Molecular Biology Reports

, Volume 41, Issue 4, pp 2427–2443 | Cite as

uORF, a regulatory mechanism of the Arabidopsis polyamine oxidase 2

  • Maria L. Guerrero-González
  • Margarita Rodríguez-Kessler
  • Juan F. Jiménez-Bremont


The translational efficiency of an mRNA can be modulated by elements located in the 5′-untranslated region. The flavin-containing polyamine oxidases catabolize oxidative deamination of spermidine and spermine, thus contributing to polyamine homeostasis as well as diverse biological processes through their reaction products. In this study, we characterized the uORF of AtPAO2 gene using the GUS reporter gene. Transgenic lines harboring the native AtPAO2 promoter or the constitutive CaMV 35S promoter show that the uORF negatively affects GUS expression. Exogenous applications of PAs positively modulate GUS expression, thus alleviating the negative effect of AtPAO2 uORF, while treatments with MGBG inhibitor show an opposite effect. Our data suggest that AtPAO2 uORF regulatory mechanism is modulated by polyamines. In addition, we present a comparative in silico study of the uORFs identified in several plant transcripts encoding polyamine oxidases in both mono- and dicotyledonous plants as well as in the Bryophyte Physcomitrella patens. The polyamine oxidase uORF-encoded peptides are conserved among families and share conserved features such as their position, length, and amino acid sequence. Our findings provide new insights into the regulatory mechanism of polyamine oxidase genes and encourage further exploration to assess the biological significance of uORFs in the polyamine catabolic pathway.


Polyamine oxidases Post-transcriptional regulation Spermidine Upstream open reading frame 



Main open reading frame


Methylglyoxal bis(guanylhydrazone)


Polyamine oxidase


Upstream open reading frame



This work was supported by CONACYT (Investigación Ciencia Básica 2008-103106) funding.

