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Publikation

Köck, M.; Groß, N.; Stenzel, I.; Hause, G.; Phloem-specific expression of the wound-inducible ribonuclease LE from tomato (Lycopersicon esculentum cv. Lukullus) Planta 219, 233-242, (2004) DOI: 10.1007/s00425-004-1227-4

Ribonuclease LE (RNaseLE) from tomato (Lycopersicon esculentum Mill. cv. Lukullus) belongs to the widespread RNase T2 family of ribonucleases. With the exception of S-RNases of the solanaceous self-incompatibility system the functions of other members of the RNase T2 family are only barely understood. Using a 2.6-kbp putative promoter sequence of RNaseLE in front of the uidA reporter gene, expression of β-glucuronidase in developing phloem tissue and, especially, in the meristematic and elongation zones at root tips was detected. The tissue-specific expression accords with the range of cis-acting elements detected in the RNaseLE promoter. RNaseLE mRNA was localized in developing phloem cells but not in mature phloem tissue, suggesting association of RNaseLE expression with phloem development. Histochemical staining of β-glucuronidase activity as well as detailed inspection of RNaseLE at mRNA, protein and enzyme activity levels revealed that the wound-induced expression of RNaseLE was also restricted to vascular tissue. RNaseLE transcript accumulation detected by in situ hybridization occurred preferentially in phloem and cambial cells of stem sections upon wounding. The data provide evidence for a role of RNaseLE in a tissue-specific wound response and in wound healing of tomato.
Publikation

Groß, N.; Wasternack, C.; Köck, M.; Wound-induced RNaseLE expression is jasmonate and systemin independent and occurs only locally in tomato (Lycopersicon esculentum cv. Lukullus) Phytochemistry 65, 1343-1350, (2004) DOI: 10.1016/j.phytochem.2004.04.036

Tomato RNaseLE is induced by phosphate deficiency and wounding and may play a role in macromolecular recycling as well as wound healing. Here, we analyzed the role of jasmonate and systemin in the wound-induced RNaseLE activation. The rapid expression of RNaseLE upon wounding of leaves leading to maximal RNase activity within 10 h, appeared only locally. Jasmonic acid (JA) or its molecular mimic ethyl indanoyl isoleucine conjugate did not induce RNaseLE expression. Correspondingly, RNaseLE was expressed upon wounding of 35S::allene oxide cyclase antisense plants known to be JA deficient. RNaseLE was not expressed upon systemin treatment, but was locally expressed in the spr1 mutant which is affected in systemin perception. In tomato plants carrying a PromLE::uidA construct, GUS activity could be detected upon wounding, but not following treatment with JA or systemin. The data indicate a locally acting wound-inducible systemin- and JA-independent signaling pathway for RNaseLE expression.RNaseLE expression was analyzed by pharmacological studies of different tomato lines and upon wounding of leaves. The gene is only locally activated via a new type of wound-induced signaling pathway in a jasmonate/systemin-independent manner.
Publikation

Miersch, O.; Weichert, H.; Stenzel, I.; Hause, B.; Maucher, H.; Feussner, I.; Wasternack, C.; Constitutive overexpression of allene oxide cyclase in tomato (Lycopersicon esculentum cv. Lukullus) elevates levels of some jasmonates and octadecanoids in flower organs but not in leaves Phytochemistry 65, 847-856, (2004) DOI: 10.1016/j.phytochem.2004.01.016

The allene oxide cyclase (AOC), an enzyme in jasmonate biosynthesis, occurs in vascular bundles and ovules of tomato flowers which exhibit a tissue-specific oxylipin signature (Plant J. 24, 113-126, 2000). Constitutive overexpression of the AOC did not led to altered levels of jasmonates in leaves, but these levels increased upon wounding or other stresses suggesting regulation of jasmonate biosynthesis by substrate availability (Plant J. 33, 577-589, 2003). Here, we show dramatic changes in levels of jasmonic acid (JA), of 12-oxo-phytodienoic acid (OPDA), their methyl esters (JAME, OPDAME), and of dinor-OPDA in most flower organs upon constitutive overexpression of AOC. Beside a dominant occurrence of OPDAME and JA in most flower organs, the ratio among the various compounds was altered differentially in the organs of transgenic flowers, e.g. OPDAME increased up to 53-fold in stamen, and JA increased about 51-fold in buds and 7.5-fold in sepals. The increase in jasmonates and octadecanoids was accompanied by decreased levels of free lipid hydro(per)oxy compounds. Except for 16:2, the AOC overexpression led to a significant increase in free but not esterified polyunsaturated fatty acids in all flower organs. The data suggest different regulation of JA biosynthesis in leaves and flowers of tomato.Constitutive overexpression of the AOC increases in all flower organs levels of some jasmonates and octadecanoids, alters the ratios among the compounds, decreases levels of free lipid hydro(per)oxy compounds and increases levels of free but not of esterified polyunsaturated fatty acids.
Publikation

Stenzel, I.; Hause, B.; Miersch, O.; Kurz, T.; Maucher, H.; Weichert, H.; Ziegler, J.; Feussner, I.; Wasternack, C.; Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana Plant Mol. Biol. 51, 895-911, (2003) DOI: 10.1023/A:1023049319723

