Disruption of Arabidopsis thaliana MYB26results in male sterility due to non-dehiscent anthers
Sabine Steiner-Lange1,?,Ulrike S.Unte1,Luca Eckstein1,Caiyun Yang2,Zoe A.Wilson2,Elmon Schmelzer3, Koen Dekker1and Heinz Saedler4
1Zentrum zur Identi?zierung von Genfunktionen durch Insertionsmutagenese in Arabidopsis thaliana(ZIGIA),
Max-Planck-Institut fu¨r Zu¨chtungsforschung,Carl-von-Linne′-Weg10,50829Ko¨ln,Germany,
2School of Biosciences,University of Nottingham,Sutton Bonington Campus,Loughborough,Leics LE125RD,UK, 3CeMic,Max-Planck-Institut fu¨r Zu¨chtungsforschung,Carl-von-Linne′-Weg10,50829Ko¨ln,Germany,and
4Abteilung Molekulare P?anzengenetik,Max-Planck-Institut fu¨r Zu¨chtungsforschung,Carl-von-Linne′-Weg10,50829 Ko¨ln,Germany
Received12September2002;revised25January2003;accepted10February2003.
?For correspondence(faxt492215062361;e-mail steiner@mpiz-koeln.mpg.de).
Summary
A male sterile mutant with a defect in anther dehiscence was identi?ed in an Arabidopsis thaliana popula-
tion mutagenized with the Zea mays transposon En-1/Spm.Mutants produce viable pollen that can fertilize when released mechanically from the anthers.Mutant stamens are of normal size and shape,but lack cell wall forti?cations in the endothecial cell layer of the anther,which are required for the dehiscence process.
The mutant phenotype was shown to be caused by a transposon insertion in AtMYB26,disrupting the putative DNA-binding domain of this R2R3-type MYB transcription factor.RT-PCR revealed that expression of AtMYB26is restricted to in?orescences.Sterility was shown to be stable under several environmental conditions.The high stability of the sterile phenotype,together with the fact that pollen is functional, makes AtMYB26and its orthologs a valuable tool for manipulating male fertility in higher plants.
Keywords:MYB transcription factor,male sterility,dehiscence,ms35,Arabidopsis thaliana,anther devel-opment.
Introduction
Many different developmental steps are required to achieve male fertility in higher plants.The ontogeny of the male organs,?laments and anthers has to be speci?ed,and the male gametophyte has to develop.Finally,pollen has to be released.Mutants defective in either of these pro-cesses provide valuable tools for studying male sterility in higher plants.In Arabidopsis thaliana,mutants have been described that are defective in meiosis or post-meiotic pollen development and therefore do not develop func-tional pollen grains(Chaudhury et al.,1994;Dawson et al., 1993;He and Mascarenhas,1998;He et al.,1996;Preuss et al.,1993;Regan and Moffatt,1990;Sanders et al.,1999; Taylor et al.,1998;Wilson et al.,2001;Yang et al.,1999). Several male sterile mutants affected in the mechanism or timing of the release of pollen from the anthers have been reported(Dawson et al.,1993,1999;Ishiguro et al.,2001; von Malek et al.,2002;Park et al.,1996,2002;Sanders et al., 1999,2000;Stintzi and Browse,2000).As pollen is at least partially functional in some of these mutants,sterility of such plants can be overcome by mechanically opening the pollen sacs,making these mutants especially attractive for an application in breeding and hybrid seed programmes. Dehiscence of the anther is a multistage process. Between the locules of the anther,the septum and stomium cells have to differentiate and then undergo a degeneration programme allowing breakage of the anther wall.Further-more,cells of the endothecium have to enlarge and their walls have to be strengthened to allow proper dehiscence (Dawson et al.,1999;Sanders et al.,1999).In self-pollinat-ing species like A.thaliana,these processes have to occur at the correct time,when the female organ pistil is receptive and in the right place,so that the?lament of the stamen is at the proper height for pollen delivery onto the stigma.
To date,all genes affecting anther dehiscence that have been characterized are involved in the jasmonic acid(JA) pathway.Mutants of these genes display defects in?lament
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elongation and timing of anther dehiscence,as well as reduced pollen viability.The mutant coi1(Xie et al., 1998)is JA insensitive,while the triple mutant fad3/fad7/ fad8(Feys et al.,1994)and the mutants opr3/dde1(Sanders et al.,2000;Stintzi and Browse,2000),dad1(Ishiguro et al., 2001)and aos/dde2-2(von Malek et al.,2002;Park et al., 2002)are defective in JA synthesis.
Three other mutants with defects in anther dehiscence have been described in A.thaliana,but the cloning of the corresponding genes has not been reported previously.In non-dehiscense1(Sanders et al.,1999),?laments elongate properly and anthers enter the dehiscence programme, including expansion of the endothecium and degeneration of the septum https://www.doczj.com/doc/5416942623.html,ter,however,the endothecium and connective tissue degenerate completely,resulting in anthers?lled with functional pollen surrounded by an anther wall consisting exclusively of the epidermal cell layer.In ms35(Dawson et al.,1999),formerly referred to as msH(Dawson et al.,1993),?laments also elongate prop-erly,but functional pollen grains are not released from the anthers,although the stomium is cleaved.This defect is associated with a lack of secondary thickening in the endothecium cells.Similarly,in the mutant por1(Park et al., 1996),anthers fail to release pollen,although the stomium is cleaved.However,in contrast to ms35,stamen?laments show a delayed growth rate and pollen grains are not functional.
Here,we present the isolation of an anther-dehiscence mutant from a population of transposon-tagged A.thaliana insertion lines and the identi?cation of the responsible mutant gene.The mutant phenotype is caused by a trans-poson insertion in the gene AtMYB26encoding a putative R2R3-type MYB-transcription factor.As in ms35,the?la-ments elongate properly but the anthers,although cleaved at the stomium,fail to release the functional pollen grains. The dehiscence defect is associated with a defect in for-ti?cation of the endothecium cell walls.Performing crosses with ms35has shown that myb26is allelic to ms35.
Results
Identification of a mutant with non-dehiscent anthers
Screening an A.thaliana population(Wisman et al., 1998a,b)mutagenized with the maize transposon En-1/ Spm(Schwarz-Sommer et al.,1985)for morphological changes,we have identi?ed a sterile mutant developing only small and empty siliques(Figure1a).Mutant plants produce normal siliques containing seeds when fertilized with wild-type pollen,indicating that female fertility is not affected.Flowers of mutant plants are of normal size and shape,and the?laments of the stamen elongate as in wild type(Figure1b).Anthers contain pollen and develop to normal size but fail to dehisce(Figure1b,c).Scanning electron micrographs revealed that mutant and wild-type pollen grains are indistinguishable(Figure1d).Further-more,if the mutant anthers were opened mechanically and the released pollen grains were used for self-pollina-tion of mutant plants,siliques containing seeds were obtained.Pollination with mutant plants was more dif?cult than with the wild type because pollen grains tended to stick together,but all seeds obtained gave rise to sterile plants,showing that the self-pollination was successful.In addition to the described male sterility phenotype,mutants show delayed apical senescence,a higher number of in?or-escences and?owers and the formation of terminal aber-rant?owers co-segregating with sterility.Such defects have been observed in various other male sterile mutants before and are thought to be a result of the lack of fertiliza-tion rather than the pleiotropic effects of a particular mutant (Chaudhury et al.,1994).
Anther development
The anthers of sterile plants appear as in the wild type and develop to normal size and shape,but directly prior to dehiscence mutant anthers appear less swollen (Figure2a,b).Cross-sections of mutant in?orescences revealed that anther development is indistinguishable from that in the wild type during phase one and early stages of phase two(as de?ned by Sanders et al.,1999;Figure2c,d). Later in phase two of anther development,which includes the dehiscence programme,differences can be seen.While maturation of pollen grains continues and the disappear-ance of the tapetum and middle layer appears normal, expansion of the endothecium layer,which occurs in wild-type anthers,cannot be observed in the mutant (Figure2i,j).At later stages,degradation of the septum and breakage of the stomium can be seen in the mutant as in wild type(Figure2e,f),but the subsequent shrinkage of the anther walls resulting in the release of pollen grains does not take place.
