elife00269.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN"  "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">eLife</journal-id><journal-id journal-id-type="hwp">elife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">00269</article-id><article-id pub-id-type="doi">10.7554/eLife.00269</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Plant biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>Sugar is an endogenous cue for juvenile-to-adult phase transition in plants</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-2449"><name><surname>Yu</surname><given-names>Sha</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2450"><name><surname>Cao</surname><given-names>Li</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2451"><name><surname>Zhou</surname><given-names>Chuan-Miao</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2452"><name><surname>Zhang</surname><given-names>Tian-Qi</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2453"><name><surname>Lian</surname><given-names>Heng</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2454"><name><surname>Sun</surname><given-names>Yue</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2455"><name><surname>Wu</surname><given-names>Jianqiang</given-names></name><xref ref-type="aff" rid="aff5"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2456"><name><surname>Huang</surname><given-names>Jirong</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2457"><name><surname>Wang</surname><given-names>Guodong</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-2335"><name><surname>Wang</surname><given-names>Jia-Wei</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">National Key Laboratory of Plant Molecular Genetics</institution>, <institution>Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences</institution>, <addr-line><named-content content-type="city">Shanghai</named-content></addr-line>, <country>China</country></aff><aff id="aff2"><institution>Graduate School of Chinese Academy of Sciences</institution>, <addr-line><named-content content-type="city">Beijing</named-content></addr-line>, <country>China</country></aff><aff id="aff3"><institution content-type="dept">State Key Laboratory of Plant Genomics and National Center for Plant Gene Research</institution>, <institution>Institute of Genetics and Developmental Biology, Chinese Academy of Sciences</institution>, <addr-line><named-content content-type="city">Beijing</named-content></addr-line>, <country>China</country></aff><aff id="aff4"><institution content-type="dept">School of Life Sciences</institution>, <institution>East China Normal University</institution>, <addr-line><named-content content-type="city">Shanghai</named-content></addr-line>, <country>China</country></aff><aff id="aff5"><institution content-type="dept">Key Laboratory of Economic Plants and Biotechnology</institution>, <institution>Kunming Institute of Botany</institution>, <addr-line><named-content content-type="city">Kunming</named-content></addr-line>, <country>China</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Amasino</surname><given-names>Richard</given-names></name><role>Reviewing editor</role><aff><institution>University of Wisconsin</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>jwwang@sibs.ac.cn</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>26</day><month>03</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e00269</elocation-id><history><date date-type="received"><day>23</day><month>09</month><year>2012</year></date><date date-type="accepted"><day>30</day><month>01</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Yu et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Yu et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife00269.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="commentary" xlink:href="10.7554/eLife.00625"/><related-article ext-link-type="doi" id="ra2" related-article-type="article-reference" xlink:href="10.7554/eLife.00260"/><abstract><object-id pub-id-type="doi">10.7554/eLife.00269.001</object-id><p>The transition from the juvenile to adult phase in plants is controlled by diverse exogenous and endogenous cues such as age, day length, light, nutrients, and temperature. Previous studies have shown that the gradual decline in microRNA156 (miR156) with age promotes the expression of adult traits. However, how age temporally regulates the abundance of miR156 is poorly understood. We show here that the expression of miR156 responds to sugar. Sugar represses miR156 expression at both the transcriptional level and post-transcriptional level through the degradation of miR156 primary transcripts. Defoliation and photosynthetic mutant assays further demonstrate that sugar from the pre-existing leaves acts as a mobile signal to repress miR156, and subsequently triggers the juvenile-to-adult phase transition in young leaf primordia. We propose that the gradual increase in sugar after seed germination serves as an endogenous cue for developmental timing in plants.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.001">http://dx.doi.org/10.7554/eLife.00269.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.00269.002</object-id><title>eLife digest</title><p>Like animals, plants go through several stages of development before they reach maturity, and it has long been thought that some of the transitions between these stages are triggered by changes in the nutritional status of the plant. Now, based on experiments with the plant <italic>Arabidopsis thaliana</italic>, Yu et al. and, independently, Yang et al. have provided fresh insights into the role of sugar in ‘vegetative phase change'—the transition from the juvenile form of a plant to the adult plant.</p><p>The new work takes advantage of the fact that vegetative phase change is controlled by two genes that encode microRNAs (MIRNAs). <italic>Arabidopsis</italic> has eight <italic>MIR156</italic> genes and both groups confirmed that supplying plants with sugar reduces the expression of two of these—<italic>MIR156A</italic> and <italic>MIR156C</italic>—while sugar deprivation increases their expression. Removing leaves also leads to upregulation of both genes, and delays the juvenile-to-adult transition. Given that this effect can be partially reversed by providing the plant with sugar, it is likely that sugar produced in the leaves—or one of its metabolites—is the signal that triggers the juvenile-to-adult transition through the reduction of miR156 levels.</p><p>Yu and co-workers confirmed that sugar also reduces the expression of <italic>MIR156</italic> in tobacco, moss, and tomato plants, suggesting that this mechanism is evolutionarily conserved. Consistent with the work of Yang and colleagues, Yu and co-workers revealed that sugar is able to reduce the transcription of <italic>MIR156A</italic> and <italic>MIR156C</italic> genes into messenger RNA. Moreover, they showed that sugar can also suppress <italic>MIR156</italic> expression by promoting the breakdown of <italic>MIR156A</italic> and <italic>MIR156C</italic> primary messenger RNA transcripts.</p><p>The work of Yu et al. and Yang et al. has thus provided key insights into the mechanisms by which a leaf-derived signal controls a key developmental change in plants. Just as fruit flies use their nutritional status to regulate the onset of metamorphosis, and mammals use it to control the onset of puberty, so plants use the level of sugar in their leaves to trigger the transition from juvenile to adult forms.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.002">http://dx.doi.org/10.7554/eLife.00269.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>microRNA</kwd><kwd>developmental timing</kwd><kwd>sugar</kwd><kwd>juvenile-to-adult phase transition</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Arabidopsis</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>National Natural Science Foundation of China</institution></institution-wrap></funding-source><award-id>31222029; 91217306</award-id><principal-award-recipient><name><surname>Wang</surname><given-names>Jia-Wei</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>State Key Basic Research Program of China</institution></institution-wrap></funding-source><award-id>2013CB127000</award-id><principal-award-recipient><name><surname>Huang</surname><given-names>Jirong</given-names></name><name><surname>Wang</surname><given-names>Jia-Wei</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Recruitment Program of Global Expects (China)</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Wang</surname><given-names>Jia-Wei</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Shanghai Pujiang Program</institution></institution-wrap></funding-source><award-id>12PJ1409900</award-id><principal-award-recipient><name><surname>Wang</surname><given-names>Jia-Wei</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution>Initiation grant from National Key Laboratory of Plant Molecular Genetics (Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences)</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Wang</surname><given-names>Jia-Wei</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution>Chinese Academy of Sciences</institution></institution-wrap></funding-source><award-id>KSCX2-YW-N-069</award-id><principal-award-recipient><name><surname>Wang</surname><given-names>Guodong</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Sugar levels in leaves act as a signal for plants to switch from their juvenile to their adult form by regulating the expression of two genes.