RESEARCH ARTICLE Open Access Genome-wide Identification And .
Stracke et al. BMC Plant Biology 2014, 9RESEARCH ARTICLEOpen AccessGenome-wide identification and characterisationof R2R3-MYB genes in sugar beet (Beta vulgaris)Ralf Stracke*, Daniela Holtgräwe, Jessica Schneider, Boas Pucker, Thomas Rosleff Sörensen and Bernd WeisshaarAbstractBackground: The R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playingregulatory roles in plant-specific developmental processes, metabolite accumulation and defense responses.Although genome-wide analysis of this gene family has been carried out in some species, the R2R3-MYBgenes in Beta vulgaris ssp. vulgaris (sugar beet) as the first sequenced member of the order Caryophyllales,have not been analysed heretofore.Results: We present a comprehensive, genome-wide analysis of the MYB genes from Beta vulgaris ssp. vulgaris(sugar beet) which is the first species of the order Caryophyllales with a sequenced genome. A total of 70R2R3-MYB genes as well as genes encoding three other classes of MYB proteins containing multiple MYBrepeats were identified and characterised with respect to structure and chromosomal organisation. Also, organspecific expression patterns were determined from RNA-seq data. The R2R3-MYB genes were functionallycategorised which led to the identification of a sugar beet-specific clade with an atypical amino acid compositionin the R3 domain, putatively encoding betalain regulators. The functional classification was verified by experimentalconfirmation of the prediction that the R2R3-MYB gene Bv iogq encodes a flavonol regulator.Conclusions: This study provides the first step towards cloning and functional dissection of the role of MYBtranscription factor genes in the nutritionally and evolutionarily interesting species B. vulgaris. In addition, it describesthe flavonol regulator BvMYB12, being the first sugar beet R2R3-MYB with an experimentally proven function.Keywords: Beta vulgaris, Caryophyllales, R2R3-MYB, Transcription factor, Gene family, Flavonol regulatorBackgroundTranscriptional control of gene expression influences almost all biological processes in eukaryotic cells or organisms. Transcription factors perform this function, alone orcomplexed with other proteins, by activating or repressing(or both) the recruitment of RNA polymerase to specificgenes. The large number and diversity of transcriptionfactors is related to their substantial regulatory complexity [1].MYB proteins are widely distributed in all eukaryoticorganisms and constitute one of the largest transcriptionfactor families in the plant kingdom. MYB proteins aredefined by a highly conserved MYB DNA-binding domain, mostly located at the N-terminus, generally consisting of up to four imperfect amino acid sequencerepeats (R) of about 52 amino acids, each forming three* Correspondence: ralf.stracke@uni-bielefeld.deChair of Genome Research, Faculty of Biology and Center for Biotechnology,Bielefeld University, Bielefeld 33615, Germanyalpha–helices [2]. The second and third helices of eachrepeat build a helix–turn–helix (HTH) structure withthree regularly spaced tryptophan (or hydrophobic) residues, forming a hydrophobic core [3]. The third helix ofeach repeat is the DNA recognition helix that makesdirect contact with DNA [4]. During DNA contact, twoMYB repeats are closely packed in the major groove, sothat the two recognition helices bind cooperatively tothe specific DNA recognition sequence motif.MYB proteins can be divided into different classes depending on the number of adjacent repeats (one, two,three or four). The three repeats of the prototypic MYBprotein c-Myb [5] are referred to as R1, R2 and R3, andrepeats from other MYB proteins are named accordingto their similarity. Plant R1R2R3-type MYB (MYB3R)proteins have been proposed to play divergent roles incell cycle control [6,7], similar to the functions of theiranimal homologs. 2014 Stracke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver ) applies to the data made available in this article,unless otherwise stated.
