Skip to main content
SpringerLink
Log in

Sequence variation in Brassica AP2 and analysis of interaction of AP2-miR172 regulatory module

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Floral organ identity in Arabidopsis thaliana is regulated via interaction of APEATALA2 (AP2) transcripts with miR172. In complex genomes of Brassicas, the impact of polyploidization on sequence and expression variation in AP2 and aspects of its interaction with Brassica miR172 is poorly understood. Herein we report sequence variation and expression pattern of Brassica AP2 homologs and demonstrate interaction of AP2 transcripts with cognate miR172. Four genomic and 3 cDNA variants of AP2 were isolated from B. rapa and B. juncea. This included a splice variant (cBju2_AP2) from B. juncea which retained first intron leading to premature termination of predicted protein. Phylogenetic analysis depicted two major clades and allowed assignment of Brassica AP2 sequences to LF and MF1 subgenomes even from uncharacterized B. juncea genome. Expression analysis of two LF specific AP2 homologs (cBju1_AP2 and cBju2_AP2) revealed near identical expression pattern. However, cBju1_AP2 exhibited significantly higher expression levels compared to cBju2_AP2 implying regulatory diversification. Higher expression levels in reproductive stages implicated Brassica AP2 in flower development. Analysis of 19 Brassica AP2 partial genomic sequences isolated from 6 Brassica species showed a conserved miR172 binding site, while considerable polymorphism was observed in flanking regions. In-silico analysis of hybridization energies between miR172 (miR172b, miR172d, miR172e) and AP2 variants revealed differing total free energy of binding suggesting complex interaction patterns in Brassicas. The predicted miR172 guided cleavage of AP2 transcript (cBju1_AP2) from B. juncea was experimentally validated through tobacco transient assays. Conservation of miR172 binding site in all Brassica AP2 sequences is indicative of them being true targets of miR172 mediated PTGS albeit with varying efficiency. This suggests that in spite of polyploidy induced polymorphisms in AP2 transcript, miR172 binding site has remained conserved to preserve dynamics of interaction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Alvarez-Buylla ER, Benitez M, Corvera-Poiré A, Chaos Cador A, de Folter S, Gamboa de Buen A, Garay-Arroyo A, García-Ponce B, Jaimes-Miranda F, Pérez-Ruiz RV, Piñeyro-Nelson A, Sánchez-Corrales YE (2010) Flower development. The Arabidopsis Book 8:e0127

    Article  PubMed  PubMed Central  Google Scholar 

  • Andrés F, Coupland G (2012) The genetic basis of flowering responses to seasonal cues. Nat Rev Genet 13:627–639

    Article  PubMed  Google Scholar 

  • Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barbazuk WB, Fu Y, McGinnis KM (2008) Genome-wide analyses of alternative splicing in plants: opportunities and challenges. Genome Res 18:1381–1392

    Article  CAS  PubMed  Google Scholar 

  • Bowman JL, Smyth DR, Meyerowitz EM (1989) Genes directing flower development in Arabidopsis. Plant Cell 1:37–52

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bowman JL, Smyth DR, Meyerowitz EM (1991) Genetic interactions among floral homeotic genes of Arabidopsis. Development 112:1–20

    CAS  PubMed  Google Scholar 

  • Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303:2022–2025

    Article  CAS  PubMed  Google Scholar 

  • Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179

    Article  PubMed  PubMed Central  Google Scholar 

  • Cheng F, Mandakova T, Xie Q, Wu J, Lysak MA, Wang X (2013) Deciphering the diploid ancestral genome of the mesohexaploid Brassica rapa. Plant cell 25:1541–1554

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chung MY, Vrebalov J, Alba R, Lee J, McQuinn R, Chung JD, Klein P, Giovannoni JJ (2010) A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening. Plant J 64:936–947

    Article  CAS  PubMed  Google Scholar 

  • Coen ES, Meyerowitz EM (1991) The war of the whorls: genetic interactions controlling flower development. Nature 353:31–37

    Article  CAS  PubMed  Google Scholar 

  • de Felippes FF, Weigel D (2010) Transient assays for the analysis of miRNA processing and function. Methods Mol Biol 592:255–264

    Article  PubMed  Google Scholar 

  • de Lima Morais DA, Harrison PM (2010) Large-scale evidence for conservation of NMD candidature across mammals. PLoS ONE 5:e11695

