Leucoanthocyanidin Dioxygenase in Arabidopsis thaliana

Leucoanthocyanidin Dioxygenase in Arabidopsis thaliana :Characterization of mutant alleles and regulation by MYB –BHLH –TTG1transcription factor complexes

Ingo Appelhagen 1,Oliver Jahns 1,Lutz Bartelniewoehner,Martin Sagasser,Bernd Weisshaar,Ralf Stracke ?

Chair of Genome Research,Department of Biology,Bielefeld University,Universitaetsstrasse 27,D-33615Bielefeld,Germany

a b s t r a c t

a r t i c l e i n f o Article history:

Accepted 30May 2011

Available online 12June 2011Keywords:

Flavonoid biosynthesis Anthocyanins LDOX

Proanthocyanidins Transparent Testa tt17

In Arabidopsis thaliana ,most mutants impaired in ?avonoid accumulation were identi ?ed through screens for altered seed pigmentation.Mutations in more than 20loci have been described that can result in a transparent testa (tt)or tannin de ?cient seed (tds)phenotype.For some of these mutants it is still unclear if they represent additional loci or if they are allelic to known mutations.In this study,we found that tt17is allelic to tt11and tds4and identi ?ed a point mutation in tt17that affects the gene encoding Leucoanthocyanidin Dioxygenase (LDOX).The mutation results in replacement of a cysteine close to the active site of the enzyme by the hydrophobic amino acid tyrosine.Effects of this mutation on protein structure and activity are discussed in the context of LDOX sequences from various genotypes.Regulation of the LDOX promoter was analyzed and found to be directly controlled by different MYB –BHLH –TTG1transcription factor complexes containing the BHLH factors EGL3and TT8.Experiments with single and double loss-of-function mutants identi ?ed EGL3and TT8as necessary regulators of anthocyanin accumulation in developing A.thaliana seedlings.

?2011Elsevier B.V.All rights reserved.

1.Introduction

Flavonoid biosynthesis is one of the best-studied secondary metabolic pathways in plants.In the model plant Arabidopsis thaliana it leads to the synthesis of three phenolic compound classes:colorless ?avonols,red to purple anthocyanins and proanthocyanidins (PAs),which appear brown in their oxidized polymeric forms (Lepiniec et al.,2006;Winkel-Shirley,2001).Flavonoids have numerous functions in plant ecology and development.They serve as pigments,signaling molecules,protectants against biotic and abiotic stresses and as regulators of fertility and auxin biosynthesis (Buer and Muday,2004;

Dixon and Paiva,1995;Santelia et al.,2008;Taylor and Grotewold,2005;Winkel-Shirley,2001).

Anthocyanins are found in epidermal and sub-epidermal cells and occur in central vacuoles either uniformly distributed or as part of sub-vacuolar pigment bodies (Gould et al.,2000;Pourcel et al.,2010).Developing A.thaliana seedlings grown on sucrose-containing media accumulate large quantities of anthocyanins,which reach their highest levels at 4to 5days after germination in cotyledons and in the upper part of the hypocotyl (Kubasek et al.,1992).In older plants,anthocyanins are present in petioles,leaves and stems,and their biosynthesis can be triggered by stresses like high light,temperature,drought or salts (Dixon and Paiva,1995).PAs,in contrast,are syn-thesized independent of external stimuli in the course of seed devel-opment in the testa of A.thaliana seeds (Debeaujon et al.,2003).Biosynthesis of anthocyanins and PAs share common enzymatic steps,from phenylalanine to anthocyanidins (Fig.1).Several mutations in the corresponding enzyme-coding genes affect anthocyanin production and show absence or reduction of PA pigments resulting in a transparent testa (tt )or tannin de ?cient seed (tds )phenotype (overview in Lepiniec et al.,2006).All known enzymes in these pathways are encoded by single copy genes in the A.thaliana genome.However,in a few cases the same locus has been named synony-mously based on independently isolated mutants.Thus,for example,TT11,TT18and TDS4are allelic,encoding Leucoanthocyanidin Dioxygenase (LDOX;also called Anthocyanidin Synthase,ANS;Abrahams et al.,2003;Lepiniec et al.,2006;Shikazono et al.,2003).

Flavonoid biosynthesis is regulated mainly at the transcriptional level (Weisshaar and Jenkins,1998)and across plant species,

Gene 484(2011)61–68

Abbreviations:AHA10,Autoinhibited H +-ATPase Isoform 10;ANR,Anthocyanidin Reductase;BAN,Banyuls;CaMV,Cauli ?ower Mosaic Virus;CHI,Chalcone Isomerase;CHS,Chalcone Synthase;CHX,Cycloheximide;DEX,Dexamethasone;DFR,Dihydro ?avonol 4-Reductase;DHQ,Dihydroquercitin;F3H,Flavanone 3β-Hydroxylase;F3′H,Flavonoid 3′-Hydroxylase;GR,Glucocorticoid Receptor;GST,Glutathione S-Transferase;GT,Glycosyl Transferase;GUS,β-Glucuronidase;LDOX,Leucoanthocyanidin Dioxygenase;MATE,Multidrug and Toxic Compound Extrusion Transporter;MBW,MYB –BHLH –WD40repeat protein;ORF,Open Reading Frame;PA,Proanthocyanidin;PAL,Phenylalanine Ammonia-Lyase;PAP,Production of Anthocyanin Pigment;PFG,Production of Flavonol Glycosides;PPO,Polyphenol Oxidase;SAAP,Single Amino Acid Polymorphism;SNP,Single Nucleotide Polymorphism;TDS,Tannin De ?cient Seed;TT,Transparent Testa .

?Corresponding author at:Universitaetsstrasse 27,D-33615Bielefeld,Germany.Tel.:+495211068759;fax:+495211066423.

E-mail addresses:appelha@cebitec.uni-bielefeld.de (I.Appelhagen),ojahns@cebitec.uni-bielefeld.de (O.Jahns),bartelniewoehner@gmx.de (L.Bartelniewoehner),sagasser@cebitec.uni-bielefeld.de (M.Sagasser),

bernd.weisshaar@uni-bielefeld.de (B.Weisshaar),ralf.stracke@uni-bielefeld.de (R.Stracke).1

These authors contributed equally to this

work.0378-1119/$–see front matter ?2011Elsevier B.V.All rights reserved.doi:

10.1016/j.gene.2011.05.031

Contents lists available at ScienceDirect

Gene

j o u r n a l h o m e p a g e :w ww.e l s ev i e r.c o m /l o c a t e /g e n e

anthocyanin and PA biosynthesis were found to be regulated by MYB –BHLH –WD40repeat protein (MBW)complexes (overview in Broun,2005).Historically,MYB –BHLH interactions were most extensively studied in Zea maize in which the MYB protein Zm C1binds different BHLH proteins like Zm R and Zm B to activate phenylpropanoid pathway genes (Mol et al.,1998).In A.thaliana ,R/B-like BHLH proteins cluster together in subgroup III of the BHLH gene family (Bailey et al.,2003;Heim et al.,2003).Within this group,the closely related members GLABRA3(GL3/BHLH001),ENHANCER OF GLABRA3(EGL3/BHLH002),TT8/BHLH042and MYC1/BHLH012were found to physically interact with R2R3–MYB proteins of subgroups 5and 6(Zimmermann et al.,2004).Gain of function experiments indicate

that R2R3–MYB subgroup 6,comprising PRODUCTION OF ANTHOCY-ANIN PIGMENT1(PAP1/MYB75),PAP2/MYB90,MYB113and MYB114,contains factors that control anthocyanin biosynthesis in vegetative tissues (Borevitz et al.,2000;Gonzalez et al.,2008;Teng et al.,2005).TT2/MYB123,the closest A.thaliana orthologue of Zm C1,belongs to subgroup 5and controls the biosynthesis of PAs in the seed coat of A.thaliana (Nesi et al.,2001).MYB5was recently proposed to be partially redundant with TT2in this respect (Gonzalez et al.,2009).Taken together,these ?ndings indicate that a particular MBW complex can regulate several target genes and,as a result of differential expression patterns,that MYB and BHLH paralogs within one subgroup can control the same metabolic pathway in different cell types (Gonzalez et al.,2008;Stracke et al.,2007).

Probably,most of the key genes involved in ?avonoid biosynthesis have been molecularly identi ?ed in A.thaliana .Nevertheless,some tt loci still require characterization.Here,we report that the tt17mutation (Bharti and Khurana,2003)does not represent a new locus but represents a new mutant LDOX allele.We also present evidence that LDOX is directly regulated by MYB –BHLH –TTG1transcription factor complexes and identify MYB and BHLH proteins contributing to sucrose-mediated anthocyanin accumulation in seedlings.