Stracke et al. BMC Plant Biology 2014, 9Most plant MYB genes encode R2R3-MYB class proteins, containing two repeats [2,8], which are thought tohave evolved from an R1R2R3-MYB gene ancestor, by theloss of the sequences encoding the R1 repeat and subsequent expansion of the gene family [9-11]. R2R3-MYBtranscription factors have a modular structure, with theN-terminal MYB domain as DNA-binding domain and anactivation or repression domain usually located at thehighly variable C-terminus. Components for the establishment of protein-protein interactions with other components of the eukaryotic transcriptional machinery havebeen detected in the N-terminal module [12-14].Based on the conservation of the MYB domain and ofcommon amino acid motifs in the C-terminal domains,R2R3-MYB proteins have been divided into several subgroups which often group proteins with functional relationship. The reliability of the subgroups defined on thebasis of phylogenetic analysis is also supported byadditional criteria, such as the gene structure and the presence and position of introns [15]. Most of these subgroups,defined first for the proteins of A. thaliana [2,16,17], arealso present, and are sometimes expanded, in other higherplants. Comparative phylogenetic studies have identifiednew R2R3-MYB subgroups in other plant species forwhich there are no representatives in A. thaliana (e.g. inrice, poplar and grapevine), suggesting that these proteinsmight have specialised functions which were either lost inA. thaliana or were acquired after divergence from thelast common ancestor [18-20].As initially described in the first plant MYB gene familyreview [21], the expansion of the plant-specific R2R3-MYBgene family is thought to be correlated with the increasein complexity of plants, particularly in Angiosperms. Consequently, the functions of R2R3-MYB genes are likely associated with regulating plant-specific processes includingprimary and secondary metabolism, developmental processes, cell fate and identity and responses to biotic andabiotic stresses [2,17,21].With the growing number of fully sequenced plant genomes, the identification of R2R3-MYB genes has increasedin recent times. Based on their well conserved MYB domains, R2R3-MYB gene families have been annotatedgenome-wide in A. thaliana (126 members) [17], Zeamays (157 members) [22], Oryza sativa (102 members)[23], Vitis vinifera (117 members) [19], Populus trichocarpa (192 members) [20], Glycine max (244 members)[15], Cucumis sativus (55 members) [24] and Malus xdomestica (222 members) [25]. Given the potential rolesof R2R3-MYB proteins in the regulation of gene expression, secondary metabolism, and responses to environmental stresses, and that Beta vulgaris ssp. vulgaris (orderCaryophyllales) is the first non-rosid, non-asterid eudicotfor which the genome has been sequenced [26], it isof interest to achieve a complete identification andPage 2 of 17classification of MYB genes in this species with respect tothe number, chromosome locations, phylogenetic relationships, conserved motifs as well as expression patterns.Particularly, since sugar beet is an important crop of thetemperate climates as a source for bioethanol as well asanimal feed and provides nearly 30% of the worlds annualsugar production [26].In the present study, we describe the R2R3-MYB genefamily by means of in silico analysis of the B. vulgarisgenome sequence, in order to predict protein domain architectures, and to assess the extent