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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

Wang, Q.; Grubb, C. D.; Abel, S.; Direct analysis of single leaf disks for chemopreventive glucosinolates Phytochem. Anal. 13, 152-157, (2002) DOI: 10.1002/pca.636

Natural isothiocyanates, produced during plant tissue damage from methionine‐derived glucosinolates, are potent inducers of mammalian phase 2 detoxification enzymes such as quinone reductase (QR). A greatly simplified bioassay for glucosinolates based on induction and colorimetric detection of QR activity in murine hepatoma cells is described. It is demonstrated that excised leaf disks of Arabidopsis thaliana (ecotype Columbia) can directly and reproducibly substitute for cell‐free leaf extracts as inducers of murine QR, which reduces sample preparation to a minimum and maximizes throughput. A comparison of 1 and 3 mm diameter leaf disks indicated that QR inducer potency was proportional to disk circumference (extent of tissue damage) rather than to area. When compared to the QR inducer potency of the corresponding amount of extract, 1 mm leaf disks were equally effective, whereas 3 mm disks were 70% as potent. The QR inducer potency of leaf disks correlated positively with the content of methionine‐derived glucosinolates, as shown by the analysis of wild‐type plants and mutant lines with lower or higher glucosinolate content. Thus, the microtitre plate‐based assay of single leaf disks provides a robust and inexpensive visual method for rapidly screening large numbers of plants in mapping populations or mutant collections and may be applicable to other glucosinolate‐producing species.
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

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

Abel, S.; Ticconi, C. A.; Delatorre, C. A.; Phosphate sensing in higher plants Physiol. Plant. 115, 1-8, (2002) DOI: 10.1034/j.1399-3054.2002.1150101.x

Phosphate (Pi) plays a central role as reactant and effector molecule in plant cell metabolism. However, Pi is the least accessible macronutrient in many ecosystems and its low availability often limits plant growth. Plants have evolved an array of molecular and morphological adaptations to cope with Pi limitation, which include dramatic changes in gene expression and root development to facilitate Pi acquisition and recycling. Although physiological responses to Pi starvation have been increasingly studied and understood, the initial molecular events that monitor and transmit information on external and internal Pi status remain to be elucidated in plants. This review summarizes molecular and developmental Pi starvation responses of higher plants and the evidence for coordinated regulation of gene expression, followed by a discussion of the potential involvement of plant hormones in Pi sensing and of molecular genetic approaches to elucidate plant signalling of low Pi availability. Complementary genetic strategies in Arabidopsis thaliana have been developed that are expected to identify components of plant signal transduction pathways involved in Pi sensing. Innovative screening methods utilize reporter gene constructs, conditional growth on organophosphates and the inhibitory properties of the Pi analogue phosphite, which hold the promise for significant advances in our understanding of the complex mechanisms by which plants regulate Pi‐starvation responses.
Publikation

Laskowski, M. J.; Dreher, K. A.; Gehring, M. A.; Abel, S.; Gensler, A. L.; Sussex, I. M.; FQR1, a Novel Primary Auxin-Response Gene, Encodes a Flavin Mononucleotide-Binding Quinone Reductase Plant Physiol. 128, 578-590, (2002) DOI: 10.1104/pp.010581

FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating thatFQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated fromEscherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathioneS-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress.
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

Grubb, C. D.; Gross, H. B.; Chen, D. L.; Abel, S.; Identification of Arabidopsis mutants with altered glucosinolate profiles based on isothiocyanate bioactivity Plant Sci. 162, 143-152, (2002) DOI: 10.1016/S0168-9452(01)00550-7