UV-illuminated cleared wild-type anthers usually reveal a net-like structure of auto?uorescent material,indicating ligni?cation of the anther wall(Figure2h),which could be seen?rst in bud no.4(buds were numbered acropetally starting with the oldest bud).These structures could not be found in the mutant(Figure2g).Cross-sections of?ower buds stained with calco?uor white revealed that cellulose deposition on radial cell walls of endothecium cells,as seen in the wild type(Figure2l),is also missing in sterile plants (Figure2k).
Cloning of AtMYB26
Segregation analysis of sister plants of the original isolate showed a3:1ratio of fertile to sterile plants,indicating that the phenotype is caused by a single recessive mutation.As
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Figure 1.Description of the phenotype.
Comparison of mutant (myb26,left)and wild-type (wt,right)plants.
(a)Mutants are sterile and thus develop only small siliques without seeds,while wild-type plants of the same age develop swollen siliques containing seeds.
(b)Flowers of mutant plants are of normal size;the ?laments of stamen elongate properly,but anthers fail to dehisce.
(c)SEM picture of anthers of open ?owers (200-fold magni ?cation).While anthers of mutant plants are still closed,anthers of the wild type are fully dehisced and have released the pollen grains.
(d)SEM of pollen grains (2000-fold magni ?ca-tion).Pollen of the mutant are indistinguishable from wild-type
pollen.
Figure 2.Anther development.
Anthers from the largest ?ower bud of myb26-2(a)appear less swollen than wild-type anthers of the same stage (b).Toluidine-blue-stained cross-sections of anthers of myb26-2(c)and the wild type (d),corresponding to stage 10according to Sanders et al .(2000),do not reveal differences between myb26-2and wild type.At stage 13,breakage of the stomium can be seen in myb26-2(e)and in the wild type (f)but the endothecium cells appear less swollen in myb26-2.UV illumination of cleared anthers of myb26-2(g)and the wild type (h)taken from open ?owers revealed a net-like structure of ligni ?ed material in wild type,which is lacking in myb26-2.The anther of myb26-2is still closed and contains pollen grains,which are visible as a result of the auto ?uorescence of sporopollenin,while the wild-type anther is dehisced and has released the pollen grains.Ligni ?ed vascular tissue is visible in myb26-2and wild type.Phloroglucinol-stained cross-sections of anthers corresponding to stage 12reveal that the endothecium cells of myb26-2(i)are not swollen while the endothecium cells of wild type (j)appear swollen and show rigid cell walls.Staining of cellulosic material with calco ?uor white using cross-sections of anthers corresponding to stage 11reveal bar-like cellulosic thickenings in the walls of wild type (l),which are not visible in myb26-2(k).
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Disruption of Arabidopsis thaliana MYB26521
the mutant was isolated from a transposon-mutagenized population,it was expected to be tagged by an En-1inser-tion.Southern analyses using the 50-end of the En-1trans-poson as probe were therefore performed with 14individuals (?ve mutants,?ve homozygous wild types and four heterozygous plants according to segregation analysis of progeny),revealing that the individuals carried between six and 10En-1insertions,one of which co-segregated with the mutation causing sterility (Figure 3a).The ?anking regions of eight different En-1insertions were isolated from one individual sterile https://www.doczj.com/doc/5416942623.html,ing these regions as probes in gel blot experiments,one of them could be shown to correspond to the transposon insertion co-segregating with the mutant phenotype (Figure 3b).The En-1used to generate our population is an autono-mous element encoding its own transposase.Therefore,the causal relation of the transposon insertion in the iso-lated locus and the sterile phenotype could be proven by investigating revertants,which could be found easily by screening sterile individuals for the rare appearance of fully developed siliques containing fertile seeds.These seeds gave rise to progeny segregating for sterility.Southern blot analyses of these plants using the isolated ?anking DNA as a probe revealed that restoration of fertility was correlated in all plants with a loss of the insertion in at least one allele of the isolated locus (data not shown),indicating that this
particular insertion is responsible for the anther dehiscence defect.
The ?anking region of the insertion causing the male sterile phenotype was sequenced,revealing that the inser-tion is located within the coding region of At3g13890,also called AtMYB26(Romero et al .,1998;Stracke et al .,2001),encoding a putative MYB-transcription factor.The En-1insertion is located behind the 14th codon of AtMYB26,thus disrupting the putative DNA-binding domain (Figure 4a).Our mutant will therefore be referred to as myb26-2.
Isolation of stable mutant alleles
As a result of the fact that the transposon inserted within AtMYB26is still active,it was necessary to isolate a stable mutant.As excision of the transposon from an integration site is often not precise leaving a footprint behind (Cardon et al .,1993),we screened for such events by crossing a sterile plant with Col-0.The resulting F 1generation was subsequently screened via PCR for the loss of the En-1insertion within AtMYB26.Of 80individuals tested,?ve were shown to have lost the En-1insertion.The AtMYB2650-region of these individuals was ampli ?ed by PCR and sequenced revealing overlapping sequences in four cases,which indicate that excision of the En-1created a deletion or insertion.In one case,the wild-type sequence had been restored.
Progeny of these plants were grown and sterile indivi-duals found among the progeny of three individuals with a putative imprecise excision,while progeny of the individual that had restored the wild-type sequence were fertile,con-?rming the causal relationship between a mutation in AtMYB26and the sterile phenotype.The AtMYB2650-regions of these sterile plants were ampli ?ed by PCR and sequenced,revealing that in one case a 9bp deletion had occurred which led to the loss of three amino acids in the MYB domain.In the other individuals,insertions of,respec-tively,four and two nucleotides could be found creating a stop codon in the open reading frame behind the 14th codon (Figure 4b).Sequence analysis
AtMYB26encodes a protein of 358amino acids,which in its N-terminal part shows homology to a MYB DNA-binding domain.This domain generally comprises up to three repeats of about 53amino acids,each forming a helix-turn-helix structure.AtMYB26,like most plant MYB factors,contains only two of these repeats covering amino acids 12–115and thus belongs to the R2R3-type of MYB tran-scription factors.The R2R3-type MYB genes have been classi ?ed into 22groups according to conserved amino acid motifs present C-terminal to the MYB domain.Classi-?cation into similar groups often also re ?ects a functional conservation.AtMYB26groups with AtMYB103
and
Figure 3.Identi ?cation of the mutant locus.
Individuals from a line segregating for sterility representing ?ve homozy-gous individuals carrying the wild-type allele (wt/wt),four heterozygous plants (wt/myb26)and ?ve homozygous mutants (myb26/myb26)and a Col-0wild-type control (C)were investigated by Southern analysis using genomic DNA cleaved with Eco RI.
(a)Segregation analysis:the blot was hybridized with an En-1-left end-speci ?c probe.One band (indicated by an arrow)is only present in the individuals carrying at least one copy of the mutant allele showing co-segregation of this En-1insertion with the mutant locus.
(b)Co-segregation of an En-1insertion site with the mutant phenotype.Hybridization was performed with an isolated En-1?anking region.All individuals not carrying the mutant allele show a single band also present in Col-0and therefore represent the wild-type allele of the isolated locus,while in the mutants this fragment is barely visible.Instead,a smaller fragment,which also hybridizes with the En-1-probe,gives a signal.In the heterozygous plants,both signals are present.The faint signal corre-sponding to the wild-type allele,which is visible in the mutants,represents excision events of the active transposon from the insertion site.
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AtMYB67(Stracke et al.,2001),displaying highest overall identity(49.1%)with AtMYB67at the amino acid level. However,AtMYB26and AtMYB67exhibit only79.8%iden-tity within the MYB domain,whilst87.3%identity within this domain can be found with a putative transcription factor from rice(gene:P0456F08.4,protein id: BAB39404.1).The overall identity with this protein,how-ever,is only43.9%.The function of these putative MYB transcription factors is not known.
Expression studies
To determine the expression pattern of AtMYB26,Northern blot analysis was https://www.doczj.com/doc/5416942623.html,ing as probe the AtMYB26 cDNA lacking the region encoding the conserved MYB domain,no transcript was detected,although RNAs from different organs were used,indicating that AtMYB26is weakly expressed.Therefore,we performed RT-PCR to examine the expression of AtMYB26.Total RNA was extracted from stem,in?orescences,cauline leaves,young and old rosette leaves,seedlings and roots of A.thaliana Col-0wild-type plants and reverse transcribed into cDNA. Both conventional RT-PCR and real-time RT-PCR ampli?ed the AtMYB26transcript only in in?orescences(Figure5a,b). RT-PCR performed on?ower buds of different stages revealed that expression is not detected in young buds (buds prior to pollen mitotic division)and?rst appears in buds3–5of the in?orescence(when buds are numbered acropetally starting with the oldest bud)containing anthers in the stages of pollen mitotic divisions and tapetal degen-eration.Transcript can still be found at a lower level in older buds and even open?owers(Figure5c).
