pp0901000142[1]

Data Mining the Arabidopsis Genome Reveals Fifteen

14-3-3Genes.Expression Is Demonstrated for Two out of Five Novel Genes1

Magnus Rosenquist*,Magnus Alsterfjord,Christer Larsson,and Marianne Sommarin

Department of Plant Biochemistry,Lund University,P.O.Box117,SE–22100Lund,Sweden

In plants,14-3-3proteins are key regulators of primary metabolism and membrane transport.Although the current dogma states that14-3-3isoforms are not very specific with regard to target proteins,recent data suggest that the specificity may be high.Therefore,identification and characterization of all14-3-3(GF14)isoforms in the model plant Arabidopsis are https://www.360docs.net/doc/9f3912697.html,ing the information now available from The Arabidopsis Information Resource,we found three new GF14 genes.The potential expression of these three genes,and of two additional novel GF14genes(Rosenquist et al.,2000),in leaves,roots,and flowers was examined using reverse transcriptase-polymerase chain reaction and cDNA library polymer-ase chain reaction screening.Under normal growth conditions,two of these genes were found to be transcribed.These genes were named grf11and grf12,and the corresponding new14-3-3isoforms were named GF14omicron and GF14iota,respec-tively.The gene coding for GF14omicron was expressed in leaves,roots,and flowers,whereas the gene coding for GF14iota was only expressed in flowers.Gene structures and relationships between all members of the GF14gene family were deduced from data available through The Arabidopsis Information Resource.The data clearly support the theory that two 14-3-3genes were present when eudicotyledons diverged from monocotyledons.In total,there are1514-3-3genes(grf s1–15) in Arabidopsis,of which12(grf s1–12)now have been shown to be expressed.

14-3-3proteins were discovered by Moore and Perez(1967)as major soluble proteins in brain tissue. Since then it has become clear that14-3-3proteins are present in all eukaryotic organisms(for review,see Rosenquist et al.,2000)and have a wide range of functions(for review,see Finnie et al.,1999;Fu et al., 2000).14-3-3s typically function as dimers and bind to particular phosphorylated motifs in other proteins, thereby regulating the activity of the respective tar-get protein.In1992,the first four plant14-3-3iso-forms were reported:one in Arabidopsis(Lu et al., 1992),one each in Spinacia oleracea and Oenothera hookeri(Hirsch et al.,1992),and one in Hordeum vul-gare(Brandt et al.,1992).The Arabidopsis isoform was identified as part of a protein/G box complex and therefore named“G box factor14-3-3,”in short “GF14.”This designation has been kept for the Ara-bidopsis isoforms,and the four additional14-3-3iso-forms reported by Lu et al.(1994)were named GF14chi,phi,psi,and upsilon,and the original iso-form received the designation GF14omega.The same year,Jarillo et al.(1994)reported two“rare cold-inducible”14-3-3s from Arabidopsis,RCI1and RCI2. The RCI1was identical to GF14psi of Lu et al.(1994), whereas RCI2was a new isoform,very closely re-lated to GF14lambda(Wu et al.,1997)and until now regarded as identical.An additional four14-3-3s, GF14epsilon,kappa,mu,and nu,were identified in 1997(Wu et al.,1997).After that,the members of the 14-3-3gene family in Arabidopsis were regarded to be10.

Although the first Arabidopsis14-3-3isoform, GF14omega,was discovered as a constituent of a protein/G box complex and implicated to be in-volved in regulation of gene transcription(Lu et al., 1992),subsequent research also has shown plant14-3-3s to have a broad range of functions(for review, see Finnie et al.,1999).In particular,plant14-3-3s have been found to be important regulators of pri-mary metabolism.For example,nitrate reductase and Suc phosphate synthase,which are key enzymes in nitrogen and carbon metabolism,respectively,are both inhibited by binding of14-3-3(Bachmann et al., 1996;Moorhead et al.,1996;Toroser et al.,1998), whereas binding of14-3-3to the plasma membrane H?ATPase activates H?pumping(Jahn et al.,1997; Oecking et al.,1997)and hence stimulates nutrient uptake.

