Fig 1.
(A) Screening of the activation tagged Arabidopsis lines identified a mutant, which was able to form roots/leaves on 4 μM Tcin. (B) The T-DNA tag was inserted into the last exon of the At5G55440 gene in trr1. (C) Expression analysis of the activation tagged AT5G55440 locus and the flanking genes, At5G55420, At5G55430, At5G55450 (AtLTP4.4), At5G55460 (AtLTP4.5) and wild type Arabidopsis Col-0 by qRT-PCR. Expression levels of AtLTP4.4 and AtLTP4.5 were significantly higher in the activation tagged trr1 line compared to the wild type Col-0. (D) Immunoblot analysis of total protein (25 μg) isolated from Col-0 and 3 independent Arabidopsis lines containing AtLTP4.4:GFP or AtLTP4.5:GFP, separated on a 12% SDS polyacrylamide gel and probed with monoclonal anti-His IgG (1:500) (GenScript) followed by Amersham ECL Plex C anti-mouse IgG (1:2500) conjugated with Cy3 and scanned using the Typhoon FLA 9500 (GE Healthcare Life Sciences). The blot was stained with Ponceau S and the ~52 kD band is shown for equal loading. (E) Analysis of AtLTP4.4 RNA expression in two independently transformed Arabidopsis lines and in the LTP4.4/LTP4.5 knockout line (SALK_207859c).
Table 1.
IC50 values for trichothecenes in yeast expressing Arabidopsis nsLTPs.
Fig 2.
Overexpression of AtLTP4.4 in Arabidopsis increases tolerance to Tcin.
(A) Detached leaves overexpressing AtLTP4.4 (line #16) relative to 35S:GFP (Col-0) control and the LTP4.4/4.5 KO line after treatment with 4 and 8 μM Tcin for 72 h. (B) Germination of AtLTP4.4 overexpressing lines # 7 and # 16 on 3 μM Tcin relative to the control, 35S:GFP. (C) Percentage of wild type and AtLTP4.4 overexpressing lines able to form roots on 4 μM Tcin in 2 weeks. Error bars represent standard error (S.E.) calculated from three independent experiments. One-way ANOVA with post hoc Bonferroni tests, *P < 0.05, **P<0.01.
Fig 3.
AtLTP4.4:GFP is localized to the cell wall/apoplast and chloroplasts in Arabidopsis leaves.
(A) Confocal microscopy analysis of line #16 expressing AtLTP4.4:GFP showing expression in the cell wall/apoplast, (B) possibly in the ER, and (C) chloroplasts (D) 35S:GFP (Col-0) control.
Fig 4.
Overexpression of AtLTP4.4 reduces Tcin-induced ROS accumulation in Arabidopsis.
Confocal microscopy analysis of ROS accumulation in wild type Arabidopsis (Col-0) leaves infiltrated with Tcin and the cell-permeable fluorogenic probe 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) after 24 h. (A,B) Mock treatment with buffer, (C,D) treatment with 8 μM Tcin (E,F) treatment with 24 μM Tcin. (G) Intact leaf assay to quantify H2O2 levels in transgenic Arabidopsis lines #7 and #16 overexpressing AtLTP4.4 using Amplex Red. Treatment with 4 μM Tcin for 24 h induced H2O2 accumulation in the vector control (35S:GFP), but not in lines #16 and #7. Error bars indicate S.E where n = 3 independent replicates. One-way ANOVA with post hoc Bonferroni tests, ***P<0.001.
Fig 5.
AtLTP4.4 expression attenuates ROS levels and increases glutathione content in yeast.
(A) Trichothecene treated or untreated wild type yeast (BY4743) cells carrying the empty vector, overexpressing AtLTP4.4, or overexpressing AtLTP4.5 were stained for ROS using DCHF and analyzed by flow cytometry. ANOVA with post hoc Bonferroni tests, *P < 0.05, **P < 0.01. (B) GSH and GSSG levels were quantified relative to vector control and normalized to total protein in yeast expressing AtLTP4.4 and AtLTP4.5 after 3 hour galactose induction. ANOVA with post hoc Bonferroni tests, *P < 0.05, **P < 0.01.
Fig 6.
AtLTP4.4 overexpression increases glutathione content of Arabidopsis leaves.
(A) Germination of 35S:GFP seedlings using media supplemented with 1 mM cysteine (Cys) or 250 μM glutathione (GSH) and 3 μM Tcin. Comparison of the effect of the addition of cys and GSH with Tcin to the Tcin treatment alone using ANOVA with post hoc Bonferroni tests, ***P<0.001. (B) Germination assay of 35S:GFP seedlings on media supplemented with 250 μM buthionine sulfoximine (BSO) and 2 μM Tcin. Unlike 3 μM Tcin which substantially reduces the germination percentage, 2 μM Tcin did not impact germination percentage. Comparison of the effect of BSO treatment on Tcin treated or untreated samples using ANOVA with post hoc Bonferroni tests, **P < 0.01. (C) GSH and GSSG levels of mature leaves from AtLTP4.4 overexpressing line #16 and 35S:GFP control (3 biological replicates each). ANOVA with post hoc Bonferroni tests, *P < 0.05. (D) GSH1 expression in lines overexpressing AtLTP4.4 relative to 35S:GFP line by qRT-PCR.