In order to determine the temporal and spatial expression patterns within in?orescences in more detail,in situ hybri-dizations were performed on in?orescences,which,how-ever,did not give a signal con?rming the weak expression level of AtMYB26.
Stability test
The stability of the sterile phenotype was tested using plants carrying the stable allele myb26-4(see Figure4b) by growing plants at228C and subjecting them for4days to low(108C)and high(308C)temperatures.Plants were visually screened for the appearance of siliques containing seeds.Only10?lled siliques were found on a total of45 plants with each plant carrying more than2000empty siliques.At228C,only one silique containing seeds was found(representing0.003%of siliques).A slightly increased proportion of siliques containing seeds was found after subjecting the plants to308C(three siliques, 0.01%of scored siliques)and108C(six siliques,0.02%of scored siliques).Although this might indicate an effect of temperature on the stability of the phenotype,it can still be considered high under different conditions,making AtMYB26and its homologues valuable tools for manipulat-ing male fertility in higher plants.
Jasmonate insensitivity of myb26
Several mutants affected in anther dehiscence are affected in synthesis or sensing of jasmonate(Ishiguro et al.,2001; von Malek et al.,2002;Park et al.,2002;Sanders et al.,2000; Stintzi and Browse,2000).Although the phenotypes described for these mutants,which show defects in?la-ment elongation and timing of dehiscence as well as defec-tive pollen,differ in many respects from the phenotype of myb26,we tested if the stable mutant myb26-5might be rescued by application of jasmonic acid and methyl jasmo-nate,respectively.As a control,we used the mutant dde2-2
Figure4.Genomic organization of the AtMYB26-locus and its mutant alleles.
(a)AtMYB26consists of three exons(I,II,III).In myb26-2,the open reading frame is disrupted by an En-1insertion within the?rst exon. (b)Sequences of the AtMYB2650-regions of the En insertion allele myb26-2and stable myb26-mutants created by imprecise excision of the En-1element,which were named myb26-3, myb26-4and myb26-5
.
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Disruption of Arabidopsis thaliana MYB26523
described by von Malek et al .(2002).While dde2-2mutants regained fertility upon treatment with both jasmonic acid and methyl jasmonate,the sterile phenotype of myb26-5could not be overcome,showing that synthesis of jasmonic acid is not affected in myb26.
MS35gene identification
The phenotype of myb26strongly resembles that described for the male sterile mutant ms35(Dawson et al .,1999).The region around the ms35mutations has been mapped using recombinants between hy2-ms35(breakpoints at approxi-mately 0.18cm intervals)and ms35-gl1(every 1.0cm;Dawson et al .,1999;Sozen,2000).The MS35gene was shown to co-segregate with the ATHCHIB and nga162markers,and to map 0.18cm away from act11,towards hy2(Sozen,2000).Using the recombinant inbred map (Lister and Dean,1993),ATHCHIB and nga162markers map to 19.1and 20.56cm,respectively.The AtMYB26gene also maps to 20.56cm and lies within 31.5kb of nga162,suggesting that MS35may be allelic to MYB26.Crosses were therefore performed between myb26-3and ms35giving rise to sterile progeny.This shows that myb26-3is allelic to ms35,which should therefore be referred to as myb26-1.
In order to determine the mutation present in ms35,we have PCR ampli ?ed and direct sequenced the AtMYB26region,from à3174upstream to 500bp beyond the tran-scriptional termination sequence,in L er and the ms35mutant.We found the coding region and downstream sequence to be identical;however,aberrations were seen in the upstream region.PCR ampli ?cations were possible throughout this region for the L er line.However,ampli ?ca-tion,including long-PCR,across a 235bp region that lies between à1288and à1523bp upstream of the translation start was not possible in the ms35mutant.The sequence up to and beyond this point is identical to that seen in L er .We therefore believe that a major chromosomal rearrangement has occurred in this region,which prevents normal MYB26gene expression,resulting in a failure of dehiscence in the ms35mutant.This is supported by RT-PCR data,which shows reduced levels of AtMYB26expression in ms35buds as compared to L er wild type (data not shown).A chromo-somal rearrangement could also explain the mapping data for ms35(Sozen,2000)that show suppression of recombi-nation within the ATHCHIB-nga162region,as no recombi-nants occurred in this region despite the fact that it spans 670kb/1.46cm and breakpoints were expected every 0.18cm.Discussion
We have isolated a male sterile mutant,myb26-2,from a population of transposon-tagged A.thaliana lines.Sterility is caused by a defect in anther dehiscence,while the pollen itself is shown to be functional.The failure of dehiscence is associated with a defect in the development of the anther walls.During the late stages of anther development,the wild-type endothecium cells appear swollen and show deposition of ligni ?ed cellulosic secondary wall thicken-ings.In myb26-2,these cell wall forti ?cations are
missing
Figure 5.Relative quanti ?cation of the transcript in different organs of Col-0wild-type plants by PCR.
(a)RT-PCR.Total RNA was isolated from different plant organs and used for a one-step RT-PCR.Ten microlitres of each of the 50m l PCR assays was loaded on a 1%agarose gel.
(b)Real-time RT-PCR.Real-time PCR was performed with AtMYB26-speci ?c probe and primers in 40cycles.The relative amounts of transcript,com-pared to the transcript level in in ?orescences,were calculated using the values given in the table.The ct-values of the different plant organs repre-sent the medium of a triplicate (SD of the three values shown).To obtain a relative view of the AtMYB26expression in the various organs,the results were normalized to the ct-value in in ?orescences:D ?ct-in ?orescence àct-x (x:ct-value in another organ).As the amount of PCR product should duplicate after each cycle,the relative amount of AtMYB26transcript was then calculated as 2D and the result converted into percentages.
(c)RT-PCR using ?ower buds of different stages (buds were numbered acropetally starting with the oldest bud):young buds correspond to buds 6-centre of in ?orescence (buds prior to pollen mitotic division);middle buds represent buds 3–5of in ?orescence (pollen mitotic divisions/tapetal degen-eration);old buds represent buds 1and 2(unopened petals visible,tricellular pollen).As control,RT-PCR was performed on the same samples using actin-speci ?c primers.
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and the endothecium cells do not expand.The endothe-cium has been proposed to contribute to two steps of anther dehiscence.First,an inwardly directed force from the anther wall,which is driven by swelling of the epidermal and endothecial cells,causes the rupture of the stomium. Subsequently,an outward bending force,leading to full opening of the stomium permitting pollen release,is caused by differential shrinkage of thickened and unthick-ened parts of the endothecium(Keijzer,1987).While the breakage of the stomium takes place in myb26-2,the retrac-tion of the anther walls does not occur,suggesting that, especially,this latter process requires the endothecium cell wall forti?cations as proposed by Dawson et al.(1999). The phenotype of the myb26mutants strongly resembles that described for the male sterile mutant ms35(Dawson et al.,1999).Furthermore,AtMYB26maps to the top arm of chromosome3at about20.5cm,while ms35maps and co-localizes with markers mapping to this region(Sozen, 2000).Crosses between myb26-3and ms35resulted in sterile progeny,demonstrating that the two mutants are allelic.PCR ampli?cation and sequence analysis indicate the presence of a major rearrangement in the AtMYB2650-region of the ms35mutant.
The sterile phenotype of our mutant was found to be because of a mutation in AtMYB26encoding a putative MYB-tanscription factor.MYB-transcription factors are found in all major eukaryotic groups and are characterized by one to three copies of imperfect helix-turn-helix motives designated R1,R2and R3,which form a DNA-binding domain.In plants,MYB genes form a large family with, e.g.136members predicted in the A.thaliana genome by Stracke et al.(2001).One hundred and twenty-?ve of them, including AtMYB26,belong to the R2R3class with two repeats,which,according to current knowledge,is plant speci?c(Riechmann et al.,2000).The importance of the MYB domain for the function of AtMYB26was shown by the fact that a three-amino acid deletion within the?rst repeat, as found in myb26-3,is suf?cient to confer the mutant phenotype.
