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. 2015 Jun 16;168(4):1762–1776. doi: 10.1104/pp.15.00736

Integrative Comparison of the Role of the PHOSPHATE RESPONSE1 Subfamily in Phosphate Signaling and Homeostasis in Rice1

Meina Guo 1,2,2, Wenyuan Ruan 1,2,2, Changying Li 1,2, Fangliang Huang 1,2, Ming Zeng 1,2, Yingyao Liu 1,2, Yanan Yu 1,2, Xiaomeng Ding 1,2, Yunrong Wu 1,2, Zhongchang Wu 1,2, Chuanzao Mao 1,2, Keke Yi 1,2, Ping Wu 1,2, Xiaorong Mo 1,2,*
PMCID: PMC4528768  PMID: 26082401

A subfamily of phosphate-responsive genes is functionally diverse in the regulation of phosphate signaling and homeostasis.

Abstract

Phosphorus (P), an essential macronutrient for all living cells, is indispensable for agricultural production. Although Arabidopsis (Arabidopsis thaliana) PHOSPHATE RESPONSE1 (PHR1) and its orthologs in other species have been shown to function in transcriptional regulation of phosphate (Pi) signaling and Pi homeostasis, an integrative comparison of PHR1-related proteins in rice (Oryza sativa) has not previously been reported. Here, we identified functional redundancy among three PHR1 orthologs in rice (OsPHR1, OsPHR2, and OsPHR3) using phylogenetic and mutation analysis. OsPHR3 in conjunction with OsPHR1 and OsPHR2 function in transcriptional activation of most Pi starvation-induced genes. Loss-of-function mutations in any one of these transcription factors (TFs) impaired root hair growth (primarily root hair elongation). However, these three TFs showed differences in DNA binding affinities and messenger RNA expression patterns in different tissues and growth stages, and transcriptomic analysis revealed differential effects on Pi starvation-induced gene expression of single mutants of the three TFs, indicating some degree of functional diversification. Overexpression of genes encoding any of these TFs resulted in partial constitutive activation of Pi starvation response and led to Pi accumulation in the shoot. Furthermore, unlike OsPHR2-overexpressing lines, which exhibited growth retardation under normal or Pi-deficient conditions, OsPHR3-overexpressing plants exhibited significant tolerance to low-Pi stress but normal growth rates under normal Pi conditions, suggesting that OsPHR3 would be useful for molecular breeding to improve Pi uptake/use efficiency under Pi-deficient conditions. We propose that OsPHR1, OsPHR2, and OsPHR3 form a network and play diverse roles in regulating Pi signaling and homeostasis in rice.

Phosphorus (P), an essential macronutrient for all living cells, is a constituent of key molecules, such as ATP, nucleic acids, and phospholipids (Rubio et al., 2001; Cheng et al., 2011). Although the overall P content in soil is high, P represents a limiting factor for plant growth because of its rapid immobilization by soil organic and inorganic components in many natural and agricultural ecosystems (for review, see Richardson et al., 2009; Rouached et al., 2010; Hinsinger et al., 2011). Consequently, plants have evolved strategies to cope with a limited phosphate (Pi) supply, including mechanisms to increase Pi uptake and use and recycle P more efficiently in the plant (Nilsson et al., 2007). PHOSPHATE STARVATION RESPONSE1 (PHR1) in Arabidopsis (Arabidopsis thaliana) and its orthologs in other species play key roles in these processes by regulating Pi signaling and Pi homeostasis to help the plant adapt to Pi deficiency by binding to a cis-element with an imperfect palindromic sequence (GnATATnC; i.e. PHOSPHATE STARVATION RESPONSE1 binding site [P1BS]; Rubio et al., 2001; Bari et al., 2006; Zhou et al., 2008; Bustos et al., 2010). PHR1 functions in three main regulatory pathways as described below.

The first (and most important) Pi regulatory pathway includes five key members: PHR1, INDUCED BY PHOSPHATE STARVATION1 (IPS1), microRNA399 (miR399), PHOSPHATE2 (PHO2), and PHOSPHATE TRANSPORTER (PT). PHR1 directly binds to the promoters of IPS1 (a noncoding RNA; Martín et al., 2000) and miR399 under Pi-deficient conditions (Rubio et al., 2001; Bustos et al., 2010; Lv et al., 2014; Wang et al., 2014). Next, miR399 represses the expression of PHO2, encoding an ubiquitin-conjugating E2 enzyme (UBIQUITIN-CONJUGATING ENZYME24) under Pi starvation conditions (Bari et al., 2006; Hu et al., 2011). In addition, IPS1 can mimic the target of miR399 to block cleavage of PHO2 when the Pi supply is limited (Franco-Zorrilla et al., 2007). Recently, PHO2 was shown to modulate Pi acquisition by regulating the abundance of PHOSPHATE TRANSPORTER1 (PHT1) in the secretory pathway destined for the plasma membrane (Huang et al., 2013).

The second pathway involves PHR1, miR827, NITROGEN LIMITATION ADAPTATION1 (NLA1), and PT. Like IPS1 and miR399, miR827 is also directly regulated by PHR1 (Lin et al., 2010). Overexpression of miR827 leads to accumulation of Pi in shoots (Wang et al., 2012) and reduces the messenger RNA (mRNA) level of NLA1, which encodes a RING FINGER-type ubiquitin E3 ligase (Hsieh et al., 2009; Kant et al., 2011). The nla1 mutant accumulates Pi under nitrogen-deficient conditions (Peng et al., 2007). Moreover, NLA1 recruits PHO2 for the degradation of Pi transporters to help maintain cellular Pi homeostasis (Lin et al., 2013; Park et al., 2014).

The third pathway involves PHR1, PT/purple acid phosphatase (PAP), or sulfoquinovosyl diacylglycerol (SQD). In this pathway, PHR1 directly binds to its target gene promoters to regulate their expression, thereby enabling the plant to adapt to changes in cellular and environmental Pi levels. OsPHT1;2 (OsPT2), a low-affinity (LA) Pi transporter that functions in the Pi translocation process, directs the expression of downstream targets of OsPHR2 in rice (Oryza sativa; Liu et al., 2010). The expression of AtPHT1;1 (containing a P1BS motif in its promoter region) is dramatically reduced in the phr1/phosphate response-like1 (phl1) double mutant (Bustos et al., 2010). Most PAPs function in the production and recycling of Pi from organic P (Tran et al., 2010a, 2010b; Zhang et al., 2011). PAP genes contain P1BS motifs in their promoters, implying that they are direct targets of PHR1 (Wu et al., 2013). In addition, the gene encoding SQD2, which functions in recycling Pi from membrane phospholipids, also contains the P1BS motif (Yu et al., 2002; Wu et al., 2013).

In addition to functioning in these regulatory pathways, PHR1 takes part in other plant growth and developmental processes. For example, PHR1 regulates shoot-to-root sulfate transport by directly binding to the promoters of sulfate transporter-encoding genes SULFATE TRANSPORT1;3 (SULTR1;3) and SULTR3;4 (Rouached et al., 2011). Because the phr1 mutant is much more sensitive to photochemical stress than the wild type, Nilsson et al. (2012) proposed that PHR1 is essential for plant adaptation to high-light levels and maintaining functional photosynthesis during Pi starvation. Arabidopsis FERRITIN1 is regulated by AtPHR1, showing a direct molecular link between iron and Pi homeostasis (Bournier et al., 2013). The responses to Pi and oxygen deficiency stress were recently shown to both be controlled by PHR1 (Klecker et al., 2014).

PHR1 subfamily transcription factors (TFs) are regulated by other factors at the protein level. AtPHR1 is sumoylated by AtSIZ1 (a SAP [for Scaffold Attachment Factors A and B, Acinus, and Protein Inhibitor of Activated Signal-transducer and activator] domain-containing and a zinc-finger Miz [for Melanocyte-stimulating homeobox2-Interacting Zinc-finger] domain-containing protein), a plant small ubiquitin-like modifier E3 ligase that is a primary controller of Pi starvation-dependent responses (Miura et al., 2005). Suppressor of yeast (Saccharomyces cerevisiae) guanine nucleotide-binding protein subunit α1, Yeast Phosphatase 81 (cyclin-dependent kinase inhibitor in yeast PHO pathway), and xenotropic and polytropic retrovirus receptor1 family proteins regulate the transcriptional activity and localization of PHRs by directly interacting with these proteins in a Pi-dependent manner (Lv et al., 2014; Puga et al., 2014; Wang et al., 2014).

