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Changes in hormone flux and signaling in white spruce (Picea glauca) seeds during the transition from… Liu, Yang; Müller, Kerstin; El-Kassaby, Yousry A; Kermode, Allison R Dec 18, 2015

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RESEARCH ARTICLE Open AccessChanges in hormone flux and signaling inwhite spruce (Picea glauca) seeds during thetransition from dormancy to germination inresponse to temperature cuesYang Liu1, Kerstin Müller2, Yousry A. El-Kassaby1 and Allison R. Kermode2*AbstractBackground: Seeds use environmental cues such as temperature to coordinate the timing of their germination,allowing plants to synchronize their life history with the seasons. Winter chilling is of central importance to alleviateseed dormancy, but very little is known of how chilling responses are regulated in conifer seeds. White spruce(Picea glauca) is an important conifer species of boreal forests in the North American taiga. The recent sequencingand assembly of the white spruce genome allows for comparative gene expression studies toward elucidating themolecular mechanisms governing dormancy alleviation by moist chilling. Here we focused on hormone metaboliteprofiling and analyses of genes encoding components of hormone signal transduction pathways, to elucidatechanges during dormancy alleviation and to help address how germination cues such as temperature and lighttrigger radicle emergence.Results: ABA, GA, and auxin underwent considerable changes as seeds underwent moist chilling and duringsubsequent germination; likewise, transcripts encoding hormone-signaling components (e.g. ABI3, ARF4and Aux/IAA) were differentially regulated during these critical stages. During moist chilling, active IAA wasmaintained at constant levels, but IAA conjugates (IAA-Asp and IAA-Glu) were substantially accumulated.ABA concentrations decreased during germination of previously moist-chilled seeds, while the precursor of bioactiveGA1 (GA53) accumulated. We contend that seed dormancy and germination may be partly mediated throughthe changing hormone concentrations and a modulation of interactions between central auxin-signaling pathwaycomponents (TIR1/AFB, Aux/IAA and ARF4). In response to germination cues, namely exposure to light and toincreased temperature: the transfer of seeds from moist-chilling to 30 °C, significant changes in gene transcripts andprotein expression occurred during the first six hours, substantiating a very swift reaction to germination-promotingconditions after seeds had received sufficient exposure to the chilling stimulus.Conclusions: The dormancy to germination transition in white spruce seeds was correlated with changes in auxinconjugation, auxin signaling components, and potential interactions between auxin-ABA signaling cascades (e.g. thetranscription factor ARF4 and ABI3). Auxin flux adds a new dimension to the ABA:GA balance mechanism that underliesboth dormancy alleviation by chilling, and subsequent radicle emergence to complete germination by warmtemperature and light stimuli.Keywords: Seed dormancy, Auxin, ABA, GAs, Moist-chilling, Seed germination, White spruce* Correspondence: kermode@sfu.ca2Department of Biological Sciences, Simon Fraser University, Burnaby, BritishColumbia V5A 1S6, CanadaFull list of author information is available at the end of the article© 2015 Liu et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Liu et al. BMC Plant Biology  (2015) 15:292 DOI 10.1186/s12870-015-0638-7BackgroundConifers are ecologically and economically importantplants, and coniferous forests cover vast tracts in theNorthern hemisphere. White spruce (Picea glauca) is akeystone species of boreal forests in the North Americantaiga. In Canada, over 100 million white spruce seedlingsare out-planted yearly for regeneration [1]. However, ourunderstanding of molecular mechanisms underlying thedormancy and germination of white spruce seeds andof conifers in general remains quite limited. As thewhite spruce genome was the first to be sequencedand assembled amongst conifer species in 2013, inter-est in investigating aspects of the molecular mecha-nisms underlying key developmental and physiologicalprocesses is mounting [2–4].Moist-chilling is a common dormancy-breaking stimu-lus for conifer seeds both in natural stands and underlaboratory conditions. Specific requirements can varyenormously amongst different conifer species, as well asamongst different clones and seed lots of a given species[5, 6]; for white spruce, the typical moist-chilling re-quirement under laboratory conditions is approximately21 days.During seed maturation, exposure of seeds on the par-ent plant to low temperatures can influence the depth ofprimary dormancy of the mature seeds. In the imbibedmature dormant seed, dormancy alleviation is often pro-moted by exposure to chilling. It is therefore assumedthat chilling plays a dual role in regulating dormancy [7].In addition, under some conditions, extended chillingcan result in secondary dormancy [8, 9]. Mechanismsthat underlie the beneficial effects of moist-chilling ondormancy alleviation undoubtedly involve plant hor-mones - with abscisic acid (ABA) and gibberellins (GAs)receiving the most attention, alone and within the con-text of their interplay or crosstalk with other hormonessuch as auxins, cytokinins, and ethylene [10–14].Although evolutionarily independent from the otherseed-bearing plants since 260 million years ago [15], theseeds of conifers exhibit conserved mechanisms regulat-ing their dormancy and germination with seeds of angio-sperms, including those mediated by ABA [16–18].Several studies demonstrate that moist-chilling invokeschanges in the levels of, and sensitivity to, ABA and GAsin conifer seeds [19–21]. ABA levels are reduced duringmoist-chilling-induced dormancy termination of yellow-cypress (Callitropsis nootkatensis) and Douglas-fir (Pseu-dotsuga menziesii) seeds [22, 23]. ABA levels of westernwhite pine (Pinus monticola) seeds also decline signifi-cantly during moist-chilling, and this decline is associ-ated with an increase in germination capacity [24]. It isnoteworthy that if dormancy-breaking conditions arenot met, seeds maintain high ABA levels; and dormancyimposition and maintenance require ABA biosynthesis[25]. For western white pine seeds, it is the ratio of ABAbiosynthesis to catabolism that appears to be the keyfactor that determines the capacity for dormancy main-tenance versus