2.Materials and methods

2.1.Plant material and growth conditions

The tt17mutant (Bharti and Khurana,2003)is in the genetic background of the A.thaliana accession Estland-1(Est-1;GenBank ID:JF681791).The dfr/tt3-1and ldox/tt11-2mutants (Debeaujon et al.,2000;Koornneef,1990;Shirley et al.,1992)are in L er background.ldox/tds4-1and tds4-2(Abrahams et al.,2003)are in Wassilewskija-4(Ws-4)and Columbia-0(Col-0)background,respectively.The pap1/myb75mutant (Teng et al.,2005)is in N?ssen-0background.Mutant seed stocks of tt8/bhlh042/GK241D05(Rosso et al.,2003),gl3/bhlh001/GK545D05(GenBank ID:EU551757;gl3-3in Jakoby et al.,2008),egl3/bhlh002/GK483D04(GenBank ID:EU551758),myc1/bhlh012/GK621F09(GenBank ID:EU551755)and pap2/SALK093731(GenBank ID:EU551756)are in the Col-0genetic background.A detailed char-acterization of the four latter alleles is given in Fig.S3.Homozygous double mutants were identi ?ed by PCR.

A.thaliana seedlings were grown for six days from surface-sterilized seeds cultivated on agar plates containing 0.5×MS medium,0.8%agar and 4%D(+)-sucrose to induce anthocyanin accumulation.Plates were placed in a phytochamber and exposed to 16h of light (white light and UV-A from Osram Lumilux coolwhite,Natura and Fluora bulbs;Osram,Munich,Germany)per day at 22°C.

2.2.Sequencing

Oligonucleotide primers were used to amplify genomic fragments of LDOX in different mutants and accessions (Table S1).DNA sequences were determined on a 3730XL sequencer using BigDye-terminator v3.1chemistry (Applied Biosystems,Weiterstadt,Germany).

2.3.Structure homology modeling

Structure homology modeling was performed with the deduced amino acid sequences of LDOX alleles from tt17and the respective Est-1wild-type.The models were generated using the web-based SWISS-MODEL Workspace (Arnold et al.,2006;Kiefer et al.,2009).Visualization of the LDOX and tt17protein structures was performed with the PyMOL Molecular Graphics System,Version 1.3r1(Schrodinger,LLC,New York,USA;https://www.wendangku.net/doc/9c12809335.html,

).

Fig.1.Simpli ?ed scheme of the ?avonoid biosynthesis pathway in A.thaliana .Arrows indicate different enzymatic steps leading to the formation of ?avonols,anthocyanins and proanthocyanidins.Dashed arrows mark multiple steps.Enzymes are indicated by bold capital letters.For full names see abbreviation list.Mutants for enzymatic steps are indicated in lowercase italic letters.Results from this work have been included.Regulatory proteins of the anthocyanin and PA branch are given in boxes next to their target.Target genes were only considered when activation was proven in vivo (Baudry et al.,2004;Zimmermann et al.,2004).Regulators that were shown to control sucrose induced anthocyanin accumulation in seedlings in the present study are marked in light gray.PAP1homologs of the R2R3–MYB subgroup 6and EGL3homologs of the BHLH subgroup IIIf,which are not involved in sucrose mediated anthocyanin accumulation in the developing seedling,are not shaded.Regulators of PA biosynthesis are highlighted in dark gray and those that are shared among anthocyanin and PA pathway are depicted in black.

62I.Appelhagen et al./Gene 484(2011)61–68

2.4.LDOX promoter analysis by co-transfection assays

Transient promoter activation assays in At7protoplasts were performed as described in Sprenger-Haussels and Weisshaar(2000). In this assay system,the activation potential of transcription factors (effectors)is tested in transient co-transfection experiments using A.thaliana protoplasts.The target gene promoters are fused to the GUS open reading frame(reporter),and standardized speci?c GUS activity(GUS',corrected by a factor describing the transformation ef?ciency)is used to measure target promoter activity.To generate the LDOX reporter construct,a1.9kb promoter fragment was PCR ampli?ed from Col-0genomic DNA with oligonucleotides S249 and S250(see Table S1)and fused to the uidA ORF by Hin dIII/Nco I insertion into pBT10-GUS(Sprenger-Haussels and Weisshaar,2000). All effector constructs used were described previously(Baudry et al., 2004;Zimmermann et al.,2004).

2.5.Flavonoid analysis

Anthocyanin quanti?cation was carried out as described by Mehrtens et al.(2005).Eight biological replicates were measured and the highest and lowest values were excluded.Vanillin staining of immature seeds(around heart stage,according to embryo develop-ment)was performed as described by Debeaujon et al.(2000). Pictures were taken using a Leica DM5500B microscope(Leica, Wetzlar,Germany)equipped with a CCD camera.

2.6.RT-PCR

RNA from seedlings was isolated using the RNeasy?Plant Mini Kit(Qiagen,Hilden,Germany)following the manufacturer's instructions. cDNA was synthesized from2μg total RNA by oligo-dT priming using SuperscriptII reverse transcriptase(Invitrogen,Karlsruhe,Germany) according to manufacturer's instructions in total volume of20μL.1μL cDNA was used as PCR template and transcripts were ampli?ed using gene speci?c primers(see Table S1).

3.Results and discussion

3.1.Transparent Testa17is allelic to LDOX

Until now,more than20different loci involved in PA biosynthesis are known in the model plant A.thaliana.Among them,a handful has not been mapped yet,including TT17(Lepiniec et al.,2006).tt17mutant plants have been reported to have a reduced PA content resulting in a pale brown seed color and a lack in anthocyanin accumulation that cannot be rescued by naringenin feeding,while the production of ?avonols is not affected(Bharti and Khurana,2003).Taken together,this makes TT17likely to act in late steps of?avonoid biosynthesis after the branching point of?avonols and anthocyanins/PAs(Fig.1).In order to identify the mutation causing the tt17phenotype,we?rst did a systematic comparison of the PA pigmentation pattern of tt17seeds with seeds from different?avonoid mutant lines(Fig.S1A).Immature seeds were treated with acidic vanillin to visualize PAs and their monomeric precursors(Deshpande et al.,1986).In developing A. thaliana wild-type seeds,PAs are synthesized in the endothelium,the innermost cell layer of the seed coat,in a few cells of the outer integument at the seed base,in the chalazal bulb and at the micropyle (Debeaujon et al.,2003).In contrast to wild-type,tt17seeds showed a sketchy and faint red coloration in the vanillin assay(Fig.2A).This indicates a reduction of PAs and thus impaired PA production.Several other loci are known to cause similar seed pigmentation phenotypes when mutated(Fig.S1A).Among them are TT6,TT7,LDOX,TT19,AHA10, TT12and the regulatory locus TT1/WIP1.Mutations in TT6and TT7, encoding the enzymes?avanone3β-hydroxylase(F3H)and?avonoid 3′-hydroxylase(F3′H)respectively,affect the formation of all three ?avonoid classes,including?avonol glycosides(Shirley et al.,1995), which are unaltered in tt17(Bharti and Khurana,2003).Autoinhibited H+-ATPASE isoform10(AHA10),TT12and TT1/WIP1mutants also differ from tt17,as they do not have an impact on anthocyanin accumulation in vegetative plant organs(Baxter et al.,2005;Debeaujon et al., 2001;Sagasser et al.,2002).In LDOX and TT19mutants however, anthocyanin and PA production are impaired as in tt17(Abrahams et al., 2003;Kitamura et al.,2004).A more detailed investigation of vanillin treated tt17seeds showed that PA staining only occurred in small circular bodies(Fig.2C;Abrahams et al.,2003).The formation of vanillin stainable vacuole-like structures was also described for tt19mutants but the structures clearly differed in size and PA content from the ones observed in ldox mutants(Fig.S1B;Abrahams et al.,2003;Kitamura et al.,2010).Thus,among the known tt mutants,only ldox mutants show a phenotype that is similar to tt17.

