Table 1.
Gene-specific primers or oligonucleotides used in this paper.
Figure 1.
The characterization of BnPHR1 gene and its deduced protein.
(A) Comparison of amino acid sequences between BnPHR1 and AtPHR1. Amino acid sequences are aligned by ClusterW. The two conserved regions (MYB domain and coiled-coil domain) are highlighted in reverse contrast and gray, respectively. A putative nuclear localization signal is shown by underline. The asterisks show the positions of the conserved amino acids residues between BnPHR1 and AtPHR1. (B) Structure of BnPHR1 gene. Exons are denoted by black boxes. Intron, 5′-untranslated region and 3′-untranslated region are denoted by lines. The length of the intron in base pairs is indicated. The number at the boundaries of each exon indicates the codon at which the intron is located. The translation initiation and termination codons are shown.
Figure 2.
Nuclear localization and transcriptional-activation of BnPHR1.
(A) Microscopic images of root cells of 35S:BnPHR1-GFP transgenic Arabidopsis. Confocal images were taken under the GFP channel (left), and with transmitted light (midst), and the images of left and midst were merged (right). (B and C) Transcriptional activation of BnPHR1. The growth of yeast strain AH109 and Y187 with BD and BD-BnPHR1 constructs under SD/−Trp, SD/−His/−Trp and SD/−Ade/−Trp nutrition-deficient medium (B). The transcription activity of BnPHR1 was measured by α-galactosidase activity assay (C).
Figure 3.
qRT-PCR analysis of BnPHR1 expression in tissues of B. napus.
(A) The wild-type B. napus plants were cultured in 1 mM Pi, and then the total RNAs isolated from stems, hypocotyls, cotyledons, roots, leaves and flowers were used in quantitative RT–PCR analysis. Error bars indicate standard deviation (n = 3). (B) The five-day-old B. napus seedling transferred to 1 µM Pi for 72 h and then total RNAs isolated from shoots and roots were used in quantitative RT–PCR analysis. Error bars indicate standard deviation (n = 3).
Figure 4.
Histochemical assay of BnPHR1 promoter activities in transgenic Arabidopsis.
(A) The five-day-old seedling germinated on 1 mM Pi. (B) The five-day-old seedling transferred to 1 µM Pi for 72 h. (C) The eight-day-old seedling transferred to 1 mM Pi for 72 h. (D) The eight-day-old seedlings transferred to 1 µM Pi for 72 h.
Figure 5.
BnPHR1 rescues the phenotypic defects of phr1.
(A) The location of the T-DNA insertion site of the phr1 mutant SALK_067629 is indicated with a triangle, and the locations of primers used for screening (LP(LP629) and RP(RP629), Table 1), and for RT–PCR (phr1-f, phr1-r, Table 1) are indicated with black and white arrows, respectively. (B) PCR analysis of the phr1 mutant to verify homozygozity line. M, DNA marker; 1, PCR products with the primers LP629 and RP629 using wild type genomic DNA as templates; 2, no PCR products with the primers LP629 and RP629 using homozygous phr1 genomic DNA as templates; 3, PCR products with the primers RP629 and LBa1 using homozygous phr1 genomic DNA as templates; 4, PCR products with the primers RP629 and LBb1 using homozygous phr1 genomic DNA as templates. (C) Relative expression of AtPHR1 and BnPHR1 in wild type (WT), phr1 and phr1/35S:BnPHR1 lines by real-time qRT–PCR using gene-specific primers (phr1-f/phr1-r and BnPHR1RT1/2, Table 1). Error bars indicate standard deviation (n = 3). ND indicates not detectable. (D) The phenotype of wild type (WT), phr1 and phr1/35S:BnPHR1 lines in MS with 1 µM Pi.
Figure 6.
The promoted growth of BnPHR1 overexpression Arabidopsis seedlings.
(A) Northern blotting analysis of BnPHR1 transcripts in 35S:BnPHR1 transgenic Arabidopsis. (B) The ten-day-old wild type (WT), phr1 and 35S:BnPHR1 transgenic Arabidopsis seedlings in 1 mM Pi. (C) The ten-day-old wild type (WT), phr1 and 35S:BnPHR1 transgenic Arabidopsis seedlings in 1 µM Pi. (D to G) The fresh weight (D), length of primary root (E), number of lateral roots (F) and anthocyanins content (G) of ten-day-old wild type (WT), phr1 and 35S:BnPHR1 transgenic Arabidopsis seedlings in 1 mM Pi and 1 µM Pi. Error bars indicate standard deviation (*0.01<p<0.05, **p<0.01, n = 20).