germination. GAs have a positive effecton dormancy alleviation and germination of coniferseeds [25]; likewise, dormancy alleviation of moist-chilled Arabidopsis seeds depends on the expression ofGA3 oxidase 1 of the GA biosynthesis pathway [19, 21].In hazel (Corylus avellana), moist-chilling has a pro-nounced effect on the capacity of the seeds for GAbiosynthesis, although active GA production does nottake place until the seeds are placed in germinationconditions [26].In the ABA signalling cascade of Arabidopsis, concertedactions of four transcription factors, i.e. ABSCISIC ACIDINSENSITIVE 3 (ABI3), FUSCA 3 (FUS3), LEAFYCOTYLEDON 1 (LEC1), and LEAFY COTYLEDON 2(LEC2), mediate various seed maturation processes andsome of these factors also participate in the transitionfrom dormancy to germination [27, 28]. Orthologs ofABI3, encoding a structurally conserved transcription fac-tor have been isolated from angiosperm and gymnosperm(conifer) species, and they act as central regulators of seeddevelopment and dormancy [10, 29]. A member of theABI3/VP1 family cloned from yellow-cypress is positivelyassociated with dormancy maintenance [30]. Throughyeast two-hybrid analyses, a yellow-cypress ABI3 Interact-ing protein (CnAIP2) that functions as a negative regula-tor of ABI3 was recently identified [31]; note that thisprotein is different from the Arabidopsis E3 ubiquitinligase, AIP2 [32]. CnAIP2, like CnABI3, acts a centralgatekeeper of important plant life cycle transitions includ-ing the seed dormancy-to-germination transition [31].GAs also modulate plant growth and development andcan act antagonistically to ABA in the control of bothseed dormancy and germination [11, 33]. Notably, regu-lation of seed germination via light and temperature iscorrelated with GA metabolism and signalling in manyspecies [10, 19, 27, 34]. Exogenous application of GAs towestern white pine seeds initiates a decrease in ABAlevels in dormant seeds by changing ABA homeostasis,i.e. promoting ABA catabolism or transport over ABAbiosynthesis [25].The hormone auxin (principally indole-3-acetic acid[IAA]) regulates many aspects of plant growth and de-velopment. Amide-linked conjugates of IAA synthesizedduring seed development [35, 36] can serve as a sourceof free IAA during seed germination [37, 38]. Severallines of evidence implicate a role for auxins in seeddormancy maintenance in Arabidopsis [39–41]; auxin-mediated seed dormancy maintenance depends on ABI3and this inhibitory effect can be nullified by moist-chilling [42]. The hub of the auxin signalling pathway isthe TRANSPORT INHIBITOR RESPONSE1 (TIR1)/Liu et al. BMC Plant Biology  (2015) 15:292 Page 2 of 14AUXIN SIGNALING F-BOX (AFB) proteins signalingsystem [43–45].In this work, we studied one white spruce (Piceaglauca) population from British Columbia, Canada, toelucidate the hormone-based mechanisms that underpindormancy alleviation and germination in response totemperature signalling (i.e. moist chilling and transfer togermination conditions). This research will help provideinsights into how winter chilling contributes to thetiming of phenology, and how conifer life histories maydevelop under new climate scenarios.MethodsSeed materials, germination testing, and seed samplingOne white spruce population from British Columbia,Canada (located at 54°26’N, 121°44’W, 850 m elevation),was selected for this study based on cumulative germin-ation performance after the standard 21-day moist-chilling treatment [46]. For germination characterization,seeds were first moist-chilled in clear plastic germinationboxes (Hoffman) lined with moistened cellulose waddingand filter paper, and moistened with 50 mL of sterilewater for 21 days at 3 °C in a dark environment. Theboxes containing seeds were then transferred into ger-mination conditions (30/20 °C, 8-h-photoperiod and70 % relative humidity). Light was provided by fluores-cence illumination at approximately 13.5 μmol · m−2s−1.Standard germination was conducted over a 21-day spanfollowing the International Seed Testing Associationstandards [47]. As controls for the transfer to thegermination-promoting conditions (30/20 °C and light),seeds were transferred to constant darkness at 30/20 °C,or were kept in moist-chilling conditions (constant 3 °C)with an 8-h photoperiod. Germination assays, scoring,and quantification were performed as previously de-scribed [46].Seed sampling for molecular and biochemical ana-lyses was conducted on 3 biological replicates and in-cluded times during moist-chilling (0, 10 and 21 d)and after transfer to germination or control condi-tions (6, 24, 80 h, and 9 d) (Fig. 1a). For seeds thathad been maintained in darkness, the sampling wasalso conducted in darkness. Samples comprising the 3replicates were collected and immediately frozen in li-quid N2 and stored at −80 °C.Reference gene selection and gene query using BLASTNThree genes were chosen and used as internal controls:CO220221 (peroxisomal targeting signal receptor),CO206996 (hypothetical protein), and AY639585 (ubi-quitin conjugating enzyme 1, UBC1); these were selecteddue to their constitutive expression during developmen-tal transitions as determined by published microarrayprofiling [48, 49]. A subset of genes specifying proteinsmediating the committed steps of ABA, GA and auxinbiosynthesis/catabolism or signalling pathways (Fig. 2),were used to query the spruce EST database (PlantGDB)and the white spruce whole genome data (NCBI) usingBLASTN. Primers (Additional file 1: Table S1) weredesigned with the primer3 tool online [50].RNA isolation, quantitative (q)RT-PCR and principlecomponent analysisRNA was isolated from seeds as previously outlined [51].Two μg of RNA was reverse-transcribed into cDNAusing the EasyScript Plus™ kit (abmGood) with oligo-dTprimers. First-strand cDNA synthesis products werediluted fivefold, and one μl of cDNA was used to carryout semi-quantitative RT-PCR for a primer specificitycheck. Quantitative RT-PCR (qRT-PCR) analyses wererun with three biological replicates per sample in 15-μlreaction volumes in an ABI7900HT machine (AppliedBiosystems) using the PerfeCTa® SYBR® Green SuperMixwith ROX (Quanta Biosciences). The reaction mixtureconsisted of 1.0 μl fivefold diluted cDNA, 7.5 μl super-mix and 1.0 μl of each primer (10 μmol · L−1). Thereaction procedure was 5 min at 95 °C, 45 cycles of 15 sat 95 °C and 60 s at 59 °C. Dissociation curves were gen-erated at the end of each qRT-PCR to validate the ampli-fication of only