Fig 1.
YUC regulates Al stress regulated root growth inhibition.
(A, B) Root growth of WT and yuc mutant seedlings after a seven-day exposure to 0 or 6 μM AlCl3. Three independent experiments were done, each with three replicates. Plants were grown at 22°C in long-day growth conditions. Bar = 1 cm. (C) Root growth of WT (DR5rev:GFP) and yucQ/DR5rev:GFP plants after a seven-day exposure of 0 to 6 μM AlCl3. (D) Effect on the Al-induced inhibition of root growth of WT by addition the YUC inhibitor yucasin. Root growth measured after a seven-day exposure to 0 or 6 μM AlCl3 in the presence of 0 to 10 μM yucasin. Three independent experiments were done, each with three replicates. Error bars represent Student’s t test confidence intervals (n = 9). Statistical difference from expected indicated by asterisks (Fisher’s exact test, **P < 0.01, ***P < 0.005).
Fig 2.
YUCs regulate Al induced local auxin response in root TZ.
(A) Five-day old DR5rev:GFP, DR5rev:GFP/yuc9, DR5rev:GFP/yuc8 yuc9 and DR5rev:GFP/yucQ seedlings were exposed or not (control) to 25 μM AlCl3 for two hours. Four-day old transgenic DR5rev:GFP seedlings were pre-treated with 10 μM yucasin for 1 day, then were co-treated with 25 μM AlCl3 for two hours or not (control). The upper row shows expression of DR5rev:GFP, DR5rev:GFP/yuc9, DR5rev:GFP/yuc8 yuc9 and DR5rev:GFP/yucQ controls while the lower row shows the expression of these transgenic seedlings exposed to 25 μM AlCl3. Cell boundaries appear red following propidium iodide staining. The root-apex TZ is marked by white arrowheads. Scale bar: 100 μm. (B) Quantification of the Al-induced fluorescence intensity in the TZ of DR5rev:GFP, DR5rev:GFP/yuc9, DR5rev:GFP/yuc8 yuc9, DR5rev:GFP/yucQ and 10 μM yucasin pre-treated DR5rev:GFP seedlings (around 30 seedlings were measured in each material). The detected fluorescence region in TZ is marked by yellow rectangles. Cell boundaries appear red following propidium iodide staining. The TZ is marked by white arrowheads. Statistical difference from detected fluorescence is indicated by asterisks (Fisher’s exact test, ***P < 0.001).
Fig 3.
Al stress up-regulated the expression of YUCs in the root-apex TZ.
The expression of the YUCp:eGFP-GUS transgenes in epidermis of the roots exposed to 25 μM AlCl3 for two hours (lower row). Controls are untreated roots (upper row). Cell boundaries appeared red following propidium iodide staining. The root-apex TZ is marked by white arrowheads. Scale bar: 100 μm.
Fig 4.
Al regulated local up-regulation of YUCs is ethylene dependent.
(A) The expression of YUCp:GFP-GUS transgenes in the root-apex epidermis in presence of Al and either ACC or AVG. Three-day old transgenic YUCp:GFP-GUS seedlings were pre-treated with 1 μM AVG or 1 μM ACC for 1 day, then the seedlings were treated with 1 μM AVG or 1 μM ACC in the presence or not of 25 μM AlCl3 for 2 hours. Cell boundaries appear red following propidium iodide staining. The root- apex TZ is marked by white arrowheads. Scale bar: 100μm. (B) Quantification of the Al-induced fluorescence intensity in the TZ of YUC8p:GFP-GUS and YUC8p:GFP-GUS in (A). Around 30 seedlings were measured in each material. The detected fluorescence region in TZ is marked by yellow rectangles. Cell boundaries appear red following propidium iodide staining. Statistical difference from detected fluorescence is indicated by asterisks (Fisher’s exact test, ***P < 0.001).
Fig 5.
EIN3 is a transcriptional activator of YUC9.
