Identification of genes expressed in the Arabidopsis female gametophyte
Identi?cation of genes expressed in the Arabidopsis female gametophyte
Joshua G.Steffen,Il-Ho Kang,Jane Macfarlane and Gary N.Drews*
Department of Biology,University of Utah,257South1400East,Salt Lake City,UT84112-0840,USA
Received08December2006;revised08March2007;accepted22March2007.
*For correspondence(fax+18015814668;e-mail drews@https://www.360docs.net/doc/713808149.html,).
Summary
The angiosperm female gametophyte typically consists of one egg cell,two synergid cells,one central cell,and
three antipodal cells.Each of these four cell types has unique structural features and performs unique functions
that are essential for the reproductive process.The gene regulatory networks conferring these four phenotypic
states are largely uncharacterized.As a?rst step towards dissecting the gene regulatory networks of the
female gametophyte,we have identi?ed a large collection of genes expressed in speci?c cells of the
Arabidopsis thaliana female gametophyte.We identi?ed these genes using a differential expression screen
based on reduced expression in determinant infertile1(dif1)ovules,which lack female gametophytes.We
hybridized ovule RNA probes with Affymetrix ATH1genome arrays and validated the identi?ed genes using
real-time RT-PCR.These assays identi?ed71genes exhibiting reduced expression in dif1ovules.We further
validated45of these genes using promoter::GFP fusions and43were expressed in the female gametophyte.In
the context of the ovule,11genes were expressed exclusively in the antipodal cells,11genes were expressed
exclusively or predominantly in the central cell,17genes were expressed exclusively or predominantly in the
synergid cells,one gene was expressed exclusively in the egg cell,and three genes were expressed strongly in
multiple cells of the female gametophyte.These genes provide insights into the molecular processes
functioning in the female gametophyte and can be used as starting points to dissect the gene regulatory
networks functioning during differentiation of the four female gametophyte cell types.
Key words:female gametophyte,embryo sac,plant reproduction,microarray.
Introduction
Plants have a two-staged life cycle that alternates between a multicellular haploid organism,the gametophyte,and a multicellular diploid organism,the sporophyte(Gifford and Foster,1988).Angiosperms have two gametophytes that are embedded within the sexual organs of the?ower.The male gametophyte(pollen grain)develops within the anther and comprises two sperm cells encased within a vegetative cell (McCormick,2004).The female gametophyte develops within the ovule and most often consists of one egg cell,two synergid cells,one central cell,and three antipodal cells (Figure1a;Drews and Yadegari,2002;Yadegari and Drews, 2004).
The female gametophyte is essential for sexual reproduc-tion in angiosperms.Sexual reproduction is initiated when the male gametophyte is transferred from the anther to the stigma of the carpel,whereupon it forms a pollen tube that grows through the carpel’s internal tissues to deliver its two sperm cells to the female gametophyte.The pollen tube enters the female gametophyte by growing into one of the synergid cells.The synergid cell penetrated by the pollen tube undergoes cell death.Soon after arrival,the pollen tube ceases growth and releases its two sperm cells into the degenerating synergid.Finally,the sperm cells migrate to and fuse with the egg cell and central cell.The fertilized egg cell and central cell give rise to the seed’s embryo and endosperm,respectively(Lord and Russell,2002).
The cells of the female gametophyte control many steps of the fertilization process.Cell ablation studies indicate that the synergid cells produce a guidance cue that directs growth of the pollen tube to the ovule(Higashiyama et al., 2001,2003;Marton et al.,2005).Structural and genetic studies suggest that the synergid cells contain factors that control the arrest of pollen tube growth and the release of the pollen tube contents(Huck et al.,2003;Rotman et al.,
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The Plant Journal(2007)51,281–292doi:10.1111/j.1365-313X.2007.03137.x
2003;Weterings and Russell,2004).Upon fertilization,the
ovule is induced to develop into a seed.Genetic and
molecular studies indicate that central cell-expressed gene
products control the activation of endosperm development
(Grossniklaus et al.,1998;Luo et al.,1999;Ohad et al.,1999).
Finally,genetic studies indicate that the female gameto-
phyte plays a role in maternal control of seed development
following fertilization (Chaudhury and Berger,2001;Evans
and Kermicle,2001;Grini et al.,2002).
The molecular processes by which the female gameto-
phyte cells acquire their unique features and functions
during cell differentiation are not understood.These pro-
cesses probably involve distinct gene expression pro?les
associated with each of the female gametophyte cell types.
Thus,understanding how the female gametophyte cells
become speci?ed and acquire their unique features and
functions requires mechanistic insight into the gene regula-
tory networks that control cell-speci?c gene expression
during development of the female gametophyte.
