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The FvPHR1 transcription factor control phosphate homeostasis by transcriptionally regulating miR399a in woodland strawberry
Plant Science 280 (2019) 258–268
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The FvPHR1 transcription factor control phosphate homeostasis by transcriptionally regulating miR399a in woodland strawberry Yan Wang1, Feng Zhang1, Weixu Cui, Keqin Chen, Rui Zhao, Zhihong Zhang
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Liaoning Key Laboratory of Strawberry Breeding and Cultivation, College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Woodland strawberry Pi-signaling FvPHR1 miR399 P content
Plants have evolved phosphate (Pi) starvation response to adapt the low-Pi environment. The regulation of adaptive responses to phosphorus deficiency by the PHR1-miR399-PHO2 module has been well studied in Arabidopsis thaliana but not in strawberry. Transcription factor PHR1 as the central regulator in the Pi starvation signaling has been revealed in a few plant species. However, the function of PHR1 homologues in strawberry is still unknown. In this study, a total of 13 MYB-CC genes were identified in the woodland strawberry (Fragaria vesca) genome and the FvPHR1 gene was characterized. FvPHR1 contains MYB domain and coiled–coil (CC) domain and is localized in the nucleus. FvPHR1 also exhibits trans-activation ability. Furthermore, the P content in leaves of FvPHR1-overexpressing woodland strawberries was significantly increased by 1.38-fold to 1.78-fold compared with that in the wild type. FvPHR1 was also demonstrated to directly bind to the FvMIR399a promoter and positively regulate the expression of FvmiR399a in woodland strawberry. These results showed that PHR1miR399 module is involved in the regulation of phosphate-signaling pathway and phosphate homeostasis in woodland strawberry.
1. Introduction Phosphorus (P), an essential macronutrient, plays a key role in the normal growth and development of plants. Plants uptake P from soil by roots [1]. In nature, P usually exists as the form of phosphate minerals, but there is a little available P for plants absorption because it is very stable or insoluble [2]. In order to adapt the low phosphate (Pi) environment, plants have evolved a number of strategies to regulate Pi homeostasis in Pi starvation, for example, the alteration of root morphology and structure [3,4], organic acid exudation [5], secretion of phosphatases [6], and upregulated expression of phosphate-starvationresponse (PSR) genes that were controlled by some transcript factors (TFs), including PHR1 (phosphate starvation response 1), WRKY75, ZAT6 and BHLH32 [7–10]. PHR1, as the central regulator in the phosphate starvation signaling [11–13], contains a MYB domain and a coiled–coil (CC) domain, and it was termed a MYB-CC protein by Rubio et al [7]. Under Pi starvation condition, PHR1 is regulated by the small ubiquitin-like modifier (SUMO E3) ligase SIZ1 at post-translational level [14]. PHR1 could bind to a cis-element of PHR1-binding sequences (P1BS: GNATATNC) in the promoter of some Pi starvation-induced (PSI) genes to regulate the expression of these genes, including several PHT1 genes and MIR399
[7,15–17]. MicroRNA399 (miR399) plays a key role in Pi-starvation signaling network in plants [18,19]. It is reported that miR399 could direct the cleavage of PHO2 mRNA in some species [15,20,21]. PHO2, an ubiquitin-conjugating E2 enzyme (UBC24), negatively regulates Pi transporters to inhibit Pi uptake and root-to-shoot translocation [15,22]. Although some components of Pi starvation signaling have been identified in plants, the overall pathway is still poorly understood. Furthermore, it was reported that AtNIGT1/HRS1 played an important role in the integration of nitrate and phosphate signals in Arabidopsis [23]. Besides, in Pi-deficient and NO3−-sufficient condition, N uptake was decreased because the PHR1 can positively regulate the expression of NIGT1 family genes in Arabidopsis, and these NIGT1s are negative regulators of NRT2.1 (a typical nitrate-inducible gene) [24]. So PHR1 also plays a significantly part in phosphorus and nitrogen nutritional regulation. Strawberry is one of the most popular fresh and processed fruits with pleasant flavor and nutritional value [25]. The growth of strawberry plants was influenced by temperature, light, water, salinity and nutrient [26]. Pi is a necessary nutrient element for strawberry plants propagation, health and vigor [27]. Thus, the study of Pi signaling in strawberry plants is crucial for strawberry production. In previous study, we reported that soluble solids content is positively correlated
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Corresponding author. E-mail address: [email protected] (Z. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.plantsci.2018.12.025 Received 16 July 2018; Received in revised form 19 December 2018; Accepted 24 December 2018 Available online 28 December 2018 0168-9452/ © 2019 Elsevier B.V. All rights reserved.
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OS06G49040: OsPHR4) [7,31–33], and performing the Protein BLAST research using the amino acid sequence of FvPHR1 as a query (PHR1s from other species). MEGA 6.06 and GeneDoc program were used in multiple sequence alignment. The phylogenetic tree was constructed using MEGA 6.06 software with the Neighbor–Joining method.
with P content in ripening strawberry fruits [28], and overexpressing miR399a in strawberry improved the fruit quality [29]. However, the TFs involved in Pi homeostasis in strawberry plants are largely unknown. In this study, 13 MYB-CC genes were identified in woodland strawberry (Fragaria vesca) genome. Further analysis showed that FvPHR1 worked as a transcription factor in woodland strawberry. And we found that the overexpression of FvPHR1 could increase the P content in woodland strawberry. In addition, FvPHR1 could positively regulate the expression of FvmiR399. The functional analysis of FvPHR1 suggests that it plays a significant role in Pi homeostasis of woodland strawberry. This study will provide a basis for illustrating the molecular regulation mechanism of P signaling network in strawberry.
2.5. Sub-cellular localization The coding sequence of FvPHR1 without the stop codon was fused to green fluorescent protein (GFP) gene, and cloned into the pAN580 vector controlled by the cauliflower mosaic virus (CaMV) 35S promoter. The primer set was shown in Supplementary Table S1. And OsMADS3-mCherry was used as a nuclear marker. We refer to the process of rice protoplast isolation and transformation as previously described [34]. GFP fluorescence was observed with Zeiss LSM700 confocal microscopy. The emission filters were 500–530 nm for GFP, 580–620 nm for mCherry.
2. Materials and methods 2.1. Plant materials and growth conditions Fifty-day-old seedlings of woodland strawberry (Fragaria vesca) accession ‘Ruegen’ were grown in pots filled with washed sand: vermiculite: perlite (2:1:1). And we watered nutrient solution (1 mM KH2PO4) and as described previously for 10 day [29]. The nutrient solution was watered every other day. Then the seedlings were subjected to Pi-deficient (0.01 mM KH2PO4) condition, and treated for 0, 6, 12, 24, 72, 120 h. After that, the leaves and roots of these seedlings were separately harvested for analysis of gene expression.
2.6. Transcriptional activation analysis in yeast cells Transactivation activity assay of FvPHR1 was performed by using Matchmaker GAL4 Two-Hybrid System 3 (Clontech, Palo Alto, CA, USA). The coding region of FvPHR1 was inserted into the pGBKT7 vector. Primers were listed in Supplementary Table S1. The pGBKT7FvPHR1 and the negative control pGBKT7 constructs were transformed into yeast strain AH109, respectively. The transformed strains were grown on the medium of SD/−Trp and selected on the medium of SD/ −Trp−His−Ade.
2.2. Identification of MYB-CC family genes in woodland strawberry
2.7. Bimolecular fluorescence complementation (BiFC) assay
To identify the MYB-CC family genes in woodland strawberry, first, we performed search program with search value “PHR1” and default parameters on PLAZA website (https://bioinformatics.psb.ugent.be/ plaza/versions/plaza_v3_dicots/basic_search/mobile_search) and download the profile named HOM03D000096 protein sequences. Next, the putative MYB-CC protein sequences from Arabidopsis thaliana and F. vesca were extracted and manually verified by NCBI Conserved Domain Database (CDD, http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi/) and SMART (http://smart.embl-heidelberg.de/) to confirm they contained a MYB-CC domain. Then, we acquired the information of strawberry MYB-CC genes from the Strawberry Genome v4.0a1 marker standard proteins databases in Genome Database for Rosaceae (GDR) by performing protein BLAST search (https://www.rosaceae.org/blast/ protein/protein). The theoretical molecular weight and isoelectric point of deductive MYB-CC proteins were predicted using ExPASy Proteomics Server (http://web.expasy.org/compute_pi/).
The coding sequence of FvPHR1 was cloned into pSPYNE173 (eYNE) and pSPYCE (M) (eYCE) vector to generate constructs of eYNE-FvPHR1 and eYCE-FvPHR1, respectively. The primers used for BiFC assays were shown in Supplementary Table S1. Agrobacterium tumefaciens strains EHA105 carrying the recombinant plasmids and ER marker were coinfiltrated into 5-week-old tobacco (Nicotiana benthamiana) leaves. The eYFP fluorescence signaling was observed with Zeiss LSM700 confocal microscopy at 72 h after infiltration. 2.8. Cloning and analysis of FvMIR399a promoter fragment First, the precursor sequence of miR399a was blasted in the woodland strawberry genome database (GDR :https://www.rosaceae. org/gb/gbrowse/fragaria_vesca_v1.0/). Next, a primer pair was designed according this presumptive sequence. And a 1440 bp fragment in the promoter of FvMIR399a was amplified from the genome of ‘Ruegen’. The obtained PCR products were cloned into pMD18-T (TaKaRa, Dalian, China). Finally, the promoter sequence of FvMIR399a was identified by sequencing at least three replicates of each cloning. The promoter sequence of FvMIR399a was shown in Supplementary Fig. 1. Several cis-acting elements were found by using the plantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/).
