Fig 1.
CBP20 phosphorylation is required for root development in ethylene response.
(A) Absolute amounts of total phosphopeptides before and after treatment with 10 ppm ethylene gas. (B) Relative phosphorylation levels of CBP20 peptides in Col-0 or ctr1-1 plants treated for 4 hours of air or 10 ppm ethylene gas. Spectral counts were computed by averaging three biological replicates. The total spectral counts from all phosphorylated proteins in each sample are indicated as an internal control. (C) Triple response phenotype of cbp20 mutant and cbp20 mutant transformed with full length of CBP20, constitutive dephosphorylated of CBP20 (CBP20S245A) and constitutive phosphorylated of CBP20 (CBP20S245E and CBP20S245D). (D-E) The length of hypocotyls (D) and roots (E) of cbp20 mutant and cbp20 mutant transformed with full length of CBP20, constitutive dephosphorylated of CBP20 (CBP20S245A) and constitutive phosphorylated of CBP20 (CBP20S245E and CBP20S245D) on 10μm ACC plate for 3 days. Bars indicates SD and n = 30 for each line. The same letters indicates no statistic significant difference; the different letters indicate statistic significant difference with p≤ 0.05.
Fig 2.
Small RNA and RNA sequencing analysis of CBP20 regulated miRNAs and RNAs in roots and shoots with ethylene treatment.
(A) The Venn diagram shows the numbers of ethylene regulated miRNAs in the roots or shoots of Col-0 and cbp20 mutant. The miRNAs, which are differentially regulated by ethylene in the shoots or roots of Col-0 or cbp20 are compared. (B) Heat map shows the expression level of differential regulated miRNAs in the root and shoot of Col-0 and cbp20 mutant with ethylene treatment. Total small RNA was prepared from the roots or shoots of 3-day-old etiolated seedlings of Col-0 or cbp20 plants treated with air or 4 h ethylene. Differentially expressed miRNA requiring a 1.5 fold change comparing the indicated conditions with P<0.05 after Benjamini–Hochberg correction. (C) The Venn diagram shows the numbers of ethylene regulated genes in the roots and shoots of Col-0 and cbp20 mutant. Total RNA was prepared from the roots or shoots of 3-day-old etiolated seedlings of Col-0 or cbp20 plants treated with air or 4 h ethylene. Differentially expressed genes were identified by fragments per kilobase per million reads (FPKM) filter<0.1, requiring a twofold change comparing the indicated conditions with P<0.05 after Benjamini–Hochberg correction (D) The Venn diagram shows the number of The CBP20 dependent differential regulated genes in Col-0 root after ethylene treatment. (E) The venn diagram shows the number of the targets of differential regulated miRNAs and the CBP20 dependent differential regulated genes in Col-0 roots after ethylene treatment. (F) Heat map shows the expression level of differential regulated miRNAs and their target genes in col root after ethylene treatment.
Fig 3.
CBP20 phosphorylation regulates miR319 biogenesis and MYB33 expression.
(A) Small RNA northern blot of miR319 in roots of 3-day old seedlings of plants indicated in the figure treated with air or 4 hours ethylene gas. Numbers indicate the relative expression level compared with the U6 control. (B) The relative gene expression of pri-miR319b in roots of 3-day old seedlings of plants indicated in the figure treated with air or 4 hours ethylene gas. (C) The relative gene expression level of MYB33 in cbp20 mutant and cbp20 mutant transformed with full length of CBP20, constitutive mimic dephosphorylated of CBP20 (CBP20S245A) and constitutive mimic phosphorylated of CBP20 (CBP20S245E and CBP20S245D) treated with ethylene. Total RNAs were extracted from the roots of 3-day-old etiolated seedlings from indicated genotypes and gene expression was analyzed by qualitative RT-PCR (3 biological replicates).
Fig 4.
miR319bOE plants and myb33 mutants show the ethylene hypersensitive phenotype.
(A) Triple response phenotype of miR319b OE plants and myb33 mutants. (B-C) The length of hypocotyls (B) and roots (C) of miR319b OE plants and myb33 mutants grown on MS or MS with 1μM ACC. (D-E) The relative expression level of pri-miR319b (D) and MYB33 (E) in miR319bOE plants. Total RNAs were extracted from the roots of 3-day-old etiolated seedlings from indicated genotypes and gene expression was analyzed by qRT-PCR (3 biological replicates).
Fig 5.
CBP20 phosphorylation does not influence expression of miR159.
(A) Small RNA northern blot of miR159 in Col-0 and cbp20 mutant treated with or without 4 hours ethylene gas. Numbers indicate the relative expression level of miR159 compared with the U6 control. (B) The relative gene expression level of pri-miR159a in cbp20 mutant and cbp20 mutant transformed with full length of CBP20 (CBP20/cbp20), constitutive dephosphorylated of CBP20 (CBP20S245A/cbp20) and constitutive phosphorylated of CBP20 (CBP20S245Ecbp20 and CBP20S245D/cbp20) treated with or without 4 hours ethylene gas. (C) The relative expression level of pri-miR159a in miR319bOE lines.
Fig 6.
Mutated MYB33 with miR319b target sites is able to rescue the root phenotype caused by miR319bOE.
(A) Triple response phenotype of miR319bOE plants, miR319bOE/mMYB33OE plants. (B-C) Measurement of hypocotyls (B) and roots (C) of miR319bOE and miR319bOE/mMYB33OE plants. (D) The relative gene expression of MYB33 in miR319bOE and miR319bOE/mMYB33OE plants. Total RNAs were extracted from the roots of 3-day-old etiolated seedlings from indicated genotypes and gene expression was analyzed by qualitative RT-PCR. Three replications have been done with similar result.
Fig 7.
(A) Luciferase assays showing that gene expression of MYB33 is down regulated in the presence of miR319b precursor. Agrobacterium containing MYB33 or mutated mMYB33 CDS fused to 35S::LUC 3’UTR with or without Agrobacterium harbouring constructs containing the miR319b precursor were injected in to N. benthamiana plants. After 3 days, The leaves were sprayed with 500 μM luciferin and placed in the dark for 5 min. Luciferase activity was observed. (B) Quantitative measurement of luciferase intensity in different treatments as indicated in the figure. 3 biology replications have been done.