Transcriptome wide Analysis Of Vernalization Reveals ...

Research Article

Transcriptome‐wide Analysis Of VernalizationReveals Conserved and Species‐specificMechanisms in BrachypodiumQing Huan1,2†, Zhiwei Mao1,2†, Jingyu Zhang1, Yunyuan Xu1 and Kang Chong1*1Key Laboratory of Plant Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China2University of Chinese Academy of Sciences, the Chinese Academy of Sciences, Beijing 100049, China†These authors contributed equally to this work.�Corresponding authorTel: þ86 10 6283 6695; Fax: þ86 10 6283 6517; E‐mail: [email protected] online on 14 March 2013 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipbdoi: 10.1111/jipb.12050

Abstract

Several temperate cereals need vernalization to promote flowering. Little, however, is known about thevernalization‐memory‐related genes, and almost no comparative analysis has been performed. Here, RNA‐Seq was used for transcriptome analysis in non‐vernalized, vernalized and post‐vernalizedBrachypodiumdistachyon (L.) Beauv. seedlings. In total, the expression of 1,665 genes showed significant changes (foldchange �4) in response to vernalization. Among them, 674 putative vernalization‐memory‐related geneswith a constant response to vernalization were significantly enriched in transcriptional regulation andmonooxygenase‐mediated biological processes. Comparative analysis of vernalization‐memory‐relatedgenes with barley demonstrated that the oxidative‐stress response was the most conserved pathwaybetween these two plant species. Moreover, Brachypodium preferred to regulate transcription and proteinphosphorylation processes, while vernalization‐memory‐related genes, whose products are cytoplasmicmembrane‐bound‐vesicle‐located proteins, were preferred to be regulated in barley. Correlation analysisof the vernalization‐related genes with barley revealed that the vernalization mechanism was conservedbetween these two plant species. In summary, vernalization, including its memory mechanism, isconserved between Brachypodium and barley, although several species‐specific features also exist. Thedata reported here will provide primary resources for subsequent functional research in vernalization.

Keywords: Brachypodium; comparative analysis; RNA‐Seq; vernalization.

Huan Q,Mao Z, Zhang J, Xu Y, Chong K (2013) Transcriptome‐wide analysis of vernalization reveals conserved and species‐specificmechanisms inBrachypodium. J. Integr. Plant Biol. xx(xx):1–14.

Introduction

Flowering, which enables plants to successfully produce theirprogeny, is important for plants (Kobayashi and Weigel 2007;Srikanth and Schmid 2011; Kumar et al. 2012). The molecularmechanisms behind flowering time control have been studied formany years (Kobayashi and Weigel 2007; He 2012), and thevernalization pathway is one of the four major floweringregulation pathways (Komeda 2004; Fornara et al. 2010;Higgins et al. 2010). Previous reports have shown that the keyfeatures of vernalization are similar between monocot and dicot

species, but the vernalization‐response pathways are different(Dennis and Peacock 2009; Srikanth and Schmid 2011). Forexample, as a repressor of flowering, FLOWERING LOCUS C(FLC) plays a central role in the vernalization pathway inArabidopsis (Sanda and Amasino 1996; Michaels 2009).However, no homolog of FLC has been reported in cereals.Instead, VRN1, a flowering activator, has been proved to act as amajor regulator of the vernalization response in barley, wheatand other temperate grasses (Yan et al. 2003; Andersenet al. 2006; Oliver et al. 2009; Sun et al. 2009). In addition,both FLC and VRN1 are regulated by histone modifications

Journal of Integrative Plant Biology 2013, 55 (9999): 1–14

© 2013 Institute of Botany, Chinese Academy of Sciences

during vernalization, although they are in two completelyopposite regulation styles. Vernalization represses the expres-sion of FLC through histone modifications in Arabidopsis, butactives VRN1 in cereals (De Lucia et al. 2008; Jiang et al. 2009;Oliver et al. 2009; Diallo et al. 2012). Another specific characterof vernalization is the vernalizationmemory phenomenon. Someplants can remember vernalization treatment and promoteflowering at a later stage. However, the exploration of genesthat are associated with vernalization memory is still underway.

Previously, Greenup et al. (2011) reported several genesshowing lasting responses to vernalization in barley. Laudencia‐Chingcuanco et al. (2011) described low temperature‐tolerantgenes in wheat, and Mutasa‐Göttgens et al. (2012) analyzed theexpression profiles in response to vernalization in sugar beet.Several genes, such as OS2 (ODDSOC2), RAV1 (related toABA‐insensitive 3/viviparous1), S‐like RNase genes and jacalin‐like lectin genes, are suggested to be vernalization‐responsive(Greenup et al. 2011; Laudencia‐Chingcuanco et al. 2011).Among them, OS2 encoding a MADS‐box floral repressor hasbeen shown to be downregulated by vernalization in temperatecereals (Greenup et al. 2010). Antisense inhibition of VER2, ajacalin‐like lectin, could delay the heading time of transgenichexaploid wheat (Yong et al. 2003; Xing et al. 2009). However, itis still not easy to further explore the vernalization‐associatedregulation mechanisms from numerous candidate genes, due tothe difficult transformation and long life periods of plant materialsand their complex genomes, and the limited knowledge ofgenome sequences.

The wild grassBrachypodium distachyon has emerged as animportant model for temperate species, including severalimportant grain crops, such as wheat (Triticum monococcum,T. durum, and T. aestivum), barley (Hordeum vulgare), and oats(Avena sativa) (Opanowicz et al. 2008; Initiative TIB 2010). Itsrelatively simple genome and releasedwhole genome sequencemake genome‐wide analysis feasible (Initiative TIB 2010).Transcriptome analysis, therefore, could be performed inBrachypodium through RNA‐Seq, which is a more precise andpowerful tool than microarray analysis (Wang et al. 2009).Furthermore, the short life cycle, efficient transformation systemand available T‐DNA mutant libraries of Brachypodium are alsohelpful for further analysis (Vain et al. 2008; Alves et al. 2009;Thole et al. 2011). Besides, the close evolutionary relationshipbetween Brachypodium and other temperate crops makescomparative studies more meaningful, and the results could bewidely applied to the enhancement of grain yield and quality(Initiative TIB 2010).

