Fig 1.
The tex1 mutation suppresses the shoot growth phenotype of the pho1-7 mutant.
(A) Phenotype of Col-0, pho1-7, pho1-7 suppressor, and pho1-7 suppressor transformed with the pTEX1:gTEX1:GFP. (B) Pi contents of 4-week-old rosettes. Data from a representative experiment shows the mean Pi content from ten different plants grown in independent pots. Errors bars represent standard deviation. Values marked with lowercase letters are statistically significantly different from those for other groups marked with different letters (P < 0.05, ANOVA with the Tukey-Kramer HSD test). Panel display a representative experiment.
Fig 2.
tex1 restores the expression of PHO1 in the pho1-7 mutant.
(A) Phenotype of 4-week-old Col-0, tex1-4, pho1-7, pho1-4, pho1-7 tex1-4 and pho1-4 tex1-4 mutants. (B) Shoot Pi contents of 4-week-old plants. Data from a representative experiment shows the means of Pi contents from six individual plants grown in independent pots. Error bars represent standard deviation. (C) Structure of truncated PHO1 mRNA produced at the PHO1 locus in the pho1-7 mutant. For the PHO1 locus, exons are indicated as black open boxes, except for the second exon, indicated as a green doted box. Exon 2 is present in Col-0 but deleted in the pho1-7 mutant as a result of T-DNA insertion. The structure of the mRNA PHO1Δ249–342 found in the pho1-7 mutant is shown below. The pairs of oligonucleotides P1 and P2 used for RT-PCR is shown. (D) Western blot showing full length and truncated PHO1 protein in roots of Col-0, pho1-2 null mutant, pho1-7 and pho1-7 tex1-4. Plants were grown for 4 weeks in a clay substrate and total protein were extracted from roots. (E) Relative expression level of PHO1 gene in the roots of 4-week-old plants grown in a clay substrate. Data are means of three samples from plants grown in independent pots and three technical replicates for each sample, with each sample being a pool of three plants. Error bars represent standard deviation. For both B and E, values marked with lowercase letters are statistically significantly different from those for other groups marked with different letters. (P < 0.05, ANOVA with the Tukey-Kramer HSD test).
Fig 3.
Truncated PHO1Δ84–114 is sufficient to restore the shoot growth phenotype of pho1-4 null mutant.
(A) Phenotype of four-week-old plants. (B) Pi contents in the rosettes of 4-week-old plants. Data from a representative experiment shows the means of Pi contents from six individual plants grown in independent pots. Error bars represent standard deviation. (C) Localization of full-length PHO1:GFP and PHO1Δ83–114:GFP in the roots of 7-day-old seedlings. Both PHO1:GFP and PHO1Δ83–114:GFP were expressed under the control of native PHO1 promoter in the pho1-4 null mutant. (D) Pi export mediated by PHO1:GFP and PHO1Δ84–114: GFP from transient expression in N. benthamiana leaf discs. As controls, Pi export was measured in leaf discs expressing either free GFP (EV) or not infiltrated (none). Data are means of 4 measurements taken from independent infiltrated leaves. Error bars represent standard deviation. For B and D, values marked with lowercase letters are statistically significantly different from those for other groups marked with different letters. (P < 0.05, ANOVA with the Tukey-Kramer HSD test).
Fig 4.
hpr1 can restore pho1-7 shoot growth but not mutations in tasiRNA biogenesis.
(A, C, D) Phenotype of four-week-old plants. (B) Pi contents in the rosettes of four-week-old plants. Data from a representative experiment shows the means of Pi contents from ten different plants grown in independent pots. Errors bars represent standard deviation. Values marked with lowercase letters are statistically significantly different from those for other groups marked with different letters (P < 0.05, ANOVA with the Tukey-Kramer HSD test).
Fig 5.
tex1 mutant impairs transcription termination at the NOS terminator.