Dissecting gene regulatory networks requires identi?ca-
tion of transcription factors conferring cell-speci?c expres-
sion,as well as the cis -regulatory elements through which
these transcription factors act to activate downstream genes
(Levine and Davidson,2005).In other systems,transcription
factors conferring cell-speci?c expression have been iden-
ti?ed using forward-genetics approaches in which mutants
were identi?ed and analyzed (Levine and Davidson,2005;
Messenguy and Dubois,2003;Schiefelbein,2003;Serna and
Martin,2006).However,most of the female gametophyte
mutants analyzed at the molecular level are affected in
cellular processes at particular stages of development of the
female gametophyte,including nuclear proliferation (Acos-
ta-Garcia and Vielle-Calzada,2004;Ebel et al.,2004;Huanca-
Mamani et al.,2005;Kwee and Sundaresan,2003;Shi et al.,
2005),cell formation (Hejatko et al.,2003;Kim et al.,2005;
Pischke et al.,2002),fusion of the polar nuclei (Christensen
et al.,2002;Niewiadomski et al.,2005;Portereiko et al.,
2006b),cell death (Christensen et al.,2002),and DNA
demethylation (Choi et al.,2002;Gehring et al.,2006),or in reproductive functions such as regulation of the initiation of endosperm development (Grossniklaus et al.,1998;Luo et al.,1999;Ohad et al.,1999),pollen tube guidance (Marton et al.,2005),and control of pollen tube growth within the female gametophyte (Huck et al.,2003;Rotman et al.,2003).To date,only two mutations,agl80(Portereiko et al.,2006a)and myb98(Kasahara et al.,2005),affecting transcription factors regulating cell differentiation during development of the female gametophyte have been identi?ed.An alternative approach to the dissection of gene regula-tory networks is to identify genes expressed in speci?c cell types,characterize the cis -regulatory elements within these genes that confer cell-speci?c expression,and analyze the transcription factors that interact with these cis -regulatory elements (Levine and Davidson,2005).However,for the female gametophyte,this approach has been hindered by a paucity of identi?ed genes that are expressed in speci?c cells of the female gametophyte.Although expression-based screens have identi?ed many transcripts present in the female gametophyte,expression within the ovule has been characterized for only 21of these genes (Cordts et al.,2001;Kumhlehn et al.,2001;Le et al.,2005;Ning et al.,2006;Sprunck et al.,2005;Yang et al.,2006;Yu et al.,2005).These and other studies have identi?ed relatively few genes exhibiting cell-speci?c expression including the central cell-expressed genes FIE (Kinoshita et al.,1999;Luo et al.,2000;Yadegari et al.,2000),FIS2(Luo et al.,2000),MEA (Luo et al.,2000),ZmEBE1and ZmEBE2(Magnard et al.,2003),FWA (Kinoshita et al.,2004),C170(Le et al.,2005),AGL80(Portereiko et al.,2006a),and ES0777and ES3441(Yang et al.,2006);the synergid cell-expressed genes MYB98(Kasahara et al.,2005),and ES5686and ES0965(Yang et al.,2006);the egg cell expressed gene E017(Le et al.,2005);and the antipodal cell-expressed genes At1g36340,At2g20070,and At4g22050(Yu et al.,2005).Here,we report the identi?cation of a large collection of genes expressed in speci?c cells of the female https://www.360docs.net/doc/713808149.html,ing microarrays and real-time RT-PCR,we identi?ed 71genes exhibiting reduced expression in mutant ovules that ec ec sc sc cc
cc (b)(c)(d)(e)
282Joshua G.Steffen et al.
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lack female https://www.360docs.net/doc/713808149.html,ing promoter::GFP fusions,we analyzed45of these genes and showed that43are expressed in the female gametophyte.Of these43,40are expressed exclusively or predominantly in speci?c cells of the female gametophyte including11in the antipodal cells, 11in the in the central cell,17in the synergid cells,and one in the egg cell.These genes can be used as starting points to dissect the gene regulatory networks controlling cell differ-entiation during development of the female gametophyte.
Results
Strategy to identify female gametophyte-expressed genes
We carried out a differential microarray screen to identify genes expressed in the female gametophyte.Our general strategy was to identify mRNAs present in ovules with female gametophytes but not in ovules lacking female gametophytes.We used male sterility1(ms1)as the source of normal ovules.ms1is a recessive sporophytic mutation that affects pollen development and homozygous mutants are male sterile(Ito and Shinozaki,2002;Thorlby et al.,1997; Wilson et al.,2001).We analyzed the development of female gametophytes in ms1and found that it was indistinguish-able from wild type(Figure1a–c).We used determinant infertile1(dif1;Bai et al.,1999;Bhatt et al.,1999;Cai et al., 2003)as the source of mutant ovules lacking female gametophytes.dif1mutants are male and female sterile, with female sterility resulting from the absence of a female gametophyte.dif1is a recessive and sporophytic mutation; thus,in homozygous mutants,all ovules lack female gametophytes.The dif1mutation affects chromosomal segregation during meiosis,and the absence of a female gametophyte in dif1ovules results from the meiosis defect. We analyzed dif1ovules at the terminal developmental stage and found that most ovules(75%)lacked a female gameto-phyte but were otherwise normal(Figure1d)and that a minority of ovules(25%)had a one-nucleate structure(Fig-ure1e)that was probably a persistent megaspore mother cell.