In this study, we used non‐vernalized control (NV), vernalized(V), and post‐vernalized (PV) seedlings to performRNA (mRNA)sequencing (RNA‐Seq), and identified a group of differentiallyexpressed genes during vernalization. Subsequently, variousexpression change patterns were clustered, among which thevernalization‐memory‐related genes showed a constant re-

sponse to vernalization, even when the seedlings were shifted tonormal conditions. The related biological processes were alsoanalyzed. Additionally, barley is a close neighbor of Brachypo-dium, and previous similar research of microarray data hasidentified a specific number of putative vernalization responsivegenes. Through comparative analysis between Brachypodiumand barley, we studied the correlation of vernalization‐memory‐related genes and found several conserved and species‐specificfeatures about their functions. These results may help us tobetter understand vernalization and its evolution.

Results

Global expression changes in Brachypodium seedlingswith or without vernalization treatment

Vernalization promotes flowering in many winter‐habit plants. InBrachypodium, flowering could be induced in plants treated withprolonged cold for four w. If the vernalization treatment wasshorter than four w, plants could not flower (Figure 1). Based onthis result, we used RNA‐Seq to examine gene expressionprofiles in NV, V, and PV Brachypodium samples (see Materialsand Methods section), and expected some new regulationmechanisms of vernalization.

After Illumina sequencing, 12,516,073, 12,670,331, and11,862,530 clean reads were obtained for NV, V, and PVsamples, respectively. Quality control of these reads demon-strated that the sequencing error rate was near 1 � 10�4

(Figure S1). TopHat/Cufflinks pipeline was then used to map theshort reads to the Brachypodium reference genome (http://www.branchypodium.org) (Initiative TIB 2010) and to evaluate thetranscript abundance (Trapnell et al. 2009, 2012). In total, 3,671and 2,255 genes were identified as differentially expressedgenes (DEGs) in vernalized and post‐vernalized samples (foldchange �2). Moreover, 967 and 427 genes (fold change �4) aswell as 466 and 266 genes (fold change �8) were identified asDEGs with more significant changes, respectively (Figure 2A,Table S1). Genes that showed altered expression in vernalizedand post‐vernalized samples were predominantly downregu-lated (Figure 2B, C). To further test the reliability of RNA‐Seq,quantitative polymerase chain reaction (qPCR) validations wereperformed with 12 randomly chosen genes, all of which yieldedresults consistent with our RNA‐Seq data (Figure 3).

Principal component analysis (PCA) and scatter plots wereperformed to evaluate the transcriptional changes in differentsamples. In the PCA, the first main two principal components,which explained more than 90% of the total variance, indicatedthat the post‐vernalized sample and non‐vernalized control weresimilar to each other, whereas the vernalized sample was quitedifferent from them (Figure 2D). In the scatter plots, theresults showed that many more genes altered their expression

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http://www.branchypodium.org/

levels in vernalized plants (R 2 ¼ 0.454) than in post‐vernalizedplants (R 2 ¼ 0.910), related to the non‐vernalized control(Figure 2E, F). This is quite consistent with the conclusions ofPCA.

Identification and functional analysis of vernalization‐related genes in Brachypodium

In order to explore the connections between expression patternsand gene functions, cluster analysis was used to analyze theexpression patterns of differentially expressed genes inBrachypodium (fold change �4; Figure 4). The gene ontology(GO) analysis of genes in each cluster was also performed usingagriGO (http://bioinfo.cau.edu.cn/agriGO/) (Du et al. 2010)(Figure 5).

According to our results, 191 genes in Cluster 2, 312 genes inCluster 5 and 362 genes in Cluster 6 exhibited significantchanges only in the vernalized sample (Figure 4, Clusters 2, 5,6). These genes were over‐represented in the GO analysis interms of transcriptional regulation, carboxyl acid metabolicprocess, metal ion transport, and photosynthesis. These genesare annotated as Homeobox‐leucine zipper proteins, C‐repeat

(CRT)‐binding factors, NAC domain proteins, glutamate decar-boxylases, metal ion binding proteins, HV90, HV58, chlorophylla‐b binding proteins, and photosystem I light‐harvesting complextype 4 protein (Figure 5).

Both Cluster 3 and Cluster 4 included 63 genes, theexpression levels of which were changed only in the post‐vernalization sample (Figure 4, Clusters 3, 4). The biologicalprocess that these genes were significantly involved in wasstress response, while most of the other genes were annotatedas hypothetical genes with unknown functions, which might beexplored in the future (Figure 5).

In total, we identified 123 genes in Cluster 1, 378 genes inCluster 7 and 173 genes in Cluster 8, which showed constantresponses to vernalization, even when seedlings were shifted tonormal conditions (Figure 4, Clusters 1, 7, 8). These genes wereconsidered putative “vernalization‐memory‐related genes” inBrachypodium, whose expression patterns were similar oropposite to those of VRN1 in barley (Oliver et al. 2009).Furthermore, Cluster 1 genes were significantly enriched intranscriptional regulation (Figure 5). These genes includeVRN1,Thioredoxin‐like 5 (TXNL5), Glutaredoxin‐C10, CBL‐interactingprotein kinase 30 (CIPK30), bHLH, and bZIP transcription factor

Figure 1. The floral phenotypes and statistics of the heading time of Brachypodium plants under differently vernalized treatments.