The functions of several R2R3-type MYB transcription factors from various plant species have been characterized, and it seems that they mainly regulate plant-speci?c pro-cesses(Stracke et al.,2001).They have been shown to act as either transcriptional activators or repressors controlling development and determination of cell fate and identity and regulating different biosynthetic pathways like tryptophan synthesis and the phenyl propanoid pathway(for review, see Jin and Martin,1999;Riechmann et al.,2000;Stracke et al.,2001).
AtMYB26is the?rst MYB transcription factor that has been shown to be required for male fertility.However, pollen itself is viable in the loss of function mutant.Sterility is caused by anther non-dehiscence associated with a lack of cell wall forti?cations consisting of lignin and cellulose material in the endothecium of the pollen sac.Therefore, the main defect seems to be a failure to properly establish the properties of this cellular layer.Other MYB factors have already been shown to control development and determi-nation of cell fate.MIXTA from Antirrhinum majus (Glover et al.,1998;Noda et al.,1994)and GL1and WER from A.thaliana(Lee and Schiefelbein,1999;Marks and Feldmann,1989;Wada et al.,1997)have been shown to confer cell-type speci?city.They are required for the deter-mination of the fate of epidermal cells being involved in the decision to form conical cells in petals,trichomes in leaf and stem and root hairs in the roots.AS1from A.thaliana (Byrne et al.,2000)and its orthologs from other species, which are required for proper meristem function,are other examples for MYB genes involved in cell differentiation. As,in myb26,ligni?cation has been shown to be missing within the endothecium,it can also be assumed that AtMYB26is involved in regulating the phenyl propanoid pathway.Several MYB transcription factors have already been shown to regulate this pathway,e.g.in A.thaliana those encoded by AtMYB4(Jin et al.,2000),PAP1and PAP2 (Borevitz et al.,2000),TT2(Nesi et al.,2001)as well as AtMYB21and AtMYB86(Shin et al.,2002).Further data come from other plant species where MYB factors have been shown to be involved in regulating the synthesis of anthocyanin,?avonol and phlobaphene(for review,see Jin and Martin,1999).An effect of MYB transcription factors on ligni?cation has been shown in the PAP1and PAP2over-expressing lines,which display an enhanced accumulation of lignin(Borevitz et al.,2000),and by expression of the A.majus genes AmMYB308and AmMYB330in tobacco, which results in the repression of phenolic acid metabolism and lignin biosynthesis(Tamagnone et al.,1998).AtMYB26 could therefore be another MYB transcription factor reg-ulating the phenyl propanoid pathway,thus affecting lig-ni?cation in the endothecium.This is supported by the fact that expression of AtMYB26was observed at those stages of anther development when secondary cell wall forti?ca-tions are formed in the endothecium.However,as other cell wall forti?cations like deposition of cellulose are also miss-ing in the endothecium of myb26,a function of AtMYB26in specifying the identity of this cellular layer seems more likely.
Sequence comparison of AtMYB26with other MYB fac-tors does not give any hint towards its molecular function. Sequence homology has,in several cases of MYB transcrip-tion factors,been shown to correspond to a similar func-tion,e.g.the A.thaliana genes WER and GL,which cluster together and are able to functionally complement each other displaying different biological functions only because of their different spatial expression patterns(Lee and Schie-felbein,2001).AtMYB26,however,does not cluster with any of the functionally characterized MYB factors(Stracke et al.,2001).
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Disruption of Arabidopsis thaliana MYB26525
Male sterile plants are useful tools for hybrid seed pro-duction.However,the use of many lines,mainly based on cytoplasmic male sterility,require the availability of appro-priate restorer lines.Furthermore,the successful applica-tion of such plants depends on whether the male-sterile female parent can be multiplied ef?ciently(Perez-Prat and van Lookeren Campagne,2002).Fertilization of myb26 mutants does not require the use of a special restorer line to obtain fertile progeny.Furthermore,as myb26pollen is functional,male sterile lines can be propagated by self-pollination after mechanically opening the anthers.There-fore,male sterility based on a mutation in AtMYB26and its orthologs in crop plants could offer some advantages over currently available male sterility systems.
Experimental procedures
Plant material
The myb26-2mutant was identi?ed in a population of A.thaliana, ecotype Columbia(Col-0),mutagenized with the En-1transposon of Zea mays(Baumann et al.,1998;Wisman et al.,1998a,b).
A.thaliana Col-0was used as wild type was used for transforma-tion and crossing.The ms35was obtained by X-ray seed mutagen-esis(Dawson et al.,1993),L er seed was obtained from the Nottingham Arabidopsis Stock Centre.Plants were grown in a cooled greenhouse in5cm clay pots?lled with soil.The medium temperature was208C.If necessary,additional light was provided during16h.
To test the sensitivity of myb26to jasmonic acid and methyl jasmonate,respectively,mutants were subjected to either of the following treatments for4days:An aqueous solution of2m M jasmonic acid(J-2500,Sigma,St Louis,USA)containing0.1% Tween20was applied as droplets of about50m l to?ower buds twice per day.Alternatively,mutants were sprayed twice per day with an aqueous solution of450m M methyl jasmonate(39,270–7, Aldrich,Steinheim,Germany)containing0.1%Tween20. Molecular biology techniques
Standard molecular biology techniques were performed as described by Sambrook et al.(1989).Isolation of genomic DNA was performed using the Plant DNA Isolation Kit(Roche Diagnos-tics,Mannheim,Germany).Ampli?cation of?anking regions was carried out according to Steiner-Lange et al.(2001).PCR products were cloned into pGem-Easy(Promega,Mannheim,Germany). Hybridization was performed with DNA fragments radioactively labelled with a32PdCTP using the Ready-To-Go DNA Labelling Beads(-dCTP;Amersham Biosciences,Freiburg,Germany).Pre-hybridization and hybridization were carried out in6?SSPE, 30g là1SDS,200mg là1PVP,200mg là1Ficoll and100mg là1 salmon sperm DNA at688C.Washes were performed in2?SSC, 0.1%SDS at688C.Probed?lters were stripped by two successive washes in0.2M NaOH,0.1%SDS at378C.
For preparation of an AtMYB26-speci?c probe lacking the MYB domain,a669bp fragment was isolated from plasmid pRD74 consisting of a PCR product representing nucleotides1272–1941 of the AtMYB26cDNA(Ralf Stracke,personal communication) cloned into the Sma I site of pCR-ScriptSKt(Stratagene,La Jolla, USA).
PCR and primers
Polymerase chain reaction was generally carried out in a total volume of50m l with0.4m M primers and200m M dNTPs using the Advantage2polymerase(Clontech,Paolo Alto,USA)according to the manufacturers’instructions.PCR conditions were:948C for 4min;35cycles of948C for30sec,648C for1min and738C for
1.5min followed by a?nal elongation step at738C for3min.
Primers MTL1(ACT CGC TGG GAA ATC TGG TAA CGC T)and MTL2(CAT GCT GCA ACA AGC AAA AGG TGA AG)were used for PCR ampli?cation of the AtMYB2650-region.MTL3(ACC GTG ATG ATG GTG GAC ATG AG)was used as the primer for sequencing the former En-1-integration sites of plants having lost the insertion and in combination with MTL2for performing RT-PCR.RTL1(ATG GGT CAT CAC TCA TGC TG)and RTR1060(GTC CAC AAG AGA TTG GCG ACG A)were also used for RT-PCR.Act2F(TGC TGA CCG TAT GAG CAA AG)and Act2R(CAG CAT CAT CAC AAG CAT CC)served as primers for the RT-PCR actin control.Con?rmation of an En-1 insertion within AtMYB26was performed with primers MTL1and En91R(TGC AGC AAA ACC CAC ACT TTT ACT TC).RS89(TTT GCT CAC AAA CCT TCC TTA TCA C)and RS88(ATG ACG TAC TGT CCA CAA GAG ATT G)were used for the preparation of an AtMYB26-speci?c probe lacking the MYB domain using an AtMYB26-cDNA clone as template.Preparation of a probe speci?c for the En-1 50-end was performed as described by Wisman et al.(1998a).
Real-time PCR and RT-PCR
Total RNA was isolated from different organs(i.e.in?orescence, young and old rosette leaf,cauline leaf,root and seedling)of
A.thaliana Col-0plants,using total RNA Isolation Reagent(biomol
GmbH,Hamburg,Germany).The isolated RNA was treated with DNase I(Roche Diagnostics GmbH,Mannheim,Germany)and precipitated in2.75M LiCl to eliminate genomic DNA contamina-tion,followed by a?nal puri?cation using RNeasy columns(Qia-gen,Hilden,Germany).