one product. Efficiency calculation andnormalization were performed using real-time PCRMiner (www.miner.ewindup.info/) [52] and data qualitywas confirmed through internal controls and no-template-controls, and by comparing the repeatability across repli-cates. An average expression value for each gene at eachtime point was generated from the normalized data.Principle component analysis (PCA) was performedusing SAS® (vers. 9.3; SAS Institute Inc., Cary, NC) basedon the expression patterns of all genes in different ger-mination conditions at 6, 24, and 80 h as described inthe text.Western blot analysisProtein extracts were generated by grinding the seed ma-terials in protein extraction buffer (50 mM Tris pH 8.0,150 mM NaCl, 1 % Triton X-100, and 100 μg · ml−1 phe-nylmethylsulfonyl fluoride) and protein concentration wasdetermined by measuring OD750 with the aid of photom-eter. Protein extracts (30 μg total soluble protein) wereseparated by 10 % SDS-PAGE and transferred onto Amer-sham Hybond-P (PVDF) membranes using wet electro-blotting. The blots were blocked overnight at 3 °C using5 % (w/v) non-fat dry milk and 0.1 % (v/v) Tween-20(PBST) followed by three washes (15 min each) withPBST. Blots were incubated with the anti-pgKS (ent-kaur-ene synthase) antibody (1:500 dilution) for 1 h at roomtemperature (provided by T-P. Sun’s lab). After threewashes with PBST (15 min each) the membrane wasLiu et al. BMC Plant Biology  (2015) 15:292 Page 3 of 14incubated with the anti-rat HRP (horseradish peroxidase)antibody (1:40,000 dilution) for 1 h at room temperature.After three washes with PBST (15 min each) the mem-brane was drained and placed within wrap film containing2 ml Supersignal West Pico solution and the membraneexposed to light. Chemiluminescent images were capturedby a CCD camera system (Fujifilm LAS 4000).Plant hormone quantification by HPLC-ESI-MS/MSMethods for quantification of multiple hormones andmetabolites, including ABA and its metabolites (cis-ABA, trans-ABA, ABA-GE, PA, DPA, 7’OH-ABA, andneoPA), gibberellins (GA53 and GA34), auxins/auxin con-jugates (IAA, IAA-Asp, and IAA-Glu), and cytokinins(iPR, cis-ZR, and cis-ZOG) followed those previouslydescribed [53, 54].Briefly, lyophilized seed samples were ground and amixture of all internal standards was added to duplicatehomogenized seed samples (~50 mg each), and extrac-tion performed using acidic isopropanol. Samples werereconstituted and purified by solid phase extraction(SPE) with Sep-Pac C18 cartridges (Waters, Mississauga,ON, Canada). Subsequently, samples were injected ontoan ACQUITY UPLC® HSS C18 SB column (2.1 ×EMB32*05101520253030°Clight30°Cdark20°Cdark0d  10d  21d       6h       24h  80h 9dTime course (days)0 5 10 15 20%noitanimreG020406080100Mc + Standard germinationMc + germination in darkness1/2Mc + Standard germinationStandard germination without McMc + germination in 3ºCRelative unitsab cFig. 1 Effect of moist chilling on the germination performance of white spruce seeds. a Schematic representation of sampling to determinegermination of white spruce seeds in different germination conditions with a 21-d or 10-d moist-chilling period (Mc or 1/2Mc) or no moist chilling.In standard germination conditions (30/20 °C and an 8-h photoperiod), seed coat rupture and radicle protrusion was observed at 80 h in the majorityof the population. The stage at which the radicle had emerged to four times of the seed length was reached on d 9. b Germination of white spruceseeds under the conditions represented in (a) and without moist chilling. Data points are means ± SE of four dishes of 100 seeds each. While biologistsdefine the completion of germination as radicle emergence, ‘germination’ percentage in the forest industry is based on the number of seeds that reachthe stage when the radicle has emerged to four times the seed length (approximately 4 mm for white spruce). In b, we used this latterdefinition. c Transcript dynamics of the dormancy marker, EMB32 during moist-chilling (0, 10, 21 d), germination (6, 24, 80 h) and seedlinggrowth (9 d) (black bars). At the 6 h time-point, transcript levels were determined under three conditions: in seeds after their transfer tostandard germination conditions (30/20 °C and 8-h photoperiod) (black bar), in seeds maintained in darkness at 30 °C (dark grey bars),and in seeds maintained in darkness at 20 °C (light grey bars). Relative expression levels as determined by RT-qPCR are shown. Each data point is theaverage of three biological replicates. Bars indicate the SEM. Note: one asterisk (*) indicates that the gene has been annotated in gymnosperms butnot in white spruceLiu et al. BMC Plant Biology  (2015) 15:292 Page 4 of 14100 mm, 1.8 μl) with an in-line filter and separated by agradient elution of water containing 0.02 % formic acidagainst an increasing percentage of a mixture of aceto-nitrile and methanol (50:50, v/v). The analysis utilizedthe Multiple Reaction Monitoring function of theMassLynx v4.1 (Waters Inc) control software. Thequality control samples and internal standard andsolvent negative controls were prepared and analyzedalong with samples.ResultsGermination profiles of white spruce seeds underdifferent germination conditionsThe mature seeds of white spruce have a relatively shal-low dormancy level, and can germinate even withoutmoist chilling. However, exposure of seeds to moistchilling led to faster and more uniform germination(Fig. 1b).To investigate the effect of light on dormancy allevi-ation and germination, white spruce seeds were placedunder different conditions following exposure to 21 daysof moist chilling. The fastest and most homogenous ger-mination occurred when seeds were subjected to light(an 8-h photoperiod) and a 30/20 °C temperature regime(standard germination conditions), compared to whenthey were kept in darkness (Fig. 1b). After the 21-daymoist-chilling, subsequent seed germination under thecombined conditions of 30/20 °C and an 8-h photo-period was more successful than in constant darknessbut with the same 30/20 °C temperature cycle. This wasthe case based on most germination parameters: dor-mancy index (i.e., area between germination curves ofno- treatment and any treatment; 15.54 ± 2.12 vs. 7.56 ±0.97), germination speed (i.e. the time required for 50 %germination, 8d vs. 10d), and lag time to germination(6 d vs. 7 d) (Fig. 1b). Germination capacities were similarfor the two treatments at the end of the 21-day studyperiod (96 % vs. 94 %) (Fig. 1b). Seeds subjected to acontrol treatment - maintaining them at 3 °C, but expos-ing them to an 8-h photoperiod - were unable to germin-ate (Fig. 1b). Regardless of the light conditions aftertransfer to germination temperatures (8-h photoperiod orAAO3**°Clight30°Cdark 20°CdarkSnRK2.2**02468CYP707A4***024681012Relative units0d  10d 21d 6h 24h  80h  9dTime courseABI30.  