Our objective was to harvest ovules containing female gametophytes at stages FG5to FG7(Christensen et al.,1997) to obtain the spectrum of stages during which the female gametophyte cells acquire their unique features and func-tions.Ovules at these stages are within?owers at stages12c (Christensen et al.,1997)to14(Smyth et al.,1990),which are self-pollinated in Arabidopsis.ms1and dif1are male sterile, which allowed us to avoid the?ower emasculations that would otherwise be required and that may result in changes in gene expression.
We developed a rapid procedure to harvest ovules of high purity from Arabidopsis.Brie?y,we harvested pistils from ?owers at stages12c to14,infused the pistils with a non-crosslinking?xative(70%ethanol),scraped out the ovules in the presence of the?xative,and removed any contaminating tissue.Ovules collected in this way were>95%pure based on microscopic inspection.We collected the ovules in batches of5000.To minimize plant-to-plant variation,each ovule batch was collected from>30plants.For each geno-type,four batches of ovules were independently collected; the RNA from these eight batches was used in the micro-array and real-time RT-PCR assays discussed below.
To determine whether the approach of using RNA from ms1and dif1ovules to identify female gametophyte-expressed genes is valid,we carried out real-time RT-PCR with two genes known to be expressed in the female gametophyte:FIS2and MEA that are both expressed speci?cally in the central cell(Luo et al.,2000).As shown in Figure S1,both genes exhibited reduced expression in dif1ovules relative to ms1ovules,indicating that our general approach is an effective means of identifying female gametophyte-expressed genes.
Identi?cation of71genes downregulated in dif1ovules
To identify female gametophyte-expressed genes,we syn-thesized probes from the ms1and dif1ovule RNAs and hybridized these probes with Affymetrix Arabidopsis ATH1 genome arrays.For each genotype,probes were generated from two biological replicates.The Pearson coef?cients were0.9977and0.9994for the ms1and dif1replicates, respectively,indicating high reproducibility of the biological replicates.We used GC-RMA(Wu and Irizarry,2004)to pro-cess the data.We then used statistical analysis of microar-rays(Tusher et al.,2001)to identify genes exhibiting reduced expression in dif1ovules relative to ms1ovules using the criteria of?2-fold change and<15%false positives. With these criteria,86genes exhibited reduced expression in dif1ovules.We refer to these as DD(downregulated in dif1) genes.The microarray data for the DD genes are summar-ized in Table S1.
To validate the expression patterns of the86DD genes,we carried out real-time RT-PCR with RNA from independently harvested ovules.The real-time RT-PCR data for these86 genes are summarized in Table https://www.360docs.net/doc/713808149.html,ing the criteria of?8-fold change,71of the86genes were validated as having reduced expression in dif1ovules.The71validated genes are listed in Table1.
The functional categories of the71validated genes are listed in Tables1and2.This classi?cation is based on the annotations provided by The Arabidopsis Information Resource(https://www.360docs.net/doc/713808149.html,).Table2shows that the majority(41of71genes)of genes encode proteins of unknown function.The predicted functions of the other30 genes span the various categories including protein meta-bolism,electron transport,energy pathways,transport,cell–cell signaling,and cell organization and biogenesis.Three of the genes have a predicted function in transcriptional Female gametophyte-expressed genes283
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284Joshua G.Steffen et al.
Table1.Genes exhibiting reduced expression in dif1ovules
Gene AGI a Description b
DD1At1g36340Ubiquitin-conjugating enzyme
DD2At5g43510Defensin-like(DEFL)family protein
DD3At3g56610Expressed protein
DD4At5g42955Expressed protein
DD6At2g42930Glycosyl hydrolase family protein
DD7At2g20595Expressed protein
DD8At5g52975Expressed protein
DD9At1g26795Self-incompatibility protein-related S3(Papaver rhoeas)
DD10At5g24316Proline-rich family protein
DD11At1g52970Expressed protein
DD12At2g21655Expressed protein
DD13At3g59260Pirin like protein
DD14At3g30540(1–4)-Beta-mannan endohydrolase family
DD15At3g17150Invertase/pectin methylesterase inhibitor family protein
DD16At2g34890Putative CTP synthase
DD17At5g34885Expressed protein
DD18At1g45190Expressed protein
DD19At2g06090Self-incompatibility protein-related S1(Papaver rhoeas)
DD20At2g03320Hypothetical protein
DD21At5g22970Expressed protein
DD22At5g38330Cysteine-rich protein
DD23At3g49300Proline-rich family protein
DD25At3g04540Defensin-like(DEFL)family protein
DD26At1g47780Acyl-protein thioesterase-related protein
DD27At3g05460Sporozoite surface protein-related protein
DD28At3g46840Subtilase family protein
DD29At2g47280Pectinesterase family protein
DD30At2g01240Reticulon family protein
DD31At1g47470Hypothetical protein
DD32At3g17080Self-incompatibility protein-related S1(Papaver rhoeas)
DD33At2g20070Expressed protein
DD34At4g07515Expressed protein
DD35At5g12380Annexin protein
DD36At3g24510Defensin-like(DEFL)family protein
DD37At3g43500Expressed protein
DD39At4g20050Expressed protein(QRT3)
DD40At1g73010Putative phosphatase protein
DD41At2g02515Expressed protein
DD42At2g20660Rapid alkalinization factor(RALF)family protein
DD43At5g47330Palmitoyl protein thioesterase family protein
