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Abstract Phosphorus (P) is an essential macronutrient for plant life, although it is frequently not readily available to crops. Arbuscular mycorrhiza fungi (AMF) can improve plant P levels by inducing the expression of some phosphate (Pi) transporters. Symbiotic Pi uptake by Pi transporters is crucial for AMF colonization and arbuscule dynamics. However, the functions of mycorrhiza-inducible maize Pi transporters are largely unclear. We focused on the interaction between the Pi concentration and AMF colonization in maize, and detecting the induction of a Pi transporter. We investigated AMF colonization and arbuscular development in maize under high and low Pi environments. Low Pi increased AMF colonization and promoted arbuscular development. Further measurement of P concentration showed that AMF significantly improved the maize P status under low Pi conditions. Here, we identified the Pi transporter gene, ZmPt9, which was induced by mycorrhiza formation. In addition, ZmPt9-overexpressing roots were difficult to colonize by AMF. Pi response analysis showed that ZmPt9 complements a yeast mutant defective in Pi transporter activity and improves the P concentration in rice. Together, these data indicated that ZmPt9 is a mycorrhiza-inducible Pi transporter gene involved in Pi uptake. Introduction Phosphorus (P) is a major mineral nutrient required for plant growth and metabolism. However, the low concentration of inorganic phosphate (Pi) in the soil is usually limited by the utilization efficiency of plants (Shen et al. 2011). To cope with Pi-deficient stress, plants must evolve multiple strategies, i.e. increasing the root–soil interface (to enhance Pi transport) and establishing symbiotic associations with microorganisms in the rhizosphere (Lopez-Arredondo et al. 2014). Two of the most extensively studied examples of beneficial microbes are arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing rhizobium bacteria (Sun et al. 2015). AMF can establish an endosymbiotic relationship with approximately 80% of terrestrial plant species, including important crop grasses such as maize (Zea mays), sorghum (Sorghum bicolor) and rice (Oryza sativa) (Xue et al. 2015). Their highly branched hyphae are contained within specialized host membrane compartments to form host–microbe interface structures called arbuscules (Pumplin et al. 2012). Each arbuscule can become enveloped by the plant cell in a membrane to form the periarbuscular membrane (Pumplin et al. 2012). The resulting symbiotic interface facilitates the exchange of nutrients, such as P and nitrogen, which promotes photosynthate production (Denison and Kiers 2011). Accumulated evidence has indicated that symbiosis with AMF can contribute up to 90% of plant Pi requirements from the soil (van der Heijden et al. 2006). Pi can be absorbed and delivered by AMF to the host in the soil and roots after hyphal networks are constructed (Kikuchi et al. 2016). This long-distance polyphosphate translocation is probably mediated by fungal aquaporin(s) via hyphal water flow (Kikuchi et al. 2016). Following acquisition of Pi delivered by AMF, multiple Pi transporters mediate root Pi uptake and homeostasis. High-affinity Pi transporters, belonging to the PHOSPHATE TRANSPORTER1 (PHT1) gene family, are involved in Pi remobilization (Rausch and Bucher 2002, Chiou and Lin 2011). The PHT1 family is part of the major facilitator superfamily, sharing the same predicted structure with 12 putative transmembrane (TM) segments (Lu et al. 2012). The 2.9 Å structure of PiPT, a fungal high-affinity phosphate transporter, was reported in an inward occlusion, with bound Pi visible in the binding site buried within the TM domain (Pedersen et al. 2013). Mycorrhiza-induced PHT1 Pi transporter genes participate in Pi transport during symbiosis between the host plant and AMF (Delaux et al. 2013). Although it is known that some plant PHT1 Pi transporter genes show decreased transcription after the establishment of AMF symbiosis (Rae et al. 2003, Nagy et al. 2005), the expression of a subgroup of PHT1 Pi transporter genes is significantly enhanced in mycorrhizal-induced roots. A marked increase of LePt1 was observed in tomato cells infected with arbuscules (Rosewarne et al. 1999). In potatoes, a high-affinity phosphate transporter, StPt3, can only be detected in mycorrhizal roots (Rausch et al. 2001). OsPt11, a mycorrhiza-specific Pi transporter gene of rice is expressed mainly in the arbuscule branch region (Kobae and Hata 2010). Plant Pi transporters located in the periarbuscular membrane play roles in import of Pi into cortical cells (Maclean et al. 2017). Some conserved plant Pi transporters play significant roles and are essential for maintaining mycorrhizal symbiosis (Maclean et al. 2017). MtPt4 is a mycorrhiza-specific Pi transporter gene of Medicago truncatula, and the loss of MtPt4 function leads to premature arbuscule degeneration and mutualistic loss of symbiosis (Javot et al. 2007a, Pumplin et al. 2012). Similarly, OsPt11 is also involved in arbuscule maintenance (Yang et al. 2012). In addition, ZEAma:Pt1;6 is a mycorrhiza-specific Pi transporter gene of maize, and a pht1;6 mutant showed reduced mycorrhiza formation in pht1;6 roots (Willmann et al. 2013). Currently, maize is considered an attractive model crop for investigating AMF symbiosis and Pi transport. Compared with many reports on symbiotic Pi transporter genes of M. truncatula and rice, less is known regarding the role of mycorrhizal-inducible Pi transporter genes in maize. In the present study, we investigated the symbiotic relationship between maize and AMF in response to low and high Pi concentrations, and aimed to shed light on the effect of AM-inducible Pi transporter genes on mycorrhiza formation and Pi uptake. The results showed that