Although the importance of AtPHR1 in Arabidopsis and OsPHR2 in rice in the Pi starvation response has been described in detail, how different OsPHRs engage in Pi signaling and homeostasis in rice is currently unclear. In this study, we showed that three OsPHR genes are functionally redundant in Pi signaling and determining root hair morphology. Overexpressing any one of these genes led to Pi accumulation in the shoot as well as root hair elongation, which results the Pi starvation response. Furthermore, the tissue-specific expression patterns and DNA binding affinity differed among OsPHRs. We also highlight the potential of using OsPHR3 in the molecular breeding of crops, because lines overexpressing this gene were tolerant to low-Pi stress.

RESULTS

Redundancy of PHR1, PHR2, and PHR3 in the Control of Pi Starvation Responses

Knockdown of OsPHR1 and OsPHR2 (PHR1-RNA interfering (Ri) and PHR2-Ri) expression leads to various degrees of reduction in Pi starvation responses as revealed by the expression of a set of Pi starvation response genes (Zhou et al., 2008). The incomplete impairment of these responses in the knockdown plants could be explained by partial gene redundancy, because OsPHR1 and OsPHR2 belong to an MYB DNA-binding domain and coiled–coil (CC) domain-containing TF family with at least four close members in cereal crops (Supplemental Fig. S1). To investigate the possibility of partial gene redundancy among these PHR1-related genes, we searched for transfer DNA (T-DNA) or Transposon of Oryza sativa17 (Tos-17) insertional mutants harboring mutations in PHR1-related genes in public databases. A T-DNA insertional mutant with a mutation in PHR2, the phr2 mutant, was previously isolated (Chen et al., 2011). Here, we identified a phylogenetically closely related rice gene, Os02g04640, termed OsPHR3, for which the mutant phr3 was available. OsPHR3 displayed a high degree of amino acid identity to OsPHR1 and OsPHR2, and the Tos-17 insertion disrupted the coding region of OsPHR3 mRNA (Supplemental Fig. S2). Because an OsPHR1 mutant was not found in the public databases, we created an OsPHR1 mutant, phr1, using the clusters of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system (Feng et al., 2013). After characterizing the CRISPR/Cas targeting site, we found a 2-base AC deletion at the first exon of OsPHR1 leading to OsPHR1 malfunction (deletion of the A and C nucleotide at 223 nucleotides from the start codon ATG; Supplemental Fig. S3).

After generating the homozygous double mutants phr1/2, phr1/3, and phr2/3 and the triple mutant phr1/2/3, we performed phenotypic and physiological tests on wild-type, phr1, phr2, phr3, phr1/2, phr1/3, phr2/3, and phr1/2/3 plants. Plant growth was retarded in the phr2 mutant, and additive inhibition of plant growth in phr1/2, phr2/3, and phr1/2/3 was observed under Pi-sufficient (200 μm Pi) and -deficient (10 μm Pi) conditions (Fig. 1, A and B). The most significant repression of growth occurred in the triple mutant phr1/2/3. There was no significant difference in Pi concentration in all single or double mutants compared with the wild type (Fig. 1C), which is different from that of the phr1 and phl1 mutants in Arabidopsis (Rubio et al., 2001; Bustos et al., 2010). However, for the triple mutant phr1/2/3, the Pi concentration was obviously reduced in both shoots and roots compared with the wild type (Fig. 1C).

Figure 1.

Figure 1.

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Effect of phr1, phr2, and phr3 mutations on Pi homeostasis. A, Phenotypic performance of 30-d-old wild-type (WT) plants; phr1, phr2 , and phr3 mutants; phr1/2, phr1/3, and phr2/3 double mutants; and phr1/2/3 triple mutant under Pi-sufficient (200 μm Pi) and -deficient (10 μm Pi) hydroponic conditions. Bars = 10 cm. B and C, Histograms of dried biomass (B) and cellular Pi concentration (C) of shoots and roots of 30-d-old wild-type plants; phr1, phr2, and phr3 single mutants; phr1/2, phr1/3, and phr2/3 double mutants; and phr1/2/3 triple mutant under Pi-sufficient (200 μm Pi) and -deficient (10 μm Pi) conditions. Values represent means ± sd of three replicates. Data significantly different from the corresponding controls are indicated. DW, Dry weight; FW, fresh weight; Nip, cv Nipponbare. *, P < 0.05 (phr1, phr2, and phr3 mutant versus the wild type); **, P < 0.01 (phr1/2, phr1/3, and phr2/3 double mutants and phr1/2/3 plants versus other plants; Student’s t test).

A previous study showed that overexpressing OsPHR2 induces results in Pi accumulation in shoots under Pi-sufficient conditions (Zhou et al., 2008). We, therefore, constructed transgenic plants overexpressing OsPHR3 and analyzed Pi concentrations in these plants. DNA gel-blot and quantitative reverse transcription (qRT)-PCR analyses confirmed that independent OsPHR3-overexpressing lines (OsPHR3-Ov) were produced (Supplemental Fig. S4). Two single-copy OsPHR3-Ov lines (PHR3-Ov1 and PHR3-Ov8) together with OsPHR1- and OsPHR2-overexpressing plants (PHR1-Ov1 and PHR2-Ov1; Zhou et al., 2008) were used for additional analysis. The accumulation of Pi in shoots was significantly increased in PHR3-Ov compared with the wild type, whereas Pi levels in PHR3-Ov were slightly higher than those in PHR1-Ov1 but lower than those in PHR2-Ov1 plants under Pi-sufficient conditions (Fig. 2). In accordance with previous results (Zhou et al., 2008), no significant difference in root Pi concentrations was observed between wild-type plants and overexpressors (Fig. 2B). We also noticed that the shoot biomass of OsPHR3-Ov was higher than that of the wild type under Pi-deficient conditions, whereas PHR2-Ov was lower on both Pi-sufficient and -deficient conditions (Fig. 2C), indicating a character for OsPHR3 in tolerance to low-Pi stress.

Figure 2.

Figure 2.

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Overexpression of OsPHR3 results in shoot Pi accumulation under Pi-sufficient conditions, like OsPHR1 and OsPHR2. A, Phenotypic performance of two independent OsPHR3-overexpressing plants (OsPHR3-Ov1 and OsPHR3-Ov8) compared with the wild type (WT) and PHR1/2-overexpressing plants (Zhou et al., 2008) under Pi-sufficient (200 μm Pi) and -deficient (10 μm Pi) conditions. Two independent lines of OsPHR3 overexpressors were confirmed by DNA gel-blot analysis (Supplemental Fig. S4). Bars = 10 cm. B, Cellular Pi levels in shoots and roots of 30-d-old wild-type and PHR1-, PHR2-, and PHR3-overexpressing plants grown in hydroponic culture under Pi-sufficient and -deficient conditions. Data significantly different from corresponding controls are indicated. FW, Fresh weight; Nip, cv Nipponbare. **, P < 0.01 (overexpressing plants versus the wild type); ++, P < 0.01 (OsPHR2-overexpressing plants versus other overexpressing plants; Student’s t test). C, Biomass of PHR1-, PHR2-, and PHR3-overexpressing lines under hydroponic culture conditions. The 30-d-old seeding biomasses of the wild type, OsPHR1-Ov1, OsPHR2-Ov1, OsPHR3-Ov1, and OsPHR3-Ov8 were measured under Pi-sufficient and -deficient conditions. Values represent means ± sd of 10 replicates. Data significantly different from corresponding controls are indicated. DW, Dry weight. **, P < 0.01 (Student’s t test).

A typical trait stimulated by Pi starvation is the elongation of root hairs, which occurs in both rice and Arabidopsis (Wissuwa, 2003; Yi et al., 2005). To determine whether all three PHR proteins play roles in determining root hair morphology, we examined the root hairs of the corresponding mutants and overexpression lines grown under both Pi-free and -replete conditions. Root hair elongation was slightly inhibited in the single mutants phr1, phr2, and phr3 and the double mutants phr1/2, phr1/3, and phr2/3 (Fig. 3, A and C). However, root hair growth was seriously repressed in the triple mutant phr1/2/3 (Fig. 3, A and C). These results are consistent with the results observed in Arabidopsis (Bustos et al., 2010). Under Pi-replete conditions, the root hairs were longer in overexpression lines than in the wild type (Fig. 3, B and D). In accordance with the root hair elongation of OsPHR2-Ov1 (Zhou et al., 2008), the OsPHR1-and OsPHR3-overexpressing lines also exhibited increased root hair elongation under Pi-replete conditions, whereas this response was weaker than that of the OsPHR2 overexpressor.

