Charakterisierung eines safenerinduzierbaren Promotors und Analyse der safenerabhängigen Genexpression in Arabidopsis thaliana (English shop)

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Safeners are added to many modern non-selective herbicides. This leads to the selective activation of metabolic processes in crop plants that result in a more rapid degradation of the herbicide. Such an effect, however, cannot be observed in the target weeds. Safeners increase the tolerance of the crop plant towards the non-selective herbicide. To date, this effect has only been demonstrated in monocotyledonous crops. A better understanding of the regulatory mechanisms leading to enhanced herbicide metabolism in crop plants is an important aspect of safener research. The aims of this work were the characterisation of elements of the safener signalling pathway as well as an analysis of safener-induced gene expression in the dicotyledonous model plant Arabidopsis thaliana.

Characterisation of safener signal transduction in A. thaliana
The safener-inducible enzymes of the plant detoxification system include, among others, glutathione S-transferases (GSTs). In order to characterise cis-regulatory elements of the safener signalling pathway, the promoter of the maize In2.1 gene was used. This gene encodes a GST of the highly safener-inducible lambda class (GSTLs) of the GST superfamily. Because of the massive induction of GSTLs by benzenesulfonamide safeners first described in maize, these GSTs are also referred to as “In2.1 proteins”. The promoter of the In2.1 gene was fused to the glucuronidase (GUS) reporter gene and integrated into the Arabidopsis thaliana genome.
It could be shown in this work that a 100 bp sequence segment is relevant for the safener inducibility of the In2.1 promoter by mefenpyr and isoxadifen. Two as-1 or ocs elements could be identified within this sequence. Deletion of one of these motifs led to a reduced safener inducibility of the promoter.
The binding of two TGA transcription factors (TGA2/5) to one of these as-1 elements in vivo was demonstrated in a chromatin immunoprecipitation experiment. The relevance of these factors for the inducibility of the In2.1 promoter was confirmed by transforming the PIn2.1:GUS construct into a TGA transcription factor knockout mutant (tga2/3/5/6).
TGA factors require a cofactor for the induction of transcription. One such interaction partner is NPR1 (non expressor of PR genes). Together with TGA factors it leads to the expression of PR1 (pathogenesis-related protein 1), a marker gene of systemic acquired resistance. The involvement of this cofactor in the safener signalling pathway was examined by transforming the PIn2.1:GUS construct into a corresponding mutant (sai1-1; salicylic insensitive 1). The safener signalling pathway could thereby be assessed as NPR1-independent.
TGA transcription factors, as-1 elements, as well as the accumulation of salicylic acid after pathogen attack play an important role in the salicylic acid signalling pathway that leads to the establishment of systemic acquired resistance. Since the In2.1 promoter is also strongly salicylic acid-inducible, the influence of the internal salicylic acid content on the inducibility of the promoter by mefenpyr and isoxadifen was examined. A low endogenous salicylic acid concentration led to a loss of safener inducibility of the maize promoter in transgenic salicylic acid biosynthesis mutants (NahG, sid2-2). The influence of the application of mefenpyr and isoxadifen on salicylic acid biosynthesis in wild-type (WT), sid2-2 and NahG plants was investigated 2-6 h after safener application. Neither in the WT nor in the salicylic acid biosynthesis mutants could a mefenpyr and isoxadifen-induced SA biosynthesis be detected.
In order to identify further, as yet unknown elements of the safener signalling pathway, a mutant screen is a suitable approach. To create the prerequisites for this, homozygous Arabidopsis plants carrying the luciferase reporter gene under the control of the In2.1 promoter were generated within the framework of this thesis. After mutagenesis of the seeds of this reporter line, both “loss-of-function” (no reporter gene expression after safener treatment) and “gain-of-function” mutations (constitutive reporter gene expression without safener treatment) can be identified.

Gene expression in A. thaliana treated with mefenpyr and isoxadifen
The effect of mefenpyr and isoxadifen on gene expression in dicotyledonous plants was investigated by a microarray experiment (ATH1 Gene Chip, Affymetrix). The wild-type and mutant plants already used for the characterisation of the In2.1 promoter (tga2/3/5/6, sid2-2, NahG) were employed for this purpose.
In wild-type plants, the induction of genes of various phases of the plant detoxification system (P450s, GSTs, GTs, transporters) as well as of an endogenous In2.1 gene could be demonstrated. The induced GSTs mainly belong to the tau class (GSTUs) of the GST superfamily. The induction of GSTs in A. thaliana can, as in monocots, be assessed as statistically significant for the safener response. This result differs from earlier investigations of safener-induced GST expression in Arabidopsis, in which mainly the induction of phi class GSTs (GSTFs) was detected after treatment with the safener benoxacor. Evidently, different safeners act selectively on the gene expression of phi and tau class GSTs in A. thaliana, which had previously also been observed in monocots.
The influence of the TGA transcription factors on the safener signalling pathway could be confirmed by the gene expression experiment with the TGA knockout mutant. About half of the genes safener-regulated in the wild type are affected by this mutation. Among the safener-regulated genes in wild-type plants, a statistically significant induction of WRKY transcription factors and an overrepresentation of WRKY transcription factor binding sites in the promoter sequences were detected. Based on these results, an involvement of WRKY transcription factors in the safener signalling pathway is very likely.
The results were summarised in a model of the safener signalling pathway. This model represents a good basis for further experiments on the characterisation of the safener-controlled signalling cascade. Owing to the many parallels regarding the safener response in mono- and dicotyledonous plants, it seems likely that safeners for dicotyledonous crops will eventually be found after all.