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Auxin response factor (OsARF12), a novel regulator for phosphate homeostasis in rice (Oryza sativa) SuiKang Wang1*, SaiNa Zhang1*, ChenDong Sun1, YanXia Xu1, Yue Chen1, ChenLiang Yu1, Qian Qian2, De-An Jiang1 and YanHua Qi1 1
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China; 2State Key Laboratory of Rice Biology, China National
Rice Research Institute, Chinese Academy of Agricultural Sciences, 359 Tiyuchang Road, Hangzhou 310006, China
Summary Authors for correspondence: YanHua Qi Tel: +86 571 88981355 Email:
[email protected] De-An Jiang Tel: +86 571 88206461 Email:
[email protected] Received: 14 July 2013 Accepted: 8 August 2013
New Phytologist (2014) 201: 91–103 doi: 10.1111/nph.12499
Key words: auxin response factor, Oryza sativa (rice), OsARF12, phosphate absorption and transport, phosphate homeostasis.
Phosphorus (P) is crucial nutrient element for crop growth and development. However, the network pathway regulating homeostasis of phosphate (Pi) in crops has many molecular breeding unknowns. Here, we report that an auxin response factor, OsARF12, functions in Pi homeostasis. Measurement of element content, quantitative reverse transcription polymerase chain reaction analysis and acid phosphatases (APases) activity assay showed that the osarf12 mutant and osarf12/25 double mutant with P-intoxicated phenotypes had higher P concentrations, up-regulation of the Pi transporter encoding genes and increased APase activity under Pi-sufficient/-deficient (+Pi/ Pi, 0.32/0 mM NaH2PO4) conditions. Transcript analysis revealed that Pi-responsive genes – Phosphate starvation (OsIPS)1 and OsIPS2, SYG1/Pho81/XPR1(OsSPX1), Sulfoquinovosyldiacylglycerol 2 (OsSQD2), R2R3 MYB transcription factor (OsMYB2P-1) and Transport Inhibitor Response1 (OsTIR1) – were more abundant in the osarf12 and osarf12/25 mutants under +Pi/ Pi conditions. Knockout of OsARF12 also influenced the transcript abundances of the OsPHR2 gene and its downstream components, such as OsMiR399j, OsPHO2, OsMiR827, OsSPX-MFS1 and OsSPXMFS2. Results from –Pi/1-naphthylphthalamic acid (NPA) treatments, and auxin reporter DR5::GUS staining suggest that root system alteration and Pi-induced auxin response were at least partially controlled by OsARF12. These findings enrich our understanding of the biological functions of OsARF12, which also acts in regulating Pi homeostasis.
Introduction Phosphate starvation ( Pi) has become one of the main limiting factors for increasing crop yield and quality improvement because phosphorus (P) fertilizer is easily fixed in soil, and utilization efficiency is very low (Lynch, 2011). Therefore, in order to improve Pi uptake and utilization efficiency in plants, the cultivation of efficient P varieties is a fundamental method to solving problems of Pi. For more than a decade, several regulators related to Pi homeostasis in Arabidopsis (Arabidopsis thaliana) were also successively found in rice (Delhaize & Randall, 1995; Rubio et al., 2001; Aung et al., 2006; Bari et al., 2006; Chiou et al., 2006; Franco-Zorrilla et al., 2007; Pant et al., 2008). These regulators in rice have very similar biological functions to Arabidopsis. For instance, the main regulatory network of Pi homeostasis in rice was revealed to be around OsPHR2, the central transcription factor controlling Pi homeostasis. OsPHR2 as a regulatory center *These authors contributed equally to this work. Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
may control at least two pathways. The first pathway is PHR2– MiRNA399–PHO2, which has a series of genes, including OsMiRNA399j and OsPHO2 (Wang et al., 2009a), OsPHR2 (Zhou et al., 2008), OsSPX1 (Wang et al., 2009b) and OsPT2 (Ai et al., 2009; Liu et al., 2010). The overexpression transgenic rice lines or repression rice mutants of these genes have P-intoxicated phenotype with a number of browning spots on leaves and increased Pi content. Overexpression of OsPHR2 results in accumulation of excess shoot Pi. OsMiR399-dependent OsPHO2 gene expression modulates Pi uptake, allocation and remobilization. OsSPX1 negatively regulates the accumulation of shoot Pi and is positively regulated by OsPHR2. OsPHR2 through OsPHO2 positively regulates the low-affinity Pi transporter gene OsPT2. The second pathway is PHR2–MiRNA827–SPX-MSF, which includes OsPHR2, OsMiRNA827 (Lin et al., 2010) and OsSPXMSF1/2 (Wang et al., 2012) genes. There are no P-intoxicated phenotypes, but P content is increased in their overexpression transgenic lines or repression mutants. The expression of OsMiRNA827 in shoots and roots was induced by the –Pi New Phytologist (2014) 201: 91–103 91 www.newphytologist.com
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condition with a concomitant decrease in its target mRNA, OsSPX-MFS1/2. The regulator network of Pi uptake and translocation needs further confirmation. Recently, the phosphate transporter traffic facilitator1 (OsPHF1), the homolog of PHF in Arabidopsis, which was not regulated by OsPHR2, was revealed in rice plants (Gonzalez et al., 2005; Chen et al., 2011). OsPHF1 regulates the localization of both the low-affinity Pi transporter OsPT2 and highaffinity Pi transporter OsPT8 to the plasma membrane and determines Pi uptake and translocation. Overexpression of OsPHF1 increased P accumulation under the +Pi condition. Furthermore, OsMYB2P-1, a novel R2R3 MYB transcriptional factor in rice, also functions in response and adaptation to Pi deficiency (Dai et al., 2012). OsMYB2P-1 overexpression transgenic lines have a larger root system and a higher P content in roots under –Pi condition. In addition, a key breakthrough in the research of Pi-deficiency tolerance was achieved (Gamuyao et al., 2012): the P-starvation tolerance 1 (PSTOL1), that is, the P-deficiency tolerance (Pup1), identified in the traditional austype rice cv Kasalath (a specific group of rice originating from a region in India with poor and problem soils) about a decade ago, whose functions were recently revealed to