high Pi reduced the mycorrhizal colonization and arbuscular development. We further identified and characterized an AMF-inducible Pi transporter, gene ZmPt9 (ZmPHT1;9, GRMZM2G154090), which responds to Pi starvation and AMF colonization. Here we revealed that ZmPt9 overexpression promoted Pi uptake and reduced the AMF colonization rate. Results Impact of AM symbiosis on the Pi status and growth of maize To investigate the effect of Pi concentration on mycorrhiza formation, colonization levels of maize growing under low Pi (LP) and high Pi (HP) conditions inoculated with Glomus etunicatum were measured. The HP condition was associated with significantly lower levels of mycorrhizal colonization, smaller arbuscules and slower arbuscular development than the LP condition, based on evaluation of the fungal colonization (Fig. 1A–E). We further investigated the effect of mycorrhiza formation on maize P content and growth under HP and LP conditions. AMF colonization had no significant effect on improving maize P concentration under HP conditions (Supplementary Fig. S1), but we observed significant differences in mycorrhizal maize under LP conditions. As Fig. 1G shows, mycorrhizal maize had a significantly higher P concentration than non-colonized maize, both in the roots and in the leaves. In addition, mycorrhizal maize under LP conditions showed taller shoots, larger roots and greater Chl contents than non-colonized maize (Fig. 1F, G;Supplementary Fig. S2). We focused on the expression of Pi transporter genes, which we have studied previously (Liu et al. 2016). In our previous study, we identified 13 Pi transporters. Among them, we found that the ZmPt9 gene represents a different member of the PHT1 gene family, which is expressed in non-colonized roots and up-regulated in both colonized and non-colonized roots of Pi-starved maize (Liu et al. 2016). The ZmPt9 expression levels were further measured in a time course experiment at 0, 30, 40 and 50 days post-inoculation (d.p.i.). ZmPt9 had a basal level of expression before AMF colonization (0 d.p.i.), but its expression level increased at 30 and 40 d.p.i., consistent with the time course of symbiosis formation. The experiment revealed that ZmPt9 has a basal gene expression in non-colonized maize roots and an increased expression level correlated with AM formation (Supplementary Fig. S3). Fig. 1 View largeDownload slide Mycorrhizal colonization and phosphate contents affect each other. (A) Trypan blue staining of maize root colonized by AMF (G. etunicatum) under the HP condition (HP + AMF) at 40 d post-inoculation. (B) Trypan blue staining of maize roots colonized by mycorrhizae under the LP condition (LP + AMF) at 40 d post-inoculation. (C) A detailed picture of the arbusculated cells shown in (A). (D) A detailed picture of the arbusculated cells shown in (B). (E) Mycorrhizal rates of maize roots under LP and HP conditions. Error bars show the SD (n = 3). Asterisks represent a significant difference of colonized roots between HP + AMF and LP + AMF (Student’s t-test; **P < 0.01). (F) Growth phenotypes of colonized (+AMF) and non-colonized (–AMF) maize under the LP condition at 40 d post-inoculation. The chart in the lower left corner shows trypan blue staining of mycorrhizae. (G) Pi contents in leaves and roots of maize colonized or not colonized by AMF under the LP condition at 40 d post-inoculation. Error bars show the SD (n = 3). An asterisk represents a significant difference between colonized and non-colonized roots under LP conditions (Student’s t-test; *P < 0.05). Scale bars: (A, B) 0.5 mm; (C, D) 0.05 mm. Fig. 1 View largeDownload slide Mycorrhizal colonization and phosphate contents affect each other. (A) Trypan blue staining of maize root colonized by AMF (G. etunicatum) under the HP condition (HP + AMF) at 40 d post-inoculation. (B) Trypan blue staining of maize roots colonized by mycorrhizae under the LP condition (LP + AMF) at 40 d post-inoculation. (C) A detailed picture of the arbusculated cells shown in (A). (D) A detailed picture of the arbusculated cells shown in (B). (E) Mycorrhizal rates of maize roots under LP and HP conditions. Error bars show the SD (n = 3). Asterisks represent a significant difference of colonized roots between HP + AMF and LP + AMF (Student’s t-test; **P < 0.01). (F) Growth phenotypes of colonized (+AMF) and non-colonized (–AMF) maize under the LP condition at 40 d post-inoculation. The chart in the lower left corner shows trypan blue staining of mycorrhizae. (G) Pi contents in leaves and roots of maize colonized or not colonized by AMF under the LP condition at 40 d post-inoculation. Error bars show the SD (n = 3). An asterisk represents a significant difference between colonized and non-colonized roots under LP conditions (Student’s t-test; *P < 0.05). Scale bars: (A, B) 0.5 mm; (C, D) 0.05 mm. Cloning and characterization of the ZmPt9 gene Using cDNA amplification techniques, we obtained the full-length coding sequence of ZmPt9 from maize root colonized by AMF. The ZmPt9 gene encodes a putative membrane-integrated protein of 541 amino acids. The molecular structure of the ZmPt9 protein was modeled using the SWISS-MODEL program (Supplementary Fig. S4). The predicted 3-D model of the ZmPt9 protein is highly conserved with respect to the PiPT protein from Piriformospora indica (Pedersen et al. 2013), resembling the molecular structure of eukaryotic Pi transporter proteins (Supplementary Fig. S4A, B). Furthermore, based on the template of the glycerol-3-phosphate transporter (GlPT) from Escherichia coli, the 3-D homology-model structure of ZmPt9 (Huang et al. 2003, Yadav et al. 2010) has 12 α-helices containing a central cytosolic tunnel required to transfer the phosphate molecule (Supplementary Fig. S4C, D). An unrooted phylogenetic tree revealed that functional Pi transporters were closely