10d 21d 6h 24h  80h  9d 0d  10d 21d 6h 24h  80h  9dca bFig. 2 Changes in ABA, ABA metabolites, and ABA signalling components during the transition from dormancy to germination of white spruceseeds. a Profiles of ABA and its metabolites as determined by UPLC/ESI-MS/MS during moist-chilling at 3 °C (0, 10, and 21 days) and during germination(6, 24 and 80 h) and seedling growth (9 d). Each data point is the average of two biological replicates. b Schematic of ABA biosynthesis, signalling, andcatabolism. c Transcript levels of ABA metabolism and signalling genes and selected downstream targets during moist-chilling (0, 10, 21 d), germination(6, 24, 80 h) and seedling growth (9 d) (black bars). Also shown are previously chilled seeds placed in two control treatments - 6 h germination conditionsunder darkness at 30 °C (dark grey bar) or 20 °C (light grey bar). Each data point is the average of three biological replicates. Bars indicate the SEM. Note:two superscript asterisks (**) indicate that the gene is only annotated in angiosperms; one asterisk (*) indicates that the gene has beenannotated in gymnosperms but not in white spruce; no asterisk indicates that the gene has been annotated in white spruceLiu et al. BMC Plant Biology  (2015) 15:292 Page 5 of 14constant darkness), germination of the population of seedsthat had been subjected to moist chilling was faster andmore synchronous than for the populations of seeds thathad not received moist-chilling; and 21-day chilling wasmore beneficial than the 10-day chilling treatment(Fig. 1b). This indicates that moist-chilling has a signifi-cant effect on dormancy alleviation, and that light cuesfollowing exposure of seeds to germination temperaturesfacilitate germination.Expression of the gene EMB32, encoding a LateEmbryogenesis Abundant (LEA) proteinDormancy status was also investigated by monitoringthe expression of the ABA-regulated gene EMB32, amember of the Late Embryogenesis Abundant (LEA)group. Dormancy maintenance bears some similaritiesto the late maturation program [55], and EMB32 andthe other LEAs have a role in ensuring seed survival inthe desiccated/dormant state. As such, EMB32 can beused as a dormancy marker [56]. Indeed, during moist-chilling of white spruce seeds, the expression of thisLEA gene was maintained at a high level, but this ex-pression decreased very quickly when seeds were trans-ferred to germination conditions, even as soon as sixhours under standard germination conditions (Fig. 1c,30 °C). Thus, the dormancy to germination transitionbegan promptly when the seeds were exposed to lightupon transfer to germination temperatures.Dynamic changes in plant hormone pathways in responseto temperature cues during moist-chilling and seedgerminationTo investigate hormone metabolism and signalling dur-ing dormancy alleviation and germination, hormonelevels and transcription of genes specifying the proteinmediators of hormone metabolism and signalling weredetermined at the various sampling stages (Fig. 1a).ABA metabolism and signallingBiologically active cis-S(+)-ABA did not substantiallychange in abundance during moist-chilling itself, but de-creased during subsequent germination of previouslychilled white spruce seeds (Fig. 2a). The so-called “trans-ABA” is in fact a product of isomerization of naturalABA under UV light, and this did not change during thetransition to germination. Generally the bioactive ABAlevels were much higher than those of the ABA catabo-lites. From the changes in ABA metabolites it was appar-ent that the main ABA metabolism pathway in whitespruce seeds is through 8’-hydroxylation (resulting inphaseic acid (PA), which is further reduced to dihydro-phaseic acid (DPA)). Nonetheless, secondary catabolismpathways such as 7’ and 9’ hydroxylation (resulting in7’hydroxy ABA and neo-PA) as well as conjugation(resulting in ABA-GE) were also represented. The vari-ous catabolites, especially PA and the 7’OH-ABAincreased during moist chilling, as well as duringgermination of moist-chilled seeds (Fig. 2a).Transcript abundance of ABI3 was markedly up-regulated during the first 10 d of moist chilling, but de-clined to a barely detectable level at 21 d (Fig. 2c).SnRK2.2 transcripts exhibited a similar expression pat-tern during the moist chilling phase (Fig. 2c). Thus,while absolute ABA levels remained constant, transcrip-tion of genes for ABA signalling components, andthereby sensitivity to ABA, started to decline during thelatter part of the moist chilling phase (Fig. 2b, c). More-over, transcripts of a putative ortholog of a negativeregulator of ABI3, CnAIP2 [31], steadily accumulatedduring moist-chilling and remained high during earlygermination (6 h). ABI3 transcripts, were high at themid-point during moist chilling, then declined precipi-tously during late moist chilling and early germination,but increased during the later stages of germination (24and 80 h) (Fig. 2c).Transcripts encoding AAO3 (ABA biosynthesis en-zyme) underwent few changes during moist chilling, butincreased dramatically during early germination understandard conditions, followed by a decline; those forCYP707A4 (encoding ABA 8’ hydroxylase) were not de-tectable (Fig. 2c). Similar to AAO3, transcripts encodingCYP707A4 and SnRK2.2 showed a significant up-regulation during the early stages when seeds were firsttransferred to standard germination conditions at 6 h,with transcripts declining at the later stages (Fig. 2c). Anactual decline in bioactive ABA was not evident until24 h of germination (Fig. 2a). At the seedling stage (9 d),transcripts for all of the monitored genes involved inABA metabolism and signalling decreased to a very lowlevel (Fig. 2c).GA metabolism and signallingOf the 14 GAs that were quantified in white spruceseeds (i.e., GA1, 3, 4, 7, 8, 9, 19, 20, 24, 29, 34, 44, 51, and 53),only GA53 and GA34 were present at detectable levels.GA53 is an early precursor in the 13-hydroxylation path-way (GA53→GA44→GA19→GA20(→GA29)→GA1→GA8) and leads to the formation of bioactive GA1 andits inactive degradation product GA8; GA34 is an inactivecatabolite of biologically active GA4 in the