Figure 3.

Figure 3.

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Effect of PHR1, PHR2, and PHR3 on root hair growth. A, Primary root hairs of 8-d-old phr1, phr2, phr3, phr1/2, phr1/3, phr2/3, and phr1/2/3 mutants were observed under Pi-free conditions (0 μm Pi). Bar = 2 mm. B, Primary root hairs of 8-d-old PHR1-, PHR2-, and PHR3-overexpressing lines under Pi-sufficient conditions (200 μm Pi). Bar = 2 mm. C and D, The root hair length measurement of related mutants and overexpression lines. The areas from 1 to 1.5 cm under the seeds were measured. Data are means ± sd (n = 6). Asterisks indicate significant differences from the wild type (WT). *, P < 0.05; **, P < 0.01 (Student’s t test).

Taken together, the physiological and morphological traits of phr mutants and their overexpression lines indicate that PHR1, PHR2, and PHR3 have functional redundancy in regulating Pi starvation response and Pi homeostasis in rice.

Molecular Analyses Reveal Partial Functional Redundancy and Functional Diversity among PHR1, PHR2, and PHR3

Now that PHR1, PHR2, and PHR3 show functional redundancy based on physiological and morphological analysis, we examined the effects of phr1, phr2, and phr3 mutants on the Pi starvation response by examining the expression of Pi starvation-induced genes downstream of OsPHR2, including OsIPS1 (Zhou et al., 2008), OsPT2 (Liu et al., 2010), and OsmiR827 (Wang et al., 2012). Although these mutations did not cause obvious changes in the expression of Pi starvation-induced genes in plants grown under a high-Pi (200 μm Pi) regimen compared with the wild type, the expression of most of these genes was significantly reduced in plants cultured in solution lacking Pi (Fig. 4, A–F). The reduced expression of OsIPS1, OsPT2, and OsmiR827 under Pi starvation conditions was less pronounced in phr3 than phr1 and phr2 (Fig. 4, A–F). We observed synergistic and additive effects of phr1, phr2, and phr3 on the expression of all Pi starvation-induced genes examined. Also, overexpression of OsPHR3, like OsPHR1 and OsPHR2, can induce a Pi starvation response (Fig. 4, G and H). As the results show, significant but lower up-regulation of the examined Pi starvation-induced genes was observed in PHR1-Ov1 and PHR3-Ov1/8 plants compared with PHR2-Ov1 plants (Fig. 4, G and H).

Figure 4.

Figure 4.

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Functional redundancy of PHR1, PHR2, and PHR3 in Pi starvation responsiveness of gene expression. qRT-PCR analysis of the effect of phr1, phr2, and phr3 mutants; phr1/2, phr1/3, and phr2/3 double mutants; and phr1/2/3 triple mutant on the expression of Pi starvation-responsive marker genes. Two-week-old plants grown in Pi-sufficient (200 μm Pi) solution were transferred to Pi-sufficient or P-lacking solution for 7 d; RNA from roots and shoots was isolated separately, and qRT-PCR was performed using specific primers (Supplemental Table S1) for OsIPS1 (A and B), OsmiR827 (C and D), and OsPT2 (E and F). The expression of the wild type under Pi-sufficient condition was set to one. Values represent means ± sd of three replicates. G and H, qRT-PCR analysis of the expression of Pi starvation-responsive genes in the PHR1-, PHR2-, and PHR3-overepressing plants. RNA from roots and shoots was isolated separately from 20-d-old plants grown in Pi-sufficient (200 μm Pi) solution, and qRT-PCR was performed using specific primers (Supplemental Table S1) for OsIPS1, OsmiR827, and OsPT2. Nip, cv Nipponbare. *, Significant differences from the wild type (P < 0.05, Student’s t test).

To further understand the functional redundancy and diversity of PHRs, we carried out comparative microarray analysis of the wild type, phr1, phr3, and phr1/2/3. The results showed that 78 transcripts were induced in the phr1 mutant compared with the wild type (among these were 2 Pi starvation-induced transcripts and 0 Pi starvation-repressed transcripts). In addition, 158 genes were repressed in the phr1 mutant compared with the wild type (including 29 and 7 that were induced and repressed, respectively, by Pi starvation). In the phr3 mutant, 169 transcripts (including 1 Pi starvation induced and 39 Pi starvation repressed) were induced and 155 transcripts (including 9 Pi starvation induced and 17 Pi starvation repressed) were repressed compared with the wild type. There were 2,115 transcripts (including 97 Pi starvation induced and111 Pi starvation repressed) induced and 2.224 transcripts (194 were Pi starvation induced and 51 were Pi starvation repressed) repressed in the triple mutant phr1/2/3 compared with the wild type (Fig. 5). Some transcripts (33 induced and 32 repressed) displayed a similar pattern in both phr1 and phr3 mutants, but most were different, indicating that OsPHR1 and OsPHR3 function differently in some processes. Thus, these results show that PHR1, PHR2, and PHR3 have functional redundancy and divergence in function as well.

Figure 5.

Figure 5.

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Comparative transcriptomes of phr mutants. A, Venn diagrams depicting the genes with altered expression in phr mutants compared with the wild type (WT). The total shoot RNA of wild-type, phr1, phr3, and phr1/2/3 seedlings transferred for 7 d to Pi conditions was isolated for microarray analysis. Three repeat microarrays were performed for each material. The number of transcripts with expression that was higher (mutation [mut] > the wild type] or lower (mut < the wild type) in phr1, phr3, and phr1/2/3 plants than in wild-type plants is shown. B and C, The number of Pi starvation-induced (green) or -repressed (red) genes with altered expression in phr1, phr3, and phr1/2/3 mutants.

Different Expression Patterns of PHR1, PHR2, and PHR3 in Time and Space

Because PHR1, PHR2, and PHR3 have functional diversity in regulating Pi starvation response and plant growth based on the above-mentioned results, we investigated their specific expression pattern. First, mRNA expression level of PHR1, PHR2, and PHR3 at different growth stages was analyzed by qRT-PCR (Supplemental Fig. S5). PHR1, PHR2, and PHR3 were expressed throughout all stages of plant growth, indicating the importance of these three PHR proteins for the growth of rice. No marked changes in the expression levels of OsPHR1 or OsPHR2 were observed in leaves between days 10 and 70. However, the expression of OsPHR3 increased dramatically in leaves before the booting stage (approximately day 50). This result is typical of the difference in expression between OsPHR3 and OsPHR1/2.

Second, to investigate the tissue-specific expression patterns of the three PHR genes in detail, we generated plants harboring PPHR1:GUS, PPHR2:GUS, and PPHR3:GUS. Examination of tissue cross sections indicated that OsPHR1, OsPHR2, and OsPHR3 were all expressed in the root cap (Fig. 6, 1C, 2C, and 3C), whereas in the remaining part of the primary root, OsPHR1 was expressed in the vascular tissues (Fig. 6, 1D), OsPHR2 was expressed in the exodermis, sclerenchyma, and vascular tissues (Fig. 6, 2D), and OsPHR3 was expressed only in the exodermis (Fig. 6, 3D). In the lateral root, OsPHR1 was expressed in the stele (Fig. 6, 1E), and OsPHR2 was expressed in the cortex cells and stele cells (Fig. 6, 2E), but no signal was detected in the lateral roots of OsPHR3, except at the root tip (Fig. 6, 3E). GUS staining of OsPHR2 and OsPHR3 was also detected in all mesophyll cells of the leaf (Fig. 6, 2F, 2G, 3F, and 3G), whereas OsPHR1 was expressed only in the mestome sheath cells and the phloem cells of the leaf (Fig. 6, 1F and 1G). In flowers, GUS staining of OsPHR1, OsPHR2, and OsPHR3 was observed in pollen, the vascular cylinder of the anther, and the veins of the lemma, palea, and pistils (Fig. 6, 1H–1J, 2H–2J, and 3H–3J). In node I, OsPHR1 was localized to the xylem and phloem regions of large vascular bundles (Fig. 6, 1K–1M), and OsPHR3 was localized to the xylem and phloem regions of large vascular bundles, small vascular bundles, and diffuse vascular bundles (Fig. 6, 3K–3M). However, OsPHR2 was expressed in all node I tissues (Fig. 6, 2K–2M). These expression pattern differences also hint that PHR1, PHR2, and PHR3 are functionally diverse in regulating crop growth.