related (Supplementary Fig. S4E). Protein sequence similarity analyses showed that ZmPt9 shares 77.06% and 85.69% sequence identity with OsPt4 and OsPt8 at the protein level, respectively (Supplementary Fig. S5A). Simulations of the TM domains of ZmPt9, OsPt4 and OsPt8 (predicted by the TMHMM server) showed that the ZmPt9 protein contains 12 TM helices and a central cytosolic tunnel, as found with OsPt4 and OsPt8 (Supplementary Fig. S5B). ZmPt9 was found to be a cytoplasm-localized transporter The subcellular localization of transporter proteins is critical for understanding the uptake of mineral nutrients and signal transduction in plants. Plant PHT1 members are predicted to localize to the cytoplasm. A ZmPt9::GFP (green fluorescent protein) vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was generated (Supplementary Fig. S6A). The ZmPt9::GFP fusion gene was transformed into Agrobacterium strain GV3101, with 35S::GFP serving as a negative control. The transformed GV3101 strain was further infiltrated into Nicotiana benthamiana leaves. Transformed N. benthamiana leaves expressing the ZmPt9::GFP fusion protein were examined. The results indicated that the ZmPt9::GFP fusion protein localized to the cytoplasm, whereas GFP alone was expressed in both the cytoplasm and the nucleus (Supplementary Fig. S6B). The results confirmed that ZmPt9 is a cytoplasm-localized transporter. The ZmPt9 promoter was activated in AMF-colonized roots Synteny analysis was conducted to test further the orthology of ZmPt9 and some AM-inducible PHT1 proteins. The results of this analysis suggested that ZmPt9 is orthologous to some AM-inducible PHT1 proteins, such as OsPt11, OsPt13, MtPt4, GmPt7, GmPt10, GmPt11 and StPt4 (Supplementary Fig. S7). Previous data showed that the CTTC element is necessary and sufficient for a transcriptional response to AMF colonization under LP conditions (Lota et al. 2013). Alignment of the CTTC element (TCCTTCTTGTTCTA) and its flanking regions with the corresponding sequences in ZmPt9 and the predicted promoters of another eight AM-inducible/specific Pi transporter genes was studied here. Unlike the phylogenetic relationship of ZmPt9 and OsPt11 based on protein sequences, promoter elements and promoter phylogenetic analysis showed that ZmPt9 is closely related to OsPt11 from rice. ZmPt9 and OsPt11 contain the conserved core sequence CTTGTT at similar regions of the corresponding promoter, 1,770 and 1,561 bp upstream of the start ATG (Fig. 2). In addition, ZmPt9 contains two CTTC motif sequences (TCTTcTT) at 1,632 and 375 bp upstream of ATG, with a G replaced by a C in the core motif (like OsPt11), where the third T is replaced by a C and a G is replaced by a C at 155 and 1,830 bp upstream of ATG (Fig. 2). Fig. 2 View largeDownload slide Phylogenetic and element analysis of promoters from mycorrhiza-inducible Pi transporter genes. The promoter fragments are 2 kb in length. The numbers on the tree represent bootstrap values. The green triangle represents a 100% match to the extended CTTC motif present (TTTCTTGTTCT). The blue square represents a motif that only matches the core motif. The purple circle represents a motif with base pair changes in the core motif. Fig. 2 View largeDownload slide Phylogenetic and element analysis of promoters from mycorrhiza-inducible Pi transporter genes. The promoter fragments are 2 kb in length. The numbers on the tree represent bootstrap values. The green triangle represents a 100% match to the extended CTTC motif present (TTTCTTGTTCT). The blue square represents a motif that only matches the core motif. The purple circle represents a motif with base pair changes in the core motif. To analyze the promoter activity, we isolated a 2 kb upstream sequence of the ZmPt9 gene and fused it to the GFP reporter gene (to generate the pZmPt9::GFP plasmid). The pZmPt9::GFP plasmid was used to create transgenic hairy roots of Lotus japonicus carrying this construct (Supplementary Fig. S8A). Plants were inoculated with Rhizophagus irregularis at an LP level (30 µM Pi). After 30 d, transformed mycorrhizae showed GFP fluorescence in pZmPt9::GFP-transformed hairy roots of L. japonicus in the colonized root area (Supplementary Fig. S8B), whereas no obvious GFP expression was observed in non-colonized transformed L. japonicus hairy roots (Supplementary Fig. S8B). Therefore, the GFP expression experiments confirmed the quantitative real-time PCR (qRT-PCR) results of colonized maize roots (Supplementary Fig. S3), showing that ZmPt9 is a mycorrhiza-inducible gene. ZmPt9 overexpression reduced AMF colonization Following R. irregularis spore germination, the fungal hyphae infected the root surfaces and formed hyphopodia. The pZmPt9-ZmPt9 and wild-type L. japonicus roots were more permissive than ZmPt9-overexpressing (OE) roots for R. irregularis colonization; we observed very little AMF colonization in the ZmPt9-OE roots. ZmPt9-OE roots showed approximately 17% of their root length colonized, equal to one-fifth of the colonization levels of wild-type roots (Fig. 3C). Rhizophagus irregularis successfully infected ZmPt9-OE roots, forming some normal arbuscules and many smaller arbuscules (Fig. 3A). We observed hyphae reaching the inner cortex in pZmPt9-ZmPt9 and wild-type roots, whereas the hyphopodia that formed on ZmPt9-OE roots were much more difficult to find. However, the hyphopodia were normal in rare colonized ZmPt9-OE roots (Fig. 3B). Fig. 3 View largeDownload slide Examination of mycorrhizal colonization of wild-type (WT; MG20), pZmPt9-ZmPt9 and ZmPt9-OE roots. (A) Trypan blue-stained roots of the WT, pZmPt9-ZmPt9 and ZmPt9-OE infected by R. irregularis, at 5 weeks post-inoculation. Scale bars represent 0.5 mm. (B) R. irregularis hyphopodia formation on the surfaces of WT, pZmPt9-ZmPt, and ZmPt9-OE roots at 5 weeks post-inoculation. Scale bars