DD44At4g30540Glutamine amidotransferase class-I domain-containing protein DD45At2g21740Expressed protein
DD46At1g22015Galactosyltransferase family protein
DD47At1g77790Glycosyl hydrolase family17protein
DD50At1g43800Putative stearoyl-ACP desaturase protein
DD52At5g65370Epsin N-terminal homology(ENTH)domain-containing protein DD53At4g18770Myb family transcription factor(MYB98)
DD54At3g28740Cytochrome P-450family protein
DD55At3g17790Acid phosphatase type5(ACP5)protein
DD56At4g30590Plastocyanin-like domain-containing protein
DD59At3g48950Glycoside hydrolase family28protein/polygalacturonase
(pectinase)family protein
DD60At4g15040Subtilase family protein
DD61At3g14630Cytochrome P-450protein
DD64At2g31030Oxysterol-binding family protein
DD65At3g10890Putative(1–4)-beta-mannan endohydrolase protein
DD66At1g60985Cysteine-rich protein
DD67At5g11940Subtilase family protein
DD70At5g41090No apical meristem(NAM)family protein
DD72At3g02040Glycerophosphoryl diester phosphodiesterase family protein DD73At5g12060Self-incompatibility protein-related S3(Papaver rhoeas)
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regulation.One of these (DD53)corresponds to the previ-ously identi?ed MYB98,which is expressed in the synergid cells (Kasahara et al.,2005).Expression within the female gametophyte To further validate the expression patterns of these genes and to determine in which cells of the female gametophyte they are expressed,we generated promoter::GFP gene fusions for 45of the 71genes and introduced these constructs into wild-type Arabidopsis plants.The results of this analysis are summarized in Figure 2and Tables 3and 4.Of the 45genes analyzed,43showed expression in the female gametophyte.The female gametophyte expression patterns of these genes fell into nine classes:egg cell only (Figure 2a,b),synergid cell only (Figure 2c,d),central cell only (Figure 2e,f),antipodal cells only (Figure 2g,h),strong expression in the synergid cells accompanied by weak expression in both the egg cell and central cell (Figure 2i,j),strong expression in the central cell accompanied by weak expression in the synergid cells (Figure 2k,l),strong expression in the synergid cells accom-panied by weak expression in the egg cell (Figure 2m,n),strong expression in both the synergid cells and central cell (Figure 2o,p),and strong expression in all female gameto-
phyte cells (Figure 2q,r).The numbers of genes within each of these expression categories are listed in Table 4.Expression throughout the plant To determine whether the 71DD genes are expressed else-where in the plant,we performed real-time RT-PCR with RNA from roots,stems,leaves,young ?owers,anthers,and young siliques.For comparison,we also performed real-time RT-PCR with RNA from ovaries,which contain the ovules and female gametophytes.The results of this analy-sis are summarized in Figure 3and Table S2.In addition,we
analyzed the 45promoter::GFP lines for expression in
developing seeds at 1–2days after pollination.The results of
this analysis are summarized in Table 3and Figure 2s–v.
Most genes (62of 71genes)are expressed in developing
siliques (Figure 3).In most cases (61of 62genes),the silique
RT-PCR signal was much weaker than the ovary signal.
Similarly,most of the promoter::GFP lines (37of 43lines)
exhibited expression in the seed (Table 3).Most of these (29
of 37genes)were expressed in the endosperm.Within the
female gametophyte,these endosperm-expressed genes
were expressed in the central cell and/or the synergid cells.
One gene (DD45)was expressed in the embryo;this gene
was expressed in the egg cell within the female gameto-
phyte.Seven genes had a diffuse signal at the chalazal end
of the embryo sac (Table 3);all of these were expressed in
the antipodal cells within the female gametophyte (Table 3),
suggesting that the chalazal signal was associated with
degenerating antipodal cells.The remaining six genes had
no detectable signal in the seed (Table 3).
Expression in the vegetative organs (root,stems,and
leaves)overall was relatively weak (Figure 3).Although no
gene exhibited strong expression in leaves,four genes
exhibited strong expression in roots (DD54,DD64,and
DD88)or stems (DD90).With two of these (DD54and
DD64),promoter::GFP lines were available to validate
expression in these organs.As shown in Figure S2a,b,Table 2.Summary of the biological functions of the DD genes
Biological process a
No.genes Protein metabolism
6Other metabolism process
9Transcription
3Electron transport or energy pathways
4Transport
2Cell–cell signaling
1Cell organization and biogenesis
1Other
4Biological process unknown
41a Based on the Arabidopsis Information Resource gene ontology biological process designation.Table 1.Continued
Gene
AGI a Description b DD75
At4g29285Cysteine-rich protein DD76
At3g47420Glycerol-3-phosphate transporter protein DD77
At3g14850Expressed protein DD78
At5g46960Invertase/pectin methylesterase inhibitor family protein DD80
At1g31530Endonuclease/exonuclease/phosphatase family protein DD81
At3g59170F-box family protein DD84
At2g39640Glycosyl hydrolase family 17protein DD86
At5g03920Expressed protein DD88
At4g23700Cation/hydrogen exchanger protein DD91
At2g37260WRKY family transcription factor (TTG2)DD96
At4g22870Eucoanthocyanidin dioxygenase
a Arabidopsis Genome Initiative number.
b Based on the Arabidopsis Information Resource annotations (https://www.360docs.net/doc/713808149.html,).