(A) Model of vernalization inducing flowering in Brachypodium. Vernalization is not effective until plants are treated for enough time. The arrow

represents the time axis of vernalization treatment.

(B) The floral phenotype of Brachypodium plants treated with gradient vernalization. The number shows the time of vernalized treatments.

W, week.

(C) The statistics of heading time in differently vernalized Brachypodium. The dotted line shows the maximal time cost of observation (200 d).

Results were calculated with plants of each treatment and the errors bars show the average ratio with SD.

Vernalization‐related Genes in Temperate Cereals 3

http://bioinfo.cau.edu.cn/agriGO/

Figure 2. Global changes in gene expression in seedlings under differently vernalized treatment.

(A) The total number of differentially expressed genes (DEGs) in different vernalization samples.

(B) The number of DEGs showing upregulation in the same comparison.

(C) The number of DEGs exhibiting downregulation in the same comparison.

(D) Principle component analysis of DEGs identified in different samples.

(E, F) Scatter plots of expression data depicting global expression patterns of vernalized (E) and post‐vernalized (F) samples versus the non‐

vernalized control. R 2 stands for the square of the correlation coefficient.

The expression levels of genes in the non‐vernalized control are plotted on the x‐axes, and the corresponding values of genes in the vernalized

and post‐vernalized plants are on the y‐axes.


genes (Table S2). On the other hand, genes in Cluster 7 andCluster 8 displayed constant downregulation, and genes withmonooxygenase, carboxylyase and nutrient reservoir activitywere over‐represented in these groups (Figure 5). Specifically,several genes in Clusters 7 and 8 are annotated as cytochromeP450, blue copper‐binding protein, tryptophan decarboxylase,and Germin (Table S2).

In Brachypodium, VRN1, VRN2, VRN3, and VRT2 are fourmajor flowering‐associated genes in the vernalization signalpathway. In our data, some of them exhibited similar expressionpatterns to their homologs in other species during and aftervernalization (Figure 6A–D). As a flowering active gene, VRN1shows a constantly upregulated response to vernalization insome temperate species (Andersen et al. 2006; Oliveret al. 2009). The transcriptional profile of BdVRN1 (Bra-di1g08340) was upregulated in the vernalized sample andremained at a high level in the post‐vernalized sample(Figure 6A). In addition, the expression of BdVRN3, thehomolog of FT, was not detected in these three samples atthe three‐leaf period, but its expression level was upregulatedsignificantly at a later stage (Figure 6C).

In addition, several homologs of putative vernalization‐memory‐related genes are also associated with flowering. Forexample, BdCO (Bradi1g43670, CONSTANS) encoded a zinc‐finger transcription factor and exhibited constant downregulation

during and after vernalization (Figure 6E, Table S3). A homologof BdCO is the integrator of the photoperiod pathway inArabidopsis (Yang and Chou 1999). BdPRR73 (Bradi1g65910,PSEUDO‐RESPONSE REGULATOR 73) and BdPRR95 (Bra-di4g36077, PSEUDO‐RESPONSE REGULATOR 95) showedconstant upregulation in vernalized and post‐vernalized samples(Figure 6F, G, Table S3). Homologs ofAtPRR7 andAtPRR9 areinvolved in the photoperiod and circadian clock pathways inArabidopsis (Nakamichi et al. 2005a, 2005b, 2007; Saloméet al. 2010). In addition, the expression profile ofBdVIL1 couldn’tbe maintained in the post‐vernalized sample, and BdVIL1 didn’tbelong to the putative vernalization‐memory‐related genes(Figure 6H). BdVIL1 was considered as a negative control.These results indicate that the vernalization signal pathway isconserved, and that the photoperiod and circadian clockpathway‐related genes may play roles in the vernalizationpathway.

Comparative analysis of vernalization‐memory‐relatedgenes between Brachypodium and barley

Microarray expression datasets of NV, V, and PV samples ofbarley were obtained from GEO (Gene Expression Ommibus,NCBI, GSE27822) to comparatively analyze the vernalization‐associated genes between Brachypodium and barley (Greenup

Figure 3. Quantitative polymerase chain reaction (PCR) validation of expression levels evaluated by RNA‐Seq.

The expression levels of 12 genes were confirmed by quantitative (q)PCR with the same RNA samples as RNA‐Seq. These genes were

randomly chosen from differentially expressed genes. The black squares represent the expression levels evaluated by RNA‐Seq, and the blank

squares represent the corresponding results by qPCR.


Figure 4. K‐means cluster analysis of differentially expressed genes.

Themeans of expression levels for each cluster are shown as red lines, and the gray lines represent the expression patterns of individual genes.

V/NV stands for the fold change of vernalized plants versus non‐vernalized samples, and PV/NV stands for that between post‐vernalized plants

and the non‐vernalized control.


et al. 2011) (Figure 7A). According to the Affymetrix 22K Barleychip probe sequences, we searched their homologs inBrachypodium with BLASTX using the protein sequencesdatabase (E‐value �1 � 10�20). With this criterion, homologswere found in Brachypodium for 12,945 (56.68%) probes ofbarley, and 9 863 (37.15%) genes of Brachypodium identifiedhomologs in barley (Figure 7B).

In an attempt to analyze the overall gene expression changefeatures in Brachypodium and barley during and after vernaliza-tion, intra‐species correlation coefficients were calculated withhomologs in these two species, respectively. Of all thehomologs, the expression levels of genes in the post‐vernalizedsample (R 2 ¼ 0.923 in Brachypodium and R 2 ¼ 0.982 inbarley) were more similar than in vernalized plants

Figure 5. Functional analysis of genes showing specific expression change patterns under differently vernalized treatments inBrachypodium.

