MicroRNA390-Directed TAS3 Cleavage Leads to the Production of tasiRNA-ARF3/4 During Somatic Embryogenesis in Dimocarpus longan Lour
Identifieur interne : 000096 ( Pmc/Corpus ); précédent : 000095; suivant : 000097MicroRNA390-Directed TAS3 Cleavage Leads to the Production of tasiRNA-ARF3/4 During Somatic Embryogenesis in Dimocarpus longan Lour
Auteurs : Yuling Lin ; Lixia Lin ; Ruilian Lai ; Weihua Liu ; Yukun Chen ; Zihao Zhang ; Xu Xuhan ; Zhongxiong LaiSource :
- Frontiers in Plant Science [ 1664-462X ] ; 2015.
Abstract
Url:
DOI: 10.3389/fpls.2015.01119
PubMed: 26734029
PubMed Central: 4680215
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">MicroRNA390-Directed <italic>TAS3</italic> Cleavage Leads to the Production of tasiRNA-<italic>ARF3/4</italic> During Somatic Embryogenesis in <italic>Dimocarpus longan</italic> Lour</title><author><name sortKey="Lin, Yuling" sort="Lin, Yuling" uniqKey="Lin Y" first="Yuling" last="Lin">Yuling Lin</name></author><author><name sortKey="Lin, Lixia" sort="Lin, Lixia" uniqKey="Lin L" first="Lixia" last="Lin">Lixia Lin</name></author><author><name sortKey="Lai, Ruilian" sort="Lai, Ruilian" uniqKey="Lai R" first="Ruilian" last="Lai">Ruilian Lai</name></author><author><name sortKey="Liu, Weihua" sort="Liu, Weihua" uniqKey="Liu W" first="Weihua" last="Liu">Weihua Liu</name></author><author><name sortKey="Chen, Yukun" sort="Chen, Yukun" uniqKey="Chen Y" first="Yukun" last="Chen">Yukun Chen</name></author><author><name sortKey="Zhang, Zihao" sort="Zhang, Zihao" uniqKey="Zhang Z" first="Zihao" last="Zhang">Zihao Zhang</name></author><author><name sortKey="Xuhan, Xu" sort="Xuhan, Xu" uniqKey="Xuhan X" first="Xu" last="Xuhan">Xu Xuhan</name></author><author><name sortKey="Lai, Zhongxiong" sort="Lai, Zhongxiong" uniqKey="Lai Z" first="Zhongxiong" last="Lai">Zhongxiong Lai</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26734029</idno><idno type="pmc">4680215</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4680215</idno><idno type="RBID">PMC:4680215</idno><idno type="doi">10.3389/fpls.2015.01119</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000096</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">MicroRNA390-Directed <italic>TAS3</italic> Cleavage Leads to the Production of tasiRNA-<italic>ARF3/4</italic> During Somatic Embryogenesis in <italic>Dimocarpus longan</italic> Lour</title><author><name sortKey="Lin, Yuling" sort="Lin, Yuling" uniqKey="Lin Y" first="Yuling" last="Lin">Yuling Lin</name></author><author><name sortKey="Lin, Lixia" sort="Lin, Lixia" uniqKey="Lin L" first="Lixia" last="Lin">Lixia Lin</name></author><author><name sortKey="Lai, Ruilian" sort="Lai, Ruilian" uniqKey="Lai R" first="Ruilian" last="Lai">Ruilian Lai</name></author><author><name sortKey="Liu, Weihua" sort="Liu, Weihua" uniqKey="Liu W" first="Weihua" last="Liu">Weihua Liu</name></author><author><name sortKey="Chen, Yukun" sort="Chen, Yukun" uniqKey="Chen Y" first="Yukun" last="Chen">Yukun Chen</name></author><author><name sortKey="Zhang, Zihao" sort="Zhang, Zihao" uniqKey="Zhang Z" first="Zihao" last="Zhang">Zihao Zhang</name></author><author><name sortKey="Xuhan, Xu" sort="Xuhan, Xu" uniqKey="Xuhan X" first="Xu" last="Xuhan">Xu Xuhan</name></author><author><name sortKey="Lai, Zhongxiong" sort="Lai, Zhongxiong" uniqKey="Lai Z" first="Zhongxiong" last="Lai">Zhongxiong Lai</name></author></analytic><series><title level="j">Frontiers in Plant Science</title><idno type="eISSN">1664-462X</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Trans</italic>-acting short-interfering RNAs (tasiRNAs) originate from <italic>TAS3</italic> families through microRNA (miRNA) 390-guided cleavage of primary transcripts and target auxin response factors (ARF3/-4), which are involved in the normal development of lateral roots and flowers in plants. However, their roles in embryo development are still unclear. Here, the pathway miR390-<italic>TAS3</italic>-<italic>ARF3</italic>/<italic>-4</italic> was identified systematically for the first time during somatic embryo development in <italic>Dimocarpus longan</italic>. We identified the miR390 primary transcript and promoter. The promoter contained <italic>cis</italic>-acting elements responsive to stimuli such as light, salicylic acid, anaerobic induction, fungal elicitor, circadian control, and heat stress. The longan <italic>TAS3</italic> transcript, containing two miR390-binding sites, was isolated; the miR390- guided cleavage site located near the 3′ end of the <italic>TAS3</italic> transcript was verified. Eight <italic>TAS3</italic>-tasiRNAs with the 21-nucleotides phase were found among longan small RNA data, further confirming that miR390-directed <italic>TAS3</italic> cleavage leads to the production of tasiRNA in longan. Among them, TAS3_5′D5+ and 5′D6+ tasiRNAs were highly abundant, and verified to target <italic>ARF3</italic> and <italic>-4</italic>, implying that miR390-guided <italic>TAS3</italic> cleavage with 21-nucleotides phase leading to the production of tasiRNA-ARF is conserved in plants. Pri-miR390 was highly expressed in friable-embryogenic callus (EC), and less expressed in incomplete compact pro-embryogenic cultures, while miR390 showed its lowest expression in EC and highest expression in torpedo-shaped embryos (TEs). <italic>DlTAS3</italic> and <italic>DlARF4</italic> both exhibited their lowest expressions in EC, and reached their peaks in the globular embryos stage, which were mainly inversely proportional to the expression of miR390, especially at the globular embryos to cotyledonary embryos (CEs) stages. While <italic>DlARF3</italic> showed little variation from the EC to TEs stages, and exhibited its lowest expression in the CEs stage. There was a general lack of correlation between the expressions of <italic>DlARF3</italic> and miR390. In addition, pri-miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> were up-regulated by 2,4-<sc>D</sc> in a concentration-dependent manner. They were also preferentially expressed in roots, pulp, and seeds of ‘Sijimi’ longan, implying their extended roles in the development of longan roots and fruit. This study provided insights into a possible role of miR390-tasiRNAs-ARF in plant somatic embryo development.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Axtell, M J" uniqKey="Axtell M">M. J. Axtell</name></author><author><name sortKey="Jan, C" uniqKey="Jan C">C. Jan</name></author><author><name sortKey="Rajagopalan, R" uniqKey="Rajagopalan R">R. Rajagopalan</name></author><author><name sortKey="Bartel, D P" uniqKey="Bartel D">D. P. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Front Plant Sci</journal-id><journal-id journal-id-type="iso-abbrev">Front Plant Sci</journal-id><journal-id journal-id-type="publisher-id">Front. Plant Sci.</journal-id><journal-title-group><journal-title>Frontiers in Plant Science</journal-title></journal-title-group><issn pub-type="epub">1664-462X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26734029</article-id><article-id pub-id-type="pmc">4680215</article-id><article-id pub-id-type="doi">10.3389/fpls.2015.01119</article-id><article-categories><subj-group subj-group-type="heading"><subject>Plant Science</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>MicroRNA390-Directed <italic>TAS3</italic> Cleavage Leads to the Production of tasiRNA-<italic>ARF3/4</italic> During Somatic Embryogenesis in <italic>Dimocarpus longan</italic> Lour</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Yuling</given-names></name></contrib><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Lixia</given-names></name></contrib><contrib contrib-type="author"><name><surname>Lai</surname><given-names>Ruilian</given-names></name></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Weihua</given-names></name></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Yukun</given-names></name></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Zihao</given-names></name></contrib><contrib contrib-type="author"><name><surname>XuHan</surname><given-names>Xu</given-names></name></contrib><contrib contrib-type="author"><name><surname>Lai</surname><given-names>Zhongxiong</given-names></name><xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/262941/overview"></uri></contrib></contrib-group><aff id="aff1"><institution>Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University</institution><country>Fuzhou, China</country></aff><author-notes><fn fn-type="edited-by"><p>Edited by: <italic>Agnieszka Ludwików, Adam Mickiewicz University in Poznań, Poland</italic></p></fn><fn fn-type="edited-by"><p>Reviewed by: <italic>Andrzej Miroslaw Pacak, Adam Mickiewicz University in Poznań, Poland; Antonio Ferrante, Università degli Studi di Milano, Italy</italic></p></fn><corresp id="fn001">*Correspondence: <italic>Zhongxiong Lai, <email xlink:type="simple">laizx01@163.com</email></italic></corresp><fn fn-type="other" id="fn002"><p>This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science</p></fn></author-notes><pub-date pub-type="epub"><day>16</day><month>12</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>6</volume><elocation-id>1119</elocation-id><history><date date-type="received"><day>22</day><month>9</month><year>2015</year></date><date date-type="accepted"><day>26</day><month>11</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015 Lin, Lin, Lai, Liu, Chen, Zhang, XuHan and Lai.