Fig 1.
ANT improves L. sativa ‘Salinas’ germination under thermoinhibition (A) ANT and fluridone reverse thermoinhibition in L. sativa ‘Salinas’. Representative images are shown. (B) Barplot summarizing germination. Germination was quantified for seeds imbibed on plates containing DMSO (mock-treated), 10 μM ANT, 10 μM fluridone or 100 μM AbamineSG (representative image in S1 Fig) and grown for 48h. (C) Structures of antabactin, abamineSG and fluridone. (D) GA and KAR1 did not reverse thermoinhibition. Germination was quantified for seeds imbibed on plates containing DMSO, 10 μM ANT, 100 μM GA or 100nM KAR1 and grown for 48h. (E) ANT is potent in the low micromolar range. Germination was quantified for seeds after growth on plates for 24h at 32°C. ET50 value inset in the graph are inferred by nonlinear fits in drc. (F) 10 μM ANT reverses thermoinhibition at temperatures of up to 40°C. Germination was quantified seeds after growth in either ANT or mock (DMSO) for 48h. In (A), (B), (D), (E), and (F), liquid sterilized seeds were grown on ½ MS, 0.7% agar plates in the dark. Error bars indicate SEM (n = 3).
Fig 2.
ANT alters the expression of thermoinhibition and ABA-regulated genes.
(A) Tree inferred using the Maximum Likelihood method and the Tamura-Nei model showing the three subgroups of MAP3Ks in Arabidopsis and the three lettuce homologs of AtMAP3K18 identified by BLAST. Bootstrap support values, derived from 500 replicate analyses, are displayed on the tree to indicate the reliability of each branch. (B) ANT treatment reduces the LsM3K18A expression (normalized to UBC21) measured by qRT-PCR of lettuce leaves in (DMSO) or 100 μM ABA or 100 μM ABA and 50 μM ANT in water. (C) ANT treatment reduces LsNCED4 expression (normalized to UBC21) measured by qRT-PCR of seeds grown for 24 hours in mock (DMSO) or 10 μM ANT. Liquid sterilized seeds were grown in ½ MS, 0.7% agar plates in the dark. * indicates p < 0.05, ** indicates p < 0.01 (pairwise two sample t-test corrected for multiple comparisons). Error bars indicate SEM (n = 3).
Fig 3.
Priming with ANT improves lettuce seed germination (A) Extracted ion chromatograms (EIC) for ANT+H m/z 611.270 (+/- 50 ppm). (B) Priming seeds with ANT improves germination rates under thermoinhibition. Germination was quantified for seeds after ripening for 7, 5, and 1.5 months (MAR) primed in -1.25 Mpa solution of PEG 8000 and either 100 μM ANT or mock (DMSO). Primed seeds were plated on ½ MS, 0.7% agar plates, and germination was quantified at 24h at 35°C. *** indicates p < 0.01 (two-sample t-tests). Error bars indicate SEM (n = 3). (C) Representative images are shown.