Octadecanoid-derived alteration of gene expression and the "oxylipin signature" in stressed barley leaves. Implications for different signaling pathways.

Stress-induced gene expression in barley (Hordeum vulgare cv Salome) leaves has been correlated with temporally changing levels of octadecanoids and jasmonates, quantified by means of gas chromatography/mass spectrometry-single ion monitoring. Application of sorbitol-induced stress led to a low and transient rise of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA), and the methyl esters JAME and OPDAME, respectively, followed by a large increase in their levels. JA and JAME peaked between 12 and 16 h, about 4 h before OPDA and OPDAME. However, OPDA accumulated up to a 2.5-fold higher level than the other compounds. Dihomo-JA and 9, 13-didehydro-OPDA were identified as minor components. Kinetic analyses revealed that a transient threshold of jasmonates or octadecanoids is necessary and sufficient to initiate JA-responsive gene expression. Although OPDA and OPDAME applied exogenously were metabolized to JA in considerable amounts, both of them can induce gene expression, as evidenced by those genes that did not respond to endogenously formed JA. Also, coronatine induces JA-responsive genes independently from endogenous JA. Application of deuterated JA showed that endogenous synthesis of JA is not induced by JA treatment. The data are discussed in terms of distinct signaling pathways.

Cellulysin from the plant parasitic fungus Trichoderma viride elicits volatile biosynthesis in higher plants via the octadecanoid signalling cascade.

Cellulysin, a crude cellulase from the plant parasitic fungus Trichoderma viride, induces the biosynthesis of volatiles in higher plants (Nicotiana plumbaginifolia, Phaseolus lunatus, and Zea mays) when applied to cut petioles by the transpiration stream. The pattern of the emitted volatiles largely resembles that from a herbivore damage or treatment of the plants with jasmonic acid (JA) indicating that cellulysin acts via activation of the octadecanoid signalling pathway. The treatment with cellulysin raises the level of endogenous JA after 30 min and is followed by a transient emission of ethylene after 2-3 h. Volatile production becomes significant after 12-24 h. Inhibitors of the JA pathway effectively block the cellulysin-dependent volatile biosynthesis.

Ethylene as a Signal Mediating the Wound Response of Tomato 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.

Localized Wounding by Heat Initiates the Accumulation of Proteinase Inhibitor II in Abscisic Acid-Deficient Plants by Triggering Jasmonic Acid Biosynthesis.

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.

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes.

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.

Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw.

Three-week-old protonemata of Funaria hygrometrica Hedw. cultivated in Petri dishes tolerate slow drying (24 h to complete dryness) but not rapid drying (1h to complete dryness). Slowly dried mosses show, on a dry-weight basis, a sixfold increase in abscisic-acid (ABA) contents during the drying process. Rehydrated, slowly dried protonemata have the ability to tolerate subsequent rapid drying. When ABA is added to three-week-old protonemata at a concentration of 10 µM for 16 h, tolerance to rapid drying is induced. These data indicate that the induction of drought tolerance in Funaria hygrometrica is mediated by ABA. Mosses treated with ABA loose their water as fast as controls do; therefore, ABA does not act via reduced water loss. However, induction of synthesis of new proteins by ABA may form an important part of the drought tolerance because 10 µM cycloheximide inhibits the ABA-mediated tolerance to rapid drying.

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots.

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA1 and GA4. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA9- as well as GA20-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod (-) and fix (-) mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.

The immunoassay of gibberellins : I. radioimmunoassays for the gibberellins A1, A 3, A 4, A 7, A 9 and A 20.

Immunological assays for the detection of picogram amounts of gibberellins A1, A3, A4, A7, A9 and A20 are described. Antisera of high affinity (Ka over 10(10) l mol(-1)) were raised in rabbits by immunization against carboxyl-coupled gibberellin-bovine-serum-albumin conjugates and used in combination with tritiated gibberellins or gibberellin derivatives of high specific radioactivity (over 10(14) Bq mol(-1)). The assays allow gibberellin quantitation in as little as 1 mg of vegetative plant tissue.

The immunoassay of gibberellins : II. Quantitation of GA3, GA 4 and GA 7 by ultra-sensitive solid-phase enzyme immunoassays.

The development of sensitive and specific solid-phase enzyme immunoassays for gibberellic acid and gibberellins A4 and A7 is reported. The use of antisera of high apparent affinity (Ka over 10(10) l mol(-1)) in conjunction with alkaline phosphatase-labeled gibberellins allows, with minimum procedural effort, the quantitative determination of sub-picogram amounts of these gibberellins. The assays reported here are applicable to most gibberellins and can be set up with 1-1.5 mg of starting material. They represent the most sensitive methods for gibberellin determination known.

The role of endogenous gibberellins in the formation of α-amylase by aleurone layers of germinating barley caryopses.

Using sensitive and selective immunological assays we have shown that in germinating caryopses of Hordeum vulgare L. cv. Himalaya, the level of gibberellin A4 (GA4) rises approximately 18-to 20-fold shortly (2-4 h) before α-amylase activity increases. Gibberellin A4 is the predominant immunoreactive gibberelin during these developmental stages and reaches a peak amount of approximately 9 pmol per caryopsis about 48 h after imbibition. Isolated aleurone layers produce GA4 in the presence of an exogenous gibberellin, such as GA1, which is not a biosynthetic precursor for GA4. Experiments with inhibitors of gibberellin biosynthesis indicate that gibberellin synthesis is required in this tissue for the induction of α-amylase. The inductive effect of exogenously applied GA1 is indirect and appears to be mediated by GA4. Embryos form predominantly GA1; however, very little of this material is released by isolated embryos into the incubation medium. The results presented make it unlikely that the role of the embryo in the process of α-amylase induction in aleurone layers is to provide gibberellins or gibberellin precursors.

Formation and distribution of sennosides in Cassia angustifolia, as determined by a sensitive and specific radioimmunoassay.

A radioimmunoassay for the quantitation of nanogram-amounts of sennoside B and related compounds in plant extracts is described. The assay makes use of [ (3)H]-8-glucosidorheinanthrone of high specific activity (5.2 Ci/mmol) whose synthesis is reported here. From this material, [ (3)H]-sennoside A and [ (3)H]-sennoside B have also been synthesized. The assay is applied to the analysis of sennoside formation and distribution in CASSIA ANGUSTIFOLIA VAHL. High levels of sennosides in dried leaves and fruits have been observed whereas the seed alone, as well as stems and roots, contain very little sennoside. In flowers, as much as 4-5% of the dry weight consists of sennoside B and other immunoreactive constituents. Sennosides have been found in cotyledons of three day old seedlings in concentrations comparable to that of the mature leaf. Upon dehydration, leaf levels of sennoside B rise steadily, this rise being inversely correlated with the water loss. The absolute levels of sennoside B formed this way are the same as compared to rapid drying at 60 degrees C.