</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Lin, Lin, Lai, Liu, Chen, Zhang, XuHan and Lai</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><italic>Trans</italic>-acting short-interfering RNAs (tasiRNAs) originate from <italic>TAS3</italic> families through microRNA (miRNA) 390-guided cleavage of primary transcripts and target auxin response factors (ARF3/-4), which are involved in the normal development of lateral roots and flowers in plants. However, their roles in embryo development are still unclear. Here, the pathway miR390-<italic>TAS3</italic>-<italic>ARF3</italic>/<italic>-4</italic> was identified systematically for the first time during somatic embryo development in <italic>Dimocarpus longan</italic>. We identified the miR390 primary transcript and promoter. The promoter contained <italic>cis</italic>-acting elements responsive to stimuli such as light, salicylic acid, anaerobic induction, fungal elicitor, circadian control, and heat stress. The longan <italic>TAS3</italic> transcript, containing two miR390-binding sites, was isolated; the miR390- guided cleavage site located near the 3′ end of the <italic>TAS3</italic> transcript was verified. Eight <italic>TAS3</italic>-tasiRNAs with the 21-nucleotides phase were found among longan small RNA data, further confirming that miR390-directed <italic>TAS3</italic> cleavage leads to the production of tasiRNA in longan. Among them, TAS3_5′D5+ and 5′D6+ tasiRNAs were highly abundant, and verified to target <italic>ARF3</italic> and <italic>-4</italic>, implying that miR390-guided <italic>TAS3</italic> cleavage with 21-nucleotides phase leading to the production of tasiRNA-ARF is conserved in plants. Pri-miR390 was highly expressed in friable-embryogenic callus (EC), and less expressed in incomplete compact pro-embryogenic cultures, while miR390 showed its lowest expression in EC and highest expression in torpedo-shaped embryos (TEs). <italic>DlTAS3</italic> and <italic>DlARF4</italic> both exhibited their lowest expressions in EC, and reached their peaks in the globular embryos stage, which were mainly inversely proportional to the expression of miR390, especially at the globular embryos to cotyledonary embryos (CEs) stages. While <italic>DlARF3</italic> showed little variation from the EC to TEs stages, and exhibited its lowest expression in the CEs stage. There was a general lack of correlation between the expressions of <italic>DlARF3</italic> and miR390. In addition, pri-miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> were up-regulated by 2,4-<sc>D</sc> in a concentration-dependent manner. They were also preferentially expressed in roots, pulp, and seeds of ‘Sijimi’ longan, implying their extended roles in the development of longan roots and fruit. This study provided insights into a possible role of miR390-tasiRNAs-ARF in plant somatic embryo development.</p></abstract><kwd-group><kwd><italic>Dimocarpus longan</italic></kwd><kwd>somatic embryo</kwd><kwd>microRNA390</kwd><kwd><italic>TAS3</italic>-tasiRNA</kwd><kwd>auxin response factors</kwd><kwd>gene regulation</kwd></kwd-group><counts><fig-count count="5"></fig-count><table-count count="4"></table-count><equation-count count="0"></equation-count><ref-count count="60"></ref-count><page-count count="15"></page-count><word-count count="0"></word-count></counts></article-meta></front><body><sec><title>Introduction</title><p>MicroRNAs and tasiRNAs are distinct classes of small RNAs, which control many aspects of development in plants by guiding silencing of target RNAs via cleavage or repression mechanisms (<xref rid="B4" ref-type="bibr">Chapman and Carrington, 2007</xref>). MiRNAs arise from endogenous transcripts that can form local fold-back structures, whereas tasiRNAs are generated by primary TAS transcripts that are initially targeted and sliced by miR173 (<italic>TAS1</italic> and <italic>TAS2</italic>), miR828 (<italic>TAS4</italic>) or miR390 (<italic>TAS3</italic>) (<xref rid="B41" ref-type="bibr">Pullman et al., 2003</xref>; <xref rid="B50" ref-type="bibr">Wilson et al., 2005</xref>; <xref rid="B1" ref-type="bibr">Axtell et al., 2006</xref>; <xref rid="B18" ref-type="bibr">Kikuchi et al., 2006</xref>; <xref rid="B39" ref-type="bibr">Montgomery et al., 2008b</xref>). The <italic>TAS3</italic> family is conserved in plants and has two sites that are complementary to miR390 (<xref rid="B19" ref-type="bibr">Krasnikova et al., 2013</xref>; <xref rid="B52" ref-type="bibr">Xia et al., 2013</xref>), while the <italic>TAS1</italic>, <italic>TAS2</italic>, and <italic>TAS4</italic> families are found only in <italic>Arabidopsis</italic><italic>thaliana</italic> or close relatives, and each of them contain only a single site complementary to miR173 or miR828 (<xref rid="B41" ref-type="bibr">Pullman et al., 2003</xref>; <xref rid="B50" ref-type="bibr">Wilson et al., 2005</xref>; <xref rid="B1" ref-type="bibr">Axtell et al., 2006</xref>; <xref rid="B18" ref-type="bibr">Kikuchi et al., 2006</xref>; <xref rid="B39" ref-type="bibr">Montgomery et al., 2008b</xref>).</p><p><italic>TAS1</italic> and <italic>TAS2</italic> tasiRNAs target PPR proteins (<xref rid="B18" ref-type="bibr">Kikuchi et al., 2006</xref>; <xref rid="B45" ref-type="bibr">Ruduś et al., 2006</xref>). <italic>TAS4</italic> tasiRNAs target MYB transcription factors (<xref rid="B42" ref-type="bibr">Rajagopalan et al., 2006</xref>). <italic>TAS3</italic> tasiRNAs target <italic>ARF3</italic> and <italic>-4</italic>, which are involved in developmental timing (<xref rid="B6" ref-type="bibr">Cho et al., 2012</xref>) and the normal development of aerial lateral organs, such as lateral roots (<xref rid="B36" ref-type="bibr">Marin et al., 2010</xref>; <xref rid="B55" ref-type="bibr">Yoon et al., 2010</xref>) and flowers (<xref rid="B37" ref-type="bibr">Matsui et al., 2014</xref>).</p><p>The roles of the pathway miR390-<italic>TAS3</italic>-<italic>ARF3</italic>/<italic>-4</italic> in root and flower development are conserved and clear in plants; however, their functions in plant embryo development are unclear. For example, miR390 peaked in mature embryos (<xref rid="B58" ref-type="bibr">Zhang et al., 2012</xref>), and three detected tasiRNAs generated from <italic>TAS3</italic>, which were triggered by miR390, peaked in mature embryos in larch (<italic>Larix leptolepis</italic>; <xref rid="B57" ref-type="bibr">Zhang et al., 2013</xref>), suggesting that the miR390-<italic>TAS3</italic>-tasiRNA pathway might play regulatory roles during CE development. In Valencia sweet orange, miR390 is involved in globular- shaped embryo formation (<xref rid="B51" ref-type="bibr">Wu et al., 2011</xref>), and over-expression of Csi- MIR390 callus lines caused lost embryogenesis capacity (<xref rid="B35" ref-type="bibr">Liu et al., 2013</xref>). During <italic>Gossypium hirsutum</italic> SE, miR390 reached the highest expression level at the EC stage, remained at moderate levels during embryo development, and one <italic>TAS3</italic> transcript was verified as the target of miR390 (<xref rid="B54" ref-type="bibr">Yang et al., 2013</xref>). As mentioned above, notably, the roles of the pathway miR390-<italic>TAS3</italic>-<italic>ARF3/-4</italic> in embryo development are somewhat confused in different plants, and their functions in plant embryos remain unclear.</p><p><italic>Dimocarpus longan</italic> (longan), a member of the Sapindaceae family, is an exotic subtropical fruit mainly planted in Northern Burma, and Northeast and Southern China. Longan fruit are preferably eaten fresh because of their sweet flavor and beneficial health effects, and usually contain a relatively large black or brown seed at maturity, with a large quantity of polysaccharides (<xref rid="B49" ref-type="bibr">Tseng et al., 2014</xref>). The seeds have also been used as bioactive ingredients in many traditional Chinese medicines to improve human health and increase the immunomodulatory capacity (<xref rid="B43" ref-type="bibr">Rangkadilok et al., 2005</xref>). However, the molecular mechanism of longan seed development remains unclear because of the extreme genetic heterozygosity exhibited and the difficulty of sampling the early embryos (<xref rid="B34" ref-type="bibr">Liu et al., 2010</xref>; <xref rid="B24" ref-type="bibr">Lai and Lin, 2013</xref>; <xref rid="B31" ref-type="bibr">Lin and Lai, 2013a</xref>,<xref rid="B32" ref-type="bibr">b</xref>; <xref rid="B33" ref-type="bibr">Lin et al., 2015</xref>). To avoid these difficulties, longan SE, which resembles zygotic embryogenesis, has previously been used widely as a model system to investigate <italic>in vitro</italic> and <italic>in vivo</italic> regulation of embryogenesis in plants (<xref rid="B25" ref-type="bibr">Lai et al., 2010</xref>; <xref rid="B30" ref-type="bibr">Lin and Lai, 2010</xref>). Our previous work indicated that dlo-miR390 a.1 and -a<sup>∗</sup>.1 accumulated during heart- and TE stages (<xref rid="B32" ref-type="bibr">Lin and Lai, 2013b</xref>). However, how miR390 directs the formation of tasiRNAs, and down- regulates the expression of target <italic>ARF3</italic> and <italic>-4</italic> during longan SE, remains unclear.</p><p>In this study, the conserved pathway miR390-<italic>TAS3</italic>-<italic>ARF3</italic>/<italic>-4</italic> was identified in longan using an integrated strategy including RT-PCR/RACE, computational, genome-wide expression profiling and experimental validation. First, the primary miR390 and <italic>TAS3</italic> transcripts were cloned by RT-PCR and RACE. The promoter of primary miR390 was isolated by Tail-PCR and its <italic>cis</italic>-acting elements were predicted using bioinformatic methods. The <italic>TAS3</italic> tasiRNAs triggered by miR390 were detected from a longan small RNA database (<xref rid="B32" ref-type="bibr">Lin and Lai, 2013b</xref>). TasiRNA-ARF targets were identified by a modified RLM-RACE. The expressions of primary miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> were analyzed in <italic>D. longan</italic> ‘Honghezi’ SE and in ‘Sijimi’s tissues. Their responses to 2,4-<sc>D</sc> stimuli were also determined. These results revealed a possible role for the conserved pathway miR390- <italic>TAS3</italic>-<italic>ARF3</italic>/<italic>-4</italic> in longan somatic embryo development.</p></sec><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>Plant Materials and Treatments</title><p>The synchronized embryogenic cultures from <italic>D. longan</italic> ‘Honghezi’ used in this study were friable-EC, ICpECs, GEs, TEs, and CEs; <xref rid="B23" ref-type="bibr">Lai and Chen, 1997</xref>). For RT-qPCR analysis, the EC were cultured on Murashige and Skoog medium (MS; <xref rid="B40" ref-type="bibr">Murashige and Skoog, 1962</xref>) supplemented with 1.0 mg/L 2,4-<sc>D</sc> and 2% sucrose (pH 5.8), for 20 days, then transferred to MS liquid medium containing 2,4-<sc>D</sc> (0, 0.5, 1.0, 1.5, and 2 mg/L) in a rotary shaker at 150 rpm for 24 h, respectively. All samples were collected and stored at -80°C for subsequent analyses.