enable plants to acquire more P and other nutrients. The overexpression of PSTOL1 in such varieties significantly enhances grain yield in P-deficient soil (Gamuyao et al., 2012). Besides these factors, the phytohormone auxin plays important roles in regulating plant responses to Pi starvation. In Arabidopsis, Pi deficiency influences lateral root (LR) development that depends on auxin receptor TIR1 and auxin response factor ARF7/19 (Ulmasov et al., 1997; Guilfoyle et al., 1998; Guilfoyle & Hagen, 2001, 2007; Perez-Torres et al., 2008). Auxin affects plant responses to Pi starvation, which may occur via local concentration changes as a result of the biosynthesis and transport of auxin (Nacry et al., 2005; Li et al., 2012; Shen et al., 2012). SIZ1 is a small ubiquitin-related modifier (SUMO) E3 ligase that is associated with Pi starvation responses (Miura et al., 2005). SIZ1 may negatively regulate root architecture modulation under low Pi by controlling auxin patterning (Miura et al., 2011). Recently, a cotton defense-related gene, GbWRKY1, was identified as positively regulating the Pi response by enhanced auxin sensitivity and resulted in modification of the root system (Xu et al., 2012). GbWRKY1 may be independent of SIZ1 and phosphate starvation response 1 (PHR1) in response to Pi. Crosstalk between auxin and Pi starvation-response in rice remains unclear. Recently, we reported that the transcription factor OsARF16 was required for auxin and Pi response in rice (Shen et al., 2012). To gain an insight into the regulatory mechanisms of Pi response and the relationship with auxin signaling, in the present study we further report the novel character of OsARF12 associated with Pi signaling. The rice mutants osarf12 (T-DNA-insertion mutant), osarf12t (Tos17-insertion mutant) and osarf12/25 double mutant, possessing higher P content and P-intoxicated phenotypes, were characterized as having enhanced responses to Pi starvation, including enhanced expression of Pi transporter and Pi starvation-induced genes, and overproduction of root-associated acid phosphatases (APases). New Phytologist (2014) 201: 91–103 www.newphytologist.com
Materials and Methods Plant materials and growth conditions Rice (Oryza sativa L.) plants – including the wildtypes (WTs) Dongjin and Nipponbare (NIP) – and mutants osarf12, osarf12t and osarf12/25 were grown using a hydroponic method in a glasshouse with a light : dark cycle of 12 : 12 h with 30 : 24°C. The hydroponic experiments used a rice culture solution described by Wang et al. (2009a). Pi starvation ( Pi), Pi supply (+Pi) and high Pi supply (+10Pi) were performed with 0, 0.32 and 3.2 mM NaH2PO4, respectively, as used by Wang et al. (2009). The treatment of a polar auxin-transport inhibitor was carried out with 0.1 lM of 1-naphthylphthalamic acid (NPA). b-Glucuronidase (GUS) staining and analysis of GUS activity The OsARF12 promoter GUS (OsARF12pro::GUS) transgenic rice and GUS staining of seedlings were performed as described by Qi et al. (2012). The DR5::GUS auxin reporter described by Ulmasov et al. (1997) was introduced into the Agrobacterium tumefaciens strain EHA105 and transformed into the Dongjin WT and osarf12 for detecting auxin distribution. Stained root tissues were observed using a Carl Zeiss laser scanning system LSM510 (http://www.zeiss.com/) and Nikon Eclipse 80i (Nikon Corporation, Tokyo, Japan). The measurement of GUS activity was performed as described by Jefferson (1987). Western blotting Total rice proteins were extracted from WTs and mutants grown in +Pi and Pi conditions with a plant protein extraction kit (KeyGeN, Shanghai, China). Of proteins, 30 lg was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane used for subsequent western blot analysis according to standard procedures. The polyclonal antibody anti-OsARF12 was generated by immunizing healthy rabbits using the synthesized peptide with 18 amino acids (including CEDVHKMGKQGNDPRYLS) from OsARF12 protein as antigens. The peptide of antigen-ARF12 was designed by the special open reading frame of the OsARF12 gene and was not homologous with other OsARF genes according to a search from DNA Data Bank of Japan (DDBJ), http://blast.ddbj.nig.ac.jp/blast/blastp?lang=en. In the hybrid of anti-OsARF12 and rice total protein, there was a unique band of size equal to the 92 kDa molecular weight of OsARF12. The protein conjugation, immunization and antiserum purification were carried out by the Beijing Protein Institute Co. Ltd (Beijing, China). The related methods are found in Li et al. (2011). Goat anti-rabbit immunoglobulin G (IgG) (H&L)-AP conjugated secondary antibody CW0111A (CWBIO, Beijing, China) was used for the detection of the OsARF12 protein. PageRuler Prestained Protein Ladder (Thermo Fisher Scientific Inc., Rockford, IL, USA) was used for determining the protein Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
New Phytologist size. Western blotting analysis was performed in at least three biological replications. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis Total RNA was isolated from leaves or roots of 7-d-old rice (WT and each mutant seedling) using various treatments. The methods, including RNA extraction, reverse transcription and qRTPCR, were perforamed according to Wang et al. (2010). The rice Actin gene was used as the reference gene. Sequences of the related primers for qRT-PCR are listed in Supporting Information, Tables S1–S3. Measurement of element contents For the measurement of the element contents, rice seedlings were grown in normal hydroponic solution with 0, 0.032 and 0.32 mM NaH2PO4 for 4 wk. P content was measured in the first to fourth leaves and in roots. For each sample in the experiment, 0.1 g was dried at 80°C for 48 h and digested with HNO3/ H2O2 at 110°C for 0.5 h using a microwave 3000 digestor (Anton Paar, Graz, Austria). These samples were diluted to 50 ml of constant volume for analysis of the element contents using an inductively coupled plasma optical emission spectrometer ICP-OES, OPTIMA 8000DV (Perkin Elmer, Waltham, MA, USA). Five independent biological replicates were used in the experiment. Analysis of root morphological traits The root morphological traits of WTs and mutants were determined for 10 biological replicates. Root hairs were observed with a Leica MZ95 stereomicroscope (Leica Instrument, Nussloch, Germany). Qualitative analysis of root-associated APase activity Root APase staining was analyzed according to Bozzo et al. (2006). The roots were excised from 7-d-old Pi-supplied and Pi-deprived seedlings and incubated with a 5-bromo-4-chloro-3indolyl-phosphate (BCIP)–agar overlay solution containing 50 mM sodium acetate (pH 5.5) with 10 mM MgCl2, 0.6% agar and 0.08% BCIP at room temperature for 20 min. The blue color on the root surface, formed by hydrolysis of BCIP, was photographed using an EOS 40D camera (Canon Corporation, Tokyo, Japan). In-gel analysis of APase activity Proteins were isolated according to Aarts et al. (1991); 1 mg soluble protein was separated on a 4% stacking and 9% resolving (w/v) native polyacrylamide gel at 4°C. After electrophoresis, the gel was washed with ice-cold distilled water several times, then washed twice (15 min each) in 50 mM NaAc (pH 5.5/10 mM MgCl2), and stained with 0.08% BCIP. Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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Protein extraction and APase activity assay Protein was isolated with ice-cold extraction buffer (100 mM potassium acetate, pH 5.5, 20 mM CaCl2, 2 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 1.5% (w/v) polyvinylpolypyrrolidone) from 0.5 mg of roots of 1-wk-old seedlings. The protein content was determined using the method of Bradford (1976), with BSA as an internal standard. APase activity was analyzed as described by Tomscha et al. (2004) with little modification. Protein (1 lg) was used for APase activity assay, and the quota of protein was added to 620 ll of reaction buffer (50 mM NaAc pH 5.5 and 10 mM MgCl2), and 10 ll of p-nitrophenol phosphate (10 mg ml 1 pNPP; Sigma). After incubation at 37°C for 10 min, the reaction was stopped by 1.2 ml of 1 M NaOH, and then absorbance was measured at 412 nm wavelength. Phosphatase activity was expressed as ng of pNPP accumulated lg–1 soluble protein min–1. These experiments were replicated three times.
Results osarf12 mutants possess higher P contents and P-intoxicated phenotypes In previous research, we reported that the osarf12 (T-DNAinsertion mutant), osarf12t (Tos17-insertion mutant) and osarf12/25 double mutant lost iron (Fe) homeostasis (Qi et al., 2012). In addition, the Fe content of these osarf12 mutants is low. The interaction between Fe and P absorption in rice (Zheng et al., 2009), and the fact that the P-intoxicated phenotype of the osarf12 mutant in outdoor pots presented a number of browning spots on leaves under high transpiration conditions (temperature, 30–37°C; relative humidity, 30–40%; Fig. S1) suggested that the osarf12 mutants may have a high P content. Hence, we studied the characteristics of the osarf12 mutant in more detail. The contents of P, divalent ions (Fe, Zn, Cu, Mn, Ca and Mg) and monovalent ions (K and Na) in roots and leaves of the WT and the osarf12 mutant were measured under +Pi/+Fe, Pi, Fe and Pi/ Fe conditions (Fig. S2) (Zheng et al., 2009). The results showed that contents of P and monovalent ions in roots and leaves of the osarf12 mutant were higher than in the WT whereas the contents of divalent ions in the osarf12 mutant were lower than in the WT. In most measured tissues, including callus, root, leaf sheath, leaf blade of 7-d-old osarf12, osarf12t and osarf12/25 seedlings, but not in individual outer glumes, the P concentrations were obviously higher than their related WT, Dongjin (for osarf12 and osarf12/25) or NIP (for osarf12t; Fig. 1a). In particular, in leaf sheaths or leaf blades of mutants, the P concentrations were close to twofold those in both WTs. Under the +Pi condition (0.32 mM NaH2PO4), compared with the WT, the tip of basal leaves of 1-month-old mutant seedlings began to wither; the P-intoxicated symptoms of leaf tip necrosis, accompanied by a large number of brown speckles and growth retardation, were more severe when seedlings were exposed to a high-Pi nutrient solution (+10Pi, 3.2 mM New Phytologist (2014) 201: 91–103 www.newphytologist.com
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NaH2PO4). However, these phenotypes exhibited in mutants did not show visual differences from their WT counterparts under –Pi conditions (Fig. 1b,c). Furthermore, the P contents from roots and first to fourth leaves of 1-month-old WTs and mutants were measured (Fig. 1d). Under Pi treatment, the P contents of roots and the fourth leaf in mutants were the same as those of individual WTs, but the P contents of first to third leaves were higher than those in the WTs. Under +Pi and +10Pi conditions, the P contents in root and first to third leaves in the mutants were c. 1.5- to twofold those of WTs. The results showed that more P might be taken up and New Phytologist (2014) 201: 91–103 www.newphytologist.com
Fig. 1 Total phosphorus (P) content of each tissue and phenotype of rice (Oryza sativa) wildtypes (WTs), osarf12 mutants and the osarf12/25 double mutant. (a) Total P content of each tissue under the phosphatesufficient (+Pi, 0.32 mM NaH2PO4) condition. Callus of 1-month-old seedlings; roots, leaf sheaths, leaf blades and glumes of 3-month-old seedlings; and mature seeds were measured. (b, c) Phenotypes of 1month-old WT and mutant seedlings grown under Pi (no Pi), +Pi and +10Pi (3.2 mM NaH2PO4) conditions (left to right): (b) basal leaf tips of Dongjin (DJ), osarf12, Nipponbare (NIP), osarf12t and osarf12/25; bars, 2 cm; (c) phenotype of 1-month-old seedlings of the five rice lines in (b); bars, 10 cm. (d) Total P content of roots and first to fourth leaves in the five rice lines. In panels (a) and (d), error bars indicate SD (n = 5, five plants) and different letters indicate a statistical difference at P < 0.05.