2.3. Cloning FvPHR1 gene We designed the primer pairs (shown in Supplementary Table S1) using the sequence of FvPHR1 (FvH4_2g20730.1) as a template. Total RNA was isolated from the leaves of ‘Ruegen’ using improved CTAB protocol [30] and cDNA was synthesized by using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Full-length coding sequence of FvPHR1 was amplified from the cDNA. The reactions was performed using the following condition: 94 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min 30 s, and a final extension at 72 °C for 10 min. The PCR products were purified with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), subcloned into a pMD18-T Vector (TaKaRa, Dalian, China), and sequenced.
2.9. Yeast one-hybrid assay The FvPHR1 coding sequence was ligated into the pB42 AD vector (pB42 AD-FvPHR1). Six fragment of the FvMIR399a promoter were amplified and fused with pLacZi vector (pLacZi-P1, pLacZi-P2, pLacZiP3, pLacZi-P4, pLacZi-P5, pLacZi-P6). These primer sets were shown in Supplementary Table S1. The vectors containing FvPHR1 coding sequence and a fragment of the FvMIR399a promoter were co-transformed into the yeast strain EGY48 according the Yeast Protocols Handbook (Clontech, Palo Alto, CA, USA). The transformants were grown on SD/- Trp/-Ura for 48 h, then transferred onto the plates
2.4. Sequence alignment and phylogenetic analysis The amino acid sequences of PHR1s were obtained from the NCBI according their accession number as reported (AT4G28610: AtPHR1, OS03g21240: OsPHR1, OS07G25710: OsPHR2, OS02G04640: OsPHR3, 259
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Fig. 1. Phylogenetic tree of MYB-CC genes from strawberry and Arabidopsis. Amino acid sequences of 15 Arabidopsis and 13 strawberry MYB-CC were aligned using MEGA 6.06 software with the Neighbor–Joining method. The Bootstrap value was 1000 replicates.
FvMIR399a promoter regions, and the third and fourth P1BS were named P1BS-3 and P1BS-4. The DNA oligonucleotide probes containing P1BS-3 and P1BS-4 motif were synthesized and labeled with biotin (Sangon, Shanghai, China), and the unlabeled sequence used as competitors. The biotin-labeled double-stranded DNA probes and unlabeled competitors were acquired by annealing complementary oligonucleotides. The EMSA was performed using the chemiliminescent EMSA Kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions.
containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) for blue color development. 2.10. Electrophoretic mobility shift assay (EMSA) To obtain MBP-FvPHR1 fusion protein, the full-length CDS of FvPHR1 was amplified using the primer pairs FvPHR1-MBPF (EcoRI)/ FvPHR1-MBPR (SalI) and cloned into the pMAL-c2X vector, and transformed the recombinant plasmid into Escherichia coli strain Rostta (DE3) (TransGen Biotech, Beijing, China). The transformant was grown in LB medium containing 60 mg/L ampicillin on rotary shaker at 37℃ until OD600 = 0.5 was reached. And then isopropyl β-D-1-thiogalactopyranoside (IPTG: 0.1 mM final concentration) was added to induce MBP-FvPHR1 fusion protein expression and the cells were cultured for 16–20 h at 16℃ on an orbital shaker. MBP-FvPHR1 fusion protein was purified using amylose resin (NEB, Beijng, China) as described previously [35]. The primer pairs were listed in Supplementary Table S1. For the EMSA assay, MBP-FvPHR1 fusion protein was expressed and purified as described above. There were four P1BS cis-elements in the
2.11. Overexpression vector construction and strawberry transformation First, full-length coding sequence of FvPHR1 was amplified using primers with NdeI and EcoRI restriction sites and cloned into pRI101AN vector. Primer pairs were listed in Supplementary Table S1. The recombinant plasmid named as pRI101-FvPHR1 was driven by the CaMV 35S promoter. Next, we transferred the recombinant plasmid into EHA105. And then ‘Ruegen’ strawberry was transformed by using Agrobacterium-mediated strawberry transformation procedure [36]. 260
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Fig. 2. Sequence alignment, domain architecture, and phylogenetic analysis of FvPHR1. A. Amino acid sequences alignment of FvPHR1 and its homologs in Arabidopsis and rice. MYB (red rectangle) and CC domain (blue rectangle) were conserved among FvPHR1 and its homologs. B. MYB and CC domain architecture in FvPHR1. FvPHR1 contained a MYB domain (amino acids 292–345) and CC domain (amino acids 382–422) on its C-terminal side. C. Phylogenetic tree of FvPHR1 with other related proteins from different plants. The phylogenetic tree was constructed with MEGA6.06 using 1000 bootstrap replicates. Scale bar: 0.1 substitutions per site (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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To explore the expression patterns of strawberry MYB-CC members in response to Pi starvation, the expression profile of all 13 strawberry MYB-CC genes in leaves and roots under different Pi starvation times was determined by qRT-PCR analysis. As shown in Supplementary Fig. 2, the expression of FvH4_6g40170.1 was up-regulated at 6 h and 12 h of Pi starvation in strawberry leaves. In roots, the expression of FvH4_4g31230.1 was up-regulated at 6 h and 12 h of Pi starvation. And, the transcript level of FvPHR1 is not responsive to Pi deprivation stress in strawberry leaves.
2.12. PCR confirmation of transgenic plants In order to confirm the positive transgenic plants, we isolated the total genomic DNA from putative transgenic and wild-type plantlets using an improved CTAB protocol [37], then PCR analysis was performed using a primer set 35S/FvPHR1-404R (Supplementary Table S1), the 35S was part sequence of the CaMV 35S promoter from pRI101AN vector, and the FvPHR1-404R was located the upstream of the FvPHR1 coding sequence at 424 bp. The amplicon size of this primer set was 570 bp. The PCR amplification was performed as follows: 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, with a final extension for 10 min at 72 °C. Negative control was the DNA from a non-transformed plant.
3.3. Identification of the transcription factor FvPHR1 in strawberry The coding sequence of FvPHR1 gene is 1542 bp, and encoded a predicted protein of 513 amino acids. The protein has a calculated molecular weight of 56.22 kDa and an isoelectric point of 5.7. Previous reports indicate that PHR1 belongs to a large protein family with a conserved MYB DNA-binding domain and a coiled coil domain [7]. Multiple sequence alignment of FvPHR1 with AtPHR1, OsPHR1, OsPHR2, OsPHR3 and OsPHR4 shows that FvPHR1 is a novel member of the MYB-CC family with high conservation in the domains (Fig. 2A). As shown in Fig. 2B, FvPHR1 contains MYB-domain and CC-domain in amino acids 292–345 and 382–422, respectively. Phylogenetic analysis of FvPHR1 and PHR1s from other plants was performed with NeighborJoining Method. Fig. 2C shows that FvPHR1 was closely related to these PHR1s of Rosaceae fruit trees (Prunus persica, P. mume, P. avium, Malus domestica and Pyrus bretschneideri). It is consistent with the result of plant evolution. In the model plant Arabidopsis and rice, PHR1 plays a significant role in Pi-starvation signaling and Pi-homeostasis [7,31–33]. So it was indicated that the new member of the family, FvPHR1, may also play a role in Pi-starvation signaling. The sequence of FvPHR1 was remarkably similar to AtPHR1, OsPHR1, OsPHR2 and OsPHR4 (Fig. 2A). It was reported that AtPHR1, OsPHR1, OsPHR2 and OsPHR4 were nuclear-localized proteins [7,31–33]. To determine whether FvPHR1 located in the nucleus like other PHR1s, the FvPHR1 coding sequence was fused to the N-terminal end of GFP and driven by CaMV 35S promoter. FvPHR1-GFP was introduced into rice protoplasts to determine the Sub-cellular localization of FvPHR1. As shown in Fig. 3A, the FvPHR1-GFP fusion protein was found in the nucleus, whereas the GFP signal of positive control was distributed throughout the entire cell. These results suggested that FvPHR1 was a nuclear protein. To determine the transcription activation of FvPHR1, a GAL4-responsive reporter system was performed in yeast cells. The full-length fragment of FvPHR1 was fused to the GAL4 DNA binding domain to create the pGBKT7-FvPHR1 construct and transformed to yeast strain AH109. The empty vector pGBKT7 was used as negative control. As shown in Fig. 3B, the yeast strain AH109 contained pGBKT7-FvPHR1 grew well in SD/-Trp and SD/-Trp-His-Ade. However, the yeast strain AH109 which contained pGBKT7 only grew in SD/-Trp. These results indicated that the FvPHR1 was a transcriptional activator. As predicted, FvPHR1 protein contained a CC-domain, which usually formed a homodimer by its helix-loop-helix structure [38]. Here, BiFC assay was used to explore whether FvPHR1 formed a homodimer in vivo. As shown in Fig. 3C, there was a strong YFP signaling in nucleus of the tobacco leaves transformed with the eYNEFvPHR1 and eYCE-FvPHR1 vectors. Whereas, there was no YFP signaling in the cell of tobacco leaves coexpressing eYNE/eYCE-FvPHR1, eYNE-FvPHR1/eYCE or eYNE/eYCE. These results suggested that FvPHR1 formed a homodimer in vivo and was located in nucleus. Together, these observations suggested that FvPHR1 worked as a transcription factor in woodland strawberry.