</p><p>Samples of roots (R), leaves (L), floral buds (FB), flowers (F), young fruits (YF), mature fruits (MF), pericarp (P1), pulp (P2), and seeds (S) collected from at least six rootstock plants of the same cultivar, <italic>D. longan</italic> ‘Sijimi,’ were provided by the experimental fields of the Fujian Academy of Agricultural Science in Putian, and used for RNA extraction.</p></sec><sec><title>Isolation of the <italic>miR390</italic> Gene from <italic>D. longan</italic></title><p>To gain a more complete understanding of the <italic>miR390</italic> gene and its evolution in plants, based on the previously published small RNA data (BioSample accession SAMN04120614, Bio-Project ID PRJNA297248; <xref rid="B32" ref-type="bibr">Lin and Lai, 2013b</xref>) and the longan EC transcriptome data (SRA050205; <xref rid="B24" ref-type="bibr">Lai and Lin, 2013</xref>), the longan primary <italic>miR390</italic> gene was obtained from longan EC DNA using PCR with sense (390F) and anti-sense (390R) primers. To determine the 5′-end, a GeneRacer Kit (Invitrogen, Carlsbad, CA, USA) was used for full-length cDNAs synthesis from a mixture of total RNAs (EC, ICpEC, GE, and HE), following the manufacturer’s instructions. 5′ RACE PCR was carried out using two nested PCR reactions and a combination of GeneRacer forward primers (5′ Primer and 5′ Nested Primer) and specific reverse primers (390-5RACE1 and 390-5RACE2). To obtain the gene regulatory sequence for the longan <italic>miR390</italic> gene, genome walking was performed to obtain flanking genomic DNA using TAIL-PCR (Takara, Japan). Its promoter was cloned by three nested PCR amplifications from a longan EC DNA template with the same forward primer, AP2, and specific reverse primers (390-SP1-SP5). The list of primers for PCR amplification is shown in <bold>Table <xref ref-type="table" rid="T1">1</xref></bold>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Primers used in pri-miR390 and <italic>TAS3</italic> cloning.</p></caption><table frame="hsides" rules="groups" cellspacing="5" cellpadding="5"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1">Primer name</th><th valign="top" align="left" rowspan="1" colspan="1">Primer sequence(5′–3′)</th><th valign="top" align="center" rowspan="1" colspan="1">Tm/°C</th><th valign="top" align="center" rowspan="1" colspan="1">Description</th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">390-F</td><td valign="top" align="left" rowspan="1" colspan="1">CTGTGTATAGAGACATACATGATGAG</td><td valign="top" align="center" rowspan="1" colspan="1">55</td><td valign="top" align="center" rowspan="1" colspan="1">RT-PCR</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">390-R</td><td valign="top" align="left" rowspan="1" colspan="1">ACCCATCAAAGATATATGATCTAGTAG</td><td valign="top" align="center" rowspan="1" colspan="1"></td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">390-5RACE1</td><td valign="top" align="left" rowspan="1" colspan="1">CATGAAACTCAGGATAGATAGCGCC</td><td valign="top" align="center" rowspan="1" colspan="1">55.6</td><td valign="top" align="center" rowspan="1" colspan="1">5′-RACE</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">390-5RACE2</td><td valign="top" align="left" rowspan="1" colspan="1">GTGGCGCTATCCCTGCTGAG</td><td valign="top" align="center" rowspan="1" colspan="1">56.6</td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">390-SP1</td><td valign="top" align="left" rowspan="1" colspan="1">CATGAAACTCAGGATAGATAGCGCC</td><td valign="top" align="center" rowspan="1" colspan="1">55.6</td><td valign="top" align="center" rowspan="1" colspan="1">Tail-PCR</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">390-SP2</td><td valign="top" align="left" rowspan="1" colspan="1">CAACAGCTCATCATCATCATCATC</td><td valign="top" align="center" rowspan="1" colspan="1">52</td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">390-SP3</td><td valign="top" align="left" rowspan="1" colspan="1">GTGGCGCTATCCCTGCTGAG</td><td valign="top" align="center" rowspan="1" colspan="1">56.6</td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">390-SP4</td><td valign="top" align="left" rowspan="1" colspan="1">GAGCTCTCATGGTGAAGGTGG</td><td valign="top" align="center" rowspan="1" colspan="1">54.8</td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">390-SP5</td><td valign="top" align="left" rowspan="1" colspan="1">GTGGTGGAGACGGTCTTGTTG</td><td valign="top" align="center" rowspan="1" colspan="1">54.8</td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TAS3-F</td><td valign="top" align="left" rowspan="1" colspan="1">TTCTTGACCTTGTAAGGCCTT</td><td valign="top" align="center" rowspan="1" colspan="1">55</td><td valign="top" align="center" rowspan="1" colspan="1">RT-PCR</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TAS3-R</td><td valign="top" align="left" rowspan="1" colspan="1">AGCTCAGGAGGGATAGAAG</td><td valign="top" align="center" rowspan="1" colspan="1"></td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TAS3-3P1</td><td valign="top" align="left" rowspan="1" colspan="1">TTCTTGACCTTGTAAGGCCTT</td><td valign="top" align="center" rowspan="1" colspan="1">53</td><td valign="top" align="center" rowspan="1" colspan="1">3′-RACE</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TAS3-3P2</td><td valign="top" align="left" rowspan="1" colspan="1">TTCCGTCCAACTCATCTTCTC</td><td valign="top" align="center" rowspan="1" colspan="1">56</td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TAS3-5P1</td><td valign="top" align="left" rowspan="1" colspan="1">AAGATGAGTTGGACGGAAAC</td><td valign="top" align="center" rowspan="1" colspan="1">54</td><td valign="top" align="center" rowspan="1" colspan="1">5′-RACE</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TAS3-5P2</td><td valign="top" align="left" rowspan="1" colspan="1">TTCTTAACGCGGGATCTTAC</td><td valign="top" align="center" rowspan="1" colspan="1">56</td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr></tbody></table></table-wrap></sec><sec><title><italic>MiR390</italic>-Targeted <italic>TAS3</italic> Gene Cloning</title><p>In <italic>Arabidopsis</italic>, miR390 has been shown to target <italic>TAS3</italic> (<xref rid="B38" ref-type="bibr">Montgomery et al., 2008a</xref>), but there is no evidence for the existence of <italic>TAS3</italic> in <italic>D. longan</italic>. To verify its existence and determine the structure of the <italic>TAS3</italic> transcript, the conserved region of the <italic>TAS3</italic> transcript was first obtained using RT-PCR with sense (TAS3-F) and anti-sense (TAS3-R) primers designed from known <italic>TAS3</italic> sequences. To determine the 5′- and 3′-ends, 5′ and 3′ RACE PCR were carried out by nested PCR reactions using a combination of GeneRacer primers and the specific primer pairs TAS3-5P1/5P2 and TAS3-3P1/TAS3-3P2. The list of primers for PCR amplification is shown in <bold>Table <xref ref-type="table" rid="T1">1</xref></bold>.</p></sec><sec><title>Bioinformatics Analysis</title><p>The <italic>miR390</italic> and <italic>TAS3</italic> genes were compared against the miRBase server (Release 21: June 2014) and NCBI using BLAST. Multiple alignment analysis and secondary structure analysis were performed using DNAMAN ver. 6.0 (Lynnon Biosoft, Pointe-Claire, QC, Canada). <italic>Cis</italic>-acting elements analysis was carried out using the PlantCARE database (<xref rid="B27" ref-type="bibr">Lescot et al., 2002</xref>). For phylogenetic analysis, MEGA 5.02 (<xref rid="B21" ref-type="bibr">Kumar et al., 2004</xref>) was performed according to the neighbor-joining method (<xref rid="B46" ref-type="bibr">Saitou and Nei, 1987</xref>) with 1000 bootstrap replicates. The psRNATarget analysis (<xref rid="B9" ref-type="bibr">Dai and Zhao, 2011</xref>) was carried out for miRNA and tasiRNA target prediction.</p></sec><sec><title>Cleaved Target mRNA Identification with 5′ RLM-RACE</title><p>TasiRNA produced from the <italic>TAS3</italic> transcript has been shown to target <italic>ARF3</italic> and <italic>-4</italic> (<xref rid="B36" ref-type="bibr">Marin et al., 2010</xref>). To discover the target transcripts of <italic>TAS3</italic> tasiRNA in longan, a modified 5′ RLM-RACE experiment was set up. Here, the transcripts of <italic>ARF3</italic> (GenBank accession No. KJ200347.1) and <italic>-4</italic> (GenBank accession No. KJ200347.1) were used to designed RACE primers. The cDNA was synthesized using a GeneRacer Kit using RNA extracted from friable-EC, ICpECs, GEs, and HEs of longan embryogenic cultures. PCR amplification of cDNA fragments using 5′ RACE outer primers and gene-specific reverse primers was performed. PCR fragments were cloned and sequenced to identify the 5′-end of the amplified target genes.</p></sec><sec><title>RNA Extraction and RT-qPCR</title><p>For RT-qPCR analysis, total RNAs were extracted from the above-mentioned samples using a TRIzol Reagent kit (Life Technologies, Grand Island, NY, USA). cDNA for mRNA quantification was subsequently synthesized using PrimeScript <sup>TM</sup> RT Reagent Kit Perfect Real Time (Takara Code, DR037A, Japan). For RT-qPCR amplification, cDNA was diluted 15 times before use. RT-qPCR was performed using LightCycler 480 equipment (Roche Applied Science, Switzerland) using the manufacturer’s instructions. Three reference genes <italic>DlFSD1a</italic>, <italic>EF-1a</italic>, and <italic>eIF-4a</italic> (<xref rid="B30" ref-type="bibr">Lin and Lai, 2010</xref>) were used to normalize our signal. All reactions were performed in triplicate. Statistical analysis was performed using SPSS 19. The gene names and primer sequences are provided in <bold>Table <xref ref-type="table" rid="T2">2</xref></bold>.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Real-time quantitative reverse transcription PCR (RT-qPCR) primers.