Figure 6.

Figure 6.

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Tissue-specific expression patterns of OsPHR1, OsPHR2, and OsPHR3. Tissue-specific expression patterns of OsPHR1, OsPHR2, and OsPHR3 indicated by expression of POsPHR1:GUS (1A–1M), POsPHR2:GUS (2A–2M), and POsPHR3:GUS (3A–3M) fusions in the transgenic plants. In 1 to 3, A to C show GUS staining of the primary root: root cap, meristematic zone, and elongation zone (A) and maturation zone (B and C). Bars = 0.5 mm. Cross sections of root elongation zone (D) and lateral root (E). cor, Cortex; ep, epidermis; exod, exodermis; edod, endodermis; sc, sclerenchyma cell; st, stele. Bars = 50 μm. Cross section of GUS-stained leaf blade (F and G). ms, Mestome sheath cell; ph, phloem. Bars = 100 μm. H to J, GUS staining of pistil, stamens, and shell. K to M, Cross section at the center of node I. DV, Diffuse vascular bundle; PL, phloem region of large vascular bundles; XL, xylem region of large vascular bundles; xm, xylem.

Different DNA Binding Affinities of PHR1, PHR2, and PHR3

Given the partial functional redundancy among OsPHR1, OsPHR2, and OsPHR3 and their differential effects on the expression of various Pi starvation-induced genes (as shown by our analysis of phr1, phr2, and phr3 single, double, and triple mutants and overexpressors), we next examined whether these proteins have similar DNA binding properties by performing yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs). The cis-element P1BS (GNATATNC) contains two variant bases. To understand the differences in DNA binding affinities between the three PHR proteins, we tested PHR proteins with different combinations of the two variant bases in the P1BS by Y1H assay. The P1BS binding affinity of OsPHR2 was the highest among the proteins, whereas that of OsPHR3 was the lowest (Fig. 7, A and B). When the first variant base of the P1BS was changed to T, C, or G, the DNA binding affinities for all three PHR proteins were significantly decreased compared with the original P1BS (GaATATcC). When the second variant base was substituted by A or G, there was no obvious change in binding affinity. By contrast, when the second variant base was substituted by T, the DNA binding affinities significantly increased for all three PHR proteins. We also observed that the P1BS variant GaATATtC was the most efficient for PHR1, PH32, and PHR3 binding, and therefore, we named it the high-affinity (HA)-P1BS. Conversely, the P1BS form GgATATgC was the lowest affinity P1BS for PHR1, PHR2, and PHR3, and we named it the LA-P1BS. To further understand the DNA binding affinity differences of PHR1, PHR2, and PHR3, DNA binding affinity competition experiments were performed using EMSAs. PHR1, PHR2, and PHR3 were individually allowed to bind biotin-labeled LA-P1BS probes, and then, different amounts of unlabeled P1BS (HA-P1BS or LA-P1BS) were added. With increasing amounts of competitor, the percentage of bound probe decreased. OsPHR3 showed the most rapid decline in binding, whereas OsPHR2 binding decreased more slowly, indicating that OsPHR3 has the lowest binding affinity and that OsPHR2 has the highest binding affinity (Fig. 7, C and D). As predicted, HA-P1BS was more efficient than LA-P1BS at competing with the bound probe. Together, these results show that all three PHR proteins bind to the P1BS motif, with different binding affinities for different types of P1BS.

Figure 7.

Figure 7.

Figure 7.

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DNA binding properties of PHR1, PHR2, and PHR3. A, Y1H assays of the DNA binding affinity of PHR1, PHR2, and PHR3 for different P1BS types. The variant bases in the P1BS motif of OsIPS2 were substituted by different bases as indicated; pLacZ2u was used as a negative control. B, β-Galactosidase of activity measurement of PHR1, PHR2, and PHR3 binding to different P1BS versions. Values represent means ± sd of five replicates. **, Data significantly different from corresponding controls (original P1BS versus the substituted P1BS; P < 0.01; Student’s t test). C, EMSA showing that PHR1, PHR2, and PHR3 preferentially bind to the P1BS GaATATtC (HA-P1BS) rather than GgATATgC (LA-P1BS). The binding between glutathione S-transferase (GST)-PHR1, GST-PHR2, and GST-PHR3 and biotin-labeled LA-P1BS was competed by different amounts of unlabeled competitor (HA-P1BS or LA-P1BS). GST protein was used as a negative control. The protein amount used was 50 ng per lane, and the biotin-labeled DNA probe was 20 fmol per lane. The fold excess of the competitor relative to the labeled probe is indicated above the lane. D, Relative binding percentage of PHR1, PHR2, and PHR3 competed by increasing amounts of unlabeled HA-P1BS or LA-P1BS quantified from experiments shown in C. The first lane without competitor was set as 100%.

OsPHR3 Displays Improved Growth Performance in Low-Pi Soils

Overexpression of OsPHR2 reduces plant growth under both normal and Pi-deficient conditions (Zhou et al., 2008). Similarly, overexpression of OsPHR1 did not confer tolerance to low-Pi stress, although it did not cause significant growth retardation under normal conditions. By contrast, OsPHR3-Ov lines did not exhibit reduced plant growth under Pi-sufficient conditions, but they exhibited enhanced growth under Pi-deficient hydroponic conditions compared with the wild type (based on biomass measurements; Fig. 2C). To rule out the possibility that the higher protein expression levels in OsPHR2-Ov lines resulted in the retarded growth, OsPHR2- and OsPHR3-tagged overexpression lines were also analyzed (Supplemental Fig. S6C). The results showed that, even when the OsPHR2 protein level was lower than that of OsPHR3, the OsPHR2 overexpressor still showed growth inhibition (Supplemental Fig. S6, A, B, and D). These results suggested that OsPHR3-Ov could be used in agricultural production systems to enhance crop tolerance of low-Pi conditions.

In another step, we examined the growth of OsPHR3-Ov in pots. The grain yield, total P concentration, and effective panicle number were higher in OsPHR3-Ov lines than in the wild type (Fig. 8), implying that OsPHR3-Ov lines tolerate low-Pi conditions, which is in accordance with our hypothesis. To further confirm that overexpression of OsPHR3 is beneficial to crop growth under Pi-deficient conditions, we performed field experiments. The results show that the biomass, production, and total P in shoots and grains were higher in the OsPHR3-Ov lines than in the wild type under three Pi gradient levels (Supplemental Fig. S7). Furthermore, the production of grain products of OsPHR3-Ov lines under moderate Pi fertilizer conditions was almost equal to that of the wild type under Pi-sufficient conditions. Taken together, these results suggest that OsPHR3 would be a useful candidate gene for breeding crops tolerant to low-Pi stress.

Figure 8.

Figure 8.

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OsPHR3-overexpressing lines tolerance of Pi stress. A, Growth performance of the wild-type (cv Nipponbare [NIP]) and transgenic plants OsPHR3-Ov-1/8 grown in pots with high-Pi fertilizer (200 mg kg−1 Pi; HP), moderate-Pi fertilizer (100 mg kg−1 Pi; MP), and low-Pi fertilizer (30 mg kg−1 Pi; LP). B and C, The grain yields and numbers of effective panicles per plant; data are means ± sd (n = 4). D and E, Total P concentration in shoot and grains; data are means ± sd (n = 4). Data significantly different from corresponding controls are indicated. DW, Dry weight. *, P < 0.05; **, P < 0.01 (Student’s t test).

DISCUSSION

The response of higher plants to Pi starvation is a complicated process, in which numerous genes are activated or repressed at the transcriptional or posttranscriptional level, leading to changes in physiological and morphological processes (Franco-Zorrilla et al., 2004; Yang and Finnegan, 2010). Although much research has focused on this process, information about the OsPHR TF network in rice is currently limited. In this study, we characterized the roles of three OsPHR proteins in regulating Pi signaling and homeostasis in rice. Functional redundancy was confirmed based on the results of physiological and morphological effects of PHR1, PHR2, and PHR3 mutants and overexpressors and the transcriptomic analysis of mutants. The different expression patterns and DNA binding affinities of these OsPHR proteins reflect their functional diversity in regulating the plant response to Pi starvation. Furthermore, the results of this study highlight the potential use of OsPHR3 for molecular breeding in agriculture systems.