represent 0.05 mm. (C) Quantification of R. irregularis colonization levels at 5 weeks post-inoculation. An asterisk indicates significant differences between the WT and pZmPt9-ZmPt9 and ZmPt9-OE, as determined by Student’s t-test, *P < 0.05. Error bars represent the SD (n = 3). Fig. 3 View largeDownload slide Examination of mycorrhizal colonization of wild-type (WT; MG20), pZmPt9-ZmPt9 and ZmPt9-OE roots. (A) Trypan blue-stained roots of the WT, pZmPt9-ZmPt9 and ZmPt9-OE infected by R. irregularis, at 5 weeks post-inoculation. Scale bars represent 0.5 mm. (B) R. irregularis hyphopodia formation on the surfaces of WT, pZmPt9-ZmPt, and ZmPt9-OE roots at 5 weeks post-inoculation. Scale bars represent 0.05 mm. (C) Quantification of R. irregularis colonization levels at 5 weeks post-inoculation. An asterisk indicates significant differences between the WT and pZmPt9-ZmPt9 and ZmPt9-OE, as determined by Student’s t-test, *P < 0.05. Error bars represent the SD (n = 3). ZmPt9 complemented Pi transport activities in pho84 mutant yeast To obtain biochemical evidence for the function of ZmPt9, we used BY4743 (a pho84 Pi transport-defective yeast mutant) for complementation analysis. ZmPt9 was transferred to BY4743 mutant yeast, and the results showed that ZmPt9 functionally complemented the mutant cultivated with 0.02 mM Pi (Fig. 4A). Yeast growth curves were measured with BY4743 and BY4743-ZmPt9 under the LP condition (0.02 mM Pi). The growth curves showed that BY4743-ZmPt9 grew much faster than BY4743 (Fig. 4B). In addition, to determine the Pi absorption rate of the ZmPt9 transporter, Pi uptake experiments was performed by measuring the remaining content of Pi in the medium at 12 h intervals. The uptake rates of Pi at a low concentration (0.02 mM Pi) showed that ZmPt9 mediated Pi uptake and detectable Pi absorption at 12–24 h, whereas Pi uptake in the control was apparent at 24–36 h (Fig. 4C). Fig. 4 View largeDownload slide Functional characterization of ZmPt9 in yeast. The BY4743 strain is a yeast mutant defective in high-affinity Pi uptake. The BY4743-ZmPt9 strain was transformed with an expression vector (pYES2.0) encoding ZmPt9. (A) Growth of yeast cells. Equal volumes of 10-fold serially diluted cells (beginning at 6 × 105 cells) were added to YNB medium supplied with 0.02 mM Pi and then incubated at 30°C for 3 d. (B) Growth curves of BY4743-ZmPt9 and BY4743 supplied with 0.02 mM Pi. (C) The remaining Pi in YNB medium was measured at different time points transported by BY4743-ZmPt9 and BY4743. Fig. 4 View largeDownload slide Functional characterization of ZmPt9 in yeast. The BY4743 strain is a yeast mutant defective in high-affinity Pi uptake. The BY4743-ZmPt9 strain was transformed with an expression vector (pYES2.0) encoding ZmPt9. (A) Growth of yeast cells. Equal volumes of 10-fold serially diluted cells (beginning at 6 × 105 cells) were added to YNB medium supplied with 0.02 mM Pi and then incubated at 30°C for 3 d. (B) Growth curves of BY4743-ZmPt9 and BY4743 supplied with 0.02 mM Pi. (C) The remaining Pi in YNB medium was measured at different time points transported by BY4743-ZmPt9 and BY4743. Involvement of ZmPt9 in Pi accumulation under both Pi-deficient and Pi-sufficient conditions The ZmPt9 overexpression vector driven by the CaMV 35S promoter was transformed into ZhongHua 11 rice (Fig. 5A). An independent transgenic line (ZmPt9-OE) showing ZmPt9 expression by Southern blotting was selected for further experimental analysis. To assess the effects of LP or HP concentration on the growth of wild-type and ZmPt9-OE lines, the seedlings were cultivated in different Pi concentrations (0.01–1.25 mM; Fig. 5B). After 16 d, the wild-type plants under the HP condition grew markedly faster than those under the LP condition, but opposite results were observed in the ZmPt9-OE line (Fig. 5B). No significant difference in primary root lengths was found between the wild-type and ZmPt9-OE lines under extremely LP conditions, such as 0.01 mM Pi (Fig. 5C). However, under HP conditions (1.25 mM), the primary root and shoot lengths of the ZmPt9-OE lines were significantly shorter than those of the wild type (Fig. 5C, D). The Pi concentration in the shoots was further measured. At 0.01, 0.1, 0.2 and 1.25 Pi supplied in the environment, the average Pi concentrations in the ZmPt9-OE lines were 3.9, 10.5, 27.2 and 42.3% higher, respectively, than those in the wild-type lines, (Fig. 5E). Fig. 5 View largeDownload slide Characterization of wild-type (WT) and ZmPt9-OE transgenic plants at different Pi concentrations at the seedling stage. (A) The p35S::ZmPt9 construct used for ZmPt9 overexpression. (B) Phenotypes of WT and ZmPt9-OE transgenic plants grown at 0.01, 0.1, 0.2 or 1.25 mM Pi. (C–F) Primary root lengths (C), shoot lengths (D), total P concentrations (E) and relative expression levels (F) in WT and ZmPt9-OE lines after 2 weeks of growth with 0.01, 0.1, 0.2 or 1.25 mM Pi. Seedlings were transferred to nutrient solution containing different Pi concentrations for 2 weeks. RNA was extracted from the shoots of the seedlings. Error bars represent the SD (n = 5). Significant differences were calculated relative to values observed with WT plants as determined by Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001. Fig. 5 View largeDownload slide Characterization of wild-type (WT) and ZmPt9-OE transgenic plants at different Pi concentrations at the seedling stage. (A) The p35S::ZmPt9 construct used for ZmPt9 overexpression. (B) Phenotypes of WT and ZmPt9-OE transgenic plants grown at 0.01, 0.1, 0.2 or 1.25 mM Pi. (C–F) Primary root lengths (C), shoot lengths (D), total P concentrations (E) and relative expression levels (F) in WT and ZmPt9-OE lines after 2 weeks of growth with 0.01, 0.1, 0.2 or 1.25 mM Pi. Seedlings were transferred to nutrient solution containing different Pi concentrations