</p></caption><table frame="hsides" rules="groups" cellspacing="5" cellpadding="5"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1">Primer name</th><th valign="top" align="left" rowspan="1" colspan="1">Primer sequence 5′–3′)</th><th valign="top" align="center" rowspan="1" colspan="1">Product size/bp</th><th valign="top" align="center" rowspan="1" colspan="1">Tm/°C</th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">miR390</td><td valign="top" align="left" rowspan="1" colspan="1">TCGCTATCCATCCTGAGTTTC</td><td valign="top" align="center" rowspan="1" colspan="1">—</td><td valign="top" align="center" rowspan="1" colspan="1">60</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">pri-miR390-QF</td><td valign="top" align="left" rowspan="1" colspan="1">GATGATGATGATGATGAGCT</td><td valign="top" align="center" rowspan="1" colspan="1">125</td><td valign="top" align="center" rowspan="1" colspan="1">56</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">pri-miR390-QR</td><td valign="top" align="left" rowspan="1" colspan="1">GCCTAGAAGAGACAAGTCGTT</td><td valign="top" align="center" rowspan="1" colspan="1"></td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TAS3-QF</td><td valign="top" align="left" rowspan="1" colspan="1">TCCATCGTCAAAAACTAGAAGG</td><td valign="top" align="center" rowspan="1" colspan="1">123</td><td valign="top" align="center" rowspan="1" colspan="1">56</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TAS3-QR</td><td valign="top" align="left" rowspan="1" colspan="1">ATGCTTGTGTCCTCCTTCATAC</td><td valign="top" align="center" rowspan="1" colspan="1"></td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">ARF3-QF</td><td valign="top" align="left" rowspan="1" colspan="1">AGATTCCGACACATCTACCG</td><td valign="top" align="center" rowspan="1" colspan="1">189</td><td valign="top" align="center" rowspan="1" colspan="1">58</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">ARF3-QR</td><td valign="top" align="left" rowspan="1" colspan="1">AGGTGGAAATGTAGCAGCAC</td><td valign="top" align="center" rowspan="1" colspan="1"></td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">ARF4-QF</td><td valign="top" align="left" rowspan="1" colspan="1">TCAAGATCCCACAATGCG</td><td valign="top" align="center" rowspan="1" colspan="1">101</td><td valign="top" align="center" rowspan="1" colspan="1">58</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">ARF4-QR</td><td valign="top" align="left" rowspan="1" colspan="1">TAACTTCATCCCCAACAGCC</td><td valign="top" align="center" rowspan="1" colspan="1"></td><td valign="top" align="center" rowspan="1" colspan="1"></td></tr></tbody></table></table-wrap></sec></sec><sec><title>Results</title><sec><title>Cloning and Analysis of the <italic>D. longan miR390</italic> Gene Sequence</title><p>To determine miR390s structure and the length of the gene in the <italic>D. longan</italic> genome, the full-length cDNA sequence of the primary miR390 transcript was obtained by RT-PCR and 5′-RACE. According to previous studies, the longan mature miR390 (<xref rid="B32" ref-type="bibr">Lin and Lai, 2013b</xref>) was predicted to derive from the transcript Contig648826, which was retrieved from longan EC transcriptome data (SRA050205; <xref rid="B24" ref-type="bibr">Lai and Lin, 2013</xref>). To verify the existence of the transcript, it was cloned from longan EC using RT-PCR and 5′ RACE. Sequence analysis showed that the transcript was 845 bp in length and had no introns (GenBank accession No. KJ372216), and the first nucleotide (transcription initiation site, TTS) of the full-length cDNA was A (<bold>Figure <xref ref-type="fig" rid="F1">1A</xref></bold>); the mature sequences dlo-miR390a-5p and dlo-miR390-3p were found to exactly match the 5′ arms of the transcript (<bold>Figure <xref ref-type="fig" rid="F1">1A</xref></bold>). A sequence of 117 bp extracted from between the positions of dlo-miR390-5p and -390-3p in the transcript was further used to predict the secondary structure using DNAMan6.0 software. The analysis showed that the sequence could form the classic stem-loop structure and the minimum free energy of the structure was -44.7 kcal/mole (<bold>Figure <xref ref-type="fig" rid="F1">1B</xref></bold>). The sequence has similarities with existing precursor miR390s in <italic>Malus domestica</italic>, <italic>G. hirsutum</italic>, <italic>Glycine max</italic>, and <italic>Cucumis melo</italic> using BLAST analysis, and is phylogenetically closely related to pre-miR390 transcripts of <italic>Arabidopsis lyrata</italic>, <italic>Populus trichocarpa</italic>, and <italic>Vitis vinifera</italic>, but was distant from pre-miR390s of <italic>C. melo</italic>, <italic>M. domestica</italic>, and <italic>Prunus persica</italic> (<bold>Figure <xref ref-type="fig" rid="F1">1C</xref></bold>).</p><fig id="F1" position="float"><label>FIGURE 1</label><caption><p><bold>MiR390 transcript in <italic>D. longan</italic>. (A)</bold> The primary miR390 sequence and its 5′ flanking sequence. Mature miR390 sequences are underlined, and the names are given under the sequences. The transcription start site (TSS) is <italic>Bold</italic> and <italic>underlined</italic>; <italic>cis</italic>-acting regulatory elements are <italic>underlined</italic>, gray shaded, and the names are given under the elements. <bold>(B)</bold> Hairpin structure of the longan miR390 precursor. <bold>(C)</bold> Bootstrapped neighbor joining phylogenetic tree of precursor miR390 in plant species. Bootstrap values are in percentages. dlo, <italic>Dimocarpus longan</italic>; ptc, <italic>Populus trichocarpa</italic>; ghr, <italic>Gossypium hirsutum</italic>; vvi<italic>, Vitis vinifera;</italic> ath, <italic>Arabidopsis thaliana</italic>; osa, <italic>Oryza sativa</italic>; <italic>cis</italic>, <italic>Citrus sinensis.</italic></p></caption><graphic xlink:href="fpls-06-01119-g001"></graphic></fig></sec><sec><title>Isolation of the Longan miR390 Promoter and Analysis of its Structural Features</title><p>Promoter analysis is an essential step in the identification of regulatory networks. Here, the partial 5′-flanking region of miR390 (927 bp) upstream of the TTS was isolated from longan friable-EC genomic DNA (GenBank accession No. KJ372219), and a promoter motif search was performed by PlantCARE (<xref rid="B27" ref-type="bibr">Lescot et al., 2002</xref>). The analysis indicated that the classical promoter elements, such as a TATA box and a number of CAAT boxes, were located in the expected position of the miR390 promoter (i.e., within -30 to -90 nucleotides upstream of the TTS). Potential <italic>cis</italic>-regulatory elements associated with light, including an ACE motif, an AE-box, an AT1-motif, three Box 4 sites, a CATT-motif, an Sp1 motif and a chs-CMA2b motif, and the <italic>cis</italic>-acting elements related to stress-related responses containing a HSE motif (involved in heat stress responses) and TC-rich repeats (involved in defense and stress responses), were also detected in the promoter region. In addition, a TCA-element involved in SA responsiveness; an ARE <italic>cis</italic>-acting regulatory element that is essential for anaerobic induction; two AT-rich sequence elements for maximal elicitor-mediated activation (two copies); a Box-W1 element involved in fungal elicitor responsiveness; a Skn-1 motif and a GCN4 motif, which are involved in endosperm expression; a circadian element involved in circadian control; a <italic>cis</italic>-acting element that confers high transcription levels (5′ UTR Py-rich stretch); and four elements of unknown function were also present in the promoter (<bold>Figure <xref ref-type="fig" rid="F1">1A</xref></bold>). These results indicated that the <italic>miR390</italic> gene could be regulated by environmental (light and stress) factors and hormones (SA).</p></sec><sec><title>Cloning and Analysis of miR390-Targeted <italic>TAS3</italic> Gene in Longan</title><p>In this study, based on the sequences of <italic>TAS3</italic> in other plants, the full-length cDNA of <italic>TAS3</italic> was obtained through RT-PCR and 5′/3′ RACE using longan EC cDNA as the template. Sequence analysis showed that the <italic>TAS3</italic> transcript, named <italic>DlTAS3</italic>, comprised 579 bp, with a 23-bp poly A tail (GenBank accession No. KJ372220). Sequence alignment analysis indicated that <italic>DlTAS3</italic> has high similarities with <italic>Arabidopsis TAS3</italic> (AT3G17185.1). The mature miRNA sequence, dlo-miR390a-5p, was predicted to bind exactly to the <italic>DlTAS3</italic> transcript at two sites, located at positions 292–312 and 472–491 bp, respectively (<bold>Figure <xref ref-type="fig" rid="F2">2A</xref></bold>). The phylogenetic analysis (<bold>Figure <xref ref-type="fig" rid="F2">2B</xref></bold>) suggested that <italic>DlTAS3</italic> in <italic>D. longan</italic> is closely related to <italic>TAS3</italic> in <italic>Citrus sinensis</italic>, <italic>Lotus japonicus</italic>, and <italic>Solanum lycopersicum</italic>, but was distant from <italic>TAS3</italic> in <italic>A. thaliana</italic> and <italic>Oryza barthii.</italic></p><fig id="F2" position="float"><label>FIGURE 2</label><caption><p><bold>Longan <italic>TAS3</italic> transcript. (A)</bold> Sequence alignment of <italic>DlTAS3</italic> in <italic>D. longan</italic> and AT3G17185.1 (<italic>TAS3</italic>) in <italic>A. thaliana</italic>. Asterisk represents the consensus nucleotide; <italic>TAS3</italic>- tasiRNAs named as 5’D1+ (reads), 5’D2+ (reads), and so on, are highlighted in different colors. The miR390 cleavage site in <italic>TAS3</italic> transcript is showed by red arrow. <bold>(B)</bold> Phylogenetic relationships among plant <italic>TAS3</italic> sequences using the neighbor-joining method. Bootstrap values are in percentages. GenBank accession numbers for each sequence represented in the tree are as follows: <italic>A. thaliana</italic> (AtTAS3a, At3g17185; AtTAS3b, At5g49615; AtTAS3c, At5g57735), <italic>V. vinifera</italic> (FQ386573); <italic>Lotus japonicus</italic> (AK338955); <italic>Malus domestica</italic> (XR_524192); <italic>C. sinensis</italic> (XR_371831); <italic>Solanum lycopersicum</italic> (JX047545); <italic>O. barthii</italic> (GQ420228); <italic>Nicotiana tabacum</italic> (FJ804751); <italic>Prunus mume</italic> (XR_513520); <italic>P. trichocarpa</italic> (XM_006378492). <bold>(C)</bold> Sequence alignment between 5’D5+ (284) and 5’D6+ (478).