(A) Structure of the wild type PHO1 gene (top) and T-DNA (red) integrated in the pho1-7 mutant (bottom). Exons are indicated as black open boxes, except for the second exon, indicated as a green doted box. Exon 2 is present in Col-0 but deleted in the pho1-7 mutant as a result of T-DNA insertion, which is shown in red. Blue lines represent introns. pNOS, NOS promoter; NPTII, neomycin phosphotransferase gene; tNOS, NOS terminator. (B) Structure of the four mRNA variants produced at the PHO1 locus in pho1-7 mutants. The thick black and blue lines represent sequences derives from PHO1 exons and introns, respectively, while the thick red lines are derived from the T-DNA. The sizes of the different transcripts are shown. Arrows indicate the location of primers used for the qPCR shown in panel D. Forward primers (discontinued arrows) in transcript 2 and 3 are at the junction of cryptic splicing sites to ensure specificity. (C) Illumina RNA sequencing reads density graph showing mRNA expression at the PHO1 locus in various genotypes. The RNA sequencing reads are mapped against the PHO1 locus in pho1-7, represented in the lower section of panel A (D) Quantification via qPCR of four different mRNA transcripts produced at the PHO1 locus in the roots of 4-week-old plants. The numbers associated with each transcript and primers used for qPCR are shown in panel B. (E) CHIP-qPCR experiments showing the density of RNAPII-S2 at the 3’end of the PHO1 gene. For D and E, data are means of three samples from plants grown in independent pots and three technical replicates for each sample. Error bars represent standard deviation. For D, statistical analysis was performed comparing each PHO1 transcript isoform in pho1-7 tex1-4 and pho1-7 hpr1-6 double mutants relative to the pho1-7 parent, with asterisks denoting statistical significance (*, P < 0.05; and ***, P < 0.001) according to Student’s t test. For E, values marked with lowercase letters are statistically significantly different from those for other groups marked with different letters (P < 0.05, ANOVA with the Tukey-Kramer HSD test).
Fig 6.
UTR extensions in endogenous genes of the pho1-7 tex1-4 and pho1-7 hpr1-6 mutants.
3′ (A) Illumina RNAseq reads density maps (blue) showing examples of 3’ UTR extensions for genes AT1G03160 (left) and AT3G11310 (right) in the pho1-7 tex1-4 and pho1-7 hpr1-6 mutants. The 3′ UTR extensions are depicted by a red rectangle. The exons of the genes are indicated as red boxes below. (B) Venn diagram showing the number and overlap in genes with 3’UTR extensions. (C) Validation of 3’ UTR extensions via qPCR. (D) Transient expression of Luciferase gene fused after the stop codon to the 3’end of genes AT1G76560 and AT1G03160. The constructs were expressed in Arabidopsis mesophyll protoplasts obtained from Col-0, hpr1-6 and tex1-4. The bar chart shows the relative ratios of long-to-short transcripts in the various genotypes. For C, data are means of three samples with each sample being a pool of seven seedlings and three technical replicates for each sample. For D, data are means of four samples with each sample being an independent transfection of protoplasts. Error bars represent standard deviation. Asterisks denote statistical significance (P < 0.05) from the Col-0 control according to Student’s t test (n.s. = not statistically different).
Fig 7.
Nucleotide composition near the 3’ cleavage site of mRNAs.
(A) Proportion of bases in a 200 nucleotide region 5’ and 3’ of the transcripts termination sites for all genes (left) or genes showing 3’UTR extensions in the hpr1-6 and tex1-4 mutant background (right). (B) Graphical representation of base enrichments found at the near upstream element sequence of all genes (left) and genes with 3’UTR extensions in the hpr1-6 and tex1-4 mutant background (right). Chi square test pvalue = 0.22 (C) Transient expression of Luciferase gene fused after the stop codon to the 3’end of the gene AT1G76560 in which the wild-type polyadenylation site AAUGAA was mutated to AAUAAA. The construct was expressed in Arabidopsis mesophyll protoplasts obtained from Col-0, hpr1-6 and tex1-4. The bar chart shows the ratios of long-to-short transcripts in the various genotypes and values are expressed relative to the wild-type construct in Col-0. Data are means of four samples with each sample being an independent transfection of protoplasts. Error bars represent standard deviation. Asterisks denote statistical significance (P < 0.05) from the Col-0 control according to Student’s t test.