT activity from SPDS. Ten amino acid exchanges in Datura stramonium SPDS dismissed the hypothesis of facile generation of PMT activity in existing SPDS proteins. Chimeric PMT and SPDS enzymes were active and indicated the necessity for a different putrescine binding site when PMT developed.

In Arabidopsis thaliana, mitogen activated protein kinase (MAPK) signaling cascades that contain MPK3, MPK4 and MPK6 have been implicated in various aspects of developmental processes and stress responses. We identified an ethylene response factor (ERF104), which controls innate immunity, to be a specific substrate of MPK6 and showed that ethylene signaling regulates the release of the ERF104 substrate from its kinase. Implications and questions that arise from our findings are addressed. To promote discussions, previously unpublished data, that are rather confounding, are presented and possible explanation provided on how these may fit into our current model.

Mitogen-activated protein kinase (MAPK)–mediated responses are in part regulated by the repertoire of MAPK substrates, which is still poorly elucidated in plants. Here, the in vivo enzyme–substrate interaction of the Arabidopsis thaliana MAP kinase, MPK6, with an ethylene response factor (ERF104) is shown by fluorescence resonance energy transfer. The interaction was rapidly lost in response to flagellin-derived flg22 peptide. This complex disruption requires not only MPK6 activity, which also affects ERF104 stability via phosphorylation, but also ethylene signaling. The latter points to a novel role of ethylene in substrate release, presumably allowing the liberated ERF104 to access target genes. Microarray data show enrichment of GCC motifs in the promoters of ERF104–up-regulated genes, many of which are stress related. ERF104 is a vital regulator of basal immunity, as altered expression in both erf104 and overexpressors led to more growth inhibition by flg22 and enhanced susceptibility to a non-adapted bacterial pathogen.

The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 Å, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties.

Lipoxygenases (LOX; linoleate:oxygen oxidoreductase EC 1.13.11.12) consist of a class of enzymes that catalyze the regio‐ and stereo specific dioxygenation of polyunsaturated fatty acids. Here we characterize two proteins that belong to the less studied class of 9‐LOXs, Solanum tuberosum StLOX1 and Arabidopsis thaliana AtLOX1. The proteins were recombinantly expressed in E. coli and the product specificity of the enzymes was tested against different fatty acid substrates. Both enzymes showed high specificity against all tested C18 fatty acids and produced (9S )‐hydroperoxides. However, incubation of the C20 fatty acid arachidonic acid with AtLOX1 gave a mixture of racemic hydroperoxides. On the other hand, with StLOX1 we observed the formation of a mixture of products among which the (5S )‐hydroperoxy eicosatetraenoic acid (5S‐ H(P)ETE) was the most abundant. Esterified fatty acids were no substrates. We used site directed mutagenesis to modify a conserved valine residue in the active site of StLOX1 and examine the importance of space within the active site, which has been shown to play a role in determining the positional specificity. The Val576Phe mutant still catalyzed the formation of (9S )‐hydroperoxides with C18 fatty acids, while it exhibited altered specificity against arachidonic acid and produced mainly (11S )‐H(P)ETE. These data confirm the model that in case of linoleate 9‐LOX binding of the substrate takes place with the carboxyl‐group first.

Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism.Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry.Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound‐hormone jasmonic acid, followed by the expression of jasmonate‐regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids.Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs.

Oxidative tailoring of C40 carotenoids by double bond-specific cleavage enzymes (carotenoid cleavage dioxygenases, CCDs) gives rise to various apocarotenoids. AtCCD1 generating C13 and C14 apocarotenoids and orthologous enzymes in other plants are the only CCDs acting in the cytosol, while the hitherto presumed C40 substrate is localized in the plastid. A new model for CCD1 action arising from a RNAi-mediated CCD1 gene silencing study in mycorrhizal hairy roots of Medicago truncatula may solve this contradiction. This approach unexpectedly resulted in the accumulation of C27 apocarotenoids but not C40 carotenoids suggesting C27 as the main substrates for CCD1 in planta. It further implies a consecutive two-step cleavage process, in which another CCD performs the primary cleavage of C40 to C27 in the plastid followed by C27 export and further cleavage by CCD1 in the cytosol. We compare the specificities and subcellular locations of the various CCDs and propose the plastidial CCD7 to be the first player in mycorrhizal apocarotenoid biogenesis.

Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5’ and 3’ termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.

Phenylpropanoid polyamine conjugates have been identified in flowers of many plant species. Their presence in Arabidopsis thaliana has only been recently established in flower buds and pollen grains. Annotation and location of a cation-dependent O-methyltransferase AtTSM1 specifically to the tapetum of young flower buds enabled the subsequent identification of several genes with a putative role in phenylpropanoid polyamine conjugate biosynthesis. Based on the analysis of several A. thaliana knockout mutants, a biosynthetic pathway of these conjugates is proposed, which involves two methylation steps catalyzed by different cation-dependent O-methyltransferases, a cytochrome P450 (CYP98A8) catalyzed hydroxylation, and a conjugating acyl transfer performed by a BAHD-like, hydroxycinnamoyl (HC)-transferase. LC/MS based metabolite profiling of the cyp98A8 knockout line identified new feruloyl- and 4-coumaroylspermidine conjugates in the corresponding flowers consistent with a role of this gene in the hydroxylation of these conjugates. A pattern of minor amounts of bis- and tris-acylspermidine conjugates, likely the products of additional HC-transferases were identified in wild type as well as in the mutant lines. Transcript suppression of the genes early in the pathway was observed in knockout or RNAi-lines of the genes encoding late enzymatic steps. The implication of these findings for spermidine conjugate biosynthesis in flower buds of A. thaliana is discussed.

Infections with Phytophthora infestans, the causal agent of potato and tomato late blight disease, are difficult to control and can lead to considerable agricultural losses. Thus, the development of new effective agents against the pathogen is of great interest. In previous work, (E)-4-oxohexadec-2-enoic acid (3) was isolated from Hygrophorus eburneus, which exhibited fungicidal activity against Cladosporium cucumerinum. Here, the inhibitory effect of 3 on P. infestans spore germination and mycelium growth in vitro is demonstrated. The in vivo effect on infections of whole potato plants was investigated by spraying plants with the sodium salt of 3, sodium (2E)-4-oxohexadec-2-enoic acid (4), prior to P. infestans inoculation. Additionally, the influence of 3 on mycelium growth of Colletotrichum coccodes, the causal agent of potato black dot disease, was analyzed. In all approaches, a significant inhibition of pathogen development was achieved. Importantly, the unsaturated fatty acid exerted no toxic effect when sprayed on plants, a prerequisite for its commercial use.