Supplementary material

11033_2014_3098_MOESM1_ESM.eps (84.5 mb)
Supplementary Fig. 1 Schematic representation of the genomic organization of plant PAO2 (A), PAO3 (B), and PAO4 (C) genes. Introns are shown as solid lines and exons are represented by boxes. Exons corresponding to the ORF region are numbered (I to IX) and exons in the UTR regions are shadowed (gray). Only the exons are drawn to scale. Most of the genomic sequences were obtained from the Phytozome database ( aSequences obtained from the TAIR database ( bSequences obtained from the NCBI database ( (EPS 86555 kb)
11033_2014_3098_MOESM2_ESM.eps (78.7 mb)
Supplementary material 2 (EPS 80545 kb)
11033_2014_3098_MOESM3_ESM.eps (46.1 mb)
Supplementary Fig. 2 RT-PCR assay of GUS mRNA in Arabidopsis uORF and non-uORF reporter lines, both under the control of native AtPAO2 promoter, treated with PAs and MGBG inhibitor. Total RNA was extracted from one-week-old seedlings of non-uORF (L2) and uORF (L4) lines treated with 0 and 100 μM of each PA (Put, Spd, and Spm) during 3 d (A). Total RNA was extracted from nine-day-old seedlings of non-uORF (L1 and L2) and uORF (L4 and L7) lines treated with 0, 2, and 4 mM MGBG during 2 d (B). Each RNA sample (1 μg) was used for RT-PCR analyses. Ten microliters of each PCR product were separated by electrophoresis on 1.2 % (w/v) agarose gel. The Arabidopsis Actin 2 and Actin 8 (ACT2/8) genes were used as an internal control. (EPS 47203 kb)
11033_2014_3098_MOESM4_ESM.eps (76.2 mb)
Supplementary Fig. 3 Effect of PAs and MGBG inhibitor on Arabidopsis 35S::GUS and 35S-UTR::GUS reporter lines. One-week-old seedlings of each line were treated for 1 d with 1 μM of each PA (Put, Spd, and Spm) or 2 mM MGBG, and controls without PAs or inhibitor were included. After each treatment, seedlings were subjected to histochemical GUS staining. Representative images of cotyledons (A), shoot apex (B), hypocotyl-root junction (C), and root tip (D) of 10 stained seedlings are shown. The scale bar corresponds to 1 μm. (EPS 77982 kb)
11033_2014_3098_MOESM5_ESM.eps (66.7 mb)
Supplementary Fig. 4 Histochemical staining of onion epidermal monolayer cells transformed with 35S::GUS (A) and 35S-UTR::GUS (B) constructs via Agrobacterium. Representative images of onion epidermal cells are shown. The scale bar corresponds to 10 µm. (EPS 68266 kb)
11033_2014_3098_MOESM6_ESM.eps (18.9 mb)
Supplementary Fig. 5 RT-PCR assay of GUS mRNA in Arabidopsis 35S::GUS and 35S-UTR::GUS lines treated with PAs and MGBG inhibitor. Total RNA was extracted from one-week-old 35S::GUS and 35S-UTR::GUS seedlings treated with 0, 1, and 100 μM of each PA (Put, Spd, and Spm) or 2 mM MGBG during 1 d. Each RNA sample (1 μg) was used for RT-PCR analyses. Ten microliters of each PCR product were separated by electrophoresis on 1.2 % (w/v) agarose gel. The Arabidopsis Actin 2 and Actin 8 (ACT2/8) genes were used as an internal control. C, 35S::GUS; L1, L2, and L3, 35S-UTR::GUS lines. (EPS 19383 kb)
11033_2014_3098_MOESM7_ESM.eps (93.9 mb)
Supplementary Fig. 6 Amino acid sequence alignments of uORFs identified in the 5′-UTR of plant polyamine oxidases 3 (PAO3) genes. Dico- and monocotyledonous PAO3 uORFs (A), dicotyledonous PAO3 uORFs (B) and monocotyledonous PAO3 uORFs (C). Sequence alignments were performed with T-Coffee (version 6.18) program and residues were shaded with BoxShade server version 3.21. Rooted phylogenetic tree of plant PAO3 uORFs (D). As outgroup the small uORF of the A. thaliana SAMDC (At3g02470) gene was used. Phylogenetic tree was created using the Parsimony method of the PHYLIP 3.67 package. Bootstrap support values out of 1,000 pseudoreplicates of the data set are provided as percentages at the corresponding nodes when > 50 %. (EPS 96157 kb)
11033_2014_3098_MOESM8_ESM.eps (80.5 mb)
Supplementary Fig. 7 Amino acid sequence alignments of uORFs identified in the 5′-UTR of plant polyamine oxidases 4 (PAO4) genes. Dico- and monocotyledonous PAO4 uORFs (A), dicotyledonous PAO4 uORFs (B) and monocotyledonous PAO4 uORFs (C). Sequence alignments were performed with T-Coffee (version 6.18) program and residues were shaded with BoxShade server version 3.21. Rooted phylogenetic tree of plant PAO4 uORFs (D). As outgroup the small uORF of the A. thaliana SAMDC (At3g02470) gene was used. Phylogenetic tree was created using the Parsimony method of the PHYLIP 3.67 package. Bootstrap support values out of 1,000 pseudoreplicates of the data set are provided as percentages at the corresponding nodes when > 50 %. (EPS 82441 kb)
11033_2014_3098_MOESM9_ESM.docx (20 kb)
Supplementary material 8 (DOCX 19 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Maria L. Guerrero-González
    • 1
  • Margarita Rodríguez-Kessler
    • 2
  • Juan F. Jiménez-Bremont
    • 1
  1. 1.Division de Biologia MolecularInstituto Potosino de Investigacion Cientifica y TecnologicaSan Luis PotosiMexico
  2. 2.Facultad de CienciasUniversidad Autonoma de San Luis PotosiSan Luis PotosiMexico

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