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>After seed germination, plants undergo two developmental transitions: juvenile-to-adult and adult-to-reproductive (<xref ref-type="bibr" rid="bib6">Bäurle and Dean, 2006</xref>). The transition from the juvenile to adult phase is marked by acquisition of reproductive competence and changes in leaf morphology (<xref ref-type="bibr" rid="bib24">Poethig, 2010</xref>). The adult to reproductive transition, also known as flowering, transforms the identity of the shoot apical meristem from vegetative into inflorescence. Physiological and genetic studies have demonstrated that both developmental transitions are regulated not only by environmental signals such as day length, light intensity, and ambient temperature, but also by endogenous signals transmitted by plant hormones and age.</p><p>microRNA156 (miR156), which targets SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcriptional factors, provides an endogenous age cue for developmental timing in plants (<xref ref-type="bibr" rid="bib24">Poethig, 2010</xref>). The expression of miR156 decreases over time, with a concomitant rise in SPL level (<xref ref-type="bibr" rid="bib42">Wu and Poethig, 2006</xref>; <xref ref-type="bibr" rid="bib37">Wang et al., 2009</xref>). Overexpression of miR156 prolongs the juvenile phase, whereas a reduction in miR156 level results in an accelerated expression of adult traits (<xref ref-type="bibr" rid="bib42">Wu and Poethig, 2006</xref>; <xref ref-type="bibr" rid="bib41">Wu et al., 2009</xref>). SPL promotes the juvenile-to-adult phase transition and flowering through activation of miR172 and MADS-box genes (<xref ref-type="bibr" rid="bib37">Wang et al., 2009</xref>; <xref ref-type="bibr" rid="bib41">Wu et al., 2009</xref>; <xref ref-type="bibr" rid="bib44">Yamaguchi et al., 2009</xref>; <xref ref-type="bibr" rid="bib16">Jung et al., 2011</xref>). Very recently, defoliation experiments and expression analyses demonstrated that the repression of miR156 in the leaf primordia is mediated by a mobile signal(s) derived from the pre-existing leaves (<xref ref-type="bibr" rid="bib45">Yang et al. 2011</xref>). However, the identity of this signal is still unknown.</p><p>In addition to being essential as prime carbon and energy sources, sugars also play critical roles as signaling molecules (<xref ref-type="bibr" rid="bib28">Rolland and Sheen, 2005</xref>; <xref ref-type="bibr" rid="bib32">Smeekens et al., 2010</xref>). In <italic>Arabidopsis thaliana</italic>, diverse sugar signals are perceived and transduced through a glucose sensor, HEXOKINASE1 (HXK1). HXK1 exerts its regulatory function through distinct molecular mechanisms including transcriptional activation, translational inhibition, mRNA decay, and protein degradation (<xref ref-type="bibr" rid="bib28">Rolland and Sheen, 2005</xref>). Analyses of two catalytic inactive <italic>HXK1</italic> alleles further indicate that the signaling activity of HXK1 is uncoupled from its catalytic activity (<xref ref-type="bibr" rid="bib21">Moore et al., 2003</xref>). Recently, a nuclear HXK1 complex has been identified (<xref ref-type="bibr" rid="bib10">Cho et al., 2006</xref>). In this complex, HXK1 binds to two unconventional partners, the vacuolar H<sup>+</sup>-ATPase B1 (VHA-B1) and the 19S regulatory particle of a proteasome subunit (RPT5B). Since neither VHA-B1 nor RPT5B has DNA binding capacity, the precise molecular mechanism by which this nuclear-localized HXK1 complex regulates gene expression remains unanswered. In addition to the HXK1-dependent pathway, some glucose-responsive genes are regulated through an HXK1-independent pathway. For instance, the expression of the genes encoding chalcone synthase, phenylalanine ammonia-lyase, and asparagine synthase responds to glucose signaling in the absence of HXK1 (<xref ref-type="bibr" rid="bib43">Xiao et al., 2000</xref>).</p><p>Here, we performed expression and mutant analyses to identify the upstream regulator of miR156. Our results demonstrate that the expression of miR156 quickly responds to sugar. Sugar reduces miR156 abundance through both transcriptional repression and transcript degradation. Thus, gradual accumulation of sugar after seed germination leads to a reduced level of miR156, which promotes the juvenile-to-adult phase transition in plants.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title><italic>MIR156A</italic> and <italic>MIR156C</italic> have dominant roles within the <italic>MIR156</italic> gene family</title><p>The transition from juvenile to adult phase in Arabidopsis is accompanied by changes in vegetative morphology. Under long day conditions, the wild type Arabidopsis plants switch from the juvenile to the adult phase from the fifth or sixth leaf. The juvenile leaves are round, smooth on their margins, and barely develop trichomes (leaf hairs) on the abaxial side (lower side). By contrast, the adult leaves are elongated, serrated, and produce abaxial trichomes (<xref ref-type="bibr" rid="bib41">Wu et al., 2009</xref>).</p><p>In the Arabidopsis genome, miR156 is encoded by eight coding loci (<italic>MIR156A</italic>–<italic>MIR156H</italic>) (<xref ref-type="bibr" rid="bib27">Reinhart et al., 2002</xref>). To understand which locus or loci play important roles within this gene family, we identified all available <italic>MIR156</italic> transfer-DNA (T-DNA) knockout plants (<xref ref-type="bibr" rid="bib29">Samson et al., 2002</xref>; <xref ref-type="bibr" rid="bib1">Alonso et al., 2003</xref>; <xref ref-type="bibr" rid="bib40">Woody et al., 2007</xref>; <xref ref-type="fig" rid="fig1">Figure 1A</xref> and <xref ref-type="supplementary-material" rid="SD1-data">supplementary file 1A</xref>). Due to functional redundancy, none of these mutants exhibited visible developmental defects (data not shown). One of the double mutants, <italic>mir156a mir156c</italic>, displayed a similar, but weak phenotype as the transgenic plant expressing a target mimicry from the constitutively active <italic>35S</italic> promoter (<italic>35S::MIM156</italic>), which reduced miR156 activity (<xref ref-type="fig" rid="fig1">Figure 1D</xref>; <xref ref-type="bibr" rid="bib14">Franco-Zorrilla et al., 2007</xref>; <xref ref-type="bibr" rid="bib34">Todesco et al., 2010</xref>). RNA gel blot demonstrated that the amount of miR156 was moderately decreased in <italic>mir156a mir156c</italic> in comparison with the wild type (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). Accordingly, the transcript levels of two miR156-target genes, <italic>SPL3</italic> and <italic>SPL9</italic>, were much higher in <italic>mir156a mir156c</italic> than in the wild type (<xref ref-type="fig" rid="fig1">Figure 1C</xref>).<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00269.003</object-id><label>Figure 1.</label><caption><title>Phenotypic analyses of the <italic>mir156a mir156c</italic> double mutant.</title><p>(<bold>A</bold>) <italic>MIR156A</italic> and <italic>MIR156C</italic> genomic regions. Arrowheads mark T-DNA insertion sites. T-DNAs are inserted 137 bp and 218 bp upstream of the stem-loops of <italic>MIR156A</italic> and <italic>MIR156C</italic>, respectively. (<bold>B</bold>) Expression of miR156 in the wild type and the <italic>mir156a mir156c</italic> double mutant. U6 was monitored as loading control. (<bold>C</bold>) Expression of <italic>SPL3</italic> and <italic>SPL9</italic> in the wild type and the <italic>mir156a mir156c</italic> double mutant. The expression level in the wild type was set to 1.0. (<bold>D</bold>) Leaf morphology of wild type, <italic>mir156a mir156c</italic>, and <italic>35S::MIM156</italic> plants. The leaves were detached and scanned. The numbers indicate leaf positions. (<bold>E</bold>) The number of juvenile and adult leaves. n=12. (<bold>F</bold>) The length-to-width ratio of the blade. Fully expanded leaves were detached and scanned. The length and width of blades were measured. n=12. Error bars indicate SE.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.003">http://dx.doi.org/10.7554/eLife.00269.003</ext-link></p></caption><graphic xlink:href="elife00269f001"/></fig></p><p>Compared to the wild type, the <italic>mir156a mir156c</italic> mutant had a shortened juvenile phase. The appearance of abaxial trichomes in <italic>mir156a mir156c</italic> was accelerated by 2.1 plastochrons (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). In addition, the length-to-width ratios of the blades in <italic>mir156a mir156c</italic> were much closer to those of the adult leaves in the wild type (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). Furthermore, <italic>mir156a mir156c</italic> flowered earlier than the wild type (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Taken together, these results indicate that <italic>MIR156A</italic> and <italic>MIR156C</italic> have dominant roles within the miR156 family in Arabidopsis.</p></sec><sec id="s2-2"><title>Sugar represses <italic>MIR156</italic> expression</title><p>To elucidate the molecular mechanism by which the level of miR156 is regulated by age, we performed time course expression assays on miR156 and the primary transcripts of <italic>MIR156A</italic> and <italic>MIR156C</italic> (<italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic>) by RNA gel blot and quantitative real-time PCR (qRT-PCR). We collected plants grown under long day conditions for 8, 9, and 16 days. As previously reported, the abundance of miR156 gradually declined (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>; <xref ref-type="bibr" rid="bib42">Wu and Poethig, 2006</xref>; <xref ref-type="bibr" rid="bib37">Wang et al., 2009</xref>). Interestingly, the transcript levels of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic>, but not mature miR156, exhibited damped oscillations with the highest level in the morning and lowest before dark (<xref ref-type="fig" rid="fig2 fig2">Figure 2C,E</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). To test whether this expression pattern is generated by the circadian clock, we grew wild type plants for 5 days in long day conditions, and then transferred them to a constant light condition. After the transfer, the oscillating expression pattern of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> was no longer observed (<xref ref-type="fig" rid="fig2">Figure 2D,F</xref>), demonstrating a negligible effect of the circadian clock on miR156 expression.