Enrichment of selected gene ontology (GO) categories in different expression patterns (P < 0.01).


Figure 6. The transcriptional profiles of floral related genes in the vernalization pathway.

(A–D) RNA‐Seq coverage profiles (upper part in NV, V, and PV samples) and transcriptional profiles (lower part in NV‐seedlings, V‐seedlings,

PV‐seedlings, mature leaves, stems and young spikes) of BdVRN1, BdVRN2, BdVRN3, and BdVRT2.

(E–H) RNA‐Seq coverage profiles (left part) and transcriptional levels (right part) of BdCO, BdPRR73, BdPRR95, and BdVIL1 in NV, V, and PV

samples.

RNA‐Seq data are shown using IGV software. The quantitative polymerase chain reaction (PCR) results are normalized to BdTubullin. Data are

the mean of the SD of three parallel samples from three biological replicates.


(R 2 ¼ 0.415 in Brachypodium and R 2 ¼ 0.957 in barley),related to their non‐vernalized control (Table 1).

Comparative analysis with fold changes could reduce theinaccuracy of expression profiles detected by different platforms(RNA‐Seq in Brachypodium and microarray in barley). Weanalyzed the inter‐species conservation of vernalized and post‐vernalized samples between these two species. The resultsshowed that the correlation of vernalization treatment(R 2 ¼ 0.260) was significantly higher than that in the post‐vernalized sample (R 2 ¼ 0.162) between Brachypodium andbarley homologs, and the correlation coefficients of correspond-ing treatments were significantly higher than those of non‐corresponding treatments (Table 1), suggesting that thevernalization response was conserved between these two plantspecies.

To further analyze the functions of differentially expressedgenes between these two species, approximately the top 25% ofthem were chosen from Brachypodium and barley datasets andclassified into nine groups, respectively, according to theirchange patterns. Among them, we identified 278 and 350 geneswith a constantly upregulated response to vernalization inBrachypodium and barley, as well as 1,314 and 434 down-

regulated genes (Table S4). Both of them were putativevernalization‐memory‐related genes at the transcriptional levels.

Gene ontology analysis of these vernalization‐memory‐related genes showed that these genes were over‐representedin the stress response and monooxygenase‐mediated process-es. Stress‐related genes were mainly downregulated in Brachy-podium, while the corresponding genes in barley were eitherupregulated or downregulated in response to vernalization(Table 2).

However, GO analysis indicated that Brachypodium vernali-zation‐memory‐related genes were enriched in the following GOterms: transcriptional regulation (P‐value ¼ 4.60 � 10�10),protein phosphorylation (P‐value ¼ 2.90 � 10�10), metal iontransport (P‐value ¼ 7.70 � 10�10), and electron carrier activity(P‐value ¼ 1.80 � 10�13). Heme‐binding was more significant-ly enriched in Brachypodium (P‐value ¼ 7.70 � 10�20) than inbarley (P‐value ¼ 8.60 � 10�2). On the other hand, barleygenes, whose products are cytoplasmic membrane‐bound‐vesicle‐located proteins, were preferentially regulated in barley(Table 2). Additionally, we also had similar results fromcomparative analysis of homologous genes with a constantresponse to vernalization between Brachypodium and barley(Table S5).

Discussion

Recently, gene expression microarray studies have identifiednumerous genes participating in vernalization in many species,such as wheat and sugar beet (Laudencia‐Chingcuancoet al. 2011; Mutasa‐Gottgens et al. 2012). But it is still difficultto separate vernalization‐memory genes from vernalization‐

Table 1. Correlation of vernalization‐related genes betweenBrachypodium and barley

Dataset 1 Dataset 2 Correlation coefficient (R 2)

Bd‐V Bd‐NV 0.415

Bd‐PV Bd‐NV 0.923

Bd‐PV Bd‐V 0.348

Hv‐V Hv‐NV 0.946

Hv‐PV Hv‐NV 0.982

Hv‐PV Hv‐V 0.957

Bd‐V/NV Hv‐V/NV 0.260

Bd‐PV/NV Hv‐PV/NV 0.162

Bd‐V/NV Hv‐PV/NV 0.116

Bd‐PV/NV Hv‐V/NV 0.044

Bd, Brachypodium distachyon; Hv, Hordeum vulgare (barley). PV/

NV, fold change between the post‐vernalized sample and the non‐

vernalized control; V/NV, fold change of the vernalized sample

versus the non‐vernalized sample.

Figure 7. Comparative transcriptome analysis of vernalization‐memory‐related genes between Brachypodium and barley.

(A) Corresponding treatments in Brachypodium and barley. RT,

room temperature; V, vernalization treatment.

(B) The number and portion of homologs identified between

Brachypodium and barley.


responsive genes. Studies have been performed in this area inbarley by Trevaskis et al. (2006) and Greenup et al. (2011).However, there are many barriers to further research in this plantspecies, including its long life period, its large and complexgenome, and the fact that its genome information is little‐knownand its genetic transformation is hard. Therefore, we choseBrachypodium, a newmodel of temperate cereals, to perform thetranscriptome analysis, and compared our results with thosefrom barley.

In searching for putative vernalization‐memory‐related genesin Brachypodium In many temperate species, VRN1 has beenreported to be upregulated by vernalization, and this upregula-tion can still be observed after vernalization (Yan et al. 2003;Andersen et al. 2006; Oliver et al. 2009). Therefore, we searchedfor genes with similar or opposite change patterns at thetranscriptional level before and after vernalization, and regardedthem as putative vernalization‐memory‐related genes in Bra-chypodium. Functional analysis of these candidate genesshowed that several transcription‐regulation‐associated genes,such as VRN1, CIPK30, and bHLH‐domain‐containing genes,were enriched (Figure 4, Cluster 1; Figure 5; Table S2). Thissuggests that flowering and cold‐stress response pathwaysmight be switched on by vernalization through transcriptionalregulation, which may exert their functions even after vernaliza-tion. These cold‐stress‐associated genes might also play someroles in the vernalization process.