RT-PCR ampli?cations were performed with2m g of total RNA in
a Peltier Thermal Cycler(model PTC-225;MJ Research)using the
OneStep RT-PCR Kit(Qiagen,Hilden,Germany).The ampli?cation products were visualized on a1%(w/v)agarose gel via ethidium bromide staining.RT-PCR conditions were:30min at508C,15min at958C;40cycles of1min at948C,1min at648C,1min at728C and a?nal extension for10min at728C.RT-PCR primers were MTL2 and MTL3.
For performing real-time PCR,the puri?ed total RNA was reverse transcribed with the Superscript First-Strand Kit(Gibco Life Tech-nologies,Karlsruhe,Germany)using250ng of oligo(dT)(Sigma, St Louis,USA)and25ng of random hexamer primer(Amersham Biosciences,Freiburg,Germany)per reaction.Reactions required
0.5m M dNTP and5m M MgCl2.To normalize the input of cDNA,
real-time PCR assays were performed in a Taqman ABI7700using the Platinum Quantitative PCR Supermix-UDG(Invitrogen Life Technologies,Karlsruhe,Germany)and pre-developed Human 18S Taqman Reagents(primers and Vic-labelled probe),which are recommended by ABI for the normalization in all eukaryotes.
According to the results,the input into each of the quantitative assays performed with the AtMYB26-speci?c probe and primers was normalized to the18S rRNA genes.The following primers and Taqman probe speci?c for the AtMYB26coding sequence were used:FP:CCA TGG ATG TTG GAG CTC TGT T;RP:GCT TCC ACG TTT AAG ATG CAG GTC T and the intron-spanning probe(50-labelled with6-Fam dye and30-labelled with TAMRA):CAT GCA GGT TTG CAG AGA TGT GGA AAG AG.Conditions for real-time PCR assays were:2min at508C,2min at958C,40cycles of15sec ?Blackwell Publishing Ltd,The Plant Journal,(2003),34,519–528
526Sabine Steiner-Lange et al.
at958C,1min at608C.Assays were performed in triplicates.The presence of residual genomic DNA in the RNA used for this assay had been excluded in advance by performing the real-time PCR assays with the puri?ed total RNA,omitting the reverse transcrip-tion step,and the AtMYB26-speci?c probe and primers. Microscopical techniques
Light microscopy was performed using a ZEISS Axiphot Fluores-cence microscope(Jena,Germany).Pictures were taken using either a digital imaging system(Leica Microsystems,Bensheim, Germany)including a JVC KV-F70digital camera,PC and imaging software or a digital imaging system(INTAS,Go¨ttingen,Germany) including a Spot Diagnostics Instruments Inc.digital camera,PC and imaging software.
For studying auto?uorescence of ligni?ed material,a?lter with the following speci?cations was used:G:365,FT:395,LP420.Prior to microscopy,anthers were cleared in70%(v/v)lactic acid for 3days at608C.
To determine the cellular structure of myb26-2and Col-0 anthers,whole in?orescences were embedded in Paraplast Plus (Sherwood,St Louis,USA)and subsequently used for preparing8 and5m m thin cross-sections(Microtome:Jung Autocut2055, Leica Microsystems,Nussloch,Germany),respectively.After transferring the sections to slides(Superfrost Plus,Menzel),they were incubated for30sec at room temperature in an aqueous 0.05%(w/v)toluidine blue solution and subsequently washed twice for5min with sterile water.After drying the sections at room temperature,the Paraplast Plus was removed by incubating the slides thrice in100%Histoclear(National diagnostics,Hessle Hull,UK).Then,the slides were treated with Entellan and subse-quently covered with a cover slip.
For studying ligni?cation,a phloroglucinol staining was estab-lished.For this purpose,the paraf?n-embedded cross-sections were treated with100%Histoclear and subsequently incubated in100%ethanol.Staining was then performed with2%(w/v) phloroglucinol in100%ethanol for1h at room temperature. Subsequently,the sections were mounted with18.5%(v/v)HCl. To show the deposition of cellulose in the endothecium,the?fth oldest bud of in?orescences of myb26-2and wild-type plants was ?xed in2%(w/v)paraformaldehyde/0.25%(v/v)glutaraldehyde for 2h and subsequently embedded in paraf?n.Semi-thin sections were stained with calco?uor white as described by Dawson et al. (1999).
For scanning electron microscopy(SEM),anthers were mounted on aluminium specimen stubs using Tissue-Tek https://www.doczj.com/doc/5416942623.html,pound (Sakura Finetek,Tokyo,Japan)and immediately shock-frozen in liquid nitrogen.Samples were subsequently transferred to a LEO DSM scanning electron microscope(LEO electron microscopy, Oberkochen,Germany)equipped with a cryo transfer chamber (Gatan GmbH,Mu¨nchen,Germany).Samples were sputter-coated with gold and examined at voltages of2–20kV. Acknowledgements
The authors would like to thank Eva-Marie Schlo¨sser,Heidrun Ha¨weker and Marc Wolff for technical assistance and Rolf Hirz for help with SEM microscopy.Anna Sorensen and Bernd Weis-shaar are acknowledged for helpful discussions and for critically reading the manuscript.We further thank Ralf Stracke for plasmid pRD74and for sharing unpublished results,Bernadette von Malek for providing us with the seeds of dde2-2and the ADIS-Unit of our Institute for performing all sequencing.This work was supported by grants from the German Federal Ministry for Education and Research(BMBF)and from the British Biotechnology and Biolo-gical Sciences Research Council(BBSRC).