Glucosinolates are a diverse class of nitrogen- and sulfur-containing secondary metabolites. They are rapidly hydrolyzed on tissue disruption to a number of biologically active compounds that are increasingly attracting interest as anticarcinogenic phytochemicals and crop protectants. Several glucosinolate-derived isothiocyanates are potent chemopreventive agents that favorably modulate carcinogen metabolism in mammals. Methylsulfinylalkyl isothiocyanates, in particular the 4-methylsulfinylbutyl derivative, are selective and potent inducers of mammalian detoxification enzymes such as quinone reductase (QR). Cruciferous plants including Arabidopsis thaliana (L.) Heyhn, synthesize methylsulfinylalkyl glucosinolates, which are derived from methionine. Using a colorimetric assay for QR activity in murine hepatoma cells and high performance liquid chromatography (HPLC) analysis of desulfoglucosinolates, we have demonstrated a strong positive correlation between leaf QR inducer potency and leaf content of methionine-derived glucosinolates in various A. thaliana ecotypes and available glucosinolate mutants. In a molecular genetic approach to glucosinolate biosynthesis, we screened 3000 chemically mutagenized M2 plants of the Columbia ecotype for altered leaf QR inducer potency. Subsequent HPLC analysis of progeny of putative mutants identified six lines with significant and heritable changes in leaf glucosinolate content and composition.
Bücher und Buchkapitel

Wasternack, C.; Hause, B.; Jasmonates and octadecanoids: Signals in plant stress responses and development Prog. Nucleic Acid Res. Mol. Biol. 72, 165-221, (2002) DOI: 10.1016/S0079-6603(02)72070-9

Plants are sessile organisms. Consequently they have to adapt constantly to fluctuations in the environment. Some of these changes involve essential factors such as nutrients, light, and water. Plants have evolved independent systems to sense nutrients such as phosphate and nitrogen. However, many of the environmental factors may reach levels which represent stress for the plant. The fluctuations can range between moderate and unfavorable, and the factors can be of biotic or abiotic origin. Among the biotic factors influencing plant life are pathogens and herbivores. In case of bacteria and fungi, symbiotic interactions such as nitrogen-fixating nodules and mycorrhiza, respectively, may be established. In case of insects, a tritrophic interaction of herbivores, carnivores, and plants may occur mutualistically or parasitically. Among the numerous abiotic factors are low temperature, frost, heat, high light conditions, ultraviolet light, darkness, oxidation stress, hypoxia, wind, touch, nutrient imbalance, salt stress, osmotic adjustment, water deficit, and desiccation.In the last decade jasmonates were recognized as being signals in plant responses to most of these biotic and abiotic factors. Signaling via jasmonates was found to occur intracellularly, and systemically as well as interorganismically. Jasmonates are a group of ubiquitously occurring plant growth regulators originally found as the major constituents in the etheric oil of jasmine, and were first suggested to play a role in senescence due to a strong senescence-promoting effect. Subsequently, numerous developmental processes were described in which jasmonates exhibited hormone-like properties. Recent knowledge is reviewed here on jasmonates and their precursors, the octadecanoids. After discussing occurrence and biosynthesis, emphasis is placed upon the signal transduction pathways in plant stress responses in which jasmonates act a signal. Finally, examples are described on the role of jasmonates in developmental processes.
Publikation

Abel, S.; Nguyen, M. D.; Theologis, A.; The PS-IAA4/5-like Family of Early Auxin-inducible mRNAs in Arabidopsis thaliana J. Mol. Biol. 251, 533-549, (1995) DOI: 10.1006/jmbi.1995.0454

The plant hormone auxin transcriptionally activates early genes. We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts inArabidopsis thaliana. The corresponding genes, IAA1 and IAA14, are homologs of PS-1AA4/5 and PS-IAA6 from pea, AUX22 and AUX28 from soybean, ARG3 and ARG4from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis. The members of the family are differentially expressed in mature Arabidopsis plants. Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility. The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cyclohexamide. Cyclohexamide alone induces all the early genes. Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cyclohexamide, indicating requirement of protein synthesis for their activation. All IAA genes display a biphasic dose response that is optimal at 10 μM IAA. However, individual genes respond differentially between 10 nM and 5μM IAA. Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2, and aux1.The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions. The spaces vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa. Overall sequence identity between members of the family is highly variable (36 to 87%). Their most significant structural features are functional nuclear transport signals, and a putative βαα-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic β-ribbon DNA recognition motif. The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins. These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development.
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