On the other hand, genes encoding monooxygenases wereconstantly downregulated in both vernalized and post‐vernalizedseedlings, most of which were cytochrome P450 genes, such asCYP71 andCYP72 (Figure 4, Clusters 7, 8; Figure 5; Table S2).In Arabidopsis, CYP71 has been reported to participate infundamental processes of chromatin assembly and histonemodification, by interacting with LHP1 (Umemoto et al. 1993;

Christoffersen et al. 1995; Li and Luan 2011). Actually, someother cytochromes P450 genes and flavonoid 30‐hydroxylasegenes were also downregulated during vernalization, buttheir expression levels returned to normal levels afterBrachypodium seedlings were shifted to room temperature(Figure 4, Clusters 5, 6; Figure 5). The homologs of thesegenes in Arabidopsis have been proven to be associatedwith the biosynthesis of glucosinolates, which might beinvolved in the plant defense response or may function as asource of flavor compounds (Chen et al. 2003; Mikkelsenet al. 2003; Tantikanjana et al. 2004). Therefore, wehypothesize that these genes probably work in response tocold stress during vernalization. Taken together, onlysome specific cytochrome P450 genes were involved in thevernalization‐memory‐related process, and further studies mightprovide new information on this regulation mechanism invernalization.

Although no homologous genes of FRI and FLC have beenfound in cereals, our data showed that, for the vernalizationpathway, one of the flowering regulation pathways BdVRN1showed the most significant change in response to vernalizationin Brachypodium (Figure 6A). According to RNA‐Seq data, theexpression profiles ofBdVRN2were almost unchanged, which isdifferent from previous results (Trevaskis et al. 2006)(Figure 6B). In addition, several flowering‐time‐related genesin Arabidopsis were also found in our datasets as putativevernalization‐memory‐related genes, such as BdPRR95 andBdPRR73 (Figure 6F, G). This implied that there might be somespecific mechanisms between vernalization and flowering timeregulation. However, the integrator of the photoperiod andcircadian clock pathway, BdCO, exhibited constant down-regulation during and after vernalization, implying that thephotoperiod pathway might be repressed during vernalization,

Table 2. Comparative analysis of GO enrichment of vernalization‐memory‐related genes between Brachypodium and barley

GO terms

Total Lasting upregulation Lasting downregulation

Bd Hv Bd Hv Bd Hv

Regulation of transcription 4.60E � 10 – 5.90E � 06 – 2.40E � 06 –

Amino acid phosphorylation 2.90E � 10 – 2.40E � 05 – 6.80E � 07 –

Metal ion transport 7.70E � 10 – 5.70E � 03 – 4.40E � 08 –

Electron carrier activity 1.80E � 13 – – – – –

Heme binding 7.70E � 20 8.60E � 02 – – 3.60E � 22 –

Nucleus 3.40E � 03 9.50E � 01 9.00E � 04 – 8.20E � 02 9.90E � 01

Membrane 1.10E � 04 7.50E � 01 – – – –

Fatty acid biosynthetic process – 1.70E � 03 – – – 1.10E � 03

Cytoplasmic membrane‐bounded

vesicle

– 1.10E � 09 – 9.20E � 06 – 2.60E � 05

Response to stress 2.70E � 06 9.20E � 06 – 2.00E � 04 1.80E � 07 8.80E � 03

Monooxygenase activity 3.30E � 12 7.80E � 07 – – 5.20E � 12 4.60E � 09

Bd, Brachypodium distachyon; Hv, Hordeum vulgare (barley). The P‐value of each category is shown in the table.


and that this repressionmight result from the feedback regulationof the activation of vernalization, in which BdVRN1 plays animportant role (Figure 6A, E).

Vernalization is conserved between Brachypodium andbarley, but several species‐specific features could alsobe found

Both the intra‐species and inter‐species correlation analysisbetween Brachypodium and barley showed that the correlationcoefficient of vernalization was significantly higher than that ofpost‐vernalization (Table 1). This suggests that regulation ofvernalization is conserved in these two species.

Furthermore, the functional comparative analysis betweenBrachypodium and barley described many conserved andspecies‐specific features of vernalization‐memory‐relatedgenes. Among them, the GO terms of stress response andmonooxygenase activity were enriched both in Brachypodiumand barley, and the expression levels of these associated geneswere mainly downregulated, with only some genes in barleyshowing slight enrichment in the upregulated group (Table 2).This implies that both of these two plant species remembervernalization through regulating stress‐related and monooxyge-nase genes.

In addition, our results also indicate that Brachypodium andbarley might have their own preferred regulation patterns toremember vernalization. The processes of transcription andprotein phosphorylation might be more important for Brachypo-dium in vernalization‐memory. Several transcription factors andtyrosine kinases are associated with these processes, suggest-ing that Brachypodium might remember vernalization throughregulating specific signaling pathways, whereas genes encodingcytoplasmic membrane‐bound‐vesicle‐located proteins andfatty‐acid‐biosynthesis‐associated genes might play someimportant roles in barley, implying that barley might remembervernalization through regulating specific physiological or meta-bolic processes (Table 2).

Genes are expressed differently at different developmentalstages in plants (Yong et al. 2003). More experiments areneeded to answer the question of whether the differentpreferences between Brachypodium and barley are caused bydifferent growth stages for plant materials used in the experi-ment. Another possible errormight be caused by there being littleknowledge of barley genome sequences, which limits homologanalysis and gene functional clustering. However, we cannotdeal with this barrier right now. Additionally, the differentmicroarray and RNA‐Seq platforms may also affect the results,although we normalized them. Although the differences wefound between Brachypodium and barley in vernalization stillrequire further verification, our data could provide some primaryclues for further research on the vernalization mechanism intemperate species.