In a previous study,using surface plasmon reso-nance,we reported strong differences in binding af-finity between nine GF14isoforms and a known tar-get,the phosphorylated C terminus of the Arabidopsis plasma membrane H?ATPase isoform2,AHA2 (Rosenquist et al.,2000).This suggests that one reason for the large number of14-3-3isoforms in multicellu-lar organisms,such as plants,is functional specificity. Thus,the regulatory role of14-3-3s in carbon and nitrogen metabolism,in nutrient uptake,etc,may be

1This work was supported by the Swedish Foundation for Strategic Research(to C.L.and M.S.),by the Swedish Natural Science Research Council(to C.L.and M.S.),by the Kungliga Fysiografiska Sa¨llskapet(to M.R.),and by the Royal Swedish Academy of Sciences(to M.R.).

*Corresponding author;e-mail magnus.rosenquist@plantbio.lu.se; fax46–462224116.

executed by different14-3-3isoforms.To determine whether this is the case,it is important to first identify and then have access to all isoforms expressed.The completion of the sequencing of the Arabidopsis ge-nome has enabled determination of gene numbers within specific gene https://www.360docs.net/doc/9f3912697.html,ing the information now available via Internet,we recently identified two novel GF14genes(Rosenquist et al.,2000).After fur-ther data mining of the Arabidopsis genome,we have now identified three additional GF14genes,bringing the total number of GF14genes https://www.360docs.net/doc/9f3912697.html,ing reverse transcriptase(RT)-PCR and cDNA library PCR screen-ing,we now demonstrate that two of the five novel genes are expressed,bringing the total number of expressed14-3-3isoforms in Arabidopsis to12.

RESULTS AND DISCUSSION

Examining the Arabidopsis genome revealed three additional novel GF14genes,grf12,grf13,and grf14, in addition to the two recently reported by Rosen-quist et al.(2000),grf11and grf15(Fig.1).One of the two previously reported novel genes,grf15(Table I), only codes for approximately30%of the length of a GF14protein(Fig.1).Furthermore,the82amino acids the gene encodes correspond to the C terminus of a functional14-3-3,and show only44%identity with its closest relatives,GF14psi and upsilon(data not shown).Grf15and its putative product were therefore excluded from further analysis.To deter-mine if the four other novel genes are expressed or not,RT-PCR and cDNA library PCR screening were performed.Thus,primers designed for the novel14-3-3genes,grf s11through14,found in BAC clones F21H2,T1K7,T16E15,and T11I11(Table I),were used to PCR screen a cDNA library made from de-veloping flowers,as well as for RT-PCR on mRNA prepared from leaves,roots,and flowers,respec-tively.Only two of these four novel GF14genes,grf11 and grf12,were found to be expressed(Fig.2).No corresponding cDNAs or ESTs were found in the database,which suggests that these mRNA species are present in low abundancy.The two novel iso-forms were named GF14omicron,encoded by grf11 (F21H2.3),and GF14iota,encoded by grf12(T1K7.15; Table I),in accordance with the designations used earlier,using Greek letters for the proteins and Ara-bic numbers for the genes.Both GF14omicron

and Figure1.Gene maps of all Arabidopsis14-3-3(GF14)genes.Exons are indicated as black boxes and introns as white boxes.

Exon and intron sizes are indicated with the number of bases within in each box.The names of the resulting proteins are

within parentheses.The genes can be divided into two groups based on exon patterns:grf s1through8,and grf s9through

14,excluding the severely truncated grf15that does not readily fit into either group.The five novel genes discussed in this

paper are grf11,grf12,grf13,grf14,and grf15,of which grf11and grf15were already reported by Rosenquist et al.(2000).

The abbreviation grf stands for G box regulating factor.

The Arabidopsis14-3-3Gene Family

GF14iota contain the expected conserved regions found in all other14-3-3s(Rosenquist et al.,2000), and most divergence is found in the N and C termini (Fig.3).The annotation of grf12(T1K7.15)in the Arabidopsis sequencing project did not correspond to the sequence we obtained for the cDNA,suggest-ing that the predicted splicing in the database is incorrect.