non-hydroxylation biosynthetic pathway (GA12→GA15→GA24→GA9(→GA51)→GA4→GA34). The presence ofintermediates from both biosynthesis routes suggeststhat both GA metabolic pathways are active in whitespruce seeds during dormancy alleviation and germin-ation. Moreover, the presence of GA34 suggests thatGA4 must have been produced at earlier stages. GA53of the early 13-hydroxylation pathway conducive toLiu et al. BMC Plant Biology  (2015) 15:292 Page 6 of 14the formation of bioactive GA1 increased steadily dur-ing germination under standard conditions after seedshad received moist chilling. During moist-chilling it-self, GA53 and GA34 were maintained at steady-statelevels, with GA53 present at ~5-fold higher levels thanGA34 (Fig. 3a). GA34 increased most substantially at 9d (i.e. during seedling growth) (Fig. 3a).Most of the GA-related genes that we monitored(those encoding mediators of GA- biosynthesis, signal-ling, or action; Fig. 3b) were expressed at low levelsbPgKS0.°Clight30°Cdark20°CdarkcPgCPS012345GA20ox1******024681012141618EXP2**  10d 21d 6h 24h  80h  9d 0d  10d 21d 6h 24h  80h  9d 0d  10d 21d 6h 24h  80h  9dRelative unitsTime courseadSPT** (ent-pgKS)50kDa ( -tubulin)0d   10d   21d 6h    24h  80h    9dMoist-chillingGermination –standard conditions6h   24h   80h 6h    24h  80hGermination in darkness with temperature cycleSeeds kept at 3°C with 8-hphotoperiod Relative units0d  10d 21d 6h 24h  80h  9dTime courseFig. 3 Changes in GAs and GA signalling components during the transition from dormancy to germination of white spruce seeds. a Profiles ofthe GA precursor GA53 and the metabolite GA34 as determined by UPLC/ESI-MS/MS during moist-chilling at 3 °C (0, 10, and 21 days), and duringgermination (6, 24 and 80 h) and seedling growth (9 d). Each data point is the average of two biological replicates. No active GAs were detectedin our analysis. b Schematic characterization of key genes and their interplays in GA signaling cascades. Connections represent positive (arrow)and negative (block) regulation. c Transcript levels of GA metabolism genes and selected downstream targets during moist-chilling (0, 10, 21 d),germination (6, 24, 80 h) and seedling growth (9 d) (black bars). Also shown are previously chilled seeds placed in two control treatments - 6 hgermination conditions under darkness at either 30 °C (dark grey bar) or 20 °C (light grey bar). Each data point is the average of three biologicalreplicates. Bars indicate the SEM. Note: see Fig. 2 note for asterisks. d Ent-pgKS protein levels during moist-chilling, germination, and growth ofwhite spruce seeds. Immunoblots show 30 μg of total protein extract per lane. Blots were probed with anti-KS antibody and anti-tubulin as aloading controlLiu et al. BMC Plant Biology  (2015) 15:292 Page 7 of 14during moist-chilling (Fig. 3c). (Note that all referencegenes were expressed during these times; Additional file1: Figure S3). The expression of SPT (SPATULA), encod-ing a mediator of ABA- and GA- signaling cross talk,decreased to a low level at 10 d of moist chilling butexhibited a 14-fold increase at 21 d (Fig. 3c).We also investigated transcript abundance of genesknown to be positively regulated by GA as indirect indi-cators of the presence of active GA. The GA-regulatedcell wall-modifying gene, expansin 2 (EXP2), exhibited a15-fold up-regulation within 6 h after transfer of moistchilled seeds to standard germination conditions (Fig. 3c).The expression of other GA-related genes was also sub-stantially increased during early germination beforeradicle protrusion; moderate expression occurred be-tween 24–80 h, while at the seedling stage, the expres-sion of all of the monitored genes was low or virtuallyundetectable (Fig. 3c). This is indicative of the presenceof active GA during completion of germination and dur-ing very early seedling growth (seedling emergence).Ent-pgKS protein levels were increased during the first10 d of moist chilling, with a decline during the latterperiod of moist chilling (Fig. 3d). Upon transfer ofmoist-chilled seeds to germination conditions, the levelsincreased by 6 h (coincident with increased transcriptlevels; Fig. 3c). The most pronounced ent-pgKS proteinlevels were evident in seeds at 80 h under standardgermination conditions; however, the control treat-ments indicated that either changing the light condi-tions or exposing seeds to germination temperatureswere sufficient to trigger the increased levels of thisprotein (Fig. 3d).Auxin metabolism and signallingActive IAA was almost at constant levels throughout themoist chilling period, and during germination (Fig. 4a).IAA conjugates (IAA-Asp and IAA-Glu) strikingly in-creased over 20-fold during the 21 d of moist-chilling(Fig. 4a). These conjugated IAAs declined markedly dur-ing the first 6 h in standard germination conditions; laterseedling growth was accompanied by an increase in bothactive and conjugated IAA (Fig. 4a).In the auxin pathway (Fig. 4b, c), the expression ofauxin biosynthesis genes (ASA1/2, ASB1, TSA1, TSB1,and AAO1) was highest at 10 d of moist chilling, thendeclined at the later stages of moist chilling (21 d). Inter-estingly, AMI1, another auxin biosynthesis gene in a par-allel pathway with AAO1, exhibited lowest expression at10 d of moist chilling (Fig. 4c). This suggests that auxinwas actively synthesized during moist-chilling and medi-ators of the two pathways that synthesize auxin wereseparately activated at early and late moist-chilling. Like-wise, ASA1/2, ASB1, TSA1, TSB1, and AAO1 exhibitedhigh expression levels at 6 and 80 h, while AMI1 had aconstant low expression level during germination. InArabidopsis, there exists a third auxin biosynthesis path-way via YUC [57]; no homolog to the Arabidopsis YUCgene was found in white spruce after an extensive data-base search, and this auxin biosynthesis route may notexist in the seeds of this conifer species. The expressionof IAR3 and ILL1/2, which specify enzymes that convertconjugated IAA to active IAA, as well as expression ofgenes for the auxin transporters PIN1-like and CUC-likewas significantly up- and then down- regulated in associ-ation with the transcript regulation of the biosynthesisgenes of the AAO1 pathway during moist chilling(Fig. 4c). In seeds placed under standard germinationconditions, the genes for the auxin transporters exhib-ited a pattern of heightened transcript abundance duringgermination, and lowered expression during seedlinggrowth (Fig. 4c).Auxin signalling primarily