for 2 weeks. RNA was extracted from the shoots of the seedlings. Error bars represent the SD (n = 5). Significant differences were calculated relative to values observed with WT plants as determined by Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001. Reduced expression of low Pi signaling genes in ZmPt9-OE transgenic rice In this study, the transcript levels of OsPHO2, OsPHR1, OsSPX1 and OsPt1 in transgenic rice under the LP condition were investigated by qRT-PCR. The expression levels of three Pi-responsive transcription factor genes (OsPHO2, OsPHR1 and OsSPX1) were 0.48-, 0.56- and 0.73-fold lower in ZmPt9-OE plants, respectively, compared with those in the wild-type plants (Fig. 5F). OsPt1, which is a Pi transporter gene responsive to Pi conditions, was also significantly down-regulated in the transgenic plants, by 0.17-fold (Fig. 5F). Excessive Pi accumulated in ZmPt9-OE transgenic rice plants in the field To understand further the function of ZmPt9 in Pi uptake, we investigated the phenotypes of wild-type and transgenic rice plants grown in the field under HP and LP conditions. Wild-type and ZmPt9-OE transgenic rice at the three-leaf stage were transplanted to the same pot containing 20 kg of air-dried soil supplied with 40 mg (LP) or 60 mg (HP) fertilizer Pi kg–1 soil. Every pot contains one wild-type plant and one ZmPt9-OE transgenic plant. Under different Pi environments, some growth parameters showed significant differences. In an HP-supplied field, growth of the ZmPt9-OE lines sharply decreased compared with the wild-type plants (Fig. 6A), and the seed-setting rates were 45.44% lower than those of the wild type (Fig. 6B, E). However, greater growth of ZmPt9-OE rice was observed when ZmPt9-OE rice and wild-type rice were grown under LP conditions, and the average seed-setting rate of wild-type plants was 66.91%, whereas that of the transgenic lines was 83.92% (Fig. 6A, E). In addition, shoot lengths in the ZmPt9-OE lines were shorter than those of the wild-type line by 10.33 cm (on average) (Fig. 6C) In contrast, in an LP environment, the shoot lengths of ZmPt9-OE lines were 4.55 cm shorter than those of the wild type on average (Fig. 6C). Furthermore, the P concentrations in the wild-type and ZmPt9-OE lines were measured under the HP condition. The shoot and root P concentrations of the ZmPt9-OE lines were significantly higher than those of the wild-type plants, whereas no difference was found in the total P concentrations in brown rice (Fig. 6D). Fig. 6 View largeDownload slide Growth performances of wild-type (WT) and ZmPt9-OE lines at different Pi levels in a pot experiment at the mature stage. (A) Growth performance of WT and ZmPt9-OE plants at 60 mg (HP) fertilizer Pi kg–1 soil and 40 mg (LP) fertilizer Pi kg–1 soil. (B) Panicle-filling performance in the panicle axis of WT and ZmPt9-OE plants under HP conditions. (C) Shoot lengths of WT and ZmPt9-OE plants in HP and LP environments. (D) Total P concentrations in shoots, roots and brown rice of WT and ZmPt9-OE plants at a HP level in the above pot experiment. (E) Percentage seed-setting rates of WT and ZmPt9-OE plants at the LP and HP levels in the pot experiment. Error bars represent the SD (n = 5). Significant differences were calculated relative to values observed with WT plants as determined by Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001. Fig. 6 View largeDownload slide Growth performances of wild-type (WT) and ZmPt9-OE lines at different Pi levels in a pot experiment at the mature stage. (A) Growth performance of WT and ZmPt9-OE plants at 60 mg (HP) fertilizer Pi kg–1 soil and 40 mg (LP) fertilizer Pi kg–1 soil. (B) Panicle-filling performance in the panicle axis of WT and ZmPt9-OE plants under HP conditions. (C) Shoot lengths of WT and ZmPt9-OE plants in HP and LP environments. (D) Total P concentrations in shoots, roots and brown rice of WT and ZmPt9-OE plants at a HP level in the above pot experiment. (E) Percentage seed-setting rates of WT and ZmPt9-OE plants at the LP and HP levels in the pot experiment. Error bars represent the SD (n = 5). Significant differences were calculated relative to values observed with WT plants as determined by Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001. Discussion The Pi status affected AMF colonization on maize roots Maize inoculated with G. etunicatum under LP and HP conditions showed significantly different levels of root colonization. The HP condition resulted in lower levels of mycorrhizal colonization, smaller arbuscules and slower arbuscular development than the LP condition (Fig. 1A–E). Research on potatoes grown under a high Pi (1 mM) condition demonstrated a similar phenotype: altered colonization, a predominance of internal hyphae and a reduced number of arbuscules (Rausch et al. 2001). These data indicated that the HP environment negatively affected maize root colonization by AMF. One hypothesis to explain these findings is that plants supplied with sufficient Pi continue to acquire Pi via a direct uptake pathway and do not deliver carbon to the fungus, resulting in decreased root colonization (Olsson et al. 2006). In addition, studies in legumes have revealed that Pi is not only a nutrient for plants, but is also necessary for reprogramming host cortex cells for symbiosis (Yang and Paszkowski 2011). Symbiotic Pi uptake is crucial for arbuscule dynamics and the development of colonization (Yang and Paszkowski 2011). AMF symbiosis improved Pi uptake in maize Under low Pi conditions, mycorrhizal maize absorbed more Pi than non-colonized maize in the roots and leaves, which was accompanied by taller shoots and larger roots (Fig. 1F, G). As an essential mineral nutrient, Pi is frequently a limiting factor in plant growth. AMF increase Pi uptake by forming root-external hyphae (Bucher 2007). When symbiosis is established, plant