Blüte U. Krämer I. Kubigsteltig M. Bernal M. Piotrowski D. Schünemann Fakultät

Project-Group: Dr. Ines Kubigsteltig

Jasmonates serve as signalling compounds in plant defence and reproduction

Jasmonic acid belongs as an important representative of the octadecanoids (octadeca = 18, derived from the three-way unsaturated C18-fatty acid α-linolenic acid, which serves as substrate for the biosynthesis) to the plant-specific signalling compounds, which steer the plant organism beside the "classical phytohormones (auxins, cytokinines, gibberellines, abscisic acid and ethylen)". Their major tasks thereby range from parasite defence to reproduction. On the basis of outside stimuli or following a genetically specified program, the content of physiologically active octadecanoids in the cells and tissues constantly change and causes thereby again the expression of octadecanoid regulated genes. Thus for example the content of 12-oxo-phytodienoic acid (OPDA, a preliminary metabolite and likewise physiologically active signal compound) rises rapidly in wounded tissue around a multiple. Consequently the metabolism of the wounded plant itself changes over on the new tasks of A) driving the attacking parasites and B) healing the wounded tissue.

pWhile jasmonate biosynthesis is well examined and almost clarified, and the number of target genes adjusted by octadecanoid signals also constantly increase, there are still large knowledge gaps in reference to the regulation of the signal biosynthesis (inclusive the release of free fatty acids out of chloroplast membranes), as well as the components taking part in further signal transduction.
 

Can new Arabidopsis mutants help to clear up the jasmonate signal transduction pathway?

In context of a project promoted by the “Sonderforschungsbereich 480” the molecular mechanisms underlying the octadecanoid production and effects in higher plants have been examined in our Labatory for a few years.

pOn the basis of transgenic Arabidopsis plants, expressing the reporter gene uidA (ß-glukuronidase) under control of the wound- and octadecanoid regulated allenoxidsynthase (AOS) promoter (Kubigsteltig et al., 1999), a mutagenesis program was conducted to select for mutants with irregular AOS expression. The enzyme AOS was particularly suitable, since it is not only transcriptionally induced as target gene by biologically active octadecanoids (JA, OPDA), but catalyzes the first specific step during jasmonate biosynthesis and thus permits direct access to the modulation of these signal compounds. After completion of the mutagenesis program 12 Arabidopsis mutants were present, from which 4 lines showed a reduced accessibility of the AOS promoter, while with 8 further lines a constitutive promoter activity could be proven. These cas (constitutive allene oxide synthase) mutants were classified on the basis of their different morphologic phenotypes and transgene expression pattern (GUS staining) into four groups (see illustration 1). The physiological investigations of the cas mutants (metabolite analyses, transcription analyses), conducted afterwards, referred to a changed regulation of the jasmonate biosynthesis and confirmed thus the success of our screening (Kubigsteltig & Weiler, 2003).

Mutanten cas1 Habitus

Fig. 1: Histochemical staining for x-glukuronidase activity in two weeks old Arabidopsis thaliana. plants. Dependend on different AOS-promoter activities one can see various staining pattern in the four groups.

Fig. 2: Phenotype of cas1 at the beginning of reproduction. Typically one can see additional rosettes at the inflorenscence axis, while the flowers are male sterile. Zoom: cas1 flower (left), wildtype flower (right).

The mutant cas1 appeared particularly promising for the following reasons:

  • homozygous cas1-mutants show a remarkable, not yet described phenotype (see illustration 2). In the vegetative growth phase the plants develop shorter roots and screwed rosette leaves. The beginning of the reproductive phase is characterized by formation of several rosettes one above the other at the likewise slightly turned inflorescence axis. Most important characteristic is however the nearly complete male sterility of the mutant. This is surprisingly similar to all jasmonate biosynthesis mutants of Arabidopsis known so far (overview see Berger, 2002), which can at least partially be compensated by exogenous application of methyljasmonate.
  • like all cas mutants cas1 exhibits increased quantities of biologically active octadecanoids (OPDA, JA). Depending on inductive stimuli (wounding, coronatine) it becomes evident that neither accessibility, nor the overall capacity of the jasmonate biosynthesis are changed in comparison to the wildtype. Also perception of exogenously applied JA is not generally prevented, which was proven by root length tests. All of this leads to the conclusion that a kind of pre-induction of the jasmonate biosynthesis takes place as result of the mutation. This supports the thesis of cas1 being a genuine mutant in regulation.
  • besides the strengthened AOS expression, cas1 shows an increased transcription of further jasmonate regulated genes. Extensive gene expression analyses have been initiated by means of microarray technology, whereby also doublemutants with coronatine insensitive 1 (coi1-1, Feys et al., 1994) were examined. It clearly showed that genexpression changed by the cas1 mutation is only partially due to the increased jasmonate content in the leaves.
  • first genetic investigations showed that the cas1 phenotype can be attributed to a recessive mutation at one individual locus. By genetic PCR supported coupling analysis, this locus could be located at a range from approximately 8 - 10 centiMorgan on chromosome IV.

 
Cited literature

Berger S (2002) Jasmonate-related mutants of Arabidopsis as tools for studying stress signaling. Planta 214: 497—504.

Feys BJF, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 6: 751—759.

Kubigsteltig I, Laudert D, Weiler EW (1999) Structure and regulation of the Arabidopsis thaliana allene oxide synthase gene. Planta 208: 463—471.

Kubigsteltig I, Weiler EW (2003) Arabidopsis mutants affected in the transcriptional control of allene oxide synthase, the enzyme catalyzing the entrance step in octadecanoid biosynthe-sis. Planta 217: 748-757.
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  • Ines Kubigsteltig
    Dr. Ines Kubigsteltig
    Project leader
    Office: 3/24 (+49 234/32-24308)
    Lab: 3/64 (+49 234/32-24296)
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