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.
Publikation

Stenzel, I.; Ziethe, K.; Schurath, J.; Hertel, S. C.; Bosse, D.; Köck, M.; Differential expression of the LePS2 phosphatase gene family in response to phosphate availability, pathogen infection and during development Physiol. Plant. 118, 138-146, (2003) DOI: 10.1034/j.1399-3054.2003.00091.x

In this study, we report the cloning of the three‐member LePS2 gene family of acid phosphatases via subtractive screening of a cDNA library of Pi‐starved cultivated tomato cells (Lycopersicon esculentum Mill. cv. Lukullus). As members of the plant Pi‐starvation response, LePS2 genes were tightly regulated in cultivated cells and tomato seedlings by Pi availability. The LePS2 enzymes which are most likely expressed in the cytoplasma could be involved in processes that are accompanied by degradation of phosphorylated organic substrates. Independently from exogenous phosphate supply LePS2 expression was detected in tomato endosperm during germination. LePS2 genes were differentially induced after infection with the bacterial pathogen Pseudomonas syringae and in the early stages of flower development. Using RT–PCR it was found that the gene LePS2B was the most abundant transcript in phosphate‐depleted cells, but a reduced expression was determined in floral buds and it was not found during pathogen interaction. In this respect, it is interesting that the promoter sequences of the LePS2 genes are also divergent. LePS2 gene products may have functions in developmental processes which are restricted to distinct plant tissues or cell types.
Bücher und Buchkapitel

Weichert, H.; Maucher, H.; Hornung, E.; Wasternack, C.; Feussner, I.; Shift in Fatty Acid and Oxylipin Pattern of Tomato Leaves Following Overexpression of the Allene Oxide Cyclase 275-278, (2003) DOI: 10.1007/978-94-017-0159-4_64

Polyunsaturated fatty acids (PUFAs) are a source of numerous oxidation products, the oxylipins. In leaves, α-linolenic acid (α-LeA) is the preferential substrate for lipid peroxidation reactions. This reaction may be catalyzed either by a 9-lipoxygenase (9-LOX) or by a 13-LOX and oxygen is inserted regioselectively as well as stereospecifically leading to formation of 13S- or 9S-hydroperoxy octadecatrienoic acid (13-/9-HPOT; Brash, 1999). At least, seven different enzyme families or reaction branches within the LOX pathway can use these HPOTs or other hydroperoxy PUFAs leading to (i) keto-PUFAs (LOX); (ii) epoxy hydroxy-PUFAs (epoxy alcohol synthase, EAS); (iii) octadecanoids and jasmonates (allene oxide synthase, AOS); (iv) leaf aldehydes and leaf alcohols (hydroperoxide lyase, HPL); (v) hydroxy PUFAs (reductase); (vi) divinyl ether PUFAs (divinyl ether synthase, DES); and (vii) epoxy- or dihydrodiol-PUFAs (peroxygenase, PDX; Fig. 1; Feussner and Wasternack, 2002).
Bücher und Buchkapitel

Stumpe, M.; Stenzel, I.; Weichert, H.; Hause, B.; Feussner, I.; The Lipoxygenase Pathway in Mycorrhizal Roots of Medicago Truncatula 287-290, (2003) DOI: 10.1007/978-94-017-0159-4_67

Mycorrhizas are by far the most frequent occurring beneficial symbiotic interactions between plants and fungi. Species in >80% of extant plant families are capable of establishing an arbuscular mycorrhiza (AM). In relation to the development of the symbiosis the first molecular modifications are those associated with plant defense responses, which seem to be locally suppressed to levels compatible with symbiotic interaction (Gianinazzi-Pearson, 1996). AM symbiosis can, however, reduce root disease caused by several soil-borne pathogens. The mechanisms underlying this protective effect are still not well understood. In plants, products of the enzyme lipoxygenase (LOX) and the corresponding downstream enzymes, collectively named LOX pathway (Fig. 1B), are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity (Feussner and Wasternack, 2002). The central reaction in this pathway is catalyzed by LOXs leading to formation of either 9- or 13-hydroperoxy octadeca(di/trien)oic acids (9/13-HPO(D/T); Brash, 1999). Thus LOXs may be divided into 9- and 13-LOXs (Fig. 1A). Seven different reaction branches within this pathway can use these hydroperoxy polyenoic fatty acids (PUFAs) leading to (i) keto PUFAs by a LOX; (ii) epoxy hydroxy-fatty acids by an epoxy alcohol synthase (EAS); (iii) octadecanoids and jasmonates via allene oxide synthase (AOS); (iv) leaf aldehydes and leaf alcohols via fatty acid hydroperoxide lyase (HPL); (v) hydroxy PUFAs (reductase); (vi) divinyl ether PUFAs via divinyl ether synthase (DES); and (vii) epoxy- or dihydrodiolPUFAs via peroxygenase (PDX; Feussner and Wasternack, 2002). AOS, HPL and DES belong to one subfamily of P450-containing enzymes, the CYP74 family (Feussner and Wasternack, 2002). Here, the involvement of this CYP74 enzyme family in mycorrhizal roots of M. truncatula during early stages of AM symbiosis formation was analyzed.
Publikation