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00269.004</object-id><label>Figure 2.</label><caption><title>Expression of miR156.</title><p>(<bold>A</bold> and <bold>B</bold>) Accumulation of miR156 in 8-, 9-, and 16-day-old long day plants. Expression of miR156 was analyzed by small RNA blot (<bold>A</bold>) and qRT-PCR (<bold>B</bold>). The plants were collected at Zeitgeber time (ZT) 24. The expression level of miR156 in 8-day-old seedlings was set to 1. (<bold>C</bold> and <bold>E</bold>) Expression of <italic>pri-MIR156A</italic> (<bold>C</bold>) and <italic>pri-MIR156C</italic> (<bold>E</bold>). The plants were collected every 4 hr and subjected to qRT-PCR analyses. Black and white boxes indicate dark and light conditions, respectively. (<bold>D</bold> and <bold>F</bold>) Expression of <italic>pri-MIR156A</italic> (<bold>D</bold>) and <italic>pri-MIR156C</italic> (<bold>F</bold>) during the shift from long day (LD) to constant light (CL) conditions. Five-day-old wild type seedlings were shifted from long day to constant light conditions. The seedlings were collected at ZT 16, 24, and 32.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.004">http://dx.doi.org/10.7554/eLife.00269.004</ext-link></p></caption><graphic xlink:href="elife00269f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00269.005</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Expression pattern of miR156.</title><p>Expression of miR156. The plants were collected every 4 hr and subjected to qRT-PCR analyses. ZT: Zeitgeber time.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.005">http://dx.doi.org/10.7554/eLife.00269.005</ext-link></p></caption><graphic xlink:href="elife00269fs001"/></fig></fig-group></p><p>In addition to the circadian clock, endogenous carbohydrates are also able to trigger the oscillation of RNA transcripts (<xref ref-type="bibr" rid="bib7">Bläsing et al., 2005</xref>). To test this possibility, we carried out sugar treatment assays. Five-day-old seedlings grown in 1/2 Murashige and Skoog (MS) liquid media were treated with sugars, including two disaccharides (maltose and sucrose) and two hexoses (glucose and fructose). The break-down of maltose results in two glucose molecules, whereas hydrolysis of sucrose produces glucose and fructose. The abundance of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> was greatly reduced after 1 day of treatment with 50 mM sucrose, glucose, or maltose (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). A reduction in <italic>pri-MIR156A</italic> or <italic>pri-MIR156C</italic> was not detected when the seedlings were treated with the same concentration of mannitol or 3-O-methyl-glucose (3-OMG), suggesting that the repression of <italic>pri-MIR156</italic> by sugars is not due to osmotic stress. Consistent with the reduction in <italic>pri-MIR156</italic> levels, mature miR156 was decreased after 1 day of sugar treatment (<xref ref-type="fig" rid="fig3">Figure 3A</xref>; <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Accordingly, the transcript levels of miR156-targeted genes, <italic>SPL9</italic> and <italic>SPL15</italic>, were markedly increased (<xref ref-type="fig" rid="fig3">Figure 3B</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00269.006</object-id><label>Figure 3.</label><caption><title>Sugar represses miR156.</title><p>(<bold>A</bold>) Expression of miR156, <italic>pri-MIR156A</italic>, and <italic>pri-MIR156C</italic> in response to sugar. Five-day-old wild type seedlings in 1/2 Murashige and Skoog (MS) liquid media were treated with 50 mM sucrose (Suc), glucose (Glc), fructose (Fru), maltose (Malt), or mannitol (Man) for 1 day. (<bold>B</bold>) Expression of <italic>SPL9</italic> and <italic>SPL15</italic> in response to sugar treatment. Five-day-old wild type seedlings were treated with 50 mM Man or Glc for 1 day. (<bold>C</bold>) <italic>pri-MIR156C</italic> quickly responds to sugar. Five-day-old wild type seedlings were treated with sugar for 30 min. The expression level in the mannitol-treated samples was set to 1. (<bold>D</bold>) Expression of <italic>pri-MIR156</italic> transcripts. Five-day-old wild type seedlings in 1/2 MS liquid media were treated with 50 mM glucose or mannitol for 1 day. (<bold>E</bold>) Expression of miR156 and <italic>pri-MIR156C</italic> during sugar starvation. Five-day-old wild type seedlings in 1/2 MS liquid media supplemented with 50 mM sucrose were transferred to 1/2 MS media without sucrose (MS<sub>0</sub>). The seedlings were grown for another 2 days and then subjected to expression analyses. Seven-day-old seedlings in 1/2 MS liquid media supplemented with 50 mM sucrose were used as control. (<bold>F</bold>) Expression of other <italic>pri-MIRNA</italic> transcripts. Five-day-old wild type seedlings in 1/2 MS liquid media were treated with 50 mM glucose or mannitol for 1 day. The expression levels of <italic>pri-MIR156</italic> and miR156 were normalized to those of <italic>TUBULIN</italic> (<italic>TUB</italic>). In the sugar treatment assays, 50 mM sugars were added at Zeitgeber time 12.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.006">http://dx.doi.org/10.7554/eLife.00269.006</ext-link></p></caption><graphic xlink:href="elife00269f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00269.007</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Sugar represses miR156.</title><p>Accumulation of miR156 in response to sugar. Five-day-old wild type seedlings were treated with sugar for 1 day and subjected to RNA blot analyses. U6 was monitored as an internal control. Sugar treatment started at Zeitgeber time 12. Man: mannitol; Suc: sucrose; Glc: glucose; Malt: maltose.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.007">http://dx.doi.org/10.7554/eLife.00269.007</ext-link></p></caption><graphic xlink:href="elife00269fs002"/></fig></fig-group></p><p>To monitor how fast miR156 responds to sugar, wild type seedlings were treated with glucose, sucrose, maltose, fructose, or mannitol for 30 min. A reduction of about 40% in <italic>pri-MIR156C</italic> was observed in the seedlings treated with glucose, sucrose, or maltose, while the level of <italic>pri-MIR156C</italic> was not altered in those treated with fructose or mannitol (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). These results, together with the fact that glucose is the common hydrolytic product shared by sucrose and maltose, suggest that glucose plays a major role in repressing miR156.</p><p>To determine whether all the miR156 coding genes are repressed by sugar, we analyzed the expression of their primary transcripts. <italic>pri-MIR156G</italic> and <italic>pri-MIR156H</italic> were not readily amplified, probably due to their very low expression level (data not shown). The expression of other <italic>pri-MIR156</italic> transcripts except <italic>pri-MIR156B</italic> was reduced after glucose treatment (<xref ref-type="fig" rid="fig3">Figure 3D</xref>).</p><p>To confirm the role of sugar in miR156 expression, we performed a sugar starvation experiment. Five-day-old wild type seedlings were transferred to 1/2 MS liquid media free of sugar and kept in the dark for 2 days. Compared to the seedlings grown in 1/2 MS liquid media supplemented with sugar under normal light conditions, the sugar-depleted seedlings exhibited a higher expression level of miR156 (<xref ref-type="fig" rid="fig3">Figure 3E</xref>).</p><p>To investigate whether sugar specifically represses miR156, we analyzed the expression of other miRNA primary transcripts, including <italic>pri-MIR159A</italic>, <italic>pri-MIR159B</italic>, and <italic>pri-MIR165A</italic>. The levels of all these transcripts were not reduced after sugar treatment (<xref ref-type="fig" rid="fig3">Figure 3F</xref>).</p></sec><sec id="s2-3"><title>Sugar promotes the juvenile-to-adult phase transition</title><p>A recent study has shown that the juvenile-to-adult phase transition is mediated by a leaf-derived mobile signal that represses the expression of miR156 in young leaf primordia (<xref ref-type="bibr" rid="bib45">Yang et al. 2011</xref>). Given the fact that sucrose is able to move within plants through the vascular tissues (<xref ref-type="bibr" rid="bib35">Truernit 2001</xref>) and that sucrose as well as its hydrolytic product, glucose, repress the expression of miR156, we speculated that sugar is a potential candidate for this mobile signal. To test this hypothesis, we first investigated the relationship between sugar content and the level of miR156 in vivo. Under long day conditions, Arabidopsis plants show a rapid life cycle with very short juvenile and adult phases. For this reason, we grew wild type plants under short day conditions to extend the vegetative phase. Then 15-day-old (in the juvenile phase) and 60-day-old (in the adult phase) plants were collected at Zeitgeber time (ZT) 16. Expression analyses demonstrated that miR156 was highly abundant in 15-day-old plants but less so in 60-day-old plants (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). In contrast to this expression pattern, 60-day-old plants exhibited a higher level of glucose, fructose, and sucrose than 15-day-old plants (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). These results are consistent with our findings that sugar represses miR156 and indicate an inverse correlation between the level of miR156 and endogenous sugar content in vivo.