of conservation anddivergence between B. vulgaris and A. thaliana genefamilies, thus leading to a functional classification of thesugar beet MYB genes on the basis of phylogenetic analyses. Furthermore, RNA-seq data was used to analyse expression in different B. vulgaris organs and to compareexpression patterns of closely grouped co-orthologs. Tovalidate the functional classification, a candidate gene waschosen for cDNA isolation and subsequent functionalanalysis by transient transactivation assays and complementation of an orthologous A. thaliana mutant. Weidentified the R2R3-MYB gene Bv iogq activating two flavonol biosynthesis enzyme promoters and complementingthe flavonol-deficient myb11 myb12 myb111 mutant, andthus encoding a functional flavonol biosynthesis regulator.Our findings provide the first step towards further investigations on the biological and molecular functions of MYBtranscription factors in the economically and evolutionarily interesting species B. vulgaris.Results and discussionThe annotated genome sequence of B. vulgaris has recently become available. It has been obtained from thedouble haploid breeding line KWS2320 [26]. The sequence has been assigned to nine chromosomes and B.vulgaris was predicted to contain 27,421 protein-codinggenes (RefBeet) in 567 Mb from which 85% are chromosomally assigned.Identification and genomic distribution of B. vulgarisR2R3-MYB genesMYB protein coding genes in B. vulgaris were identifiedusing a consensus R2R3-MYB DNA binding domain sequence as protein query in TBLASTN searches on theRefBeet genome sequence. The putative MYB sequenceswere manually analysed for the presence of an intactMYB domain to ensure that the gene models containedtwo or more (multiple) MYB repeats, and that they mappedto unique loci in the genome. We identified six B. vulgarisMYB (BvMYB) genes which had been missed in the automatic annotation [26] and two which had been annotatedwith incomplete open reading frames. We created a primary data set of 70 R2R3-MYB proteins and three typesof atypical multiple repeat MYB proteins distantly related
Stracke et al. BMC Plant Biology 2014, 9to the typical R2R3-MYB proteins: three R1R2R3-MYB(MYB3R) proteins, one MYB4R protein and one CDC5like protein from the B. vulgaris genome (Table 1). Thenumber of atypical multiple repeat MYB genes identifiedin B. vulgaris is in the same range as those reported forother plant species, with for example up to six MYB3Rand up to two MYB4R and CDC5-like genes. However,the number of R2R3-MYB genes is one of the smallestamong the species that have been studied (ranging from55 in C. sativus to in 244 G. max). As discussed below,this is probably due to the absence of recent genomeduplication events in B. vulgaris.A keyword search in the NCBI database (http://www.ncbi.nlm.nih.gov/) revealed three previously annotatedB. vulgaris MYB proteins from different sugar beet cultivars: AET43456 and AET43457, both corresponding toBvR2R3-MYB Bv jkkr in this work and AEL12216, corresponding to Bv nqis in this work. The identified 75BvMYB genes, constituting approximately 0.27% of the27,421 predicted protein-coding B. vulgaris genes and5.9% of the 1271 putative B. vulgaris transcription factorgenes [26], were subjected for further analyses. Similarto all other genes in the annotated B. vulgaris genome(RefBeet), a unique, immutable four-letter identifier (ID)was assigned to each BvMYB gene (Table 1). This immutable ID is part of the gene designator