    Article  PubMed  PubMed Central  Google Scholar 

  • Dhakate P, Shivaraj SM, Singh A (2014) Design of artificial miRNA for redundant silencing of Brassica SHP1 and SHP2: transient assay-based validation of transcript cleavage from polyploid Brassicas. Acta Physiol Plant 36:2125–2135

    Article  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissues. Focus 12:13–15

    Google Scholar 

  • Drews GN, Bowman JL, Meyerowitz EM (1991) Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product. Cell 65:991–1002

    Article  CAS  PubMed  Google Scholar 

  • Drummond AJ, Rambaut A (2007) BEAST: bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7:214

    Article  PubMed  PubMed Central  Google Scholar 

  • Ehrenreich IM, Purugganan MD (2008) Sequence variation of Arabidopsis microRNAs and their binding sites. Plant Physiol 146:1974–1982

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fankhauser C, Yeh KC, Lagarias JC, Zhang H, Elich TD, Chory J (1999) PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis. Science 284:1539–1541

    Article  CAS  PubMed  Google Scholar 

  • Fedorov A, Merican AF, Gilbert W (2002) Large-scale comparison of intron positions among animal, plant, and fungal genes. Proc Natl Acad Sci USA 99:16128–16133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fornara F, de Montaigu A, Coupland G (2010) SnapShot: control of flowering in Arabidopsis. Cell 141:550.e1–550.e2

    Article  Google Scholar 

  • Gil-Humanes J, Pistón F, Martín A, Barro F (2009) Comparative genomic analysis and expression of the APETALA2-like genes from barley, wheat, and barley—wheat amphiploids. BMC Plant Biol 29:66

    Article  Google Scholar 

  • Grigorova B, Mara C, Hollender C, Sijacic P, Chen X et al (2011) LEUNIG and SEUSS co-repressors regulate miR172 expression in Arabidopsis flowers. Development 138:2451–2456

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gu W, Wang X, Zhai C, Xie X, Zhou T (2012) Selection on synonymous sites for increased accessibility around miRNA binding sites in plants. Mol Biol Evol 29:3037–3044

    Article  CAS  PubMed  Google Scholar 

  • Gu W, Wang X, Zhai C, Zhou T, Xie X (2013) Biological basis of miRNA action when their targets are located in human protein coding region. PLoS ONE 8:e63403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Hughes TE, Langdale JA, Kelly S (2014) The impact of widespread regulatory neofunctionalization on homeolog gene evolution following whole genome duplication in maize. Genome Res 24:1348–1355

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jofuku KD, Den Boer BG, Van Montagu M, Okamuro JK (1994) Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell 6:1211–1225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jofuku K, Omidyar P, Gee Z, Okamuro J (2005) Control of seed mass and seed yield by the floral homeotic gene APETALA2. Proc Natl Acad Sci USA 102:3117–3122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kalyna M, Simpso CG, Syed NH, Lewandowska D, Marquez Y, Kusenda B, Marshall J, Fuller J, Cardle L, McNicol J et al (2012) Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis. Nucleic Acids Res 40:2454–2469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kapustin Y, Souvorov A, Tatusova T, Lipman D (2008) Splign: algorithms for computing spliced alignments with identification of paralogs. Biol Direct 3:20

    Article  PubMed  PubMed Central  Google Scholar 

  • Karlova R, Rosin FM, Busscher-Lange J, Parapunova V, Do PT et al (2011) Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. Plant Cell 23:923–941

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Keck E, McSteen P, Carpenter R, Coen E (2003) Separation of genetic functions controlling organ identity in flowers. EMBO J 22:1058–1066

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Keele BF, Giorgi EE, Salazar-Gonzalez JF, Decker JM, Pham KT, Salazar MG, Sun C et al (2008) Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci USA 105:7552–7557

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kertesz M, Iovino N, Unnerstall U, Gaul U, Segal E (2007) The role of site accessibility in microRNA target recognition. Nat Genet 39:1278–1284

    Article  CAS  PubMed  Google Scholar 

  • Kim S, Soltis PS, Wall K, Soltis DE (2006) Phylogeny and domain evolution in the APETALA2-like gene family. Mol Biol Evol 23:107–120

    Article  CAS  PubMed  Google Scholar 

  • Koch MA, Haubold B, Mitchell-Olds T (2001) Molecular systematics of the Brassicaceae: evidence from coding plastidic matK and nuclear Chs sequences. Am J Bot 88:534–544