(A) Left panel: Schematic diagrams of effector and reporter constructs used in the transient dual-luciferase assays. CaMV 35S promoter driving EIN3 (35S:EIN3) was used as effector, and empty vector as a negative control. Promoter fragments of YUC9 were used to make the YUC9p-LUC reporter. Right panel: Transient dual-luciferase assay shows that EIN3 transactivates the promoter of YUC9 in Arabidopsis protoplasts. Data represent the means of three biological replicates. Error bars represent Student’s t test confidence intervals (n ≥ 9). Statistical significant difference indicated by asterisks (Fisher’s exact test, **P < 0.01). (B) Physical interactions of EIN3 with YUC9 promoter in Y1H assays. Yeast expression plasmids pGADT7-EIN3 were reintroduced into yeast strain Y1H Gold carrying the reporter gene AbAr under the control of the YUC9 promoter. The transformants were screened for their growth on the yeast synthetic defined media (SD/-Leu) in the presence of 50 or 100 ng ml-1 Aureobasidin A (AbA) for stringent selection. The empty vector pGADT7 was included as a negative control. Yeast cultures were diluted (1:10 successive dilution series) spotted onto plates. (C) EIN3 associated with the promoter of YUC9 in ChIP-qPCR assay. Up panel: Schematic diagrams of YUC9 promoter of showing the potential EIN3 binding site (black triangles). The translational start sites (ATG) are shown as +1. Numbers above the diagram indicated the distance away from ATG. DNA fragments (P1 and P2) were used for ChIP. Chromatins isolated from 35S:EIN3-GFP transgenic line and 35S:GFP control were immunoprecipitated with anti-GFP antibody followed by qPCR to amplify P1 and P2 regions. Segment C located in the coding region was used as negative control. Input sample was used to normalize the qPCR results in each ChIP. Fold enrichment was presented as a ratio of normalized results from 35S:EIN3-GFP plants and 35S:GFP. Data are mean ± SD.
Fig 6.
YUCs-dependent auxin biosynthesis contributes to ethylene enhanced local auxin signaling in root-apex TZ under Al stress.
(A) The expression of DR5rev:GFP, DR5rev:GFP/yuc9, DR5rev:GFP/yuc8 yuc9 and DR5rev:GFP/yucQ transgenes, or of DR5rev:GFP after yucasin treatment, in the epidermis of the root-apex in the presence of Al and/or ACC. Single treatments are performed on five-day old seedlings with 25 μM AlCl3 for 2 hours or on three-day old seedlings with 1 μM ACC or 10 μM yucasin for 2 hours. Co-treatments were done on three-day old transgenic seedlings (30 seedlings were analysed for each genotype/treatment combination) with pre-treatment with 1 μM ACC or 10 μM yucasin for 2 days, and co-treatment with 1 μM ACC or 10 μM yucasin and 25 μM AlCl3 for 2 hours. Cell boundaries appear red following propidium iodide staining. The root-apex TZ is marked by white arrowheads. Scale bar: 100μm. (B) Quantification of the Al-induced fluorescence intensity in the TZ of DR5rev:GFP, DR5rev:GFP/yuc9, DR5rev:GFP/yuc8 yuc9 and DR5rev:GFP/yucQ transgenes, or of DR5rev:GFP after yucasin treatment in (A). Around 30 seedlings were measured in each material. The detected fluorescence region in TZ is marked by yellow rectangles. Cell boundaries appear red following propidium iodide staining. Statistical difference from detected fluorescence is indicated by asterisks (Fisher’s exact test, ***P < 0.001).
Fig 7.
YUCs mediate ethylene signaling to enhance root growth inhibition in response to Al stress.
(A, B) Root growth of WT (Col-0), yuc9 and yuc8 yuc9 seedlings after a seven-day exposure to 6 μM AlCl3, 50 nM ACC or co-treatment. Three independent experiments were done, each with three replicates. Plants were grown at 22°C in long days growth conditions. Bar = 1 cm. Error bars in (B) indicate mean ±SD (n≥30). Statistical significance was determined by two-way ANOVA with multiple comparison correction by Tukey HSD. Different letters indicate significance groups (P<0.05). (C) Root growth of WT (DR5rev:GFP), DR5rev:GFP/yucQ seedlings after a seven-day exposure to 6 μM AlCl3, 50 nM ACC or co-treatment. Three independent experiments were done, each with three replicates. Error bars indicate mean ±SD (n≥20). Statistical significance was determined by two-way ANOVA with multiple comparison correction by Tukey HSD. Different letters indicate significance groups (P<0.05). (D) Root growth of WT (Col-0) plants after a seven-day exposure to 6 μM AlCl3 in the presence or not of 10 μM yucasin and/or 50 nM ACC. Three independent experiments were done, each with three replicates. Bar = 1 cm. Error bars in indicate mean ±SD (n≥12). Statistical significance was determined by one-way ANOVA with multiple comparison correction by Tukey HSD. Different letters indicate significance groups (P<0.05).
Fig 8.
PIF4 regulates expression of YUCs in Al stress conditions.
