Publikationen - Molekulare Signalverarbeitung

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.

In barley leaves, there is a dramatic alteration of gene expression upon treatment with jasmonates leading to the accumulation of newly formed proteins, designated as jasmonate‐inducible proteins (JIPs). In the present study, a new jasmonate‐inducible cDNA, designated pHvJS37, has been isolated by differential screening of a γgt10 cDNA library constructed from mRNA of jasmonate‐treated barley leaf segments. The open reading frame (ORF) encodes a 39‐9 kDa polypeptide which cross‐reacts with antibodies raised against the in vivo JIP‐37. The hydropathic plot suggests that the protein is mainly hydrophilic, containing two hydrophilic domains near the C‐terminus. Database searches did not show any sequence homology of pHv.JS37 to known sequences. Southern analysis revealed at least two genes coding for JIP‐37 which map to the distal portion of the long arm of chromosome 3 and are closely related to genes coding for JIP‐23. The expression pattern of the JIP‐37 genes over time shows differential responses to jasmonate, abscisic acid (ABA), osmotic stress (such as sorbitol treatment) and desiccation stress. No expression was found under salt stress. From experiments using an inhibitor and intermediates of jasmonate synthesis such as α‐linolenic acid and 12‐oxophytodienoic acid, we hypothesize that there is a stress‐induced lipid‐based signalling pathway in which an endogenous rise of jasmonate switches on JIP‐37 gene expression. Using immunocytochemical techniques, JIP‐37 was found to be simultaneously located in the nucleus, the cytoplasm and the vacuoles.

BarJey leaf segments treated with jasmonate respond with the synthesis of specific proseins, referred to as jasmonate‐induced proteins (JIPs). Application of abscisic acid (ABAl also induced JIP synthesis (Weidhase et al. 1987). In this study the effects of inhibitors on sorbitol‐induced increases of endogenous jasmonates and ABA were investigated. The promotion of jasmonates by sorbitol was inhibited by the growth retardant tetcyclacis at concentrations as low as 1 ftM. In parallel with the decrease of jasmonates, JIP gene expression was reduced as reflected by a decline in the level of a 23‐kDa protein UIP‐23) and mRNAs of JIP‐6 and JIP‐23. 12‐Oxo‐phytodienoic acid, an inlermediale in the lipoxygenase (LOX) pathway leading to jasmonic acid was able to overcome the inhibition by tetcyclacis and increases both the endogenous jasmonate content and transcript accumulation. This suggests that tetcyclacis acts upstream of 12‐oxo‐phytodienoic acid and in keeping with this proposal, an increase in relative LOX activity was detected after tetcyclacis treatment. Although tetcyclacis was shown to inhibit the degradation of ABA to phaseic acid, its effect on jasmonate synthesis is much more pronounced.

The accumulation of abundant proteins and their respective transcripts, induced by 10−4 M cisabscisic acid or 10−5 M jasmonic acid methyl ester, was studied in barley (Hordeum vulgare L.) leaf segments and compared to that resulting from osmotic stress caused by floating the segments on solutions of sorbitol, glucose, polyethyleneglycol (PEG)-6000 or NaCl. Osmotic stress or treatment with abscisic acid led to the synthesis of novel proteins which were identical to jasmonateinduced proteins (JIPs) with respect to immunological properties and molecular masses. The most prominent polypeptides were characterized by molecular masses of 66, 37 and 23 kDa and were newly synthesized. Whereas sorbitol, mannitol, sucrose, glucose and PEG provoked the synthesis of JIPs, 2deoxyglucose and NaCl did not. We provide evidence that the synthesis of JIPs induced by osmotic stress is directly correlated with a preceding rise in endogenous jasmonates. These jasmonates, quantified by an enzyme immunoassay specific for (−)jasmonic acid and its aminoacid conjugates, increased remarkably in leaf segments treated with sorbitol, glucose or other sugars. In contrast, no increase in jasmonates could be observed in tissues exposed to salts (NaCl). The results strengthen the hypothesis that the accumulation of jasmonates, probably by de-novo synthesis, is an intermediate and essential step in a signalling pathway between (osmotic) stress and activation of genes coding for polypeptides of high abundance.

The effect of osmotically active substances on the alteration of endogenous jasmonates was studied in barley (Hordeum vulgare L. cv. Salome) leaf tissue. Leaf segments were subjected to solutions of d-sorbitol, d-mannitol, polyethylene glycol 6000, sodium chloride, or water as a control. Alterations of endogenous jasmonates were monitored qualitatively and quantitatively using immunoassays. The structures of jasmonates isolated were determined on the basis of authentic substances by capillary gas chromatography-mass spectrometry. The stereochemistry of the conjugates was confirmed by high performance liquid chromatography with diastereoisomeric references. In barley leaves, jasmonic acid and its amino acid conjugates, for example, with valine, leucine, and isoleucine, are naturally occurring jasmonates. In untreated leaf segments, only low levels of these native jasmonates were found. After treatment of the leaf tissues with sorbitol, mannitol, as well as with polyethylene glycol, an increase of both jasmonic acid and its conjugates could be observed, depending on the stress conditions used. In contrast, salt stress was without any stimulating effect on the levels of endogenous jasmonates. From barley leaf segments exposed to sorbitol (1m) for 24 h, jasmonic acid was identified as the major accumulating compound. Jasmonic acid-amino acid conjugates increased likewise upon stress treatment.