translocated from roots to leaves as a result of mutation of OsARF12, resulting in overaccumulation of P in old leaves and P-toxicity symptoms in the osarf12 mutants and the osarf12/25 double mutant. Increased P content in osarf12 mutants and the osarf12/25 double mutant was related to up-regulated Pi transporter genes Compared with WTs, the P contents in the mutant roots and leaves were markedly elevated under normal (+Pi) and high Pi Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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(+10Pi) supply conditions. Thus, to clarify the function of OsARF12 in P accumulation, total P content over a time-course was monitored in roots of 7-d-old Pi-deprived rice plants during a 6 h period when resupplied with 0.32 mM Pi (Fig. 2a); and total P content was measured in leaves during a 48 h period after Pi supply (Fig. 2b). In the mutant roots, the P content was always higher than both WTs during the 6 h period and the P content in the mutant leaves was also higher than the WT during the 48 h period. These results imply that OsARF12 might help to alleviate P excess accumulation in roots and leaves. The PHOSPHATE TRANSPORTER1 (PHT1) gene family functions in Pi uptake, translocation and homeostasis (Ai et al., 2009; Liu et al., 2010; Jia et al., 2011; Sun et al., 2012). To determine whether the increased P content in the mutants was related to the gene family, the expression of several members of the rice PHT1 gene family was analyzed by qRT-PCR. The transcripts of most analyzed members (including OsPT2, 3, 6, 8, 9 and 10) in both roots and leaves of mutant plants were more abundant than in WTs under the +Pi condition. Under the Pi condition, upregulation of OsPT3, 6, 8 and 9 was greater in mutant roots, although the amount of up-regulation in leaves was less than in WTs (Fig. 2c,d). However, OsPT1 did not show significant changes in roots and leaves under both +Pi and Pi conditions. These results indicate that OsARF12 negatively controlled P accumulation through regulating the PHT1 gene family in rice. OsARF12 expression at transcript abundance or protein level was inhibited by Pi starvation To elucidate the regulatory mechanism of OsARF12 under +Pi and Pi conditions, the expression of OsARF12 was first investigated using qRT-PCR, GUS staining/activity and western blotting (Fig. 3). qRT-PCR showed that the expression of OsARF12 at transcript abundance in roots and leaves was reduced by the Pi treatment (Fig. 3a). The activity of OsARF12::GUS was inhibited under the Pi condition (Fig. 3b). These results were
Fig. 2 Analysis of total phosphorus (P) content over a time-course and expression of phosphate (Pi) transporter genes. (a, b) Total P content in rice (Oryza sativa) Dongjin, Nipponbare (NIP), osarf12, osarf12t and osarf12/25 after Pi resupply from Pi for 7 d: (a) in roots; (b) in leaves. The statistics of P content were calculated for five independent biological replications. (c, d) Expression of Pi transporter genes in Pi-sufficient (+Pi) and Pideficient ( Pi) treatments: (c) in roots; (d) in leaves. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis used three independent biological replicates and was performed according to Wang et al. (2010). The Actin gene of rice was used as the reference gene for qRT-PCR. Error bars indicate SD (n = 3, three plants), and followed by different letters indicate a statistical difference at P < 0.05. Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
consistent with the GUS staining in primary roots (PRs), LRs and leaves (Fig. 3c–h). Similarly, the expression of OsARF12 at the protein level was also inhibited by the Pi condition (Fig. 3i, j). The results imply that the reduction of OsARF12 expression during the Pi condition may be useful for absorbing or translocating more Pi, which was consistent with the knockout of OsARF12 enhancing P content (Fig. 2a,b). Altered response of the roots of osarf12 mutants and the osarf12/25 double mutant to Pi starvation The dicot model plant, Arabidopsis, displays a number of root developmental responses to Pi, including PR growth inhibition, greater formation of LR and increased root hair (RH) elongation (Lopez-Bucio et al., 2005). By contrast with Arabidopsis, in the monocots rice and maize, the PR elongation was enhanced (Zhou et al., 2008; Li et al., 2012). In the present study, the growth of PR in WTs was slightly promoted, while the promotion of PR growth in the mutants was greater after 10 d of the Pi condition (Fig. 4a,b,e). Similarly to the PR growth trend, RH elongation, adventitious root (AR) number, and LR number and density were more highly induced in mutants than in the WTs under the Pi condition (Fig. 4a–d, f–h). These results suggest that roots of osarf12 mutants and the osarf12/25 double mutant were more responsive to Pi deficiency than was the WT. APase activity was increased in the roots of the osarf12 mutants and the osarf12/25 double mutant under both +Pi and Pi conditions Acid phosphatases help to catalyze inorganic phosphate (Pi) hydrolysis from organophosphates, and APases are involved in Pi acquisition and homeostasis under the Pi condition (Duff et al., 1994; del Pozo et al., 1999). Increased production of APase is an important strategy for plants responding to Pi deficiency (Duff et al.,
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Fig. 3 OsARF12 expression in transcription and protein levels in rice (Oryza sativa) wildtype (WT), Dongjin under phosphate-sufficient (+Pi) and phosphate-deficient ( Pi) conditions. Each experiment was performed with three independent biological replicates. (a) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of OsARF12; qRT-PCR analysis used three independent biological replicates and was performed according to Wang et al. (2010), as in Fig. 2(c). (b) Analysis of OsARF12-Promoter::GUS activity. Error bars indicate SD (n = 3, three plants). *, P < 0.05; **, P < 0.01. (c–h) OsARF12-Promoter::GUS staining: each tissue with +Pi and Pi treatments was stained simultaneously at 25°C: (c) in primary root (PR) under +Pi; (d) in PR under Pi; (e) in lateral root (LR) under +Pi; (f) in LR under Pi; (g) in leaf under +Pi; (h) in leaf under Pi. Bars: 200 lm (c–h). For western blotting of OsARF12 under +Pi and Pi treatments, 40, 30 and 20 lg of plant proteins were extracted from roots or leaves (i) under +Pi and Pi conditions, respectively. (j) The bands in western blotting (panel i) were quantified by software image-J (http://developer.imagej.net/development).