In conclusion, our study has identified several putativevernalization‐memory‐related genes at the transcriptional levelin Brachypodium, and comparative analysis between Brachy-podium and barley described some conserved and species‐specific features in the aspect of vernalization memory. Ourresults provide insight into the pathways in which Brachypodiumvernalization‐associated genes are probably involved, and pointout several differences between Brachypodium and barley inthe vernalization regulating mechanism, which will provideuseful clues for future functional research in other related plantspecies.

Materials and Methods

Plant materials and growth conditions

To isolate RNA, Brachypodium distachyon (L.) Beauv. seeds(ABR1 ecotype) were germinated and grown on wet sterile filterpaper at 24 °C for 14 d (at the three‐leaf period). Then, seedlingswere respectively treated at normal conditions for 3 d (NV) andwere subjected to vernalization‐treatment at 4 °C for 30 d (V).Post‐vernalized seedlings (PV) were first grown under long‐dayconditions for 7 d, followed by 30 d of vernalization‐treatment and7 d of normal treatment. Under the described conditions, thepost‐vernalized plants would be at the same developmentalstage as the vernalized and control plants. The aerial parts of theseedlings were harvested for experiments.

RNA extraction and RNA‐Seq library construction

A mirVanami RNA Isolation Kit (Ambion, Austin, TX, USA) wasused to isolate the total RNA from the aerial part of ABR1seedlings, following the manufacturer’s protocol. The isolatedRNA was used for RNA‐Seq library construction and furtherquantification by real‐time PCR.

To construct the RNA‐Seq library, the extracted mRNA wasenriched and interrupted into short fragments (about 200 bp),and the second strand was synthesized by reverse transcription.Sequencing adaptors were ligated with purified doublestrand cDNA and enriched by PCR amplification. Finally,approximately 20 mg of the library products were used forsequencing analysis.

Quantitative PCR

An MX3000P real time PCR instrument (Stratagene, La Jolla,CA, USA) was used to perform quantitative PCR. Reactionswere carried out in a total volume of 15 mL with SYBR GreenRealtime PCR Master Mix (TOYOBO, Osaka, Japan). Cyclingconditions were 2 min at 95 °C, 45 cycles of 10 s at 95 °C, 10 sat ambient temperature, and 15 s at 72 °C, followed by a


melting‐curve program. The transcriptional levels were normal-ized by BdTubllin. Each set of experiments was repeated threetimes, and the error bars show standard error.

Reads mapping and gene abundance evaluation

TopHat was used to map these short reads to the Brachypodiumgenome (http://www.brachypodium.org, version 1.2) (InitiativeTIB 2010) with the default parameters except maximum intronlength was set to 2 � 10 4 nt and minimum intron length was setto 20 nt (Trapnell et al. 2009). Cufflinks 2.0.1 software was usedto evaluate the transcriptional abundance ofBrachypodium genemodels with the default parameters (Trapnell et al. 2012). Finally,Cuffdiff 2.0.1 software was used to compare the datasets underdifferent vernalization treatments and to identify the differentiallyexpressed genes. SAMtools 0.1.18 software was used to sortand index themapped reads (Li et al. 2009). The reads coverageof specific genes were visualized by Integrative GenomicsViewer (IGV) 2.2.2 (IGV) (Robinson et al. 2011; Thorvaldsdottiret al. 2012). The scatter plots were performed using R 2.15.2(http://www.r‐project.org/). Expression pattern analysis wasperformed using Mev 4.0 software (http://www.tm4.org/mev/MEV_4_0) (Saeed et al. 2006).

Homolog searching and transcriptional correlationanalysis between Brachypodium and barley

We used the dataset provided in HarvEST: Barley GeneChip2009 (http://harvest.ucr.edu/) (Close et al. 2004). It containseach of the Affymetrix probe set target sequences. We usedBLASTX to search the Brachypodium gene models. Theexpectation value of homologs in barley towards Brachypodiumwas smaller than or equal to 1 � 10�20. The intra‐species andinter‐species correlation coefficients were calculated in thesetwo species. Genes used for functional comparison were chosenfrom the top 25% of differentially expressed genes both inBrachypodium (fold change �2) and barley (fold change �1.5),respectively.

Acknowledgments

The authors thank Professor Xiaoquan Qi in the Institute ofBotany, the Chinese Academy of Sciences, for his kind gift of thediploid line Brachypodium seeds (ABR1). This work wassupported by the Main Direction Program of KnowledgeInnovation of the Chinese Academy of Sciences (KSCX2‐EW‐

J‐1), as well the National Natural Science Foundation of China(30970241).

Received 4 Feb. 2013 Accepted 7 Mar. 2013

References

Alves SC,WorlandB, Thole V, Snape JW,BevanMW, Vain P (2009) A

protocol for Agrobacterium‐mediated transformation of Brachypo-

dium distachyon community standard line Bd21. Nat. Protoc. 4,

638–649.

Andersen JR, Jensen LB, Asp T, Lubberstedt T (2006) Vernalization

response in perennial ryegrass (Lolium perenne L.) involves

orthologues of diploid wheat (Triticum monococcum) VRN1 and

rice (Oryza sativa) Hd1. Plant Mol. Biol. 60, 481–494.

Chen S, Glawischnig E, Jorgensen K, Naur P, Jorgensen B, Olsen

CE, Hansen CH, Rasmussen H, Pickett JA, Halkier BA (2003)

CYP79F1 andCYP79F2 have distinct functions in the biosynthesis of

aliphatic glucosinolates in Arabidopsis. Plant J. 33, 923–937.