Function Redundancy of PHR1, PHR2, and PHR3

The regulatory mechanisms of PHR1 proteins in Arabidopsis and rice are similar (for review, see Chiou and Lin, 2011; Wu et al., 2013). Nonetheless, there are some differences in the functions of PHR1 family members. AtPHR1 and OsPHR2 are central regulators in Arabidopsis and rice, respectively (Bustos et al., 2010; Wu et al., 2013). Like the phr1 mutant in Arabidopsis (Rubio et al., 2001), the phr2 mutant in rice exhibited dramatically inhibited Pi starvation signaling (Fig. 4, A–F). Although mutations in the two other OsPHR genes also disrupted Pi starvation signaling, the degree of reduction was weaker compared with that of the phr2 mutant. Furthermore, examination of the double and triple OsPHR mutants revealed that OsPHR1, OsPHR2, and OsPHR3 are functionally redundant in regulating Pi starvation signaling, which is similar to the roles of AtPHR1 and TRANSPORTER TRAFFIC FACILITATOR1 (AtPHF1) in Arabidopsis (Bustos et al., 2010). However, there are some differences in Pi homeostasis between AtPHRs and OsPHRs. For example, the phr1, phl1, and phr1/phl1 mutants in Arabidopsis exhibit disrupted Pi homeostasis, leading to reduced Pi levels in the plant, but this was not observed in phr1, phr2, or phr3 in rice. Pi homeostasis was affected only when three OsPHR genes were mutated (Fig. 1C). In Arabidopsis, two highly important Pi transporter genes, AtPht1;1 and AtPht1;4 (Shin et al., 2004), as well as AtPHF1 (González et al., 2005; Bayle et al., 2011) are directly regulated by AtPHRs, which may explain why AtPHR mutants exhibit reduced Pi levels. Of 13 Pi transporter genes in the rice genome (Goff et al., 2002; Paszkowski et al., 2002), OsPT8 is one of the most important, because one-half of the Pi uptake capacity depends on the presence of OsPT8, an HA Pi transporter (Chen et al., 2011; Jia et al., 2011). In addition, OsPHF1 plays a key role in regulating the plasma membrane localization of Pi transporters. Mutation of OsPHF1 also severely reduces Pi accumulation (Chen et al., 2011). However, OsPHF1 is not directly regulated by OsPHR2 in rice (Chen et al., 2011). These results also show that there was little reduction in the expression of OsPHF1 and OsPT8, even in the triple mutant phr1/2/3 (Supplemental Fig. S8), perhaps because mutation of rice PHR genes does not obviously affect Pi homeostasis as it does in Arabidopsis. We also noticed that the growth of triple mutant phr1/2/3 was greatly inhibited under both Pi-sufficient and -deficient conditions (Fig. 1, A and B), although there was little reduction in Pi levels in the triple phr1/2/3 mutant. These results suggest that other unknown factors in addition to Pi levels affect the growth of phr1/2/3. Indeed, OsPHRs mediate other regulatory pathways that are essential for the normal growth of rice, such as light, iron, and oxygen signaling pathways (Nilsson et al., 2012; Bournier et al., 2013; Klecker et al., 2014).

PHR1/PHL1 plays a key role in regulating the root-to-shoot ratio, because this ratio is higher in the Arabidopsis phr1/phl1 mutant than in the wild type (Bustos et al., 2010). However, this ratio was not obviously altered in rice mutants phr1, phr2, phr3, phr1/2, phr1/3, phr2/3, and phr1/2/3 (Supplemental Fig. S9). Although the triple mutant phr1/2/3 exhibited greatly reduced growth, no obvious changes in this ratio were observed compared with the wild type (Supplemental Fig. S9), because both shoot and root growth were inhibited in phr1/2/3.

PHR1, PHR2, and PHR3 Show Functional Diversity

In general, more than one functionally similar TF family member plays the same role in responses to a given abiotic or biotic stress. For example, Basic helix-loop-helix transcription factors MYC2 (for Jasmonate-zim-domain protein-interacting transcription factor), MYC3, and MYC4 function in regulating jasmonate responses (Boter et al., 2004; Lorenzo et al., 2004; Devoto et al., 2005; Dombrecht et al., 2007; Fernández-Calvo et al., 2011). Similarly, a group of PHR TFs has similar functions in regulating the Pi starvation response by binding to the P1BS motif to regulate downstream genes that function in these responses (Rubio et al., 2001; Zhou et al., 2008; Bustos et al., 2010; Liu et al., 2010; Wang et al., 2014). Although all three OsPHRs take part in the same Pi regulatory pathway, there are clear differences between them. OsPHR1 is expressed mainly in the vascular bundle, suggesting that it plays a role in regulating Pi translocation from roots to shoots and Pi signal transduction (Fig. 6).

In addition, OsPHR3 and OsPHR1/2 exhibit the differences in their mRNA expression levels in response to Pi-deficient conditions; OsPHR3 was induced under Pi starvation conditions, whereas OsPHR1 and OsPHR2 were not (Supplemental Fig. S10). These results indicate that OsPHR1 and OsPHR2 are transcribed throughout normal plant growth, whereas OsPHR3 is only transcribed when plants are faced with Pi deficiency, perhaps explaining why OsPHR3 was induced at the flowering stage, during which more energy and Pi are needed for flower production (Supplemental Fig. S5). Indeed, Brassica napus PHR1 (BnPHR1) and wheat (Triticum aestivum) PHR1 (TaPHR1) are induced under Pi-deficient conditions, whereas AtPHR1 and AtPHL1 are not (Rubio et al., 2001; Zhou et al., 2008; Bustos et al., 2010; Ren et al., 2012; Wang et al., 2013). We also noticed that all of the three OsPHR genes were expressed in the root tip (Fig. 6), which is the most important tissue for Pi sensing and uptake from the environment (Zhang et al., 2014), implying their coregulation in this tissue. Moreover, most of the genes regulated by OsPHR1 and OsPHR3 are different based on the microarray analysis (Fig. 5), indicating that OsPHR1 and OsPHR3 mediate in different regulation processes in rice.

TF binding sites and flanking sequences can affect TF DNA binding affinity (Rajkumar et al., 2013). In this study, OsPHR proteins exhibited different P1BS binding affinities (Fig. 7), and their binding abilities were affected by variant bases in P1BS as reported by Ruan et al. (2015). How the P1BS variant bases influence these affinities will be a subject of another study.

Overexpression of OsPHR3 Confers Tolerance to Low-Pi Stress in Rice

Overexpression of AtPHR1, OsPHR2, and BnPHR1 leads to Pi accumulation in shoots, producing a necrotic phenotype in leaves and growth repression under normal conditions (Rubio et al., 2001; Zhou et al., 2008; Bustos et al., 2010; Ren et al., 2012). By contrast, overexpression of TaPHR1 promotes the growth of wheat under Pi-deficient conditions (Wang et al., 2013). These studies show that there are species-specific differences in PHR TFs. In this study, three PHR-overexpressing lines exhibited different phenotypes in the face of various Pi levels (Figs. 2A and 8A; Supplemental Fig. S6, A and B). OsPHR2 overexpressors exhibited retarded plant growth, whereas OsPHR3-Ov lines exhibited low-Pi stress tolerance under hydroponic, pot, and field conditions (Figs. 4A and 8A; Supplemental Fig. S6, A and B). Perhaps the better growth of the OsPHR3-Ov lines is because of the low DNA binding affinity of OsPHR3 compared with OsPHR2. Some genes that play positive roles in abiotic stress (such as Pi deficiency) resistance pathways may have negative effects on growth and development; OsPHR3 might bind less effectively to such genes than OsPHR2. Considering the different binding affinities of OsPHRs with different types of P1BS, this explanation seems to be reasonable, and it may account for the observation that overexpression of OsPHR2 inhibited plant growth, even under low-Pi conditions, in which the Pi content was below toxic levels that repress plant growth (Zhou et al., 2008). These findings imply that not only do the Pi levels affect the growth of OsPHR2-Ov plants, but also, other factors triggered by OsPHR2 also affect plant growth under normal conditions. Furthermore, transcriptomic analysis of phr mutants showed that the expression levels of some auxin-responsive, photosystem, and redox system-related genes were induced or repressed, indicating that PHR is a sophisticated regulation system in regulating plant growth.

