Mode of Action of Jasmonates

Grain development of the maternal effect shrunken endosperm mutant seg8 was analysed by comprehensive molecular, biochemical and histological methods. The most obvious finding was de‐regulation of ABA levels, which were lower compared to wild‐type during the pre‐storage phase but higher during the transition from cell division/differentiation to accumulation of storage products. Ploidy levels and ABA amounts were inversely correlated in the developing endosperms of both mutant and wild‐type, suggesting an influence of ABA on cell‐cycle regulation. The low ABA levels found in seg8 grains between anthesis and beginning endosperm cellularization may result from a gene dosage effect in the syncytial endosperm that causes impaired transfer of ABA synthesized in vegetative tissues into filial grain parts. Increased ABA levels during the transition phase are accompanied by higher chlorophyll and carotenoid/xanthophyll contents. The data suggest a disturbed ABA‐releasing biosynthetic pathway. This is indicated by up‐regulation of expression of the geranylgeranyl reductase (GGR) gene, which may be induced by ABA deficiency during the pre‐storage phase. Abnormal cellularization/differentiation of the developing seg8 endosperm and reduced accumulation of starch are phenotypic characteristics that reflect these disturbances. The present study did not reveal the primary gene defect causing the seg8 phenotype, but presents new insights into the maternal/filial relationships regulating barley endosperm development.

To elucidate the molecular mechanisms underlying pathogen‐associated molecular pattern (PAMP)‐induced defense responses in potato (Solanum tuberosum ), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep‐13, a PAMP from Phytophthora , induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive‐like cell death. We have previously shown that SA is required for Pep‐13‐induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12‐oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F‐box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep‐13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep‐13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA‐deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans .

The benzylisoquinoline alkaloids are a highly diverse group of about 2500 compounds which accumulate in a species‐specific manner. Despite the numerous compounds which could be identified, the biosynthetic pathways and the participating enzymes or cDNAs could be characterized only for a few selected members, whereas the biosynthesis of the majority of the compounds is still largely unknown. In an attempt to characterize additional biosynthetic steps at the molecular level, integration of alkaloid and transcript profiling across Papaver species was performed. This analysis showed high expression of an expressed sequence tag (EST) of unknown function only in Papaver somniferum varieties. After full‐length cloning of the open reading frame and sequence analysis, this EST could be classified as a member of the class II type O ‐methyltransferase protein family. It was related to O ‐methyltransferases from benzylisoquinoline biosynthesis, and the amino acid sequence showed 68% identical residues to norcoclaurine 6‐O ‐methyltransferase. However, rather than methylating norcoclaurine, the recombinant protein methylated norreticuline at position seven with a K m of 44 μm using S ‐adenosyl‐l ‐methionine as a cofactor. Of all substrates tested, only norreticuline was converted. Even minor changes in the benzylisoquinoline backbone were not tolerated by the enzyme. Accordingly, the enzyme was named norreticuline 7–O ‐methyltransferase (N7OMT). This enzyme represents a novel O ‐methyltransferase in benzylisoquinoline metabolism. Expression analysis showed slightly increased expression of N7OMT in P. somniferum varieties containing papaverine, suggesting its involvement in the partially unknown biosynthesis of this pharmaceutically important compound.

The first step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by two isoforms of 1‐deoxy‐d‐ xylulose 5‐phosphate synthase (DXS1 and DXS2). In Medicago truncatula , MtDXS1 and MtDXS2 genes exhibit completely different expression patterns. Most prominently, colonization by arbuscular mycorrhizal (AM) fungi induces the accumulation of certain apocarotenoids (cyclohexenone and mycorradicin derivatives) correlated with the expression of MtDXS2 but not of MtDXS1. To prove a distinct function of DXS2, a selective RNAi approach on MtDXS2 expression was performed in transgenic hairy roots of M. truncatula. Repression of MtDXS2 consistently led to reduced transcript levels in mycorrhizal roots, and to a concomitant reduction of AM‐induced apocarotenoid accumulation. The transcript levels of MtDXS1 remained unaltered in RNAi plants, and no phenotypical changes in non‐AM plants were observed. Late stages of the AM symbiosis were adversely affected, but only upon strong repression with residual MtDXS2‐1 transcript levels remaining below approximately 10%. This condition resulted in a strong decrease in the transcript levels of MtPT4 , an AM‐specific plant phosphate transporter gene, and in a multitude of other AM‐induced plant marker genes, as shown by transcriptome analysis. This was accompanied by an increased proportion of degenerating and dead arbuscules at the expense of mature ones. The data reveal a requirement for DXS2‐dependent MEP pathway‐based isoprenoid products to sustain mycorrhizal functionality at later stages of the symbiosis. They further validate the concept of a distinct role for DXS2 in secondary metabolism, and offer a novel tool to selectively manipulate the levels of secondary isoprenoids by targeting their precursor supply.