</p></caption><graphic xlink:href="fpls-06-01119-g002"></graphic></fig></sec><sec><title>MiR390 Guides In-phase Processing of <italic>DlTAS3</italic> Transcripts</title><p><italic>TAS3</italic> tasiRNAs originate from sequences between the two miR390 target sites (<xref rid="B18" ref-type="bibr">Kikuchi et al., 2006</xref>; <xref rid="B38" ref-type="bibr">Montgomery et al., 2008a</xref>). Here, the sequence of <italic>DlTAS3</italic> between the two binding sites was 158 bp, and the dlo-miR390-guided cleavage site was predicted to be located near the 3′ end of the <italic>DlTAS3</italic> transcript, while dlo-miR390 interacts in a non-cleavage mode with a second site near the 5′ end, which was found to have a conserved mismatch at the center of the binding region (<bold>Figure <xref ref-type="fig" rid="F2">2A</xref></bold>).</p><p>MicroRNAs direct tasiRNA biogenesis in plants, and miR390-guided cleavage was shown to set the 21-nucleotides phase for tasiRNA precursor processing (<xref rid="B18" ref-type="bibr">Kikuchi et al., 2006</xref>). Here, according to the dlo-miR390-guided cleavage site, eight potential tasiRNAs with the 21-nucleotides phase were predicted to be produced from miR390-guided <italic>DlTAS3</italic> cleavage (<bold>Figure <xref ref-type="fig" rid="F2">2A</xref></bold>; <bold>Table <xref ref-type="table" rid="T3">3</xref></bold>). In-phase, the 21-nucleotides positions on the 5′ side of the miR390 cleavage site were named 5′D1+, 5′D2+, and so on. Among these tasiRNAs, the TAS3_ 5′D5+ and 5′D6+ have a high similarity, with only four nucleotides differences at the 3′ end of the tasiRNAs (<bold>Figure <xref ref-type="fig" rid="F2">2C</xref></bold>), and they are also very similar to the 5′D7+ and 5′D8+ of the <italic>TAS3</italic> in <italic>A. thaliana</italic> (<xref rid="B18" ref-type="bibr">Kikuchi et al., 2006</xref>), <italic>G. max</italic> (<xref rid="B16" ref-type="bibr">Hu et al., 2013</xref>), <italic>M. domestica</italic>, <italic>C. sinensis</italic>, and <italic>P. trichocarpa</italic> (<bold>Figure <xref ref-type="fig" rid="F3">3</xref></bold>). These results suggested that miR390- directed <italic>TAS3</italic> cleavage leading to the production of tasiRNA is conserved in plants.</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>The sequence abundance of longan <italic>TAS3</italic> tasiRNA.</p></caption><table frame="hsides" rules="groups" cellspacing="5" cellpadding="5"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1">No.</th><th valign="top" align="center" rowspan="1" colspan="1">tasiRNA sequence and color</th><th valign="top" align="center" rowspan="1" colspan="1">Reads in smallRNA database</th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">D1+</td><td valign="top" align="center" rowspan="1" colspan="1">TTCTCCTTCCTTGTCTATCCC</td><td valign="top" align="center" rowspan="1" colspan="1">8</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D2+</td><td valign="top" align="center" rowspan="1" colspan="1">CCCGTTTCCGTCCAACTCATC</td><td valign="top" align="center" rowspan="1" colspan="1">1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D2-</td><td valign="top" align="center" rowspan="1" colspan="1">CTACTCAACCTGCCTTTGCCC</td><td valign="top" align="center" rowspan="1" colspan="1">26</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D3+</td><td valign="top" align="center" rowspan="1" colspan="1">TTCTAGTTTTTTTAAAGACTC</td><td valign="top" align="center" rowspan="1" colspan="1">No hits found</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D4+</td><td valign="top" align="center" rowspan="1" colspan="1">CGTTAAGAATTGGTCGCTTAT</td><td valign="top" align="center" rowspan="1" colspan="1">80</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D5+</td><td valign="top" align="center" rowspan="1" colspan="1">TCTTGACCTTGTAAGATCCCG</td><td valign="top" align="center" rowspan="1" colspan="1">284</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D6+</td><td valign="top" align="center" rowspan="1" colspan="1">TCTTGACCTTGTAAGACCTTT</td><td valign="top" align="center" rowspan="1" colspan="1">478</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D7+</td><td valign="top" align="center" rowspan="1" colspan="1">TTTTATTTCTATTTTTAGTCT</td><td valign="top" align="center" rowspan="1" colspan="1">1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D8+</td><td valign="top" align="center" rowspan="1" colspan="1">TTCTACCACCCTCTCCTTTAT</td><td valign="top" align="center" rowspan="1" colspan="1">1</td></tr></tbody></table></table-wrap><fig id="F3" position="float"><label>FIGURE 3</label><caption><p><bold>Alignment of <italic>TAS3</italic> sequences corresponding to the <italic>TAS3</italic> tasiRNAs in orthologs of 11 species</bold>. GenBank accession numbers are as follows: <italic>V. vinifera</italic> (FQ386573), <italic>P. trichocarpa</italic> (XM_006378492), <italic>M. domestica</italic> (XR_ 524192), <italic>C. sinensis</italic> (XR_371831), <italic>Prunus mume</italic> (XR_513520), <italic>S. lycopersicum</italic> (JX047545), <italic>L. japonicus</italic> (AK338955), <italic>Nicotiana tabacum</italic> (FJ804751), <italic>A. thaliana</italic> (At3g17185), <italic>O. barthii</italic> (GQ420228).</p></caption><graphic xlink:href="fpls-06-01119-g003"></graphic></fig></sec><sec><title>Longan tasiRNA Abundance Analysis and their Target Prediction</title><p>To verify the existence of <italic>DlTAS3</italic> tasiRNAs in longan, the tasiRNAs predicted in Section “MiR390 Guides In-phase Processing of <italic>DlTAS3</italic> Transcripts” were searched for in a longan small RNA dataset using local BLAST (<xref rid="B32" ref-type="bibr">Lin and Lai, 2013b</xref>). Among the eight tasiRNAs, the reads of TAS3_5′D5+ and 5′D6+ siRNAs were more abundant, with 284 and 478 reads. This result was similar to previous findings in which the reads of <italic>TAS3</italic> 5′D7+/5′D8+ tasiRNAs were also high in <italic>G. max</italic> (<xref rid="B16" ref-type="bibr">Hu et al., 2013</xref>). The signatures of 5′D2- and 5′D4+ siRNAs were 14 and 44 reads, respectively, while the expressions of other tasiRNAs were very low, and the 5′D3+ siRNA was not detected in longan SE (<bold>Figure <xref ref-type="fig" rid="F2">2A</xref></bold>). These results further proved that dlo-miR390 was sufficient to trigger secondary tasiRNA biogenesis, and miR390-guided <italic>TAS3</italic> cleavage with the 21-nucleotides phase was conserved in plants.</p><p>TasiRNAs interact with target homologous mRNAs and guide cleavage by the same mechanism as plant miRNAs (<xref rid="B13" ref-type="bibr">Hao et al., 2006</xref>; <xref rid="B45" ref-type="bibr">Ruduś et al., 2006</xref>). Here, to identify the targets of <italic>DlTAS3</italic>_tasiRNAs, the three with the highest abundances, TAS3_5′D4+, 5′D5+, and 5′D6+ siRNAs, were matched against nucleic acid sequences of the longan <italic>ARF</italic> gene families and the transcripts library removed the miRNA gene of <italic>A. thaliana</italic> (TAIR, version 10, released on 2010.12.14). The psRNATarget analysis indicated that 10 mRNAs were predicted to be targets of TAS3_5′D4+, 5′D5+ and 5′D6+ (<bold>Table <xref ref-type="table" rid="T4">4</xref></bold>). The TAS3_5′D4+ targets RNA-dependent RNA polymerase 6 (RDR6) and TRS120 mRNAs for cleavage. The 5′D5+ and 5′D6+ tasiRNAs both target <italic>ARF3/-4</italic> from <italic>A. thaliana</italic> or <italic>D. longan</italic>, indicating that these tasiRNA binding sites are highly conserved among different plants. In addition, <italic>DlARF3</italic> and <italic>-4</italic> contained two complementary sites to the <italic>TAS3</italic>_5′D5+/5′D6+ tasiRNAs, which result was consistent with previous studies in <italic>A. thaliana</italic> (<xref rid="B18" ref-type="bibr">Kikuchi et al., 2006</xref>) and <italic>G. max</italic> (<xref rid="B16" ref-type="bibr">Hu et al., 2013</xref>). Moreover, TAS3_5′D5+ also targets PXA1 (peroxisomal ABC transporter 1), NAC (No Apical Meristem, domain transcriptional regulator super family protein), SPL5 (squamosa promoter binding protein-like 5), and UPL2 (ubiquitin-protein ligase 2); and TAS3_5′D6+ also targets MEI1 (transcription coactivators), Core-2/I-branching beta-1, and 6-<italic>N</italic>-acetylglucosaminyltransferase family protein. These results suggested that tasiRNAs are broadly involved in plant development by guiding the cleavage of different targets, similar to plant miRNAs.</p><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Identified potential target genes of longan <italic>TAS3</italic>-tasiRNA.</p></caption><table frame="hsides" rules="groups" cellspacing="5" cellpadding="5"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1">tasiRNA</th><th valign="top" align="left" rowspan="1" colspan="1">Target accession No.