Robert Kramell

(+)-7- iso -Jasmonoyl- L -isoleucine is the endogenous bioactive jasmonate

Hormone-triggered activation of the jasmonate signaling pathway in Arabidopsis thaliana requires SCF(COI1)-mediated proteasome degradation of JAZ repressors. (-)-JA-L-Ile is the proposed bioactive hormone, and SCF(COI1) is its likely receptor. We found that the biological activity of (-)-JA-L-Ile is unexpectedly low compared to coronatine and the synthetic isomer (+)-JA-L-Ile, which suggests that the stereochemical orientation of the cyclopentanone-ring side chains greatly affects receptor binding. Detailed GC-MS and HPLC analyses showed that the (-)-JA-L-Ile preparations currently used in ligand binding studies contain small amounts of the C7 epimer (+)-7-iso-JA-L-Ile. Purification of each of these molecules demonstrated that pure (-)-JA-L-Ile is inactive and that the active hormone is (+)-7-iso-JA-L-Ile, which is also structurally more similar to coronatine. In addition, we show that pH changes promote conversion of (+)-7-iso-JA-L-Ile to the inactive (-)-JA-L-Ile form, thus providing a simple mechanism that can regulate hormone activity through epimerization.

Induction of Jasmonate Biosynthesis in Arbuscular Mycorrhizal Barley Roots

Colonization of barley (Hordeum vulgare cv Salome) roots by an arbuscular mycorrhizal fungus, Glomus intraradices Schenck & Smith, leads to elevated levels of endogenous jasmonic acid (JA) and its amino acid conjugate JA-isoleucine, whereas the level of the JA precursor, oxophytodienoic acid, remains constant. The rise in jasmonates is accompanied by the expression of genes coding for an enzyme of JA biosynthesis (allene oxide synthase) and of a jasmonate-induced protein (JIP23). In situ hybridization and immunocytochemical analysis revealed that expression of these genes occurred cell specifically within arbuscule-containing root cortex cells. The concomitant gene expression indicates that jasmonates are generated and act within arbuscule-containing cells. By use of a near-synchronous mycorrhization, analysis of temporal expression patterns showed the occurrence of transcript accumulation 4 to 6 d after the appearance of the first arbuscules. This suggests that the endogenous rise in jasmonates might be related to the fully established symbiosis rather than to the recognition of interacting partners or to the onset of interaction. Because the plant supplies the fungus with carbohydrates, a model is proposed in which the induction of JA biosynthesis in colonized roots is linked to the stronger sink function of mycorrhizal roots compared with nonmycorrhizal roots.

Disruption of adenosine-5'-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 5′-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 3′-phosphoadenosine 5′-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normal growth rates.

Agrobacterium tumefaciens Promotes Tumor Induction by Modulating Pathogen Defense in Arabidopsis thaliana

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis–Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria.

Structure–activity relations of substituted, deleted or stereospecifically altered jasmonic acid in gene expression of barley leaves

Abstract Jasmonic acid and 66 structurally related compounds were tested to find the structural requirements which induce the expression of jasmonate-responsive genes in barley. An intact cyclopentanone ring as well as a pentenyl side chain exhibiting only minor alterations are necessary for this activity. The (−)-enantiomeric and the (+)-7- iso -enantiomeric structure increase activity of jasmonoyl compounds.

Amino acid conjugates of jasmonic acid induce jasmonate-responsive gene expression in barley (Hordeum vulgare L.) leaves

Leaves of barley (Hordeum vulgare L. cv. Salome) treated with jasmonic acid (JA), its methyl ester (JM), or its amino acid conjugates exhibit up‐regulation of specific genes and down‐regulation of house‐keeping genes. This transcriptional regulation exhibits several specificities. (i) The (−)‐enantiomers are more active, and conjugates are mainly active if they carry an l‐amino acid moiety. (ii) The various JA‐responsive genes respond differentially to enantiomeric and chiralic forms. (iii) Both JA and its amino acid conjugates exhibiting no or negligible interconversion induce/repress genes.