1994; Trull & Deikman, 1998). To determine whether OsARF12 affects Pi acquisition and homeostasis through altering the activity of APase, the APase activity was measured in WTs and mutants. For both the +Pi and Pi conditions, the APase activity of the surface of mutant roots was higher, as indicated by the stronger blue staining of osarf12 and osarf12/25 roots compared with the WT (Fig. 5a). Similar results were gained from the analysis of root intracellular APase content (Fig. 5b) and hydrolytic activity on substrate pNPP (Fig. 5c). In addition, the expression of the 10 purple APase (PAPases) genes, which were induced by the Pi condition, was also studied in WTs and mutants (Wang et al., 2009a). The expression of six of the 10 PAPase genes (OsPAP10a, OsPAP1c, OsPAP20b, OsPAP21b, OsPAP23 and OsPAP27a) was increased in osarf12 roots compared with Dongjin under the +Pi condition. Under the Pi condition, the expression of the nine PAPase genes (OsPAP1d, OsPAP3b, OsPAP9b, OsPAP10a, OsPAP10c, OsPAP20b, OsPAP21b, OsPAP23 and OsPAP27a) was induced in Dongjin and osarf12. Notably, four of them (OsPAP9b, OsPAP10a, OsPAP10c and OsPAP27a) were more up-regulated in mutants (Fig. 5d). Similar results were also observed in the osarf12t mutant and the osarf12/25 double mutant. This evidence is sufficient to indicate that the knockout of OsARF12 enhanced APase activity, suggesting that ARF12 influenced transcript abundance from PAP genes under both the +Pi and Pi conditions and that the effects differed depending on the Pi supply. New Phytologist (2014) 201: 91–103 www.newphytologist.com
Expression of Pi starvation-response genes in the osarf12 mutants and the osar12/25 double mutant was up-regulated under the +Pi and Pi conditions To test whether the genes related to Pi starvation-response were affected by loss-of-function of OsARF12, the transcript abundance of six genes (OsIPS1, OsIPS2, OsSPX1, OsSQD2, OsMYB2P-1 and OsTIR1) was analyzed in WTs, the osarf12 mutants and the osarf12/25 double mutant under +Pi, Pi and Pi resupply conditions (Fig. 6). Transcript analysis revealed that both Pi-responsive genes, OsIPS1 and OsIPS2, were more highly induced in the osarf12 mutants and the osarf12/25 double mutant than in the WT (Hou et al., 2005). OsSPX1 is involved in the Pi signaling pathway, and was induced by the Pi condition. Suppression of OsSPX1 resulted in toxicity and overaccumulation of Pi, similar to that in OsPHR2 overexpressors, the ospho2 mutant and the osarf12 mutants (Wang et al., 2009). OsSQD2 was highly homologous with AtSQD2, encoding sulfolipid synthase, which is also involved in Pi signaling (Yu et al., 2002; Wang et al., 2009). OsSQD2 was more up-regulated by Pi starvation in the osarf12 mutants than in the WT. OsMYB2P-1 is a transcriptional activator localized in the nuclei (Dai et al., 2012). Expression of OsMYB2P-1 was unregulated by the Pi condition. Overexpression of OsMYB2P-1 enhanced the tolerance to Pi starvation and produced shorter PRs than the WT. These characteristics were Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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Fig. 4 The growth characteristics and statistical parameters of roots in rice (Oryza sativa) Dongjin (DJ), osarf12, Nipponbare (NIP), osarf12t and osarf12/25 mutants under phosphate-sufficient (+Pi) and phosphate-deficient ( Pi) conditions. (a) Root phenotype under the +Pi condition; bar, 2 cm. (b) Root phenotype under the Pi condition; bar, 2 cm. (c) Root hair phenotype under the +Pi condition; bar, 200 lm. (d) Root hair phenotype under the Pi condition; bar, 200 lm. (e) Length of primary root (PR). (f) Number of adventitious roots (AR). (g) Number of lateral roots (LR). (h) Density of LR. (e–h) + Pi, open bars; Pi, closed bars; error bars indicate SD (n = 10, 10 plants).*, P < 0.05; **, P < 0.01. The phenotype statistics were performed on 10 biological replications.