Cation‐ and S ‐adenosyl‐l ‐methionine (AdoMet)‐dependent plant natural product methyltransferases are referred to as CCoAOMTs because of their preferred substrate, caffeoyl coenzyme A (CCoA). The enzymes are encoded by a small family of genes, some of which with a proven role in lignin monomer biosynthesis. In Arabidopsis thaliana individual members of this gene family are temporally and spatially regulated. The gene At1g67990 is specifically expressed in flower buds, and is not detected in any other organ, such as roots, leaves or stems. Several lines of evidence indicate that the At1g67990 transcript is located in the flower buds, whereas the corresponding CCoAOMT‐like protein, termed AtTSM1, is located exclusively in the tapetum of developing stamen. Flowers of At1g67990 RNAi‐suppressed plants are characterized by a distinct flower chemotype with severely reduced levels of the N  ′,N  ′′‐ bis‐(5‐hydroxyferuloyl)‐N  ′′′‐sinapoylspermidine compensated for by N1 ,N5 ,N10 ‐tris‐(5‐hydroxyferuloyl)spermidine derivative, which is characterized by the lack of a single methyl group in the sinapoyl moiety. This severe change is consistent with the observed product profile of AtTSM1 for aromatic phenylpropanoids. Heterologous expression of the recombinant protein shows the highest activity towards a series of caffeic acid esters, but 5‐hydroxyferuloyl spermidine conjugates are also accepted substrates. The in vitro substrate specificity and the in vivo RNAi‐mediated suppression data of the corresponding gene suggest a role of this cation‐dependent CCoAOMT‐like protein in the stamen/pollen development of A. thaliana .

Plants of the order Ranunculales, especially members of the species Papaver , accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum ) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum , 69 show increased expression in morphinan alkaloid‐containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7‐epi ‐salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo‐keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.

Gene expression patterns covering over 10 000 seed‐expressed sequences were analyzed by macroarray technology in maternal tissue (mainly pericarp) and filial endosperm and embryo during barley seed development from anthesis until late maturation. Defined sets of genes showing distinct expression patterns characterized both tissue type and major developmental phases. The analysis focused on regulatory networks involved in programmed cell death (PCD) and abscisic acid (ABA)‐mediated maturation. These processes were similar in the different tissues, but typically involved the expression of alternative members of a common gene family. The analysis of co‐expressed gene sets and the identification of cis regulatory elements in orthologous rice gene ‘promoter’ regions suggest that PCD in the pericarp is mediated by distinct classes of proteases and is under the hormonal control of both jasmonic acid (JA) and ethylene via ethylene‐responsive element binding protein (EREBP) transcription factors (TFs). On the other hand, PCD in endosperm apparently involves only the ethylene pathway, but employs distinct gene family members from those active in the pericarp, and a different set of proteases and TFs. JA biosynthetic genes are hardly activated. Accordingly, JA levels are high in the pericarp but low in the endosperm during middle and late developmental stages. Similarly, genes acting in the deduced ABA biosynthetic pathway and signaling network differ between endosperm and embryo. ABA in the endosperm appears to exert an influence over storage product synthesis via SNF1 kinase. In the embryo, ABA seems to influence the acquisition of desiccation tolerance via ABA response element binding factors, but the data also suggest the existence of an ABA‐independent but interactive pathway acting via the dehydration‐responsive element binding (DREB) 2A TF.

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2), a non‐radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of 1O2. Vitamin B6 that quenches 1O2 in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of 1O2, indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild‐type level. The flu mutant was also crossed with the jasmonic acid (JA)‐depleted mutant opr3 , and with the JA, OPDA and dinor OPDA (dnOPDA)‐depleted dde2‐2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA‐induced suppression of H2O2/superoxide‐dependent cell death reported earlier, JA promotes singlet oxygen‐mediated cell death in flu , whereas other oxylipins such as OPDA and dnOPDA antagonize this death‐inducing activity of JA.

The allene oxide cyclase (AOC)‐catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue‐specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i) activation of systemin‐dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii) the tissue‐specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii) the tissue‐specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.

Thionins are a group of antimicrobial polypeptides that form part of the plant's defense mechanism against pathogens. The Thi 2.1 thionin gene of Arabidopsis thaliana has been shown to be inducible by jasmonic acid (JA), an oxylipin‐like hormone derived from oxygenated linolenic acid and synthesized via the octadecanoid pathway. The JA‐dependent regulation of the Thi 2.1 gene has been exploited for setting up a genetic screen for the isolation of signal transduction mutants that constitutively express the Thi 2.1 gene. Ten cet‐mutants have been isolated which showed a c onstitutive e xpression of the t hionin gene. Allelism tests revealed that they represent at least five different loci. Some mutants are dominant, others recessive, but all cet mutations behaved as monogenic traits when backcrossed with Thi 2.1‐GUS plants. Some of the mutants overproduce JA and its bioactive precursor 12‐oxophytodienoic acid (OPDA) up to 40‐fold while others have the same low levels as the control wildtype plants. Two of the mutants showed a strong induction of both the salicylic acid (SA)‐ and the JA‐dependent signaling pathways, while the majority seems to be affected only in the octadecanoid pathway. The Thi 2.1 thionin gene and the Pdf 1.2 defensin gene are activated independently, though both are regulated by JA. The cet‐mutants, except for one, also show a spontaneous leaf cell necrosis, a reaction often associated with the systemic acquired resistance (SAR) pathway.