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Publikation

Floß, D. S.; Hause, B.; Lange, P. R.; Küster, H.; Strack, D.; Walter, M. H.; Knock-down of the MEP pathway isogene 1-deoxy-d-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes Plant J. 56, 86-100, (2008) DOI: 10.1111/j.1365-313X.2008.03575.x

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.
Publikation

Vigliocco, A.; Alemano, S.; Miersch, O.; Alvarez, D.; Abdala, G.; Endogenous jasmonates in dry and imbibed sunflower seeds from plants grown at different soil moisture contents Seed Sci. Res. 17, 91-98, (2007) DOI: 10.1017/S0960258507708371

In this study, we characterized two sunflower (Helianthus annuus L.) lines with differential sensitivity to drought, the sensitive line B59 and the tolerant line B71. Using both lines, we compared the content of endogenous jasmonates (JAs) in dry and imbibed seeds from plants grown under irrigation and drought. Jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), 11-hydroxyjasmonate (11-OH-JA) and 12-hydroxyjasmonate (12-OH-JA) were detected in dry and imbibed sunflower seeds. Seeds from plants grown under drought had a lower content of total JAs and exhibited higher germination percentages than seeds from irrigated plants, demonstrating that environmental conditions have a strong influence on the progeny. OPDA and 12-OH-JA were the main compounds found in dry seeds of both lines. Imbibed seeds showed an enhanced amount of total JAs with respect to dry seeds produced by plants grown in both soil moisture conditions. Imbibition triggered a dramatic OPDA increase in the embryo, suggesting a role of this compound in germination. We conclude that JAs patterns vary during sunflower germination and that the environmental conditions experienced by the mother plant modify the hormonal content of the seed progeny.
Publikation

Andrade, A.; Vigliocco, A.; Alemano, S.; Miersch, O.; Botella, M. A.; Abdala, G.; Endogenous jasmonates and octadecanoids in hypersensitive tomato mutants during germination and seedling development in response to abiotic stress Seed Sci. Res. 15, 309-318, (2005) DOI: 10.1079/SSR2005219

Although jasmonates (JAs) are involved in germination and seedling development, the regulatory mechanism of JAs, and their relation with endogenous level modifications in these processes, is not well understood. We report here the detection of 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), 11-hydroxyjasmonate (11-OH-JA), 12-hydroxyjasmonate (12-OH-JA) and methyljasmonate (JAME) in unimbibed seeds and seedlings of tomato Lycopersicon esculentum Mill cv. Moneymaker (wild type) and tss1, tss2, tos1 mutants. The main compounds in wild-type and tss1, tss2, tos1 seeds were the hydroxylate-JAs; 12-OH-JA was the major component in dry seeds of the wild type and in tss2 and tos1. The amounts of these derivatives were higher in seeds than in seedlings. Changes in JAs during wild-type and tss1 imbibition were analysed in seeds and the imbibition water. In wild-type imbibed seeds, 11-OH-JA content was higher than in tss1. 12-OH-JA showed a different tendency with respect to 11-OH-JA, with high levels in the wild type at early imbibition. In tss1, levels of 12-OH-JA rose from 24 to 48 h of imbibition. At 72 h of imbibition, when radicles had emerged, the amounts of both hydroxylates in wild-type and tss1 seeds were minimal. An important release of the hydroxylate forms was observed in the imbibition water. 11-OH-JA decreased in the imbibition water of wild-type seeds at 48 h. On the contrary, a high and sustained liberation of this compound was observed in tss1 after 24 h. 12-OH-JA increased in wild-type as well in tss1 until 24 h. Thereafter, a substantial reduction in the content of this compound was registered. NaCl-treated wild-type seedlings increased their 12-OH-JA, but tss1 seedlings increased their JA in response to salt treatment. In tss2 seedlings, NaCl caused a slight decrease in 11-OH-JA and JAME, whereas tos1 seedlings showed a dramatic OPDA and 12-OH-JA decrease in response to salt treatment. Under salt stress the mutant seedlings showed different patterns of JAs according to their differential hypersensitivity to abiotic stress. The JA-hydroxylate forms found, and the differential accumulation of JAs during germination, imbibition and seedling development, as well as their response to NaCl stress, provide new evidence about the control of many developmental processes by JA.
Publikation

Isayenkov, S.; Mrosk, C.; Stenzel, I.; Strack, D.; Hause, B.; Suppression of Allene Oxide Cyclase in Hairy Roots of Medicago truncatula Reduces Jasmonate Levels and the Degree of Mycorrhization with Glomus intraradices Plant Physiol. 139, 1401-1410, (2005) DOI: 10.1104/pp.105.069054