The moss Physcomitrella patens contains cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology.

• Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)-12-oxo-phytodienoic acid (cis-(+)-OPDA), were isolated from the moss Physcomitrella patens. • Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13-hydroperoxy linolenic acid (13-HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12-hydroperoxy arachidonic acid (12-HPETE). • In protonema and gametophores the occurrence of cis-(+)-OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis-(+)-OPDA was detected. • Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.

Coronalon: a powerful tool in plant stress physiology

Coronalon, a synthetic 6‐ethyl indanoyl isoleucine conjugate, has been designed as a highly active mimic of octadecanoid phytohormones that are involved in insect and disease resistance. The spectrum of biological activities that is affected by coronalon was investigated in nine different plant systems specifically responding to jasmonates and/or 12‐oxo‐phytodienoic acid. In all bioassays analyzed, coronalon demonstrated a general strong activity at low micromolar concentrations. The results obtained showed the induction of (i) defense‐related secondary metabolite accumulation in both cell cultures and plant tissues, (ii) specific abiotic and biotic stress‐related gene expression, and (iii) root growth retardation. The general activity of coronalon in the induction of plant stress responses together with its simple and efficient synthesis suggests that this compound might serve as a valuable tool in the examination of various aspects in plant stress physiology. Moreover, coronalon might become employed in agriculture to elicit plant resistance against various aggressors.

Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis

Benzylisoquinoline alkaloids constitute a group of about 2,500 structures and are mainly produced by plants of the order Ranunculales. But only the opium poppy, Papaver somniferum, and Papaver setigerum are able to produce morphine. In this study, we started to investigate by gene expression analysis the molecular basis for this exceptional biosynthetic ability. A sequencing project from P. somniferum seedlings was initiated using a method based on the amplified fragment length polymorphism technique that resulted in 849 UniGenes. These cDNAs were analysed on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species, which accumulate other benzylisoquinolines instead of morphine. Three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species were identified. Whereas two showed no significant homology to any known protein, one putatively encoded an O-methyltransferase. Analysis of substrate specificity of the heterologously expressed protein and mass spectrometric identification of the enzymatic products identified this protein as S-adenosyl-L-methionine:(R,S)-3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (EC 2.1.1.116). Unlike other O-methyltransferases of different positional specificities implicated in benzylisoquinoline metabolism, the enzyme only accepted tetrahydroxylated tetrahydrobenzylisoquinolines as substrates; methylation was tolerated only at the 6-hydroxy position.

Evolution of morphine biosynthesis in opium poppy.

Benzylisoquinoline alkaloids (BIAs) are a group of nitrogen-containing plant secondary metabolites comprised of an estimated 2500 identified structures. In BIA metabolism, (S)-reticuline is a key branch-point intermediate that can be directed into several alkaloid subtypes with different structural skeleton configurations. The morphinan alkaloids are one subclass of BIAs produced in only a few plant species, most notably and abundantly in the opium poppy (Papaver somniferum). Comparative transcriptome analysis of opium poppy and several other Papaver species that do not accumulate morphinan alkaloids showed that known genes encoding BIA biosynthetic enzymes are expressed at higher levels in P. somniferum. Three unknown cDNAs that are co-ordinately expressed with several BIA biosynthetic genes were identified as enzymes in the pathway. One of these enzymes, salutaridine reductase (SalR), which is specific for the production of morphinan alkaloids, was isolated and heterologously overexpressed in its active form not only from P. somniferum, but also from Papaver species that do not produce morphinan alkaloids. SalR is a member of a class of short chain dehydrogenase/reductases (SDRs) that are active as monomers and possess an extended amino acid sequence compared with classical SDRs. Homology modelling and substrate docking revealed the substrate binding site for SalR. The amino acids residues conferring salutaridine binding were compared to several members of the SDR family from different plant species, which non-specifically reduce (-)-menthone to (+)-neomenthol. Previously, it was shown that some of these proteins are involved in plant defence. The recruitment of specific monomeric SDRs from monomeric SDRs involved in plant defence is discussed.