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如何写先进个人事迹 篇一：如何写先进事迹材料 如何写先进事迹材料 一般有两种情况：一是先进个人，如先进工作者、优秀党员、劳动模范等；一是先进集体或先进单位，如先进党支部、先进车间或科室，抗洪抢险先进集体等。无论是先进个人还是先进集体，他们的先进事迹，内容各不相同，因此要整理材料，不可能固定一个模式。一般来说，可大体从以下方面进行整理。 (1)要拟定恰当的标题。先进事迹材料的标题，有两部分内容必不可少，一是要写明先进个人姓名和先进集体的名称，使人一眼便看出是哪个人或哪个集体、哪个单位的先进事迹。二是要概括标明先进事迹的主要内容或材料的用途。例如《王鬃同志端正党风的先进事迹》、《关于评选张鬃同志为全国新长征突击手的材料》、《关于评选鬃处党支部为省直机关先进党支部的材料》等。 (2)正文。正文的开头，要写明先进个人的简要情况，包括：姓名、性别、年龄、工作单位、职务、是否党团员等。此外，还要写明有关单位准备授予他(她)什么荣誉称号，或给予哪种形式的奖励。对先进集体、先进单位，要根据其先进事迹的主要内容，寥寥数语即应写明，不须用更多的文字。 然后，要写先进人物或先进集体的主要事迹。这部分内容是全篇材料
的主体，要下功夫写好，关键是要写得既具体，又不繁琐；既概括，又不抽象；既生动形象，又很实在。总之，就是要写得很有说服力，让人一看便可得出够得上先进的结论。比如，写一位端正党风先进人物的事迹材料，就应当着重写这位同志在发扬党的优良传统和作风方面都有哪些突出的先进事迹，在同不正之风作斗争中有哪些突出的表现。又如，写一位搞改革的先进人物的事迹材料，就应当着力写这位同志是从哪些方面进行改革的，已经取得了哪些突出的成果，特别是改革前后的．经济效益或社会效益都有了哪些明显的变化。在写这些先进事迹时，无论是先进个人还是先进集体的，都应选取那些具有代表性的具体事实来说明。必要时还可运用一些数字，以增强先进事迹材料的说服力。 为了使先进事迹的内容眉目清晰、更加条理化，在文字表述上还可分成若干自然段来写，特别是对那些涉及较多方面的先进事迹材料，采取这种写法尤为必要。如果将各方面内容材料都混在一起，是不易写明的。在分段写时，最好在每段之前根据内容标出小标题，或以明确的观点加以概括，使标题或观点与内容浑然一体。 最后，是先进事迹材料的署名。一般说，整理先进个人和先进集体的材料，都是以本级组织或上级组织的名义；是代表组织意见的。因此，材料整理完后，应经有关领导同志审定，以相应一级组织正式署名上报。这类材料不宜以个人名义署名。 写作典型经验材料-般包括以下几部分： (1)标题。有多种写法，通常是把典型经验高度集中地概括出来，一
商业地产是一个综合的领域，涉及商业、经济、建筑、设计等多方面的知识。刚入门的小白常常会被一些专业术语弄得一头雾水。小编在这里整理了一份商业地产相关术语及解释，助你快速掌握该领域，给你最全面的指引。 商业地产概念 商业地产: 作为商业用途的地产，用于各种零售、批发、餐饮、娱乐、健身、休闲等经营用途的房地产形式，国外用的比较多的词汇是零售地产的概念。 1.商圈：是指商业中心以其所在地点为圆心，沿着一定的方向和距离扩展，吸引顾客的辐射范围。 2. 所有权：是所有人依法对自己财产所享有的占有,使用,收益和处分的权利。 3.产权：产权是经济所有制关系的法律表现形式。它包括财产的所有权、占有权、支配权、使用权、收益权和处置权。 4. 经营权：经营者对其经营财产的一种占有、使用和收益的权利。 5.零售业：是一种交易形式,可定义为将商品或劳务直接出售给最终消费者的销售活动,也是向消费者提供销售商品的一种商业活动环境，直接关系到居民的生活质量和生活方式，商业行为的最后一步。 6. 开发商：开发项目，做为项目的第一承担人、所有人。 7.商业用地：商业用地是指规划部门根据城市规划所规定该宗地块的用地性质是用于建设商业用房屋，出让后用地的使用年限为40年。
8. 国内一线城市：指对本国经济、政治具有重要作用的大都市。在城市规模、基建、财政收入、消费、对人才吸引力等各层面，领先于其他城市。如：上海、北京、广州、深圳、天津 9. 国内二线城市：指除一线城市以外，省会、旅游城市，京广、京沪等主要干线旁边的城市等，如：成都、重庆、武汉、南京、苏州、无锡、济南、长沙、 郑州、西安、哈尔滨、杭州、沈阳、青岛、大连、宁波、厦门等 10. 国内三线城市：昆山、江阴、常熟、张家港、义乌、洛阳、柳州、株洲、 长春、温州、佛山、东莞等其它较发达城市。 11. 五证一书：“五证一书”指的是商品房屋预售许可证、建设工程规划许可证、建设用地许可证、国有土地使用规划许可证、建筑工程施工许可证及项目 选址意见书。 商业建筑 1.占地面积：占地面积是指建筑物所占有或使用的土地水平投影面积，计算一般按底层建筑面积。 2. 建筑面积：建筑展开面积，它是指建筑外墙外围线测定的各层平面面积之和。它是表示一个建筑物建筑规模大小的经济指标。 3. 经营面积（实用面积、柜台面积）：它是“建筑面积”扣除公共分摊面积后的余额。
How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.
英语演讲稿：未来的工作 这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！ 热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn
小学生个人读书事迹简介怎么写800字 书,是人类进步的阶梯,苏联作家高尔基的一句话道出了书的重要。书可谓是众多名人的“宠儿”。历来,名人说出关于书的名言数不胜数。今天小编在这给大家整理了小学生个人读书事迹,接下来随着小编一起来看看吧! 小学生个人读书事迹1 “万般皆下品,惟有读书高”、“书中自有颜如玉,书中自有黄金屋”,古往今来,读书的好处为人们所重视,有人“学而优则仕”,有人“满腹经纶”走上“传道授业解惑也”的道路……但是,从长远的角度看,笔者认为读书的好处在于增加了我们做事的成功率,改善了生活的质量。 三国时期的大将吕蒙,行伍出身,不重视文化的学习,行文时,常常要他人捉刀。经过主君孙权的劝导,吕蒙懂得了读书的重要性,从此手不释卷,成为了一代儒将,连东吴的智囊鲁肃都对他“刮目相待”。后来的事实证明,荆州之战的胜利,擒获“武圣”关羽,离不开吕蒙的“运筹帷幄,决胜千里”,而他的韬略离不开平时的读书。由此可见,一个人行事的成功率高低,与他的对读书,对知识的重视程度是密切相关的。 的物理学家牛顿曾近说过,“如果我比别人看得更远,那是因为我站在巨人的肩上”,鲜花和掌声面前,一代伟人没有迷失方向,自始至终对读书保持着热枕。牛顿的话语告诉我们,渊博的知识能让我们站在更高、更理性的角度来看问题,从而少犯错误,少走弯路。
读书的好处是显而易见的,但是,在社会发展日新月异的今天,依然不乏对读书,对知识缺乏认知的人,《今日说法》中我们反复看到农民工没有和用人单位签订劳动合同,最终讨薪无果;屠户不知道往牛肉里掺“巴西疯牛肉”是犯法的;某父母坚持“棍棒底下出孝子”,结果伤害了孩子的身心,也将自己送进了班房……对书本,对知识的零解读让他们付出了惨痛的代价,当他们奔波在讨薪的路上,当他们面对高墙电网时,幸福,从何谈起?高质量的生活,从何谈起? 读书,让我们体会到“锄禾日当午,汗滴禾下土”的艰辛;读书,让我们感知到“四海无闲田,农夫犹饿死”的无奈;读书,让我们感悟到“为报倾城随太守,西北望射天狼”的豪情壮志。 读书的好处在于提高了生活的质量,它填补了我们人生中的空白,让我们不至于在大好的年华里无所事事,从书本中,我们学会提炼出有用的信息,汲取成长所需的营养。所以,我们要认真读书,充分认识到读书对改善生活的重要意义,只有这样,才是一种负责任的生活态度。 小学生个人读书事迹2 所谓读一本好书就是交一个良师益友,但我认为读一本好书就是一次大冒险,大探究。一次体会书的过程,真的很有意思,咯咯的笑声,总是从书香里散发;沉思的目光也总是从书本里透露。是书给了我启示,是书填补了我无聊的夜空,也是书带我遨游整个古今中外。所以人活着就不能没有书,只要爱书你就是一个爱生活的人,只要爱书你就是一个大写的人,只要爱书你就是一个懂得珍惜与否的人。可真所谓
商场商业术语营销术语大全 1、返券：即在促销活动期间内，购物满一定金额赠送不同或相同金额的抵用券，如满500元返20元券。 2、折扣：折扣是一种最常见、也最容易操作的促销方式。即全场商品或部分商品进行不同程度的折扣销售。 3、特价：即限定某个时间段内某些商品执行特价销售，超出时间自动恢复，一般建议在客流比较少的时段做，这样可以拉平卖场人气。 4、限定条件优惠：如，限会员购买优惠或会员达到一定消费积分时优惠，还有买购多少额度优惠或返利。 5、限总量优惠：即在优惠的商品出售够一定数量时，特价终止，恢复原价销售。 6、限客单量优惠：即限定每客最多购买特价商品的数量，若超过限量则超过的部分按原价销售计算。 7、购买超过一定量优惠：即某个单品一次购买超过几个，就按设定的优惠价计算销售。 8、商品碰头分组促销：此项促销事先设定好分组的商品（必须是两个单品），定好优惠价格，只有消费者在购买时，在规定的时间同时找到两个对应的单品时则按优惠价销售；此活动一般适应与卖场布局死角多的地方，将两个分组商品放置在不同的区域，让消费者去寻找，以提升卖场动线和消费者购物的乐趣。 9、捆绑销售：即将一些关联性强的商品放在一起，并打包给予一定的优惠进行销售。 10、消费购买一定额度，可以购买超低价商品，如：购物满50元可以1元钱买一斤色拉油等。 11、买赠促销：即买指定的东西送赠品，还有买够多少钱送不同的赠品；赠品可以是商品也可以是企业的广告礼品（如：茶杯、围君、广告衫、广告伞、遮阳帽等）。 12、有奖销售：即购物满一定条件可以参加摸奖或抽奖活动，主要是奖品一定要丰富，并且有公众吸引力。 13、商家联盟促销：即购物满一定条件或消费达到某种条件，可以提供消费者到其他商家消费的打折权利，比如：在超市买够500元送餐饮或娱乐项目的赠票或折扣券，或者在某餐饮或娱乐项目消费单位满多少元可以送超市的优惠卡或折扣券。 14、购物送服务：购物满一定条件可以免费送货、报销车费、代大扫除、免费般移大件物品、免费维修家具或电器和通讯工具等。