In summary, we compared and analyzed the integrated regulatory effects of three OsPHR proteins on Pi signaling and Pi homeostasis in rice. Our findings provide evidence that their functional redundancy and diversity enable them to coregulate the Pi response of rice.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The phr1 mutant of rice (Oryza sativa ssp. japonica ‘Nipponbare’) was obtained by the Crisper/Cas technique (Feng et al., 2013; Mao et al., 2013), and the recognition site (5′-GATTCTGTGTCTAGCCATGA-3′) was designed at the first exon. The mutation site of phr1 was identified by sequencing analysis (Supplemental Fig. S2). The primers used for identification of phr1 are listed in Supplemental Table S1. The phr3 T-DNA insertional mutant (NE3009; ssp. japonica cv Nipponbare) was obtained from Centre de Coopération International en Recherche Agronomique pour le Développement (http://orygenesdb.cirad.fr). The phr3 T-DNA insertion site at the seventh exon was characterized by sequencing analysis (Supplemental Fig. S3). The primers used to identify the phr3 mutant are listed in Supplemental Table S1. The identification of the phr2 T-DNA insertional mutant was performed as described (Chen et al., 2011). The phr2 mutant was backcrossed for three generations with cv Nipponbare before analysis. The primers are listed in Supplemental Table S1.

To generate a double mutant containing mutations or insertions in OsPHR1 and OsPHR2, a phr1 homozygous allele was crossed with a phr2 homozygous allele. Double mutants, designated phr1/2, were obtained from the F1 population. The other double mutants, designated phr1/3 and phr2/3, were obtained using the same method along with the triple mutant, designated phr1/2/3. The primers used to identify the mutations are listed in Supplemental Table S1.

The hydroponic experiments were performed using rice solution culture (Zhou et al., 2008). Rice plants were grown in a greenhouse with a 12-h-light/12-h-dark cycle (200 mol−2 s−1 of photon density) at 30°C/22°C after germination. Humidity was maintained at approximately 60%. The soil pot experiments were performed as described previously (Zhou et al., 2008).

RNA Extraction, Reverse Transcription PCR, and Quantitative PCR Assay

RNA was isolated from roots and shoots using Trizol Reagent (Invitrogen) following the manufacturer’s instructions. Reverse transcription was performed using a Moloney Murine Leukemia Virus Reverse Transcriptase cDNA Synthesis Kit (Promega) according to the manufacturer’s instructions. Real-time qRT-PCR was performed as previously described (Zhou et al., 2008); briefly, wild-type expression was normalized to one. Detection and quantification of mature miR399 and miR827 were performed as previously described (Varkonyi-Gasic et al., 2007). The primers used for qRT-PCR analysis are listed in Supplemental Table S1.

Construction of Vectors and Plant Transformation

The OsPHR3 overexpression vector was constructed as follows. Full-length OsPHR3 was cloned and inserted into the pFLAG122 vector after digestion with KpnI and XbaI. The primers are listed in Supplemental Table S1.

To produce transgenic plants expressing the GUS reporter gene, POsPHR1-GUS, POsPHR2-GUS, and POsPHR3-GUS, the cv Nipponbare genomic DNA fragments containing promoter regions less than 3 kb in size (from start codon ATG) were amplified with the primer pairs OsPHR1-P-GUS-F (BamHI)/OsPHR1-P-GUS-R (KpnI), OsPHR2-P-GUS-F (SalI)/OsPHR2-P-GUS-R (BamHI), and OsPHR3-P-GUS-F (BamHI)/OsPHR3-P-GUS-R (KpnI), respectively, and cloned into the modified pCAMBIA1300-GUS plasmid (Lv et al., 2014), respectively. The primers are listed in Supplemental Table S1.

The CRISPR/Cas9 endonuclease system is a site-specific genomic editing tool used in many organisms (Hale et al., 2009; Jore et al., 2011; Carroll, 2012; Jinek et al., 2012), including plants (Feng et al., 2014). The OsPHR1 CRISPR vector was constructed following previously published protocols (Feng et al., 2013) using the primers listed in Supplemental Table S1. The constructed vectors were transformed into rice (‘Nipponbare’) by Agrobacterium tumefaciens EHA105-mediated transformation as described previously (Chen et al., 2003).

GUS Histochemical Analysis

The T1 seeds of POsPHR1:GUS, POsPHR2:GUS, and POsPHR3:GUS transgenic plants were grown in standard rice culture solution. The roots and shoots of 10-d-old seedlings, stamens, pistils, shells, and nodes were subjected to histochemical GUS analysis as described (Jefferson et al., 1987). After staining, the sections were washed in 70% (v/v) ethanol and observed under a ZEISS Microscope. The roots, shoots, and nodes were embedded in 2% (w/w) agar, cut into 30-μm sections using a vibrating microtome (Leica), and observed/photographed through a microscope (ZEISS).

DNA Gel-Blotting Analysis

DNA was isolated from transgenic plants using a Plant Genomic DNA Kit (TIANGEN), and the DNA was digested with HindIII and EcoRI. DNA gel blotting was performed as previously described (Zhou et al., 2008) using G418 as the hybridization probe.

EMSA

To express the recombinant GST:OsPHR1, GST:OsPHR2, and GST:OsPHR3 proteins in Escherichia coli strain BL21(DE3) (Novagen), the full-length CDS of OsPHR1, OsPHR2, and OsPHR3 was amplified with primer pairs OsPHR1-GST-F (EcoRI)/OsPHR1-GST-R (SalI), OsPHR2-GST-F (BamHI)/OsPHR2-GST-R (SalI), and OsPHR3-GST-F (BamHI)/OsPHR3-GST-R (SmaI) and cloned into the pGEX-4T-1 vector (GE Healthcare), resulting in the GST-PHR1, GST-PHR2, and GST-PHR3 vectors. The recombinant proteins were extracted and purified according to the manufacturer’s instructions (GE Healthcare Life Science). The promoter regions containing the PIBS motifs were amplified using primer pairs OsIPS1-probe-F/OsIPS1-probe-R, OsPT2-probe-F/OsPT2-probe-R, and OsmiR827-probe-F/OsmiR827-probe-R, which were used to obtain biotin-labeled probes. The primer sequences are listed in Supplemental Table S1. The EMSA was performed using a LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to the manufacturer’s instructions.

Y1H Assay

To identify and characterize the interactions between PHR1, PHR2, and PHR3 and the promoter regions of OsIPS1 and OsmiR827 in yeast (Saccharomyces cerevisiae), full-length PHR1, PHR2, and PHR3 complementary DNA was cloned into the pB42AD vector (Clontech), thus generating pB42AD-OsPHR1 (AD:OsPHR1), pB42AD-OsPHR2 (AD:OsPHR2), and pB42AD-OsPHR3 (AD:OsPHR3) for the Y1H assay. The promoter regions of OsIPS1 (from −1 to −1,678) and OsmiR827 (from −1 to −1,827) were cloned into pLAZ2u (Clontech). Yeast strain EGY48 was transformed with AD:OsPHR1, AD:OsPHR2, and AD:OsPHR3 and grown on synthetic medium lacking urea and Trp (Clontech). The primers and restriction endonucleases used for Y1H analysis are listed in Supplemental Table S1.

β-galactosidase activity measurement was performance according to Calvenzani et al. (2012). Briefly, transformed yeast cultures grown overnight were injected in a fresh Trp/Ura culture solution until the yeast grew to optical density at 600 nm = 0.4. The cells were then collected to extract total proteins, and β-galactosidase activity was measured after treatment with reaction buffer (Z buffer: 60 mm NaH2PO4, 40 mm Na2HPO4 anhydrous, 10 mm KCl, 1 mm Mg2SO4, and 50 mm β-mercaptoethanol), stopping buffer (1.5 m CaCO3), and substrate o-nitrophenyl-β-d-galactopyranoside (1.4 mg mL−1). The chromogenic reaction and plate reading were carried out essentially following the MatchMaker One Hybrid System Manual (Clontech).

