Fig 1.
The root of sr5 mutant growth is restricted and its root meristem size is reduced.
(A) The phenotype of five-day old WT and sr5 seedlings. Bar = 1 cm. (B) Primary root growth over the first ten days following germination. Data shown are mean±SE (n = 25). (C) Longitudinal zonation pattern in the primary roots of five-day old WT and sr5 seedlings. Cell boundaries appear red following Propidium Iodide staining. The meristem zone (MZ) and transition zone (TZ), which together form the root apical meristem (RAM), and the elongation zone (EZ) are indicated. Bar = 50 μm. (D) Root cortical cell length in the maturation zone of five-day old WT and sr5 seedlings. Data shown are mean±SE (n = 100), **: means of sr5 and WT differ significantly (P<0.01). (E) Cell number in the proliferation domain of five-day old WT and sr5 seedlings. Data shown are mean±SE (n = 25), **: means of sr5 and WT differ significantly (P<0.01). (F) pCYCB1;1::GUS expression in root tips of five-day old WT and sr5 seedlings. Bar = 50 μm.
Table 1.
WT and sr5 root growth and comparative analysis of their RAM activity.
Fig 2.
RNA-Seq analysis of the det2-9 and WT root transcriptome.
(A) Differential transcription in det2-9 and WT. Genes, which were significantly induced or repressed in det2-9 compared with WT, appear in, respectively, the upper left (in red) and bottom right (in green) hand portions of the plot. RPKM: reads per kilobase per million reads, DEGs: differentially expressed genes. (B, C) A heat map representation of the transcriptional behavior of genes associated with ethylene (B) and responding to ROS (C) based on the GO analysis. *: means more than two times up change in the expression genes of det2-9 compared to WT. (D) A qRT-PCR expression analysis of a selection of genes related to ethylene in det2-9 compared to WT. Data shown are mean±SE (n = 3), **: means of det2-9 and WT differ significantly (P<0.01).
Fig 3.
The det2-9 mutant accumulates more ethylene than WT.
(A) Ethylene-induced GUS activity (pEBS::GUS) in det2-9 and WT. Seedlings of det2-9 and WT were grown for five days either in the presence or absence of 10 nM eBL, and were stained for GUS activity analysis. Each treatment involved 20–30 seedlings; here, representative samples are presented. Bar = 1 cm. (B) Ethylene content in indicated BR-related transgenic and WT nine-day old seedlings exposed to either eBL (10 nM) or propiconazole (2 μM) under light conditions. Data shown are mean±SE (n = 5). **: means of det2-9, bes1-D, WT+eBL, WT+PPZ differ significantly from mean of WT (P<0.01). (C) Ethylene content in five-day old WT, det2-9 and pDET2::DET2-GFP-GUS/det2-9 seedlings in light conditions. Data shown are mean±SE (n = 5). **: means of det2-9 and WT differ significantly (P<0.01).
Fig 4.
BR affects root growth and ethylene production in Arabidopsis.
(A) and (B) Phenotype of five-day old Col-0 seedlings grown in different concentrations of BR. Bar = 1 cm. Data shown are mean±SE (n = 30). **: means significantly different in treated seedlings versus control (P<0.01). (C) Ethylene-induced GUS activity (pEBS::GUS) in seedlings grown on different concentration of eBL. Seedlings of pEBS::GUS were grown for five days in the presence of different concentrations of eBL, after which they were stained for GUS activity. Each treatment involved 20–30 seedlings; here, representative samples are presented. Bar = 1 cm. (D) Ethylene levels in seedlings grown in the presence of different concentration of eBL. Data shown are mean±SE (n = 30). **: means in treated seedlings significantly differ from control (P<0.01). (E) Transcript abundance of ERFs in WT grown in different concentration of eBL. **: means in treated seedling significantly differ from untreated samples (P<0.01).
Fig 5.
Enhanced ethylene synthesis is involved in the inhibitory effect of BR on root growth.