Pi uptake mainly occurs via the AMF route, rather than by direct uptake by the plant (Yang and Paszkowski 2011). Consequently, when plants were colonized by AMF, some growth stimulation occurred (Javot et al. 2007b). Many plant PHT1 Pi transporter genes have been found to play roles in direct or mycorrhizal pathways. In M. truncatula, the MtPt4 protein, localized to the peri-arbuscular membrane, mainly functioned in Pi uptake from fungi to plants (Harrison et al. 2002, Sawers et al. 2017). In addition, mycorrhiza-specific PHT1 genes, OsPt11 from rice (Paszkowski et al. 2002), StPt4 from potatoes (Nagy et al. 2005) and LePt4 from tomatoes (Nagy et al. 2005), are strongly induced by AMF symbiosis and show basal expression in non-colonized roots (Javot et al. 2007b). In maize, we previously identified several AM-responsive PHT1 genes (Liu et al. 2016). The ZmPt9 gene is a special member of the PHT1 genes in maize. In contrast to ZEAma:Pt1;6 whose expression in root was only induced by mycorrhiza formation (Nagy et al. 2006), ZmPt9 is expressed in both colonized and non-colonized roots, and is induced by mycorrhiza formation and Pi starvation (Liu et al. 2016). The different expression patterns of ZmPt9 might reflect that ZmPt9 plays role in acquiring Pi via both an direct AM-independent and AM-dependent pathway. Therefore, ZmPt9 is an excellent candidate gene for Pi metabolism analysis in mycorrhizal maize. ZmPt9 was activated by AMF colonization ZmPt9 is orthologous to some AMF-inducible PHT1 proteins from rice, M. truncatula, soybeans and potatoes (Supplementary Fig. S7), indicating that ZmPt9 is probably an AM-inducible transporter and may have similar functions in AMF symbiosis. Promoter phylogenetic analysis showed that ZmPt9 is closely related to OsPt11 from rice, which is an AMF-specific Pi transporter gene and is important for AM symbiosis (Paszkowski et al. 2002). Similar to OsPt11, ZmPt9 contains the conserved core sequence CTTGTT and two CTTC motif sequences (TCTTcTT), with a G replaced by a C in the core motif (Fig. 2), which is necessary and sufficient for AMF colonization (Lota et al. 2013). In addition to the mycorrhizal-specific conserved CTTC motif, both of their promoters contain additional AMF-related elements, such as NODCON2GM (Supplementary Fig. S9). Previous data showed that the 2 kb OsPt11 promoter::GUS fusions in transgenic rice roots inoculated with R. irregularis were strongly indicative of GUS activity in arbusculated cortical cells of roots. In this study, the 2 kb ZmPt9 promoter::GFP vector showed clear GFP expression after AMF colonization (Supplementary Fig. S8B), suggesting that mycorrhizal may have the same inducing effect on ZmPt9 and OsPt11. However, unlike promoter phylogenetic analysis, ZmPt9 protein had distant relationships to OsPt11 protein in the phylogenetic tree (Supplementary Fig. S4E). In addition, ZmPt9 was not an AMF-specific gene like OsPt11, as it was expressed in colonized roots, non-colonized roots and other tissues (Liu et al. 2016), indicating that ZmPt9 may have different functions from OsPt11 although both of them were induced by AMF. Studies have revealed that Pi transporters always play roles in Pi uptake and translocation in different growth situations. For example, OsPt1 constitutively expressed in root and leaf tissues plays a role in Pi uptake and translocation under Pi-sufficient conditions (Sun et al. 2012). OsPt6 expressed in the epidermis, cortex and stelar tissue under Pi-deficient conditions is broadly involved in Pi uptake and translocation through the plants (Ai et al. 2009). OsPt8 expressed constitutively in root tips, lateral roots, leaves, stamens, caryopses and germinated seeds functions in Pi homeostasis (Jia et al. 2011). Thus, ZmPt9 expressed in many tissues may broadly be involved in Pi uptake and translocation in maize. Moreover, mycorrhizal rice relies on AMF symbiosis to improve Pi absorption and meet Pi needs, and OsPt11 plays a significant role in the process of mycorrhizal Pi uptake (Yang et al. 2012). Evaluation of the P concentration in the leaves of six maize lines grown with or without inoculation with AMF (R. irregularis) under P-limiting conditions showed that maize obtained more P via the mycorrhizal pathway under low Pi condiions (Sawers et al. 2017). The study reported that Pi uptake in mycorrhizal maize is correlated with the accumulation of specific PHT1 transcripts (Sawers et al. 2017). Accumulation of ZmPt1 in maize was positively correlated with Pi uptake from the hyphal compartment (Sawers et al. 2017). In addition, accumulation of ZmPt1, ZmPt3, ZmPt4 and ZmPt5 transcripts in maize under LP conditions is positively correlated with shoot biomass or dry weight among mycorrhizal maize (Sawers et al. 2017), which may be an effect of P accumulation. These ZmPHT1 genes are expressed well in seedlings and respond to Pi conditions and/or AMF (Sawers et al. 2017). In this study, as ZmPt9 was also expressed well in seedlings, responded to Pi conditions and AMF inoculation, and promoted Pi uptake, we speculated that ZmPt9 may be a Pi transporter partly involved in mycorrhizal Pi uptake during AMF symbiosis similar to ZmPt1. ZmPt9 overexpression reduced AMF colonization In HP-level soil, maize could obtain sufficient Pi by direct uptake during maize colonization by AMF, which resulted in reduced colonization. We found a similar phenomenon in ZmPt9-OE transgenic L. japonicus hairy roots with a dramatic reduction of AMF colonization. On the one hand, we speculated that ZmPt9 overexpression in L. japonicus hairy roots caused excessive Pi accumulation, resulting in the absence of symbiotic Pi uptake and reduced symbiotic signaling. The reduced symbiotic signaling might generate an unsustainable environment and cause rejection of AMF symbiosis, and further reduced the colonization rate (Yang