used in the B. vulgarisnomenclature system and should be stable, in contrast tothe designator elements describing the chromosomal assignment and position on pseudochromosomes which maychange when currently unassigned or unanchored scaffoldsare integrated into the pseudochromosomes. Hereafter thefour-letter-ID is used to name individual BvMYB genesand the deduced proteins. On the basis of RefBeet, 67 ofthe 75 BvMYB genes could be assigned to the nine chromosomes. On average, one R2R3-MYB gene was presentevery 10.5 Mb. The chromosomal distribution of BvMYBgenes on the pseudochromosomes is shown in Figure 1and revealed that B. vulgaris MYB genes were distributedthroughout all chromosomes. Although each of the nineB. vulgaris chromosomes contained MYB genes, the distribution appeared to be uneven (Figure 1). The BvMYBgene density per chromosome was patchy, with only twoBvR2R3-MYB genes present on chromosome 9, while 16were found on chromosome 5. In general, the centralsections of chromosomes including the centromeres andthe pericentromere regions, lack MYB genes. Relativelyhigh densities of BvMYB genes were observed at thechromosome ends, with highest densities observed at thetop of chromosome 2 and at the bottom of chromosome 5(Figure 1). This uneven distribution was previously observed for Z. mays, G. max and M. x domestica R2R3MYB genes [15,22,25].The total number of identified MYB genes was, compared to other plant species, low in B. vulgaris. Even ifPage 3 of 17some MYB genes may have been missed due to gaps inthe reference sequence, this does not adequately explainthe small number. High numbers of MYB genes in aspecies are mainly attributed to ancestral whole genomeduplication events as known for A. thaliana, O. sativa,P. trichocarpa, G. max and M. x domestica [27-31]. Theabsence of a recent lineage-specific whole genome duplication event in B. vulgaris [32] is further substantiatedby the detection of only 70 R2R3-MYB genes, because alack of this duplication event can easily explain the smallnumber of MYB genes in this species. This interpretation is in accordance with the findings in cucumber,where the number of R2R3-MYB genes has been reported to be 55 [24].We further determined physically linked sister BvMYBgene pairs along the nine chromosomes (Figure 1, markedwith vertical black bars), which form clusters and mayhave evolved from local intrachromosomal duplicationevents that result in tandem arrangement of the duplicated gene. Three gene pairs have been identified: one onchromosome 7 consisting of the closely related genesBv ahzu and Bv eztu, a second on the top of chromosome 8 with Bv dxny and Bv zguf and a third on anunlinked scaffold (0254.scaffold00675) constituted ofBv dani and Bv sjwa. The BvMYB genes of the two latter pairs were physically located near to each other without intervening annotated genes between. In total, about5% (6 of 75) of BvMYBs were involved in tandem duplication, which is the same value as reported for MYB genesin soybean [15]. Moreover, an incomplete gene pair wasobserved on chromosome 2, where a solitary typicallyR2R3-MYB "third exon" containing sequences encoding apart of a R3 repeat and the C-terminal region was foundabout 18.7 kb downstream of Bv jkkr showing 88% identity on cDNA- and 82% identity on deduced protein levelto the third exon of the near Bv jkkr.Gene structure analysis revealed that most BvR2R3MYB genes (53 of 70, 76%) follow the previously reported rule of having