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Identification of transcription factors that bind to the 5′-UTR of the barley PHO2 gene

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Abstract

In barley and other higher plants, phosphate homeostasis is maintained by a regulatory network involving the PHO2 (PHOSPHATE2) encoding ubiquitin-conjugating (UBC) E2 enzyme, the PHR1 (PHOSPHATE STARVATION RESPONSE 1) transcription factor (TF), IPS1 (INDUCED BYPHOSPHATESTARVATION1) RNA, and miR399. During phosphate ion (Pi) deprivation, PHR1 positively regulates MIR399 expression, after transcription and processing mature miR399 guides the Ago protein to the 5′-UTR of PHO2 transcripts. Non-coding IPS1 RNA is highly expressed during Pi starvation, and the sequestration of miR399 molecules protects PHO2 mRNA from complete degradation. Here, we reveal new cis- and trans-regulatory elements that are crucial for efficient PHO2 gene expression in barley. We found that the 5′-UTR of PHO2 contains two PHR1 binding sites (P1BSs) and one Pi-responsive PHO element. Using a yeast one-hybrid (Y1H) assay, we identified two candidate proteins that might mediate this transcriptional regulation: a barley PHR1 ortholog and a TF containing an uncharacterized MYB domain. Additional results classified this new potential TF as belonging to the APL (ALTERED PHLOEM DEVELOPMENT) protein family, and we observed its nuclear localization in barley protoplasts. Pi starvation induced the accumulation of barley APL transcripts in both the shoots and roots. Interestingly, the deletion of the P1BS motif from the first intron of the barley 5′-UTR led to a significant increase in the transcription of a downstream β-glucuronidase (GUS) reporter gene in tobacco leaves. Our work extends the current knowledge about putative cis- and trans-regulatory elements that may affect the expression of the barley PHO2 gene.

Key Message

The 5′-UTR of the barley PHOSPHATE 2 gene contains two P1BS motifs that can bind the transcription factor (TF) PHR1 (PHOSPHATE STARVATION RESPONSE 1) and the newly identified TF APL (ALTERED PHLOEM DEVELOPMENT)

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Acknowledgements

The authors wish to thank Dr. Iver Jakobsen (University of Copenhagen, Copenhagen, Denmark) for providing low-P soil and Prof Tzyy-Jen Chiou (Agricultural Biotechnology Research Center, Academia Sinica, Taiwan) for the construct containing the AtPHO2 promoter and the 5′-UTR. We thank Michał Taube and Przemysław Wieczorek for their help with the protein overexpression and purification protocols and Mateusz Bajczyk for all his advice.

Funding

This work was funded by the National Science Centre, Poland, on the basis of DEC-2013/11/B/NZ9/01761, UMO-2016/23/B/NZ9/00857, and UMO-2015/19/N/NZ9/00218 and by KNOW RNA Research Centre in Poznan 01/KNOW2/2014.

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Authors and Affiliations

  1. Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61-614, Poznań, Poland

    Paweł Sega, Katarzyna Kruszka, Łukasz Szewc, Zofia Szweykowska-Kulińska & Andrzej Pacak

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  1. Paweł Sega
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  2. Katarzyna Kruszka
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  3. Łukasz Szewc
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  4. Zofia Szweykowska-Kulińska
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  5. Andrzej Pacak
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Contributions

PS performed most of the experiments and wrote the manuscript under the supervision of AP. AP prepared the degradome and transcriptome libraries. LS performed the protein subcellular localization. All authors reviewed the manuscript.

Corresponding author

Correspondence to Andrzej Pacak.

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All authors declare that they have no conflict of interest.

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Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries (AtNSR1 (AAF05867), MYB-1 (AK373855), MYB-2/APL (AK371403), MADS57 (AK363243)) and the Ensembl Plants database for the barley genome (PHR1 (HORVU4Hr1G051080.5), PHR2 (HORVU4Hr1G051080.1), MYB-2/APL (HORVU6Hr1G031470), PHO2 (HORVU1Hr1G085570.2)).