<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00269.008</object-id><label>Figure 4.</label><caption><title>Sugar as a mobile signal to trigger vegetative phase transition.</title><p>(<bold>A</bold>) Expression of miR156 in 15-day-old and 60-day-old wild type plants grown under short day conditions. (<bold>B</bold>) Sugar measurement. Fifteen-day-old and 60-day-old short day plants were collected at Zeitgeber time 16. The fructose (Fru), glucose (Glc), and sucrose (Suc) content was analyzed by GC-MS and quantified. **Significant difference from 15-day-old wild type plants, Student t-test, p&lt;0.001. Error bars indicate SD. n.d.: undetected; FW: fresh weight. (<bold>C</bold>) Seven-day-old wild type Arabidopsis seedlings before and after defoliation. Arrows indicate where the lanolin-sucrose (Suc) paste was applied. Scale bar indicates 0.5 cm. (<bold>D</bold> and <bold>E</bold>) Seven-day-old wild type seedlings before and after defoliation. Appearance of the first abaxial trichome (<bold>D</bold>) and the length-to-width ratios of blades (<bold>E</bold>) were measured. n=10. **Significant difference from wild type, Student t-test, p&lt;0.001. Error bars indicate SE. defol: defoliated; Suc: sucrose. (<bold>F</bold>) Expression of miR156. Seven-day-old wild type seedlings were defoliated and sucrose (Suc) or mannitol (Man) was applied to the defoliated petioles. The shoot apices were collected for expression analyses 2 days after defoliation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.008">http://dx.doi.org/10.7554/eLife.00269.008</ext-link></p></caption><graphic xlink:href="elife00269f004"/></fig></p><p>We then performed defoliation assays. The blades of the first two leaves of 7-day-old wild type seedlings were manually removed. Then 50 mM sucrose or mannitol (as control) was applied to the petioles of the defoliated leaves (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). Consistent with the previous report (<xref ref-type="bibr" rid="bib45">Yang et al. 2011</xref>), the removal of the first two leaves resulted in an increased level of miR156 in the shoot apices (<xref ref-type="fig" rid="fig4">Figure 4F</xref>). The expression of adult-specific traits was accordingly delayed. Compared to intact plants, the production of abaxial trichomes in the defoliated plants was delayed by 1.0 plastochrons (<xref ref-type="fig" rid="fig4">Figure 4D</xref>), and the increase in the length-to-width ratio of the lamina was slower (<xref ref-type="fig" rid="fig4">Figure 4E</xref>).</p><p>Sucrose application partially suppressed the delay in the juvenile-to-adult phase transition caused by defoliation. The sucrose-treated plants produced the abaxial trichomes 0.8 plastochrons later than intact wild type plants, but 1.6 plastochrons earlier than the mannitol-treated plants (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). In addition, the length-to-width ratios of the fifth, seventh, and ninth leaves in the sucrose-treated plants were higher than those in the mannitol-treated plants (<xref ref-type="fig" rid="fig4">Figure 4E</xref>). In agreement with these phenotypic differences, the expression of miR156 was reduced in the apices of the sucrose-treated plants but not in those treated with mannitol (<xref ref-type="fig" rid="fig4">Figure 4F</xref>).</p></sec><sec id="s2-4"><title>A reduced photosynthetic rate delays the juvenile-to-adult phase transition</title><p>To confirm the role of sugar in the juvenile-to-adult phase transition, we analyzed the Arabidopsis <italic>cao</italic>/<italic>chlorina1</italic> (<italic>ch1</italic>) mutant. A mutation in <italic>CAO</italic>/<italic>CH1</italic> (At1g44446), which encodes chlorophyll (Chl) <italic>a</italic> oxygenase, causes a reduced level of Chl <italic>b</italic> and low efficiency of photosynthesis (<xref ref-type="bibr" rid="bib13">Espineda et al., 1999</xref>). Compared to the wild type, the <italic>cao</italic>/<italic>ch1</italic> mutant developed smaller pale green leaves and had a prolonged juvenile phase (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). The rosette leaves in the <italic>cao</italic>/<italic>ch1</italic> mutant were rounder than those in the wild type plant (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>). Additionally, the appearance of abaxial trichomes in the <italic>cao</italic>/<italic>ch1</italic> mutant was delayed (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). Expression analyses indicated that higher levels of miR156 accumulated in the <italic>cao</italic>/<italic>ch1</italic> mutant than in the wild type plant (<xref ref-type="fig" rid="fig5">Figure 5D</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.00269.009</object-id><label>Figure 5.</label><caption><title><italic>cao/ch1</italic> mutant impairs vegetative phase transition.</title><p>(<bold>A</bold>) Leaf morphology of wild type, <italic>cao</italic>/<italic>ch1</italic>, and <italic>35S::MIM156 cao</italic>/<italic>ch1</italic> plants under long day conditions. The leaves from 15-day-old plants were detached and scanned. The numbers indicate leaf positions. (<bold>B</bold> and <bold>C</bold>) The length-to-width ratio of the blade (<bold>B</bold>) and the appearance of the first abaxial trichome (<bold>C</bold>). n=12. (<bold>D</bold>) Expression of miR156 during development. Wild type plants and <italic>cao</italic>/<italic>ch1</italic> mutants were collected at 7, 9, or 12 days after germination under long day conditions. (<bold>E</bold>) Expression of miR156, <italic>pri-MIR156A</italic>, and <italic>pri-MIR156C</italic>. Five-day-old wild type and <italic>cao</italic>/<italic>ch1</italic> mutants in 1/2 Murashige and Skoog (MS) liquid media were treated with 50 mM glucose or mannitol for 1 day. The expression levels in the mannitol-treated wild type or <italic>cao</italic>/<italic>ch1</italic> were set to 1. The treatment was started at Zeitgeber time 12. **Significant difference from wild type, Student t-test, p&lt;0.001. Error bars indicate SE.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.009">http://dx.doi.org/10.7554/eLife.00269.009</ext-link></p></caption><graphic xlink:href="elife00269f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00269.010</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Phenotype of <italic>cao</italic> mutant.</title><p>Plant morphology of wild type (wt), <italic>cao/ch1</italic>, and <italic>35S::MIM156 cao/ch1</italic>. Scale bar indicates 1.0 cm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.010">http://dx.doi.org/10.7554/eLife.00269.010</ext-link></p></caption><graphic xlink:href="elife00269fs003"/></fig></fig-group></p><p>To examine whether the delayed phase transition in <italic>cao</italic>/<italic>ch1</italic> depends on miR156 function, we crossed <italic>35S::MIM156</italic> into <italic>cao</italic>/<italic>ch1</italic>. Similarly to <italic>35S::MIM156</italic>, <italic>35S::MIM156 cao</italic>/<italic>ch1</italic> produced the abaxial trichomes on the first leaf, and the leaves were elongated and serrated (<xref ref-type="fig" rid="fig5">Figure 5A–C</xref>; <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). Compared to the wild type, the <italic>cao</italic>/<italic>ch1</italic> mutants exhibited higher glucose sensitivity. Treatment of <italic>cao</italic>/<italic>ch1</italic> seedlings with 50 mM glucose significantly reduced the level of miR156 (<xref ref-type="fig" rid="fig5">Figure 5E</xref>). Taken together, we conclude that sugar from the pre-existing leaves acts as a mobile signal to trigger the juvenile-to-adult phase transition through repression of miR156 in the young leaf primordia.</p></sec><sec id="s2-5"><title>Repression of miR156 by sugar is evolutionarily conserved</title><p>miR156 is present in all major plant taxa (<xref ref-type="bibr" rid="bib4">Axtell and Bowman, 2008</xref>). To test whether the regulation of miR156 by sugar is evolutionarily conserved, we examined the expression of miR156 in response to sugar in other plants, including <italic>Nicotiana benthamiana</italic> (tobacco), <italic>Physcomitrella patens</italic> (moss), and <italic>Solanum lycopersicum</italic> (tomato).</p><p><italic>N. benthamiana</italic> and <italic>S. lycopersicum</italic> were grown in 1/2 MS liquid media without sugar. After the first two leaves appeared, the seedlings were treated with 50 mM sucrose for 2 days. The seedlings of <italic>N. benthamiana</italic> and <italic>S. lycopersicum</italic> were collected and used for expression analyses. For <italic>P. patens</italic>, the sugar treatment was conducted during the protonema stage. Compared to those treated with mannitol, the amount of miR156 was greatly reduced in all the sucrose-treated plants (<xref ref-type="fig" rid="fig6">Figure 6</xref>), indicating that repression of miR156 by sugar is evolutionarily conserved.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.00269.011</object-id><label>Figure 6.</label><caption><title>Repression of miR156 by sugar is evolutionarily conserved.</title><p>Expression of miR156 in <italic>Physcomitrella patens</italic>, <italic>Solanum lycopersicum</italic>, and <italic>Nicotiana benthamiana</italic>. The plants were treated with 50 mM sucrose (Suc) or mannitol (Man) for 2 days. U6 was monitored as the loading control. Treatment was started at Zeitgeber time 12.