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consistent with that of the osarf12 mutants. AtTIR1 (transport inhibitor response1), an auxin receptor in Arabidopsis, is specifically induced by low Pi availability (Perez-Torres et al., 2008). OsTIR1, the closest homolog of AtTIR1 (Bian et al., 2012), was obviously increased in the osarf12 mutants and the osarf12/25 double mutant compared with the WT. Considered together, the expressions of the six genes were more highly induced in the osarf12 mutants and the osarf12/25 double mutant than in the WT under the Pi condition. Their expressions quickly decreased to normal expression levels in all OsARF12-related mutants compared with the WT with increasing time of Pi resupply. These results implied that OsARF12 could regulate these Pi response genes and was also involved in the Pi starvation-response. OsARF12 influenced the transcript abundance of genes around the OsPHR2 center, but OsARF12 expression was not dependent on OsPHR2 under the +Pi and Pi conditions The osarf12 mutants had leaf toxicity symptoms under high transpiration, which was similar to the phenotype of the OsPHR2overexpression line (Zhou et al., 2008). To determine in detail the relationship between OsARF12 and OsPHR2, the central regulator in phosphate homeostasis, the expression of genes related to the OsPHR2 regulatory pathway, including OsPHR2, OsMiR399j, OsPHO2, OsMiR827, OsSPX-MFS1 and OsSPXMFS2, were studied in WTs and all OsARF12-related mutants under +Pi and Pi conditions by qRT-PCR (Fig. 7a). OsPHR2 Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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was up-regulated in the osarf12 mutants and the osarf12/25 double mutant compared with the WTs under +Pi and Pi conditions. The results suggest that OsARF12 might negatively regulate OsPHR2. In all mutants, the expression of OsPHO2, a downstream component negatively regulated by OsMiR399j, was more dramatically inhibited under the –Pi condition, although the transcripts of OsMiR399j and OsPHO2 did not differ from that in WTs under the +Pi condition. The expression of OsMiR827 was higher in mutants and its one target, OsSPX-MFS1, showed an opposite trend under +Pi and Pi conditions. However, another target, OsSPX-MFS2, in mutants was inhibited under the +Pi condition but more induced under the Pi condition. In addition, OsARF12 expression in OsPHR2-overexpression and OsPHR2-RNAi transgenic lines was investigated by qRTPCR and western blotting (Fig. 7b,c). Although OsARF12 transcripts and protein were inhibited under the –Pi condition, there were no significant changes in OsARF12 expression in OsPHR2overexpression in comparison with WT and OsPHR2-RNAi at both transcript abundance and protein level, indicating that OsARF12 may not be directly downstream of OsPHR2 even though there is a PHR1-binding site (P1BS) site upstream of the OsARF12 promoter, 842 bp from start codon ATG (Fig. S2). Enhanced auxin signaling in rice root under the –Pi condition was related to OsARF12 The underlying molecular mechanism of auxin and Pi response in plants is poorly understood. In Arabidopsis, the New Phytologist (2014) 201: 91–103 www.newphytologist.com
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Fig. 5 The activity of acid phosphatase (APase) in rice (Oryza sativa) Dongjin (DJ), osarf12, Nipponbare (NIP), osarf12t and osarf12/25 roots. (a) The activity of acid phosphatase (APase) in the root surface. (b) In-gel analysis of APase activity: the blue bands show the major part of APase in a native polyacrylamide gel. (c) Root-associated APase activity. (d) Expression of 10 genes encoding purple APase. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis used three independent biological replicates and was performed according to Wang et al. (2010), as in Fig. 2(c). The statistical analysis in (c) was performed on five biological replications. Error bars indicate SD (n = 5, five plants) and different letters indicate a statistical difference at P < 0.05.
alteration of auxin content under the Pi condition is controversial, according to Nacry et al. (2005), Sanchez-Calderon et al. (2005) and Lopez-Bucio et al. (2005). To explore whether OsARF12 affects crosstalk of auxin and Pi response, the PR phenotype of 7-d-old seedlings of WTs and mutants were observed for +Pi, +Pi+NPA (an auxin polar transport inhibitor), Pi and –Pi+NPA treatments (Fig. 8a). The PR phenotype and auxin reporter DR5::GUS activity under Pi and NPA treatments showed an antagonism in WT. The PR length in WTs under +Pi+NPA was significantly shorter than only +Pi treatments; and under –Pi+NPA, it was also much shorter than only Pi treatments (Fig. 8a,b). The changes in PR length in mutants showed similar trends to WTs under these four treatments. However, the knockout of OsARF12 led the osarf12 mutants to lose sensitivity to NPA treatment, which showed less PR growth inhibition than that in WTs under +Pi+NPA or –Pi+NPA treatment (Fig. 8b). Furthermore, auxin reporter gene DR5::GUS staining in Dongjin and osarf12 was also observed under +Pi, +Pi+NPA, Pi and – Pi+NPA treatments (Fig. 8c). The DR5::GUS expression in New Phytologist (2014) 201: 91–103 www.newphytologist.com
WT was obviously repressed by +Pi+NPA and significantly increased by the Pi condition, as compared with the +Pi condition, while it had an intermediate level under –Pi+NPA treatment similar to +Pi. Nevertheless, the DR5::GUS expression in osarf12 was stable under +Pi+NPA compared with the +Pi condition. The DR5::GUS expression in osarf12 was less induced by Pi and –Pi+NPA than by +Pi treatment. These results suggested that DR5::GUS activity under the Pi condition was at least partially controlled by OsARF12.
Discussion OsARF12 is a negative regulator of Pi homeostasis through some PHT1 genes OsARF12 as a transcriptional activator was highly homologous with ARF19 in Arabidopsis, which has also been implicated in Pi homeostasis (Wang et al., 2007; Perez-Torres et al., 2008; Qi et al., 2012). Compared with WTs, the osarf12 mutants had greater Pi concentrations and P-toxicity symptoms, especially in Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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Fig. 6 Expression analysis of phosphate (Pi) starvation-induced (PSI) genes, OsIPS1, OsIPS2, OsSPX1, OsSQD2, OsMYB2P-1 and OsTIR1 in rice (Oryza sativa) wildtype (WT), osarf12, osarf12t and osarf12/25 under phosphate-sufficient (+Pi), phosphatedeficient ( Pi) and Pi resupply conditions (R6h, R12h, R18h and R24h refer to resupply times of 6, 12, 18 and 24 h, respectively). Seven-day-old seedlings of WTs and mutants grown under +Pi and Pi conditions were used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. qRT-PCR analysis used three independent biological replicates and was performed according to Wang et al. (2010). Error bars indicate SD (n = 3, three plants).
