Publikationen - Molekulare Signalverarbeitung

Jasmonates are lipid-derived signals that mediate plant stress responses and development processes. Enzymes participating in biosynthesis of jasmonic acid (JA) (1, 2) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants of Arabidopsis and tomato have helped to define the pathway for synthesis of jasmonoyl-isoleucine (JA-Ile), the active form of JA, and to identify the F-box protein COI1 as central regulatory unit. However, details of the molecular mechanism of JA signaling have only recently been unraveled by the discovery of JAZ proteins that function in transcriptional repression. The emerging picture of JA perception and signaling cascade implies the SCFCOI1 complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ repressors for degradation by the 26S-proteasome pathway, thereby allowing the transcription factor MYC2 to activate gene expression. The fact that only one particular stereoisomer, (+)-7-iso-JA-l-Ile (4), shows high biological activity suggests that epimerization between active and inactive diastereomers could be a mechanism for turning JA signaling on or off. The recent demonstration that COI1 directly binds (+)-7-iso-JA-l-Ile (4) and thus functions as JA receptor revealed that formation of the ternary complex COI1-JA-Ile-JAZ is an ordered process. The pronounced differences in biological activity of JA stereoisomers also imply strict stereospecific control of product formation along the JA biosynthetic pathway. The pathway of JA biosynthesis has been unraveled, and most of the participating enzymes are well-characterized. For key enzymes of JA biosynthesis the crystal structures have been established, allowing insight into the mechanisms of catalysis and modes of substrate binding that lead to formation of stereospecific products.

Arabidopsis thaliana contains a large number of genes encoding carboxylic acid-activating enzymes, including long-chain fatty acyl-CoA synthetase (LACS), 4-coumarate:CoA ligases (4CL), and proteins closely related to 4CLs with unknown activities. The function of these 4CL-like proteins was systematically explored by applying an extensive substrate screen, and it was uncovered that activation of fatty acids is the common feature of all active members of this protein family, thereby defining a new group of fatty acyl-CoA synthetase, which is distinct from the known LACS family. Significantly, four family members also displayed activity towards different biosynthetic precursors of jasmonic acid (JA), including 12-oxo-phytodienoic acid (OPDA), dinor-OPDA, 3-oxo-2(2′-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8), and OPC-6. Detailed analysis of in vitro properties uncovered significant differences in substrate specificity for individual enzymes, but only one protein (At1g20510) showed OPC-8:CoA ligase activity. Its in vivo function was analysed by transcript and jasmonate profiling of Arabidopsis insertion mutants for the gene. OPC-8:CoA ligase expression was activated in response to wounding or infection in the wild type but was undetectable in the mutants, which also exhibited OPC-8 accumulation and reduced levels of JA. In addition, the developmental, tissue- and cell-type specific expression pattern of the gene, and regulatory properties of its promoter were monitored by analysing promoter::GUS reporter lines. Collectively, the results demonstrate that OPC-8:CoA ligase catalyses an essential step in JA biosynthesis by initiating the β-oxidative chain shortening of the carboxylic acid side chain of its precursors, and, in accordance with this function, the protein is localized in peroxisomes.

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function. Because of their high structural and sequence similarity with bona fide 4CLs and their highly hydrophobic putative substrate-binding pockets, the 4CL-like proteins At4g05160 and At5g63380 were selected for detailed analysis. Following heterologous expression, the purified proteins were subjected to a large scale screen to identify their preferred in vitro substrates. This study uncovered a significant activity of At4g05160 with medium-chain fatty acids, medium-chain fatty acids carrying a phenyl substitution, long-chain fatty acids, as well as the jasmonic acid precursors 12-oxo-phytodienoic acid and 3-oxo-2-(2′-pentenyl)-cyclopentane-1-hexanoic acid. The closest homolog of At4g05160, namely At5g63380, showed high activity with long-chain fatty acids and 12-oxo-phytodienoic acid, the latter representing the most efficiently converted substrate. By using fluorescent-tagged variants, we demonstrated that both 4CL-like proteins are targeted to leaf peroxisomes. Collectively, these data demonstrate that At4g05160 and At5g63380 have the capacity to contribute to jasmonic acid biosynthesis by initiating the β-oxidative chain shortening of its precursors.

The synthesis of jasmonate-induced proteins in leaves of tobacco (Nicotiana plumbaginifolia) and tomato (Lycopersicon esculentum) was studied in order to find a possible functional link in the actions of abscisic acid (ABA) and jasmonates. ABA-deficient mutants of tobacco (CKR1) and of tomato (sitiens, flacca), and their corresponding wild-types, were compared with respect to endogenous contents of jasmonates and ABA, and polypeptide and transcript patterns in water- or jasmonate-floated leaves, leaves stressed by floating on sorbitol, or by weak desiccation. Our results indicate that in tobacco the synthesis of proteins induced by jasmonate differed from those induced by ABA, whereas in tomato some jasmonate-induced proteins were also induced by ABA. The results provide further evidence that different signalling pathways exist for jasmonate/ABA-responsive gene expression in various plant species.

The role of systemin in Pin2 gene expression was analyzed in wild-type plants of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum Mill.), as well as in abscisic acid (ABA)-deficient tomato (sitiens) and potato (droopy) plants. The results showed that systemin initiates Pin2 mRNA accumulation only in wildtype tomato and potato plants. As in the situation after mechanical wounding,Pin2 gene expression in ABA-deficient plants was not activated by systemin. Increased endogenous levels of jasmonic acid (JA) and accumulation of Pin2 mRNA were observed following treatment with α-linolenic acid, the precursor of JA biosynthesis, suggesting that these ABA mutants still have the capability to synthesize de novo JA. Measurement of endogenous levels of ABA and JA showed that systemin leads to an increase of both phytohormones (ABA and JA) only in wild-type but not in ABA-deficient plants.