关于坚持的英语演讲稿 Results are not important, but they can persist for many years as a commemoration of. Many years ago, as a result of habits and overeating formed one of obesity, as well as indicators of overall physical disorders, so that affects my work and life. In friends to encourage and supervise, the participated in the team Now considered to have been more than three years, neither the fine rain, regardless of winter heat, a day out with 5:00 time. The beginning, have been discouraged, suffering, and disappointment, but in the end of the urging of friends, to re-get up, stand on the playground. 成绩并不重要，但可以作为坚持多年晨跑的一个纪念。多年前，由于庸懒习惯和暴饮暴食，形成了一身的肥胖，以及体检指标的全盘失常，以致于影响到了我的工作和生活。在好友的鼓励和督促下，参加了晨跑队伍。现在算来，已经三年多了，无论天晴下雨，不管寒冬酷暑，每天五点准时起来出门晨跑。开始时，也曾气馁过、痛苦过、失望过，但最后都在好友们的催促下，重新爬起来，站到了操场上。 In fact, I did not build big, nor strong muscles, not a sport-born people. Over the past few years to adhere to it, because I have a team behind, the strength of a strongteam here, very grateful to our team, for a long time, we encourage each other, and with sweat, enjoying common health happy. For example, Friends of the several run in order to maintain order and unable to attend the 10，000 meters race, and they are always concerned about the brothers and promptly inform the place and time, gives us confidence and courage. At the same time, also came on their own inner desire and pursuit for a good health, who wrote many of their own log in order to refuel for their own, and inspiring. 其实我没有高大身材，也没健壮肌肉，天生不属于运动型的人。几年来能够坚持下来，因为我的背后有一个团队，有着强大团队的力量，在这里，非常感谢我们的晨跑队，长期以来，我们相互鼓励着，一起流汗，共同享受着健康带来的快
常用的商业术语 口部（部口）：地下商城的专用名词，即出入口。 坪（平）效:指单位面积的销售额或租金额。 动线: 指商场的布局，使顾客自然行走、购物的轨迹。良好的动线规划可诱导顾客在店内顺畅地选购商品，避免卖场产生死角，提高卖场坪效。动线设计对超市尤其重要。 主动线：也称主通道，顾客在一走进商场就可以看到的货架间的通道，主要指入口到末端也指顾客经过的主要通道，也是商品陈列的最佳地段。主通道的宽度应在3米以上。 副动线：也称副通道，指专柜之间的通道。副通道宽度应在2米以上。水平动线：指在一个层面中,人们行走的最佳路线。 垂直动线：是指不同标高空间或楼层的垂直联系如楼梯、电梯和自动扶梯。 引导动线：指通过一定的手段（比如刀旗、横幅等）将顾客引导到指定目的地的路线安排。 开间：又称面宽，即商铺对外可见的宽度距离。 进深：指商铺纵深的长度，与面宽线段到商铺墙面垂直的距离。 建筑面积：建筑面积亦称建筑展开面积，包含主要设备间室、动线、出入口等。 使用面积：指商铺中直接供用户经营使用的净平面面积之和。 套内建筑面积：成套商铺的套内建筑面积，成套商铺的套内建筑面积由套内商铺使用面积，套内墙体面积，套内公共面积三部分组成。
层高：层高是指顶板到地面的垂直距离。 内净高：指装修落成后的地面与形成最终铺设的棚顶间的间距。 门楣：门楣，就是商铺正门上方的横梁，用于悬挂或粘贴牌匾的地点。一装：一般指商场公共区间的原始装修，如墙面、地面、柱体、卫生间等，一装将确立商场的主题定位思想，也将引导商户店面装饰的方向。 二装：即商户进场后对其商铺的装修、装饰。 均价：某一个个案的总销金额除以总建面得出的单价。 基价：某一个个案的最低价。 订金：商铺订购金，又称协议金、诚意金等等，可退还给客户，或抵用购买、租赁商铺的费用。 定金：商铺定购金，又称大定金等。如购买者超出定购期未付全款，该定金归出售方所有，不可退还给认购人；如签订全款合同可抵用购买商铺的费用。 经销商：是产品的经销环节，直接面向销售终端。 供应商：为零售案场提供商品，销售终端的货品供给商。 代理商：是货品生产厂家的合作者。生产厂家在某一区域内设立代理商，代理商负责该区域的品牌宣传、推广、分销等活动，拥有该商品品牌的独家经销权，及该区域内市场拓扑分布的规划、统筹权。 渠道商：渠道商是指连接制造商和消费者之间的众多中间商家,包括:批发商,零售商,代理商和佣金商等。 码单：指供货商提供给经销商货品的尺码单据。
1.How to build a business that lasts100years 0:11Imagine that you are a product designer.And you've designed a product,a new type of product,called the human immune system.You're pitching this product to a skeptical,strictly no-nonsense manager.Let's call him Bob.I think we all know at least one Bob,right?How would that go? 0:34Bob,I've got this incredible idea for a completely new type of personal health product.It's called the human immune system.I can see from your face that you're having some problems with this.Don't worry.I know it's very complicated.I don't want to take you through the gory details,I just want to tell you about some of the amazing features of this product.First of all,it cleverly uses redundancy by having millions of copies of each component--leukocytes,white blood cells--before they're actually needed,to create a massive buffer against the unexpected.And it cleverly leverages diversity by having not just leukocytes but B cells,T cells,natural killer cells,antibodies.The components don't really matter.The point is that together,this diversity of different approaches can cope with more or less anything that evolution has been able to throw up.And the design is completely modular.You have the surface barrier of the human skin,you have the very rapidly reacting innate immune system and then you have the highly targeted adaptive immune system.The point is,that if one system fails,another can take over,creating a virtually foolproof system. 1:54I can see I'm losing you,Bob,but stay with me,because here is the really killer feature.The product is completely adaptive.It's able to actually develop targeted antibodies to threats that it's never even met before.It actually also does this with incredible prudence,detecting and reacting to every tiny threat,and furthermore, remembering every previous threat,in case they are ever encountered again.What I'm pitching you today is actually not a stand-alone product.The product is embedded in the larger system of the human body,and it works in complete harmony with that system,to create this unprecedented level of biological protection.So Bob,just tell me honestly,what do you think of my product? 2:47And Bob may say something like,I sincerely appreciate the effort and passion that have gone into your presentation,blah blah blah-- 2:56(Laughter) 2:58But honestly,it's total nonsense.You seem to be saying that the key selling points of your product are that it is inefficient and complex.Didn't they teach you 80-20?And furthermore,you're saying that this product is siloed.It overreacts, makes things up as it goes along and is actually designed for somebody else's benefit. I'm sorry to break it to you,but I don't think this one is a winner.
