Figure 3.

Overexpression of PHR1, PHL2, or PHL3 enhances jasmonate-induced anthocyanin accumulation and root growth inhibition. A) Phenotypes of 8-d-old wild-type (WT), PHR1-HA-L5, PHL2-HA-L3, and PHL3-HA-L7 seedlings grown on modified half-strength Murashige and Skoog (MS) medium containing different concentrations of Pi with or without (Mock) 15 µM methyl jasmonate (MeJA). In the absence of MeJA (mock), an equal volume of 10% (v/v) ethanol was added. The media with 0.65 mM and 1 µM KH₂PO₄ were used as the Pi-sufficient (Pi+) and Pi-deficient (Pi−) media, respectively. Experiments were performed four times with similar results by analyzing different batches of seedlings (more than 60 seedlings for each sample per replicate). Bars = 5 mm. B) Anthocyanin contents in 8-d-old WT, PHR1-HA-L5, PHL2-HA-L3, and PHL3-HA-L7 seedlings grown on Pi+ or Pi− media with or without 15 µM MeJA. Experiments were performed eight times with more than 100 seedlings for each sample per replicate. Data are means ± Sd from eight independent experiments (n = 8 times). FW, fresh weight. C) Root length of 8-d-old WT, PHR1-HA-L5, PHL2-HA-L3, and PHL3-HA-L7 seedlings grown on Pi+ or Pi− media with or without 15 µM MeJA. Experiments were performed four times with similar results by analyzing different batches of seedlings (more than 60 seedlings for each sample per replicate). The root length of 20 representative seedlings was measured for each sample per replicate. Data are means ± Sd (n = 20 representative plants). D) Anthocyanin content changes in 8-d-old WT, PHR1-HA-L5, PHL2-HA-L3, and PHL3-HA-L7 seedlings induced by 15 µM MeJA. Data represent the anthocyanin content differences of seedlings grown on Pi+ or Pi− media. The differences mean the anthocyanins of seedlings with 15 µM MeJA treatment minus those of seedlings without MeJA treatment. The data used to calculate these difference values are from panel B. Data are means ± Sd from eight independent experiments (n = 8 times). E) Percentage of root length changes in 8-d-old WT, PHR1-HA-L5, PHL2-HA-L3, and PHL3-HA-L7 seedlings induced by 15 µM MeJA. Data represent the percentages of root length changes of seedlings grown on Pi+ or Pi− media. The percentages mean the differences of root length with or without 15 µM MeJA treatment divided by root length without MeJA exposure. The data used to calculate percentage are from panel C. Data are means ± Sd (n = 20 representative plants). F) RT-qPCR analyses of LDOX, DFR, AOS, and LOX2 expression levels in WT, PHR1-HA-L5, PHL2-HA-L3, and PHL3-HA-L7 seedlings. For LDOX and DFR, total RNA was extracted from 8-d-old seedlings (more than 60 seedlings for each sample per replicate) grown on Pi+ or Pi− media with or without 15 µM MeJA. For AOS and LOX2, total RNA was extracted from 8-d-old seedlings (more than 60 seedlings for each sample per replicate grown on Pi+ or Pi− media) which were treated with or without 100 μM MeJA for 6 h. Data are means ± Sd from five independent experiments (n = 5 times). Bars with different letters are significantly different from each other (P < 0.05). Data were analyzed by a two-way ANOVA using Tukey's HSD test.