Overexpression of ERF106 and ERF107 leads to tolerance to 9-HOT.

<p>(A) Root growth of Est-inducible XVE-ERF106 and XVE-ERF107 lines was quantified after application of Est (10μM) or Est + 9-HOT (25μM). For each genotype, two independent transgenic lines are analyzed. The untreated wt (Col-0) is set to 100%. Given are mean values +SE (n≥22). The indicated differences are calculated using the student’s <i>t</i>-test: *p ≤ 0.05, **p≤0.01, ***p ≤ 0.001. (B) Immunoblot using an α-HA antibody to detect HA-ERF fusion proteins (arrows). Seedlings were grown for 5d on Medium supplemented with 10μM Est. * background signal. (C) The single and double <i>erf</i> mutants indicated show a root growth on 9-HOT which is comparable to the wt (Col-0). Significant differences between treatments are designated by different letters (one-way analysis of variance with Bonferroni post hoc test; p>0.05).</p

Overview of the screening results.

<p>TF transgenes were PCR-amplified from those <i>At</i>TORF-Ex lines which display restored root growth on 9-HOT. Frequency: TFs found 3–5 time were given in bold.</p

Down-regulation of bZIP11-related TFs enhance root growth upon 9-HOT treatment.

<p>Root growth of Est-inducible bZIP11 overexpressing plants (XVE-bZIP11) or artificial micro RNA lines (XVE-amibZIP2/11/44) was quantified after application of Est (10μM) or Est + 9-HOT (25μM). The untreated wt (Col-0) is set to 100%. Given are mean values +SE (n≥63). The indicated differences are calculated using the student’s <i>t</i>-test: *p ≤ 0.05, **p≤0.01, ***p ≤ 0.001.</p

Root growth responses of XVE-ERF lines upon treatment with various oxylipin and xenobiotic compounds.

<p>Root growth of Est-induced XVE-ERF106 and ERF-107 lines treated or untreated (control) with the compounds indicated. (A) 9-LOX-oxylipins 9-HOT and 9-KOT, (B) JA and its precursor OPDA, (C) the xenobiotic compound TIBA and the synthetic auxin NAA. The untreated wt (Col-0) is set to 100%. Given are mean values +SE (n≥18). The indicated differences are calculated using the student’s <i>t</i>-test: *p ≤ 0.05, **p≤0.01, ***p ≤ 0.001.</p

Expression of TGA5 and its target gene <i>CYP81D11</i> are mediating tolerance to 9-HOT.

<p>Root growth of several <i>Arabidopsis</i> transgenic lines has been quantified comparing 9-HOT treated and untreated roots. The untreated wt (Col-0) is set to 100%. Results obtained from the <i>Arabidopsis</i> triple TGA mutant <i>tga2</i>,<i>5</i>,<i>6</i> and lines expressing TGA5 or TGA2 under control of the 35S promoter in the <i>tga2</i>,<i>5</i>,<i>6</i> background (A) or 35S:CYP81D11 and <i>cyp81d11</i> (B) are shown. Given are mean values +SE (n≥22). The indicated statistical differences are calculated using the student’s <i>t</i>-test: *p ≤ 0.05, **p≤0.01, ***p ≤ 0.001.</p

Screen Identifying <i>Arabidopsis</i> Transcription Factors Involved in the Response to 9-Lipoxygenase-Derived Oxylipins

<div><p>13-Lipoxygenase-derived oxylipins, such as jasmonates act as potent signaling molecules in plants. Although experimental evidence supports the impact of oxylipins generated by the 9-Lipoxygenase (9-LOX) pathway in root development and pathogen defense, their signaling function in plants remains largely elusive. Based on the root growth inhibiting properties of the 9-LOX-oxylipin 9-HOT (9-hydroxy-10,12,15-octadecatrienoic acid), we established a screening approach aiming at identifying transcription factors (TFs) involved in signaling and/or metabolism of this oxylipin. Making use of the <i>At</i>TORF-Ex (<u><i>A</i></u><i>rabidopsis</i> <u><i>t</i></u><i>haliana</i> <u>T</u>ranscription Factor <u>O</u>pen <u>R</u>eading <u>F</u>rame <u>Ex</u>pression) collection of plant lines overexpressing TF genes, we screened for those TFs which restore root growth on 9-HOT. Out of 6,000 lines, eight TFs were recovered at least three times and were therefore selected for detailed analysis. Overexpression of the basic leucine Zipper (bZIP) TF TGA5 and its target, the monoxygenase CYP81D11 reduced the effect of added 9-HOT, presumably due to activation of a detoxification pathway. The highly related ETHYLENE RESPONSE FACTORs ERF106 and ERF107 induce a broad detoxification response towards 9-LOX-oxylipins and xenobiotic compounds. From a set of 18 related group S-bZIP factors isolated in the screen, bZIP11 is known to participate in auxin-mediated root growth and may connect oxylipins to root meristem function. The TF candidates isolated in this screen provide starting points for further attempts to dissect putative signaling pathways involving 9-LOX-derived oxylipins.</p></div

An Iron 13<i>S</i>-Lipoxygenase with an α-Linolenic Acid Specific Hydroperoxidase Activity from <i>Fusarium oxysporum</i>

<div><p>Jasmonates constitute a family of lipid-derived signaling molecules that are abundant in higher plants. The biosynthetic pathway leading to plant jasmonates is initiated by 13-lipoxygenase-catalyzed oxygenation of α-linolenic acid into its 13-hydroperoxide derivative. A number of plant pathogenic fungi (e.g. <i>Fusarium oxysporum</i>) are also capable of producing jasmonates, however, by a yet unknown biosynthetic pathway. In a search for lipoxygenase in <i>F. oxysporum</i>, a reverse genetic approach was used and one of two from the genome predicted lipoxygenases (FoxLOX) was cloned. The enzyme was heterologously expressed in <i>E. coli</i>, purified via affinity chromatography, and its reaction mechanism characterized. FoxLOX was found to be a non-heme iron lipoxygenase, which oxidizes C₁₈-polyunsaturated fatty acids to 13<i>S</i>-hydroperoxy derivatives by an antarafacial reaction mechanism where the bis-allylic hydrogen abstraction is the rate-limiting step. With α-linolenic acid as substrate FoxLOX was found to exhibit a multifunctional activity, because the hydroperoxy derivatives formed are further converted to dihydroxy-, keto-, and epoxy alcohol derivatives.</p></div