Although tt17was previously described as not allelic to LDOX (At4g22880;Bharti and Khurana,2003),we assayed their relation by complementation tests.We crossed tt17plants with different ldox alleles and analyzed F1seedlings for their ability to produce anthocyanins when grown on sucrose containing media(Fig.2D). While seedlings of all wild-types accumulated anthocyanins under these conditions,we did not observe visible anthocyanins in all ldox mutants,in tt17and in F1seedlings of crosses with tt17.We further analyzed PA accumulation in F2seeds,whose testa is genetically F1 due to the maternal inheritance of the seed coat(Figs.2A and B). Consistent with the F1plants'defects to produce anthocyanins, F2seeds from all tt17x ldox crosses showed PA reduction similar to the homozygous parental lines and did not lead to phenotypical complementation(Fig.2B).As a control we performed crosses of tt17with tt3,a mutant that also does not accumulate PAs and anthocyanins.F1seedlings from this cross accumulated anthocyanins and produced F2seeds with a PA pattern comparable to wild-type (Figs.2A and D).This experiment shows that the tt17mutation is recessive and that the mutant phenotype can be overcome by the TT17wild-type allele in heterozygous conditions.The failed comple-mentation of tt17by different ldox alleles indicates that all of these alleles represent the same locus and leads to the conclusion that tt17 is a new ldox allele.

3.2.tt17contains a point mutation in LDOX

The tt17allele originated from a population mutagenized by fast neutrons(Bharti and Khurana,2003).In order to check for variation in the LDOX gene,we sequenced the locus in tt17,in the corresponding wild-type Est-1,and in selected other lines(tds4-1,Ws-4,tt11-2 and L er).Comparison of the deduced LDOX amino acid sequences revealed natural variation between the different wild-type accessions. Four single amino acid polymorphisms(SAAPs)were detected between Est-1and the other accessions(Q53K,S65N,D151E and I189V,Fig.S2).LDOX from Ws-4differed from the other LDOX variants by the SAAP I157J.These SAAPs apparently do not have major impact on LDOX function,as anthocyanin and PA accumulation were similar in the A.thaliana accessions tested.

LDOX sequence comparison between Est-1and tt17revealed a single nucleotide polymorphism(SNP,G to A)in the coding region that results in an amino acid substitution in the deduced protein sequence(C220Y,Fig.3A).Fast neutron mutagenesis usually leads to chromosomal deletions in the range of some basepairs to several kilobases(Li and Zhang,2002).Since such deletions were not found at the tt17locus,we assume that the mutation was not caused by the mutagen itself.A similar observation has been described for the tds4-1mutant,which originated from a T-DNA insertion population, whereas the phenotype was found to result from a point mutation (Abrahams et al.,2003).A SNP in the coding region of LDOX is also in accordance with the observation that LDOX expression is not changed

63

I.Appelhagen et al./Gene484(2011)61–68

in tt17mutant seedlings in comparison to wild-type,as reported by Bharti and Khurana (2003).

Replacement of cysteine 220,present in Est-1,by the hydrophobic amino acid tyrosine in tt17may lead to conformational changes in enzyme structure that reduce its catalytic activity and result in the described mutant phenotype.Cysteine 220is conserved in most ?avonoid 2-oxoglutarate-dependent dioxygenases (data not shown).However,due to the large distances between the other cysteines in the protein (C66-C220:39.69?and C220-C305:32.35?,as deter-mined using PyMOL software)it seems unlikely that the C220Y mutation changes structural enzyme integrity by affecting a cysteine bridge.Substrate binding in 2-oxoglutarate-dependent dioxygenases is,among others,mediated through Y217(Wilmouth et al.,2002).A mutation at position 220may therefore have an impact on the positioning of the substrates 2-oxoglutarate,dihydroquercetin and ascorbate at the active site (Fig.3C),de ?ned by H232,D234and H288(HXD …H;Abrahams et al.,2003;Turnbull et al.,2001;Wilmouth

et al.,2002).A multiple alignment of LDOX sequences encoded in tt17,tt11-2,tds4-1and the corresponding wild-types Est-1,L er and Ws-4(Fig.S2)showed that tt11-2(Debeaujon et al.,2000)contains a G78E exchange.The single amino acid exchange G228D in tds4-1(Fig.3B)is also located close to the active site motif and causes a mutant phenotype which is indistinguishable from tt17(Fig.2and S1).From these considerations on structure together with our complementation experiments,which showed that the tt17locus encodes a non-functional LDOX protein,we conclude that the SNP in the LDOX ORF represents a very good candidate to cause the tt phenotype of the tt17mutant.

3.3.LDOX activation is controlled by MYB –BHLH –TTG1transcription factor complexes

Results from expression analysis in loss-of-function and gain-of-function mutants indicate that anthocyanin and PA biosynthesis

are

Fig.2.PA and anthocyanin accumulation in tt17and ldox mutants.(A)Immature seeds at heart stage of embryo development were treated with acidic vanillin.Red color indicates presence of PA precursors.Genotypes are indicated (for details see Materials and Methods).Scale bar,100μm;cb,chalazal bulb;mi,micropyle.All analyzed ldox alleles (tt11-2,tds4-1,tds4-2),tt17and F2seeds (with testa from the F1mother plant)of crosses with tt17showed reduced PA accumulation.All wild-type seeds corresponding to the respective mutants showed the same staining as Est-1.(B)Mature seeds of the lines shown in (A).The upper right panel shows seeds of the different accessions representing the genetic backgrounds of the mutants.Scale bar,2mm.(C)Enlarged images of selected lines shown in (A).In contrast to huge PA ?lled vacuoles seen in wild-type seeds,small vanillin stained structures (marked by white arrowheads)are observed in endothelial cells of tt17and tds4-2seeds.Single endothelial cells are outlined with dotted lines.Scale bar,10μm.(D)Sucrose-induced anthocyanin accumulation in 5day old seedlings.Genotypes are indicated.All ldox ,tt17and F1seedlings of crosses with tt17do not show visible anthocyanins.Seedlings of all wild-types accumulate anthocyanins as shown for Est-1.Scale bar,1mm.

64I.Appelhagen et al./Gene 484(2011)61–68

transcriptionally regulated by MBW complexes (Borevitz et al.,2000;Gonzalez et al.,2008;Teng et al.,2005;Tohge et al.,2005).However,experimental data on direct activation of the promoters of anthocyanin and PA biosynthesis genes by MYB –BHLH –TTG1com-plexes is still limited.So far,only the promoters of DFR and BAN have been tested in vivo using protoplast transactivation assays.ProDFR was successfully assayed for activation in this system,using different MYB –BHLH effector combinations (PAP1or PAP2with GL3,EGL3,MYC1or TT8,respectively;Zimmermann et al.,2004).ProBAN reporter constructs were shown to be active in simultaneous presence of TT2,TT8and TTG1(Baudry et al.,2004).LDOX is also thought to be a primary target of TTG1complexes,as it was found to be strongly expressed in experiments with Pro35S:TTG1-GR lines treated with cycloheximide (CHX)and induced with dexamethasone (DEX;Gonzalez et al.,2008).These results prompted us use protoplast co-transfection assays to analyze ProLDOX activation (see Materials and methods ).Different R2R3–MYB and BHLH proteins were tested for their potential to directly activate a 1.9kb LDOX promoter fragment (Fig.4).As mutations in PAP1,EGL3and TT8are known to have an impact on anthocyanin accumulation (Gonzalez et al.,2008;Shirley et al.,1995;Teng et al.,2005),we chose combinations of these factors as candidate regulators of LDOX in the anthocyanin branch of the ?avonoid pathway.The combination of TT8and TT2was selected to test ProLDOX activation by regulators of the PA branch.None of the tested effectors exhibited a signi ?cant activation potential on ProLDOX when assayed alone.However,activation was observed for all combinations of MYB and BHLH effectors tested (Fig.4).TTG1,the third factor in MBW complexes,is endogenously expressed in the cultured At7cells used for the experiments.In order to show that the observed ProLDOX activation is not exclusively caused by MYB and BHLH factors,we co-transfected an additional plasmid,encoding Pro35S:TTG1,and observed an up to 10-fold increase in reporter gene activity as compared to activation obtained with endogenous TTG1expression alone (Fig.4).Direct interactions between the R2R3–MYB proteins PAP1and TT2and the BHLH factors EGL3and TT8as well as between these BHLH factors and TTG1have been shown by yeast-two-hybrid experiments (Baudry et al.,2004;Zimmermann et al.,2004).Analysis of publicly available microarray data using Geneves-tigator (Hruz et al.,2008)revealed co-expression of LDOX and DFR in anthocyanin-producing tissues in seedlings and of LDOX ,DFR and BAN in developing seeds.Based on these ?ndings,we conclude from our transactivation data that ProLDOX is regulated by MYB –BHLH –TTG1complexes and propose that LDOX expression in planta is controlled by the same mechanisms as DFR and BAN .