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DD54and DD64were expressed in the elongation zone and
root tip,respectively.Many (54)of the DD genes had
detectable RT-PCR signals in young ?owers and/or anthers
(Figure 3).In most cases (41of 54genes),the signal in young
?owers was weaker than that in anthers,suggesting that the
?ower signal is due to expression in anthers.We the
analyzed expression of the promoter::GFP lines with the
three genes (DD39,DD40,DD46)displaying strong anther
expression and found that two (DD40and DD46)were
expressed in pollen (Figure 2w,x).By contrast,expression of
DD39::GFP was not detected in pollen,suggesting that this
gene is expressed in the sporophytic tissue of the anther.Analysis of GFP expression within the anther proved dif?cult due to high auto?uorescence.Discussion Identi?cation of genes expressed in the female gametophyte We performed a differential expression screen to identify genes expressed in the female gametophyte.This screen identi?ed 71genes exhibiting reduced expression in dif1ovules relative to ms1ovules (Table S1).Of the 45genes analyzed,43show expression in the female gametophyte sc sc sc cc
sc
cc sc cc ac ac ac sc cc ac cc
cc ec sc sc
ec
ec ec e e sc cc
sc
cc ec
ec ec ec ce
ce sc cc sc cc K (a)(c)(f)
(b)(d)(h)(g)(i)
(o)(e)(m)
(n)(p)(q)(k)(l)(j)(s)
(t)(v)(x)(u)
(w)(r)286Joshua G.Steffen et al.
a2007The Authors Journal compilation a2007Blackwell Publishing Ltd,The Plant Journal ,(2007),51,281–292
(Table3),suggesting that most of the remaining26genes also are expressed in the female gametophyte.
Most of the genes we identi?ed are expressed exclusively or predominantly in speci?c cells of the female gameto-phyte.In the context of the ovule,17genes are expressed exclusively(three genes)or predominantly(14genes)in the synergid cells,11genes are expressed exclusively in the antipodal cells,11genes are expressed exclusively(eight genes)or predominantly(three genes)in the central cell,and one gene is expressed exclusively in the egg cell(Table4).In addition,three genes are expressed at high levels in two or more of the female gametophyte cells including two in the synergids and central cell,and one in the synergids,egg, central,and antipodal cells(Table4).
Egg-expressed genes are underrepresented in our collec-tion of genes expressed in the female gametophyte:of the43 genes analyzed,only one gene(DD45)is expressed exclu-sively in the egg cell.This observation suggests that the egg cell is relatively inactive and that alternative approaches will be required to identify egg-expressed genes.For example, several groups have generated expressed sequence tag (EST)collections from isolated egg cells(Cordts et al.,2001; Kumhlehn et al.,2001;Le et al.,2005;Ning et al.,2006; Sprunck et al.,2005;Yang et al.,2006),which should lead to the identi?cation of additional egg-expressed genes.DD45 shares signi?cant sequence similarity with egg cell tran-scripts belonging to the ECA1family in barley(Vrinten et al., 1999)and the EC1family in wheat(Sprunck et al.,2005),and with other genes in the Arabidopsis genome(IHK,JGS,and GND,unpublished data).These observations suggest that DD45belongs to a family of proteins with important functions in the egg cell.
Genes expressed in all cells of the female gametophyte are also underrepresented in our collection of genes expressed in the female gametophyte:only one such gene (DD33)was identi?ed.It is likely that most genes expressed throughout the female gametophyte confer general func-tions,and thus are also expressed in the surrounding sporophytic cells of the ovule.Such genes would be eliminated in our differential expression screen.
With most(37of45genes)of our promoter::GFP lines, GFP is detected in developing seeds,as well as the female gametophyte(Table3).Although analysis of promoter::GFP fusions does not distinguish between perdurance of mater-nally derived GFP and de novo expression,expression in siliques was detected for most(31of37genes)of these genes using real-time RT-PCR(Figure3).These data suggest that the post-fertilization GFP patterns represent post-fertil-ization expression of the corresponding genes.
No expression in the female gametophyte was detected in transgenic plants containing the DD40::GFP and DD54::GFP constructs.These two constructs could be lacking sequences essential for expression within the female gametophyte.Alternatively,these two genes could be false positives.In support of the latter possibility,DD40 and DD54exhibited ms1-dif1fold change values that were only slightly larger than our cut-off value of eightfold (Table S1).Furthermore,expression of these transgenes was detected elsewhere in the plant(Figure2w,x and Figure S2a,b).