2.13. Gene expression analysis Gene expression level was analyzed by quantitative RT-PCR (qRTPCR). For the detection of all strawberry MYB-CC genes, FvPHR1, preFvmiR399a, pre-FvmiR399b and pre-FvmiR399c, cDNA was synthesized using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Quantitative PCR was performed with SYBR Premix Ex TaqII (TaKaRa, Dalian, China) using Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The specific stem-loop reverse primer of FvmiR399a was used for the mature FvmiR399a synthesis. The expression level of mature miR399a was detected by a TaqMan PCR using the method described previously [29]. The relative expression levels of genes were calculated with 2−ΔΔCT method. The strawberry 26S rRNA gene was used as the reference gene. Specific primers were listed in Supplementary Table S1. qRT-PCR was performed with three biological replicates. 2.14. Measurement of P content The P content in the leaves of wild type (WT) and transgenic plants was determined using the Mo-Sb colorimetric method as described previously [29]. 3. Results 3.1. Genome-wide identification of strawberry MYB-CC family genes in woodland strawberry We identified 26 putative strawberry MYB-CC genes by searching on the PLAZA 3.0 Dicots, and then excluded some predicted protein sequences without a MYB-CC domain using NCBI’s CDD. Subsequently, we acquired the information of these predicted strawberry MYB-CC genes from GDR, such as GDR number, chromosomal location and the number of amino acid. As Table S2, a total of 13 MYB-CC genes were identified in the woodland strawberry (F. vesca) genome. These MYBCC proteins ranged from 273 (FvH4_6g22540.1) to 513 (FvH4_2g20730.1) amino acids in length. The predicted molecular weights of FvMYB-CC proteins ranged from 30.71 (FvH4_6g22540.1) to 56.21 (FvH4_2g20730.1) KDa and the isoelectric points ranged from 5.05 (FvH4_6g01670.1) to 8.76 (FvH4_1g29370.1). 3.2. Phylogenetic and expression patterns analysis of strawberry MYB-CC genes We acquired 13 and 15 MYB-CC genes which contain MYB-CC domain from F. vesca and A. thaliana, respectively. In order to analyze evolutionary relationships between the MYB-CC members in strawberry and Arabidopsis, phylogenetic analysis was performed using MEGA 6.06 software with the Neighbor–Joining method. As shown in Fig. 1, we found there was a very close homology between AtPHR1 (AT4G28610) and strawberry MYB-CC gene FvH4_2g20730.1. Therefore, we named FvH4_2g20730.1 as FvPHR1.
3.4. Expression patterns of the FvPHR1 in different organs of woodland strawberry In order to investigate the spatial expression pattern of FvPHR1 gene 262
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Fig. 3. Transcription factor characteristics of FvPHR1. A. Subcellular localization of FvPHR1.The vectors of FvPHR1-GFP and GFP positive control were infiltrated into rice protoplast, respectively. OsMADS3-mCherry as a nuclear marker. Scale bars = 4 μm. B. Transcriptional activities of the FvPHR1 gene. The transformed yeast strain with constructs of FvPHR1-BD and BD empty (pGBKT7) were grown on SD/-Trp and SD/-Trp/-His/-Ade media, respectively. C. The homodimer of FvPHR1 in vivo. The eYFP and m-Cherry fluorescence signaling was observed in tobacco leaves. There was a protein–protein interaction between FvPHR1 and itself.
pattern of FvmiR399a was similar to FvPHR1, higher in flowers, leaves and roots and lower in green fruits [29].
in strawberry, qRT-PCR analysis was used to measure that the expression of FvPHR1 in roots, stems, old leaves, young leaves, petioles, flowers, different stages fruits (green fruits, white fruits, turn fruits, red fruits) of woodland strawberry. As shown in Fig. 4, the FvPHR1 was expressed in all organs, with higher expression level in roots, old leaves, and lower in green fruits. Interestingly, we found that the expression
3.5. FvPHR1 binds to the promoter of FvMIR399a PHR1 acts as a central regulator in Pi starvation signaling by 263
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into five parts and assayed with FvPHR1 protein. The results indicated that FvPHR1 could bind to P3 of FvMIR399a promoter containing two P1BS (Fig. 5B). As shown in Fig. 5B, two P1BS motif were found in the P3 of FvMIR399a promoter. Here, to identify which P1BS was essential for the binding of FvPHR1, EMSA was performed. The biotin-labeled probe sequence containing P1BS-3 and P1BS-4 motif were shown in Fig. 6A. The EMSA results indicated that FvPHR1 was able to bind to P1BS1-3 and P1BS-4 biotin-labeled probes (Fig. 6B). Furthermore, non-labeled competitive probe could specifically compete with the biotin-labeled probe in binding to FvPHR1. These results suggested that FvPHR1 could bind to both the P1BS-3 and P1BS-4 motif in the promoter of FvMIR399a. Fig. 4. Expression patterns of FvPHR1 in different organs revealed by qRT-PCR. Errors bars were acquired from three biological repeats.
3.6. Overexpression of FvPHR1 increases P content in the woodland strawberry
regulating some PSI genes in Arabidopsis [7]. In addition, previous study has shown that the overexpression of miR399a could improve the P content in the leaves of the woodland strawberry [29]. So, we hypothesized that FvPHR1 might mediate the change of P content in woodland strawberry by regulate the expression of miR399a. To determine whether PHR1 could involve in the regulation of Pi starvation response by binding to the promoter of FvMIR399a gene, the promoter of FvMIR399a was analyzed. As shown in Supplementary Table S3, these cis-acting elements included some responsive to light, abscisic acid, heat stress, drought, salicylic acid, defense and stress. In addition, 4 PHR1-binding sites (P1BS: GNATATNC) were found in the FvMIR399a promoter region. Yeast one-hybrid assay showed that FvPHR1 protein could bind to the FvMIR399a promoter (Fig. 5A). As shown in supplementary Fig. 1, there were 4 P1BS in the FvMIR399a promoter. To identify the position that FvPHR1 could bind to, the promoter of FvMIR399a was divided
To investigate the function of FvPHR1 in strawberry, we developed FvPHR1 overexpression plants of woodland strawberry ‘Ruegen’ by transformation with the construct pRI101-FvPHR1 (Fig. 7A). PCR analysis was performed to detect transgenic plantlets using the specific primer set (35S and FvPHR1-424R), a 570 bp band was amplified from genomic DNA of three independent transgenic lines and no corresponding bands was found in non-transformed WT plants (Fig. 7B). To further confirm the three transgenic lines, qRT-PCR was performed to determine the transcript level of FvPHR1. As shown in Fig. 7C, the expression of FvPHR1 was significantly increased in these transgenic lines. To further verified the relationship between FvPHR1 and FvmiR399 in strawberry. The expression of pre-FvmiR399a, pre-FvmiR399b, preFvmiR399c and mature FvmiR399a was detected in FvPHR1-overexpressing and WT plants by qRT-PCR [39]. As shown in Fig. 8, preFvmiR399a, pre-FvmiR399b and pre-FvmiR399c were significantly upregulated in leaves of transgenic lines as compared with the WT. And the expression of mature FvmiR399a was increased by 11.0-fold to 59.6-fold in FvPHR1-overexpressing lines compared with the WT. Therefore, these results indicated that FvPHR1 positively regulates the expression of FvmiR399 in woodland strawberry. Our previous research showed that the overexpression of miR399a could increase the P content in woodland strawberry leaves [29]. So we measured the P content in the leaves of WT and FvPHR1-overexpressing woodland strawberry plants under Pi-sufficient conditions. As shown in Fig. 9, the P contents in FvPHR1-overexpressing lines were significantly increased by 1.38-fold to 1.78-fold compared with that in WT. Thus, these results suggested that the overexpression of FvPHR1 could increase P content in the woodland strawberry. 4. Discussion P is an essential macronutrient to sustain the normal plant growth and development. Plants make a series of response under Pi starvation. Meanwhile, a complicated Pi signaling network is formed in plants. Although Pi starvation signaling is well studied in Arabidopsis and rice, it is still largely unknown in strawberry. In strawberry, Pi plays a significant role in fruit quality [28,40,41]. Thus, to further understand the Pi starvation signaling in strawberry, it is important to identify the key factor in the network. In Arabidopsis and rice, PHR1 acts as a central transcriptional activator regulating PSI genes in Pi starvation response [7,42]. Here, we identified a MYB-CC family gene FvPHR1, as a key transcription factor involved in the regulation of phosphate-signaling in woodland strawberry. Like the PHR1 in Arabidopsis, rice and maize [7,12,31], FvPHR1 is located in cell nucleus. TaPHR1 forms a homodimer to activate the expression of Pi transporter TaPHT1.2 in wheat [43]. In this study, we also found that FvPHR1 would work by forming a homodimer (Fig. 3C), which suggested that FvPHR1 may regulate the expression of Pi
Fig. 5. Yeast one-hybrid assay showing that FvPHR1 binds to the promoter of FvMIR399a. A. pB42 AD-FvPHR1 represented FvPHR1 coding sequence was inserted into pB42 AD as an effector. pLacZi-FvMIR399apro represented the FvMIR399a promoter (1440 bp) fused with LacZ as a reporter. The empty vectors pLacZi and pB42 AD were used as negative control. B. The interaction between pB42 AD and pB42 AD-FvPHR1 with the different fragments of the FvMIR399a promoter. The black boxes represented the four P1BS. 264
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Fig. 6. EMSA assay showing that FvPHR1 bind directly to the FvMIR399a promoter. A. Diagram showing the P1BS element in FvMIR399a promoter used for EMSA. Two biotin-labeled probe sequences containing PIBS-3 and P1BS-4 motif are shown and the P1BS motif are highlighted in bold. B. EMSA results showing that FvPHR1 specifically bind to the P1BS-3 and P1BS-4 motif in FvMIR399a promoter. Biotin-labeled sequences containing PIBS-3 and P1BS-4 motif are used as hot probes, while non-labeled sequences are used as cold probe (100-fold higher concentration than that of biotin-labeled probe), respectively. MBP-FvPHR1 fusion protein was purified from Escherichia coli Rostta (DE3) and used for the DNAbinding assays.