The exact regulons of MYB –BHLH –TTG1complexes regulating ?avonoid biosynthesis however,remain to be elucidated.Stable ectopic over-expression of PAP1led to upregulation of a large number of genes whose products are involved in anthocyanin formation (Borevitz et al.,2000;Tohge et al.,2005).Expression analysis in DEX induced and CHX treated TTG1over-expressors in contrast,identi ?ed only DFR ,LDOX and TT8as primary targets of MBW-complexes (Gonzalez et al.,2008).Experiments like co-transactivation-or chromatin immunoprecipitation studies would help to clarify the range of the regulons of different MYB –BHLH –TTG1

complexes.

Fig.3.The tt17mutation.(A)Schematic drawing of the LDOX gene illustrating mutations in tds4-1,tds4-2,tt11-2and tt17.Exons are indicated by boxes,introns by thin lines.The bended arrow tags the translation start;the triangle marks a T-DNA insertion.(B)Multiple alignment of LDOX protein fragments surrounding the C220Y mutation in tt17(?lled arrow).Amino acids of the HXD …H motive involved in Fe(II)binding (Wilmouth et al.,2002)are indicated in green and by black circles.Amino acids involved in substrate binding (Wilmouth et al.,2002)are marked with triangles.The G228D mutation in tds4-1is printed in bold face.(C)Stereo views of LDOX structures from tt17and the corresponding Est-1wild-type (TT17).Views show overall structures and close-ups of active site regions.The genuine C220in the TT17protein (left panel)is indicated in blue.Y220in tt17is marked in red (right panel).

65

I.Appelhagen et al./Gene 484(2011)61–68

3.4.PAP-like R2R3–MYBs and subgroup IIIf BHLH members contribute differentially to sucrose induced LDOX gene activity in planta

Different combinations of R2R3–MYB and BHLH factors were able to activate the LDOX promoter in transiently co-transfected pro-toplasts (Fig.4).However,in this test-system,as well as in studies using transgenic plants (Borevitz et al.,2000;Gonzalez et al.,2008;Tohge et al.,2005),expression of the transcription factors was controlled by the constitutive CaMV 35S promoter,which may lead to activation levels that are not comparable with those provided by the native promoters.To investigate the contribution of each regulator to LDOX activation in the anthocyanin pathway in planta ,we made use of loss-of-function mutants for various candidate regulators and chose sucrose-induced anthocyanin accumulation in developing seedlings as an indicator for LDOX activity (Fig.1).Since we had strong evidence that,among the R2R3–MYB genes from subgroup 6,MYB113and MYB114are not expressed in developing seedlings (unpublished results),we concentrated on the R2R3–MYBs PAP1and PAP2.pap1mutant seedlings (Teng et al.,2005)showed anthocyanin accumula-tion in a small distinct area in the transition zone between hypocotyl and cotyledons (see arrowheads in Fig.5B).pap2mutants (this work,Fig.S3)were indistinguishable from wild-type seedlings and had a similar anthocyanin content (Figs.5A and D).No anthocyanins were detected in pap1pap2double mutant seedlings,indicating that among the R2R3–MYB factors PAP1and PAP2are necessary and suf ?cient to activate the enzyme-coding genes required for sucrose-mediated anthocyanin production in developing seedlings,including LDOX .This result is consistent with PAP1and PAP2transcript accumulation data,showing predominant expression of PAP1in seedlings grown under these conditions,and with data on RNAi knock-down lines of PAP2(Gonzalez et al.,2009).

The test system of sucrose-induced anthocyanin accumulation in seedlings of loss-of-function mutants was also used to investigate the contribution of different BHLH factors to LDOX activation.The alleles characterized and used here are proven NULL alleles (gl3-3,egl3,myc1,this work,Fig.S3;tt8,Rosso et al.,2003).Among the bhlh single mutants analyzed,only egl3seedlings showed a clearly visible reduction in purple colouration (Fig.5C).Anthocyanin quanti ?cation (Fig.5D)con ?rmed a reduction to ca.30%in the egl3mutant

compared to wild-type and additionally revealed a reduction to ca.60%in tt8,that was not visible in intact seedlings.This result prompted us to combine both mutations by crossing.The resulting egl3tt8double mutant showed an additive effect of both single mutations and yielded seedlings completely devoid of anthocyanins (Figs.5C and D).Therefore,both,EGL3and TT8,unambiguously contribute to sucrose-induced anthocyanin accumulation in seedlings,whereas GL3or MYC1are not required under these conditions.Expression analysis (Fig.5E)showed that BHLHs are not induced by sucrose.These factors are present in the tissue and determine the ability of the cells to accumulate anthocyanin when the respective R2R3–MYB partner factor is expressed,for example in response to elevated sucrose concentrations.

The stronger reduction in anthocyanin content in egl3in com-parison to tt8can be correlated with the observation that the com-bination of PAP1–EGL3led to higher activation of the LDOX promoter in co-transfection experiments than PAP1–TT8(Fig.4).EGL3exclusively regulates the anthocyanin branch of ?avonoid biosynthe-sis,whereas TT8is required for both,anthocyanin and PA pathways.TT8represents the only BHLH factor needed for PA formation (Baudry et al.,2006)and may therefore have a subordinate role in anthocyanin accumulation in planta .It is tempting to speculate that this re ?ects a weaker af ?nity or reduced activation capacity of PAP1–TT8–TTG1complexes on promoters of anthocyanin speci ?c genes.

4.Conclusions

The oxidation of leucoanthocyanidins to anthocyanidins catalyzed by the LDOX enzyme is one of the central steps of anthocyanin and PA biosynthesis.In this study,we identi ?ed the LDOX gene as the locus of tt17and presented evidence that the tt17phenotype is linked to a point mutation in LDOX .Furthermore,we showed that the LDOX promoter is directly controlled by TTG1-containing MYB –BHLH complexes and identi ?ed PAP1,PAP2,EGL3and TT8as the determining factors for sucrose-induced anthocyanin accumulation in developing seedlings.

Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2011.05.031

.

Fig.4.LDOX promoter analysis.LDOX promoter activation assay in transiently co-transfected At7protoplasts.A 1.9kb LDOX promoter fragment was fused to the GUS open reading frame (reporter construct)and assayed for its responsiveness to different combinations of 35S promoter-driven PAP1,TT2,EGL3and TT8effectors.Mean values and standard deviations of normalized GUS'activities of 6experiments are given.Activation was only observed for combinations of MYB and BHLH factors.TTG1is endogenously expressed in At7protoplasts;addition of a TTG1effector leads to increased GUS'activity.

66I.Appelhagen et al./Gene 484(2011)61–68

Fig.5.Sucrose mediated anthocyanin accumulation in pap1/2and subgroup IIIf bhlh mutants.(A)5day old Col-0and N?-0seedlings grown on MS media lacking sucrose (left panel)and on MS supplemented with 4%sucrose (right panel).Sucrose induced anthocyanin accumulation is observed in the upper hypocotyl and in cotyledons of both accessions.Suc,sucrose;Scale bar,1mm.(B)Different pap R2R3–MYB mutant seedlings,grown on MS with 4%sucrose.pap1seedlings (N?-0background)showed reduced anthocyanin accumulation restricted to a small area in the upper hypocotyl (marked by white arrowheads).pap2seedlings (Col-0background)showed anthocyanin accumulation similar to wild-type.pap1pap2double mutants were free of visible purple anthocyanin pigments.(C)Seedlings of different bhlh subgroup IIIf mutants grown on MS with 4%sucrose.gl3,tt8and myc1seedlings showed no visible differences compared to wild-type lines.egl3showed a drastic reduction in anthocyanin accumulation.No visible anthocyanin pigments were found in egl3tt8double mutants.(D)Quanti ?cation of sucrose-induced anthocyanins in lines shown in (A,B,C).Relative anthocyanin contents were calculated using the quotient (A 530?0.25×A 657)/M,where A 530and A 657is the absorption at the indicated wavelengths and M is the weight of the plant material (in g)used for extraction.The tt4mutant that lacks all ?avonoids (Shirley et al.,1995)was included as negative https://www.wendangku.net/doc/9c12809335.html,parable to the tt4mutant,anthocyanins were undetectable in egl3tt8and pap1pap2double mutants.Results are means ±standard deviation of six experiments.(E)Results from RT-PCR showing ACT2,PAP and BHLH expression in 5days old seedlings grown on MS without and with 4%sucrose.PAP1and PAP2transcripts were undetectable in seedlings grown on media without sucrose.Numbers of ampli ?cation cycles are indicated.