and Paszkowski 2011). On the other hand, previous studies indicated that the plant hormones strigolactone and ABA/gibberellin influence and maintain the colonization and arbuscules (Bonfante and Genre 2010, Martín-Rodríguez et al. 2016, Pimprikar et al. 2016). Strigolactone-mediated signaling is necessary for a normal level of root colonization (Gomez-Roldan et al. 2008), and high Pi supply has a strong negative effect on strigolactone production (Yoneyama et al. 2007a, Yoneyama et al. 2007b). The balance between ABA and gibberellin is essential for AM formation, but they have a negative interaction in metabolism (Martín-Rodríguez et al. 2016). Gibberellin is an important physiological signal which inhibits arbuscule formation (Pimprikar et al. 2016). Pi starvation causes a decrease in the level of bioactive gibberellin (Jiang et al. 2007). Therefore, if ZmPt9 overexpression produced sufficient Pi for roots this would reduce the strigolactone production and increase the gibberellin level, and as a result the extent of AM symbiosis would decrease (Bouwmeester et al. 2007, Yoneyama et al. 2007b). Unlike ZmPt9-OE roots (and similar to wild-type roots), the pZmPt9-ZmPt9 transgenic roots showed similar colonization and normal arbuscular development to the wild type. Lotus japonicus roots were more permissive than ZmPt9-OE roots for R. irregularis colonization (Fig. 3C). We speculated that ZmPt9 is expressed at a low level in pZmPt9-ZmPt9 transgene root before AMF colonization, but its expression was induced (Supplementary Fig. S8B) during symbiosis formation. Then Pi absorption was promoted through the symbiotic pathway. In return, plants supplied carbon for AMF to maintain symbiosis, which resulted in slightly increased arbuscular development in transgenic pZmPt9-ZmPt9 L. japonicus hairy roots. These results also further indicated that adequately controlled expression of ZmPt9 required AMF induction. ZmPt9 as a functional Pi transporter OsPt13, a second AM-specific PHT1 protein from rice, is induced during AM symbiosis (Yang et al. 2012). As a homolog of OsPt13, ZmPt9 may have a similar function. OsPt13 is necessary for arbuscule development in rice, but functional studies on OsPt13 in plants and yeast showed that OsPt13 probably does not encode a functional Pi transporter (Yang et al. 2012). However, in contrast to OsPt13, ZmPt9 functionally complemented the pho84 yeast mutant cultivated under the LP condition (Fig. 4A), with faster Pi absorption than with the pho84 mutant (Fig. 4B) under LP conditions. ZmPt9-OE transgenic rice lines contained more Pi than the wild type under different Pi conditions. Studies of transcriptional responses to Pi deprivation have implicated an increasing number of genes in plant adaptation to Pi deficiency. However, the expression of these regulatory genes can also be affected by feedback from transcriptional changes in PHT1 genes. ZmPt9-OE lines reduced LP signaling genes (OsPHO2, OsPHR1 and OsPHR2) in transgenic rice, which means that ZmPt9 promoted Pi absorption, and excessive Pi accumulation resulted in suppressed Pi starvation signaling and decreased Pi starvation responses (Fig. 5F). These functional studies demonstrated that ZmPt9 encodes a functional Pi transporter. Materials and Methods Plant materials and growth conditions Maize seeds were surface sterilized with 15% bleach for 15 min and washed with ddH2O three times. After washing, seeds were germinated on filter paper for 7 d at 28°C with a photoperiod of 16 h light and 8 h dark. Non-mycorrhizal treatments (under HP and LP conditions) and inoculation with G. etunicatum (under HP and LP conditions) were set up. Pairs of two uniform seedlings were transferred to one pot (28 cm diameter×25 cm, with three small holes in the bottom) filled with sterilized sand, and a total of 48 pots were prepared. Seedlings transferred to 24 of the 48 pots were spread with 8 g of G. etunicatum inoculum (provided by Sun Yat-Sen University) on each root. The 24 pots inoculated with G. etunicatum were watered with modified Hoagland solution containing 5 mM Pi (HP) and 50 μM Pi (LP), respectively. Another 24 pots with transferred maize seedlings with no G. etunicatum inoculum were watered with modified Hoagland solution containing 5 mM Pi (HP) and 50 μM Pi (LP), respectively. Plants were grown in a greenhouse at 16 h light/8 h dark, 28°C/25°C (day/night), relative humidity 65%. All the pots were watered once a week with 500 ml of modified Hoagland solution. The leaves and roots of maize were harvested from the seedlings at 0, 30, 40 and 50 d after four treatments. Leaves were used for measuring P and Chl content. Roots were used for measuring P content, evaluating mycorrhizal colonization and isolating RNA. There were three biological replicates for each treatment. Construction of ZmPt9 promoter–GFP fusion vectors, overexpression and plant transformation The putative ZmPt9 promoter was amplified from a 2 kb region upstream of the coding region within maize genomic DNA, using specific primers (pZmPt9P-F/R; Supplementary Table S1). Using gene-specific primers (ZmPt9 F/R; Supplementary Table S1), the full-length coding sequence was amplified. The pCAMBIA1302 vector was used for GFP analysis, whereas the pCAMBIA1301 vector was used to generate a ZmPt9-overexpression vector. The pCAMBIA1302 constructs were transferred into Agrobacterium rhizogenes strain LBA9402 by electroporation and then transformed into L. japonicus hairy roots. The pCAMBIA1301 constructs were transferred into A. tumefaciens strain GV3101 by electroporation and then transformed into rice. Transgenic plant growth conditions The rice ZhongHua 11 (Oryza sativa L. ssp. japonica) was used for all physiological experiments. Various concentrations of Pi (0.02, 0.1, 0.2 or 1.25 mM KH2PO4) were supplied in growth experiments performed with Murashige