    Article  CAS  PubMed  Google Scholar 

  • Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396

    Article  CAS  Google Scholar 

  • Krasileva KV, Buffalo V, Bailey P, Pearce S, Ayling S, Tabbita F, Dubcovsky J (2013) Separating homeologs by phasing in the tetraploid wheat transcriptome. Genome Biol 14:R66

    Article  PubMed  PubMed Central  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948

    Article  CAS  PubMed  Google Scholar 

  • Lauter N, Kampani A, Carlson S, Goebel M, Moose SP (2005) microRNA172 down-regulates glossy15 to promote vegetative phase change in maize. Proc Natl Acad Sci USA 102:9412–9417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452

    Article  CAS  PubMed  Google Scholar 

  • Liu SL, Adams KL (2010) Dramatic change in function and expression pattern of a gene duplicated by polyploidy created a paternal effect gene in the Brassicaceae. Mol Biol Evol 27:2817–2828

    Article  CAS  PubMed  Google Scholar 

  • Long D, Lee R, Williams P, Chan CY, Ambros V, Ding Y (2007) Potent effect of target structure on microRNA function. Nat Struct Mol Biol 14:287–294

    Article  CAS  PubMed  Google Scholar 

  • Lukens LN, Quijada PA, Udall J, Pires JC, Schranz ME, Osborn TC (2004) Genome redundancy and plasticity within ancient and recent Brassica crop species. Biol J Linn Soc 82:665–674

    Article  Google Scholar 

  • Lysak MA, Koch MA, Pecinka A, Schubert I (2005) Chromosome triplication found across the tribe Brassiceae. Genome Res 15:516–525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Maes T, Van de Steene N, Zethof J, Karimi M, D’Hauw M, Mares G, van Montagu M, Gerats T (2001) Petunia AP2-like genes and their role in flower and seed development. Plant Cell 13:229–244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Muckstein U, Tafer H, Hackermuller J, Bernhart SH, Stadler PF, Hofacker IL (2006) Thermodynamics of RNA–RNA binding. Bioinformatics 22:1177–1182

    Article  PubMed  Google Scholar 

  • Nair SK, Wang N, Turuspekov Y et al (2010) Cleistogamous flowering in barley arises from the suppression of microRNA-guided HvAP2 mRNA cleavage. Proc Natl Acad Sci USA 107:490–495

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol 140:411–432

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nilsson L, Carlsbecker A, Sundas-Larsson A, Vahala T (2007) APETALA2 like genes from Picea abies show functional similarities to their Arabidopsis homologues. Planta 225:589–602

    Article  CAS  PubMed  Google Scholar 

  • Ohto MA, Fischer RL, Goldberg RB, Nakamura K, Harada JJ (2005) Control of seed mass by APETALA2. Proc Natl Acad Sci USA 102:3123–3128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Okamuro JK, Caster B, Villarroel R, Van Montagu M, Jofuku KD (1997) The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc Natl Acad Sci USA 94:7076–7081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF (2000) B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405:200–203

    Article  CAS  PubMed  Google Scholar 

  • Reddy ASN, Marquez Y, Kalyna M, Bartab A (2013) Complexity of the alternative splicing landscape in plants. Plant Cell 25:3657–3683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ripoll JJ, Roeder AH, Ditta GS, Yanofsky MF (2011) A novel role for the floral homeotic gene APETALA2 during Arabidopsis fruit development. Development 138:5167–5176

    Article  CAS  PubMed  Google Scholar 

  • Rosloski SM, Singh A, Jali SS, Balasubramanian S, Weigel D, Grbic V (2013) Functional analysis of splice variant expression of MADS AFFECTING FLOWERING 2 of Arabidopsis thaliana. Plant Mol Biol 81:57–69

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sayani S, Janis M, Lee CY, Toesca I, Chanfreau GF (2008) Widespread impact of nonsense-mediated mRNA decay on the yeast intronome. Mol Cell 31:360–370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schranz ME, Osborn TC (2000) Novel flowering time variation in the resynthesized polyploid Brassica napus. J Hered 91:242–246

    Article  CAS  PubMed  Google Scholar 

  • Schranz ME, Lysak MA, Mitchell-Olds T (2006) The ABC’s of comparative genomics in the Brassicaceae: building blocks of crucifer genomes. Trends Plant Sci 11:535–542