Fig. 7. Molecular confirmation of FvPHR1 overexpressing transgenic lines of woodland strawberry. A. Structure diagram of FvPHR1 overexpressing construct pRI101-FvPHR1, the FvPHR1 gene was driven by CaMV35S promoter. B. PCR amplification of the specific fragment (570bp) from the genomic DNA of FvPHR1 transgenic lines, lanes from left to right: DL2000 marker, FvPHR1 transgenic lines OE#5, #6, #10 and wild type (WT) plants. C. Transcript level of FvPHR1 was examined in three independent positive transgenic lines and WT plants by qRT–PCR. Vertical bars represent the SD (n = 3).
condition through the post-translational modification. In addition, we found FvPHR1 positively regulates the expression of FvMIR399a through directly binding to its promoter, and our previous study showed that the overexpression of miR399a increased the P content in woodland strawberry [29], so we infer that FvPHR1 could control P content through positively regulate the expression of FvMIR399. The results in this study indicate that the PHR1-miR399 signaling pathway is conserved among plant species, and this pathway is important for modulating P homeostasis in strawberry as it is in Arabidopsis and rice. Although Pi signaling is very important for plant growth, it does not work alone. During the regulation of Pi homeostasis in plants, the Pi starvation response is associated with other signaling pathways, including sugar and hormone-mediated signaling pathways [46–53].
starvation induced genes by the homodimer. The overexpression of FvPHR1 can increase the P content in woodland strawberry. This result is similar to the previous reports, which the overexpression of AtPHR1, OsPHR1/2/3/4, TaPHR1, ZmPHR1 and BnPHR1 enhanced Pi accumulation in shoot [12,31–3343–45]. It was reported that the transcript levels of OsPHR3, OsPHR4, BnPHR1 and PvPHR1 were increased by Pi starvation in leaves and roots [11,33,45]. And under low Pi treatment, the expression level of ZmPHR1 was up-regulated by Pi starvation in roots of maize, while down-regualted in leaves [12]. While, under Pistarvation condition, the transcript level of FvPHR1 was not significantly changed in leaves of woodland strawberry. These results are similar to that of AtPHR1, AtPHL1, OsPHR1 and OsPHR2 [7,16,31]. But it is speculated that PHR1 may become more active under Pi-starvation 265
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Fig. 8. The expression levels of pre-FvmiR399a (A), pre-FvmiR399b (B), pre-FvmiR399c (C) and mature FvmiR399a (D) in FvPHR1-OE lines and WT plants of woodland strawberry. Vertical bars represent the SD (n = 3).
Fig. 9. The P content in FvPHR1-OE lines and WT plants of woodland strawberry. Vertical bars represent the SD (n = 3). Different lowercase letters above the bars indicate significant difference compared with the WT (n = 3, P < 0.05).
Recently, research showed that light and ethylene coordinately regulate PSRs through regulating the PHR1 expression (Fig. 10) [54]. Besides, PHR1, a potential general integrator, regulates the expression of some genes involved in P, S, Fe, Zn homoeostasis and transport [55]. In rice, OsmiR399 as a key miRNA involved in Pi starvation response [15]. It was reported that OsmiR399 also takes part in the regulation of Na, K, Ca, Fe starvation responses [19]. In A. thaliana, the miR399-PHO2 module involved in the regulation of ambient temperature-responsive flowering [56]. Further, there was a link between Pi homeostasis and flowering time regulation by confirming an interaction between OsPHO2 and OsGI in rice [57]. In summary, a hypothetical model for phosphate-signaling pathway in woodland strawberry was proposed by combining our results and previous reported in model plant (Fig. 10). Here, we identify the PHR1miR399 module control phosphate homeostasis in woodland
Fig. 10. A hypothetical model for the role of the PHR1-miR399 module in woodland strawberry. The network marked in red indicate the results in this work. Other pathways indicate the results have reported in Arabidopsis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
strawberry plants. Our research provided theoretical basis for the study of FvPHR1 function in Pi signaling. Besides, in order to improve the low P tolerance ability in strawberry by agronomic strategies, the crosstalk between Pi signaling and other signaling pathway should be explored in the future.
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Conflict of interest
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The authors declare no conflict of interest. Contributions YW and FZ conceived and designed the experiments. YW and FZ contributed to the experimental work. WC, RZ and KC contributed to coordinated the study. YW wrote manuscript. ZZ helped perform the analysis with constructive discussions and approved the final version. All the authors read and approved the final manuscript. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (31872072) and the Key R&D and Technology Transfer Program (Z17-0-035) Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2018.12.025. References [1] D.P. Schachtman, R.J. Reid, S.M. Ayling, Phosphorus uptake by plants: from soil to cell, Plant Physiol. 116 (1998) 447–453. [2] I.C.R. Holford, Soil phosphorus: its measurement, and its uptake by plants, Aust. J. Soil Res. 35 (1997) 227–240. [3] T.R. Bates, J.P. Lynch, Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability, Plant Cell Environ. 19 (1996) 529–538. [4] B.I. Linkohr, L.C. Williamson, A.H. Fitter, H.M. Leyser, Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis, Plant J. 29 (2002) 751–760. [5] L. Cheng, B. Bucciarelli, J. Shen, D. Allan, C.P. Vance, Update on white lupin cluster root acclimation to phosphorus deficiency, Plant Physiol. 156 (2011) 1025–1032. [6] X.F. Ma, E. Wright, Y. Ge, J. Bell, Y. Xi, J.H. Bouton, Z.Y. Wang, Improving phosphorus acquisition of white clover (Trifolium repens L.) by transgenic expression of plant-derived phytase and acid phosphatase genes, Plant Sci. 176 (2009) 479–488. [7] V. Rubio, F. Linhares, R. Solano, A.C. Martín, J. Iglesias, A. Leyva, J. Paz-Ares, A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plant and unicellular algae, Gene Dev. 15 (2001) 2122–2133. [8] B.N. Devaiah, A.S. Karthikeyan, K.G. Raghothama, WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis, Plant Physiol. 143 (2007) 1789–1801. [9] B.N. Devaiah, V.K. Nagarajna, K.G. Raghothama, Phosphate homeostasis and root development in Arabidopsis are synchronized by the zing finger transcription factor ZAT6, Plant Physiol. 145 (2007) 147–159. [10] Z.H. Chen, G.A. Nimmo, G.I. Jenkins, H.G. Nimmo, BHLH32 modulates several biochemical and morphological process that respond to Pi starvation in Arabidopsis, Biochem. J. 405 (2007) 191–198. [11] O. Valdés-López, C. Arenas-Huertero, M. Ramírez, L. Girard, F. Sánchez, C.P. Vance, J. Luis Reyes, G. Hernández, Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deficiency signalling in common bean roots, Plant Cell Environ. 31 (2008) 1834–1843. [12] X.H. Wang, J.R. Bai, H.M. Liu, Y. Sun, X.Y. Shi, Z.Q. Ren, Overexpression of a Maize transcription factor ZmPHR1 improves shoot inorganic phosphate content and growth of Arabidopsis under low-phosphate conditions, Plant Mol. Biol. Rep. 31 (2013) 665–677. [13] T.J. Chiou, S.I. Lin, Signaling network in sensing phosphate availability in plants, Annu. Rev. Plant Biol. 62 (2011) 185–206. [14] K. Miura, A. Rus, A. Sharkhuu, S. Yokoi, A.S. Karthikeyan, K.G. Raghothama, D. Baek, Y.D. Koo, J.B. Jin, R.A. Bressan, D.J. Yun, P.M. Hasegawa, The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses, P. Natl. Acad. Sci. U. S. A. 102 (2005) 7760–7765. [15] R. Bari, B.D. Pant, M. Stitt, W.R. Scheible, PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants, Plant Physiol. 141 (2006) 988–999. [16] R. Bustos, G. Castrillo, F. Linhares, M.I. Puga, V. Rubio, J. Pérez-Pérez, R. Solano, A. Leyva, J. Paz-Ares, A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis, PLoS Genet. 6 (2010) e1001102. [17] S. Okumura, N. Mitsukawa, Y. Sgurano, D. Shibata, Phosphate transporter gene family of Arabidopsis thaliana, DNA Res. 5 (1998) 261–269. [18] H. Fujii, T.J. Chiou, S.I. Lin, K. Aung, J.K. Zhu, A miRNA involved in phosphatestarvation response in Arabidopsis, Curr. Biol. 15 (2005) 2038–2043. [19] B. Hu, W. Wang, K. Deng, H. Li, Z.H. Zhang, L.H. Zhang, C.C. Chu, MicroRNA399 is involved in multiple nutrient starvation responses in rice, Front. Plant Sci. 6 (2015) 188.