</th><th valign="top" align="left" rowspan="1" colspan="1">Target description</th><th valign="top" align="left" rowspan="1" colspan="1">Target_end</th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">D4+</td><td valign="top" align="left" rowspan="1" colspan="1">AT5G11040</td><td valign="top" align="left" rowspan="1" colspan="1">RDR6, RNA-dependent RNA polymerase 6</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 UAUUCGCUGGUUAAGAAUUGC 1 Target 1284 AUAAACGACCAGUUUUUGAUG 1304</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT5G11040</td><td valign="top" align="left" rowspan="1" colspan="1">TRS120</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 20 AUUCGCUGGUUAAGAAUUGC 1 Target 2904 UAAGCUACCGGUUCUUGAUG 2923</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D5+</td><td valign="top" align="left" rowspan="1" colspan="1">AT2G33860</td><td valign="top" align="left" rowspan="1" colspan="1">ARF3, Transcriptional factor B3 family protein / auxinresponsive factor AUX/ IAA- relate</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 20 CCCUAGAAUGUUCCAGUUCU 1 Target 1674 AGGGUCUUGCAAGGUCAAGA 1693</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">KJ200347</td><td valign="top" align="left" rowspan="1" colspan="1">ARF3, auxin-responsive factor AUX/IAA- relate</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 20 CCCUAGAAUGUUCCAGUUCU 1 Target 1470 AAGGUCUUGCAAGGUCAAGA 1489 miRNA 20 CCCUAGAAUGUUCCAGUUCU 1 Target 1680 AAGGUCUUGCAAGGUCAAGA 1699</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT5G60450</td><td valign="top" align="left" rowspan="1" colspan="1">ARF4, auxin response factor 4</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 20 CCCUAGAAUGUUCCAGUUCU 1 Target 2084 AGGGUCUUGCAAGGUCAAGA 2103</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">KJ200347</td><td valign="top" align="left" rowspan="1" colspan="1">ARF4, auxin response factor 4</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 20 CCCUAGAAUGUUCCAGUUCU 1 Target 1462 AAGGUCUUGCAAGGUCAAGA 1481 miRNA 20 CCCUAGAAUGUUCCAGUUCU 1 Target 1663 AAGGUCUUGCAAGGUCAAGA 1682</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT3G12910</td><td valign="top" align="left" rowspan="1" colspan="1">NAC (no apical meristem) domain transcriptional regulator superfamily protein</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 GCCCUAGAAUGUUCCAGUUCU 1 Target 238 UGGGAUCUUCCAAGGUCGAGG 258</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT4G39850</td><td valign="top" align="left" rowspan="1" colspan="1">Peroxisomal ABC transporter 1</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 GCCCUAGAAUGUUCCAGUUCU 1 Target 374 CGGGGUCUUGUAGCGUCAAGA 394</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT3G15270</td><td valign="top" align="left" rowspan="1" colspan="1">SPL5, squamosa promoter binding protein-like 5</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 GCCCUAGAAUGUUCCAGUUCU 1 Target 704 UGGCAUCUAACAAUGUCAAGA 724</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT1G70320</td><td valign="top" align="left" rowspan="1" colspan="1">UPL2 | ubiquitin-protein ligase 2</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 GCCCUAGAAUGUUCCAGUUCU 1 Target 3848 UGGGAUUUU-CAAGGUCAAGG 3867</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">D6+</td><td valign="top" align="left" rowspan="1" colspan="1">AT2G33860</td><td valign="top" align="left" rowspan="1" colspan="1">ARF3, transcriptional factor B3 family protein/auxinresponsive factor AUX/IAA- relate</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 20 UUCCAGAAUGUUCCAGUUCU 1 Target 1794 AAGGUCUUGCAAGGUCAAGA 1813</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">KJ200347</td><td valign="top" align="left" rowspan="1" colspan="1">ARF3, transcriptional factor B3 family protein/auxinresponsive factor AUX/ IAA- relate</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 UUUCCAGAAUGUUCCAGUUCU 1 Target 1469 GAAGGUCUUGCAAGGUCAAGA 1489 miRNA 20 UUCCAGAAUGUUCCAGUUCU 1 Target 1680 AAGGUCUUGCAAGGUCAAGA 1699</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT5G60450</td><td valign="top" align="left" rowspan="1" colspan="1">ARF4, auxin response factor 4</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 UUUCCAGAAUGUUCCAGUUCU 1 Target 2083 AAGGGUCUUGCAAGGUCAAGA 2103</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">KJ200347</td><td valign="top" align="left" rowspan="1" colspan="1">ARF4, auxin response factor 4</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 UUUCCAGAAUGUUCCAGUUCU 1 Target 1662 GAAGGUCUUGCAAGGUCAAGA 1682 miRNA 20 UUCCAGAAUGUUCCAGUUCU 1 Target 1462 AAGGUCUUGCAAGGUCAAGA 1481</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT1G77320</td><td valign="top" align="left" rowspan="1" colspan="1">MEI1, transcription coactivators</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 21 UUUCCAGAAUGUUCCAGUUCU 1 Target 66 GAAGCUCUUACAAAGUCGAGA 86</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">AT5G57270</td><td valign="top" align="left" rowspan="1" colspan="1">Core-2/I-branching beta-1,6- <italic>N</italic>acetylglucosaminyltransferase family protein</td><td valign="top" align="left" rowspan="1" colspan="1">miRNA 20 UUCCAGAAUGUUCCAGUUCU 1 Target 516 AAGUUUUUCCAGGGUCAAGA 535</td></tr></tbody></table></table-wrap></sec><sec><title>Validation of tasiRNA-Guided Cleavage of Target Gene <italic>DlARF3</italic> and <italic>-4</italic> mRNAs in Longan</title><p>Four <italic>ARF</italic> genes have been validated as the target of TAS3 5_D7+/5_D8+ siRNAs in <italic>Arabidopsis</italic> (<xref rid="B22" ref-type="bibr">Kumria et al., 2003</xref>). In our study, only <italic>DlARF3</italic> and <italic>-4</italic> were predicted to be targets of <italic>TAS3</italic>_5′D5+/5′D6+ tasiRNAs, which are similar to TAS3 5_D7+/5_D8+ siRNAs in <italic>Arabidopsis</italic>. Therefore, to further verify the cleavage of <italic>DlARF3</italic> and <italic>-4</italic> by the <italic>TAS3</italic>_5′D5+/5′D6+ tasiRNAs, a modified RLM-RACE analysis was performed, which resulted in the detection of fragments of <italic>DlARF3</italic> and <italic>-4</italic> mRNAs from longan embryogenic tissues (<bold>Figure <xref ref-type="fig" rid="F4">4</xref></bold>). Fragment sequence analysis showed that the cleavage sites in <italic>DlARF3</italic> and <italic>-4</italic> mRNAs are located at positions corresponding to the amino acid sequences KVLQGQE; in addition, <italic>DlARF3</italic> was cleaved between the 10th and 11th nucleotides complementary to miR390, while <italic>DlARF4</italic> was cleaved between the 9th and 10th nucleotides of miR390 (<bold>Figure <xref ref-type="fig" rid="F4">4</xref></bold>). These results clearly indicated that <italic>TAS3</italic>_5′D5+/5′D6+ tasiRNAs cleaved the <italic>DlARF3</italic> and <italic>-4</italic> mRNAs during longan SE, which further proved that the tasiRNA-directed <italic>ARF</italic> mRNA cleavage was conserved in plants.</p><fig id="F4" position="float"><label>FIGURE 4</label><caption><p><bold>Validation of tasiRNA guided cleavage of target genes <italic>DlARF3</italic>, <italic>-4</italic> and mapping of the cleavage sites in target gene mRNAs</bold>. Numbers indicate the fraction of cloned PCR products. The B3 DNA binding domain (B3), Auxin_resp, and AUX_IAA, are highlighted in green, red, and blue in the ARF3 and -4 proteins, respectively. The tasiRNA complementary sequence in the <italic>DlARF3</italic> and <italic>-4</italic> mRNA and the corresponding amino acid sequences are shown.</p></caption><graphic xlink:href="fpls-06-01119-g004"></graphic></fig></sec><sec><title>Expression Profiling of miR390-<italic>DlTAS3</italic>-<italic>DlARF3/-4</italic> during Longan SE</title><p>To systematically analyze the function of miR390-<italic>TAS3</italic>-<italic>ARF3/-4</italic> during longan SE, expression profiling was performed using five embryogenic cultures. RT-qPCR analysis showed that the pri-miR<italic>390</italic>, miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> exhibited different temporal and spatial expressions (<bold>Figure <xref ref-type="fig" rid="F5">5A</xref></bold>). Pri-miR390 was highly expressed in EC, and less expressed in ICpECs, while it had a stable expression level with no fluctuations from GEs to CEs, suggesting that the accumulated pri-miR390 in the EC stage may be necessary for the maintenance of embryonic callus in an undifferentiated state in longan. miR390 showed its lowest expression in EC and highest expression in TEs, with a general lack of correlation of pri-miRNAs at the transcription level. It is worth noting that <italic>DlTAS3</italic> and <italic>-4</italic> showed similar expression patterns. They both exhibited their lowest expressions in EC, and reached their peaks in the GEs stage, which were mainly inversely proportional to the expression of miR390, especially at the GEs to CEs stages. <italic>DlARF3</italic> showed little variation from the EC to TEs stages, and exhibited its lowest expression in the CEs stage. There was a general lack of correlation between the expressions of <italic>DlARF3</italic> and <italic>TAS3</italic>. We concluded that miR390 down-regulation of <italic>TAS3</italic> leading to the production of tasiRNAs via cleavage of <italic>DlARF3/-4</italic> mRNAs during somatic embryo development in longan.</p><fig id="F5" position="float"><label>FIGURE 5</label><caption><p><bold>Expression profiles of miR390, pri-miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> in <italic>D. longan</italic>. (A)</bold> Relative expressions of miR390, pri-miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> during longan SE. Samples: EC, friable-embryogenic callus; ICpEC, incomplete compact pro-embryogenic cultures; GE, globular embryos; TE, torpedo-shaped embryos; CE, cotyledonary embryos. Expression level was normalized to the reference genes <italic>DlFSD1a</italic>, <italic>EF-1a</italic>, and <italic>eIF-4a</italic>. <bold>(B)</bold> Auxin control of primary miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> expression levels. The expression levels were normalized to the reference gene <italic>EF-1a</italic>. <bold>(C)</bold> Differential expression analysis of primary miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> in vegetative and reproductive tissues of ‘Sijimi’ longan. Samples: R, roots; L, leaves; FB, floral buds; F, flowers; YF, young fruit; MF, mature fruit; P1, pericarp; P2, pulp; S, seeds. The expression level was normalized to the reference genes <italic>DlFSD1a</italic>, <italic>EF-1a</italic>, and <italic>eIF-4a</italic>; The y-axes represent the relative expression values; the x-axes represent the vegetative and reproductive samples of ‘Sijimi’ longan. Different lowercase letters above the bars indicate a statistically significant difference, and identical lowercase letters denote no significant difference among different samples (<italic>P</italic> < 0.05).