(A) Root growth of WT, pif4 and 35S:PIF4 plants after a seven-day exposure to 0 or 6 μM AlCl3. Three independent experiments were done, each with three replicates. Plants were grown at 22°C in long days growth conditions. Error bars represent Student’s t test confidence intervals (n≥30). Statistical difference from expected indicated by asterisks (Fisher’s exact test, *P<0.05, **P<0.01). (B) Relative transcript abundance of YUC3, YUC5, YUC7, YUC8 and YUC9 genes in 7-d-old WT and 35S:PIF4 seedlings roots. Data represent the mean of three biological replicates. Statistical difference is indicated by asterisks (Fisher’s exact test, *P < 0.05, ***P < 0.005). (C) Left panel: Schematic diagrams of effector and reporter constructs used in the transient dual-luciferase assays. CaMV 35S promoter driving PIF4 (35S:PIF4) was used as effector, and empty vector as a negative control. Promoter fragments of YUC5 and YUC9 were used to make the YUC5p-LUC and YUC9p-LUC reporters. Right panel: Transient dual-luciferase assay shows that PIF4 transactivates the promoters of YUC5 and YUC9 in Arabidopsis protoplasts. Data represent the mean of three biological replicates. Statistical difference are indicated by asterisks (Fisher’s exact test, *P < 0.05, **P < 0.01).
Fig 9.
Al regulated local induction of YUCs in root-apex TZ is regulated by PIF4.
(A) Five-day old YUC8p:eGFP-GUS, YUC8p:eGFP-GUS/pif4-101, YUC9p:eGFP-GUS, YUC9p:eGFP-GUS/pif4101 transgenes were exposed or not (control) to 25 μM AlCl3 for two hours. Cell boundaries appear red following propidium iodide staining. The TZ is marked by white arrowheads. Scale bar: 100μm. (B) Quantification of the Al-induced fluorescence intensity in the TZ of YUC8p:eGFP-GUS, YUC8p:eGFP-GUS/pif4-101, YUC9p:eGFP-GUS, YUC9p:eGFP-GUS/pif4-101 seedlings. The detected fluorescence region in TZ is marked by yellow rectangles. Cell boundaries appear red following propidium iodide staining. Error bars indicate mean ±SD (n≥20). Statistical difference from detected fluorescence is indicated by asterisks (Fisher’s exact test, *P<0.05). (C) The expression of PIF4p:GFP transgenes in the root-apex epidermis in presence of 25 μM AlCl3 for 2 hours. Cell boundaries appear red following propidium iodide staining. The TZ is marked by white arrowheads. Scale bar: 100μm.
Fig 10.
PIF4 regulates Al-induced local auxin signaling in root-apex TZ.
(A) Five-day old DR5rev:GFP and DR5rev:GFP/pif4-101 transgenes were exposed or not (control) to 25 μM AlCl3 for 2hours. Cell boundaries appear red following propidium iodide staining. The root-apex TZ is marked by white arrowheads. Scale bar: 100μm. (B) Quantification of the Al-induced fluorescence intensity in the root-apex TZ of DR5rev:GFP and DR5rev:GFP/pif4-101 seedlings. The detected fluorescence region in TZ is marked by yellow rectangles. Cell boundaries appear red following propidium iodide staining. The TZ is marked by white arrowheads. Error bars indicate mean ±SD (n≥25). Statistical difference from detected fluorescence is indicated by asterisks (Fisher’s exact test, **P < 0.01).
Fig 11.
The Al-induced local expression of PIF4 acts downstream of ethylene signaling.
(A) The expression of PIF4p:GFP transgenes in the root apex epidermis in presence of Al and either ACC or/and AVG. Four-day old transgenic PIF4p:GFP seedlings were pre-treated with 1 μM AVG or 0.5 μM ACC for 1 day, then the seedlings were treated with 1 μM AVG, 0.5 or 1 μM ACC in the presence or not of 25 μM AlCl3 for 2 hours. Cell boundaries appear red following propidium iodide staining. The TZ is marked by white arrowheads. Scale bar: 100μm. (B) Relative transcript abundance of PIF4 in 7-d-old Col, 35S:EIN3 and 35S:EIL1 seedlings roots. (C) Transient dual-luciferase assay shows that EIN3 or EIL1 transactivates the promoter of PIF4 in Arabidopsis protoplasts. In (B) and (C), data represent the means of three biological replicates. Error bars represent Student’s t test confidence intervals (n = 3). Statistical significant difference indicated by asterisks (Fisher’s exact test, *P < 0.05, **P<0.01).
Fig 12.
A Working model of YUC regulated root growth inhibition in response to Al stress.
Al stress induces the local up-regulation of TAA1 and YUCs in the root TZ through an ethylene-dependent pathway. PIF4, which is specifically up-regulated in root TZ in response to Al stress, also participates into the local up-regulation of YUCs. The Al–induced expression of PIF4 in the root TZ acts downstream of ethylene signaling. The locally induced TAA1 and YUCs in response to Al stress contribute to auxin accumulation in the root TZ, which suppresses primary root growth. Green represents GFP signaling; EZ: Elongation zone; TZ: Transition zone; MZ: Meristem zone.