depends on the TIR1/AFBauxin receptor (TAAR), Aux/IAA, and ARF4. The ex-pression of TIR1, AFB3 and Aux/IAA was significantlyup and then down regulated during moist-chilling andthat of ARF4 appeared to follow the same pattern but ata lower absolute level (Fig. 4c). At 6 h in germinationconditions, the expression of TIR1, AFB3, Aux/IAA, andARF4 significantly increased but only TIR1 and AFB3continued to increase at 24 h. At the seedling stage,ARF4 along with TIR1, AFB3, and Aux/IAA wasexpressed at a fairly low level (Fig. 4c). Likewise, tran-script for CUL1, a component of SCF ubiquitin ligasecomplexes, was substantially produced during 80 h ingermination but not during seedling growth (Fig. 4c).Dynamic changes of hormone signalling pathways afterdormancy termination during germination and radicleprotrusionTo separate the contributions of optimal germinationtemperature and light signalling to germination comple-tion (i.e. radicle protrusion), two additional germinationconditions in place of the standard conditions were usedafter seeds had received 21 days of moist chilling. Ascontrols, seeds were not exposed to light (i.e. kept indarkness) but were exposed to either an optimal germin-ation temperature (30/20 °C) (Fig. 1a) or a non-optimalgermination temperature (constant 20 °C) (not shown inFig. 1a). Transferring seeds to standard (i.e. optimal)germination conditions led to greater fold transcriptchanges than transferring seeds to the same temperatureregime but keeping them in darkness. Transferring seedsto 20 °C in darkness further reduced transcript induction(Figs. 2c, 3c, and 4c). This effect was particularly obviousfor the studied genes of the GA pathway.PCA analysis for all studied genes in different germin-ation conditions was conducted (Fig. 5 and Additionalfile 1: Figure S4). Gene expression variations (68.96 andLiu et al. BMC Plant Biology  (2015) 15:292 Page 8 of 14AAO1**012345AMI1**012345(a)ASA1/2**0123456730°Clight30°Cdark20°CdarkASB1**0246810TSA1**012345TSB1**0246810121416(b)ARF40.**0246810PIN1-like0.***02468101214Aux/IAA0. unitsTime course0d  10d 21d 6h 24h  80h  9d 0d  10d 21d 6h 24h  80h  9dAFB3**02468101214ILL1/2**0246810CUL1**01234560d  10d 21d 6h 24h  80h  9d(c)Fig. 4 (See legend on next page.)Liu et al. BMC Plant Biology  (2015) 15:292 Page 9 of 1427.08 %) were explained by PC1 and PC2, respectively,and the PCA grouped the samples into five clusters(Fig. 5). Based on PCA analysis, we found that: 1) ger-mination initiation (6 h) and radicle protrusion (80 h)under standard germination conditions (30/20 °C and 8-h photoperiod) were associated with similar gene expres-sion patterns. The same was true of 6 h and 24 h indarkness with a 30/20 °C temperature cycle and of 24and 80 h in constant low temperature (3 °C) with an 8-hphotoperiod; 2) gene expression patterns at 80 h in con-stant darkness were similar to those at 24 h with an 8-hphotoperiod; 3) six h in low temperature was associatedwith unique gene expression patterns. Therefore, seedsin constant darkness with temperature cycles displayed asimilar expression pattern but were delayed in time,compared with those seeds placed under both optimalgermination temperature and photoperiod cycles, Con-versely, seeds in constant low temperature with an 8-hphotoperiod exhibited different gene expression patternsat 6 h, and at 24 and 80 h, despite not completinggermination (visible radicle protrusion) (Fig. 5). Takentogether, temperature and light jointly promoted ger-mination mediated by ABA, GA, and auxin pathways.DiscussionPlant hormones co-ordinately respond to temperaturecuesMoist-chilling is associated with changes in hormone fluxIAA biosynthesis was active during moist-chilling (Fig. 4c),but active IAA levels were maintained at constant levels,while conjugated IAA-Asp and IAA-Glu steadily andsignificantly increased (Fig. 4a). Conjugated IAAs areregarded as storage compounds, which, in seeds, areeither stored to be activated by de-conjugation andserve in early seedling growth, or are used for an entryroute into subsequent catabolism [58]. IAA conjugatedto amino acids such as aspartate and glutamate maybe largely degraded [59]. Although the function of IAAconjugates and the genes that regulate their formation isscarcely investigated, the large amount of IAA conjugatesthat accumulated during moist-chilling likely has bio-logical significance. More information is required con-cerning the cellular distribution of the different auxinforms as well as their relative dependence on specifictransport mechanisms [60].The PIN family proteins and the recently discoveredPIN-LIKES are important as IAA efflux carriers in IAAtransport between the cytosol and the endoplasmicreticulum [60, 61]. We observed that, at 10 d of moist-chilling, and during subsequent germination, transcriptsof PIN1-like and CUC-like were markedly up-regulated(Fig. 4c) while active IAA remained relatively constant(Fig. 4a). Auxin-induced cell expansion connected to theacidification of the cell wall, is thought to invoke an in-crease in the activity of the wall loosening proteins,expansins [62], which can disrupt the non-covalentbonds that form between cellulose and hemicellulose inthe wall and thus promote cell expansion [63]. Despiteno substantial overall increase in IAA during germin-ation, IAA may nonetheless be redistributed within seedtissues to active areas of cell expansion due to the actionof various transporters (Fig. 4c). Polar transport sets upauxin gradients in specific cell types, and such gradientscan provide developmental cues during key processes in-cluding embryogenesis and root development [64, 65].The auxin response not only depends on auxin levelsand locations, but also on the specificity and strength ofthe TIR1-Aux/IAA and Aux/IAA-ARF interactions [45].S80PC1 (68.96 )0.70 0.75 0.80 0.85 0.90 0.95PC2 (27.08)-1.0- D80L24 L80D06D24L06Fig. 5 The results of principle component analysis applied to theexpression of all the genes used in previous qPCR analysis in ABA andGA pathways over three different germination conditions. S06/S24/S80,D06/D24/D80, and L06/L24/L80 represent standard, darkness, and lowtemperature (3 °C) germination conditions corresponding to D/E/F,H/I/J, and K/L/M in Fig. 1a, respectively(See figure on previous page.)Fig. 4 Changes in IAA, IAA conjugates, and auxin-related gene expression during the transition from dormancy to germination of white spruceseeds. a IAA and IAA conjugates in seeds as determined by UPLC/ESI-MS/MS