Electronic supplementary material

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Supplementary Table 1

List of proteins obtained from Y1H experiments. Supplementary Table 2 Synthetic oligonucleotides used for EMSA experiments. Supplementary Table 3 List of selected proteins used for the phylogenetic analysis of the APL TF. Supplementary Table 4 Primers used in this study. Supplementary Table 5 Y1H “bait” sequences derived from PHO2 gene fragments used for screening. (DOCX 29 kb)

Supplementary Fig. S1

The graph showing all six PHO2 transcript variants from the Ensembl Plants database and RNA-Seq results. There are six PHO2 protein-coding transcripts that are present in the barley Ensembl Plants database: HORVU1Hr1G085570.1 (544 aa), HORVU1Hr1G085570.2 (847 aa), HORVU1Hr1G085570.3 (445 aa), HORVU1Hr1G085570.4 (847 aa), HORVU1Hr1G085570.5 (606 aa), and HORVU1Hr1G085570.6 (491 aa). The lower panel displays the cDNA sequences of the first 180 bp from the HORVU1Hr1G085570.1 variant and the last 26 bp of the HORVU1Hr1G085570.2 variant to show the 5′ (blue arrow) and 3′ (red arrows) ends obtained in our RNA-Seq analysis. The white boxes indicate the untranslated region, and the gray boxes indicate the coding region. Scale bar = 1000 bp. Supplementary Fig. S2 The genomic sequence of the barley PHO2 5′-UTR region used in this study. The validated genomic sequence of the barley PHO2 5′-UTR region (2742 bp) that was cloned and sequenced from the Morex genotype (Ensembl Plants database: chr1H:535891650:535894391‬:1). Denoted are the cis-regulatory motifs that were identified in this study, in addition to six miR399 potential cleavage sites. The yellow boxes indicate the locations of exons. The black star indicates the additional exon within the 5′-UTR that is present only in barley PHO2 isoforms 5 and 6. Supplementary Fig. S3 The P1BS motif is evolutionarily conserved and present in various PSI genes in barley. The genomic localization of P1BS motifs within regulatory sequences of the barley Pi-starvation-responsive genes PHO2, MIR399c, IPS1, RNS1, and PHT1;1. The Arabidopsis thaliana (AtPHO2) and Nicotiana benthamiana (NbPHO2) PHO2 gene orthologs were used as a reference to show the P1BS motif positions within barley PHO2 regulatory sequences relative to those within the other plant species used in this study. The gray box depicts the 5′-UTR; the white box depicts the promoter; the red line indicates the position of the P1BS motif. Scale bar = 1000 bp (left). Comparison of the sequences of all P1BS motifs that are present in the left panel (right). The yellow box indicates the specific nucleotide within the motif consensus; the consensus match connects barley PHO2 motifs with P1BS motifs that are the same but present within the regulatory sequences of other genes. Supplementary Fig. S4 The barley PHO2 transcript is cleaved within its 5′-UTR. The red vertical line shows the cleavage position directed by miR399; the cleavage position 1203 is within exon No. 2 in the 5′-UTR of the PHO2 transcript (HORVU1Hr1G085570.2, length of 4347 nt). The black vertical lines on the graph show the positions within the PHO2 cDNA to which 20 nt degradome fragments (reads) were mapped. The number of such reads (fragment abundance) is depicted by the height of the red and black lines. Below the graph, the structure of the PHO2 transcript is presented. The white boxes denote UTRs, the gray boxes denote CDSs, and the dotted vertical lines denote cleavage sites within the 5′-UTR. Supplementary Fig. S5 The multiple sequence alignment for PHR-like TFs in which the AtNSR1 protein sequence was used as a query. On the basis of the results published by Todd and his group in 2004, we selected two MYB-like TFs (temporarily named MYB-1 and MYB-2) that exhibited the highest homology to the AtNSR1 protein (At3g04030) and analyzed them in a Y1H screening assay. The orange box indicates the SANT/MYB protein domain, and the green box indicates the MYB-CC domain. Supplementary Fig. S6 The barley TFs PHR1 and APL interact with the second exon “bait” fragment originating from the PHO2 