</p></caption><graphic xlink:href="fpls-06-01119-g005"></graphic></fig></sec><sec><title>MiR390-<italic>DlTAS3</italic>-<italic>DlARF3/-4</italic> Expressions in Response to Auxin in Longan EC</title><p>A previous study showed that miR390 and tasiRNA ARF are regulated by auxin concentration (IAA; <xref rid="B55" ref-type="bibr">Yoon et al., 2010</xref>). Embryogenesis is inhibited by exogenously supplied 2,4-<sc>D</sc> (>10<sup>-9</sup> M) or indoleacetic acid (IAA; >10<sup>-10</sup> M) in plants (<xref rid="B44" ref-type="bibr">Ruduś et al., 2002</xref>). Here, to examine how the pathway of miR390-<italic>TAS3</italic>- <italic>ARF3/-4</italic> expression is regulated by 2,4-<sc>D</sc>, their expression levels were monitored in longan EC exposed to different 2,4-<sc>D</sc> concentrations (<bold>Figure <xref ref-type="fig" rid="F5">5B</xref></bold>). The results showed that their levels all accumulated at 1.0 mg/L 2,4-<sc>D</sc> compared with auxin-free medium under the same conditions, but decreased at higher concentrations, except for <italic>DlTAS3</italic>. In contrast to miR390-<italic>ARF3/-4</italic>, <italic>DlTAS3</italic> was expressed at its lowest level at 0.5 mg/L 2,4-<sc>D</sc> compared with auxin-free, increased at 1.0 mg/L, but decreased at 1.5 mg/L, and reached its peak at 2.0 mg/L (<bold>Figure <xref ref-type="fig" rid="F5">5B</xref></bold>). This result demonstrated that the presence of auxin in the medium controlled the level of miR390-<italic>TAS3</italic>-<italic>ARF3/-4</italic>, which is similar to the results of a previous study (<xref rid="B55" ref-type="bibr">Yoon et al., 2010</xref>).</p></sec><sec><title>MiR390-<italic>DlTAS3</italic>-<italic>DlARF3/-4</italic> Expressions in ‘Sijimi’ Longan Tissue Types</title><p>To more precisely pinpoint the locations of miR390-<italic>TAS3-ARF</italic>3/-4 expressions, the tissues of the cultivar ‘Sijimi’ longan at nine stages of development, roots (R), leaves (L), floral buds (FB), flowers (F), young fruits (YF), mature fruits (MF), pericarp (P1), pulp (P2), and seeds (S) were used to assay RNA accumulation.</p><p>The results showed that primary transcripts of miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> exhibited more or less expression in vegetative and reproductive organs, and were preferentially expressed in roots (<bold>Figure <xref ref-type="fig" rid="F5">5C</xref></bold>), suggesting they might affect ‘Sijimi’ root development. In addition, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> were also expressed at relatively high levels in pulp (P2) and seeds (S), while the primary miR390 transcript expression was an exception, implying that <italic>TAS3-ARF3/-4</italic> may also be involved in the fruit development of longan. Furthermore, the expressions of pri-miR390 in young fruits (YF), mature fruits (MF), and pericarp (P1), <italic>DlTAS3</italic> in pericarp (P1), and <italic>DlARF4</italic> in leaves (L) and flower buds (FB) in mature fruits were low. These data revealed extensive regulation roles of miR390-<italic>TAS3</italic>-<italic>ARF3/-4</italic> signal in the developmental stages of vegetative and reproductive growth in ‘Sijimi’ longan.</p></sec></sec><sec><title>Discussion</title><p>MiRNAs and tasiRNAs are small RNAs of ∼21 nucleotides in length that have various roles as negative regulators of mRNA targets in plant physiology and development (<xref rid="B7" ref-type="bibr">Chuck et al., 2009</xref>). TasiRNAs originate from <italic>TAS3</italic> families through miR390-guided initiation-cleavage of primary transcripts and target <italic>ARF2/-3/-4</italic>, which are involved in the normal development of lateral roots (<xref rid="B36" ref-type="bibr">Marin et al., 2010</xref>;<xref rid="B55" ref-type="bibr">Yoon et al., 2010</xref>) and flowers in plants (<xref rid="B37" ref-type="bibr">Matsui et al., 2014</xref>); however, their roles in embryo development are still unclear. Here, we cloned, identified and examined the expressions of primary miR390 and its promoter, <italic>DlTAS3</italic>, tasiRNA, and its targets <italic>DlARF3/-4</italic> in longan. This study was the first systematic investigation of the conserved pathway miR390-<italic>TAS3</italic>- <italic>ARF3</italic>/<italic>-4</italic> in longan SE, thereby providing insights into a possible role in plant somatic embryo development.</p><sec><title>Characteristics of Longan Primary miR390 and its Promoter</title><p>MicroRNAs and tasiRNAs are both produced from larger capped and polyadenylated precursor non-coding transcripts of a few hundred base pairs in size (<xref rid="B20" ref-type="bibr">Krasnikova et al., 2009</xref>). However, the precursors of miRNAs or tasiRNAs are little studied and cloned, and have only been reported in some genome-sequenced or model plants, such as <italic>Arabidopsis</italic> (<xref rid="B53" ref-type="bibr">Xie et al., 2005</xref>) and <italic>G. max</italic> (<xref rid="B12" ref-type="bibr">Han et al., 2014</xref>). In non-genome- sequenced or non-model plants, it is hard to clone the precursors of small RNA. Here, based on the transcriptome data and RT-PCR/RACE, we successfully cloned the primary transcripts of miR390 and <italic>TAS3</italic>, and identified their TTS without genome data. These results laid the foundation for further experiments to identify the functions of longan miR390 and <italic>TAS3</italic>.</p><p>MiR390 has two genomic loci, on chromosome 2 (<italic>MIR390a</italic>) and 5 (<italic>MIR390b</italic>) in <italic>Arabidopsis</italic> (<xref rid="B38" ref-type="bibr">Montgomery et al., 2008a</xref>); the mature miR390 mostly originates from the <italic>MIR390a</italic> and not from the <italic>MIR390b</italic> locus in roots (<xref rid="B36" ref-type="bibr">Marin et al., 2010</xref>). <italic>MIR390a</italic> exhibited higher processing accuracy and efficiency of miR390 than the <italic>MIR390b</italic> (<xref rid="B8" ref-type="bibr">Cuperus et al., 2010</xref>). In longan, only one primary miR390 was cloned and its precursor was closest to pre-miR390s identified from <italic>C. sinensis</italic> and <italic>O. sativa</italic>, but closer to pre-miR390a and close to pre-miR390b in <italic>Arabidopsis</italic>, suggesting that the longan pre-miR390 cloned in our study perhaps has a similar function to the <italic>Arabidopsis MIR390a</italic>.</p><p>Previously, miR390, which has been reported to respond to UV-B in <italic>Populus tremula</italic> (<xref rid="B17" ref-type="bibr">Jia et al., 2009</xref>), Cd (<xref rid="B10" ref-type="bibr">Ding et al., 2011</xref>) and As (<xref rid="B47" ref-type="bibr">Srivastava et al., 2013</xref>) stress in rice, was down-regulated in response to Al (<xref rid="B5" ref-type="bibr">Chen et al., 2012</xref>) and Hg-toxicity (<xref rid="B60" ref-type="bibr">Zhou et al., 2012</xref>) in <italic>Medicago truncatula</italic>, and up-regulated under drought stress in <italic>Vigna unguiculata</italic> (<xref rid="B2" ref-type="bibr">Barrera-Figueroa et al., 2011</xref>) and <italic>Brachypodium distachyon</italic> (<xref rid="B3" ref-type="bibr">Budak and Akpinar, 2011</xref>). In our study, the <italic>cis</italic>-acting elements of miR390 promoter related to TC-rich repeats (defense and stress responses), and a Box-W1 element, which is a binding site for the WRKY transcriptional regulators that control pathogen defense, wound response, and senescence (<xref rid="B11" ref-type="bibr">Eulgem et al., 2000</xref>), were also identified, indicating the potential role of miR390 in stress responses in longan. In addition, miR390 was differentially regulated under heat conditions in switchgrass (<xref rid="B14" ref-type="bibr">Hivrale et al., 2015</xref>) and <italic>Brassica rapa</italic> (<xref rid="B56" ref-type="bibr">Yu et al., 2012</xref>). In longan, a <italic>cis</italic>-acting element related to heat stress responses (HSE motif) was identified, which was also present in miR390 promoter in strawberry (<xref rid="B29" ref-type="bibr">Li et al., 2014</xref>), indicating a potential role for heat in the control of miR390 accumulation in plants.</p><p>Previous studies showed that miR390 and tasiRNA ARF were positively regulated by auxin (IAA) concentration, but were not sensitive to abscisic acid, gibberellins, and cytokinin (<xref rid="B55" ref-type="bibr">Yoon et al., 2010</xref>; <xref rid="B28" ref-type="bibr">Li et al., 2013</xref>). Here, although no auxin-related <italic>cis</italic>-elements were identified in the longan miR390 promoter, pri-miR390 was up-regulated by 2,4-<sc>D</sc> treatment, suggesting that ARF elements may be present outside of the 927 bp promoter region; in addition, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> expressions were also up-regulated by 2,4-<sc>D</sc> in a concentration-dependent manner, implying that 2,4-<sc>D</sc> may influence the activity of the <italic>tasiRNA-ARF</italic> pathway through its concentration-dependent regulation of <italic>miR390</italic> expression. However, the negative regulatory relationship was not observed among <italic>miR390</italic>, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> under 2,4-<sc>D</sc> treatment, suggesting that the molecular mechanism of these genes response to 2,4-<sc>D</sc> is more complicated than expected, this needs further experimental verify. It is worth noting that the primary <italic>miR390</italic>, whose promoter contains a SA- responsive element (TCA element), was not sensitive to SA treatment (50, 75, and 100 mg/L) compared with hormone free (data not shown). A previous study showed that the mature miR390s response to auxin was significantly broader than that of the miR390 promoter GUS expression (<xref rid="B55" ref-type="bibr">Yoon et al., 2010</xref>), leading to a possible interpretation that SA affects the expression of mature miR390 but does not affect the expression of primary miR390 in longan.</p></sec><sec><title><italic>TAS3</italic> and tasiRNA Expressed in Longan SE</title><p>MiR390 targets <italic>TAS3</italic>. Here, a 579-bp cDNA was cloned from longan EC cDNA, which was homologous to <italic>TAS3</italic> in plants. The data presented here provide further evidence for the existence of <italic>TAS3</italic> in <italic>D. longan</italic>. Three <italic>TAS3</italic> loci have been identified in <italic>Arabidopsis</italic>: TAS3a (At3g17185), TAS3b (At5g49615), and TAS3c (At5g57735; <xref rid="B15" ref-type="bibr">Howell et al., 2007</xref>). In our study, the <italic>DlTAS3</italic> is phylogenetically closer to <italic>AtTAS3a</italic> than <italic>AtTAS3b/c</italic>, suggesting that <italic>DlTAS3</italic> may have a similar function to <italic>AtTAS3a</italic>. <italic>TAS3</italic> tasiRNAs arise from the <italic>TAS3</italic> family, all members of which have conserved two miR390-guided target sites; for each member, the 3′ miR390 target site, but not the 5′ target site, was cleaved (<xref rid="B15" ref-type="bibr">Howell et al., 2007</xref>). Recently, however, the 5′ miR390 binding site in <italic>TAS</italic> transcripts was demonstrated to undergo cleavage for the initiation of processing in dicots (<xref rid="B20" ref-type="bibr">Krasnikova et al., 2009</xref>). In this study, two miR390 target sites also existed in <italic>DlTAS3</italic>, and the cleaved site was predicted to be located near the 3′ end of the <italic>DlTAS3</italic>. Moreover, the longan small RNA data analysis showed that tasiRNAs of 21 nucleotides formed by miR390-guided cleavage on the 3′ side of <italic>DlTAS3</italic> were also found in longan SE, which further proved that the roles of miR390-cleaved <italic>TAS3</italic> in the production of tasiRNAs are also functional and conserved in longan.