Weichert, H.; Kolbe, A.; Kraus, A.; Wasternack, C.; Feussner, I.; Metabolic profiling of oxylipins in germinating cucumber seedlings - lipoxygenase-dependent degradation of triacylglycerols and biosynthesis of volatile aldehydes Planta 215, 612-619, (2002) DOI: 10.1007/s00425-002-0779-4

A particular isoform of lipoxygenase (LOX) localized on lipid bodies was shown by earlier investigations to play a role in initiating the mobilization of triacylglycerols during seed germination. Here, further physiological functions of LOXs within whole cotyledons of cucumber (Cucumis sativus L.) were analyzed by measuring the endogenous amounts of LOX-derived products. The lipid-body LOX-derived esterified (13S)-hydroperoxy linoleic acid was the dominant metabolite of the LOX pathway in this tissue. It accumulated to about 14 µmol/g fresh weight, which represented about 6% of the total amount of linoleic acid in cotyledons. This LOX product was not only reduced to its hydroxy derivative, leading to degradation by β-oxidation, but alternatively it was metabolized by fatty acid hydroperoxide lyase leading to formation of hexanal as well. Furthermore, the activities of LOX forms metabolizing linolenic acid were detected by measuring the accumulation of volatile aldehydes and the allene oxide synthase-derived metabolite jasmonic acid. The first evidence is presented for an involvement of a lipid-body LOX form in the production of volatile aldehydes.
Publikation

Bachmann, A.; Hause, B.; Maucher, H.; Garbe, E.; Vörös, K.; Weichert, H.; Wasternack, C.; Feussner, I.; Jasmonate-Induced Lipid Peroxidation in Barley Leaves Initiated by Distinct 13-LOX Forms of Chloroplasts Biol. Chem. 383, 1645-1657, (2002) DOI: 10.1515/BC.2002.185

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [Vörös et al., Eur. J. Biochem. 251 (1998), 36 44], two fulllength cDNAs (LOX2:Hv:2, LOX2:Hv:3) were isolated from barley leaves (Hordeum vulgare cv. Salome) and characterized. Both of them encode 13-lipoxygenases with putative target sequences for chloroplast import. Immunogold labeling revealed preferential, if not exclusive, localization of lipoxygenase proteins in the stroma. The ultrastructure of the chloroplast was dramatically altered following methyl jasmonate treatment, indicated by a loss of thylakoid membranes, decreased number of stacks and appearance of numerous osmiophilic globuli. The three 13-lipoxygenases are differentially expressed during treatment with jasmonate, salicylate, glucose or sorbitol. Metabolite profiling of free linolenic acid and free linoleic acid, the substrates of lipoxygenases, in water floated or jasmonatetreated leaves revealed preferential accumulation of linolenic acid. Remarkable amounts of free 9- as well as 13-hydroperoxy linolenic acid were found. In addition, metabolites of these hydroperoxides, such as the hydroxy derivatives and the respective aldehydes, appeared following methyl jasmonate treatment. These findings were substantiated by metabolite profiling of isolated chloroplasts, and subfractions including the envelope, the stroma and the thylakoids, indicating a preferential occurrence of lipoxygenasederived products in the stroma and in the envelope. These data revealed jasmonateinduced activation of the hydroperoxide lyase and reductase branch within the lipoxygenase pathway and suggest differential activity of the three 13-lipoxygenases under different stress conditions.
Publikation

BERGER, S.; Weichert, H.; Porzel, A.; Wasternack, C.; Kühn, H.; Feussner, I.; Enzymatic and non-enzymatic lipid peroxidation in leaf development BBA-Mol. Cell Biol. Lipids 1533, 266-276, (2001) DOI: 10.1016/S1388-1981(01)00161-5

Enzymatic and non-enzymatic lipid peroxidation has been implicated in programmed cell death, which is a major process of leaf senescence. To test this hypothesis we developed a high-performance liquid chromatography (HPLC) method for a simultaneous analysis of the major hydro(pero)xy polyenoic fatty acids. Quantities of lipid peroxidation products in leaves of different stages of development including natural senescence indicated a strong increase in the level of oxygenated polyenoic fatty acids (PUFAs) during the late stages of leaf senescence. Comprehensive structural elucidation of the oxygenation products by means of HPLC, gas chromatography/mass spectrometry and 1H nuclear magnetic resonance suggested a non-enzymatic origin. However, in some cases a small share of specifically oxidized PUFAs was identified suggesting involvement of lipid peroxidizing enzymes. To inspect the possible role of enzymatic lipid peroxidation in leaf senescence, we analyzed the abundance of lipoxygenases (LOXs) in rosette leaves of Arabidopsis. LOXs and their product (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid were exclusively detected in young green leaves. In contrast, in senescing leaves the specific LOX products were overlaid by large amounts of stereo-random lipid peroxidation products originating from non-enzymatic oxidation. These data indicate a limited contribution of LOXs to total lipid peroxidation, and a dominant role of non-enzymatic lipid peroxidation in late stages of leaf development.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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