and Skoog medium containing 0.8% agar in a greenhouse (12 h photoperiod, 30°C). Soil pot experiments were performed with five replications for each treatment of Pi supply in a greenhouse. One wild-type plant and one ZmPt9-OE plant each were grown in separate pots containing 20 kg of air-dried soil. The Pi levels supplied to the plants were 40 and 60 mg fertilizer Pi kg–1 soil. qRT-PCR experiments Total RNAs from maize and rice were isolated using the guanidine thiocyanate extraction method with the RNAiso Plus Kit (TAKARA BIO INC.). RNA samples were treated with DNase after extraction to eliminate potential trace contaminants of genomic DNA. Approximately 1 µg of RNA was used for first-strand cDNA synthesis with the Transcriptor First Strand cDNA Synthesis Kit (Roche Molecular Systems, Inc.), and the synthesized cDNAs were used as templates for qRT-PCR. The qRT-PCR was conducted on an Applied Biosystems 7300 instrument using the SYBR Green Kit (Roche Molecular Systems, Inc.). The primers used in this experiment are shown in Supplementary Table S1. Other primers used in this study were described previously (Zhou et al. 2008). Expression of the reference genes ZmACTIN1 and ZmGAPDH was detected as an internal control (Supplementary Table S1). Relative expression levels were calculated using the 2–ΔΔCt method (Zhang et al. 2017). Yeast manipulations The yeast Pi uptake-defective mutant BY4743 and the expression vector pYES2 were used in this study. The coding sequence of ZmPt9 was cloned into pYES2. The yeast strain BY4743, as well as BY4743 containing pYES2-ZmPt9, were grown to logarithmic phase on yeast nitrogen base (YNB) medium. The strains were then harvested and washed in Pi-free YNB medium. Finally, the yeast strains were incubated in liquid medium containing different Pi concentrations (0.02 and 0.1 mM). Measuring the total P and Chl content in plants To measure the total P concentrations in plants, the plants were dried at 80°C for 3 d to determine their dry weights (approximately 0.2 g), and total phosphate measurements in the dry samples were performed as previously described (Chen et al. 2007). Chl contents in maize leaves were estimated according to Lichtenthaler and Wellburn (1985). Detection of mycorrhizal colonization Initially, maize and L. japonicus roots segments were fixed in FAA for >4 h, treated with 10% KOH and heated at 90°C for 1 h. Next, the roots were further treated with a 2% HCl solution for 5 min. Root samples were stained with 0.05% Trypan blue solution and made transparent by treatment with lactic acid and glycerin. Fifty root fragments for each 1 cm long root sample were mounted on slides and examined for the specific AMF structures at ×100–400 magnification using a Leica DM5000B microscope. The percentage of the root colonized by AMF was calculated using the root segment colonization weighting method (Biermann and Linderman 1981). Bioinformatics analysis Multiple alignments of ZmPt9 and other PHT1 protein sequences were performed using the DNAMAN software package (version 8.0; Lynnon Biosoft). The different statistical parameter settings used were described previously (Pudake et al. 2017). The phylogenic tree of PHT1 proteins was drawn using MEGA6 with the Neighbor–Joining method (Tamura et al. 2013). Prediction of TM helices of proteins was conducted using the online TMHMM Server, version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Molecular modeling of the ZmPt9 protein was conducted using the online SWISS-MODEL server (https://swissmodel.expasy.org/). The phylogenic tree of PHT1 genes promoters was drawn by MEGA6 based on 2.0 kb promoter sequences with the maximum likelihood method (Tamura et al. 2013). Cis-acting elements in the ZmPt9 promoter were analyzed and plotted using the RSAT website (http://floresta.eead.csic.es/rsat/) (Turatsinze et al. 2008, Medina-Rivera et al. 2015). Synteny between ZmPt9 and other PHT1 genes was analyzed using CoGe:GEvo (https://genomevolution.org/CoGe/GEvo.pl) (Xue et al. 2015). Statistical analysis Student’s t-test was performed for comparisons of means of colonization rates, plant growth parameters, P content and relative expression levels of genes using the Excel software program (Microsoft). The statistical information, including numbers of replicates and error bars, is described in each figure legend. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Key Research and Development Program of China [No. 2016YFD0300300]; the National Science Foundation of China [Nos. 31470465 and 31640057]; and the Project of Natural Science Foundation of Anhui Province [No. 1708085MC53]. Acknowledgments We are grateful to Dr. Xiang Xiao for providing the Ri T-DNA-transferred carrot hairy roots with R. irregularis. We also thank Dr. Yuancheng Peng for revising our paper. Disclosures The authors have no conflicts of interest to declare. References Ai P. , Sun S. , Zhao J. , Fan X. , Xin W. , Guo Q. ( 2009 ) Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation . Plant J . 57 : 798 – 809 . Google Scholar CrossRef Search ADS PubMed Biermann B. , Linderman R.G. ( 1981 ) Quantifying vercular-arbuscular mycorrhizas: a proposed method towards standardization . New Phytol. 87 : 63 – 67 . 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations AMF arbuscular mycorrhiza fungi d.p.i. days post-inoculation; GFP green fluorescent protein HP high phosphate LP low phosphate OE overexpressing P phosphorus PHO2 phosphate 2 PHR1 phosphate starvation response1 PHT1 phosphate transporter 1 Pi phosphate qRT-PCR quantitative real-time PCR TM transmembrane © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: firstname.lastname@example.org This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
Plant and Cell Physiology – Oxford University Press
Published: Aug 1, 2018
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