67

I.Appelhagen et al./Gene 484(2011)61–68

Acknowledgments

We thank Melanie Kuhlmann for excellent technical assistance, Antje Bluemke for help with preparation and vanillin staining of seeds and Gunnar Huep for critical reading of the manuscript. References

Abrahams,S.,Lee,E.,Walker,A.R.,Tanner,G.J.,Larkin,P.J.,Ashton,A.R.,2003.The Arabidopsis TDS4gene encodes leucoanthocyanidin dioxygenase(LDOX)and is essential for proanthocyanidin synthesis and vacuole development.Plant J.35, 624–636.

Arnold,K.,Bordoli,L.,Kopp,J.,Schwede,T.,2006.The SWISS-MODEL workspace:a web-based environment for protein structure homology modelling.Bioinformatics22, 195–201.

Bailey,P.C.,et al.,2003.Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana.Plant Cell15,2497–2502.

Baudry,A.,Heim,M.A.,Dubreucq,B.,Caboche,M.,Weisshaar,B.,Lepiniec,L.,2004.TT2, TT8,and TTG1synergistically specify the expression of BANYULS and proantho-cyanidin biosynthesis in Arabidopsis thaliana.Plant J.39,366–380.

Baudry,A.,Caboche,M.,Lepiniec,L.,2006.TT8controls its own expression in a feedback regulation involving TTG1and homologous MYB and bHLH factors,allowing a strong and cell-speci?c accumulation of?avonoids in Arabidopsis thaliana.Plant J.

46,768–779.

Baxter,I.R.,et al.,2005.A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana.Proc.Natl.

Acad.Sci.U.S.A.102,2649–2654.

Bharti,A.,Khurana,J.,2003.Molecular characterization of transparent testa(tt)mutants of Arabidopsis thaliana(ecotype Estland)impaired in?avonoid biosynthetic pathway.

Plant Sci.165,1321–1332.

Borevitz,J.O.,Xia,Y.J.,Blount,J.,Dixon,R.A.,Lamb,C.,2000.Activation tagging identi?es

a conserved MYB regulator of phenylpropanoid biosynthesis.Plant Cell12,

2383–2393.

Broun,P.,2005.Transcriptional control of?avonoid biosynthesis:a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis.

Curr.Opin.Plant Biol.8,272–279.

Buer, C.S.,Muday,G.K.,2004.The transparent testa4mutation prevents?avonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light.Plant Cell16,1191–1205.

Debeaujon,I.,Leon-Kloosterziel,K.,Koornneef,M.,2000.In?uence of the testa on seed dormancy,germination,and longevity in Arabidopsis.Plant Physiol.122,403–413. Debeaujon,I.,Peeters, A.J.M.,Léon-Kloosterziel,K.M.,Koornneef,M.,2001.The TRANSPARENT TESTA12gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for?avonoid sequestration in vacuoles of the seed coat endothelium.Plant Cell13,853–872.

Debeaujon,I.,et al.,2003.Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of differentiation and role in seed development.Plant Cell15,2514–2531. Deshpande,S.S.,Cheryan,M.,Salunkhe,D.K.,1986.Tannin analysis of food products.

Crit.Rev.Food Sci.Nutr.24,401–449.

Dixon,R.A.,Paiva,N.L.,1995.Stress-induced phenylpropanoid metabolism.Plant Cell7, 1085–1097.

Gonzalez,A.,Zhao,M.,Leavitt,J.M.,Lloyd,A.M.,2008.Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabi-dopsis seedlings.Plant J.53,814–827.

Gonzalez,A.,Mendenhall,J.,Huo,Y.,Lloyd,A.,2009.TTG1complex MYBs,MYB5and TT2,control outer seed coat differentiation.Dev.Biol.325,412–421.

Gould,K.S.,Markham,K.R.,Smith,R.H.,Goris,J.J.,2000.Functional role of anthocyanins in the leaves of Quintinia serrata A.Cunn.J.Exp.Bot.51,1107–1115.

Heim,M.A.,Jakoby,M.,Werber,M.,Martin,C.,Weisshaar,B.,Bailey,P.C.,2003.The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity.Mol.Biol.Evol.20,735–747.

Hruz,T.,et al.,2008.Genevestigator v3:a reference expression database for the meta-analysis of transcriptomes.Adv.Bioinf.2008,420747.

Jakoby,M.J.,et al.,2008.Transcriptional pro?ling of mature Arabidopsis trichomes reveals that NOECK encodes the MIXTA-like transcriptional regulator MYB106.

Plant Physiol.148,1583–1602.Kiefer,F.,Arnold,K.,Kunzli,M.,Bordoli,L.,Schwede,T.,2009.The SWISS-MODEL repository and associated resources.Nucleic Acids Res.37,D387–D392. Kitamura,S.,Shikazono,N.,Tanaka,A.,2004.TRANSPARENT TESTA19is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis.Plant J.

37,104–114.

Kitamura,S.,et al.,2010.Metabolic pro?ling and cytological analysis of proanthocya-nidins in immature seeds of Arabidopsis thaliana?avonoid accumulation mutants.

Plant J.62,549–559.

Koornneef,M.,1990.Mutations affecting the testa colour in Arabidopsis.Arabidopsis Inf.

Serv.27,1–4.

Kubasek,W.L.,Shirley,B.W.,McKillop,A.,Goodman,H.M.,Briggs,W.,Ausubel,F.M., 1992.Regulation of?avonoid biosynthetic genes in germinating Arabidopsis seedlings.Plant Cell4,1229–1236.

Lepiniec,L.,et al.,2006.Genetics and biochemistry of seed?avonoids.Annu.Rev.Plant Biol.57,405–430.

Li,X.,Zhang,Y.,2002.Reverse genetics by fast neutron mutagenesis in higher plants.

Funct.Integr.Genomics2,254–258.

Mehrtens,F.,Kranz,H.,Bednarek,P.,Weisshaar,B.,2005.The Arabidopsis transcription factor MYB12is a?avonol-speci?c regulator of phenylpropanoid biosynthesis.

Plant Physiol.138,1083–1096.

Mol,J.,Grotewold,E.,Koes,R.,1998.How genes paint?owers and seeds.Trends Plant Sci.3,212–217.

Nesi,N.,Jond,C.,Debeaujon,I.,Caboche,M.,Lepiniec,L.,2001.The Arabidopsis TT2gene encodes an R2R3MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed.Plant Cell13,2099–2114. Pourcel,L.,Irani,N.G.,Lu,Y.,Riedl,K.,Schwartz,S.,Grotewold,E.,2010.The formation of anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments.Mol.Plant3,78–90.

Rosso,M.G.,Li,Y.,Strizhov,N.,Reiss,B.,Dekker,K.,Weisshaar,B.,2003.An Arabidopsis thaliana T-DNA mutagenised population(GABI-Kat)for?anking sequence tag based reverse genetics.Plant Mol.Biol.53,247–259.

Sagasser,M.,Lu,G.-H.,Hahlbrock,K.,Weisshaar,B.,2002.A.thaliana TRANSPARENT TESTA1is involved in seed coat development and de?nes the WIP subfamily of plant zinc?nger proteins.Genes Dev.16,138–149.

Santelia,D.,et al.,2008.Flavonoids redirect PIN-mediated polar auxin?uxes during root gravitropic responses.J.Biol.Chem.283,31218–31226.

Shikazono,N.,et al.,2003.Mutation rate and novel tt mutants of Arabidopsis thaliana induced by carbon ions.Genetics163,1449–1455.

Shirley,B.W.,Hanley,S.,Goodman,H.M.,1992.Effects of ionizing radiation on a plant genome:analysis of two Arabidopsis transparent testa mutations.Plant Cell4, 333–347.

Shirley, B.W.,et al.,1995.Analysis of Arabidopsis mutants de?cient in?avonoid biosynthesis.Plant J.8,659–671.

Sprenger-Haussels,M.,Weisshaar,B.,2000.Transactivation properties of parsley proline rich bZIP transcription factors.Plant J.22,1–8.

Stracke,R.,et al.,2007.Differential regulation of closely related R2R3-MYB transcription factors controls?avonol accumulation in different parts of the Arabidopsis thaliana seedling.Plant J.50,660–677.

Taylor,L.P.,Grotewold,E.,2005.Flavonoids as developmental regulators.Curr.Opin.

Plant Biol.8,317–323.

