Fig. 6: Dose-dependent effect of proline-2′-deoxymugineic acid and iron toxicity in Brachypodium distachyon under high-temperature conditions.

a B. distachyon Bd21 grown hydroponically with 2.5 mg Fe L⁻¹ non-chelated iron [Fe₂(SO₄)₃] in the absence (−) or presence (+) of proline-2′-deoxymugineic acid (PDMA) at the indicated concentrations under HC for 3 weeks. For chelated iron (Fe-EDTA) treatment, plants were grown with 2.5 mg Fe L⁻¹ of Fe-EDTA under HC for 3 weeks. Scale bars, 5 cm. b Simple ratio pigment index (SRPI) and photochemical reflectance index (PRI) measured from the first fully expanded leaf blade. *P < 0.05, **P < 0.001 (two-tailed Student’s t test, comparing plants with and without PDMA treatment; n = 9, biological replicates). c Expression of the iron deficiency–marker gene BdIRO2.1 and the iron overload-marker gene BdFerritin in roots. Plants were grown with 2.5 mg Fe L⁻¹ non-chelated iron ± PDMA (3, 30, 150, or 300 µM) or Fe-EDTA under HC for 3 weeks. *P < 0.05, **P < 0.001 (two-tailed Student’s t test, comparing plants with PDMA treatment at various levels to those without PDMA; n = 3, biological replicates). d High-temperature treatment increases susceptibility to iron overload. Plants were grown under NC or HC with Fe-EDTA at a normal (45 µM, 2.5 mg Fe L⁻¹) or 100-fold higher concentration (4.5 mM, 250 mg Fe L⁻¹) for 2 weeks. H₂O₂ accumulation was detected in younger leaves using 3,3′-diaminobenzidine staining. Scale bars, 5 cm. e Expression of iron overload-marker gene BdFerritin in the first fully expanded leaves grown under NC or HC with chelated iron at normal (×1, 45 µM) or 100-fold higher concentration (×100, 4.5 mM) for 1 week. *P < 0.05 (two-tailed Student’s t test, comparing plants grown under normal and higher iron concentrations; n = 6, biological replicates). Data are means ± SEM. Exact P values for significant differences are shown on each graph. Source data are provided as a Source Data file.