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.011">http://dx.doi.org/10.7554/eLife.00269.011</ext-link></p></caption><graphic xlink:href="elife00269f006"/></fig></p></sec><sec id="s2-6"><title>Sugar regulates miR156 expression at both the transcriptional and post-transcriptional level</title><p>To investigate at which level sugar represses miR156, we performed chromatin immunoprecipitation analyses (ChIP) using anti-RNA polymerase II (anti-Pol II) antibody, which recognizes the C-terminal heptapeptide repeat of RNA Pol II and has been used to correlate RNA Pol II binding with gene expression. Enrichment of the promoter fragments of <italic>MIR156A</italic> and <italic>MIR156C</italic> was compared between the seedlings treated with mannitol and those treated with glucose. As shown in <xref ref-type="fig" rid="fig7">Figure 7A</xref>, the promoter fragments (harboring TATA boxes) of <italic>MIR156A</italic> and <italic>MIR156C</italic> were substantially enriched in the mannitol-treated seedlings, but not in those treated with glucose, indicating that glucose induces transcriptional repression of <italic>MIR156A</italic> and <italic>MIR156C</italic> (<xref ref-type="fig" rid="fig7">Figure 7A</xref>).<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.00269.012</object-id><label>Figure 7.</label><caption><title>Sugar promotes the degradation of miR156 primary transcripts.</title><p>(<bold>A</bold>) Chromatin immunoprecipitation (ChIP) analyses. Five-day-old wild type seedlings were treated with 50 mM glucose (Glc) or mannitol (Man) for 1 day. Anti-Pol II was used for ChIP analyses. The genomic fragments near the <italic>MIR156A</italic> or <italic>MIR156C</italic> TATA box were amplified. Relative enrichment was calculated by the ratio of bound DNAs after ChIP to input DNAs. (<bold>B</bold>) Expression of <italic>HXK1</italic> in response to glucose. Five-day-old wild type seedlings in 1/2 Murashige and Skoog (MS) liquid media were pre-treated with or without actinomycin (ActD) for 12 hr. The seedlings were harvested at 0, 1, and 3 hr after 50 mM glucose or mannitol was added. The expression level at 0 hr was set to 1. (<bold>C</bold> and <bold>D</bold>) Expression of <italic>pri-MIR156A</italic> (<bold>C</bold>) and <italic>pri-MIR156C</italic> (<bold>D</bold>) in the wild type and <italic>se-</italic>3 mutant. Five-day-old wild type seedlings in 1/2 MS liquid media were pre-treated with ActD for 12 hr. The seedlings were then treated with 50 mM glucose or mannitol. The expression levels of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> in the wild type at 0 hr were set to 1. Sugar treatment was started at Zeitgeber time 12.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.012">http://dx.doi.org/10.7554/eLife.00269.012</ext-link></p></caption><graphic xlink:href="elife00269f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00269.013</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Effect of CHX on sugar-induced <italic>pri-MIR156C</italic> degradation.</title><p>Five-day-old wild type seedlings in 1/2 Murashige and Skoog liquid media were pre-treated with actinomycin-D (ActD) for 12 hr. Glucose was added 1 h after 100 µM cycloheximide (CHX). The levels in the mannitol-treated samples (mock) were set to 1. Glucose (50 mM) was added at Zeitgeber time 12.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.013">http://dx.doi.org/10.7554/eLife.00269.013</ext-link></p></caption><graphic xlink:href="elife00269fs004"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00269.014</object-id><label>Figure 7—figure supplement 2.</label><caption><title>Expression analyses of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> in <italic>upf</italic> mutants.</title><p>(<bold>A</bold>) Expression of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> in <italic>upf1-</italic>5 and <italic>upf3-</italic>1 mutants. Seven-day-old wild type (WT), <italic>upf1-</italic>5, and <italic>upf3-</italic>1 seedlings were used for expression analyses. (<bold>B</bold>) Glucose response in <italic>upf</italic> mutants. Five-day-old wild type and <italic>upf1-</italic>5 seedlings in 1/2 Murashige and Skoog liquid media were pre-treated with actinomycin-D (ActD) for 12 h. The transcript level of <italic>pri-MIR156C</italic> was monitored at 0, 3, and 6 hr after glucose (Glc) or mannitol (Man) treatment. Glucose (50 mM) was added at Zeitgeber time 12.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.014">http://dx.doi.org/10.7554/eLife.00269.014</ext-link></p></caption><graphic xlink:href="elife00269fs005"/></fig></fig-group></p><p>We next examined the effect of actinomycin-D (ActD), which blocks transcription. To test transcription blocking efficiency, we analyzed the expression of <italic>HXK1</italic>, which is rapidly induced by glucose (<xref ref-type="bibr" rid="bib25">Price et al., 2004</xref>). The transcript level of <italic>HXK1</italic> was increased about fourfold after 3 h of glucose treatment. By contrast, the expression of <italic>HXK1</italic> was not altered in the seedlings treated with glucose and ActD (<xref ref-type="fig" rid="fig7">Figure 7B</xref>).</p><p>The addition of ActD did not affect repression of <italic>pri-MIR156C</italic> by glucose. The transcript level of <italic>pri-MIR156C</italic> was reduced by about 75% after 3 hr in the presence of glucose, compared to a 30% reduction in the presence of mannitol (<xref ref-type="fig" rid="fig7">Figure 7D</xref>). A similar expression pattern was observed in <italic>pri-MIR156A</italic> (<xref ref-type="fig" rid="fig7">Figure 7C</xref>), suggesting that glucose modulates miR156 expression at the post-transcriptional level through the degradation of <italic>pri-MIR156</italic>.</p><p>To investigate whether the reduction in miR156 primary transcripts after glucose treatment was caused by an increase in the processing efficiency of <italic>pri-MIR156</italic>, we performed the glucose treatment assay using the <italic>serrate</italic> (<italic>se</italic>) mutant which is defective in miRNA biogenesis (<xref ref-type="bibr" rid="bib15">Grigg et al., 2005</xref>; <xref ref-type="bibr" rid="bib20">Lobbes et al., 2006</xref>; <xref ref-type="bibr" rid="bib46">Yang et al., 2006</xref>; <xref ref-type="bibr" rid="bib19">Laubinger et al., 2008</xref>). Similar to the wild type, the amount of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> was markedly decreased in the ActD/glucose-treated <italic>se-</italic>3 mutant (<xref ref-type="fig" rid="fig7">Figure 7C,D</xref>), indicating that glucose regulates the abundance of <italic>pri-MIR156</italic> independently of the miRNA processing machinery.</p><p><italic>HXK1</italic> encodes a glucose sensor that transduces diverse sugar signals. <italic>gin2-</italic>1, the HXK1-null mutant (<xref ref-type="bibr" rid="bib21">Moore et al. 2003</xref>), exhibited a lower level of miR156 than the wild type (<xref ref-type="fig" rid="fig8">Figure 8A</xref>). The expression of miR156 still decreased over time in the <italic>gin2</italic>-1 mutant (<xref ref-type="fig" rid="fig8">Figure 8B</xref>). To test whether the repression of miR156 by sugar is mediated by HXK1, we compared the glucose response between the wild type and the <italic>gin2-</italic>1 mutant. The expression of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> was reduced after sugar treatment in both the wild type and the <italic>gin2</italic>-1 mutant (<xref ref-type="fig" rid="fig8">Figure 8C,D</xref>). Similarly, an evident decrease in <italic>pri-MIR156C</italic> was observed in the <italic>gin2</italic>-1 seedlings treated with ActD/glucose (<xref ref-type="fig" rid="fig8">Figure 8E</xref>). These results suggest that HXK1 plays a role in miR156 expression but is not absolutely required for the repression of miR156 by sugar.<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.00269.015</object-id><label>Figure 8.</label><caption><title>The role of <italic>HXK1</italic> in sugar-induced miR156 repression.</title><p>(<bold>A</bold>) Expression of miR156 in the 5-day-old wild type (ecotype Ler) and <italic>gin2</italic>-1 mutant. The expression level of miR156 in Ler was set to 1. (<bold>B</bold>) Time course analyses of miR156 in the <italic>gin2</italic>-1 mutant. (<bold>C</bold> and <bold>D</bold>) Expression of <italic>pri-MIR156A</italic> (<bold>C</bold>) and <italic>pri-MIR156C</italic> (<bold>D</bold>) in response to glucose in the wild type (ecotype Ler) and <italic>gin2</italic>-1 mutant. Five-day-old seedlings in 1/2 Murashige and Skoog (MS) liquid media were treated with 50 mM glucose (Glc) or mannitol (Man) for 6 hr. The expression level in Ler at 0 h was set to 1. (<bold>E</bold>) Expression of <italic>pri-MIR156C</italic> in Ler and <italic>gin2-</italic>1. Five-day-old seedlings in 1/2 MS liquid media were pre-treated with actinomycin-D (ActD) for 12 hr and then treated with 50 mM glucose or mannitol. The expression level of <italic>pri-MIR156C</italic> in Ler at 0 hr was set to 1. Sugar treatment was started at Zeitgeber time 12.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00269.015">http://dx.doi.org/10.7554/eLife.00269.015</ext-link></p></caption><graphic xlink:href="elife00269f008"/></fig></p><p>We then performed the sugar treatment assay in the presence of ActD and cycloheximide (CHX), an inhibitor of protein synthesis. The level of <italic>pri-MIR156C</italic> transcripts was greatly reduced in the ActD-treated samples, but not in those treated with both ActD and CHX (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>), suggesting that sugar-induced <italic>pri-MIR156C</italic> degradation requires de novo protein synthesis.