The GF14omicron gene grf11was expressed in leaves,roots,and flowers,whereas the GF14iota gene grf12was only expressed in flowers(Fig.2).The expression of these isoforms in flowers,and particu-larly the exclusive expression of GF14iota in this tissue,is interesting because when examining bind-ing of the10previously identified GF14s to the phos-phorylated C terminus of the flower-specific Arabi-dopsis plasma membrane H?ATPase isoform9, AHA9,using surface plasmon resonance,all10 GF14s were classified as nonbinding(data not shown).The C terminus of AHA9differs from the one in AHA2(which showed different degrees of binding to the nine GF14isoforms tested;Rosenquist et al., 2000)on three out of six amino acid positions,and the 14-3-3binding motifs of AHA9and AHA2thus are sufficiently different to suggest that they are activated by different GF14s.Therefore,GF14omicron and par-ticularly GF14iota are good candidates as regulators of AHA9.

The14-3-3genes grf13(T11I11.16;Table I)and grf14 (T16E15.9;Table I)were not expressed in any of the examined tissues.The coding sequence of gene grf14 contains a single nucleotide insertion in exon4 (which we have confirmed by additional sequencing [data not shown])causing a frame shift,which cre-ates a stop codon and loss of the last45amino acids (Fig.1).Given the fact that seven of the17conserved target-binding amino acid residues,as well as the nuclear export signal(Rittinger et al.,1999),are lo-cated within the missing area,grf14does not likely produce a functional14-3-3isoform,although possi-ble expression cannot be ruled out.The fact that no expression of the gene grf13was observed is intrigu-ing.The amino acid sequence it encodes is the most divergent of all GF14s,suggesting that if it is ex-pressed it might have a very specific function.There-fore,our failure to detect transcription may be due to a rather specific expression of this isoform,either regarding developmental stage,tissue,or stress con-dition.We have refrained from naming the gene

Table I.Summary of available database information on Arabidopsis14-3-3(GF14)genes and proteins

The chromosomes and bacteria artificial chromosome(BAC)clones the different genes are found on are indicated,as well as the accession no.of the BAC clone,the gene no.in the BAC,and the accession no.of the mRNA.Accession nos.to putative and expressed gene products are also indicated.

GF14Isoform BAC Gene

mRNA

Accession No.

BAC Accession

No.

Protein

Accession No.

Chromosome1

Putative14-3-3T11I11grf13a(T11I11.16)–AC012680AAG52105b Similar to epsilon T16E15grf14(T16E15.9c)–AC068562AAF87262d Iota T1K7grf12(T1K7.15e)AF335544AC013427AAF98570 Omicron F21H2grf11(F21H2.3)AF323920AC007894AAG47840 Epsilon T16E15grf10(T16E15.8)U36446AC068562P48347 Omega F3F9grf2(F3F9.16)M96855AC013430Q01525 Phi T32G9grf4(T32G9.30)L09111AC079605P46077 Chromosome2

Mu F14N22grf9(F14N22.14)U60444AC007087AAD51784 14-3-3Like F12P23grf15(F12P23.4)–AC007264AAD28654 Chromosome3

Nu F16B3grf7(F16B3.15)U60445AC021640AAD51782 Chromosome4

Chi Contig.25(F23J3)grf1L09112AL161513P42643 Chromosome5

Upsilon F1N13grf5(F1N13?190)L09109AL391145AAB62225

Psi f P1Clone MXI10?

MBB18grf3(MXI10.21)L09110AB005248?

AB005231

P42644

Kappa P1Clone MNA5grf8(MNA5.16)U36447AB011479AAD51783 Lambda(or AFT1)F12B17grf6U68545AL353995P48349 14-3-3-Like protein AFT1g F12B17grf6(F12B17?200)–AL353995CAB89398 RCI2h–RCIIB X74141–CAA52238

a The abbreviation grf stands for G-box regulating factor.

b Incorrect annotation resulted in a shortened N terminus(first exon and5?part of second exon were not included).

c T16E15.9is located1,329bp from grf10(T16E15.8)in the same BAC clone.

d A singl

e nucleotide was counted as a fifth intron when sequence was annotated in the original entry.e Splicing suggested in database is incorrect.

f The isoform GF14psi is identical to the entry RCI1(protein accession no.S47969).

g Putative product due to alternative splicing.

h An additional thymine at the end of exon3of grf6causes a frame shift resulting in this putative isoform,which is probably the result of erroneous sequence analysis.

Rosenquist et al.

products of grf13and grf14as no expression has been shown so far.