</p><p>In the longan small RNA data, eight <italic>DlTAS3</italic> tasiRNAs were found, and the reads of TAS3_5′D5+ and 5′D6+ tasiRNAs, which are similar to the tasiRNAs of <italic>TAS3</italic> 5′D7+/5′D8+ identified from <italic>G. max</italic> (<xref rid="B16" ref-type="bibr">Hu et al., 2013</xref>) and <italic>L. leptolepis</italic> (<xref rid="B57" ref-type="bibr">Zhang et al., 2013</xref>), were more abundant during longan SE, suggesting that their roles might be related to the development of longan SE. TasiRNAs guide homologous mRNAs cleavage, as do plant miRNAs (<xref rid="B13" ref-type="bibr">Hao et al., 2006</xref>; <xref rid="B45" ref-type="bibr">Ruduś et al., 2006</xref>). Plants <italic>TAS3</italic> 5′D7+/5′D8+ both target <italic>ARF3</italic> and <italic>-4</italic> (<xref rid="B18" ref-type="bibr">Kikuchi et al., 2006</xref>). The longan 5′D5+ and 5′D6+ tasiRNAs, which target <italic>ARF</italic>3 and <italic>-4</italic>, were also verified, suggesting that the miR390-tasiRNA-<italic>ARF3/-4</italic> pathway is conserved in different plants. It is worth mentioning that the longan TAS3_5′ D4+ targets RNA-dependent RNA polymerase 6 (RDR6), which is required for the production of tasiRNAs in <italic>Arabidopsis</italic> (<xref rid="B13" ref-type="bibr">Hao et al., 2006</xref>), suggesting the existence of an interactive relationship between tasiRNA and RDR6 during longan SE.</p></sec><sec><title>MiR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> Define an Auto-regulatory Network in Longan Somatic Embryo Development</title><p>We established that the pathway miR390-TAS3-<italic>ARF3/-4</italic> operates in longan, like other plants, but its roles during plant SE remained unclear. In our study, primary miR390 reached a peak in EC, which is consistent with the mature miR390 expression in <italic>G. hirsutum</italic> (<xref rid="B54" ref-type="bibr">Yang et al., 2013</xref>). The mature miR390 showed its lowest expression in EC and highest expression in TEs in longan, and reached a peak in globular-shaped embryo in Valencia sweet orange (<xref rid="B51" ref-type="bibr">Wu et al., 2011</xref>). This implied that miR390 has different expression patterns during SE, suggesting that miR390 has different roles during SE in different plants. In addition, there was a general lack of correlation between the expression of pri-miRNAs and the corresponding mature miR390s in longan, which is a common phenomenon in eukaryotes (<xref rid="B26" ref-type="bibr">Lee et al., 2008</xref>), suggesting that unknown mechanisms that control processing play a critical role in regulating the mature miRNA expression.</p><p>Previous studies showed that the miR390-<italic>TAS3</italic> tasiRNA pathway might play regulatory roles in the development of mature embryos in larch (<xref rid="B58" ref-type="bibr">Zhang et al., 2012</xref>, <xref rid="B57" ref-type="bibr">2013</xref>). Here, <italic>DlTAS3</italic> exhibited the lowest expressions in EC, and reached its peaks in the GEs stage, which was mostly the reverse of the expression of miR390, further confirming that miR390 cleaved <italic>DlTAS3</italic>, leading to the production of tasiRNAs in longan. A previous study observed that the expression pattern of TAS3_5′D7+ was identical to that of miR390 during SE in <italic>B. napus</italic> (<xref rid="B59" ref-type="bibr">Zhao et al., 2012</xref>), suggesting that the expression of <italic>TAS3</italic> tasiRNA was induced by miR390. In addition, <italic>TAS3</italic> mRNA reached its peak at EC after 1–5 days of sub-culture in larch (<xref rid="B58" ref-type="bibr">Zhang et al., 2012</xref>), which was distinct from that of longan SE, implying that the roles of the miR390-<italic>TAS3</italic> signal are not conserved between angiosperm and gymnosperm species. Moreover, parallel expression of <italic>DlTAS3</italic> and <italic>DlARF4</italic> during longan SE was observed, while <italic>DlARF3</italic> showed little variation among the tissues. Obviously, the expression levels between <italic>DlTAS3</italic> and <italic>DlARF3</italic> were largely not correlated at the transcript levels, which was also found between TAS3a-5′D6+ and its target ARFs in <italic>Triticum aestivum</italic> (<xref rid="B48" ref-type="bibr">Tang et al., 2012</xref>). Taken together, we propose that the pathway of miR390-<italic>TAS3</italic>-tasiRNA- <italic>ARF3/-4</italic> operates in the development of longan embryo. MiR390-TAS3 tasiRNAs and ARFs define an autoregulatory network quantitatively by regulating lateral root growth (<xref rid="B36" ref-type="bibr">Marin et al., 2010</xref>). Here, the primary transcripts of miR390, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> were all preferentially expressed in ‘Sijimi’ longan roots; therefore, we propose that their roles in roots are also conserved in longan. Besides, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> were also expressed at relatively high levels in the pulp and seeds, implying extended roles in the development of longan fruit.</p><p>In summary, this study was the first to identify the longan miR390-<italic>TAS3</italic> tasiRNA- <italic>ARF3/-4</italic> axis, using a RACE strategy and the expression profiles of small RNA and their targets in longan SE. We demonstrated that <italic>MiR390</italic>, <italic>DlTAS3</italic>, <italic>DlARF3</italic> and <italic>-4</italic> define an autoregulatory network duing longan somatic embryo development.</p></sec></sec><sec><title>Author Contributions</title><p>YL participated in the study design, carried out the experimental work and wrote the manuscript. LL carried out the experimental work. ZL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. LL, RL, WL, ZZ, YC, and XX prepared the materials. All authors read and approved the final version of the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.</p></sec><sec><title>Conflict of Interest Statement</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type="financial-disclosure"><p><bold>Funding</bold>. This work was funded by Research Funds for the National Natural Science Foundation of China (31201614), the Doctoral Program of Higher Education of the Chinese Ministry of Education (20123515120008), the Natural Science Funds for Distinguished Young Scholar in Fujian Province (2015J06004), the Natural Science Funds for Distinguished Young Scholar in the Fujian Agriculture and Forestry University (xjq201405), and the Outstanding Youth Fund of the Fujian Provincial Department of Education Teacher Education of Young and Middle-aged Teachers in Science and Technology Research (Science and Technology Class A; JA14099).</p></fn></fn-group><ref-list><title>References</title><ref id="B1"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Axtell</surname><given-names>M. 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M.</given-names></name></person-group> (<year>2012</year>). <article-title>Genome-wide identification of <italic>Medicago truncatula</italic> microRNAs and their targets reveals their differential regulation by heavy metal.</article-title><source><italic>Plant Cell Environ.</italic></source><volume>35</volume><fpage>86</fpage>–<lpage>99</lpage>. <pub-id pub-id-type="doi">10.1111/j.1365-3040.2011.02418.x</pub-id><pub-id pub-id-type="pmid">21895696</pub-id></mixed-citation></ref></ref-list><glossary><title>ABBREVIATIONS</title><def-list id="DL1"><def-item><term>2,4-D</term><def><p>2,4-dichlorophenoxyacetic acid</p></def></def-item><def-item><term>ARF</term><def><p>auxin response factor</p></def></def-item><def-item><term>EC</term><def><p>friable-embryogenic callus</p></def></def-item><def-item><term>cDNA</term><def><p>complementary DNA</p></def></def-item><def-item><term>CDS</term><def><p>coding DNA sequence</p></def></def-item><def-item><term>CEs</term><def><p>cotyledonary embryos</p></def></def-item><def-item><term>GEs</term><def><p>globular embryos</p></def></def-item><def-item><term>ICpECs</term><def><p>incomplete compact pro-embryogenic cultures</p></def></def-item><def-item><term>miRNAs</term><def><p>microRNAs</p></def></def-item><def-item><term>PPRs</term><def><p>pentatricopeptide repeat proteins</p></def></def-item><def-item><term>pre-miRNA</term><def><p>precursor miRNA</p></def></def-item><def-item><term>primiRNAs</term><def><p>primary miRNA transcripts</p></def></def-item><def-item><term>RLM-RACE</term><def><p>RNA ligase- mediated rapid amplification of cDNA ends</p></def></def-item><def-item><term>RT-PCR</term><def><p>reverse transcription-polymerase chain reaction</p></def></def-item><def-item><term>RT-qPCR</term><def><p>real-time quantitative reverse transcription PCR</p></def></def-item><def-item><term>SA</term><def><p>salicylic acid</p></def></def-item><def-item><term>SE</term><def><p>somatic embryogenesis</p></def></def-item><def-item><term>Tail-PCR</term><def><p>thermal asymmetric interlaced PCR</p></def></def-item><def-item><term>tasiRNAs</term><def><p>trans-acting short-interfering RNAs</p></def></def-item><def-item><term>TEs</term><def><p>torpedo-shaped embryos</p></def></def-item><def-item><term>TSSs</term><def><p>transcription start sites</p></def></def-item><def-item><term>UTR</term><def><p>un-translated region</p></def></def-item></def-list></glossary></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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