Table3.Expression of the DD genes within mature ovules and during early seed development
Gene Expression of promoter::GFP lines
Mature ovule Early seed development
DD1AC(Chalazal)
DD2SC Endo
DD3SC,(EC),(CC)Endo
DD4SC,(EC),(CC)Endo
DD6AC(Chalazal)
DD7CC Endo
DD8SC,(EC),(CC)Endo
DD9CC Endo
DD11SC,(EC),(CC)Endo
DD12SC,(EC),(CC)Endo
DD13AC NE
DD16AC(Chalazal)
DD17SC,(EC),(CC)Endo
DD18SC,(EC),(CC)Endo
DD19(SC),CC Endo
DD22CC Endo
DD23AC NE
DD25CC Endo
DD26AC NE
DD27SC,CC Endo
DD28(SC),CC Endo
DD29AC NE
DD31SC NE
DD32SC,(EC),(CC)Endo
DD33AC,(SC),(EC),(CC)Endo
DD34SC,(EC),(CC)Endo
DD35SC Endo
DD36CC Endo
DD39SC,(EC)NE
DD40NE NE
DD41CC Endo
DD42SC,(EC),(CC)Endo
DD45EC Emb.
DD46SC,CC Endo
DD47AC(Chalazal)
DD52AC(Chalazal)
DD53SC,(EC),(CC)Endo
DD54NE NE
DD56SC,(EC),(CC)Endo
DD61AC(Chalazal)
DD64AC(Chalazal)
DD65CC Endo
DD66CC Endo
DD67SC,(EC),(CC)Endo
DD73(SC),CC Endo
AC,antipodal cells;CC,central cell;EC,egg cell;Emb,embryo;
Endo,endosperm;NE,not expressed;SC,synergid cells;(),weak
expression.
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Comparison with other studies A number of other expression-based screens have been performed to identify female gametophyte-expressed genes.Most signi?cantly,Yu et al.(2005)performed a screen for genes with reduced expression in mutant sporo-cyteless /nozzel ovules in Arabidopsis.That study identi?ed 225genes exhibiting reduced expression in mutant ovules based on microarray analysis,and six of these were valid-ated through analysis of promoter::GUS fusions.Of the six genes characterized,three are expressed exclusively in the antipodal cells and three are expressed in multiple cells of the female gametophyte.In addition,several groups have identi?ed mRNAs from isolated female gametophytes (Yang et al.,2006),isolated egg cells (Dresselhaus et al.,1994;Kumhlehn et al.,2001;Le et al.,2005;Ning et al.,2006;Sprunck et al.,2005;Yang et al.,2006),and isolated central cells (Le et al.,2005).These studies identi?ed 3850female gametophyte transcripts from corn (Yang et al.,2006),963(Yang et al.,2006)and 31(Le et al.,2005)egg cell transcripts from corn,350(Kumhlehn et al.,2001)and 404(Sprunck et al.,2005)egg cell transcripts from wheat,61egg cell transcripts from tobacco (Ning et al.,2006),and 31central cell transcripts from corn (Le et al.,2005).Expression within the ovule was characterized for 15of these genes and these studies identi?ed one gene expressed exclusively in the egg cell (Le et al.,2005),two genes
expressed exclusively in the synergid cells (Yang et al.,
2006),three genes expressed exclusively in the central cell
(Le et al.,2005;Yang et al.,2006),and nine genes expressed
in multiple cells of the female gametophyte (Cordts et al.,
2001;Le et al.,2005;Marton et al.,2005;Yang et al.,2006).
Of the 43genes we analyzed by promoter::GFP fusions,
only four had been identi?ed previously including DD1
(At1g36340),DD9(At1g26795),DD33(At2g20070),and DD56
(At4g30590;Yu et al.,2005).Of these,DD1and DD33exhibit
the same expression patterns as reported previously (Yu
et al.,2005).By contrast,we report expression exclusively in Table 4.Summary of the expression patterns of the DD genes
Expression pattern
No.genes Gene identity SC
3DD2,DD31,DD35EC
1DD45CC
8DD7,DD9,DD22,DD25,DD36,DD41,DD65,DD66AC
11DD1,DD6,DD13,DD16,DD26,DD29,DD47,DD52,DD61,DD64,DD23SC,(EC)
1DD39CC,(SC)
3DD19,DD28,DD73SC,CC
2DD27,DD46SC,(EC),(CC)
13DD3,DD4,DD8,DD11,DD12,DD17,DD18,DD32,DD34,DD42,DD53,DD56,DD67AC,(SC),(EC),(CC)1DD33AC,antipodal cells;CC,central cell;EC,egg cell;SC,synergid cells;(),weak expression.100%10%1%288Joshua G.Steffen et al.
a2007The Authors Journal compilation a2007Blackwell Publishing Ltd,The Plant Journal ,(2007),51,281–292
the central cell for DD9and expression predominantly in the synergid cells for DD56(Table3),whereas Yu et al.(2005) report strong expression in the egg cell and antipodal cells for both genes.The differences in the expression patterns reported for DD9and DD56may have a number of explana-tions including differences in ecotype,reporter gene,or promoter size used to make the promoter fusion.