during moist-chilling at 3 °C (0, 10, and 21 d) and during germination (6,24 and 80 h) and seedling growth (9 d). Each data point is the average of two biological replicates. b Schematic characterization of key genes and theirinterplays in auxin signalling cascade. Connections represent positive (arrow) regulation. c Transcript levels of auxin metabolism genes, auxin signallinggenes and selected downstream targets during moist-chilling (0, 10, 21 d), germination (6, 24, 80 h) and seedling growth (9 d) (black bars). Also shownare previously chilled seeds placed in two control treatments - 6 h germination conditions under darkness at either 30 °C (dark grey bar) or 20 °C (lightgrey bar). Each data point is the average of three biological replicates. Bars indicate the SEM. Notes: 1) see Fig. 2 note for asterisk in c; 2) no other ARFhomologs (such as ARF16) and GH3 (converting active IAA to IAA-aa) homologs were found in white spruce by BLASTNLiu et al. BMC Plant Biology  (2015) 15:292 Page 10 of 14The decreased availability of TIR1 could lead to in-creased levels of free Aux/IAAs, which would com-bine with ARF4, thereby eventually decreasing freeARF4 levels. Hence, it is possible that prior to 10 dof moist chilling, Aux/IAA is predominantly com-bined with TIR1/AFB3 rather than with ARF4, andthat the free ARF4 contributes to the increase ofABI3 transcripts at 10 d (Figs. 4c and 2c), becauseARFs may bind to putative auxin response elements(AuxREs) of the ABI3 gene promoter [42]. Conversely,after 10 d, ARF4 may be more likely to interact withAux/IAA [45], thus lowering free ARF4 and contrib-uting to the decreasing expression of ABI3 at 21 d(Figs. 4c and 2c). These changing interactions be-tween components of the ABA and auxin signallingpathways may promote dormancy alleviation [42].Our analyses were confined to monitoring transcriptlevels and so we can only speculate as to changes atthe level of the proteins that mediate auxin and ABAaction. Changes in transcript levels for the ABI3 an-tagonist – CnAIP2 – may also be relevant here as theCnAIP2 promoter is exquisitely regulated by auxin.Active ABA did not decrease during moist-chillingitself, but did decrease substantially during subse-quent germination (Fig. 2a), at a time when GA53 ofthe early 13-hydroxylation pathway conducive to theformation of bioactive GA1, increased steadily (Fig. 3aand c). Thus an increased GA/ABA ratio, was clearlyassociated with germination of white spruce seeds[66]. We did not detect any bioactive cytokinin inour samples (Additional file 1: Figure S1). Cytokininand auxin have long been known to interact antagon-istically, and the past five years have seen significantadvances in our understanding of the extensive cross-talk between cytokinin and various other hormones,particularly auxin (reviewed in [67]). In our study, none ofbioactive free base cytokinins (zeatin, dihydrozeatin, andisopentenyladenine) was detected during moist chilling.However, the biosynthesis precursors cis-zeatin roboside(cis-ZR) and isopentenyladenine riboside (iPR) were mark-edly increased, while the catabolism product cis-zeatin-O-glucoside (cis-ZOG) was detected only at very low levelsduring moist-chilling (Additional file 1: Figure S1). Thismay indicate that a small amount of cis-zeatin was transi-ently produced as a result of moist chilling.SPT (SPATULA) is thought to be involved in both ABAand GA signaling cross talk and may drive two antagonistroles in mature seeds of Arabidopsis – ‘dormancy-pro-moting’ and ‘dormancy-repressing’ – depending on theecotype background [68, 69]. In white spruce seeds, SPTexpression decreased to a low level at 10 d of moist chill-ing but exhibited a 14-fold increase at 21 d (Fig. 3c). Itsrole in white spruce dormancy alleviation and germinationremains to be determined.Germination conditionsWhen seeds were transferred to germination conditions,we observed remarkably strong changes in expression ofour monitored genes typically by only 6 h. Transcriptsencoding the GA-regulated cell wall-modifying proteinexpansin 2 (EXP2) were 15-fold up-regulated, indicatinga strong up-regulation in GA signalling (Fig. 3c). ABAdeclined especially after 24 h (Fig. 2a). ABI3 wasexpressed at low levels at 6 h but was up regulated at24 h, perhaps relevant to a ‘stress sensing’ function atthis critical stage (Fig. 2c). At the radicle protrusiontime-point (80 h), transcripts of genes specifying auxinbiosynthesis enzymes (ASA1/2, ASB1, TSA1, and AAO1),or proteins mediating conversion to active IAA (IAR3and ILL1/2), and signaling (Aux/IAA) were substantiallyproduced, and these pathways may have acted in concertwith those of the GA and ABA signaling pathways(Fig. 4c). Thus auxin likely also plays a pivotal role ingermination of white spruce seeds.Plants have evolved a battery of photoreceptors tosense ambient light and transduction of light signals[70]. In the control of seed dormancy and germination,phytochromes represent the most investigated photore-ceptors. Phytochromes are temperature- and light-dependent in association with the GA pathway via SPT[71]. The expression of SPT significantly decreased at6 h and the transcript levels were almost the same asthose in the seeds exposed to light or kept in darkness.However, the seeds placed in 30 °C had a lower level ofSPT transcripts than seeds placed in 20 °C (Fig. 3c). Inwhite spruce, as in Arabidopsis, SPT may be a light-stable repressor of seed germination and may play a rolein the germination response to temperature throughtemperature-sensitive changes in its transcription [68].Winter chilling under new climate scenarios and itseffects on conifer life historiesWinter chilling is an important signal for regulatingplant life histories; chilling leads to a competence forflowering through vernalization in winter annuals, andalleviates both bud and seed dormancy, allowing the on-set of growth in the spring [9, 72]. It is noteworthy thatclimate change may ultimately result in winter short-ening and an increase in the growing season length[73, 74]. In North America, the number of winterchilling days has become insufficient for bud dormancybreak from 40°N southward as climate changes, leading todelayed vegetation green-up, but it has remained sufficientfrom 40°N northwards as earlier springs lead to anadvanced green-up onset [75]. A similar geographic pat-tern as observed in budburst may also occur for germin-ation in temperate regions and two possible scenariosexist depending on whether moist-chilling requirementsare minimally met; namely, fast and prompt germinationLiu et al. BMC Plant Biology  (2015) 15:292 Page 11 of 14leading to greater recruitment (adequate chilling) oran extended germination span leading to adverse con-ditions during dry summers (inadequate chilling) (seeFig. 