Teng,S.,Keurentjes,J.,Bentsink,L.,Koornneef,M.,Smeekens,S.,2005.Sucrose-speci?c induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene.Plant Physiol.139,1840–1852.

Tohge,T.,et al.,2005.Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor.

Plant J.42,218–235.

Turnbull,J.J.,Prescott, A.G.,Scho?eld, C.J.,Wilmouth,R.C.,2001.Puri?cation, crystallization and preliminary X-ray diffraction of anthocyanidin synthase from Arabidopsis thaliana.Acta Crystallogr.,Sect.D:Biol.Crystallogr.57,425–427. Weisshaar,B.,Jenkins,G.I.,1998.Phenylpropanoid biosynthesis and its regulation.Curr.

Opin.Plant Biol.1,251–257.

Wilmouth,R.C.,Turnbull,J.J.,Welford,R.W.,Clifton,I.J.,Prescott,A.G.,Scho?eld,C.J.,2002.

Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana.

Structure10,93–103.

Winkel-Shirley,B.,2001.Flavonoid biosynthesis.A colorful model for genetics,biochemistry, cell biology,and biotechnology.Plant Physiol.126,485–493.

Zimmermann,I.M.,Heim,M.A.,Weisshaar, B.,Uhrig,J.F.,https://www.wendangku.net/doc/9c12809335.html,prehensive identi?cation of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins.Plant J.40,22–34.

68I.Appelhagen et al./Gene484(2011)61–68

高中英语每日一题第3周playapartin含解析新人教版必修120171011143

play a part (in) 重要程度：★☆☆☆☆难易程度：★★☆☆☆ Have you realized the part computers have ___________ in the daily life? A. made B. given C. caused D. played 【参考答案】D 【拓展延伸】 1. play a part (in) 在……中扮演一个角色；参与；在……中起作用 2. play the role of 扮演……的角色 play an important role / part in...在……中起重要作用 play the leading role / part主演；起带头作用 3. take part in 参加 for the most part多半；在很大程度上 for one’s part就某人而言，对某人来说 1. Colors play an important ___________ in the way you look. A. part B. form C. effect D. pride 2. Mr. Huang will ___________ in the movement. A. play a leading part B. take parts C. play leading part D. take a part 3. __________ part that women ___________ in society is great. A. The; plays B. A; takes C. A; plays

黄自艺术歌曲钢琴伴奏及艺术成就

【摘要】黄自先生是我国杰出的音乐家，他以艺术歌曲的创作最为代表。而黄自先生特别强调了钢琴伴奏对于艺术歌曲组成的重要性。本文是以黄自先生创作的具有爱国主义和人道主义的艺术歌曲《天伦歌》为研究对象，通过对作品分析，归纳钢琴伴奏的弹奏方法与特点，并总结黄自先生的艺术成就与贡献。 【关键词】艺术歌曲；和声；伴奏织体；弹奏技巧 一、黄自艺术歌曲《天伦歌》的分析 （一）《天伦歌》的人文及创作背景。黄自的艺术歌曲《天伦歌》是一首具有教育意义和人道主义精神的作品。同时，它也具有民族性的特点。这首作品是根据联华公司的影片《天伦》而创作的主题曲，也是我国近代音乐史上第一首为电影谱写的艺术歌曲。作品创作于我国政治动荡、经济不稳定的30年代，这个时期，这种文化思潮冲击着我国各个领域，连音乐艺术领域也未幸免――以《毛毛雨》为代表的黄色歌曲流传广泛，对人民大众，尤其是青少年的不良影响极其深刻，黄自为此担忧，创作了大量艺术修养和文化水平较高的艺术歌曲。《天伦歌》就是在这样的历史背景下创作的，作品以孤儿失去亲人的苦痛为起点，发展到人民的发愤图强，最后升华到博爱、奋起的民族志向，对青少年的爱国主义教育有着重要的影响。 （二）《天伦歌》曲式与和声。《天伦歌》是并列三部曲式，为a+b+c，最后扩充并达到全曲的高潮。作品中引子和coda所使用的音乐材料相同，前后呼应，合头合尾。这首艺术歌曲结构规整，乐句进行的较为清晰，所使用的节拍韵律符合歌词的特点，如三连音紧密连接，为突出歌词中号召的力量等。 和声上，充分体现了中西方作曲技法融合的创作特性。使用了很多七和弦。其中，一部分是西方的和声，一部分是将我国传统的五声调式中的五个音纵向的结合，构成五声性和弦。与前两首作品相比，《天伦歌》的民族性因素增强，这也与它本身的歌词内容和要弘扬的爱国主义精神相对应。 （三）《天伦歌》的伴奏织体分析。《天伦歌》的前奏使用了a段进唱的旋律发展而来的，具有五声调性特点，增添了民族性的色彩。在作品的第10小节转调入近关系调，调性的转换使歌曲增添抒情的情绪。这时的伴奏加强和弦力度，采用切分节奏，节拍重音突出，与a段形成强弱的明显对比，突出悲壮情绪。 c段的伴奏采用进行曲的风格，右手以和弦为主，表现铿锵有力的进行。右手为上行进行，把全曲推向最高潮。左手仍以柱式和弦为主，保持节奏稳定。在作品的扩展乐段，左手的节拍低音上行与右手的八度和弦与音程对应，推动音乐朝向宏伟、壮丽的方向进行。coda 处，与引子材料相同，首尾呼应。 二、《天伦歌》实践研究 《天伦歌》是具有很强民族性因素的作品。所谓民族性，体现在所使用的五声性和声、传统歌词韵律以及歌曲段落发展等方面上。 作品的整个发展过程可以用伤感――悲壮――兴奋――宏达四个过程来表述。在钢琴伴奏弹奏的时候，要以演唱者的歌唱状态为中心，选择合适的伴奏音量、音色和音质来配合，做到对演唱者的演唱同步，并起到连接、补充、修饰等辅助作用。 作品分为三段，即a+b+c+扩充段落。第一段以五声音阶的进行为主，表现儿童失去父母的悲伤和痛苦，前奏进入时要弹奏的使用稍凄楚的音色，左手低音重复进行，在弹奏完第一个低音后，要迅速的找到下一个跨音区的音符；右手弹奏的要有棱角，在前奏结束的时候第四小节的t方向的延音处，要给演唱者留有准备。演唱者进入后，左手整体的踏板使用的要连贯。随着作品发展，伴奏与旋律声部出现轮唱的形式，要弹奏的流动性强，稍突出一些。后以mf力度出现的具有转调性质的琶音奏法，要弹奏的如流水般连贯。在重复段落，即“小

我国艺术歌曲钢琴伴奏-精

我国艺术歌曲钢琴伴奏-精 2020-12-12 【关键字】传统、作风、整体、现代、快速、统一、发展、建立、了解、研究、特点、突出、关键、内涵、情绪、力量、地位、需要、氛围、重点、需求、特色、作用、结构、关系、增强、塑造、借鉴、把握、形成、丰富、满足、帮助、发挥、提高、内心 【摘要】艺术歌曲中，伴奏、旋律、诗歌三者是不可分割的重 要因素，它们三个共同构成一个统一体，伴奏声部与声乐演唱处于 同样的重要地位。形成了人声与器乐的巧妙的结合，即钢琴和歌唱 的二重奏。钢琴部分的音乐使歌曲紧密的联系起来，组成形象变化 丰富而且不中断的套曲，把音乐表达的淋漓尽致。 【关键词】艺术歌曲；钢琴伴奏；中国艺术歌曲 艺术歌曲中，钢琴伴奏不是简单、辅助的衬托，而是根据音乐 作品的内容为表现音乐形象的需要来进行创作的重要部分。准确了 解钢琴伴奏与艺术歌曲之间的关系,深层次地了解其钢琴伴奏的风 格特点,能帮助我们更为准确地把握钢琴伴奏在艺术歌曲中的作用 和地位，从而在演奏实践中为歌曲的演唱起到更好的烘托作用。 一、中国艺术歌曲与钢琴伴奏 “中西结合”是中国艺术歌曲中钢琴伴奏的主要特征之一，作 曲家们将西洋作曲技法同中国的传统文化相结合，从开始的借鉴古 典乐派和浪漫主义时期的创作风格，到尝试接近民族乐派及印象主 义乐派的风格，在融入中国风格的钢琴伴奏写作，都是对中国艺术 歌曲中钢琴写作技法的进一步尝试和提高。也为后来的艺术歌曲写 作提供了更多宝贵的经验，在长期发展中，我国艺术歌曲的钢琴伴 奏也逐渐呈现出多姿多彩的音乐风格和特色。中国艺术歌曲的钢琴