    Article  CAS  PubMed  Google Scholar 

  • Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8:517–527

    Article  CAS  PubMed  Google Scholar 

  • Shigyo M, Hasebe M, Ito M (2006) Molecular evolution of the AP2 subfamily. Gene 2:256–265

    Article  Google Scholar 

  • Shivaraj SM, Dhakate P, Mayee P, Negi MS, Singh A (2014) Natural genetic variation in MIR172 isolated from Brassicas. Biol Plant 58:627–640

    Article  CAS  Google Scholar 

  • Simons KJ, Fellers JP, Trick HN, Zhang ZC, Tai YS, Gill BS, Faris JD (2006) Molecular characterization of the major wheat domestication gene Q. Genetics 172:547–555

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Srikanth A, Schmid M (2011) Regulation of flowering time: all roads lead to Rome. Cell Mol Life Sci 68:2013–2037

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Wang H, Wang J, Sun R, Wu J et al (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039

    Article  CAS  PubMed  Google Scholar 

  • Warwick SI, Black LD (1991) Molecular systematics of Brassica and allied genera (subtribe Brassicinae Brassicae) chloroplast genome and cytodeme congruence. Theor Appl Genet 82:81–92

    Article  CAS  PubMed  Google Scholar 

  • Warwick SI, Francis A, La Fleche J (2000) Guide to wild germplasm of Brassicaceae, 2nd edn. Technical Bulletin 1993-14E, Center for Land and Biological Resources Research, Research Branch, Agriculture Canada

  • Wellmer F, Graciet E, Riechmann JL (2014) Specification of floral organs in Arabidopsis. J Exp Bot 65:1–9

    Article  CAS  PubMed  Google Scholar 

  • Wollmann H, Mica E, Todesco M, Long JA, Weigel D (2010) On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development. Development 137:3633–3642

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wurschum T, Gross-Hardt R, Laux T (2006) APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem. Plant Cell 18:295–307

    Article  PubMed  PubMed Central  Google Scholar 

  • Yan X, Zhang L, Chen B, Xiong Z, Chen C et al (2012) Functional identification and characterization of the Brassica napus transcription factor gene BnAP2, the ortholog of Arabidopsis Thaliana APETALA2. PLoS ONE 7:e33890

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu QH, Helliwell CA (2011) Regulation of flowering time and floral patterning by miR172. J Exp Bot 62:487–495

    Article  CAS  PubMed  Google Scholar 

  • Zhu QH, Upadhyaya NM, Gubler F, Helliwell CA (2009) Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa). BMC Plant Biol 9:149

    Article  PubMed  PubMed Central  Google Scholar 

  • Ziolkowski PA, Kaczmarek M, Babula D, Sadowski J (2006) Genome evolution in Arabidopsis/Brassica: conservation and divergence of ancient rearranged segments and their breakpoints. Plant J 47:63–74

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants (BT/PR10071/AGR/36/31/2007 and BT/PR628/AGR/36/674/2011) received from Department of Biotechnology, Government of India. Contribution from Tanu Sri and Shikha Tyagi (Department of Biotechnology, TERI University) towards critical reading of manuscript is acknowledged. The contribution of Mr. Hari Ram Gupta in field-related activities is duly recognized. Financial assistance as SRF to S.M. Shivaraj from Department of Biotechnology, Government of India is gratefully acknowledged. Infrastructural support from TERI and TERI University are duly acknowledged.

Authors contribution

A.S. conceived the study. A.S. and S.M.S. designed the experiments, analyzed the data and prepared the manuscript. S.M.S. performed the experiments. Both authors have seen the manuscript and agree to the same.

Author information

Authors and Affiliations

  1. Department of Biotechnology, TERI University, 10 Institutional Area, Vasant Kunj, Delhi, 110070, India

    S. M. Shivaraj & Anandita Singh

Authors
  1. S. M. Shivaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anandita Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anandita Singh.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 595 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shivaraj, S.M., Singh, A. Sequence variation in Brassica AP2 and analysis of interaction of AP2-miR172 regulatory module. Plant Cell Tiss Organ Cult 125, 191–206 (2016). https://doi.org/10.1007/s11240-015-0938-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-015-0938-5

Keywords

Search

Navigation