</p><p>mRNAs can be degraded through several partially independent pathways, including nonsense-mediated mRNA decay (NMD), 5′-to-3′ mRNA degradation via exonucleases, and 3′-to-5′ mRNA degradation via the exosome. The UP-frameshift (UPF) proteins, UPF1, UPF2, and UPF3, are essential for the NMD function in plants (<xref ref-type="bibr" rid="bib3">Arciga-Reyes et al., 2006</xref>; <xref ref-type="bibr" rid="bib18">Kurihara et al., 2009</xref>). It has been shown that <italic>upf1</italic> and <italic>upf3</italic> mutants impair the sugar response and over-accumulate sugar-inducible mRNAs (<xref ref-type="bibr" rid="bib47">Yoine et al. 2006</xref>). Therefore, we investigated the role of UPF in the sugar-mediated repression of miR156. Compared to the wild type, the expression of <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> was slightly increased in <italic>upf1-</italic>5 and <italic>upf3-</italic>1 mutants (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2A</xref>). Glucose was still able to repress the accumulation of <italic>pri-MIR156C</italic>, albeit to a lesser extent (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2B</xref>), indicating that sugar promotes <italic>pri-MIR156</italic> degradation independently of canonical NMD.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><sec id="s3-1"><title>Sugar as an endogenous timer for the juvenile-to-adult phase transition</title><p>Based on expression analyses, defoliation experiments, and photosynthetic mutant characterization, we show that sugar acts upstream of miR156. We propose a model explaining how sugar regulates the juvenile-to-adult phase transition through modulation of miR156 expression as follows. After seed germination, plants start accumulating sugars through photosynthesis. Sucrose, the major transportable sugar, moves from the pre-existing leaves to the young leaf primordia, where its hydrolytic hexose product, glucose, represses the expression of miR156. As a result, the level of <italic>SPL</italic> increases and the expression of adult traits is promoted.</p><p>Identification of sugar as the endogenous developmental timing cue explains the irreversible nature of the age pathway. The level of miR156 is destined to decrease because the gradual accumulation of carbohydrates is inevitable and essential for plant growth and development. In <italic>Caenorhabditis elegans</italic>, the transitions between the stages of larval development are controlled by the sequential action of two miRNAs, <italic>lin-4</italic> and <italic>let-7</italic> (<xref ref-type="bibr" rid="bib23">Pasquinelli and Ruvkun, 2002</xref>; <xref ref-type="bibr" rid="bib22">Moss, 2007</xref>; <xref ref-type="bibr" rid="bib2">Ambros, 2011</xref>). In contrast to miR156, the expression of these two miRNAs is increased with age. It will be intriguing to examine whether sugar/carbohydrates or nutrients from the diet triggers the upregulation of <italic>lin-4</italic> and <italic>let-7</italic> in worms.</p><p>Cellular carbon (C) and nitrogen (N) are tightly coordinated to sustain optimal plant growth (<xref ref-type="bibr" rid="bib26">Raven et al., 2004</xref>; <xref ref-type="bibr" rid="bib48">Zheng, 2009</xref>). C compounds including many carbohydrates such as sucrose and glucose are synthesized in the leaf, while N nutrients such as nitrate <inline-formula><mml:math id="inf1"><mml:mrow><mml:mo>(</mml:mo><mml:msubsup><mml:mrow><mml:mtext>NO</mml:mtext></mml:mrow><mml:mn>3</mml:mn><mml:mo>-</mml:mo></mml:msubsup><mml:mo>)</mml:mo></mml:mrow></mml:math></inline-formula> and ammonium <inline-formula><mml:math id="inf2"><mml:mrow><mml:mo>(</mml:mo><mml:msubsup><mml:mrow><mml:mtext>NH</mml:mtext></mml:mrow><mml:mn>4</mml:mn><mml:mo>+</mml:mo></mml:msubsup><mml:mo>)</mml:mo></mml:mrow></mml:math></inline-formula> are assimilated by the root system. Biochemical and physiological studies have demonstrated long-distance sensing and signaling of the C/N balance in plants. When soil is short of <inline-formula><mml:math id="inf3"><mml:mrow><mml:msubsup><mml:mrow><mml:mtext>NH</mml:mtext></mml:mrow><mml:mn>4</mml:mn><mml:mo>+</mml:mo></mml:msubsup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id="inf4"><mml:mrow><mml:msubsup><mml:mtext>NO</mml:mtext><mml:mn>3</mml:mn><mml:mo>-</mml:mo></mml:msubsup></mml:mrow></mml:math></inline-formula>, photosynthesis in the leaf is inhibited. Whether miR156 and the juvenile-to-adult transition respond to N excess or deficient conditions is another interesting topic awaiting investigation.</p></sec><sec id="s3-2"><title>Does sugar promote flowering through miR156?</title><p>In addition to the juvenile-to-adult transition, flowering is of great importance for reproductive success in plants. Previous studies revealed that the floral transition is regulated by diverse environmental factors, such as photoperiod, temperature, and light, in combination with the endogenous signal derived from nutritional status. The nutrient-dependent regulation of flowering is likely dependant on the rate of sucrose export from source leaves (<xref ref-type="bibr" rid="bib11">Corbesier et al., 1998</xref>; <xref ref-type="bibr" rid="bib31">Sivitz et al., 2007</xref>). This notion is supported by our observations that sugar from pre-existing leaves acts as a long-distance signal to repress the expression of miR156 in young leaf primordia, and that a high level of miR156 delays flowering (<xref ref-type="bibr" rid="bib37">Wang et al., 2009</xref>). Intriguingly, a recent study has demonstrated that INDETERMINATE DOMAIN transcription factor AtIDD8 regulates photoperiodic flowering by modulating sugar transport and metabolism (<xref ref-type="bibr" rid="bib30">Seo et al., 2011</xref>), suggesting that additional sugar-mediated flowering pathways exist.</p><p>Sugar, produced in mesophyll cells in leaves, is transported from source tissues to sink tissues through vascular bundles (<xref ref-type="bibr" rid="bib17">Kuhn and Grof, 2010</xref>; <xref ref-type="bibr" rid="bib5">Ayre, 2011</xref>). In Arabidopsis, sucrose transporters are involved in loading sucrose into the phloem in source leaves and the uptake of sucrose into the cells of sink tissues such as roots, fruit, and developing leaves (<xref ref-type="bibr" rid="bib39">Williams et al., 2000</xref>). Very recently, the sucrose effluxers, SWEET11 and SWEET12, which facilitate sucrose efflux into the cell wall of companion cells, have been identified (<xref ref-type="bibr" rid="bib9">Chen et al., 2011</xref>). It is therefore interesting to investigate whether impairment of sucrose transport from leaf cells into the vascular system causes a defect in miR156 expression and developmental transitions.</p></sec><sec id="s3-3"><title>Regulation of miR156 by sugar in a complex manner</title><p>There are several means by which sugar regulates gene expression. For example, sugar decreases the transcript level of rice <italic>AMY3</italic> at both the transcriptional and post-transcriptional level. It was shown that destabilization of the mRNAs of <italic>AMY3</italic> is mediated by its 3′ untranslated region (UTR) (<xref ref-type="bibr" rid="bib8">Chan and Yu, 1998</xref>). Similarly, we found that <italic>pri-MIR156A</italic> and <italic>pri-MIR156C</italic> are subjected to transcriptional repression as well as transcript degradation in response to glucose. This two-level expression control by sugar might contribute to robust repression of miR156, which leads to irreversible transition from the juvenile to adult phase in plants. In Arabidopsis, HXK1 is a glucose sensor that transduces diverse aspects of sugar response. For example, the <italic>gin2</italic>-1 mutant reduces shoot and root growth, delays flowering, increases apical dominance, and alters sensitivity to auxin and cytokinin (<xref ref-type="bibr" rid="bib21">Moore et al., 2003</xref>). However, we did not observe an obvious juvenile-to-adult phase phenotype in the <italic>gin2-</italic>1 mutant under long day conditions (data not shown). Further studies will determine if the transcriptional repression of miR156 by sugar is mediated by the previously identified nuclear-localized HXK1-VHA-B1-RPT5B complex.</p><p>The level of miR156 is greatly reduced when plants are treated with both glucose and sucrose. Since these sugars can be easily interconverted, it remains unclear whether the repression of miR156 is hexose or sucrose-dependent. Moreover, based on pharmacological treatment and mutant analyses, we show that sugar is able to trigger the degradation of <italic>pri-MIR156A/C</italic> independently of the canonical glucose sensor, HXK1. Thus, investigation of the molecular mechanism by which sugar in particular recognizes <italic>pri-MIR156</italic> and promotes their degradation is an important goal for future research.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Plant materials</title><p><italic>A. thaliana</italic>, <italic>P. patens</italic>, <italic>S. lycopersicum</italic>, and <italic>N. benthamiana</italic> were grown at 21°C (day)/19°C (night) under long day (16 hr light/8 hr dark) or short day (8 hr light/16 hr dark) conditions. White light was provided by a 4:2 mixture of cool white fluorescent lamps (Lifemax cool daylight 36W/865; Philips Lighting Co., Shangai, China) and warm white fluorescent lamps (Lifemax warm white 36W/830; Philips Lighting Co.). Light intensity was 80 µmol/m<sup>2</sup>/s in long day and 90 µmol/m<sup>2</sup>/s in short day conditions. <italic>mir156a</italic> (SALK_056809), <italic>mir156c</italic> (SALK_004679), <italic>cao</italic>/<italic>ch1</italic>, and <italic>gin2-</italic>1 mutants were ordered from the Arabidopsis Biological Resource Center (Columbus, OH). <italic>35S::MIM156</italic> was described (<xref ref-type="bibr" rid="bib38">Wang et al., 2008</xref>).