Regulation of the alkaloid biosynthesis by miRNA in opium poppy.

Opium poppy (Papaver somniferum) is an important medicinal plant producing benzylisoquinoline alkaloids (BIA). MicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) of approximately 21 nucleotides. They are noncoding, but regulate gene expression in eukaryotes. Although many studies have been conducted on the identification and functions of plant miRNA, scarce researches on miRNA regulation of alkaloid biosynthesis have been reported. In this study, a total of 316 conserved and 11 novel miRNAs were identified in opium poppy using second-generation sequencing and direct cloning. Tissue-specific regulation of miRNA expression was comparatively analysed by miRNA microarray assays. A total of 232 miRNAs were found to be differentially expressed among four tissues. Likewise, 1469 target transcripts were detected using in silico and experimental approaches. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates carbohydrate metabolism and genetic-information processing. Additionally, miRNA target transcripts were mostly involved in response to stress against various factors and secondary-metabolite biosynthesis processes. Target transcript identification analyses revealed that some of the miRNAs might be involved in BIA biosynthesis, such as pso-miR13, pso-miR2161 and pso-miR408. Additionally, three putatively mature miRNA sequences were predicted to be targeting BIA-biosynthesis genes.

[1]  R. Shearman,et al.  Genome-wide profiling and analysis of Festuca arundinacea miRNAs and transcriptomes in response to foliar glyphosate application , 2010, Molecular Genetics and Genomics.

[2]  Sean R. Eddy,et al.  Rfam: annotating non-coding RNAs in complete genomes , 2004, Nucleic Acids Res..

[3]  Aili Li,et al.  Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in bread wheat (Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv , 2009, Functional & Integrative Genomics.

[4]  D. Bartel,et al.  MicroRNAS and their regulatory roles in plants. , 2006, Annual review of plant biology.

[5]  T. Unver,et al.  Genome-Wide Identification of miRNAs Responsive to Drought in Peach (Prunus persica) by High-Throughput Deep Sequencing , 2012, PloS one.

[6]  P. Hernández,et al.  Genome-wide identification of alternate bearing-associated microRNAs (miRNAs) in olive (Olea europaea L.) , 2013, BMC Plant Biology.

[7]  N. Chua,et al.  MicroRNA Directs mRNA Cleavage of the Transcription Factor NAC1 to Downregulate Auxin Signals for Arabidopsis Lateral Root Development , 2005, The Plant Cell Online.

[8]  P. Facchini,et al.  The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy. , 2006, The Plant journal : for cell and molecular biology.

[9]  H. Budak,et al.  Conserved microRNAs and their targets in model grass species Brachypodium distachyon , 2009, Planta.

[10]  L. Luo,et al.  Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa. , 2010, Journal of experimental botany.

[11]  T. Unver,et al.  Plant microRNAs: new players in functional genomics , 2013 .

[12]  J. Page.  Silencing nature's narcotics: metabolic engineering of the opium poppy. , 2005, Trends in biotechnology.

[13]  J. Leebens-Mack,et al.  Large-scale identification of microRNAs from a basal eudicot (Eschscholzia californica) and conservation in flowering plants. , 2007, The Plant journal : for cell and molecular biology.

[14]  Ruiqiang Li,et al.  SOAP: short oligonucleotide alignment program , 2008, Bioinform..

[15]  Uwe Ohler,et al.  High-resolution experimental and computational profiling of tissue-specific known and novel miRNAs in Arabidopsis. , 2012, Genome research.

[16]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[17]  X. Deng,et al.  Global identification of miRNAs and targets in Populus euphratica under salt stress , 2013, Plant Molecular Biology.

[18]  P. Zielenkiewicz,et al.  High-throughput sequencing identification of novel and conserved miRNAs in the Brassica oleracea leaves , 2013, BMC Genomics.

[19]  P. Facchini,et al.  Alkaloid biosynthesis: metabolism and trafficking. , 2008, Annual review of plant biology.

[20]  M. Gu,et al.  Role of microRNAs in aluminum stress in plants , 2014, Plant Cell Reports.

[21]  F. Bilka,et al.  Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures. , 2010, Plant physiology and biochemistry : PPB.

[22]  Stijn van Dongen,et al.  miRBase: tools for microRNA genomics , 2007, Nucleic Acids Res..

[23]  C. Wijekoon,et al.  Systematic knockdown of morphine pathway enzymes in opium poppy using virus-induced gene silencing. , 2012, The Plant journal : for cell and molecular biology.

[24]  K. Chong,et al.  Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response , 2009, BMC Genomics.

[25]  Baohong Zhang,et al.  MicroRNA‐Based Biotechnology for Plant Improvement , 2015, Journal of cellular physiology.