5′-UTR in yeast cells. The structure of the barley PHO2 gene with marked a Y1H “bait” fragment. The pPHO2_5 fragment is 27 bp in length and includes the P1BS.2 motif. The blue triangle indicates positions 447916090 (+) and 447916332 (+) on barley chromosome 1; the two TSSs of the PHO2 transcripts were identified using 5′RLM-RACE. The lower panel contains images of the growing colonies of the tested Y1HGold yeast strain having the “bait” fragment from the PHO2 5′-UTR second exon. The barley MADS57 TF was used as a non-binding negative control. Supplementary Fig. S7 The first intron originating from the barley PHO2 5′-UTR contains signals that help increase gene expression. The distribution of IMEter scores for either (A) all nine PHO2-related introns (the first two introns are located within 5′-UTR region) or (B) seven fragments approximately 200 bp in length chopped from the first intron of the PHO2 5′-UTR. Each ~200 bp fragment is designated with an additional prefix from p1 to p7, where the p1 fragment is located on the first intron 5′-end, and p7 is located on its 3′-end. The plots show percentage scores, which were calculated on the basis of the results from three monocotyledonous plant species: Brachypodium distachyon, Oryza sativa, and Zea mays. Supplementary Fig. S8PHO2, PHR1, and APL transcript coverage. RNA-Seq paired reads were mapped to the PHO2 (HORVU1Hr1G085570.2, Ensembl Plants database), PHR1 (HORVU4Hr1G051080.5), and APL (HORVU6Hr1G031470.1) transcripts. Blue indicates the reverse paired reads, green indicates forward unpaired reads, and red indicates reverse unpaired reads; the black vertical line denotes the ATG start codon. Below the graph, the structures of the transcript are presented. The white boxes denote UTRs, and the gray boxes denote CDSs. Supplementary Fig. S9 The new protein identified in Y1H screening has two highly conserved protein domains and is likely related to the APL TFs. Phylogenetic analysis was carried out using the neighbor-joining method in CLC Main Workbench software (QIAGEN). To select proteins for the cladogram, we used the APL protein sequence as a query, and proteins showing more than 60% homology were chosen for analysis. Additionally, the pool of chosen proteins was enriched with several well-characterized plant PHR-like proteins. Each bootstrap value was calculated with 100 replications. All accession numbers of the protein sequences are listed in Table S3. The black arrow shows the APL position on the tree. The lower panel presents the protein structures of the barley APL, PHR1, and PHR2 proteins. The APL protein contains 368 aa residues (39.85 kDa) that comprise two domains: a SANT/MYB domain (orange box, PF00249) at aa residues 45–93 and a MYB-CC-type LHEQLE-containing domain (blue box, PF14379) at aa residues 139–193. The PHR1 protein contains 451 aa residues (49.48 kDa), and the same domains are present at aa residues 233–284 and residues 314–360; however, the PHR2 protein contains 438 aa residues (47.17 kDa), and the domains are located at residues 256–307 and residues 340–385, respectively. Scale bar = 100 aa. Supplementary Fig. S10 The cellular localization of PHR1–eGFP fusion proteins in barley protoplasts. Exclusive images that show the localization of recombinant proteins transiently expressed in protoplasts isolated from 6-day-old barley leaves. Slight expression of PHR1–eGFP proteins in the cytoplasm was observed in one out of every five cells. The protoplasts shown here were incubated overnight in standard W5 buffer (without extra KH2PO4, a source of Pi). Microscopic analyses were repeated three times, and similar patterns were imaged. Scale bars = 20 µm. (PDF 12523 kb)

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Sega, P., Kruszka, K., Szewc, Ł. et al. Identification of transcription factors that bind to the 5′-UTR of the barley PHO2 gene. Plant Mol Biol 102, 73–88 (2020). https://doi.org/10.1007/s11103-019-00932-9

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  • DOI: https://doi.org/10.1007/s11103-019-00932-9

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