OsARF16 Is Involved in Cytokinin-Mediated Inhibition of Phosphate Transport and Phosphate Signaling in Rice (<i>Oryza sativa L.</i>)

<div><p>Background</p><p>Plant responses to phytohormone stimuli are the most important biological features for plants to survive in a complex environment. Cytokinin regulates growth and nutrient homeostasis, such as the phosphate (Pi) starvation response and Pi uptake in plants. However, the mechanisms underlying how cytokinin participates in Pi uptake and Pi signaling are largely unknown. In this study, we found that OsARF16 is required for the cytokinin response and is involved in the negative regulation of Pi uptake and Pi signaling by cytokinin.</p><p>Principal Findings</p><p>The mutant <i>osarf16</i> showed an obvious resistance to exogenous cytokinin treatment and the expression level of the <i>OsARF16</i> gene was considerably up-regulated by cytokinin. Cytokinin (6-BA) application suppressed Pi uptake and the Pi starvation response in wild-type <i>Nipponbare</i> (NIP) and all these responses were compromised in the <i>osarf16</i> mutant. Our data showed that cytokinin inhibits the transport of Pi from the roots to the shoots and that OsARF16 is involved in this process. The Pi content in the <i>osarf16</i> mutant was much higher than in NIP under 6-BA treatment. The expressions of <i>PHOSPHATE TRANSPORTER1</i> (<i>PHT1</i>) genes, <i>phosphate (Pi) starvation-induced</i> (<i>PSI</i>) genes and <i>purple PAPase</i> genes were higher in the <i>osarf16</i> mutant than in NIP under cytokinin treatment.</p><p>Conclusion</p><p>Our results revealed a new biological function for OsARF16 in the cytokinin-mediated inhibition of Pi uptake and Pi signaling in rice.</p></div

Analysis of total phosphorus (P) contents over a time-course in both NIP and <i>osarf16</i> mutant.

<p>(A,B) Total P contents in NIP and <i>osarf16</i> 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 <i>ACTIN</i> 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 <i>OsARF16</i> from treatments to mock at 1% by student's <i>t</i> test. “b” indicated significant difference in expression levels of <i>OsARF16</i> from treatments to mock at 5% by student's <i>t</i> test.</p

Physiological evidences for involvement of OsARF16 in cytokinin (6-BA) Responses.

<p>(A–E) Phenotype of NIP and <i>osarf16</i> 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 <i>osarf16</i> and NIP are indicated by an asterisk.</p

Effects of cytokinin on regulation of root system architecture (RSA).

<p>(A) 0.1 µM 6-BA treatment reduced the number of lateral root in primary root and induced root hair of NIP and <i>osarf16</i> 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 <i>osarf16</i> and NIP are indicated by an asterisk.</p

Analysis of acid phosphatase (APase) activity and expression of six purple APase coding genes.

<p>(A) Acid phosphatase (APase) activity in NIP and <i>osarf16</i> 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 <i>osarf16</i> 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 <i>osarf16</i> mutant and NIP are indicated by an asterisk. “a” indicated significant difference in expression levels of <i>OsPAPs</i> from treatments to mock at 1% by student's <i>t</i> test. “b” indicated significant difference in expression levels of <i>OsPAPs</i> from treatments to mock at 5% by student's <i>t</i> test.</p

Analysis of <i>OsARF16</i> expression pattern under cytokinin treatment using <i>OsARF16</i>-Promoter: GUS lines.

<p>The expression pattern of <i>OsARF16</i> in primary root tips under (A) control and (B) 0.1 µM 6-BA treatment for 3 hr; The expression pattern of <i>OsARF16</i> in root hairs of primary root under (C) control and (D) 0.1 µM 6-BA treatment for 3 hr; The expression pattern of <i>OsARF16</i> in lateral roots of primary root under (E) control and (F) 0.1 µM 6-BA treatment for 3 hr. The expression pattern of <i>OsARF16</i> 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 <i>OsARF16</i> 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 <i>OsARF16</i> between 6-BA treatment and mock treatment is indicated by an asterisk.</p

OsARF16 affects cytokinin signaling.

<p>(A, B) <i>pCYCB1;1:Dbox-GUS</i> staining was performed in primary root tips, lateral roots and lateral root primordia of NIP and <i>osarf16</i> mutant under −6-BA/+6-BA treatment. Concentration of 6-BA in the treatments was 0.1 µM. <i>pCYCB1;1:Dbox-GUS</i> staining in root tips of NIP (A-1) and <i>osarf16</i> mutant (A-2) under −6-BA treatment; <i>pCYCB1;1:Dbox-GUS</i> staining in lateral roots of NIP (A-3) and <i>osarf16</i> mutant (A-4) under −6-BA treatment; <i>pCYCB1;1:Dbox-GUS</i> staining in lateral root primordia of NIP (A-5) and <i>osarf16</i> mutant (A-6) under −6-BA treatment; <i>pCYCB1;1:Dbox-GUS</i> staining in primary root tips of NIP (B-1) and <i>osarf16</i> mutant (B-2) under +6-BA treatment; <i>pCYCB1;1:Dbox-GUS</i> staining in lateral roots of NIP (B-3) and <i>osarf16</i> mutant(B-4) under +6-BA treatment; <i>pCYCB1;1:Dbox-GUS</i> staining in lateral root primordia of NIP (B-5) and <i>osarf16</i> 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 <i>osarf16</i> mutant roots. Data are shown as the mean ± SD (n = 5). Significant (P<0.05) differences in GUS activity between <i>osarf16</i> and NIP are indicated by an asterisk. (D) Analysis of the expression levels of cytokinin signaling related genes in <i>osarf16</i> mutant and NIP. The data of 2^{−ΔΔ<i>Ct</i>} (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^{−ΔΔ<i>Ct</i>} 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.</p

Expression analysis of <i>phosphate (Pi) starvation-induced</i> (<i>PSI</i>) genes, <i>OsIPS1</i>, <i>OsIPS2</i>, <i>OsSPX1</i>, <i>OsSQD2</i> in NIP and <i>osarf16</i> mutant.

<p>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 <i>osarf16</i> mutant were used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Value ± SD of five independent replicates.</p

Additional file 4 of Genome wide association analysis for yield related traits in maize

Additional file 4

Additional file 3 of Genome wide association analysis for yield related traits in maize

Additional file 3