Christoffersen RE, Percival FW, BozakKR (1995) Functional andDNA

sequence divergence of theCYP71 gene family in higher plants.Drug

Metabol. Drug Interact. 12, 207–219.

Close TJ, Wanamaker SI, Caldo RA, Turner SM, Ashlock DA,

Dickerson JA, Wing RA, Muehlbauer GJ, Kleinhofs A, Wise RP

(2004) A new resource for cereal genomics: 22K barley GeneChip

comes of age. Plant Physiol. 134, 960–968.

De Lucia F, Crevillen P, Jones AM, Greb T, Dean C (2008) A

PHD‐polycomb repressive complex 2 triggers the epigenetic silenc-

ing of FLC during vernalization. Proc. Natl. Acad. Sci. USA 105,

16831–16836.

Dennis ES, Peacock WJ (2009) Vernalization in cereals. J. Biol. 8, 57.

Diallo AO, Ali‐Benali MA, Badawi M, Houde M, Sarhan F (2012)

Expression of vernalization responsive genes in wheat is associated

with histone H3 trimethylation. Mol. Genet. Genomics 287, 575–590.

Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: A GO analysis

toolkit for the agricultural community. Nucleic Acids Res. 38, W64–

W70.

Fornara F, de Montaigu A, Coupland G (2010) SnapShot: Control of

flowering in Arabidopsis. Cell 141, 550.

Greenup AG, Sasani S, Oliver SN, Talbot MJ, Dennis ES, Hemming

MN, Trevaskis B (2010) ODDSOC2 is a MADS box floral repressor

that is down‐regulated by vernalization in temperate cereals. Plant

Physiol. 153, 1062–1073.

GreenupAG, Sasani S,Oliver SN,WalfordSA,Millar AA, TrevaskisB

(2011) Transcriptome analysis of the vernalization response in barley

(Hordeum vulgare) seedlings. PLoS ONE 6, e17900.

HeY (2012) Chromatin regulation of flowering.TrendsPlant Sci. 17, 556–

562.

Higgins JA, Bailey PC, Laurie DA (2010) Comparative genomics of

flowering time pathways using Brachypodium distachyon as a model

for the temperate grasses. PLoS ONE 5, e10065.

Initiative TIB (2010) Genome sequencing and analysis of the model

grass Brachypodium distachyon. Nature 463, 763–768.

Jiang D, Gu X, He Y (2009) Establishment of the winter‐annual growth

habit via FRIGIDA‐mediated histone methylation at FLOWERING

LOCUS C in Arabidopsis. Plant Cell 21, 1733–1746.


http://www.brachypodium.org/

http://www.r-project.org/

http://www.tm4.org/mev/MEV_4_0

http://www.tm4.org/mev/MEV_4_0

http://harvest.ucr.edu/

Kobayashi Y,Weigel D (2007) Move on up, it’s time for change�mobile

signals controlling photoperiod‐dependent flowering.Genes Dev. 21,

2371–2384.

Komeda Y (2004) Genetic regulation of time to flower in Arabidopsis

thaliana. Annu. Rev. Plant Biol. 55, 521–535.

Kumar S, Sharma V, Chaudhary S, Tyagi A, Mishra P, Priyadarshini

A, Singh A (2012) Genetics of flowering time in bread wheat Triticum

aestivum: complementary interaction between vernalization‐insensi-

tive and photoperiod‐insensitive mutations imparts very early flower-

ing habit to spring wheat. J. Genet. 91, 33–47.

Laudencia‐Chingcuanco D, Ganeshan S, You F, Fowler B, Chibbar

R, Anderson O (2011) Genome‐wide gene expression analysis

supports a developmental model of low temperature tolerance

gene regulation in wheat (Triticum aestivum L.) BMC Genomics

12, 299.

Li H, Luan S (2011) The cyclophilin AtCYP71 interacts with CAF‐1 and

LHP1 and functions inmultiple chromatin remodeling processes.Mol.

Plant 4, 748–758.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G,

Abecasis G, Durbin R (2009) The Sequence Alignment/Map format

and SAMtools. Bioinformatics 25, 2078–2079.

Michaels SD (2009) Flowering time regulation producesmuch fruit.Curr.

Opin. Plant Biol. 12, 75–80.

Mikkelsen MD, Petersen BL, Glawischnig E, Jensen AB, Andreas-

son E, Halkier BA (2003) Modulation of CYP79 genes and

glucosinolate profiles in Arabidopsis by defense signaling pathways.

Plant Physiol. 131, 298–308.

Mutasa‐Gottgens ES, Joshi A, Holmes HF, Hedden P, Gottgens B

(2012) A new RNASeq‐based reference transcriptome for sugar beet

and its application in transcriptome‐scale analysis of vernalization

and gibberellin responses. BMC Genomics 13, 99.

Nakamichi N, Kita M, Ito S, Sato E, Yamashino T, Mizuno T (2005a)

The Arabidopsis pseudo‐response regulators, PRR5 and PRR7,

coordinately play essential roles for circadian clock function. Plant

Cell Physiol. 46, 609–619.

Nakamichi N, Kita M, Ito S, Yamashino T, Mizuno T, (2005b)

PSEUDO‐RESPONSE REGULATORS, PRR9, PRR7 and PRR5,

together play essential roles close to the circadian clock of

Arabidopsis thaliana. Plant Cell Physiol. 46, 686–698.

Nakamichi N, Kita M, Niinuma K, Ito S, Yamashino T, Mizoguchi T,

Mizuno T (2007) Arabidopsis clock‐associated pseudo‐response

regulators PRR9, PRR7 and PRR5 coordinately and positively

regulate flowering time through the canonical CONSTANS‐

dependent photoperiodic pathway. Plant Cell Physiol. 48, 822–832.

