Abstract
Phytohormone salicylic acid (SA) is a crucial component of plant-induced defense against biotrophic pathogens. Although the key players of the SA pathway are known, there are still gaps in the understanding of the molecular mechanism and the regulation of particular steps. In our previous research, we showed in Arabidopsis suspension cells that n-butanol, which specifically modulates phospholipase D activity, significantly suppresses the transcription of the pathogenesis related (PR-1) gene, which is generally accepted as the SA pathway marker. In the presented study, we have investigated the site of n-butanol action in the SA pathway. We were able to show in Arabidopsis plants treated with SA that n-butanol inhibits the transcription of defense genes (PR-1, WRKY38). Fluorescence microscopy of Arabidopsis thaliana mutants expressing 35S::NPR1-GFP (nonexpressor pathogenesis related 1) revealed significantly decreased nuclear localization of NPR1 in the presence of n-butanol. On the other hand, n-butanol did not decrease the nuclear localization of NPR1 in 35S::npr1C82A-GFP and 35S::npr1C216A-GFP mutants constitutively expressing NPR1 monomers. Mass spectrometric analysis of plant extracts showed that n-butanol significantly changes the metabolic fingerprinting while t-butanol had no effect. We found groups of the plant metabolites, influenced differently by SA and n-butanol treatment. Thus, we proposed several metabolites as markers for n-butanol action.
Highlights
The resistance of plants to pathogens relies on a sophisticated immune system comprising an orchestra of defense mechanisms
We have shown that n-butanol is a molecule with a high impact on the Salicylic acid (SA) signaling pathway in A. thaliana seedlings. n-butanol has been for a long time accepted by the “phospholipase D (PLD) community” as a modulator of PLD activity due to its preference for primary alcohols as substrates (Yang et al, 1967; Munnik et al, 1995)
As n-butanol treatment revealed no effect on the nuclear localization of nonexpressor of pathogenesis related 1 (NPR1) in the 35S::npr1C82A-GFP and 35S::npr1C216A-GFP mutants (Figure 3), we can assume that n-butanol acts in the cytosol in the SA pathway before or during NPR1 translocation to the nucleus. n-butanol could either affect the transmission of the monomer from the cytosol to the nucleus e.g., by direct effect of n-butanol on the nucleopores or by active transport which can be mediated by phosphatidic acid (PA)
Summary
The resistance of plants to pathogens relies on a sophisticated immune system comprising an orchestra of defense mechanisms. The monomeric NPR1, in the nucleus, is continuously degraded by proteasome, a process which plays a dual function in the induction of transcription of the SA related genes (e.g., PR-1) (Wang et al, 2005; Spoel et al, 2009). Phospholipase D affects salicylic acid signaling the nucleus, but considering that newly formed NPR1 is needed for the induction of PR-1 transcription, the proteasome plays a key role in the regulation of NPR1 turnover (Spoel et al, 2009). This work provides evidence that n-butanol, the most effective primary alcohol modulating the activity of PLD, is involved in the regulation of PR-1 transcription in the seedlings of A. thaliana. The non-targeted metabolomic fingerprinting provides evidence that n-butanol has a substantial impact on metabolome whereas t-butanol remains ineffective
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