关于工作的优秀英语演讲稿 Different people have various ambitions. Some want to be engineers or doctors in the future. Some want to be scientists or businessmen. Still some wish to be teachers or lawers when they grow up in the days to come. Unlike other people, I prefer to be a farmer. However, it is not easy to be a farmer for Iwill be looked upon by others. Anyway,what I am trying to do is to make great contributions to agriculture. It is well known that farming is the basic of the country. Above all, farming is not only a challenge but also a good opportunity for the young. We can also make a big profit by growing vegetables and food in a scientific way. Besides we can apply what we have learned in school to farming. Thus our countryside will become more and more properous. I believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. All the working position can provide him with a good chance to become a talent. 1 ————来源网络整理，仅供供参考
个人先进事迹简介 01 在思想政治方面,xxxx同学积极向上,热爱祖国、热爱中国共产党,拥护中国共产党的领导.利用课余时间和党课机会认真学习政治理论,积极向党组织靠拢. 在学习上,xxxx同学认为只有把学习成绩确实提高才能为将来的实践打下扎实的基础,成为社会有用人才.学习努力、成绩优良. 在生活中,善于与人沟通,乐观向上,乐于助人.有健全的人格意识和良好的心理素质和从容、坦诚、乐观、快乐的生活态度,乐于帮助身边的同学,受到师生的好评. 02 xxx同学认真学习政治理论,积极上进,在校期间获得原院级三好生,和校级三好生,优秀团员称号,并获得三等奖学金. 在学习上遇到不理解的地方也常常向老师请教,还勇于向老师提出质疑.在完成自己学业的同时,能主动帮助其他同学解决学习上的难题,和其他同学共同探讨,共同进步. 在社会实践方面,xxxx同学参与了中国儿童文学精品“悦”读书系,插画绘制工作,xxxx同学在班中担任宣传委员,工作积极主动,认真负责,有较强的组织能力.能够在老师、班主任的指导下独立完成学院、班级布置的各项工作. 03 xxx同学在政治思想方面积极进取,严格要求自己.在学习方面刻苦努力,不断钻研,学习成绩优异,连续两年荣获国家励志奖学金；作
为一名学生干部,她总是充满激情的迎接并完成各项工作,荣获优秀团干部称号.在社会实践和志愿者活动中起到模范带头作用. 04 xxxx同学在思想方面,积极要求进步,为人诚实,尊敬师长.严格 要求自己.在大一期间就积极参加了党课初、高级班的学习,拥护中国共产党的领导,并积极向党组织靠拢. 在工作上,作为班中的学习委员,对待工作兢兢业业、尽职尽责 的完成班集体的各项工作任务.并在班级和系里能够起骨干带头作用.热心为同学服务,工作责任心强. 在学习上,学习目的明确、态度端正、刻苦努力,连续两学年在 班级的综合测评排名中获得第1.并荣获院级二等奖学金、三好生、优秀班干部、优秀团员等奖项. 在社会实践方面,积极参加学校和班级组织的各项政治活动,并 在志愿者活动中起到模范带头作用.积极锻炼身体.能够处理好学习与工作的关系,乐于助人,团结班中每一位同学,谦虚好学,受到师生的好评. 05 在思想方面,xxxx同学积极向上,热爱祖国、热爱中国共产党,拥护中国共产党的领导.作为一名共产党员时刻起到积极的带头作用,利用课余时间和党课机会认真学习政治理论. 在工作上,作为班中的团支部书记,xxxx同学积极策划组织各类 团活动,具有良好的组织能力. 在学习上,xxxx同学学习努力、成绩优良、并热心帮助在学习上有困难的同学,连续两年获得二等奖学金. 在生活中,善于与人沟通,乐观向上,乐于助人.有健全的人格意 识和良好的心理素质.
商场商业术语营销术语大全（不断更新） 商场商业术语营销术语大全 1、返券：即在促销活动期间内，购物满一定金额赠送不同或相同金额的抵用券，如满500元返20元券。 2、折扣：折扣是一种最常见、也最容易操作的促销方式。即全场商品或部分商品进行不同程度的折扣销售。 3、特价：即限定某个时间段内某些商品执行特价销售，超出时间自动恢复，一般建议在客流比较少的时段做，这样可以拉平卖场人气。 4、限定条件优惠：如，限会员购买优惠或会员达到一定消费积分时优惠，还有买购多少额度优惠或返利。 5、限总量优惠：即在优惠的商品出售够一定数量时，特价终止，恢复原价销售。 6、限客单量优惠：即限定每客最多购买特价商品的数量，若超过限量则超过的部分按原价销售计算。 7、购买超过一定量优惠：即某个单品一次购买超过几个，就按设定的优惠价计算销售。 8、商品碰头分组促销：此项促销事先设定好分组的商品（必须是两个单品），定好优惠价格，只有消费者在购买时，在规定的时间同时找到两个对应的单品时则按优惠价销售；此活动一般适应与卖场布局死角多的地方，将两个分组商品放置在不同的区域，让消费者去寻找，以提升卖场动线和消费者购物的乐趣。
9、捆绑销售：即将一些关联性强的商品放在一起，并打包给予一定的优惠进行销售。 10、消费购买一定额度，可以购买超低价商品，如：购物满50元可以1元钱买一斤色拉油等。 11、买赠促销：即买指定的东西送赠品，还有买够多少钱送不同的赠品；赠品可以是商品也可以是企业的广告礼品（如：茶杯、围君、广告衫、广告伞、遮阳帽等）。 12、有奖销售：即购物满一定条件可以参加摸奖或抽奖活动，主要是奖品一定要丰富，并且有公众吸引力。 13、商家联盟促销：即购物满一定条件或消费达到某种条件，可以提供消费者到其他商家消费的打折权利，比如：在超市买够500元送餐饮或娱乐项目的赠票或折扣券，或者在某餐饮或娱乐项目消费单位满多少元可以送超市的优惠卡或折扣券。 14、购物送服务：购物满一定条件可以免费送货、报销车费、代大扫除、免费般移大件物品、免费维修家具或电器和通讯工具等。 15、POP：POP是英文point of purchase的缩写，意为“卖点广告”,其主要商业用途是刺激引导消费和活跃卖场气氛。她的形式有户外招牌，展板，橱窗海报，店内台牌，价目表，吊旗，甚至是立体卡通模型等等。常用的POP为短期的促销使用，其表现形式夸张幽默，色彩强烈，能有效地吸引顾客的视点唤起购买欲，她作为一种低价高效的广告方式已被广泛应用。
尊敬的领导，老师，亲爱的同学们， 大家好！我是5班的梁浩东。今天早上我坐车来学校的路上，我仔细观察了路上形形色色的人，有开着小车衣着精致的叔叔阿姨，有市场带着倦容的卖各种早点的阿姨，还有偶尔穿梭于人群中衣衫褴褛的乞丐。于是我问自己，十几年后我会成为怎样的自己，想成为社会成功人士还是碌碌无为的人呢，答案肯定是前者。那么十几年后我怎样才能如愿以偿呢，成为一个受人尊重，有价值的人呢？正如我今天演讲的题目是：自主管理。 大家都知道爱玩是我们孩子的天性，学习也是我们的责任和义务。要怎样处理好这些矛盾，提高自主管理呢？ 首先，我们要有小主人翁思想，自己做自己的主人，要认识到我们学习，生活这一切都是我们自己走自己的人生路，并不是为了报答父母，更不是为了敷衍老师。 我认为自主管理又可以理解为自我管理，在学习和生活中无处不在，比如通过老师，小组长来管理约束行为和同学们对自身行为的管理都属于自我管理。比如我们到一个旅游景点，看到一块大石头，有的同学特别兴奋，会想在上面刻上：某某某到此一游话。这时你就需要自我管理，你需要提醒自己，这样做会破坏景点，而且是一种素质低下的表现。你设想一下，如果别人家小孩去你家墙上乱涂乱画，你是何种感受。同样我们把自主管理放到学习上，在我们想偷懒，想逃避，想放弃的时候，我们可以通过自主管理来避免这些，通过他人或者自己的力量来完成。例如我会制定作息时间计划表，里面包括学习，运动，玩耍等内容的完成时间。那些学校学习尖子，他们学习好是智商高于我们吗，其实不然，在我所了解的哪些优秀的学霸传授经验里，就提到要能够自我管理，规范好学习时间的分分秒秒，只有辛勤的付出，才能取得优异成绩。 在现实生活中，无数成功人士告诉我们自主管理的重要性。十几年后我想成为一位优秀的，为国家多做贡献的人。亲爱的同学们，你们们？让我们从现在开始重视和执行自主管理，十几年后成为那个你想成为的人。 谢谢大家！
关于工作的英语演讲稿 【篇一：关于工作的英语演讲稿】 关于工作的英语演讲稿 different people have various ambitions. some want to be engineers or doctors in the future. some want to be scientists or businessmen. still some wish to be teachers or lawers when they grow up in the days to come. unlike other people, i prefer to be a farmer. however, it is not easy to be a farmer for iwill be looked upon by others. anyway,what i am trying to do is to make great contributions to agriculture. it is well known that farming is the basic of the country. above all, farming is not only a challenge but also a good opportunity for the young. we can also make a big profit by growing vegetables and food in a scientific way. besides we can apply what we have learned in school to farming. thus our countryside will become more and more properous. i believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. all the working position can provide him with a good chance to become a talent. 【篇二：关于责任感的英语演讲稿】 im grateful that ive been given this opportunity to stand here as a spokesman. facing all of you on the stage, i have the exciting feeling of participating in this speech competition. the topic today is what we cannot afford to lose. if you ask me this question, i must tell you that i think the answer is a word---- responsibility. in my elementary years, there was a little girl in the class who worked very hard, however she could never do satisfactorily in her lessons. the teacher asked me to help her, and it was obvious that she expected a lot from me. but as a young boy, i was so restless and thoughtless, i always tried to get more time to play and enjoy myself. so she was always slighted over by me. one day before the final exam, she came up to me and said, could you please explain this to me? i can not understand it. i