Figure 7.
The retarded growth of BnPHR1 overexpressing Arabidopsis in soil.
(A) The one-week-old wild type (WT), phr1 mutant and 35S:BnPHR1 transgenic Arabidopsis in soil. (B) The two-week-old wild type (WT), phr1 mutant and 35S:BnPHR1 transgenic Arabidopsis in soil. (C) The bolting wild type (WT) and 35S:BnPHR1 transgenic Arabidopsis in soil. (D) The fresh weight of two-week-old wild type (WT), phr1 and 35S:BnPHR1 transgenic Arabidopsis seedlings in soil. (E) The Pi content in shoot and root of wild type (WT) and 35S:BnPHR1 transgenic Arabidopsis. Error bars indicate standard deviation (*0.01<p<0.05, **p<0.01, n = 10).
Figure 8.
The retarded growth of BnPHR1 overexpressing B. napus in soil.
(A) The expressing analysis of BnPHR1 in wild type (WT) and 35S:BnPHR1 transgenic B. napus by semi-quantitative RT-PCR. (B)–(F) The growth of wild type and 35S:BnPHR1 transgenic B. napus in high Pi conditions. (B) The five-week old wild type (left) and 35S:BnPHR1 transgenic (right) B. napus grew in soil with high Pi. The seven-week old wild type (C) and transgenic (D) B. napus grew in high Pi conditions in soil. The nodes of basal leaves in wild type (E) and 35S:BnPHR1 transgenic (F) plants. (G) The fresh weight of seven-week-old wild type (WT) and 35S:BnPHR1 transgenic B. napus plants in soil. (H) Anthocyanins content in shoots of wild type (WT) and 35S:BnPHR1 transgenic B. napus. (I) Phosphate content in shoots of wild type (WT) and 35S:BnPHR1 transgenic B. napus. Error bars indicate standard deviation (*0.01<p<0.05, **p<0.01, n = 10).
Figure 9.
qRT-PCR analysis of Pi starvation response genes in transgenic Arabidopsis.
The wild type (WT) and 35S:BnPHR1 transgenic Arabidopsis was cultured in 1 mM Pi, and then the total RNAs isolated from the seedlings were used in real time qRT-PCR analysis. The data are presented as means standard deviation (n = 3).
Figure 10.
BnPHR1 directly regulating ATPT2 encoding high-affinity Pi transporter in transgenic Arabidopsis.
(A) The partial sequence of ATPT2 promoter containing two overlapped P1BS cis-elements (GNATATNC) (uppercase and underlined). (B) Western blotting assay of 35:GFP and 35:BnPHR1-GFP transgenic Arabidopsis with GFP antibody. (C–D) ChIP-qPCR analysis of the ATPT2 promoter sequence. ChIP assay was performed with chromatin prepared from 35:GFP and 35:BnPHR1-GFP transgenic Arabidopsis roots. Genomic DNA fragments that coimmunoprecipitated with GFP antibody were analyzed by real time qPCR using primers amplifying ATPT2 promoter fragment (C) and ATPT2 coding fragment (D) respectively. The experiments were repeated three times, and three replicates were included for each sample in each experiment. The data are presented as means standard deviation (n = 3).
Figure 11.
BnPT2 is the target gene of BnPHR1 in B. napus.
(A) The partial sequence of BnPT2 promoter. By Genome Walker PCR, a 1 276 bp DNA fragment of upstream region BnPT2 gene was isolated from B. napus genomic DNA. By motif scanning, two P1BS cis-elements (GNATATNC) (uppercase and underlined) were found in BnPT2 promoter sequence. (B–C) EMSA of BnPHR1 binding to DNA fragments from the promoter of BnPT2. (B) The gel was stained with SYBR® Green EMSA stain. (C) The same gel as in (B) stained with SYPRO® Ruby EMSA stain. The MBP-BnPHR1/DNA complex is observed in both DNA and protein staining. The MBP protein was used as negative control. (D) Interaction between BnPHR1 and the BnPT2 promoter sequence in yeast one-hybrid assays. (E) BnPT2 expression in wild type (WT) and 35S:BnPHR1 trangenic B. napus by real time qRT-PCR analysis. Error bars indicate standard deviation (n = 3).