When examining the 10previously characterized grf s,we found that the gene coding for GF14psi,grf3(MXI10.21),is divided between P1clone MXI10and MBB18(Table I).A striking discovery was that the annotation in the Arabidopsis genome project of BAC clone F12B17corresponds to a putative protein,“14-3-3-like protein AFT1,”and not to the expressed GF14lambda of Wu et al.(1997).Thus,an alternative splicing of the pre-mRNA of the GF14lambda coding gene,grf6,is suggested,resulting in the putative GF14protein “14-3-3-like protein AFT1”(CAB89398)with a completely different C terminus from the pre-viously reported GF14lambda (P48349).The C termi-nus of the putative “14-3-3-like protein AFT1”is 25amino acids longer than the C terminus of GF14lambda.Our attempts to detect mRNA corre-sponding to “14-3-3-like protein AFT1”have been negative,suggesting that the gene grf6codes for no other product than GF14lambda,and that the pre-dicted splicing in the database is incorrect.There is,in fact,no experimental evidence to date that alter-native splicing of 14-3-3genes exists.Thus,the an-notation in the database of the grf6gene (F12B17_200)in the BAC F12B17should be changed to correspond to GF14lambda.The RCIIB gene reported by Jarillo et al.(1994)results in a protein,RCI2(CAA52238),that only differs from GF14lambda in the last eight amino acids.The RCIIB gene cannot be found among the data from the Arabidopsis genome project,but is due to an additional thymine at the end of exon 3in grf6(Fig.1).Because this is most likely the result of erroneous sequence analysis,RCIIB has been ex-cluded from the present analysis.

Examining the gene maps of all GF14genes,one can observe two major groups based on exon pat-terns;grf s 1through 8form one group,where all genes are expressed,and grf s 9through 14form a second group (Fig.1).Unrooted phylogenetic trees based on either the resulting mRNA or amino acid sequences (including the putative products of grf13and grf14)show that the gene products of grf s 1through 8form three branches in one part of the tree,and that the products of grf s 9through 14form the other,more divergent,part of the tree (Fig.4;mRNA data not shown).It is intriguing that a branch-wise specificity was found when the affinity between nine of the GF14s and the phosphorylated C terminus of AHA2was measured (Rosenquist et al.,2000).

In Figure 5,the locations of the different BAC clones containing GF14genes are indicated on

the

Figure 2.Detection of novel 14-3-3gene transcripts in flower (A,B,and F),leaf (C and E),and root (D)tissues by RT-PCR and cDNA library PCR.Primary RT-PCR reactions were performed with primers corresponding to exon 2(see Fig.1)of grf11(omicron;A and C),grf12(iota;A and C),grf13(A,C,and D),and grf14(B,C,and D).Subsequent RT-PCR reactions were performed with full-length prim-ers for grf11on leaf (E)and root (D)cDNA,and for grf12on flower (F)and root (D)cDNA.Products were run on 1.5%(w/v)agarose gel (inverted image)and bands of interest are indicated with black arrowheads.The identities of these bands were confirmed by se-quencing.Sizes of standard (std)markers are indicated to the left.Note that the band grf12(iota)exon2(A)only corresponds to 290bp of the exon due to optimization of primer positions,and therefore is 17bp shorter than grf11(omicron)exon2.

The Arabidopsis 14-3-3Gene Family

chromosomes,and two major duplication events 170and 50million years ago (Vision et al.,2000),which may be responsible for the formation of some of the isoforms,are indicated.The genes grf5(upsilon)and grf7(nu)are located in regions of chromosome 5and 3,respectively,that were duplicated 170million years ago.However,considering the very close rela-tion between GF14upsilon and nu (Fig.4),it is more likely that the duplication leading to the formation of grf5(upsilon)and grf7(nu)was a much more recent event.The gene grf2(omega)and grf13are situated close together on chromosome 1,and this is also the case for grf4(phi)and grf11(omicron).These two

gene pairs,as well as grf1(chi)on chromosome 4,are located within areas that were duplicated 50million years ago.It is notable that no other GF14gene is located close to grf1(chi),suggesting that a gene related to grf11(omicron)and grf13has been lost.The fact that GF14phi,chi,and omega are much more closely related than GF14Grf13(the putative product of grf13)is to omicron (Fig.4)is noteworthy because they all seem to originate from the major duplication event 50million years ago (Fig.5).Considering their common evolutionary history,one would expect GF14Grf13and omicron to show about the same high homology as GF14phi,chi,and omega (Fig.4).