two introns and three exons, anddisplay the highly conserved splicing arrangement thathas also been reported for other plant species [15,22,24].Eleven BvR2R3-MYB genes (16%) have one intron andtwo exons and four (6%) were one exon genes. Only twoBvR2R3-MYB genes have more than three exons:Bv mxck with four exons and Bv zeqy with twelve exons(Table 1). The complex exon-intron structure of Bv zeqyis known from its A. thaliana orthologs AtMYB88 andAtMYB124/FOUR LIPS (FLP) containing ten and elevenexons, respectively, and more than the typically zero totwo introns in the MYB domain coding sequences[18,19]. This supports their close evolutionary relationship, but also indicates the conservation of this intronpattern in evolution since the split of the Caryophyllalesfrom the precursor of rosids and asterids.
Stracke et al. BMC Plant Biology 2014, 9Page 4 of 17Table 1 List of annotated MYB genes with two or more repeats in the B. vulgaris ssp. vulgaris (KWS2320) genomeClade(subgroup)Landmark MYBin cladeFunctional assignmentProteinExonlength [aa] nr.1,381,156C14 (S4)AtMYB4, HvMYB5Metabolism34131,903,2391,898,047C8 (S3)AtMYB58, AtMYB63Metabolism31633,065,4343,067,536C18 I2Development3033132,690,615 32,692,641 C12 (S14)SlBLIND, AtRAX1Development36331un38,330,908 38,328,715 C28 (S18)HvGAMYB, AtDUO1 Development30331un45,201,741 45,199,866 C36 (S21)AtLOF1, AtMYB523502Bv2g023560 uksi2127,507126,341C35 (S23)2531Bv2g024650 wdyc21,373,9121,378,227C35 g030925 ralf28,431,7188,435,595Bv2g031800 ghua29,813,4079,800,544Bv2g039110 huqyGeneIDGene codeChr.Position onpseudochr.iqucBv1g001230 iquc11,379,505owzxBv1g001750 owzx1dwkiBv1g002800 dwki1qxpiBv1g006050 qxpiksfiBv1g014750 ksfizqorBv1ug018140 zqorjxgtBv1ug021520 jxgtuksiwdycihfgBv2g027580 ihfgjkkrBv2g027795 jkkrmxckBv2g027990 mxckxprdBv2g029260 xprdralfghuahuqyFaMYB1Devel., 4222C21(metabolism)2373C37 (MYB3R) AtMYB3R1Cell cycle105011EgMYB2, PtMYB4227,806,443 27,798,295 C26AtMYB26Development3292dcmm Bv2g040720 dcmm233,641,162 33,643,072 C1 (S9)AmMIXTA, AtNOKdifferentiation4543mxwzBv2g041120 mxwz234,717,448 34,719,429 C11AtTDF1Development3313nqisBv2ug047120 nqis2un45,488,410 45,491,366 C4 (S1)AtMYB30Defense3443NtMYB1, AtMYB13DefenseurrgBv3g049510 urrg31,126,3501,124,818C9 (S2)hwccBv3g050090 hwcc31,847,1951,845,526C6 (S24)28833603cwttBv3ug070140 cwtt3un35,695,445 35,697,294 C292423cjuqBv4g071740 g073190 yruo41,667,4141,671,378C27EgMYB2, PtMYB4Metabolism3972ygxgBv4g074860 ygxg43,396,3143,392,041C28 (S18)HvGAMYB, AtDUO1 Development5563skuhBv4g078900 skuh47,533,9437,547,807C42 (MYB4R) AtMYB4R191912zfigBv4g079610 zfig48,546,4548,544,269C25 (S13)AtMYB61Metabolism4493orefBv4g079670 oref48,669,2848,678,138C41 (CDC5)AtCDC5Cell cyclejoshBv4g083815 josh416,552,338 16,566,03599142093rwwjBv4g084340 rwwj418,292,096 18,285,979 C14 (S4)AtMYB4, HvMYB5Metabolism3223xwneBv4g091510 xwne432,888,798 32,889,966 3jofqBv5g098940 jofq5834,523837,220C10sskdBv5g100530 sskd52,767,0182,770,045C233,804,868C37 (MYB3R) AtMYB3R1mhxhBv5g101320 mhxh53,800,529zkefBv5g107260 zkef514,451,259 14,465,332Cell cycle52572722ztydBv5g110930 ztyd525,553,310 25,538,334 C32 (S19)AtMYB21Development2313udmhBv5g110960 udmh525,780,619 25,782,807 C4 (S1)AtMYB30Defense2933tcwdBv5g112510 tcwd531,754,754 31,740,788 C37 (MYB3R) AtMYB3R1Cell cycle5497nmrgBv5g115970 nmrg542,841,475 42,839,260 C4 (S1)AtMYB30Defense3343oaxtBv5g116880 oaxt544,348,080 44,350,042 C11AtTDF1Development3563tfkhBv5g118200 tfkh546,474,363 46,471,416 C38 (S25)AtPGA37Development5013ahtjBv5g118320 ahtj546,667,594 46,670,167 C38 (S25)AtPGA37DevelopmentcfqeBv5g118940 cfqe547,478,672 47,484,50645633543