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Fig. 7 Relationship between OsARF12 and the OsPHR2 regulatory pathway. (a) Expression analysis of the genes involved in the OsPHR2 regulatory pathway in roots of 7-d-old rice (Oryza sativa) (wildtypes (WTs), osarf12 mutants and osarf12/25 double mutant) under phosphate-sufficient (+Pi) and phosphate-deficient ( Pi) conditions. Error bars indicate SD (n = 3, three plants) and different letters indicate a statistical difference at P < 0.05. (b, c) Analysis of OsARF12 expression in Nipponbare (NIP), OsPHR2-overexpression and OsPHR2-RNAi transgenic lines under +Pi and Pi conditions: (b) in transcription level with quantitative reverse transcription polymerase chain reaction (qRT-PCR), error bars indicate SD (n = 3, three plants) and different letters indicate a statistical difference at P < 0.05; (c) in protein level with western blotting. Plant proteins (40/ 20 lg) were extracted from roots of 7-d-old NIP, OsPHR2-overexpression and OsPHR2RNAi lines under +Pi and Pi conditions, respectively. The related binds in western blotting were quantified using software image-J (http://developer.imagej.net/ development). Each experiment was performed with three independent biological replicates.
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leaves – with a similar phenotype to OsPHR2-overexpression or ospho2 mutant (Zhou et al., 2008; Hu et al., 2011). These results indicated that control of Pi absorption and translocation was lost after the regulatory factor OsARF12 was knocked out. For absorption and translocation of Pi, plants require numerous transporters in a complex process (Misson et al., 2004). OsPT1, Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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a key member of the PHT1 family, is involved in Pi transportation from roots to shoots (Sun et al., 2012). OsPHR2 regulates the expression of several PHT1 coding genes under Pi starvation in rice (Zhou et al., 2008). The expression of OsPT1 was not strongly altered in the mutants, which was consistent with that in the OsPHR2- overexpression line. This suggests that OsPT1 New Phytologist (2014) 201: 91–103 www.newphytologist.com
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Fig. 8 Antagonism of Pi (phosphate) and 1-naphthylphthalamic acid (NPA) treatments to primary root (PR) phenotype and auxin distribution:.(a) PR phenotype of 7-d-old rice (Oryza sativa) Dongjin (DJ), osarf12, Nipponbare (NIP), osarf12t, osarf12/25 seedlings under phosphate-sufficient (+Pi), phosphate + NPA (+Pi+NPA), phosphate-deficient ( Pi) and no phosphate + NAP ( Pi+NPA) treatments; bars, 2 cm. (b) Relative PR length of wildtypes (WTs) and mutants under +Pi+NPA, Pi and Pi+NPA treatments compared with +Pi treatment. Error bars indicate SD (n = 10, 10 plants), and when followed by different letters indicate a statistical difference at P < 0.05. (c) The reporter gene of auxin distribution, DR5::GUS staining in Dongjin and osarf12 under the described treatments; bars, 200 lm.
was not directly regulated by OsARF12. OsPT2, a low-affinity Pi transporter, was responsible for most of the overaccumulation of P in shoots of the OsPHR2-overexpression line under the +Pi condition (Liu et al., 2010). Overexpression of OsPT2 can cause overaccumulation of shoot P in rice and thus a P-toxicity phenotype (Liu et al., 2010). In the osarf12 mutants, OsPT2 transcripts were increased under the +Pi condition, which was consistent with the expression pattern in the OsPHR2-overexpression line. This result suggests that regulation of OsPT2 expression by OsARF12 might share the same pathway as OsPHR2. OsPT6 plays a broad role in Pi uptake and translocation throughout the plant, and OsPT6 is strongly activated by Pi starvation, with distinct localization and transport functions (Ai et al., 2009). Our experiments clearly showed that OsPT6 New Phytologist (2014) 201: 91–103 www.newphytologist.com
was induced in roots and leaves of osarf12 mutants under +Pi and Pi conditions (Fig. 2c,d). OsPT8 encodes a high-affinity Pi transporter and acts downstream of OsPHR2 (Jia et al., 2011). Overexpression of OsPT8 resulted in excessive Pi and Pi-toxicity symptoms under high-Pi conditions. OsPT8 was also up-regulated in roots and leaves of osarf12 mutants under both +Pi and Pi conditions. The expression of OsPT9 was also enhanced in both roots and shoots of osarf12 mutants, which was similar to that in the OsPHR2-overexpression line (Zhou et al., 2008). Previous reports showed that up-regulation of Pi transporter genes can enhance Pi uptake and transport efficiency (Liu et al., 1998; Karthikeyan et al., 2002; Xiao et al., 2006; Hu et al., 2011). The present study strongly indicates that the knockout of OsARF12 led to P toxicity as a result of increased Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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P transport through these OsPHT1 genes, and that OsARF12 was a negative regulator for Pi uptake and translocation in rice under both +Pi and Pi conditions. Alteration of OsARF12-regulated phosphatase activity might be associated with increases in OsPAP10a transcripts The expression of OsPHR1 and OsPHR2 in both roots and shoots was not altered by Pi deprivation (Zhou et al., 2008). Similarly, the expression of OsARF12 was not induced by Pi starvation and the osarf12 mutants showed sensitivity to Pi starvation (Figs 2–4). Under Pi-sufficient conditions, typical Pi starvation responses such as APase activity were also improved in the roots of the osarf12 mutants (Fig. 5). The increased APases can help to catalyze Pi hydrolysis from organophosphates. Interestingly, an OsPAP10a gene encoding an important member of rice PAPases was up-regulated about fivefold in the osarf12 mutants under Pi compared with the WT under the +Pi condition (Fig. 5d). The OsPAP10a transcript was controlled by OsPHR2 and specifically induced by Pi deficiency (Tian et al., 2012). OsPAP10a is a root-associated APase, and OsPAP10a overexpression resulted in a significant increase of phosphatase activity and improved ATP hydrolysis and utilization. These results strongly demonstrated that the alteration of OsARF12-regulated phosphatase activity may be related to OsPAP10a. OsARF12 was related to the OsPHR2-mediated response pathway