In addition to expression-based screens,mutant screens have identi?ed many genes functioning in the female gametophyte(Acosta-Garcia and Vielle-Calzada,2004;Aga-she et al.,2002;Choi et al.,2002;Christensen et al.,2002; Ebel et al.,2004;Grossniklaus et al.,1998;Hejatko et al., 2003;Huanca-Mamani et al.,2005;Kim et al.,2005;Kiyosue et al.,1999;Kohler et al.,2003;Kwee and Sundaresan,2003; Luo et al.,1999;Marton et al.,2005;Niewiadomski et al., 2005;Ohad et al.,1999;Pischke et al.,2002;Portereiko et al., 2006a,b;Shi et al.,2005).None of the DD genes have been previously characterized as a result of female gametophyte mutant analysis.Thus,the genes we have identi?ed repre-sent a valuable resource for future mutant analysis.
Dissecting the gene regulatory networks of the female gametophyte
The genes we have identi?ed can be used as starting points to dissect the gene regulatory networks functioning in each cell of the female gametophyte.For example,analysis of the promoters of these genes should lead to the identi?cation of cis-regulatory elements and transcription factors conferring cell-speci?c expression within the female gametophyte (Deplancke et al.,2006).Alternatively,the promoter::GFP lines could be used in screens to identify mutants with changes in cell fate(Kwak et al.,2005).These genes also will be useful as markers for the female gametophyte cell types in the analysis of female gametophyte mutants(Huck et al., 2003;Portereiko et al.,2006a)or for the isolation of these cells using cell-sorting approaches(Galbraith and Birn-baum,2006).
Our screen has identi?ed relatively few regulatory mole-cules.Of the71genes identi?ed,only three encode putative transcription factors.These results suggest that genes encoding transcription factors are expressed at low levels in Arabidopsis,as has been demonstrated in yeast(Holland, 2002).Identi?cation of such genes will probably require alternative approaches such as directed analysis of genes encoding transcription factors using RT-PCR with RNA from ms1and dif1ovules(Kasahara et al.,2005).
Experimental procedures
Plant materials and growth conditions
Seeds were surface sterilized using chlorine gas for3h and ger-minated on plates containing0.5·Murashige and Skoog salts,0.05%2-[N-morpholino]ethane-sulfonic acid(MES),0.5%sucrose, and0.8%Phytagar(Life Technologies,https://www.360docs.net/doc/713808149.html,/). Ten-day-old seedlings were transferred to Scott’s Redi-Earth(http:// https://www.360docs.net/doc/713808149.html,)and grown under24-h illumination.
Confocal analysis of female gametophyte structure Analysis of female gametophyte structure in dif1,ms1,and wild-type ovules was performed as described previously(Christensen et al.,1997,1998,2002).
Ovule isolation and RNA extraction
Ovules were harvested from?owers at stages12c(Christensen et al.,1997)and13(Smyth et al.,1990).Sepals,petals,and stamens were removed from?owers.Pistils were removed from the plant and cut at the top of the ovary to remove the stigma and style,and the remaining ovary tissue was placed into cold70%ethanol and 0.1%Tween20.The ovaries were incubated in this solution for 18h at4°C with rotation.The ovules then were dissected from the ovaries under a solution of70%ethanol and0.1%Tween20.The isolated ovules were stored in70%ethanol until RNA extraction. The RNA was extracted from the ovules using the Arcturus PicoPure RNA isolation kit and following the manufacturer’s instructions (https://www.360docs.net/doc/713808149.html,).Typically, 4l g of RNA was obtained from5000ovules.
Microarray hybridization and data analysis
Two biological replicates were collected for both ms1and dif1 ovules,and RNA was extracted from these four ovule samples. Microarray probes were generated from the RNA according to the manufacturer’s recommendations(Affymetrix,http://www. https://www.360docs.net/doc/713808149.html,),except that the Invitrogen Superscript II kit(http:// https://www.360docs.net/doc/713808149.html,/)was used for?rst strand synthesis and the Enzo BioArray High Yield RNA transcript labeling kit was used for probe labeling(Enzo,https://www.360docs.net/doc/713808149.html,).Fifteen micrograms of biotin-labeled cDNA was added to300l l of hybrid-ization cocktail,and this mixture was hybridized with the arrays for 20h at45°C.Standard Affymetrix post-hybridization wash and stain protocols were then followed using an Affymetrix GeneChips Fluidics Station450.Arrays were scanned on an Affymetrix Gene-Chip Scanner3000.Scanned intensities were pre-processed using GC-RMA(https://www.360docs.net/doc/713808149.html,/jhubiostat/paper1)in the plat-form R(https://www.360docs.net/doc/713808149.html,/).Gene expression values were compared using the Signi?cance Analysis of Microarrays(SAM) program(Tusher et al.,2001)in Microsoft Excel and using the cri-teria of more than twofold change and<15%false positives. Real-time RT-PCR
Ovule tissue was harvested as described above from two independ-ent biological samples.Tissue used for whole-plant expression pro-?ling was harvested from plants and placed immediately into liquid nitrogen.Ovaries were harvested from ms1at?ower stages12c (Christensen et al.,1997)and13(Smyth et al.,1990).Floral cluster tissue includes the in?orescence meristem and?owers at stages1–10 (Smyth et al.,1990).Silique tissue includes siliques at1–2days after pollination.Leaf tissue includes leaves of sizes5–12mm.Roots were harvested from seedlings at11days after germination.Floral stem tissue includes internodes from4-week-old plants.Anthers were collected from?owers at stages11–13(Smyth et al.,1990).