Stracke et al. BMC Plant Biology 2014, 9Page 5 of 17Table 1 List of annotated MYB genes with two or more repeats in the B. vulgaris ssp. vulgaris (KWS2320) genome(Continued)roaoBv5g122000 roao550,845,046 50,849,238 C9 (S2)NtMYB1, AtMYB13Defense3043iogqBv5g122370 iogq551,297,529 51,287,534 C13 (S7)ZmP, AtPFG1Metabolism3873ijmcBv5g123335 ijmc552,180,596 52,182,338 C33 (S20)TaPIMP1, AtBOS1Def., devel.3133ohkkBv5ug126300 ohkk5un58,923,676 58,920,132 C39AmPHAN, AtAS1Development3691knacBv5ug126380 knac5un59,366,406 59,371,048 C36 (S21)AtLOF1, AtMYB52Devel., metab.3543qcwxBv5ug126530 qcwx 5un60,109,952 60,111,819 C7 (S11)AtMYB102Defense3342suchBv6g128620 such6508,583510,104C34 (S22)AtMYB44Def., devel.3131hwmtBv6g129790 hwmt62,064,4962,062,852C12 (S14)SlBLIND, AtRAX1DevelopmentoypcBv6g136060 oypc69,418,7729,413,559C6 (S24)32133263usyiBv6g142590 usyi624,870,820 24,874,245 C33 (S20)TaPIMP1, AtBOS1Def., devel.2753qttnBv6g154730 qttn658,819,129 58,822,997 C36 (S21)AtLOF1, AtMYB52Devel., metab.4613zeqyBv6g155340 zeqy659,766,073 59,770,527 C40AtFLPDifferentiation44512yejrBv7g162730 yejr77,720,067SlBLIND, AtRAX1Development3803ahzsBv7g172570 ahzs737,491,045 37,494,304 C26AtMYB26Development4053eztuBv7g172590 eztu737,594,009 37,596,132 C26AtMYB26Development4173qzmsBv7g174540 qzms740,066,216 40,070,163 C38 (S25)AtPGA37Development4813ksgeBv7g176420 ksge742,070,277 42,071,595 722,320C12 (S14)qzfyBv7ug180860 qzfy7un49,349,681 49,351,461 C31dxnyBv8g183050 dxny8719,286716,855C7 (S11)zgufBv8g183060 zguf8734,446721,513C7 (S11)AtMYB102Defense3963jonaBv8g199535 jona837,703,170 37,704,769 C12 (S14)SlBLIND, AtRAX1Development2713khqqBv8g200250 khqq838,542,747 38,540,562 C12 (S14)SlBLIND, AtRAX1Development3683gjwrBv9g216350 gjwr933,882,588 33,884,593 C14 (S4)AtMYB4, HvMYB5Metabolism276244,634,979 44,633,906 C34 (S22)AtMYB44Def., devel.25314103krezBv9g225930 krez9ezheBvg229250 ezhernd0020 411,935416,451C28 (S18)HvGAMYB, AtDUO1 DevelopmentcraeBvg229400 craernd0039 39,53540,725C17 (S5)AtTT2Metabolism2823entgBvg229850 entgrnd0043 141,170143,052C33 (S20)TaPIMP1, AtBOS1Def., devel.3103swwiBvg235150 swwirnd0157 176,581179,1811913oyjzBvg238960 oyjzrnd0254 47,16149,928C3AtLMI2Development2393daniBvg239075 danirnd0254 156,586162,101C24 (S16)AtLAF1Development2853sjwaBvg239080 sjwarnd0254 178,734182,264C24 (S16)AtLAF1Development2783pgyaBvg243050 pgyarnd0446 12,40714,2143263The genes are ordered by RefBeet pseudochromosomes, from north to south. The unique, immutable four-letter identifier (gene ID) is given in the first column.The modifiable, annotation-version-specific gene code describing the chromosomal assignment and position on pseudochromosomes is given in the secondcolumn. "un" indicates the assignment to a chromosome without position and "rnd" indicates scaffolds without chromosomal assignment. Clade classificationand functional assignment is based on the NJ tree presented in Figure 3.Sequence features of the MYB domainsTo investigate the R2R3-MYB domain sequence features,and the frequencies of the most prevalent amino acids ateach position within each repeat of the B. vulgaris R2R3MYB domain, sequence logos were produced throughmultiple alignment analysis (ClustalW) using the 70 deduced amino acid sequences of R2 and R3 repeats, respectively. In general, the two MYB repeats covered about104 amino acid residues (including the linker region), withrare