写作中，不可忽略的是钢琴伴奏织体的作用，因此作曲家们通常都以丰富的伴奏织体来烘托歌曲的意境，铺垫音乐背景，增强音乐感染力。和声织体，复调织体都在许多作品中使用，较为常见的是综合织体。这些不同的伴奏织体的歌曲，极大限度的发挥了钢琴的艺术表现力，起到了渲染歌曲氛围，揭示内心情感，塑造歌曲背景的重要作用。钢琴伴奏成为整体乐思不可缺少的部分。优秀的钢琴伴奏织体，对发掘歌曲内涵，表现音乐形象，构架诗词与音乐之间的桥梁等方面具有很大的意义。在不断发展和探索中，也将许多伴奏织体使用得非常娴熟精确。 二、青主艺术歌曲《我住长江头》中钢琴伴奏的特点 《我住长江头》原词模仿民歌风格，抒写一个女子怀念其爱人的深情。青主以清新悠远的音乐体现了原词的意境，而又别有寄寓。歌调悠长，但有别于民间的山歌小曲；句尾经常出现下行或向上的拖腔，听起来更接近于吟哦古诗的意味，却又比吟诗更具激情。钢琴伴奏以江水般流动的音型贯穿全曲，衬托着气息宽广的歌唱，象征着绵绵不断的情思。由于运用了自然调式的旋律与和声，显得自由舒畅，富于浪漫气息，并具有民族风味。最有新意的是，歌曲突破了“卜算子”词牌双调上、下两阕一般应取平行反复结构的惯例，而把下阕单独反复了三次，并且一次比一次激动，最后在全曲的高音区以ff结束。这样的处理突出了思念之情的真切和执著，并具有单纯的情歌所没有的昂奋力量。这是因为作者当年是大革命的参加者，正被反动派通缉，才不得不以破格的音乐处理，假借古代的

playapartin的用法

play a part in的用法 Do you know this pretty girl Right! She is Audrey Hepburn who played many classic roles in a great many famous films. For example, < Roman Holiday> is the one of the films which earned Hepburn her first Academy Award for Best Actress. Audrey Hepburn played the part/role of Princess Ann in this film. play the part/role of…扮演……角色

Audrey Hepburn played a leading part in directed by Steven Spielberg.( Always 直到永远/天长地久) play a part in…在……中扮演角色，在……中起作用

Audrey Hepburn also played leading roles in < Funny Face>and < My Fair Lady〉.( My Fair Lady 窈窕淑女影片讲述下层卖花女被语言学教授改造成优雅贵妇的故事，从头至尾洋溢着幽默和雅趣 .Funny Face 甜姐儿) Play a role in…在……中扮演角色，在……中起作用 play a part/role in doing sth. 在做某事方面起作用,参与做某事 We can?all?play?a?role/part?in?reducing?our?dependence?on?plastic, if we started to?take some small?steps?in?our?everyday?lives?to?be

Unit 7 Will people have robots-知识点整理

Unit 7 Will people have robots?知识 点整理 Unit7willpeoplehaverobots?知识点整理 一、词组、短语： 、oncomputers在电脑上， 2、onpaper在纸上， 3、livetobe200yearsold活到200岁， 4、freetime空闲时间， 5、indanger 在危险中， 6、ontheearth在世界上 7、playapartinsth在某方面出力/做贡献， 8、spacestation太空站， 8、lookfor寻找， 9、computerprogrammer电脑程序师， 10、inthefuture 在将来， 11、hundredsof成百上千的， 12、thesame…as与…一样， 13、overandoveragain反复， 14、getbored 无聊，

15、wakeup醒来/唤醒， 16、looklike 看起来像， 17、falldown倒下/落下 二、重要句子（语法） 、will+动词原形 将要做 2、fewer/more+可数名词复数更少/更多… 3、less/more+不可数名词 更少/更多 4、trytodosth. 尽力做某事 5、havetodosth 不得不做某事 6、agreewithsb. 同意某人的意见 7、such+名词（词组） 如此

8、playapartindoingsth 参与做某事 9、makesbdosth 让某人做某事 10、helpsbwithsth 帮助某人做某事 11、Therewillbe+主语+其他 将会有…. 12、Thereis/are+sb.+doingsth 有…正在做… 13、Itis +形容词+forsb+todosth 做某事对某人来说… 语法： whatwillthefuturebelike? citieswillbemorepolluted.Andtherewillbefewertrees. willpeopleusemoneyin100years?

【K12学习】人教版八年级英语上册Unit7知识点归纳整理

人教版八年级英语上册Unit7知识点归纳整 理 Unit7illpeoplehaverobots? 短语归纳 onputer在电脑上2.onpaper在纸上3.aeup醒 livetodo200yearsold活动200岁5.freetie空闲时间indanger处于危险之中7.ontheearth在地球上 playapartinsth.参与某事9.inthefuture在未 0spacestation太空站11.puterprograer电脑编程员 loofor寻找13.hundredsof许多；成百上千 thesae…as…与……一样15.getbored感到厌烦的 overandoveragain多次；反复地17.falldon倒塌 ill+动词原形将要做…… feer/ore+可数名词复数更少/更多…… 0.less/ore+不可数名词更少/更多…… 1.havetodosth.不得不做某事 2.agreeithsb.同意某人的意见 3.such+名词如此…… playapartindoingsth.参与做某事 Thereillbe+主语+其他将会有…… Thereis/are+sb./sth.+doingsth.有……正在做某事

aesb.dosth.helpsb.ithsth.帮助某人做某事 trytodosth.尽力做某事 It’s+ad+forsb.todosth.对某人来说，做某事……的。 语法讲解 Boosillonlybeonputers,notonpaper.书将只在电脑里，而不是在纸上。 Thereillbeorepollution.将会有更多的污染。 ).Thereillbe+n=Thereis/aregoingtobe+n将会有…ThereisgoingtobeafootballatchthisFriday. ).pollution:污染；公害pollute：污染；弄脏polluted：受污染的 Everyoneshouldplayapartinsavingtheearth.每个人应该参与挽救地球。 Todaytherearealreadyrobotsoringinfactories.现在已经有机器人在工厂里工作了。 Therebesb.doingsth.有某人正在做…Thereisabirdsinginginthetree. Theyagreeitaytaehundredsofyears.他们同意这可能花费几百年的时间。 Ittaes+时间+todosth.某人花费时间区做某事。 Ittooehalfanhourtofinishyhoeor. agreetodosth.eagreetoeetuplaterandtalthingsover

XX九年级英语上册Unit13教案(新版人教版)

XX九年级英语上册Unit13教案（新版人教 版） 学科English年级9班级 课型fresh课时1/6媒体ataperecorder,cAI 课题Unit13e’retryingtosavetheearth!SectionA1a~1c 话题Protectingtheenvironent 功能Talaboutpollutionandenvironentalprotection 教 学 目 标 知识 技能1.Targetlanguage: e’retryingtosavetheearth. Peoplearethroinglitterintotheriver. Theriverusedtobesoclean. Everyoneshouldhelptocleanuptheriver. Graar: Presentprogressive,usedto,odalverbs ordsandexpressions;

curriculuords:litter,botto,fisheran Usefulexpressions:befullof,put…into,thro…into,cleanup,playapartin,closedon 过程 方法Accordingtodesigningsoetass,totrainstudents’listeningabilityandtotrainstudents’unicativepetence． 情感 态度Everyoneshouldeepourriversclean. 学习策略Listeningforeyords,transforinginforation. 重点TargetLanguage 难点1．Hototrainstudents’listeningability． ．Hototrainstudents’unicativepetence． 教学内容及问题情境学生活动设计意图 a.TointroduceSstotheunitgoal,talaboutpollutionanden vironentalprotection. Picture: Thefourpicturesshodifferentforsofenvironentalpollut

人教版八年级英语上册单元知识点归纳

八年级上册英语单元知识点归纳 Unit1Wheredidyougoonvacation? 短语归纳 1.goonvacation去度假 2.stayathome待在家里 3.gotothemountains去爬山 4.gotothebeach去海滩 5.visitmuseums参观博物馆 6.gotosummercamp去参加夏令营 7.quiteafew相当多 8.studyfortests为测验而学习 9.goout出去10.mostofthetime大部分时间11.haveagoodtimedoing=havefundoing=enjoyoneself玩得高兴 去购 物because+句子 查明22.goon上上下下 用法： 3.nothing 4.seem+ 13.dislikedoingsth.不喜欢做某事 14.keepdoingsth.继续做某事keepondoingsth不停做某事 15.Whynotdo.sth.=whydon’tyoudosth为什么不做……呢？ 16.so+adj.+that+从句如此……以至于…… 17.tellsb.(not)todosth.告诉某人（不要）做某事 18.enough+名词，形容词+enough 19.notreally.真的没有。 20.seemtodosth似乎好像做某事 21.Byefornow!到这该说再见了。