Oliver SN, Finnegan EJ, Dennis ES, PeacockWJ, Trevaskis B (2009)

Vernalization‐induced flowering in cereals is associated with

changes in histone methylation at the VERNALIZATION1 gene.

Proc. Natl. Acad. Sci. USA 106, 8386–8391.

Opanowicz M, Vain P, Draper J, Parker D, Doonan JH (2008)

Brachypodium distachyon: Making hay with a wild grass. Trends

Plant Sci. 13, 172–177.

Robinson JT, Thorvaldsdottir H,WincklerW,GuttmanM, Lander ES,

Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat.

Biotechnol. 29, 24–26.

Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA,

Li J, Thiagarajan M, White JA, Quackenbush J (2006) TM4

microarray software suite. Methods Enzymol. 411, 134–193.

Salomé PA, Weigel D, McClung CR (2010) The role of the Arabidopsis

morning loop components CCA1, LHY, PRR7, and PRR9 in

temperature compensation. Plant Cell 22, 3650–3661.

Sanda SL, Amasino RM (1996) Interaction of FLC and late‐flowering

mutations in Arabidopsis thaliana. Mol. Gen. Genet. 251, 69–74.

SrikanthA, SchmidM (2011) Regulation of flowering time: All roads lead

to Rome. Cell Mol. Life Sci. 68, 2013–2037.

Sun QM, Zhou RH, Gao LF, Zhao GY, Jia JZ (2009) The characteriza-

tion and geographical distribution of the genes responsible for

vernalization requirement in Chinese bread wheat. J. Integr. Plant

Biol. 51, 423–432.

Tantikanjana T, Mikkelsen MD, Hussain M, Halkier BA, Sundaresan

V (2004) Functional analysis of the tandem‐duplicated P450 genes

SPS/BUS/CYP79F1 andCYP79F2 in glucosinolate biosynthesis and

plant development by Ds transposition‐generated double mutants.

Plant Physiol. 135, 840–848.

Thole V, Peraldi A, Worland B, Nicholson P, Doonan JH, Vain P

(2011) T‐DNAmutagenesis inBrachypodium distachyon. J. Exp. Bot.

63, 567–576.

Thorvaldsdottir H, Robinson JT, Mesirov JP (2012) Integrative

Genomics Viewer (IGV): High‐performance genomics data visuali-

zation and exploration. Brief Bioinfor. 14, 178–192.

Trapnell C, Pachter L, Salzberg SL (2009) TopHat: Discovering splice

junctions with RNA‐Seq. Bioinformatics 25, 1105–1111.

Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel

H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and

transcript expression analysis of RNA‐seq experiments with TopHat

and Cufflinks. Nat. Protoc. 7, 562–578.

Trevaskis B, Hemming MN, Peacock WJ, Dennis ES (2006) HvVRN2

responds to daylength, whereas HvVRN1 is regulated by vernaliza-

tion and developmental status. Plant Physiol. 140, 1397–1405.

Umemoto N, Kobayashi O, Ishizaki‐Nishizawa O, Toguri T (1993)

cDNAs sequences encoding cytochrome P450 (CYP71 family) from

eggplant seedlings. FEBS Lett. 330, 169–173.

Vain P, Worland B, Thole V, McKenzie N, Alves SC, Opanowicz M,

Fish LJ, Bevan MW, Snape JW (2008) Agrobacterium‐mediated

transformation of the temperate grass Brachypodium distachyon

(genotype Bd21) for T‐DNA insertional mutagenesis. Plant Biotech-

nol. J. 6, 236–245.

WangZ,GersteinM, SnyderM (2009) RNA‐Seq: A revolutionary tool for

transcriptomics. Nat. Rev. Genet. 10, 57–63.

Xing L, Li J, Xu Y, Xu Z, Chong K (2009) Phosphorylation modification

of wheat lectin VER2 is associated with vernalization‐induced

O‐GlcNAc signaling and intracellular motility. PLoS ONE 4,

e4854.


Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T,

Dubcovsky J (2003) Positional cloning of the wheat vernalization

gene VRN1. Proc. Natl. Acad. Sci. USA 100, 6263–6268.

YangCH, ChouML (1999) FLD interacts with CO to affect both flowering

time and floral initiation inArabidopsis thaliana.Plant Cell Physiol. 40,

647–650.

Yong WD, Xu YY, Xu WZ, Wang X, Li N, Wu JS, Liang TB, Chong K,

Xu ZH, Tan KH, Zhu ZQ (2003) Vernalization‐induced flowering in

wheat is mediated by a lectin‐like gene VER2. Planta 217, 261–270.

(Co‐Editor: Qi Xie)

Supporting Information

Additional supporting information may be found in the onlineversion of this article at the publisher’s web site:

Figure S1. The quality analysis of RNA‐Seq data.

(A) The quality scores across all bases. X‐axes show theposition of nucleotides in each read, and each read contained49 bp of nucleotides. Y‐axes show the quality scores. Scoreswere classified into three categories: high (green region), middle(orange region) and low (red region).

(B) The distribution of quality scores over all sequences. X‐axesshow the mean of sequence quality (Phred score). Y‐axes standfor the quality scores. Most of them are enriched at ascore of ¼ 37, which means that the sequencing error rateis near 1 � 10�4

sQ ¼ �10 log10 E

Q, sequencing quality value; E, sequencing error rate.

Table S1. Differentially expressed genes in vernalizedand post‐vernalized samples related to non‐vernalizedcontrol.

Table S2. Genes that significantly enriched in Clusters 1, 7and 8.

Table S3. Flowering associated genes that showed aconstant response to vernalization.

Table S4. The statistics of genes that changed in similarpatterns between Brachypodium and barley.

Table S5. Homologs with a constant response to vernaliza-tion between Brachypodium and barley.

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