Measurement of Pi Levels, Total Pi, and Pi Uptake Rate

Measurements of Pi levels, total Pi, biomass, and Pi uptake ability in transgenic plants were performed as described previously (Zhou et al., 2008; Wu et al., 2011).

Microarray Analysis

Fifteen-day-old plants of the wild type, phr1, phr3, and phr1/2/3 were treated with P for 7 d, and shoots of plants from three biological repeats were sampled for Affymetrix Microarray Analysis. Microarray and data analyses were performed as described (Bustos et al., 2010). The raw microarray data can be accessed in the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo) through accession number GSE69010. The Pi starvation-induced or -repressed genes were referred from published microarray analysis data (accession no. GSE60823; Wang et al., 2014).

Statistics

Student’s two-tailed t test was used for all experiments to determine the significance of differences between two groups.

Genes from this article can be found in the Rice Genome Initiative under locus identifiers Os03g21240 (OsPHR1), Os07g25710 (OsPHR2), Os02g04640 (OsPHR3), Os07g09000 (OsPHF1), Os03g05640 (OsPT2), Os10g30790 (OsPT8), and OsACTIN (Os03g50890), and in Supplemental Text S1. Data from this article can be found in the GenBank/EMBL data libraries under accession numbers AY568759 (OsIPS1) and AK240849 (OsIPS2) and the miRNA Database under accession number MI0010490 (OsmiR827).

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_168_4_1762__index.html (1.2KB, html)

Glossary

CRISPR

clusters of regularly interspaced short palindromic repeats

EMSA

electrophoretic mobility shift assay

Pi

phosphate

qRT

quantitative reverse transcription

T-DNA

transfer DNA

TF

transcription factor

Y1H

yeast one-hybrid

Footnotes

1

This work was supported by the National Basic Research and Development Program of China (grant no. 2011CB100303), the National High Technology Research and Development Program of China 863 (grant no. 2012AA10A302), the Ministry of Agriculture of China (grant no. 2014ZX08001005), and the Ministry of Education and Bureau of Foreign Experts of China.