During the symbiotic interaction between Medicago truncatula and the arbuscular mycorrhizal (AM) fungus Glomus intraradices, an endogenous increase in jasmonic acid (JA) occurs. Two full-length cDNAs coding for the JA-biosynthetic enzyme allene oxide cyclase (AOC) from M. truncatula, designated as MtAOC1 and MtAOC2, were cloned and characterized. The AOC protein was localized in plastids and found to occur constitutively in all vascular tissues of M. truncatula. In leaves and roots, MtAOCs are expressed upon JA application. Enhanced expression was also observed during mycorrhization with G. intraradices. A partial suppression of MtAOC expression was achieved in roots following transformation with Agrobacterium rhizogenes harboring the MtAOC1 cDNA in the antisense direction under control of the cauliflower mosaic virus 35S promoter. In comparison to samples transformed with 35S∷uidA, roots with suppressed MtAOC1 expression exhibited lower JA levels and a remarkable delay in the process of colonization with G. intraradices. Both the mycorrhization rate, quantified by fungal rRNA, and the arbuscule formation, analyzed by the expression level of the AM-specific gene MtPT4, were affected. Staining of fungal material in roots with suppressed MtAOC1 revealed a decreased number of arbuscules, but these did not exhibit an altered structure. Our results indicate a crucial role for JA in the establishment of AM symbiosis.
Publikation

Abdala, G.; Miersch, O.; Kramell, R.; Vigliocco, A.; Agostini, E.; Forchetti, G.; Alemano, S.; Jasmonate and octadecanoid occurrence in tomato hairy roots. Endogenous level changes in response to NaCl Plant Growth Regul. 40, 21-27, (2003) DOI: 10.1023/A:1023016412454

Jasmonic acid biosynthesis occurs in leaves and there is also evidence of a similar pathway in roots. The expression of lipoxygenase, allene oxide cyclase and low amounts of transcripts of allene oxide synthase in tomato roots indicates that some steps of the jasmonate synthesis may occur in these organs. Thus, the aim of the present work was to study the jasmonate and octadecanoid occurrence in tomato roots using isolated cultures of hairy roots. These were obtained by the transformation of cv. Pera roots with Agrobacterium rhyzogenes. Also we investigated the effect of NaCl stress on the endogenous levels of these compounds. Jasmonic acid, 12-oxophytodienoic acid and their methylated derivatives, as well as a jasmonate-isoleucine conjugate, were present in control hairy roots of 30 d of culture. The 12-oxophytodienoic acid and its methylated derivative showed higher levels than jasmonic acid and its methylated form, although the content of the conjugate was the same as that of jasmonic acid. After salinization of hairy roots for 14, 20 and 30 d, free jasmonates and octadecanoids were measured. Fourteen days after salt treatment, increased levels of these compounds were found, jasmonic acid and 12-oxophytodienoic acid showed the most remarkable rise. 11-OH-jasmonic acid was found at 14 d of culture in control and salt-treated hairy roots; whereas the 12-OH- form of jasmonic acid was only detected in the salt-treated hairy roots. Agrobacterium rhizogenes cultures did not produce jasmonates and/or octadecanoids.
Publikation

Vigliocco, A.; Bonamico, B.; Alemano, S.; Miersch, O.; Abdala, G.; Stimulation of jasmonic acid production in Zea Mays L. infected by the maize rough dwarf virus - Río Cuarto. Reversion of symptoms by salicylic acid Biocell 26, 369-374, (2002)

In the present paper we study the possible biological relevance of endogenous jasmonic acid (JA) and exogenous salicylic acid (SA) in a plant-microbial system maize-virus. The virus disease "Mal de Río Cuarto" is caused by the maize rough dwarf virus - Río Cuarto. The characteristic symptoms are the appearance of galls or "enations" in leaves, shortening of the stem internodes, poor radical system and general stunting. Changes in JA and protein pattern in maize control and infected plants of a virus-tolerant cultivar were investigated. Healthy and infected-leaf discs were collected for JA measurement at different post-infection times (20, 40, 60 and 68 days). JA was also measured in roots on day 60 after infection. For SDS-PAGE protein analysis, leaf discs were also harvested on day 60 after infection. Infected leaves showed higher levels of JA than healthy leaves, and the rise in endogenous JA coincided with the enation formation. The soluble protein amount did not show differences between infected and healthy leaves; moreover, no difference in the expression of soluble protein was revealed by SDS-PAGE. Our results show that the octadecanoid pathway was stimulated in leaves and roots of the tolerant maize cultivar when infected by this virus. This finding, together with fewer plants with the disease symptoms, suggest that higher foliar and roots JA content may be related to disease tolerance. SA exogenous treatment caused the reversion of the dwarfism symptom.
Publikation

Hause, B.; Maier, W.; Miersch, O.; Kramell, R.; Strack, D.; Induction of Jasmonate Biosynthesis in Arbuscular Mycorrhizal Barley Roots Plant Physiol. 130, 1213-1220, (2002) DOI: 10.1104/pp.006007

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.
Publikation

Ziegler, J.; Vogt, T.; Miersch, O.; Strack, D.; Concentration of Dilute Protein Solutions Prior to Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Anal. Biochem. 250, 257-260, (1997) DOI: 10.1006/abio.1997.2248

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