9(S)-HOTrE

Chemical treatments

The following compounds were tested in root growth assays using the indicated concentrations. 9-HOT (9(S)-hydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid), 9-KOT (9-keto-10(E),12(Z),15(Z)-octadecatrienoic acid) and OPDA (12-Oxo-Phytodienoic Acid) were prepared as described in [,], respectively. TIBA (2,3,5-triiodobenzoic acid), Naphthalene-Acetic Acid (NAA) and Jasmonic Acid (JA) were obtained from Sigma (Munich, Germany).

Screening for transcription factors involved in oxylipin signaling and detoxification

Aiming at TF function involved in the responses to 9-LOX-oxylipins, we assayed the root growth of 2-week-old Arabidopsis seedlings on MS-Agar supplemented with 25 μM 9-HOT. In contrast to [], a “wavy” root growth phenotype was not reproducibly observed under our experimental conditions, but root apical growth was severely impaired (). Based on the hypothesis that oxylipin signaling or detoxification programs are transcriptionally controlled, a screen based on overexpression of TFs should identify crucial regulators involved in these processes. Here, we established a screen using the AtTORF-Ex resource [], a collection of GATEWAY®-derived Arabidopsis TF overexpression lines employing the 35S promoter and scored plants showing wildtype (wt) root growth after 9-HOT application to the media. Screening of more than 6000 seedlings recovered 201 individuals (3%) which display tolerance to 9-HOT (). According to the calculations in [], the sample size should be statistically sufficient to cover all possible TFs in the screen with a probability of 99%. Due to the conserved GATEWAY®-att sequences flanking the TF orfs in the AtTORF-Ex transgenic lines, the respective overexpressed TF genes can easily be recovered by PCR (). As summarized in , several TF genes have been isolated up to 5 times, and hence their overexpression leads reproducibly to 9-HOT tolerant root growth (e.g. ERF106, ERF107, MADS-box factor AGL86). Members of the bZIP family are clearly overrepresented among the TFs leading to 9-HOT tolerance. This is particularly interesting, as bZIP members have been described as regulators of protective pathways towards endogenous or exogenous oxylipin molecules in plant and non-plant species [,,,–]. However, evolutionary conservation is not supported by homology outside the bZIP domain. Frequently, we observed that expression of related family members resulted in the same growth phenotype. Presumably, these TFs share related binding properties and functional redundancies. This was particularly true for bZIP factors of the group S [], which overall were found 18 times in the screen. The most abundant bZIP members are the highly related TFs bZIP11 and bZIP2 (overall 7 times). Furthermore, CRFs are a group of TFs which modulate cytokinine signaling [,]. Although single CRFs were found only twice, overall 3 related group members were detected. Cytokinine has a crucial function in controlling root meristem size [] and hence, CRF overexpression may overcome the block in root growth caused by 9-HOT. In contrast, TFs identified only once or twice are less likely candidates. Nevertheless, less abundant TFs may also be important but might have missed for technical reasons. We have selected several TF candidates to study their impact in the tolerance mechanism to 9-HOT in more detail.

Expression of the bZIP transcription factor TGA5 and its target gene CYP81D11 provoke tolerance to 9-HOT

TGAs have been proposed to be involved in controlling detoxification pathways with respect to oxylipins or xenobiotics. The cytochrome P450 monoxygenase gene CYP81D11 is transcriptionally induced by lipid peroxidation products like phytoprostanes, OPDA and JA as well as electrophilic xenobiotics (e.g. 2,4-dichlorophenoxyacetic acid, benoxacor, or the auxin-transport inhibitor TIBA (2,3,5-triiodobenzoic acid) [,]. Along this line, plants constitutively expressing CYP81D11 show significantly increased root growth on 9-HOT containing media (), demonstrating that the CYP81D11 enzyme is sufficient to provide full tolerance to 9-HOT. Hence, although the enzymatic function of CYP81D11 is not well-defined yet, these and previous data propose a function in metabolizing both 9- and 13-LOX-derived oxylipins. However, in comparison to wt, root growth of the cyp81d11 mutant on 9-HOT is slightly but not significantly reduced. Most likely, other CYP450 proteins may share redundant functions. Further studies are needed to define the substrate specificity of CYP81D11 with respect to xenobiotics and endogenous compounds.