</p></sec><sec id="s4-2"><title>Plant treatment</title><p>All treatment assays were carried out under long day conditions. Defoliation assays were performed as described (<xref ref-type="bibr" rid="bib45">Yang et al., 2011</xref>). For the sugar treatment assay, Arabidopsis seeds were sterilized with 20% bleach and germinated in 50 ml 1/2 MS liquid media with shaking at 140 rpm. The seedlings were then transferred to 1/2 MS media supplemented with sugar. For the sugar starvation assay, 5-day-old wild type seedlings grown in 1/2 MS liquid media supplemented with 50 mM sucrose were transferred to 1/2 MS liquid media free of sugar and grown in the dark for 2 days. For the ActD and CHX assay, 20 µg/ml ActD (Sigma-Aldrich, Beijing, China) or 100 µM CHX (Sigma-Aldrich) was used. <italic>P. patens</italic> was cultured as described (<xref ref-type="bibr" rid="bib12">Cove et al., 2009</xref>). The plants in the protonema stage were used for the sugar treatment assay. Seedlings of <italic>S. lycopersicum</italic> and <italic>N. benthamiana</italic> were treated with 50 mM sucrose for 2 days.</p></sec><sec id="s4-3"><title>Expression analyses</title><p>Total RNA was extracted with Trizol reagent (Invitrogen, Life Technologies, Shanghai, China). Then 1 µg of total RNA was DNase I-treated and used for cDNA synthesis with an oligo (dT) primer. The qRT-PCR primers for <italic>SPL3</italic>, <italic>SPL9</italic>, <italic>SPL15</italic>, and <italic>TUB</italic> have been described (<xref ref-type="bibr" rid="bib38">Wang et al., 2008</xref>; <xref ref-type="bibr" rid="bib37">Wang et al., 2009</xref>). The primer sequences for other genes are shown in <xref ref-type="supplementary-material" rid="SD1-data">supplementary file 1B</xref>. A small RNA blot was performed as described (<xref ref-type="bibr" rid="bib37">Wang et al., 2009</xref>). qRT-PCR on mature miR156 was performed according to a published protocol (<xref ref-type="bibr" rid="bib36">Varkonyi-Gasic et al., 2007</xref>).</p></sec><sec id="s4-4"><title>ChIP analyses</title><p>ChIP analysis was performed according to protocol (<xref ref-type="bibr" rid="bib37">Wang et al., 2009</xref>). Crude chromatin extract was pulled down with anti-Pol II antibodies (Abcam, Hong Kong, China). ChIP DNAs were reverse crosslinked and purified using a PCR purification kit (Qiagen, Shanghai, China). A 1 μl sample of DNA was used for real-time PCR analyses. The relative enrichment of each fragment was calculated by the ratio of bound DNAs after ChIP to input DNAs.</p></sec><sec id="s4-5"><title>Sugar measurement</title><p>Wild type plants were grown under short day conditions. Then 15-day-old juvenile or 50-day-old adult plants were collected at ZT 16. Sugar was measured using 50 mg (fresh weight) of tissue. Sample extraction, preparation, and analyses were performed as previously described (<xref ref-type="bibr" rid="bib33">Tan et al., 2011</xref>). The individual sugar was identified based on the retention time and mass spectrometry standards. Quantification was performed by an external standard method.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Li Yang and Scott Poethig for the provision of unpublished data; Hong-Tao Liu for discussion; Han Xiao for <italic>S. lycopersicum</italic> seeds; and the Arabidopsis Biological Resource Center for <italic>cao</italic>/<italic>ch1</italic>, <italic>gin2-</italic>1, and <italic>MIR156</italic> T-DNA insertion mutants.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>SY, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con2"><p>HL, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>LC, Acquisition of data, Analysis of data (sugar measurement)</p></fn><fn fn-type="con" id="con4"><p>T-QZ, Acquisition of data, Analysis of data (tobacco small RNA blot)</p></fn><fn fn-type="con" id="con5"><p>C-MZ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>YS, Analysis and interpretation of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con7"><p>JW, Analysis and interpretation of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con8"><p>JH, Analysis and interpretation of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con9"><p>GW, Experiment design, Analysis and interpretation of data (sugar measurement)</p></fn><fn fn-type="con" id="con10"><p>J-WW, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.00269.016</object-id><label>Supplementary file 1.</label><caption><p>(A) <italic>MIR156</italic> T-DNA insertion mutants. 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person-group-type="author"><name><surname>Yoine</surname><given-names>M</given-names></name><name><surname>Ohto</surname><given-names>MA</given-names></name><name><surname>Onai</surname><given-names>K</given-names></name><name><surname>Mita</surname><given-names>S</given-names></name><name><surname>Nakamura</surname><given-names>K</given-names></name></person-group><year>2006</year><article-title>The lba1 mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay causes pleiotropic phenotypic changes and altered sugar signalling in Arabidopsis</article-title><source>Plant J</source><volume>47</volume><fpage>49</fpage><lpage>62</lpage></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zheng</surname><given-names>ZL</given-names></name></person-group><year>2009</year><article-title>Carbon and nitrogen nutrient balance signaling in plants</article-title><source>Plant Signal Behav</source><volume>4</volume><fpage>584</fpage><lpage>91</lpage></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00269.017</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Amasino</surname><given-names>Richard</given-names></name><role>Reviewing editor</role><aff><institution>University of Wisconsin</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://www.elifesciences.org/the-journal/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for choosing to send your work entitled “Sugar is an Endogenous Cue for Developmental Timing in Plants” for consideration at eLife. Your article has been evaluated by a Senior editor (Detlef Weigel) and 2 reviewers, one of whom (Rick Amasino) is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments based on the reviewers' reports.</p><p>The finding that sugar may be the age signal that acts via the miR156/SPL module is a significant advance in plant biology that will stimulate further research.</p><p>There are, however, some issues that need to be resolved, in particular the inconsistencies between your paper and the related paper submitted by the Poethig lab.</p><p>[Editors' note: these two studies were conducted independently; each was reviewed on its own merits, on the understanding that it would be published alongside the related study if both were accepted.]</p><p>Although the major conclusion of both papers is the same, namely that sugar treatment reduces the levels of miR156, three major differences are: 1) effect or lack thereof of cycloheximide, 2) hexokinase dependence (i.e., whether or not HXK1 is a glucose sensor for the age effect), and 3) transcriptional versus post-transcriptional regulation of <italic>MIR156</italic> expression. Some of these inconsistencies might result from studying different promoters (<italic>MIR156A</italic> vs <italic>MIR156C</italic>), measuring miR156 versus <italic>pri-MIR156C</italic> levels, the use of liquid culture versus solid media, or different temperature and light regimens.</p><p>Two other issues are:</p><p>4) The possible difference in effect of the <italic>cao/chlorina1</italic> mutant is due to short day versus long day conditions. In addition to day length, light intensity, and temperature are likely to play a role. (There also needs to be agreement on the nomenclature of the mutant.)</p><p>5) Differences in glucose concentrations used.</p><p>We hope that you will exchange information with Scott Poethig and colleagues on experimental design, repeat a number of crucial experiments under common conditions (which should not take long), and determine if the inconsistencies may in fact be resolvable. We will relay a corresponding message to the Poethig lab.</p><p>A final note: the editors and reviewers discussed the lack of measurement of endogenous sugars during phase change and concluded that, although this would be a valuable addition to the body of work you have presented, it would be beyond the scope of your study.</p><p>Comments specific to your paper:</p><p>A) The Materials and methods often do not provide sufficient detail for the reader to evaluate your work. A major example is the lack of detail of plant growth conditions. You simply note LD and SD. But for the type of research you are presenting – an effect of sugar – a detailed description of light intensity and light quality, as well as temperature is critical.</p><p>B) You should attempt to quantify GUS activity for the MIR156 promoter response experiments.</p><p>C) The ActD treatment should have a control (e.g., from another miRNA gene, which presumably should have no effect).</p><p>D) Another useful control would be measurements from wild-type plants alongside the sugar-response result for the <italic>gin2-1</italic> mutant. <italic>gin2-1</italic> still responds to sugar, but does it respond as much as wild type in the same experiment?</p><p>E) Removal of the first two leaves in 7d old Arabidopsis seedlings resulted in a delayed expression of adult traits and this can be partially overcome by sucrose application to the remaining petioles. This indicates that sugars may be the mobile signal. However, the effects of defoliation and subsequent sugar feeding on miR156 expression were not investigated, leaving part of the question unresolved.</p><p>F) In the <italic>cao</italic> mutant and in norflurazon-treated plants, it is important to determine whether all aspects of the vegetative phase change phenotype (morphological as well as molecular) can be rescued by application of glucose or sucrose. This is crucial, especially in the norflurazon experiment, as, contrary to what is claimed, not only are pre-existing leaves affected by the treatment, but the newly formed leaves are too, as can be seen in Figure 3H. Therefore it cannot be ruled out that there is a direct norflurazon effect on miR156 expression.