deletions or insertions (Additional file 1). As shownin Figure 2, the distribution of conserved amino acidsamong the B. vulgaris MYB domain was very similar tothose of A. thaliana, Z. mays, O. sativa, V. vinifera, P. trichocarpa, G. max and C. sativus. The R2 and R3 MYB repeats of the B. vulgaris R2R3-MYB family containedcharacteristic amino acids, including the most prominentseries of regularly spaced and highly conserved tryptophan(W) residues, which are known to play a key role insequence-specific DNA binding, serving as landmarks ofplant MYB proteins. As known from orthologs in other
Stracke et al. BMC Plant Biology 2014, rdralfghua3Page 6 of uchhwmt79 eqy(6)(6)70Figure 1 Chromosomal distribution of BvMYB genes. R2R3-MYB genes are present on all nine chromosomes in the B. vulgaris genome. Eachbroad vertical bar represents one chromosome drawn to scale. The black parts indicate concatenated scaffolds and grey parts mark scaffoldsassigned to a chromosome without detailed position. The positions of centromeres (white dots) are roughly estimated from repeats distributiondata. The chromosomal positions of the MYB genes (given in four-letter-ID) are indicated by horizontal lines. R2R3-MYB genes are given in blackletters and other MYB genes are given in grey letters. The bracketed numbers below the chromosomes show the number of MYB genes on thischromosome. Eight R2R3-MYB genes could not be localised to a specific chromosome (unanchored). Vertical black lines indicate R2R3-MYB geneswhich are located in close proximity (sister gene pairs).plant species, the first conserved tryptophan residue inthe R3 repeat (position 60, W60) could be replaced by F orless frequently by isoleucine (I), leucine (L) or tyrosine(Y). Interestingly, the position 98 of the MYB repeat,which contains the last of the conserved tryptophan residues (W98), is not completely conserved in the B. vulgarisR2R3-MYBs (Figure 2, Additional file 1). A phenylalanine(F) residue, found in Bv ralf and Bv zeqy, has been reported very rarely at this position (e.g. in ZmMYB29) [22],but an atypical cysteine (C) at this important position, asfound in Bv jkkr, has not been described yet. This makesthe R2R3-MYB protein Bv jkkr interesting for furtheranalyses in respect to DNA-binding and target sequencespecificities.In addition to the highly conserved tryptophan residues,we observed amino acid residues that are conserved in allB. vulgaris R2R3-MYBs: D11, C43, R46 in the R2 repeat andE64, G76, R89 and T90 in the R3 repeat. Further highlyconserved residues of the B. vulgaris R2R3-MYB domainsare: G4, E10, L14, G35, R38, K41, R44, N49, L51 and P53 inthe R2 repeat and I82, A83, N92, K95 and N96 in the R3 repeat (Figure 2). These highly conserved amino acid residues are mainly located in the third helix and the turn ofthe helix-turn-helix (HTH) motif, which is in good accordance with the findings in other plant species.Phylogenetic analysis of the B. vulgaris MYB familyTo explore the putative function of the predicted B. vulgarisMYBs, we assigned them to functional clades known fromA. thaliana, which was chosen because most of our knowledge about plant MYB genes has been obtained from studiesof this major plant model. As known from similar studies,most MYB proteins sharing similar functions cluster in thesame phylogenetic clades, suggesting that most closelyrelated MYBs could recognise similar target genes and possess redundant, overlapping, and/or cooperative functions.