22.Howdoyoulike…=Whatdoyouthinkof…=Whatdoyouthinkabout… Unit2Howoftendoyouexercise? 短语 1.helpwithhousework帮助做家务 2.onweekends=ontheweekend在周末 3.howoften多久 一次howsoon多久（回答in10years）4.hardlyever几乎从不 5.onceaweek每周一次twiceamonth每月两次everyday每天three/four…timesaweek每周三四…次 6.befree=beavailable有空 7.gotothemovies去看电影 https://www.wendangku.net/doc/9c12809335.html,etheInternet用互联网ontheInternet在网上surftheInternet 上网 9.swingdance摇摆舞10.playtennis打网球play+ 球类/棋类/中国乐器playthe+西洋乐器11.stayuplate熬夜；睡得很晚12.atleast至少atmost最多 擅长 根本firstofall 21.suchas看牙医 用法 少…? 5.主语 7.It’ 10.What Unit3I 短语 和……相同；与……一致bedifferentfrom与……不同5.careabout关心；介意takecareof=lookafter照顾6.belikeamirror像一面镜子7.themostimportant最重要的8.aslongas只要；既然 9.bringout使显现；使表现出10.getbettergrades取得更好的成绩11.reachfor伸手取12.infact 事实上；实际上13.makefriends交朋友14.one…,theother…一个…，另一个15.touchone’sheart 感动某人16.betalentedinmusic有音乐天赋 17.shareeverything分享一切18.talkabout谈论19.primaryschoolstudents小学生middleschoolstudent中学生https://www.wendangku.net/doc/9c12809335.html,rmation是不可数名词，一则消息：apieceofinformation 21.theEnglishstudycenter英语学习中心 用法

XX八年级英语上册第七单元导学案(人教版)

XX八年级英语上册第七单元导学案（人 教版） 本资料为woRD文档，请点击下载地址下载全文下载地址Unit7 willpeoplehaverobots? Period8 SectionBSelfcheck 教师复备栏或 学生笔记栏 【学习目标】 .熟练掌握本单元词汇： 2.熟练掌握本单元句型： 5） In20years，IthinkI’llbeanewspaperreporter. ontheweekend,I’lllooklesssmartbutIwillbemorecomfortable. whatwillyour…belike? 【学习重点 难点】 本单元的单词、短语、语法 【学法指导】 及时练习与巩固

【教学过程】 一、 导入（启发探究 3分钟） 对话复习： Nick:whatareyoureading,jill? jill:It’sbookaboutfuture. Nick:Soundscool.Sowhatwillthefuturebelike? jill:well,citieswillbemorecrowdedandpolluted.Therew illbefewertreesandtheenvironmentwillbeingreatdanger. Nick:Thatsoundsbad!willwehavetomovetootherplanets. jill:maybe.ButIwanttoliveontheearth. Nick:me,too.Thenwhatcanwedo? jill:wecanuselesswaterandplantsmoretrees.Everyonesh ouldplayapartinsavingtheearth. 二、自学（自主探究 6分钟） 用法：

八年级英语上册Unit7 Will people have robots-知识点归纳

八年级英语上册Unit7 Will people have robots?知识点归纳 课 件www.5y https://www.wendangku.net/doc/9c12809335.html,八年级英语上册Unit7willpeoplehaverobots?知识点归纳 八年级上Unit7willpeoplehaverobots? oncomputer在电脑上 onpaper在纸上 livetodo200yearsold活动200岁 freetime空闲时间 indanger处于危险之中 ontheearth在地球上 playapartinsth.参与某事 spacestation太空站 lookfor寻找 computerprogrammer电脑编程员 inthefuture在未来 hundredsof许多；成百上千 thesame…as…与……一样 overandoveragain多次；反复地 getbored感到厌烦的

wakeup醒来 falldown倒塌will+动词原形 将要做…… fewer/more+可数名词复数 更少/更多……less/more+不可数名词 更少/更多…… havetodosth.不得不做某事 agreewithsb.同意某人的意见 such+名词（词组） 如此…… playapartindoingsth.参与做某事 Therewillbe+主语+其他 将会有…… Thereis/are+sb./sth.+doingsth.有……正在做某事makesb.dosth.使某人做某事 helpsb.withsth.帮助某人做某事 trytodosth.尽力做某事 It’s+adj.+forsb.todosth. 对某人来说，做某事……的。 1.ThestudentsinourschoollearnEnglish computers. A.at

八年级英语上册Unit7Willpeoplehaverobots-知识点归纳

八年级英语上册Unit7Willpeoplehaverobots?知识点归纳puters在电脑上， 2、onpaper在纸上， 3、livetobe200yearsold活到200岁， 4、freetime空闲时间， 5、indanger在危险中， 6、ontheearth在世界上 7、playapartinsth在某方面出力/做贡献， 8、spacestation太空站， 8、lookfor寻找， 9、computerprogrammer电脑程序师， 10、inthefuture在将来， 11、hundredsof成百上千的， 12、thesame…as与…一样， 13、overandoveragain反复， 14、getbored无聊， 15、wakeup醒来/唤醒， 16、looklike看起来像， 17、falldown倒下/落下 二、重要句子（语法） 1、will+动词原形将要做

2、fewer/more+可数名词复数更少/更多… 3、less/more+不可数名词更少/更多 4、trytodosth.尽力做某事 5、havetodosth不得不做某事 6、agreewithsb.同意某人的意见 7、such+名词（词组）如此 8、playapartindoingsth参与做某事 9、makesbdosth让某人做某事 10、helpsbwithsth帮助某人做某事 11、Therewillbe+主语+其他将会有…. 12、Thereis/are+sb.+doingsth有…正在做… 13、Itis+形容词+forsb+todosth做某事对某人来说…语法： Whatwillthefuturebelike? Citieswillbemorepolluted.Andtherewillbefewertrees. Willpeopleusemoneyin100years? No,theywon’t.Everythingwillbefree. Willtherebeworldpeace? Yes,Ihopeso. Kidswillstuffyathomeoncomputers. Theywon’tgotoschool.

八上英语复习重点总结

八上英语复习重点总结 Unit1Wheredidyougoonvacation? 单元重点： 1）一般过去时 2）不定代词的用法： a)不定代词作主语，谓语动词用单三 b)形容词修饰不定代词要放在其后 c)something一般用在肯定句中，anything一般用在否定句和疑问句中重点词组： goonvacation去度假stayathome待在家里gotothemountains去爬山gotothebeach去海滩visitmuseums参观博物馆gotosummercamp去参观夏令营quiteafew相当多 studyfor为……而学习 goout出去 mostofthetime大部分时间tastegood尝起来很好吃ofcourse当然 feellike给……的感觉/想要inthepast在过去walkaround四处走走becauseof因为onebowlof…一碗……thenextday第二天 drinktea喝茶 findout找出；查明 goon继续 takephotos照相somethingimportant重要的事upanddown上上下下 comeup出来

用法集锦： 1）buysth.forsb./buysb.sth.为某人买某物 2）taste+adj.尝起来……look+adj.看起来…… 3）nothing…but+动词原形除了……之外什么都没有 4）seem+（tobe）+adj.看起来…… 你还能想到seem的什么用法？ 5）arrivein+大地点/arriveat+小地点到达某地 6）decidetodosth.决定去做某事 7）try的用法： 作动词：trydoingsth.尝试做某事/trytodosth.尽力去做某事tryone’sbesttodosth. 作名词：haveatry 8)forgetdoingsth.忘记做过某事/forgettodosth.忘记做某事 9)enjoydoingsth.喜欢做某事 10)dislikedoingsth.不喜欢做某事 11)Whynotdo.sth.?为什么不做……呢？ 12)So+adj.+that+从句如此……以至于…… 13)tellsb.(not)todosth.告诉某人（不要）做某事 14)总结keep的用法 Unit2Howoftendoyouexercise? 单元重点： 1）关于how的词组 2）频率副词的位置：放在be动词、助动词、情态动词之后，实意动词之前 3)Howoften与Howman ytimes的区别？ 重点词组： helpwithhousework帮助做家务onweekends/ontheweekend在周末