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The FvPHR1 transcription factor control phosphate homeostasis by transcriptionally regulating miR399a in woodland strawberry Yan Wang1, Feng Zhang1, Weixu Cui, Keqin Chen, Rui Zhao, Zhihong Zhang
T
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Liaoning Key Laboratory of Strawberry Breeding and Cultivation, College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Woodland strawberry Pi-signaling FvPHR1 miR399 P content
Plants have evolved phosphate (Pi) starvation response to adapt the low-Pi environment. The regulation of adaptive responses to phosphorus deficiency by the PHR1-miR399-PHO2 module has been well studied in Arabidopsis thaliana but not in strawberry. Transcription factor PHR1 as the central regulator in the Pi starvation signaling has been revealed in a few plant species. However, the function of PHR1 homologues in strawberry is still unknown. In this study, a total of 13 MYB-CC genes were identified in the woodland strawberry (Fragaria vesca) genome and the FvPHR1 gene was characterized. FvPHR1 contains MYB domain and coiled–coil (CC) domain and is localized in the nucleus. FvPHR1 also exhibits trans-activation ability. Furthermore, the P content in leaves of FvPHR1-overexpressing woodland strawberries was significantly increased by 1.38-fold to 1.78-fold compared with that in the wild type. FvPHR1 was also demonstrated to directly bind to the FvMIR399a promoter and positively regulate the expression of FvmiR399a in woodland strawberry. These results showed that PHR1miR399 module is involved in the regulation of phosphate-signaling pathway and phosphate homeostasis in woodland strawberry.
1. Introduction Phosphorus (P), an essential macronutrient, plays a key role in the normal growth and development of plants. Plants uptake P from soil by roots [1]. In nature, P usually exists as the form of phosphate minerals, but there is a little available P for plants absorption because it is very stable or insoluble [2]. In order to adapt the low phosphate (Pi) environment, plants have evolved a number of strategies to regulate Pi homeostasis in Pi starvation, for example, the alteration of root morphology and structure [3,4], organic acid exudation [5], secretion of phosphatases [6], and upregulated expression of phosphate-starvationresponse (PSR) genes that were controlled by some transcript factors (TFs), including PHR1 (phosphate starvation response 1), WRKY75, ZAT6 and BHLH32 [7–10]. PHR1, as the central regulator in the phosphate starvation signaling [11–13], contains a MYB domain and a coiled–coil (CC) domain, and it was termed a MYB-CC protein by Rubio et al [7]. Under Pi starvation condition, PHR1 is regulated by the small ubiquitin-like modifier (SUMO E3) ligase SIZ1 at post-translational level [14]. PHR1 could bind to a cis-element of PHR1-binding sequences (P1BS: GNATATNC) in the promoter of some Pi starvation-induced (PSI) genes to regulate the expression of these genes, including several PHT1 genes and MIR399
[7,15–17]. MicroRNA399 (miR399) plays a key role in Pi-starvation signaling network in plants [18,19]. It is reported that miR399 could direct the cleavage of PHO2 mRNA in some species [15,20,21]. PHO2, an ubiquitin-conjugating E2 enzyme (UBC24), negatively regulates Pi transporters to inhibit Pi uptake and root-to-shoot translocation [15,22]. Although some components of Pi starvation signaling have been identified in plants, the overall pathway is still poorly understood. Furthermore, it was reported that AtNIGT1/HRS1 played an important role in the integration of nitrate and phosphate signals in Arabidopsis [23]. Besides, in Pi-deficient and NO3−-sufficient condition, N uptake was decreased because the PHR1 can positively regulate the expression of NIGT1 family genes in Arabidopsis, and these NIGT1s are negative regulators of NRT2.1 (a typical nitrate-inducible gene) [24]. So PHR1 also plays a significantly part in phosphorus and nitrogen nutritional regulation. Strawberry is one of the most popular fresh and processed fruits with pleasant flavor and nutritional value [25]. The growth of strawberry plants was influenced by temperature, light, water, salinity and nutrient [26]. Pi is a necessary nutrient element for strawberry plants propagation, health and vigor [27]. Thus, the study of Pi signaling in strawberry plants is crucial for strawberry production. In previous study, we reported that soluble solids content is positively correlated
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Corresponding author. E-mail address: [email protected] (Z. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.plantsci.2018.12.025 Received 16 July 2018; Received in revised form 19 December 2018; Accepted 24 December 2018 Available online 28 December 2018 0168-9452/ © 2019 Elsevier B.V. All rights reserved.
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OS06G49040: OsPHR4) [7,31–33], and performing the Protein BLAST research using the amino acid sequence of FvPHR1 as a query (PHR1s from other species). MEGA 6.06 and GeneDoc program were used in multiple sequence alignment. The phylogenetic tree was constructed using MEGA 6.06 software with the Neighbor–Joining method.
with P content in ripening strawberry fruits [28], and overexpressing miR399a in strawberry improved the fruit quality [29]. However, the TFs involved in Pi homeostasis in strawberry plants are largely unknown. In this study, 13 MYB-CC genes were identified in woodland strawberry (Fragaria vesca) genome. Further analysis showed that FvPHR1 worked as a transcription factor in woodland strawberry. And we found that the overexpression of FvPHR1 could increase the P content in woodland strawberry. In addition, FvPHR1 could positively regulate the expression of FvmiR399. The functional analysis of FvPHR1 suggests that it plays a significant role in Pi homeostasis of woodland strawberry. This study will provide a basis for illustrating the molecular regulation mechanism of P signaling network in strawberry.
2.5. Sub-cellular localization The coding sequence of FvPHR1 without the stop codon was fused to green fluorescent protein (GFP) gene, and cloned into the pAN580 vector controlled by the cauliflower mosaic virus (CaMV) 35S promoter. The primer set was shown in Supplementary Table S1. And OsMADS3-mCherry was used as a nuclear marker. We refer to the process of rice protoplast isolation and transformation as previously described [34]. GFP fluorescence was observed with Zeiss LSM700 confocal microscopy. The emission filters were 500–530 nm for GFP, 580–620 nm for mCherry.
2. Materials and methods 2.1. Plant materials and growth conditions Fifty-day-old seedlings of woodland strawberry (Fragaria vesca) accession ‘Ruegen’ were grown in pots filled with washed sand: vermiculite: perlite (2:1:1). And we watered nutrient solution (1 mM KH2PO4) and as described previously for 10 day [29]. The nutrient solution was watered every other day. Then the seedlings were subjected to Pi-deficient (0.01 mM KH2PO4) condition, and treated for 0, 6, 12, 24, 72, 120 h. After that, the leaves and roots of these seedlings were separately harvested for analysis of gene expression.
2.6. Transcriptional activation analysis in yeast cells Transactivation activity assay of FvPHR1 was performed by using Matchmaker GAL4 Two-Hybrid System 3 (Clontech, Palo Alto, CA, USA). The coding region of FvPHR1 was inserted into the pGBKT7 vector. Primers were listed in Supplementary Table S1. The pGBKT7FvPHR1 and the negative control pGBKT7 constructs were transformed into yeast strain AH109, respectively. The transformed strains were grown on the medium of SD/−Trp and selected on the medium of SD/ −Trp−His−Ade.
2.2. Identification of MYB-CC family genes in woodland strawberry
2.7. Bimolecular fluorescence complementation (BiFC) assay
To identify the MYB-CC family genes in woodland strawberry, first, we performed search program with search value “PHR1” and default parameters on PLAZA website (https://bioinformatics.psb.ugent.be/ plaza/versions/plaza_v3_dicots/basic_search/mobile_search) and download the profile named HOM03D000096 protein sequences. Next, the putative MYB-CC protein sequences from Arabidopsis thaliana and F. vesca were extracted and manually verified by NCBI Conserved Domain Database (CDD, http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi/) and SMART (http://smart.embl-heidelberg.de/) to confirm they contained a MYB-CC domain. Then, we acquired the information of strawberry MYB-CC genes from the Strawberry Genome v4.0a1 marker standard proteins databases in Genome Database for Rosaceae (GDR) by performing protein BLAST search (https://www.rosaceae.org/blast/ protein/protein). The theoretical molecular weight and isoelectric point of deductive MYB-CC proteins were predicted using ExPASy Proteomics Server (http://web.expasy.org/compute_pi/).
The coding sequence of FvPHR1 was cloned into pSPYNE173 (eYNE) and pSPYCE (M) (eYCE) vector to generate constructs of eYNE-FvPHR1 and eYCE-FvPHR1, respectively. The primers used for BiFC assays were shown in Supplementary Table S1. Agrobacterium tumefaciens strains EHA105 carrying the recombinant plasmids and ER marker were coinfiltrated into 5-week-old tobacco (Nicotiana benthamiana) leaves. The eYFP fluorescence signaling was observed with Zeiss LSM700 confocal microscopy at 72 h after infiltration. 2.8. Cloning and analysis of FvMIR399a promoter fragment First, the precursor sequence of miR399a was blasted in the woodland strawberry genome database (GDR :https://www.rosaceae. org/gb/gbrowse/fragaria_vesca_v1.0/). Next, a primer pair was designed according this presumptive sequence. And a 1440 bp fragment in the promoter of FvMIR399a was amplified from the genome of ‘Ruegen’. The obtained PCR products were cloned into pMD18-T (TaKaRa, Dalian, China). Finally, the promoter sequence of FvMIR399a was identified by sequencing at least three replicates of each cloning. The promoter sequence of FvMIR399a was shown in Supplementary Fig. 1. Several cis-acting elements were found by using the plantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/).