[26]  P. Facchini,et al.  Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy. , 2012, The Plant journal : for cell and molecular biology.

[27]  H. Budak,et al.  Regulation of barley miRNAs upon dehydration stress correlated with target gene expression , 2010, Functional & Integrative Genomics.

[28]  Z. Chen,et al.  cis- and trans-Regulation of miR163 and Target Genes Confers Natural Variation of Secondary Metabolites in Two Arabidopsis Species and Their Allopolyploids[W][OA] , 2011, Plant Cell.

[29]  Zongrang Liu,et al.  Apple miRNAs and tasiRNAs with novel regulatory networks , 2012, Genome Biology.

[30]  Jonathan D. G. Jones,et al.  The microRNA miR393 re-directs secondary metabolite biosynthesis away from camalexin and towards glucosinolates. , 2011, The Plant journal : for cell and molecular biology.

[31]  Hajime Sakai,et al.  Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016238. , 2003, The Plant Cell Online.

[32]  T. Unver,et al.  Identification of conserved micro-RNAs and their target transcripts in opium poppy (Papaver somniferum L.) , 2010, Plant Cell Reports.

[33]  A. Covarrubias,et al.  Conserved and novel miRNAs in the legume Phaseolus vulgaris in response to stress , 2009, Plant Molecular Biology.

[34]  P. Facchini,et al.  Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. , 2004, The Plant journal : for cell and molecular biology.

[35]  I. Sharma,et al.  MicroRNA mediated regulation of metal toxicity in plants: present status and future perspectives , 2013, Plant Molecular Biology.

[36]  Javier F. Palatnik,et al.  Specific effects of microRNAs on the plant transcriptome. , 2005, Developmental cell.

[37]  T. Unver,et al.  Transcriptome Profiling of Alkaloid Biosynthesis in Elicitor Induced Opium Poppy , 2014, Plant Molecular Biology Reporter.

[38]  D. Schriemer,et al.  Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures , 2010, BMC Plant Biology.

[39]  M. Cariaso,et al.  Identification of conserved and novel microRNAs that are responsive to heat stress in Brassica rapa , 2011, Journal of experimental botany.

[40]  Patrick Achard,et al.  Modulation of floral development by a gibberellin-regulated microRNA , 2004, Development.

[41]  Giorgio Valle,et al.  High throughput approaches reveal splicing of primary microRNA transcripts and tissue specific expression of mature microRNAs in Vitis vinifera , 2009, BMC Genomics.

[42]  Baohong Zhang,et al.  Boron Stress Responsive MicroRNAs and Their Targets in Barley , 2013, PloS one.

[43]  Jonathan D. G. Jones,et al.  A Plant miRNA Contributes to Antibacterial Resistance by Repressing Auxin Signaling , 2006, Science.

[44]  Adam M. Gustafson,et al.  microRNA-Directed Phasing during Trans-Acting siRNA Biogenesis in Plants , 2005, Cell.

[45]  Baohong Zhang,et al.  MicroRNAs and their regulatory roles in animals and plants , 2007, Journal of cellular physiology.

[46]  D. Weigel,et al.  Transcriptional Control of Gene Expression by MicroRNAs , 2010, Cell.

[47]  Yuki Moriya,et al.  KAAS: an automatic genome annotation and pathway reconstruction server , 2007, Nucleic Acids Res..

[48]  Dustin J Cram,et al.  Integration of deep transcript and targeted metabolite profiles for eight cultivars of opium poppy , 2012, Plant Molecular Biology.

[49]  Xuemei Chen,et al.  A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development , 2004, Science.

[50]  Jack A. M. Leunissen,et al.  Turning CFCs into salt. , 1996, Nucleic Acids Res..

[51]  Patrick Xuechun Zhao,et al.  psRNATarget: a plant small RNA target analysis server , 2011, Nucleic Acids Res..

[52]  J. Ziegler,et al.  The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[53]  Baohong Zhang,et al.  Identification of 188 conserved maize microRNAs and their targets , 2006, FEBS letters.

[54]  M. Atak,et al.  Genome-wide fungal stress responsive miRNA expression in wheat , 2014, Planta.

[55]  M. Robles,et al.  University of Birmingham High throughput functional annotation and data mining with the Blast2GO suite , 2022 .

[56]  Yiyong Zhu,et al.  Role of microRNAs in plant responses to nutrient stress , 2013, Plant and Soil.

[57]  M. Willmann,et al.  Global Regulation of Embryonic Patterning in Arabidopsis by MicroRNAs1[W][OPEN] , 2014, Plant Physiology.

[58]  Siu-Ming Yiu,et al.  SOAP2: an improved ultrafast tool for short read alignment , 2009, Bioinform..