Fig. 5

Nitrate deprivation induces ROS at the root hair differentiation zone (RHDZ) and H₂O₂ treatment alters the gene expression of ROXYs. a Pseudo-color images of ROS fluorescence are shown for nitrate-sufficient (4 mM NO₃⁻) wild type roots and for nitrate-starved (no NO₃⁻) wild type roots. Three-day-old seedlings grown on complete nutrient medium were floated onto liquid nutrient media containing either 4 mM nitrate or no nitrate. After 24 h, ROS images were collected following staining of roots with 20 μM DFFDA for 20 min. Yellow and red colors indicate higher ROS production (see a bottom inset for pixel intensity). Top insets show magnification of the RHDZ. White lines were drawn by hand to outline the root. Bar = 200 μm; bar in inset = 100 μm. b Quantified data of ROS in the RHDZ from representative images shown in (a) (n = 10 images of individual seedlings ± SE). Results from one of two independent experiments are shown here. Different letters above the bars indicate values that are statistically different (P < 0.05; t test). c H₂O₂ treatment upregulates ROXY9,19,21 while (d) H₂O₂ treatment downregulates ROXY10–15,17. Expression levels of ROXY genes in the wild type seedlings treated with H₂O₂ relative to those in the wild type seedlings not treated with H₂O₂ (set to 1) are shown. Seven-day-old seedlings grown on complete nutrient medium were floated onto the same complete liquid nutrient media containing 10 mM H₂O₂ for 1, 3, and 6 h. Gene expression levels were quantified using qRT-PCR with ACTIN7 as a reference gene. The fold change of ROXY expression levels in seedlings treated with H₂O₂ was compared with the ROXY expression levels in seedlings that were not treated with H₂O₂. Different letters above the bars indicate values that are significantly different (n = 3 biological replicates, P < 0.05; t test)