2.3. Cloning FvPHR1 gene We designed the primer pairs (shown in Supplementary Table S1) using the sequence of FvPHR1 (FvH4_2g20730.1) as a template. Total RNA was isolated from the leaves of ‘Ruegen’ using improved CTAB protocol [30] and cDNA was synthesized by using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Full-length coding sequence of FvPHR1 was amplified from the cDNA. The reactions was performed using the following condition: 94 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min 30 s, and a final extension at 72 °C for 10 min. The PCR products were purified with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), subcloned into a pMD18-T Vector (TaKaRa, Dalian, China), and sequenced.
2.9. Yeast one-hybrid assay The FvPHR1 coding sequence was ligated into the pB42 AD vector (pB42 AD-FvPHR1). Six fragment of the FvMIR399a promoter were amplified and fused with pLacZi vector (pLacZi-P1, pLacZi-P2, pLacZiP3, pLacZi-P4, pLacZi-P5, pLacZi-P6). These primer sets were shown in Supplementary Table S1. The vectors containing FvPHR1 coding sequence and a fragment of the FvMIR399a promoter were co-transformed into the yeast strain EGY48 according the Yeast Protocols Handbook (Clontech, Palo Alto, CA, USA). The transformants were grown on SD/- Trp/-Ura for 48 h, then transferred onto the plates
2.4. Sequence alignment and phylogenetic analysis The amino acid sequences of PHR1s were obtained from the NCBI according their accession number as reported (AT4G28610: AtPHR1, OS03g21240: OsPHR1, OS07G25710: OsPHR2, OS02G04640: OsPHR3, 259
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Fig. 1. Phylogenetic tree of MYB-CC genes from strawberry and Arabidopsis. Amino acid sequences of 15 Arabidopsis and 13 strawberry MYB-CC were aligned using MEGA 6.06 software with the Neighbor–Joining method. The Bootstrap value was 1000 replicates.
FvMIR399a promoter regions, and the third and fourth P1BS were named P1BS-3 and P1BS-4. The DNA oligonucleotide probes containing P1BS-3 and P1BS-4 motif were synthesized and labeled with biotin (Sangon, Shanghai, China), and the unlabeled sequence used as competitors. The biotin-labeled double-stranded DNA probes and unlabeled competitors were acquired by annealing complementary oligonucleotides. The EMSA was performed using the chemiliminescent EMSA Kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions.
containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) for blue color development. 2.10. Electrophoretic mobility shift assay (EMSA) To obtain MBP-FvPHR1 fusion protein, the full-length CDS of FvPHR1 was amplified using the primer pairs FvPHR1-MBPF (EcoRI)/ FvPHR1-MBPR (SalI) and cloned into the pMAL-c2X vector, and transformed the recombinant plasmid into Escherichia coli strain Rostta (DE3) (TransGen Biotech, Beijing, China). The transformant was grown in LB medium containing 60 mg/L ampicillin on rotary shaker at 37℃ until OD600 = 0.5 was reached. And then isopropyl β-D-1-thiogalactopyranoside (IPTG: 0.1 mM final concentration) was added to induce MBP-FvPHR1 fusion protein expression and the cells were cultured for 16–20 h at 16℃ on an orbital shaker. MBP-FvPHR1 fusion protein was purified using amylose resin (NEB, Beijng, China) as described previously [35]. The primer pairs were listed in Supplementary Table S1. For the EMSA assay, MBP-FvPHR1 fusion protein was expressed and purified as described above. There were four P1BS cis-elements in the
2.11. Overexpression vector construction and strawberry transformation First, full-length coding sequence of FvPHR1 was amplified using primers with NdeI and EcoRI restriction sites and cloned into pRI101AN vector. Primer pairs were listed in Supplementary Table S1. The recombinant plasmid named as pRI101-FvPHR1 was driven by the CaMV 35S promoter. Next, we transferred the recombinant plasmid into EHA105. And then ‘Ruegen’ strawberry was transformed by using Agrobacterium-mediated strawberry transformation procedure [36]. 260
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Fig. 2. Sequence alignment, domain architecture, and phylogenetic analysis of FvPHR1. A. Amino acid sequences alignment of FvPHR1 and its homologs in Arabidopsis and rice. MYB (red rectangle) and CC domain (blue rectangle) were conserved among FvPHR1 and its homologs. B. MYB and CC domain architecture in FvPHR1. FvPHR1 contained a MYB domain (amino acids 292–345) and CC domain (amino acids 382–422) on its C-terminal side. C. Phylogenetic tree of FvPHR1 with other related proteins from different plants. The phylogenetic tree was constructed with MEGA6.06 using 1000 bootstrap replicates. Scale bar: 0.1 substitutions per site (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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To explore the expression patterns of strawberry MYB-CC members in response to Pi starvation, the expression profile of all 13 strawberry MYB-CC genes in leaves and roots under different Pi starvation times was determined by qRT-PCR analysis. As shown in Supplementary Fig. 2, the expression of FvH4_6g40170.1 was up-regulated at 6 h and 12 h of Pi starvation in strawberry leaves. In roots, the expression of FvH4_4g31230.1 was up-regulated at 6 h and 12 h of Pi starvation. And, the transcript level of FvPHR1 is not responsive to Pi deprivation stress in strawberry leaves.
2.12. PCR confirmation of transgenic plants In order to confirm the positive transgenic plants, we isolated the total genomic DNA from putative transgenic and wild-type plantlets using an improved CTAB protocol [37], then PCR analysis was performed using a primer set 35S/FvPHR1-404R (Supplementary Table S1), the 35S was part sequence of the CaMV 35S promoter from pRI101AN vector, and the FvPHR1-404R was located the upstream of the FvPHR1 coding sequence at 424 bp. The amplicon size of this primer set was 570 bp. The PCR amplification was performed as follows: 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, with a final extension for 10 min at 72 °C. Negative control was the DNA from a non-transformed plant.
3.3. Identification of the transcription factor FvPHR1 in strawberry The coding sequence of FvPHR1 gene is 1542 bp, and encoded a predicted protein of 513 amino acids. The protein has a calculated molecular weight of 56.22 kDa and an isoelectric point of 5.7. Previous reports indicate that PHR1 belongs to a large protein family with a conserved MYB DNA-binding domain and a coiled coil domain [7]. Multiple sequence alignment of FvPHR1 with AtPHR1, OsPHR1, OsPHR2, OsPHR3 and OsPHR4 shows that FvPHR1 is a novel member of the MYB-CC family with high conservation in the domains (Fig. 2A). As shown in Fig. 2B, FvPHR1 contains MYB-domain and CC-domain in amino acids 292–345 and 382–422, respectively. Phylogenetic analysis of FvPHR1 and PHR1s from other plants was performed with NeighborJoining Method. Fig. 2C shows that FvPHR1 was closely related to these PHR1s of Rosaceae fruit trees (Prunus persica, P. mume, P. avium, Malus domestica and Pyrus bretschneideri). It is consistent with the result of plant evolution. In the model plant Arabidopsis and rice, PHR1 plays a significant role in Pi-starvation signaling and Pi-homeostasis [7,31–33]. So it was indicated that the new member of the family, FvPHR1, may also play a role in Pi-starvation signaling. The sequence of FvPHR1 was remarkably similar to AtPHR1, OsPHR1, OsPHR2 and OsPHR4 (Fig. 2A). It was reported that AtPHR1, OsPHR1, OsPHR2 and OsPHR4 were nuclear-localized proteins [7,31–33]. To determine whether FvPHR1 located in the nucleus like other PHR1s, the FvPHR1 coding sequence was fused to the N-terminal end of GFP and driven by CaMV 35S promoter. FvPHR1-GFP was introduced into rice protoplasts to determine the Sub-cellular localization of FvPHR1. As shown in Fig. 3A, the FvPHR1-GFP fusion protein was found in the nucleus, whereas the GFP signal of positive control was distributed throughout the entire cell. These results suggested that FvPHR1 was a nuclear protein. To determine the transcription activation of FvPHR1, a GAL4-responsive reporter system was performed in yeast cells. The full-length fragment of FvPHR1 was fused to the GAL4 DNA binding domain to create the pGBKT7-FvPHR1 construct and transformed to yeast strain AH109. The empty vector pGBKT7 was used as negative control. As shown in Fig. 3B, the yeast strain AH109 contained pGBKT7-FvPHR1 grew well in SD/-Trp and SD/-Trp-His-Ade. However, the yeast strain AH109 which contained pGBKT7 only grew in SD/-Trp. These results indicated that the FvPHR1 was a transcriptional activator. As predicted, FvPHR1 protein contained a CC-domain, which usually formed a homodimer by its helix-loop-helix structure [38]. Here, BiFC assay was used to explore whether FvPHR1 formed a homodimer in vivo. As shown in Fig. 3C, there was a strong YFP signaling in nucleus of the tobacco leaves transformed with the eYNEFvPHR1 and eYCE-FvPHR1 vectors. Whereas, there was no YFP signaling in the cell of tobacco leaves coexpressing eYNE/eYCE-FvPHR1, eYNE-FvPHR1/eYCE or eYNE/eYCE. These results suggested that FvPHR1 formed a homodimer in vivo and was located in nucleus. Together, these observations suggested that FvPHR1 worked as a transcription factor in woodland strawberry.
