Publikationen - Molekulare Signalverarbeitung

Despite the vital roles of jasmonoyl-isoleucine (JA-Ile) in governing plant growth and environmental acclimation, it remains unclear what intracellular processes lead to its induction. Here, we provide compelling genetic evidence that mechanical and osmotic regulation of turgor pressure represents a key elicitor of JA-Ile biosynthesis. After identifying cell wall mutant alleles in KORRIGAN1 (KOR1) with elevated JA-Ile in seedling roots, we found that ectopic JA-Ile resulted from cell nonautonomous signals deriving from enlarged cortex cells compressing inner tissues and stimulating JA-Ile production. Restoring cortex cell size by cell type–specific KOR1 complementation, by isolating a genetic kor1 suppressor, and by lowering turgor pressure with hyperosmotic treatments abolished JA-Ile signaling. Conversely, hypoosmotic treatment activated JA-Ile signaling in wild-type plants. Furthermore, constitutive JA-Ile levels guided mutant roots toward greater water availability. Collectively, these findings enhance our understanding on JA-Ile biosynthesis initiation and reveal a previously undescribed role of JA-Ile in orchestrating environmental resilience.

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

Over the next decade, wheat grain production must increase to meet the demand of a fast growing human population. One strategy to meet this challenge is to raise wheat productivity by optimizing plant stature. The Reduced height 8 (Rht8) semi-dwarfing gene is one of the few, together with the Green Revolution genes, to reduce stature of wheat (Triticum aestivum L.), and improve lodging resistance, without compromising grain yield. Rht8 is widely used in dry environments such as Mediterranean countries where it increases plant adaptability. With recent climate change, its use could become increasingly important even in more northern latitudes. In the present study, the characterization of Rht8 was furthered. Morphological analyses show that the semi-dwarf phenotype of Rht8 lines is due to shorter internodal segments along the wheat culm, achieved through reduced cell elongation. Physiological experiments show that the reduced cell elongation is not due to defective gibberellin biosynthesis or signalling, but possibly to a reduced sensitivity to brassinosteroids. Using a fine-resolution mapping approach and screening 3104 F2 individuals of a newly developed mapping population, the Rht8 genetic interval was reduced from 20.5 cM to 1.29 cM. Comparative genomics with model genomes confined the Rht8 syntenic intervals to 3.3 Mb of the short arm of rice chromosome 4, and to 2 Mb of Brachypodium distachyon chromosome 5. The very high resolution potential of the plant material generated is crucial for the eventual cloning of Rht8.

Coffea arabica is susceptible to several pests and diseases, some of which affect the leaves and roots. Systemic acquired resistance (SAR) is the main defence mechanism activated in plants in response to pathogen attack. Here, we report the effects of benzo(1,2,3)thiadiazole-7-carbothioic acid-s-methyl ester (BTH), a SAR chemical inducer, on the expression profile of C. arabica. Two cDNA libraries were constructed from the mRNA isolated from leaves and embryonic roots to create 1587 nonredundant expressed sequence tags (ESTs). We developed a cDNA microarray containing 1506 ESTs from the leaves and embryonic roots, and 48 NBS-LRR (nucleotide-binding site leucine-rich repeat) gene fragments derived from 2 specific genomic libraries. Competitive hybridization between untreated and BTH-treated leaves resulted in 55 genes that were significantly overexpressed and 16 genes that were significantly underexpressed. In the roots, 37 and 42 genes were over and underexpressed, respectively. A general shift in metabolism from housekeeping to defence occurred in the leaves and roots after BTH treatment. We observed a systemic increase in pathogenesis-related protein synthesis, in the oxidative burst, and in the cell wall strengthening processes. Moreover, responses in the roots and leaves varied significantly.

Racemic jasmonic acid (3R,7R/3S,7S)-(±)-JA) was chemically conjugated with different biogenic amines originating from aliphatic and aromatic α-amino acids by decarboxylation. The resulting isomeric compounds were subjected to reversed-phase high-performance liquid chromatography (HPLC) and to HPLC on the chiral stationary phases Chiralpak AS and Nucleodex β-PM. Under reversed-phase conditions, all the homologous amine derivatives tested could be separated from each other except the JA-conjugates containing 2-phenyl-ethylamine and 3-methylbutylamine. On both chiral supports the (3R,7R)-(−)-JA conjugates eluted earlier than those of the enantiomeric counterpart (3S,7S)-(+)-JA. On Chiralpak AS all the isomers studied could be separated to baseline with a mobile phase containingn-hexane and 2-propanol. The calculated resolution factors were between 1.80 and 4.17. The pairs of isomers were also chromatographed on the cyclodextrin stationary phase Nucleodex β-PM with methanol-triethylammonium acetate buffer as mobile phase. Under these conditions resolution factors were between 0.74 and 1.29. The individual isomers were chiroptically characterized by measurement of their circular dichroism.

The chiral separation of peptide-like conjugates of jasmonic acid and of cucurbic acid isomers was investigated by liquid chromatography on Chiralpak AS and Nucleodex β-PM. The retention sequences reflect distinct chromatographic properties with respect to the chirality of the jasmonic acid part or of the cucurbic acid isomers. The chromatographic behaviour of the amide conjugates on a reversed-phase C18 column provides evidence for the resolution of diastereomeric conjugates depending on the chirality of both constituents of the conjugate molecule. The chromatographic procedures are suitable for the analytical and preparative separation of such conjugates.

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Synthetic amide conjugates of (−)-jasmonic acid and its (+)-enantiomer were resolved by means of chiral liquid chromatography. The diastereomeric pairs prepared by chemical reaction of (±)-jasmonic acid with a series of (S)- or (R)-amino acids and with some (S)-amino acid alcohols were completely separated on Chiralpak AS using a mixture of n-hexane/2-propanal as mobile phase. The retention data indicate that the (−)-jasmonic acid conjugates eluted faster than those of the (+)-enantiomer, independent on the configuration of the bound amino acid. Likewise, enantiomeric derivatives of (±)-jasmonic acid and non-chiral amino acids were completely separated on the chiral stationary phase and showed the same elution sequence. The resolution factors,Rs, were found to range between 1.13 and 6.64. The separated compounds were chiropatically analyzed by measurement of the circular dichroism.

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.