Female gametophyte-expressed genes289
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The RNA was extracted from these tissues using the Qiagen RNeasy kit according to the manufacturer’s instructions(http:// https://www.360docs.net/doc/713808149.html,).Ovule RNA was extracted as described above. DNA contamination was removed from the RNAs using the Ambion TURBO DNA-freeòDNase kit following the manufacturer’s instruc-tions(https://www.360docs.net/doc/713808149.html,/).Following DNase treatment,RNA samples were repuri?ed using the Qiagen RNeasy kit following the manufacturer’s instructions.Aliquots of RNA(1l g)were reverse transcribed using the RETROscriptòKit(Ambion)following the manufacturer’s instructions.
Real-time RT-PCR was performed using the Roche FastStart DNA Master SYBR Green I master mix(https://www.360docs.net/doc/713808149.html,)in a volume of10l l on a Roche LightCycler system.The PCR reaction mixture consisted of0.05l l of cDNA,0.5l M primers,and1·master mix.The PCR primers are listed in Table S3.With analysis of ovule expression,0.2l l of cDNA per reaction was used.In every real-time RT-PCR run,ACTIN2was used as an internal control to normalize for variation in the amount of cDNA template.
The PCR program consisted of a?rst step of denaturation and Taq activation(95°C for5min)followed by45cycles of denaturation (95°C for15sec),annealing(60°C for15sec),and extension(72°C for10sec).To determine the speci?city of the PCR,the ampli?ed products were subjected to melt curve analysis using the machine’s standard method.
For each gene analyzed,three real-time RT-PCR reactions were carried out,including one technical replicate(identical reactions) and one biological replicate(using RNA from independently harvested tissue).The C T values reported in the tables are averages of three independent trials(including technical and biological replicates).
With some genes,expression was not detected in dif1ovules in at least one of the triplicates.In these cases,a C T value of40was used to calculate average fold change.Higher template concentrations were used in each PCR reaction when assaying gene expression throughout the plant therefore we de?ned a C T of36as the absence of detectable signal and this value was used to calculate fold change.
Promoter fusion constructs
The promoter::GFP constructs included400–2000bp of sequence upstream of the translational start codon.These promoter frag-ments were obtained by PCR ampli?cation using the primers listed in Table S4.The resulting PCR products were cloned into pBI101.1-GFP(Clontech,https://www.360docs.net/doc/713808149.html,/)using either the BD InFusion Cloning Kit following the manufacturer’s recommended protocol or through the use of unique5￠and3￠restriction sites introduced during PCR.
Plant transformation
The T-DNA constructs were introduced into Agrobacterium turme-faciens strain LBA4404by electroporation.Arabidopsis plants (ecotype Columbia)were transformed using the?oral dip method (Clough and Bent,1998).Transformed plants were selected by germinating seeds on growth medium containing30l g ml)1 kanamycin.
Analysis of promoter::GFP expression patterns
Tissue from plants containing the promoter::GFP constructs were initially analyzed using an Olympus SXZ12compound UV dissect-ing microscope with epi?uorescence.Ovules were then analyzed using a Zeiss LSM510confocal or a Zeiss Axioplan microscope (https://www.360docs.net/doc/713808149.html,/)to determine which cell types express GFP. Using the confocal microscope,GFP was excited with an argon laser at a wavelength of488nm and emission was detected between 500nm https://www.360docs.net/doc/713808149.html,ing the Zeiss Axioplan microscope,GFP was excited using a UV lamp and a38HE enhanced(e)GFP?lter set?lter, and images were captured using an Axiocam MRm REV2camera (Zeiss).
For analysis of GFP expression in female gametophytes,we emasculated?owers at stage12c,waited24hours,and removed the?owers from the plants.We then removed sepals,petals,and stamen,and dissected off the carpel walls using a30-gauge syringe needle.The pistils were then mounted on a slide in10m M phosphate buffer(pH7.0).Similar procedures were used to analyze siliques from?owers1to2days after pollination.For analysis of GFP expression in female gametophytes,we analyzed5–15T1lines for each construct.
Acknowledgements
We thank Ramin Yadegari,Karen Schumaker,and members of the Drews lab for critical review of this manuscript.We thank the University of Utah Microarray Facility for performing the micro-arrary hybridization and assisting with data processing.We thank Christopher L.Plaisier and Ellen L.Radike for technical assistance. This work was supported by a National Science Foundation(grant no.IOB-0520008)grant to GND and a National Institutes of Health Developmental Biology Training Grant appointment to JGS. Supplementary Material
The following supplementary material is available for this article online:
Figure S1.Real-time RT-PCR Analysis of EF1a,MEA,and FIS2 Figure S2.Expression DD54::GFP and DD64::GFP in the root
Table S1.Genes exhibiting reduced expression in dif1ovules Table S2.Whole plant real-time RT-PCR
Table S3.Real-time RT-PCR primers
Table S4.Promoter fusion information
This material is available as part of the online article from http:// https://www.360docs.net/doc/713808149.html,.
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a2007The Authors
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