Two-week-old wild-type-lines were treated with 100 nM NAA, 10 M NPA, 200 M ZnPPIX, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 100 nM CORM2, 100 nM CORM2+10 M NPA, 30 M SNAP, 30 M GSNO, and 30 M SNAP+10 M NPA, respectively, for 1 week, and the FCR activity was measured. GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S5: Carbon monoxide (CO) modulates root hair and lateral root development. (A,B) Root hair phenotype of Col-0, lines produced in +Fe, CFe, and CFe+CORM2 media for 1 week. (C) Col-0, lines produced on +Fe, CFe, or CFe+CORM2 media for 1 week. Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S6: The effects of different inhibitors on FCR activity in the wild-type Col-0 SR 18292 lines with or without CFe treatment. Two-week-old wild-type-lines were treated with 100 nM NAA, 10 M NPA, 200 M ZnPPIX, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 100 nM CORM2, 100 nM CORM2+10 M NPA, 30 M SNAP, 30 M GSNO, and 30 M SNAP+10 M NPA, respectively, for 1 week, and the FCR activity was measured. Data are the means SD (= 12), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S7: The effects of different inhibitors on auxin transport in the roots of wild-type Col-0 lines with or without CFe treatment. Two-week-old wild-type Col-0 lines were treated with 100 nM NAA, 10 M NPA, 200 M ZnPPIX, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 100 nM CORM2, 100 nM CORM2+10 M NPA, 30 M SNAP, 30 M GSNO, and 30 M SNAP+10 M NPA, respectively, for 1 week, and basipetal auxin transport was measured. Data are the means SD (= 9), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S8: The effects of different inhibitors on Fe content in roots of wild-type Col-0 lines with or without CFe treatment. Two-week-old wild-type Col-0 lines were treated with 100 nM NAA, 10 M NPA, 200 M ZnPPIX, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 100 nM CORM2, 100 nM CORM2+10 M NPA, 30 M SNAP, 30 M GSNO, and 30 M SNAP+10 M NPA, respectively, for 1 week, and the Fe content was measured. Data are the means SD (= 9), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S9: The effect of various inhibitors on NO generation after CFe stress. Two-week-old wild-type Col-0 lines were treated with 100 nM NAA, 10 mM NPA, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 200 M ZnPPIX, 100 nM CORM2, 100 nM CORM2+10 M NPA, and 30 M SNAP, respectively, for 1 day, and the green fluorescence (A) and the corresponding relative fluorescence intensity (B) in the roots were recorded. Data are the means SD (= 9), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S10: The effects of various inhibitors on and expression in the roots of wild-type Col-0 after CFe stress. Two-week-old Col-0 seedlings were treated with the indicated inhibitors for 1 day, and = 3), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Table_1.DOC (53K) GUID:?CD72E7D7-FD12-479F-A71D-F27AC99C9D29 Abstract To clarify the roles of carbon monoxide (CO), nitric oxide (NO), and auxin in the plant response to iron deficiency (CFe), and to establish how the signaling molecules interact to enhance Fe acquisition, we conducted physiological, genetic, and molecular analyses that compared the responses of various mutants, including (CO deficient), (NO deficient), (NO deficient), (auxin over-accumulation), and (NO over-accumulation) to CFe stress. We also generated a HY1 over-expression line (named HY1-OX) in which CO is over-produced compared to wild-type. We found that the suppression of CO and NO generation using various inhibitors enhanced the sensitivity of wild-type plants to Fe depletion. Similarly, the mutants were more sensitive to Fe deficiency. By contrast, the mutant with low CO content exhibited no induced expression of the Fe uptake-related genes and as compared to wild-type plants. On the other hand, the treatments of exogenous CO and NO enhanced Fe uptake. Likewise, and HY1-OX lines with increased endogenous content of NO and CO, respectively, also exhibited enhanced Fe uptake and increased expression of bHLH transcriptional factor FIT1as compared to wild-type.The wild-type Col-0, lines were grown on +Fe media for 2 weeks and then shifted to CFe media (CFe), or +Fe media (+Fe) for 1 week before measurements were taken. were measured. Data are the means SD (= 3). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S4: Expression patterns of in the root tip of wild-type Col-0, lines, and line treated with CORM2. The seedlings were treated with 100 nM CORM2 after 12 h of CFe stress, and YFP fluorescence were recorded. Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S5: Carbon monoxide (CO) modulates root hair and lateral root development. (A,B) Root hair phenotype of Col-0, lines grown in +Fe, CFe, and CFe+CORM2 media for 1 week. (C) Col-0, lines grown on +Fe, CFe, or CFe+CORM2 media for 1 week. Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S6: The effects of different inhibitors on FCR activity in the wild-type Col-0 lines with or without CFe treatment. Two-week-old wild-type-lines were treated with 100 nM NAA, 10 M NPA, 200 M ZnPPIX, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 100 nM CORM2, 100 nM CORM2+10 M NPA, 30 M SNAP, 30 M GSNO, and 30 M SNAP+10 M NPA, respectively, for 1 week, and the FCR activity was measured. Data are the means SD (= 12), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S7: The effects of different inhibitors on auxin transport in the roots of wild-type Col-0 lines with or without CFe treatment. Two-week-old wild-type Col-0 lines were treated with 100 nM NAA, 10 M NPA, 200 M ZnPPIX, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 100 nM CORM2, 100 nM CORM2+10 M NPA, 30 M SNAP, 30 M GSNO, and 30 M SNAP+10 M NPA, respectively, for 1 week, and basipetal auxin transport was measured. Data are the means SD (= 9), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S8: The effects of different inhibitors on Fe content in roots of wild-type Col-0 lines with or without CFe treatment. Two-week-old wild-type Col-0 lines were treated with 100 nM NAA, 10 M NPA, 200 M ZnPPIX, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 100 nM CORM2, 100 nM CORM2+10 M NPA, 30 M SNAP, 30 M GSNO, and 30 M SNAP+10 M NPA, respectively, for 1 week, and the Fe content was measured. Data are the means SD (= 9), SR 18292 and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S9: The effect of various inhibitors on NO generation after CFe stress. Two-week-old wild-type Col-0 lines were treated with 100 nM NAA, 10 mM NPA, 300 M cPTIO, 1 mM L-NAME, 10 mM tungstate, 200 M ZnPPIX, 100 nM CORM2, 100 nM CORM2+10 M NPA, and 30 M SNAP, respectively, for 1 day, and the green fluorescence (A) and the corresponding relative fluorescence intensity (B) in the roots were recorded. Data are the means SD (= 9), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 FIGURE S10: The effects of various inhibitors on and expression in the roots of wild-type Col-0 after CFe stress. Two-week-old Col-0 seedlings were treated with the indicated inhibitors for 1 day, and = 3), and bars with different letters are significantly different at 0.05 (Tukeys test). Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Image_1.PDF (522K) GUID:?C46C40F5-EA08-4E28-966C-94239B7350E9 Table_1.DOC (53K) GUID:?CD72E7D7-FD12-479F-A71D-F27AC99C9D29 Abstract To clarify the roles of SR 18292 carbon monoxide (CO), nitric oxide (NO), and auxin in the plant response to iron deficiency (CFe), and to establish how the signaling molecules interact to enhance Fe acquisition, we conducted physiological, genetic, and molecular analyses that compared the responses of various mutants, including (CO deficient), (NO deficient), (NO deficient), (auxin over-accumulation), and (NO over-accumulation) to CFe stress. We also generated a HY1 over-expression line (named HY1-OX) in which CO is over-produced compared to wild-type. We found that the suppression of CO and NO generation using various inhibitors enhanced the sensitivity of wild-type plants to Fe depletion. Similarly, the mutants were IL2RA more sensitive to Fe deficiency. By contrast, the mutant with low CO content exhibited no induced expression of the Fe uptake-related genes and as compared to.