2.13. Gene expression analysis Gene expression level was analyzed by quantitative RT-PCR (qRTPCR). For the detection of all strawberry MYB-CC genes, FvPHR1, preFvmiR399a, pre-FvmiR399b and pre-FvmiR399c, cDNA was synthesized using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Quantitative PCR was performed with SYBR Premix Ex TaqII (TaKaRa, Dalian, China) using Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The specific stem-loop reverse primer of FvmiR399a was used for the mature FvmiR399a synthesis. The expression level of mature miR399a was detected by a TaqMan PCR using the method described previously [29]. The relative expression levels of genes were calculated with 2−ΔΔCT method. The strawberry 26S rRNA gene was used as the reference gene. Specific primers were listed in Supplementary Table S1. qRT-PCR was performed with three biological replicates. 2.14. Measurement of P content The P content in the leaves of wild type (WT) and transgenic plants was determined using the Mo-Sb colorimetric method as described previously [29]. 3. Results 3.1. Genome-wide identification of strawberry MYB-CC family genes in woodland strawberry We identified 26 putative strawberry MYB-CC genes by searching on the PLAZA 3.0 Dicots, and then excluded some predicted protein sequences without a MYB-CC domain using NCBI’s CDD. Subsequently, we acquired the information of these predicted strawberry MYB-CC genes from GDR, such as GDR number, chromosomal location and the number of amino acid. As Table S2, a total of 13 MYB-CC genes were identified in the woodland strawberry (F. vesca) genome. These MYBCC proteins ranged from 273 (FvH4_6g22540.1) to 513 (FvH4_2g20730.1) amino acids in length. The predicted molecular weights of FvMYB-CC proteins ranged from 30.71 (FvH4_6g22540.1) to 56.21 (FvH4_2g20730.1) KDa and the isoelectric points ranged from 5.05 (FvH4_6g01670.1) to 8.76 (FvH4_1g29370.1). 3.2. Phylogenetic and expression patterns analysis of strawberry MYB-CC genes We acquired 13 and 15 MYB-CC genes which contain MYB-CC domain from F. vesca and A. thaliana, respectively. In order to analyze evolutionary relationships between the MYB-CC members in strawberry and Arabidopsis, phylogenetic analysis was performed using MEGA 6.06 software with the Neighbor–Joining method. As shown in Fig. 1, we found there was a very close homology between AtPHR1 (AT4G28610) and strawberry MYB-CC gene FvH4_2g20730.1. Therefore, we named FvH4_2g20730.1 as FvPHR1.
3.4. Expression patterns of the FvPHR1 in different organs of woodland strawberry In order to investigate the spatial expression pattern of FvPHR1 gene 262
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Fig. 3. Transcription factor characteristics of FvPHR1. A. Subcellular localization of FvPHR1.The vectors of FvPHR1-GFP and GFP positive control were infiltrated into rice protoplast, respectively. OsMADS3-mCherry as a nuclear marker. Scale bars = 4 μm. B. Transcriptional activities of the FvPHR1 gene. The transformed yeast strain with constructs of FvPHR1-BD and BD empty (pGBKT7) were grown on SD/-Trp and SD/-Trp/-His/-Ade media, respectively. C. The homodimer of FvPHR1 in vivo. The eYFP and m-Cherry fluorescence signaling was observed in tobacco leaves. There was a protein–protein interaction between FvPHR1 and itself.
pattern of FvmiR399a was similar to FvPHR1, higher in flowers, leaves and roots and lower in green fruits [29].
in strawberry, qRT-PCR analysis was used to measure that the expression of FvPHR1 in roots, stems, old leaves, young leaves, petioles, flowers, different stages fruits (green fruits, white fruits, turn fruits, red fruits) of woodland strawberry. As shown in Fig. 4, the FvPHR1 was expressed in all organs, with higher expression level in roots, old leaves, and lower in green fruits. Interestingly, we found that the expression
3.5. FvPHR1 binds to the promoter of FvMIR399a PHR1 acts as a central regulator in Pi starvation signaling by 263
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into five parts and assayed with FvPHR1 protein. The results indicated that FvPHR1 could bind to P3 of FvMIR399a promoter containing two P1BS (Fig. 5B). As shown in Fig. 5B, two P1BS motif were found in the P3 of FvMIR399a promoter. Here, to identify which P1BS was essential for the binding of FvPHR1, EMSA was performed. The biotin-labeled probe sequence containing P1BS-3 and P1BS-4 motif were shown in Fig. 6A. The EMSA results indicated that FvPHR1 was able to bind to P1BS1-3 and P1BS-4 biotin-labeled probes (Fig. 6B). Furthermore, non-labeled competitive probe could specifically compete with the biotin-labeled probe in binding to FvPHR1. These results suggested that FvPHR1 could bind to both the P1BS-3 and P1BS-4 motif in the promoter of FvMIR399a. Fig. 4. Expression patterns of FvPHR1 in different organs revealed by qRT-PCR. Errors bars were acquired from three biological repeats.
3.6. Overexpression of FvPHR1 increases P content in the woodland strawberry
regulating some PSI genes in Arabidopsis [7]. In addition, previous study has shown that the overexpression of miR399a could improve the P content in the leaves of the woodland strawberry [29]. So, we hypothesized that FvPHR1 might mediate the change of P content in woodland strawberry by regulate the expression of miR399a. To determine whether PHR1 could involve in the regulation of Pi starvation response by binding to the promoter of FvMIR399a gene, the promoter of FvMIR399a was analyzed. As shown in Supplementary Table S3, these cis-acting elements included some responsive to light, abscisic acid, heat stress, drought, salicylic acid, defense and stress. In addition, 4 PHR1-binding sites (P1BS: GNATATNC) were found in the FvMIR399a promoter region. Yeast one-hybrid assay showed that FvPHR1 protein could bind to the FvMIR399a promoter (Fig. 5A). As shown in supplementary Fig. 1, there were 4 P1BS in the FvMIR399a promoter. To identify the position that FvPHR1 could bind to, the promoter of FvMIR399a was divided
To investigate the function of FvPHR1 in strawberry, we developed FvPHR1 overexpression plants of woodland strawberry ‘Ruegen’ by transformation with the construct pRI101-FvPHR1 (Fig. 7A). PCR analysis was performed to detect transgenic plantlets using the specific primer set (35S and FvPHR1-424R), a 570 bp band was amplified from genomic DNA of three independent transgenic lines and no corresponding bands was found in non-transformed WT plants (Fig. 7B). To further confirm the three transgenic lines, qRT-PCR was performed to determine the transcript level of FvPHR1. As shown in Fig. 7C, the expression of FvPHR1 was significantly increased in these transgenic lines. To further verified the relationship between FvPHR1 and FvmiR399 in strawberry. The expression of pre-FvmiR399a, pre-FvmiR399b, preFvmiR399c and mature FvmiR399a was detected in FvPHR1-overexpressing and WT plants by qRT-PCR [39]. As shown in Fig. 8, preFvmiR399a, pre-FvmiR399b and pre-FvmiR399c were significantly upregulated in leaves of transgenic lines as compared with the WT. And the expression of mature FvmiR399a was increased by 11.0-fold to 59.6-fold in FvPHR1-overexpressing lines compared with the WT. Therefore, these results indicated that FvPHR1 positively regulates the expression of FvmiR399 in woodland strawberry. Our previous research showed that the overexpression of miR399a could increase the P content in woodland strawberry leaves [29]. So we measured the P content in the leaves of WT and FvPHR1-overexpressing woodland strawberry plants under Pi-sufficient conditions. As shown in Fig. 9, the P contents in FvPHR1-overexpressing lines were significantly increased by 1.38-fold to 1.78-fold compared with that in WT. Thus, these results suggested that the overexpression of FvPHR1 could increase P content in the woodland strawberry. 4. Discussion P is an essential macronutrient to sustain the normal plant growth and development. Plants make a series of response under Pi starvation. Meanwhile, a complicated Pi signaling network is formed in plants. Although Pi starvation signaling is well studied in Arabidopsis and rice, it is still largely unknown in strawberry. In strawberry, Pi plays a significant role in fruit quality [28,40,41]. Thus, to further understand the Pi starvation signaling in strawberry, it is important to identify the key factor in the network. In Arabidopsis and rice, PHR1 acts as a central transcriptional activator regulating PSI genes in Pi starvation response [7,42]. Here, we identified a MYB-CC family gene FvPHR1, as a key transcription factor involved in the regulation of phosphate-signaling in woodland strawberry. Like the PHR1 in Arabidopsis, rice and maize [7,12,31], FvPHR1 is located in cell nucleus. TaPHR1 forms a homodimer to activate the expression of Pi transporter TaPHT1.2 in wheat [43]. In this study, we also found that FvPHR1 would work by forming a homodimer (Fig. 3C), which suggested that FvPHR1 may regulate the expression of Pi
Fig. 5. Yeast one-hybrid assay showing that FvPHR1 binds to the promoter of FvMIR399a. A. pB42 AD-FvPHR1 represented FvPHR1 coding sequence was inserted into pB42 AD as an effector. pLacZi-FvMIR399apro represented the FvMIR399a promoter (1440 bp) fused with LacZ as a reporter. The empty vectors pLacZi and pB42 AD were used as negative control. B. The interaction between pB42 AD and pB42 AD-FvPHR1 with the different fragments of the FvMIR399a promoter. The black boxes represented the four P1BS. 264
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Fig. 6. EMSA assay showing that FvPHR1 bind directly to the FvMIR399a promoter. A. Diagram showing the P1BS element in FvMIR399a promoter used for EMSA. Two biotin-labeled probe sequences containing PIBS-3 and P1BS-4 motif are shown and the P1BS motif are highlighted in bold. B. EMSA results showing that FvPHR1 specifically bind to the P1BS-3 and P1BS-4 motif in FvMIR399a promoter. Biotin-labeled sequences containing PIBS-3 and P1BS-4 motif are used as hot probes, while non-labeled sequences are used as cold probe (100-fold higher concentration than that of biotin-labeled probe), respectively. MBP-FvPHR1 fusion protein was purified from Escherichia coli Rostta (DE3) and used for the DNAbinding assays.