References

  1. Bari R, Datt Pant B, Stitt M, Scheible WR (2006) PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol 141: 988–999 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bayle V, Arrighi JF, Creff A, Nespoulous C, Vialaret J, Rossignol M, Gonzalez E, Paz-Ares J, Nussaume L (2011) Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of posttranslational regulation. Plant Cell 23: 1523–1535 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boter M, Ruíz-Rivero O, Abdeen A, Prat S (2004) Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis. Genes Dev 18: 1577–1591 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bournier M, Tissot N, Mari S, Boucherez J, Lacombe E, Briat JF, Gaymard F (2013) Arabidopsis ferritin 1 (AtFer1) gene regulation by the phosphate starvation response 1 (AtPHR1) transcription factor reveals a direct molecular link between iron and phosphate homeostasis. J Biol Chem 288: 22670–22680 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bustos R, Castrillo G, Linhares F, Puga MI, Rubio V, Pérez-Pérez J, Solano R, Leyva A, Paz-Ares J (2010) A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis. PLoS Genet 6: e1001102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Calvenzani V, Testoni B, Gusmaroli G, Lorenzo M, Gnesutta N, Petroni K, Mantovani R, Tonelli C. (2012) Interactions and CCAAT-binding of Arabidopsis thaliana NF-Y subunits. PLoS One 7: e42902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carroll D. (2012) A CRISPR approach to gene targeting. Mol Ther 20: 1658–1660 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen J, Liu Y, Ni J, Wang Y, Bai Y, Shi J, Gan J, Wu Z, Wu P (2011) OsPHF1 regulates the plasma membrane localization of low- and high-affinity inorganic phosphate transporters and determines inorganic phosphate uptake and translocation in rice. Plant Physiol 157: 269–278 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen S, Jin W, Wang M, Zhang F, Zhou J, Jia Q, Wu Y, Liu F, Wu P (2003) Distribution and characterization of over 1000 T-DNA tags in rice genome. Plant J 36: 105–113 [DOI] [PubMed] [Google Scholar]
  10. Cheng L, Bucciarelli B, Liu J, Zinn K, Miller S, Patton-Vogt J, Allan D, Shen J, Vance CP, (2011) White lupin cluster root acclimation to phosphorus deficiency and root hair development involve unique glycerophosphodiester phosphodiesterases. Plant Physiol 156: 1131–1148 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chiou TJ, Lin SI (2011) Signaling network in sensing phosphate availability in plants. Annu Rev Plant Biol 62: 185–206 [DOI] [PubMed] [Google Scholar]
  12. Devoto A, Ellis C, Magusin A, Chang HS, Chilcott C, Zhu T, Turner JG (2005) Expression profiling reveals COI1 to be a key regulator of genes involved in wound- and methyl jasmonate-induced secondary metabolism, defence, and hormone interactions. Plant Mol Biol 58: 497–513 [DOI] [PubMed] [Google Scholar]
  13. Dombrecht B, Xue GP, Sprague SJ, Kirkegaard JA, Ross JJ, Reid JB, Fitt GP, Sewelam N, Schenk PM, Manners JM, et al. (2007) MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis. Plant Cell 19: 2225–2245 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang DL, Wang Z, Zhang Z, Zheng R, Yang L, et al. (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111: 4632–4637 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, Cao F, Zhu S, Zhang F, Mao Y, et al. (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23: 1229–1232 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fernández-Calvo P, Chini A, Fernández-Barbero G, Chico JM, Gimenez-Ibanez S, Geerinck J, Eeckhout D, Schweizer F, Godoy M, Franco-Zorrilla JM, et al. (2011) The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses. Plant Cell 23: 701–715 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Franco-Zorrilla JM, González E, Bustos R, Linhares F, Leyva A, Paz-Ares J (2004) The transcriptional control of plant responses to phosphate limitation. J Exp Bot 55: 285–293 [DOI] [PubMed] [Google Scholar]
  18. Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI, Rubio-Somoza I, Leyva A, Weigel D, García JA, Paz-Ares J (2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet 39: 1033–1037 [DOI] [PubMed] [Google Scholar]
  19. Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296: 92–100 [DOI] [PubMed] [Google Scholar]
  20. González E, Solano R, Rubio V, Leyva A, Paz-Ares J (2005) PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 is a plant-specific SEC12-related protein that enables the endoplasmic reticulum exit of a high-affinity phosphate transporter in Arabidopsis. Plant Cell 17: 3500–3512 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139: 945–956 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hinsinger P, Betencourt E, Bernard L, Brauman A, Plassard C, Shen J, Tang X, Zhang F (2011) P for two, sharing a scarce resource: soil phosphorus acquisition in the rhizosphere of intercropped species. Plant Physiol 156: 1078–1086 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hsieh LC, Lin SI, Shih ACC, Chen JW, Lin WY, Tseng CY, Li WH, Chiou TJ (2009) Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing. Plant Physiol 151: 2120–2132 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hu B, Zhu C, Li F, Tang J, Wang Y, Lin A, Liu L, Che R, Chu C (2011) LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice. Plant Physiol 156: 1101–1115 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Huang TK, Han CL, Lin SI, Chen YJ, Tsai YC, Chen YR, Chen JW, Lin WY, Chen PM, Liu TY, et al. (2013) Identification of downstream components of ubiquitin-conjugating enzyme PHOSPHATE2 by quantitative membrane proteomics in Arabidopsis roots. Plant Cell 25: 4044–4060 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jia H, Ren H, Gu M, Zhao J, Sun S, Zhang X, Chen J, Wu P, Xu G (2011) The phosphate transporter gene OsPht1;8 is involved in phosphate homeostasis in rice. Plant Physiol 156: 1164–1175 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816–821 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, et al. (2011) Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18: 529–536 [DOI] [PubMed] [Google Scholar]
  30. Kant S, Peng M, Rothstein SJ (2011) Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in Arabidopsis. PLoS Genet 7: e1002021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Klecker M, Gasch P, Peisker H, Dörmann P, Schlicke H, Grimm B, Mustroph A (2014) A shoot-specific hypoxic response of Arabidopsis sheds light on the role of the phosphate-responsive transcription factor PHOSPHATE STARVATION RESPONSE1. Plant Physiol 165: 774–790 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lin SI, Santi C, Jobet E, Lacut E, El Kholti N, Karlowski WM, Verdeil JL, Breitler JC, Périn C, Ko SS, et al. (2010) Complex regulation of two target genes encoding SPX-MFS proteins by rice miR827 in response to phosphate starvation. Plant Cell Physiol 51: 2119–2131 [DOI] [PubMed] [Google Scholar]
  33. Lin WY, Huang TK, Chiou TJ (2013) Nitrogen limitation adaptation, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis. Plant Cell 25: 4061–4074 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu F, Wang Z, Ren H, Shen C, Li Y, Ling HQ, Wu C, Lian X, Wu P (2010) OsSPX1 suppresses the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in shoots of rice. Plant J 62: 508–517 [DOI] [PubMed] [Google Scholar]
  35. Lorenzo O, Chico JM, Sánchez-Serrano JJ, Solano R (2004) JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16: 1938–1950 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lv Q, Zhong Y, Wang Y, Wang Z, Zhang L, Shi J, Wu Z, Liu Y, Mao C, Yi K, et al. (2014) SPX4 negatively regulates phosphate signaling and homeostasis through its interaction with PHR2 in rice. Plant Cell 26: 1586–1597 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mao Y, Zhang H, Xu N, Zhang B, Gou F, Zhu JK (2013) Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant 6: 2008–2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Martín AC, del Pozo JC, Iglesias J, Rubio V, Solano R, de La Peña A, Leyva A, Paz-Ares J (2000) Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis. Plant J 24: 559–567 [DOI] [PubMed] [Google Scholar]
  39. Miura K, Rus A, Sharkhuu A, Yokoi S, Karthikeyan AS, Raghothama KG, Baek D, Koo YD, Jin JB, Bressan RA, et al. (2005) The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc Natl Acad Sci USA 102: 7760–7765 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nilsson L, Lundmark M, Jensen PE, Nielsen TH (2012) The Arabidopsis transcription factor PHR1 is essential for adaptation to high light and retaining functional photosynthesis during phosphate starvation. Physiol Plant 144: 35–47 [DOI] [PubMed] [Google Scholar]
  41. Nilsson L, Müller R, Nielsen TH (2007) Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant Cell Environ 30: 1499–1512 [DOI] [PubMed] [Google Scholar]
  42. Park BS, Seo JS, Chua NH (2014) NITROGEN LIMITATION ADAPTATION recruits PHOSPHATE2 to target the phosphate transporter PT2 for degradation during the regulation of Arabidopsis phosphate homeostasis. Plant Cell 26: 454–464 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Paszkowski U, Kroken S, Roux C, Briggs SP (2002) Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci USA 99: 13324–13329 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Peng M, Hannam C, Gu H, Bi YM, Rothstein SJ (2007) A mutation in NLA, which encodes a RING-type ubiquitin ligase, disrupts the adaptability of Arabidopsis to nitrogen limitation. Plant J 50: 320–337 [DOI] [PubMed] [Google Scholar]
  45. Puga MI, Mateos I, Charukesi R, Wang Z, Franco-Zorrilla JM, de Lorenzo L, Irigoyen ML, Masiero S, Bustos R, Rodríguez J, et al. (2014) SPX1 is a phosphate-dependent inhibitor of Phosphate Starvation Response 1 in Arabidopsis. Proc Natl Acad Sci USA 111: 14947–14952 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rajkumar AS, Dénervaud N, Maerkl SJ (2013) Mapping the fine structure of a eukaryotic promoter input-output function. Nat Genet 45: 1207–1215 [DOI] [PubMed] [Google Scholar]
  47. Ren F, Guo QQ, Chang LL, Chen L, Zhao CZ, Zhong H, Li XB (2012) Brassica napus PHR1 gene encoding a MYB-like protein functions in response to phosphate starvation. PLoS One 7: e44005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Richardson AE, Barea JM, McNeill AM, Prigent-Combaret C (2009) Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganism. Plant Soil 321: 305–339 [Google Scholar]
  49. Rouached H, Arpat AB, Poirier Y (2010) Regulation of phosphate starvation responses in plants: signaling players and cross-talks. Mol Plant 3: 288–299 [DOI] [PubMed] [Google Scholar]
  50. Rouached H, Secco D, Arpat B, Poirier Y (2011) The transcription factor PHR1 plays a key role in the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis. BMC Plant Biol 11: 19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ruan W, Guo M, Cai L, Hu H, Li C, Liu Y, Wu Z, Mao C, Yi K, Wu P, et al. (2015) Genetic manipulation of a high-affinity PHR1 target cis-element to improve phosphorous uptake in Oryza sativa L. Plant Mol Biol 87: 429–440 [DOI] [PubMed] [Google Scholar]
  52. Rubio V, Linhares F, Solano R, Martín AC, Iglesias J, Leyva A, Paz-Ares J (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev 15: 2122–2133 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Secco D, Jabnoune M, Walker H, Shou H, Wu P, Poirier Y, Whelan J (2013) Spatio-temporal transcript profiling of rice roots and shoots in response to phosphate starvation and recovery. Plant Cell 25: 4285–4304 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shin H, Shin HS, Dewbre GR, Harrison MJ (2004) Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. Plant J 39: 629–642 [DOI] [PubMed] [Google Scholar]
  55. Tran HT, Hurley BA, Plaxton WC (2010a) Feeding hungry plants: the role of purple acid phosphatases in phosphate nutrition. Plant Sci 179: 14–27 [Google Scholar]
  56. Tran HT, Qian W, Hurley BA, She YM, Wang D, Plaxton WC (2010b) Biochemical and molecular characterization of AtPAP12 and AtPAP26: the predominant purple acid phosphatase isozymes secreted by phosphate-starved Arabidopsis thaliana. Plant Cell Environ 33: 1789–1803 [DOI] [PubMed] [Google Scholar]
  57. Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3: 12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang C, Huang W, Ying Y, Li S, Secco D, Tyerman S, Whelan J, Shou H (2012) Functional characterization of the rice SPX-MFS family reveals a key role of OsSPX-MFS1 in controlling phosphate homeostasis in leaves. New Phytol 196: 139–148 [DOI] [PubMed] [Google Scholar]
  59. Wang J, Sun J, Miao J, Guo J, Shi Z, He M, Chen Y, Zhao X, Li B, Han F, et al. (2013) A phosphate starvation response regulator Ta-PHR1 is involved in phosphate signalling and increases grain yield in wheat. Ann Bot (Lond) 111: 1139–1153 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wang Z, Ruan W, Shi J, Zhang L, Xiang D, Yang C, Li C, Wu Z, Liu Y, Yu Y, et al. (2014) Rice SPX1 and SPX2 inhibit phosphate starvation responses through interacting with PHR2 in a phosphate-dependent manner. Proc Natl Acad Sci USA 111: 14953–14958 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wissuwa M. (2003) How do plants achieve tolerance to phosphorus deficiency? Small causes with big effects. Plant Physiol 133: 1947–1958 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wu P, Shou H, Xu G, Lian X (2013) Improvement of phosphorus efficiency in rice on the basis of understanding phosphate signaling and homeostasis. Curr Opin Plant Biol 16: 205–212 [DOI] [PubMed] [Google Scholar]
  63. Wu ZC, Ren HY, McGrath SP, Wu P, Zhao FJ (2011) Investigating the contribution of the phosphate transport pathway to arsenic accumulation in rice. Plant Physiol 157: 498–508 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yang XJ, Finnegan PM (2010) Regulation of phosphate starvation responses in higher plants. Ann Bot (Lond) 105: 513–526 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yi K, Wu Z, Zhou J, Du L, Guo L, Wu Y, Wu P (2005) OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice. Plant Physiol 138: 2087–2096 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yu B, Xu C, Benning C (2002) Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth. Proc Natl Acad Sci USA 99: 5732–5737 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zhang Q, Wang C, Tian J, Li K, Shou H (2011) Identification of rice purple acid phosphatases related to phosphate starvation signalling. Plant Biol (Stuttg) 13: 7–15 [DOI] [PubMed] [Google Scholar]
  68. Zhang Z, Liao H, Lucas WJ (2014) Molecular mechanisms underlying phosphate sensing, signaling, and adaptation in plants. J Integr Plant Biol 56: 192–220 [DOI] [PubMed] [Google Scholar]
  69. Zhou J, Jiao F, Wu Z, Li Y, Wang X, He X, Zhong W, Wu P (2008) OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants. Plant Physiol 146: 1673–1686 [DOI] [PMC free article] [PubMed] [Google Scholar]

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