Stracke et al. BMC Plant Biology 2014, 9Page 7 of 17R2***Helix 1Helix 2Turn***Helix 3R3***Helix 1**Helix 2TurnHelix 3Figure 2 Sequence conservation of the R2R3-MYB domain. The R2 and R3 MYB repeats are highly conserved across all BvR2R3-MYB proteins.The logos base on alignments of all R2 and R3 MYB repeats of BvR2R3-MYBs. The overall height of each stack indicates the conservation of thesequence at the given position within the repeat, while the height of symbols within the stack indicates the relative frequency of each aminoacid at that position. The asterisks indicate positions of the conserved amino acids that are identical among all 70 B. vulgaris R2R3-MYB proteins.Arrowheads indicate the typical, conserved tryptophan residues (W) in the MYB domain.We performed a phylogeny reconstruction of 75BvMYBs, the complete A. thaliana MYB family (133 members, including 126 R2R3-MYB, five MYB3R, one MYB4Rand one CDC5-MYB) and 51 well-characterised landmarkR2R3-MYBs from other plant species, using theneighbour-joining (NJ) method (Figure 3) and the maximum parsimony (MP) method (Additional file 2) inMEGA5 [33]. With the exception of some inner nodeswith low bootstrap support values, the phylogenetictrees derived from each method displayed very similartopologies. We took this as an indication of reliability ofour clade- and subgroup designations. The phylogenetictree topology allowed us to classify the analysed MYB proteins into 42 clades (C1 to C42) (Figure 3). In our classification of the MYB genes, we also considered the subgroup(S) categories from A. thaliana [2,17]. Our classificationresulted in the same clusters as those presented in previous studies for grape and soybean [15,19]. As shownin Figure 3, 34 out of 42 clades were present both inB. vulgaris and A. thaliana. Thus, it is likely that theappearance of most MYB genes in these two speciespredates the branch-off of Caryophyllales before theseparation of asterids and rosids [26].We also observed species-specific clades and clades containing B. vulgaris or A. thaliana MYB proteins togetherwith landmark MYBs from other species. It should benoted that we use the term "species-specific" in the context of the current set of species with sequenced genomes.Significantly more genome sequences would be requiredto resolve the presence and absence of genes or clades atthe genus or family level. As indicated also from otherstudies, the observation of species-specific clades may betaken as a hint that MYB genes may have been acquiredor been lost in a single species, during the following divergence from the most recent common ancestor. Forexample, members of the clade C2 (subgroup S12, withHIGH ALIPHATIC GLUCOSINOLATE1 (AtHAG1),HIGH INDOLIC GLUCOSINOLATE1 (AtHIG1),ALTERED TRYPTOPHAN REGULATION1 (AtATR1))have been identified as glucosinolate biosynthesis regulators
Stracke et al. BMC Plant Biology 2014, 9Page 8 of 17Figure 3 Phylogenetic Neighbor Joining (NJ) tree (1000 bootstraps)with MYB proteins from Beta vulgaris (Bv), Arabidopsis thaliana(At) and landmark MYBs from other plant species built withMEGA5.2. Clades (and Subgroups) are labeled with differentalternating tones of grey background. Functional annotation ofclade members are given. The numbers at the branches give bootstrapsupport values from 1000 replications.[34-37]. No BvMYBs were grouped within this clade,containing members which are predominantly presentin plants of the glucosinolate compounds accumulatingBrassicaceae family. A previous study indicated that thisclade was derived fro
BvMYB genes, constituting approximately 0.27% of the 27,421 predicted protein-coding B. vulgaris genes and 5.9% of the 1271 putative B. vulgaris transcription factor genes [26], were subjected for further analyses. Similar to all other genes in the annotated B. vulgaris genome (RefBeet), a unique, immutable four-letter identifier (ID)
The human genome is the first genome entirely sequenced. b. The human genome is about the same size as the genome of E. coli. c. Researchers completed the genomes of yeast and fruit flies during the same time they sequenced the human genome. d. The sequence of the human genome was completed in June 2000. 10.
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The human genome is the first genome entirely sequenced. b. The human genome is about the same size as the genome of E. coli. c. Researchers completed the genomes of yeast and fruit flies during the same time they sequenced the human genome. d. Aworking copy of the human genome was completed in June 2000. 10.
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Keywords: Open access, open educational resources, open education, open and distance learning, open access publishing and licensing, digital scholarship 1. Introducing Open Access and our investigation The movement of Open Access is attempting to reach a global audience of students and staff on campus and in open and distance learning environments.
Thanks to the Human Genome Project, scientists now know the DNA sequence of the entire human genome. The Human Genome Project is an international project that includes scientists from around the world. It began in 1990, and by 2003, scientists had sequenced all 3 billion base pairs of human
Paramecium tetraurelia that lack epigenetic modulation of excision frequently do (Duret et al. 2008). cing Project, we used high-throughput T. thermophila MIC genome se-quencing to initiate the genome-scale investigation of nuclear differ-entiation from MIC to MAC. By aligning MIC genome Sanger
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