Fig. 7. Molecular confirmation of FvPHR1 overexpressing transgenic lines of woodland strawberry. A. Structure diagram of FvPHR1 overexpressing construct pRI101-FvPHR1, the FvPHR1 gene was driven by CaMV35S promoter. B. PCR amplification of the specific fragment (570bp) from the genomic DNA of FvPHR1 transgenic lines, lanes from left to right: DL2000 marker, FvPHR1 transgenic lines OE#5, #6, #10 and wild type (WT) plants. C. Transcript level of FvPHR1 was examined in three independent positive transgenic lines and WT plants by qRT–PCR. Vertical bars represent the SD (n = 3).
condition through the post-translational modification. In addition, we found FvPHR1 positively regulates the expression of FvMIR399a through directly binding to its promoter, and our previous study showed that the overexpression of miR399a increased the P content in woodland strawberry [29], so we infer that FvPHR1 could control P content through positively regulate the expression of FvMIR399. The results in this study indicate that the PHR1-miR399 signaling pathway is conserved among plant species, and this pathway is important for modulating P homeostasis in strawberry as it is in Arabidopsis and rice. Although Pi signaling is very important for plant growth, it does not work alone. During the regulation of Pi homeostasis in plants, the Pi starvation response is associated with other signaling pathways, including sugar and hormone-mediated signaling pathways [46–53].
starvation induced genes by the homodimer. The overexpression of FvPHR1 can increase the P content in woodland strawberry. This result is similar to the previous reports, which the overexpression of AtPHR1, OsPHR1/2/3/4, TaPHR1, ZmPHR1 and BnPHR1 enhanced Pi accumulation in shoot [12,31–3343–45]. It was reported that the transcript levels of OsPHR3, OsPHR4, BnPHR1 and PvPHR1 were increased by Pi starvation in leaves and roots [11,33,45]. And under low Pi treatment, the expression level of ZmPHR1 was up-regulated by Pi starvation in roots of maize, while down-regualted in leaves [12]. While, under Pistarvation condition, the transcript level of FvPHR1 was not significantly changed in leaves of woodland strawberry. These results are similar to that of AtPHR1, AtPHL1, OsPHR1 and OsPHR2 [7,16,31]. But it is speculated that PHR1 may become more active under Pi-starvation 265
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Fig. 8. The expression levels of pre-FvmiR399a (A), pre-FvmiR399b (B), pre-FvmiR399c (C) and mature FvmiR399a (D) in FvPHR1-OE lines and WT plants of woodland strawberry. Vertical bars represent the SD (n = 3).
Fig. 9. The P content in FvPHR1-OE lines and WT plants of woodland strawberry. Vertical bars represent the SD (n = 3). Different lowercase letters above the bars indicate significant difference compared with the WT (n = 3, P < 0.05).
Recently, research showed that light and ethylene coordinately regulate PSRs through regulating the PHR1 expression (Fig. 10) [54]. Besides, PHR1, a potential general integrator, regulates the expression of some genes involved in P, S, Fe, Zn homoeostasis and transport [55]. In rice, OsmiR399 as a key miRNA involved in Pi starvation response [15]. It was reported that OsmiR399 also takes part in the regulation of Na, K, Ca, Fe starvation responses [19]. In A. thaliana, the miR399-PHO2 module involved in the regulation of ambient temperature-responsive flowering [56]. Further, there was a link between Pi homeostasis and flowering time regulation by confirming an interaction between OsPHO2 and OsGI in rice [57]. In summary, a hypothetical model for phosphate-signaling pathway in woodland strawberry was proposed by combining our results and previous reported in model plant (Fig. 10). Here, we identify the PHR1miR399 module control phosphate homeostasis in woodland
Fig. 10. A hypothetical model for the role of the PHR1-miR399 module in woodland strawberry. The network marked in red indicate the results in this work. Other pathways indicate the results have reported in Arabidopsis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
strawberry plants. Our research provided theoretical basis for the study of FvPHR1 function in Pi signaling. Besides, in order to improve the low P tolerance ability in strawberry by agronomic strategies, the crosstalk between Pi signaling and other signaling pathway should be explored in the future.
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Conflict of interest
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The authors declare no conflict of interest. Contributions YW and FZ conceived and designed the experiments. YW and FZ contributed to the experimental work. WC, RZ and KC contributed to coordinated the study. YW wrote manuscript. ZZ helped perform the analysis with constructive discussions and approved the final version. All the authors read and approved the final manuscript. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (31872072) and the Key R&D and Technology Transfer Program (Z17-0-035) Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2018.12.025. References [1] D.P. Schachtman, R.J. Reid, S.M. Ayling, Phosphorus uptake by plants: from soil to cell, Plant Physiol. 116 (1998) 447–453. [2] I.C.R. Holford, Soil phosphorus: its measurement, and its uptake by plants, Aust. J. Soil Res. 35 (1997) 227–240. [3] T.R. Bates, J.P. Lynch, Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability, Plant Cell Environ. 19 (1996) 529–538. [4] B.I. Linkohr, L.C. Williamson, A.H. Fitter, H.M. Leyser, Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis, Plant J. 29 (2002) 751–760. [5] L. Cheng, B. Bucciarelli, J. Shen, D. Allan, C.P. Vance, Update on white lupin cluster root acclimation to phosphorus deficiency, Plant Physiol. 156 (2011) 1025–1032. [6] X.F. Ma, E. Wright, Y. Ge, J. Bell, Y. Xi, J.H. Bouton, Z.Y. Wang, Improving phosphorus acquisition of white clover (Trifolium repens L.) by transgenic expression of plant-derived phytase and acid phosphatase genes, Plant Sci. 176 (2009) 479–488. [7] V. Rubio, F. Linhares, R. Solano, A.C. Martín, J. Iglesias, A. Leyva, J. Paz-Ares, A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plant and unicellular algae, Gene Dev. 15 (2001) 2122–2133. [8] B.N. Devaiah, A.S. Karthikeyan, K.G. Raghothama, WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis, Plant Physiol. 143 (2007) 1789–1801. [9] B.N. Devaiah, V.K. Nagarajna, K.G. Raghothama, Phosphate homeostasis and root development in Arabidopsis are synchronized by the zing finger transcription factor ZAT6, Plant Physiol. 145 (2007) 147–159. [10] Z.H. Chen, G.A. Nimmo, G.I. Jenkins, H.G. Nimmo, BHLH32 modulates several biochemical and morphological process that respond to Pi starvation in Arabidopsis, Biochem. J. 405 (2007) 191–198. [11] O. Valdés-López, C. Arenas-Huertero, M. Ramírez, L. Girard, F. Sánchez, C.P. Vance, J. Luis Reyes, G. Hernández, Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deficiency signalling in common bean roots, Plant Cell Environ. 31 (2008) 1834–1843. [12] X.H. Wang, J.R. Bai, H.M. Liu, Y. Sun, X.Y. Shi, Z.Q. Ren, Overexpression of a Maize transcription factor ZmPHR1 improves shoot inorganic phosphate content and growth of Arabidopsis under low-phosphate conditions, Plant Mol. Biol. Rep. 31 (2013) 665–677. [13] T.J. Chiou, S.I. Lin, Signaling network in sensing phosphate availability in plants, Annu. Rev. Plant Biol. 62 (2011) 185–206. [14] K. Miura, A. Rus, A. Sharkhuu, S. Yokoi, A.S. Karthikeyan, K.G. Raghothama, D. Baek, Y.D. Koo, J.B. Jin, R.A. Bressan, D.J. Yun, P.M. Hasegawa, The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses, P. Natl. Acad. Sci. U. S. A. 102 (2005) 7760–7765. [15] R. Bari, B.D. Pant, M. Stitt, W.R. Scheible, PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants, Plant Physiol. 141 (2006) 988–999. [16] R. Bustos, G. Castrillo, F. Linhares, M.I. Puga, V. Rubio, J. Pérez-Pérez, R. Solano, A. Leyva, J. Paz-Ares, A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis, PLoS Genet. 6 (2010) e1001102. [17] S. Okumura, N. Mitsukawa, Y. Sgurano, D. Shibata, Phosphate transporter gene family of Arabidopsis thaliana, DNA Res. 5 (1998) 261–269. [18] H. Fujii, T.J. Chiou, S.I. Lin, K. Aung, J.K. Zhu, A miRNA involved in phosphatestarvation response in Arabidopsis, Curr. Biol. 15 (2005) 2038–2043. [19] B. Hu, W. Wang, K. Deng, H. Li, Z.H. Zhang, L.H. Zhang, C.C. Chu, MicroRNA399 is involved in multiple nutrient starvation responses in rice, Front. Plant Sci. 6 (2015) 188.
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