(A, B) Root growth of WT and det2-9 in the presence of either AVG, AgNO3 or ACC under light conditions. Data shown in B are mean±SE (n = 30); **: means of det2-9 and WT differ significantly (P<0.01). Bar = 1 cm. (C) Root growth in the presence of propiconazole (2 μM) of the octuple acs mutant CS16651, the ein2-5 mutant and the ein3/eil1-1 double mutant. Data shown are mean±SE (n = 30); **: means of CS16651, ein2-5, ein3/eil1-1 and WT differ significantly (P<0.01). The root phenotype (D) and root length measurement (F) of five-day old WT, ein3/eil1-1, det2-9 and four lines of det2-9/ein3/eil1-1 triple mutant seedlings. Bar = 1 cm. Data shown are mean±SE (n = 30); **: means significantly differ from WT (P<0.01). The root phenotype (E) and root length measurement (G) of five-day old WT, acs9, det2-9 and det2-9/acs9 double mutant seedlings. Bar = 1 cm. Data shown are mean±SE (n = 30); **: means significantly differ from WT (P<0.01).
Fig 6.
Interaction of BZR1/BES1 transcription factors with various ACS promoters.
(A) ChIP/qPCR assay. A scheme of the promoters of ACS6, 7, 9 and 11 are shown with the position of BRRE-box (black) and E-box (gray). Graphs show the ratio of bound promoter fragments (P1-P5) versus total input detected by qPCR after immuno-precipitation in p35S::BZR1-YFP and pNP::BES1-FLAG seedlings by YFP or FLAG antibodies. Data shown are mean±SE (n = 9). (B) Yeast one-hybrid binding assay involving BZR1/BES1 and ACS6, 7, 9 and 11 promoters. (C) Transient expression in A. thaliana protoplasts. BZR1 or BES1 transcription factors were co-transfected with either ACS7, 9 or 11 promoters. The LUC to REN ratio is shown and indicated the activity of the transcription factors on the expression level of the promoters. LUC: firefly luciferase activity, REN: renilla luciferase activity. Data shown are mean±SE (n = 9); **: means significant difference compared to control (P<0.01).
Fig 7.
BR represses the accumulation of superoxide anions.
(A) Five-day old det2-9 and WT seedlings grown in the presence or absence of eBL (10 nM), then assayed for the superoxide anion using NBT. Bar = 1 cm. (B) Superoxide anion accumulation in the root tips of BR-signaling enhanced plants (p35S::BRI1-GFP and bes1-D) and BR signaling deficient plants (bri1-116). Bar = 50 μm. (C, D) Elongation of the primary root of WT and det2-9 seedlings exposed to either (C) superoxide dismutase (SOD) or (D) 1,3-dimethyl-2-thiourea (DMTU). Data shown are mean±SE (n = 30); Asterisks means significant difference from the control-treated plants (**P<0.01).
Fig 8.
BR inhibits the synthesis of superoxide anions through the peroxidase pathway.
The length of the primary root of WT and det2-9 seedlings when exposed to inhibitors of NADPH oxidase (A) diphenylene iodonium, (B) ZnCl2. Asterisks means significant difference from the control-treated plants (**P<0.01). (C) NBT staining of WT and det2-9 when exposed to salicylhydroxamic acid (SHAM), 1,10-phenanthroline (1,10-Phe), diphenylene iodonium (DPI) and ZnCl2. Bar = 50 μm. (D) Peroxidase activity in WT and det2-9 nine-day old seedlings treated or not with eBL (10 nM). Data shown are mean±SE (n = 6); **: means differ significantly (P<0.01).
Fig 9.
Relationship between ethylene and superoxide anion.
(A) NBT staining of WT and det2-9 seedlings exposed to either AVG or ACC. Bar = 50 μm. (B) Peroxidase activity (POD) in WT, p35S::EIN3-GFP and the ein3/eil1-1 double mutant seedlings. Data shown are mean±SE (n = 6); no significant differences calculated. (C) Ethylene-induced GUS activity (pEBS::GUS) in the WT when treated or not with methyl viologen (MV). Bar = 1 cm. (D) Primary root length of WT, ein2-5 and ein3/eil1-1 when treated with various MV concentrations. Data shown are mean±SE (n = 30); **: means significant difference compared to WT (P<0.01).
Fig 10.
Proposed model to explain how BR regulates root growth in A. thaliana.
BR inhibits ethylene synthesis by activating the transcription factors BZR1 and BES1 to repress the transcription of ACSs under low levels. While high levels of BR induce ethylene biosynthesis either through increasing the stability to ACSs or influencing auxin signaling regulated ethylene. At the same time, BR inhibits the synthesis of O2- via the peroxidase pathway, but not NADPH oxidase pathway, which serves to regulate the growth of the A. thaliana seedling root.