1b). Thus shorter winters may delay or advancegermination [76].On the other hand, the range of spruce trees and otherconifers cover large climatic gradients while their sub-population can be adapted to their local environments[77, 78]. These populations may draw on alternativemolecular solutions to respond to local environmentalconditions [79, 80]. Presumably, variations in gene ex-pression contribute to phenotypic diversity includingdormancy variation and, therefore sustain the adaptabil-ity of conifer populations [81]. As such, our results ofgene expression during moist-chilling may help predictfuture seed recruitments in response to climate change.Finally, it is important to note that the seeds of certainother conifer species (yellow cypress, western white pineand white bark pine) exhibit much deeper dormancy atmaturity than white spruce seeds. These seeds requireseveral months of moist chilling to alleviate their dor-mancy, and may well be more substantially impacted byclimate change, as the extended cold period is so criticalfor their ability to germinate.ConclusionsIn addition to classic ABA and GA mechanisms, auxinappears to be actively involved in dormancy terminationand germination of white spruce seeds. We hypothesizethat auxin signalling plays a role in these processespartly by interacting with ABA signalling. This is inaccordance with recent findings regarding the crosstalkof auxin and ABA in the regulation of seed dormancy inangiosperms [42]. Auxin has a dominant role in plantmorphogenesis and is an inescapable player in manydevelopmental processes and a central component ofcrosstalk networks. Our findings now point to auxin as akey player that likely works in conjunction with theABA and GA signal pathways previously investigated inmechanisms underlying dormancy alleviation by chillingin conifer seeds. Our study also yields insights into thespeed with which imbibed seeds can adjust their tran-scription to environmental conditions, as demonstratedwhen seeds were transferred from moist chilling to ger-mination conditions. After only six hours in light athigher temperatures, significant changes in transcriptabundance were observed.Additional fileAdditional file 1: Table S1. Description of genes and primer pairs usedfor qPCR. Figure S1. Profiles of cytokinins and their metabolites in seeds ofwhite spruce (as determined by UPLC/ESI-MS/MS) during moist-chilling at3 °C (0, 10, and 21 d), and during germination (6, 24 and 80 h) and seedlinggrowth (9 d). Each data point is the average of two biological replicates.cis-ZR, cis-Zeatin riboside; iPR, Isopentenyladenine roboside; cis-ZOG, cis-Zea-tin-O-glucoside. Figure S2. Transcript levels of various marker genes at 6,24, and 80 h following transfer of seeds to standard germination conditions(i.e. 8-h photoperiod and 30/20 °C) (black bars), constant darkness with a30/20 °C cycle (light grey bars), or constant 3 °C with an 8-h photoperiod(dark grey bars). Each data point is the average of three biological replicates.Bars indicate the SEM. Figure S3. C(t) values for all three referencegenes across studied time-points. Figure S4. Repeatability of hormonequantification analyses. Note: variation between two experimental replicatesin four metabolites (IAA-Asp, IAA, PA, and ABA) was distinguished by coloursin the panel. (DOCX 475 kb)AbbreviationsABA: Abscisic acid; ABA-GE: abscisic acid glucose ester; PA: Phaseic acid;DPA: Dihydrophaseic acid; neo-PA: neo-phaseic acid; 7’OH-ABA: 7’-hydroxyabscisic acid; GA: Gibberellin; GA53/34: Gibberellin 53 and 34; IAA: Indole-3-aceticacid; IAA-Asp: N-(Indole-3-yl-acetyl)-aspartic acid; IAA-Glu: N-(Indole-3-yl-acetyl)-glutamic acid; AAO3: Abscisic acid aldehyde oxidase 3; CYP707As: Aba 8’-hydroxylases; PYR/RCAR: Pyrbactin resistance 1-like/ regulatory component of abareceptor; PP2C: Protein phosphatase 2C; SnRK2: Sucrose nonfermenting 1(SNF1)- Related protein kinase 2; ABI3/5: Aba insensitive 3/5; AIP2: ABI3-interactingprotein 2; LEC1: Leafy cotyledon 1; ABF/AREB: Abscisic acid responsive element-binding factor; CPS: Ent-copalyl diphosphate synthase; KS: Ent- kaurene synthase;GA20ox: Gibberellin 20 oxidase; GA3ox: Gibberellin 3 oxidase; GA2ox: Gibberellin 2Oxidase; GID1: Gibberellin insensitive dwarf 1; BME3: Blue micropylar end 3;SPY: Spindly; EXP2: Expansin A2; SPT: Spatula; ASA1/2: Anthranilate synthasecomponent I-1/2; ASB1: Anthranilate synthase beta subunit 1; TSA1: Tryptophansynthase alpha chain 1; TSB1: Tryptophan synthase beta subunit 1; AAO1: Aldehydeoxidase 1; AMI1: Amidase 1; IAR3: IAA-alanine resistant 3 (IAA-amino acidhydrolase); ILL1/2: IAA-leucine resistant (ILR)-like 1/2 (IAA-amino acid hydrolase);PIN1-like: Pin formed 1-like; CUC-like: Cup-shaped cotyledon-like; CUL1: Cullin1;TIR1: Transport inhibitor response 1; AFB3: Auxin signaling F-box 3; Auxin/IAA: Auxin-induced protein 2 (AUXIN/IAA); ARF4: Auxin responsive factor 4.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsConceived and designed the experiments: YL ARK KM YAK. Performed theexperiments: YL. Analyzed the data: YL. Wrote the paper: YL ARK KM YAK.All authors have read and approved the final version of the manuscript.AcknowledgementsWe would like to extend our thanks to Dr. T-P. Sun (Duke University) for theKS antibody, to Dr. L. Irina Zaharia (Plant Biotechnology Institute) for thehormone quantification data, and to Mr. D. Kolotelo (British Columbia Ministryof Forests, Land and Natural Resource Operations) for seed supply. We gratefullyacknowledge funding by the European Commission to KM through a MarieCurie IOF Fellowship, and from NSERC Discovery grants to ARK and YEK.Author details1Department of Forest and Conservation Sciences, Faculty of Forestry,University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.2Department of Biological Sciences, Simon Fraser University, Burnaby, BritishColumbia V5A 1S6, Canada.Received: 26 August 2015 Accepted: 5 October 2015References1. 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Mol Ecol. 2013;22(9):2369–79.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitLiu et al. BMC Plant Biology  (2015) 15:292 Page 14 of 14


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