Figure 1.
Physiological evidences for involvement of OsARF16 in cytokinin (6-BA) Responses.
(A–E) Phenotype of NIP and osarf16 mutant under different concentration of 6-BA treatments (0/0.01/0.1/1/10 µM) using 7-day-old seedlings (Bar represents 2 cm). The graphs represent statistics data of main root length (F) and main shoot length (G). Data are shown as the mean ± SD (n = 5). Significant (P<0.05) differences in length of main root and main shoot between osarf16 and NIP are indicated by an asterisk.
Figure 2.
Effects of cytokinin on regulation of root system architecture (RSA).
(A) 0.1 µM 6-BA treatment reduced the number of lateral root in primary root and induced root hair of NIP and osarf16 mutant in primary root tips (The bar represents 5 mm). The graphs represent statistics data of lateral root number (B) and average root hairs length (C). Data are shown as the mean ± SD (n = 5). Significant (P<0.05) differences in number of lateral root in primary root and average length of root hairs in primary root tips between osarf16 and NIP are indicated by an asterisk.
Figure 3.
OsARF16 affects cytokinin signaling.
(A, B) pCYCB1;1:Dbox-GUS staining was performed in primary root tips, lateral roots and lateral root primordia of NIP and osarf16 mutant under −6-BA/+6-BA treatment. Concentration of 6-BA in the treatments was 0.1 µM. pCYCB1;1:Dbox-GUS staining in root tips of NIP (A-1) and osarf16 mutant (A-2) under −6-BA treatment; pCYCB1;1:Dbox-GUS staining in lateral roots of NIP (A-3) and osarf16 mutant (A-4) under −6-BA treatment; pCYCB1;1:Dbox-GUS staining in lateral root primordia of NIP (A-5) and osarf16 mutant (A-6) under −6-BA treatment; pCYCB1;1:Dbox-GUS staining in primary root tips of NIP (B-1) and osarf16 mutant (B-2) under +6-BA treatment; pCYCB1;1:Dbox-GUS staining in lateral roots of NIP (B-3) and osarf16 mutant(B-4) under +6-BA treatment; pCYCB1;1:Dbox-GUS staining in lateral root primordia of NIP (B-5) and osarf16 mutant (B-6) under +6-BA treatment. (C) Analysis of GUS activity. The entire root of the seedling was used for analysis. The graph represents statistics data of GUS activity in NIP and osarf16 mutant roots. Data are shown as the mean ± SD (n = 5). Significant (P<0.05) differences in GUS activity between osarf16 and NIP are indicated by an asterisk. (D) Analysis of the expression levels of cytokinin signaling related genes in osarf16 mutant and NIP. The data of 2−ΔΔCt (qRT data) refers to the fold difference in expression of cytokinin-related genes under cytokinin treatment (3 hr) compared with the untreated seedlings. Heat map representation was performed using the normalized 2−ΔΔCt values with Treeview 1.6 to visualize the analysis data. The different colors correspond to the values of the gene change-fold ratio shown in the bar. The data were analyzed by three independent repeats.
Figure 4.
Analysis of OsARF16 expression pattern under cytokinin treatment using OsARF16-Promoter: GUS lines.
The expression pattern of OsARF16 in primary root tips under (A) control and (B) 0.1 µM 6-BA treatment for 3 hr; The expression pattern of OsARF16 in root hairs of primary root under (C) control and (D) 0.1 µM 6-BA treatment for 3 hr; The expression pattern of OsARF16 in lateral roots of primary root under (E) control and (F) 0.1 µM 6-BA treatment for 3 hr. The expression pattern of OsARF16 in leaves under (G) control and (H) 0.1 µM 6-BA treatment for 3 hr. (I) qRT-PCR method was used to confirm the OsARF16 expression pattern changes in roots under 0.1 µM 6-BA treatment for 3 hr. Significant (P<0.05) differences in the expression level of OsARF16 between 6-BA treatment and mock treatment is indicated by an asterisk.
Figure 5.
Analysis of total phosphorus (P) contents over a time-course in both NIP and osarf16 mutant.
(A,B) Total P contents in NIP and osarf16 mutant after Pi resupply using 7-day-old Pi-deprived seedlings: (A) in roots and (B) in shoots. The statistics of P contents were calculated for five independent biological replications. (C, D) Expression of Pi transporter coding genes in Pi-sufficient (+Pi) and Pi-deficient (−Pi) treatments with or without 6-BA (C) in roots and (D) in shoots. Concentration of 6-BA in the treatments was 0.1 µM. The ACTIN gene of rice was used as the reference gene for qRT-PCR. Value ± SD of five independent replicates. “a” indicated significant difference in expression levels of OsARF16 from treatments to mock at 1% by student's t test. “b” indicated significant difference in expression levels of OsARF16 from treatments to mock at 5% by student's t test.
Figure 6.
Expression analysis of phosphate (Pi) starvation-induced (PSI) genes, OsIPS1, OsIPS2, OsSPX1, OsSQD2 in NIP and osarf16 mutant.
Analysis of expressions were performed under phosphate-sufficient (+Pi), phosphate-deficient (−Pi) and Pi resupply conditions (R6h, R12h, R18h and R24h refer to resupply times of 6, 12, 18 and 24 h, respectively) with or without 6-BA treatments. Concentration of 6-BA in the treatments was 0.1 µM. 7-day-old seedlings of NIP and osarf16 mutant were used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Value ± SD of five independent replicates.
Figure 7.
Analysis of acid phosphatase (APase) activity and expression of six purple APase coding genes.
(A) Acid phosphatase (APase) activity in NIP and osarf16 mutant under +Pi/−6BA, +Pi/+6BA, −Pi/−6BA and −Pi/+6BA conditions respectively. Concentration of 6-BA in the treatments was 0.1 µM. The graph represents statistics data of root-associated APase activity. (B) Acid phosphatase (APase) activity in the root surface of NIP and osarf16 mutant under +Pi/−6BA, +Pi/+6BA, −Pi/−6BA and −Pi/+6BA conditions respectively. (C) Expression levels of six purple APase coding genes under +Pi/−6BA, +Pi/+6BA, −Pi/−6BA and −Pi/+6BA conditions respectively. Data are shown as the mean ± SD (n = 5). Significant (P<0.05) differences in APase activity between osarf16 mutant and NIP are indicated by an asterisk. “a” indicated significant difference in expression levels of OsPAPs from treatments to mock at 1% by student's t test. “b” indicated significant difference in expression levels of OsPAPs from treatments to mock at 5% by student's t test.