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In the present study, the osarf12 mutants showed high similarity to the OsPHR2-overexpression line or the ospho2 mutant (Zhou et al., 2008; Hu et al., 2011). The genes related to Pi response around the OsPHR2 center were also altered in the osarf12 mutants, suggesting that OsARF12 may be involved in an OsPHR2-mediated Pi response. However, subsequent experiments showed that OsARF12 expression at both transcript abundance and protein level was not controlled by OsPHR2 even though there was a P1BS site upstream of the OsARF12 promoter, close to start codon ATG (Figs 7b,c, S3). The results contributed a complexity to the relationship between OsARF12 and OsPHR2 or other genes involved in the OsPHR2-mediated Pi signaling pathway. For example, Hu proposed that the targets of LTN1 (OsPHO2) were most likely transcription factors that could activate the expression of PHT genes and Pi starvationresponsive genes (Hu et al., 2011). OsARF12 as a transcription factor that met the conditions and the osarf12 mutant had similar phenotypes to ospho2, which had greater Pi concentrations and P-toxicity symptoms. There were three auxin response elements, AuxRE: TGTCT(A,C)C sites upstream of the OsPHO2 promoter (Fig. S3). Therefore, a detailed regulating network and genetic study on cooperation between OsARF12 and OsPHO2 should be further explored. OsMiR827 and OsSPX-MFS2 transcripts in the osarf12 mutants were significantly altered under both +Pi and Pi conditions, suggesting that OsARF12 may control both genes to a certain degree. The bioinformational prediction also supported this Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
suggestion, because there were three AuxRe sites, 569, 459 and 322 bp upstream of the promoter of the OsMiR827 precursor (Fig. S2). There were four AuxRE sites, 2185, 1490, 437 and 315 bp upstream of the OsSPX-MFS2 start codon ATG. Consequently, the direct downstream factor controlled by OsARF12 and the direct upstream factor regulating OsARF12 should be investigated in detail by chromatin immunoprecipitation assay (ChIP) and more genetic experiments. OsARF12 is an important regulating factor for crosstalk between auxin and Pi signaling High P content was observed in the osarf12 mutants even under the Pi condition, suggesting that Pi homeostasis was altered in the mutants. These results demonstrate a requirement of auxin signaling mediated by OsARF12 for Pi adaptation in rice. Pi starvation affects the auxin content in root tissues (Nacry et al., 2005). In our experiments, the auxin reporter DR5::GUS in the WT was markedly induced under the Pi condition (Fig. 8c) – consistent with the report of Nacry et al. (2005). However, in osarf12, the DR5::GUS was less induced by Pi, implying that knockout of OsARF12 affected auxin accumulation under the Pi condition. Based on these results and previous investigation (Qi et al., 2012), we propose a model in which OsARF12 participates in a crosstalk between auxin and Pi response (Fig. 9). OsARF12, a target of OsmiR167 (Liu et al., 2012; Qi et al., 2012), repressed the central regulator of Pi signaling – OsPHR2 (up-regulated in the osarf12 mutants) – so mutation of OsARF12
Fig. 9 Model of OsARF12 regulating crosstalk between auxin and phosphate-deficient ( Pi) signaling in rice (Oryza sativa). Solid arrows denote direct upstream/downstream of genes. Dashed arrows denote a regulating relationship but it is not determined whether they are directly upstream/downstream. ↓ indicates positive control, while ⊥ indicates negative control. The blue dashed line indicates the connections established by the present study and the black lines indicate already known interactions. New Phytologist (2014) 201: 91–103 www.newphytologist.com
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led to great expressing disturbance of downstream components of OsPHR2, including two well-documented members, OsmiR399 and OsmiR827, that direct the cleavage of OsPHO2 and OsSPX1MFS1/2 mRNA, respectively (Lin et al., 2010; Hu et al., 2011; Wang et al., 2012). Expression of root-secreted exudates (mainly PAPase) and Pi transporter coding genes were elevated, resulting in increased Pi concentration in osarf12 mutants. In addition, OsARF12, as a transcription factor, plays an essential role in root growth through maintaining correct polarization of the auxintransport machinery, such as PIN proteins (Qi et al., 2012). Taken together, we found that the knockout of OsARF12 improved the absorption and translocation of Pi, and that OsARF12 participated in OsPHR2-mediated Pi response through controlling several -Pi response genes. Our data support OsARF12 playing a critical role not only in a wide field regulating Pi uptake, translocation and homeostasis, but also with pivotal functions in crosstalk between auxin and Pi signaling. However, the direct downstream factors of OsARF12-regulated Pi signaling should be researched in detail. The discovery of the negative regulator, OsARF12, of P uptake and translocation should have a significant impact on Pi-use efficiency of rice.
Acknowledgements This research is supported by the National Natural Science Foundation of China (grant nos. 31071392, 31171462 and 31271692), the National Science and Technology Support Plan (2012BAC09B01) and the Natural Science Foundation for Distinguished Young Scholars of ZheJiang Province, China (LR13C130002). We gratefully acknowledge Professor A. Miyao in the Rice Genome Resource Center in Japan for providing the full-length cDNA clones of the OsARF12 gene and the osarf12-TOS17 mutant; Professor G. An in the Plant Functional Genomics Laboratory in Korea, for contributing T-DNA insertion mutants of osarf12; and Professor P. Wu in Zhejiang University for providing the PHR2-RNAi and OsPHR2-Ov transgenic lines.
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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Phenotypes of WT, Dongjin and osarf12 planting in outdoor pots. Fig. S2 Analysis of element contents. Fig. S3 Promoter analysis of genes related to OsARF12. Table S1 Primer sequences for phosphorus transporters Table S2 Primer sequences for the phosphate starvation-induced genes (PSIs) Table S3 Primer sequences for purple acid phosphatase genes Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. New Phytologist (2014) 201: 91–103 www.newphytologist.com