</p><p>G) The title “Sugar is an endogenous cue for developmental timing in plants” is too general as only one aspect of developmental timing is studied in detail (vegetative phase change).</p><p>[Editors' note: the authors were also asked to address the following comment before acceptance.]</p><p>Before acceptance, it is necessary for you to compare Wt and <italic>gin2</italic> mutant directly because only the immediate response in the presence of ActD has been examined. Specifically you need to show the explicit comparison by presenting the data in Figure 7C+D, 7E+F, 7 G+H and normalized to the same standard. If another round of experiments are needed, these can be performed rapidly as you are working with seedlings.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00269.018</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>Although the major conclusion of both papers is the same, namely that sugar treatment reduces the levels of miR156, three major differences are: 1) effect or lack thereof of cycloheximide, 2) hexokinase dependence (i.e., whether or not HXK1 is a glucose sensor for the age effect), and 3) transcriptional versus post-transcriptional regulation of</italic> MIR156 <italic>expression. Some of these inconsistencies might result from studying different promoters (</italic>MIR156A <italic>vs</italic> MIR156C<italic>), measuring miR156 versus</italic> pri-MIR156C <italic>levels, the use of liquid culture versus solid media, or different temperature and light regimens</italic>.</p><p>We have discussed our results with Scott Poethig. In the revision, most of the inconsistencies between our papers have been resolved. The conclusion is that sugar represses miR156 at both transcriptional level (partially dependent on HXK1), and post-transcriptional level through promoting degradation of <italic>pri-MIR156</italic> transcripts.</p><p>In the last version, we did not exclude the possibility that sugar regulates miR156 expression through transcriptional level. In the revision, we performed ChIP assay using anti-Pol II antibody. The enrichment of the genomic fragment of <italic>MIR156A</italic> and <italic>MIR156C</italic> was markedly reduced after glucose treatment, demonstrating that sugar is able to repress miR156 expression at transcriptional level (Figure 7A).</p><p>In the revised version, our paper focuses on how sugar represses miR156 at the post-transcriptional level in an HXK1-independent manner, while the paper from the Poethig lab emphasizes the role of sugar in the transcriptional control of <italic>MIR156A</italic>.</p><p>After blocking transcription by ActD, we found that sugar is able to repress miR156 through promoting the degradation of primary transcripts of <italic>MIR156A</italic> and <italic>MIR156C</italic>. This sugar-induced pri-miRNA degradation is not dependent on HXK1 but requires de novo protein synthesis.</p><p><italic>4) The possible difference in effect of the</italic> cao/chlorina1 <italic>mutant is due to short day versus long day conditions. In addition to day length, light intensity</italic><italic>, and temperature are likely to play a role. (There also needs to be agreement on the nomenclature of the mutant.)</italic></p><p>We did observe a defect in juvenile-to-adult phase transition in the <italic>cao/ch1</italic> mutant under our growth conditions. This inconsistency could be due to different growth conditions. In the revision, the nomenclature of the <italic>cao</italic> mutant has been unified by using <italic>cao/ch1</italic>.</p><p><italic>5) Differences in glucose concentrations used</italic>.</p><p>The inconsistencies between our papers were not due to the different glucose concentration that we used. We repeated our experiment using 10 mM glucose and got the same results as those from Poethig lab.</p><p><italic>A final note: the editors and reviewers discussed the lack of measurement of endogenous sugars during phase change and concluded that, although this would be a valuable addition to the body of work you have presented, it would be beyond the scope of your study</italic>.</p><p>According to your suggestions, we performed sugar measurements. We compared the sugar content between juvenile and adult plants. We could not obtain reliable results in long day, probably due to a rapid life cycle under this condition. We then performed our measurement in short day. It appears that the adult plants accumulated more sugar (glucose, fructose, and sucrose) than juvenile plants. Consistently, we found that there was a nice correlation between miR156 level and sugar content in these samples (Figure 4A and 4B).</p><p><italic>Comments specific to your paper:</italic></p><p><italic>A) The Materials and methods often do not provide sufficient detail for the reader to evaluate your work. A major example is the lack of detail of plant growth conditions. You simply note LD and SD. But for the type of research you are presenting – an effect of sugar – a detailed description of light intensity and light quality, as well as temperature is critical</italic>.</p><p>We have included further details in the Materials and methods. For example: “<italic>A. thaliana</italic>, <italic>P. patens</italic>, <italic>S. lycopersicum</italic>, and <italic>N. benthamiana</italic> plants were grown at 21 °C (day)/19 °C (night) in long days (16 hours light/8 hours dark). White light was provided by a 4:2 mixture of cool white fluorescent lamps (Lifemax cool daylight 36W/865; Philips Lighting Co., China) and warm white fluorescent lamps (Lifemax warm white 36W/830, Philips Lighting Co.). Light intensity is 80 µmol/m<sup>2</sup>/s.”</p><p><italic>B) You should attempt to quantify GUS activity for the MIR156 promoter response experiments</italic>.</p><p>We communicated with Scott Poethig. It turned out that the <italic>MIR156A/C</italic> promoter GUS reporters from Peter Huijser's lab lack the essential sugar responsive element in the 3'UTR. Therefore, we removed this result in the revision.</p><p><italic>C) The ActD treatment should have a control (e.g., from another miRNA gene, which presumably should have no effect)</italic>.</p><p>We used HXK1 as a control (Figure 7B). HXK1 is rapidly induced by glucose. This effect is abolished after the addition of ActD, indicating that our treatment is effective.</p><p><italic>D) Another useful control would be measurements from wild-type plants alongside the sugar-response result for the</italic> gin2-1 <italic>mutant</italic>. gin2-1 <italic>still responds to sugar, but does it respond as much as wild type in the same experiment?</italic></p><p><italic>gin2-1</italic> is in the Ler genetic background. Therefore, we performed the same treatment in wild-type Ler accession. There is no difference in miR156 expression between Ler and <italic>gin2-1</italic> (Figure 7E).</p><p><italic>E) Removal of the first two leaves in 7d old Arabidopsis seedlings resulted in a delayed expression of adult traits and this can be partially overcome by sucrose application to the remaining petioles. This indicates that sugars may be the mobile signal. However, the effects of defoliation and subsequent sugar feeding on miR156 expression were not investigated, leaving part of the question unresolved</italic>.</p><p>We performed the expression analyses of miR156. Compared to intact plants, miR156 is increased after defoliation. This effect is suppressed by sucrose application (Figure 4F). This expression pattern is consistent with our phenotypic characterizations.</p><p><italic>F) In the</italic> cao <italic>mutant and in norflurazon-treated plants, it is important to determine whether all aspects of the vegetative phase change phenotype (morphological as well as molecular) can be rescued by application of glucose or sucrose. This is crucial, especially in the norflurazon experiment, as, contrary to what is claimed, not only are pre-existing leaves affected by the treatment, but the newly formed leaves are too, as can be seen in Figure 3H. Therefore it cannot be ruled out that there is a direct norflurazon effect on miR156 expression</italic>.</p><p>We agree with this argument. Therefore we removed the norflurazon treatment assay in the revision.</p><p><italic>G) The title “Sugar is an endogenous cue for developmental timing in plants” is too general as only one aspect of developmental timing is studied in detail (vegetative phase change)</italic>.</p><p>We have changed it to “Sugar is an endogenous cue for juvenile-to-adult phase transition in plants”.</p><p><italic>Before acceptance, it is necessary for you to compare Wt and</italic> gin2 <italic>mutant directly because only the immediate response in the presence of ActD has been examined. Specifically you need to show the explicit comparison by presenting the data in Figure 7C+D, 7E+F, 7 G+H and normalized to the same standard. If another round of experiments are needed, these can be performed rapidly as you are working with seedlings</italic>.</p><p>We compared the expression of miR156 and <italic>pri-MIR156</italic> between Wt and <italic>gin2-1</italic> (Figure 8). Compared to Wt, the <italic>gin2-1</italic> mutant has a lower level of miR156 in seedlings. However, the <italic>gin2-1</italic> mutant still exhibited the gradual decreased expression pattern of miR156 as Wt. Consistently, the expression of <italic>pri-MIR156</italic> was reduced in response to sugar treatment. We normalized the data to the same standard (Figure 7C, 7D, and 8E).</p></body></sub-article></article>