Mode of Action of Jasmonates

The plant growth substance jasmonic acid and its methyl ester (JA‐Me) induce a set of proteins (jasmonate‐induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one‐dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno‐gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP‐23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP‐37 was detected in vacuoles and in the nucleoplasm; JIP‐66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP‐100 was found within the stromal fraction of chloroplasts; JIP‐70 is present in the peroxisome and the nucleus; JIP‐50 and JIP‐6 accumulate in vacuoles. The location of JIP‐66 and JIP‐6 confirms their possible physiological role deduced from molecular analysis of their cDNA.

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.

Barley leaves respond to application of (−)‐jasmonic acid (JA), or its methylester (JM) with the synthesis of abundant proteins, so‐called jasmonate induced proteins (JIPs). Here Western blot analysis is used to show a remarkable increase upon JM treatment of a 100 kDa lipoxygenase (LOX), and the appearance of two new LOX forms of 98 and 92 kDa. The temporal increase of LOX‐100 protein upon JM treatment was clearly distinguishable from the additionally detectable LOX forms. JM‐induced LOX forms in barley leaves were compared with those of Arabidopsis and soybean leaves. Both dicot species showed a similar increase of one LOX upon JM induction, whereas, leaves from soybean responded with additional synthesis of a newly formed LOX of 94 kDa.Using immunofluorescence analysis and isolation of intact chloroplasts, it is demonstrated that JM‐induced LOX forms of barley leaves are exclusively located in the chloroplasts of all chloroplast‐containing cells. Analogous experiments carried out with Arabidopsis and soybean revealed a similar plastidic location of JM‐induced LOX forms in Arabidopsis but a different situation for soybean. In untreated soybean leaves the LOX protein was mainly restricted to vacuoles of paraveinal mesophyll cells. Additionally, LOX forms could be detected in cytoplasm and nuclei of bundle sheath cells. Upon JM treatment cytosolic LOX was detectable in spongy mesophyll cells, too. The intracellular location of JM‐induced LOX is discussed in terms of stress‐related phenomena mediated by JM.

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.

Onset of acquired resistance of barley (Hordeum vulgare) chemically induced by 2,6-dichloroisonicotinic acid (DCINA) correlated with the accumulation of mRNA homologous to cDNA pHvJ256 which codes for a soluble leaf-thionin with a Mr. of 6 kDa [Wasternacket al., 1994a]. In the present work, we extend this finding by showing that the thionin transcript also accumulated following treatment of barley with the resistance-inducing compounds 3,5-dichlorosalicylic acid (DCSA), salicylic acid (SA), and an extract fromBacillus subtilis. The polypeptide showed antifungal activity against the biotrophic cereal pathogensErysiphe graminis f.sp.hordei andPuccinia graminis f.sp.tritici which may indicate a possible role in the mechanism of acquired resistance in barley. A thionin transcript hybridizing to pHvJ256 accumulated also in response to application of jasmonates, or treatments that elevated endogenous amounts of the plant growth substance, pointing to the possibility that signaling mediating defense responses in barley involves jasmonates. However, a topical spray application of jasmonic acid (JA) or jasmonate methyl ester (JM) did not protect barley leaves against infection byE. graminis. Performing a kinetic analysis by an enzyme immunoassay specific for (−)-JA, (−)-JM, and its amino acid conjugates, accumulation of jasmonates was detected in osmotically stressed barley but not at the onset of chemically induced or genetically based resistance governed by the powdery mildew resistance genesMlg, Mla 12, ormlo 5. Furthermore, the jasmonate-inducible proteins JIP-23 and JIP-60 were strongly induced following JM- but not DCINA-treatment or inoculation withE. graminis. Hence, in barley, no indications were found in favour for the previously proposed model of a lipid-based signaling pathway via jasmonates mediating expression of resistance in plants against pathogens.

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.

To test whether the response to electrical current and heat treatment is due to the same signaling pathway that mediates mechanical wounding, we analyzed the effect of electric-current application and localized burning on proteinase inhibitor II (Pin2) gene expression in both wild-type and abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) and potato (Solanum phureja) plants. Electric-current application and localized burning led to the accumulation of Pin2 mRNA in potato and tomato wild-type plants. Among the treatments tested, only localized burning of the leaves led to an accumulation of Pin2 mRNA in the ABA-deficient plants. Electric-current application, like mechanical injury, was able to initiate ABA and jasmonic acid (JA) accumulation in wild-type but not in ABA-deficient plants. In contrast, heat treatment led to an accumulation of JA in both wild-type and ABA-deficient plants. Inhibition of JA biosynthesis by aspirin blocked the heat-induced Pin2 gene expression in tomato wild-type leaves. These results suggest that electric current, similar to mechanical wounding, requires the presence of ABA to induce Pin2 gene expression. Conversely, burning of the leaves activates Pin2 gene expression by directly triggering the biosynthesis of JA by an alternative pathway that is independent of endogenous ABA levels.

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.

Plants respond to physical injury, such as that caused by foraging insects, by synthesizing proteins that function in general defense and tissue repair. In tomato plants, one class of wound-responsive genes encodes proteinase inhibitor (pin) proteins shown to block insect feeding. Application of many different factors will induce or inhibit pin gene expression. Ethylene is required in the transduction pathway leading from injury, and ethylene and jasmonates act together to regulate pin gene expression during the wound response.