As

Figure 3.DNA sequences of the two expressed novel Arabidopsis 14-3-3(GF14)genes,grf11and grf12,and corresponding amino acid sequences for the proteins,GF14omicron and GF14iota,respectively.Conserved identity between all expressed GF14s is indicated with gray boxes,and conserved homology is indicated with white boxes.

Rosenquist et al.

proteins involved in complex formation,14-3-3s are subject to a high selection pressure,which acts to conserve these proteins.Thus,if GF14phi,chi,and omega have retained their functional specificity this will have limited their evolution.In a converse man-ner,if GF14omicron and the putative gene product of grf13(GF14Grf13)have obtained different functions this may explain their low homology,or if grf13is not expressed at all,it has of course evolved without any constraints.

Previous phylogenetic analyses of plant 14-3-3iso-forms indicate that at the time of the split into eud-icotyledons and monocotyledons,about 200million years ago,two 14-3-3isoforms were already present (Wang and Shakes,1996;Rosenquist et al.,2000).This is in accordance with both the gene map in Figure 1and the phylogenetic tree in Figure 4.The gene map (Fig.1)divides the Arabidopsis 14-3-3genes into two major groups;grfs 1through 8,and grfs 9through 14,respectively.The phylogenetic tree (Fig.4)similarly suggests two major branches:the GF14upsilon/nu/psi,phi/chi/omega,and lambda/kappa branches forming a major branch in the lower part of the tree,and the GF14mu,iota,omicron,epsilon,Grf14,and Grf13forming an upper major branch.One of the gene pairs,grf2(omega)/grf13,grf4(phi)/grf11(omi-cron),or grf1(chi)and its putative aborted neighbor,is probably the direct descendant of the two ancestral 14-3-3genes (Fig.5).

The completion of the sequencing of the Arabidop-sis genome finally enabled the determination of the total number of 14-3-3genes to be 15.The challenge now is to examine the expression pattern of all GF14isoforms,to identify new target proteins,and to de-termine the functional specificity of all GF14iso-forms,in order to reveal all the cellular processes involving regulatory 14-3-3proteins in the model plant Arabidopsis.

MATERIALS AND METHODS Examination of Database Entries

GF14database entries were obtained by a simple key-word search at the National Center for Biotechnology In-formation (https://www.360docs.net/doc/9f3912697.html,).A blast search of the Arabidopsis BAC clones at The Arabidopsis Infor-mation Resource (https://www.360docs.net/doc/9f3912697.html,)revealed additional isoforms,and allowed mapping of the https://www.360docs.net/doc/9f3912697.html,putational Analysis of GF14Gene Sequences DNA sequences corresponding to GF14genes were ob-tained from the GenBank database via the National Center for Biotechnology Information.Clustal W multiple align-ments of sequences were performed using the MacVector 7.0software (Oxford Molecular Group Plc,Oxford).The alignments were carried out using the Blossum series ma-trix,with an open gap penalty of 10and an extend-gap penalty of 0.05.Alignments were examined and adjusted manually.A heuristic search using the maximum parsi-mony method was done on the alignment using the PAUP 4.0b4a software (Sinauer Associates,Inc.Publishers),with gaps treated as missing data.The tree bisection reconnec-tion branch-swapping algorithm was used.Tree building and calculation of Bootstrap values,with 12,000replicates,were also carried out using the PAUP 4.0b4a software.Gene maps were drawn from splicing patterns indicated in the respective BAC entries.The splicing patterns of grf13(T11I11.16)and grf14(T16E15.9)suggested in the database were incorrect,and hence adjusted.Plant Material

Arabidopsis ecotype Columbia-0was grown on soil at 22°C with a 9-h-light/15-h-dark photoperiod (170?E)and 70%relative humidity.Root cultures were grown in dark-ness in flasks with 100mL of Murashige and Skoog me-dium,pH 5.7,supplemented with 3%(w/v)Suc (Malamy and Benfey,1997).Plants for flower RNA preparation were grown under a long-day light regime (16h light).mRNA Isolation and RT-PCR

Leaves were harvested after 4weeks and total RNA was prepared with a conventional phenol/chloroform extrac-tion.Subsequent mRNA isolation was carried out accord-ing to the manufacturer ’s protocol using the QIAGEN Oli-gotex mRNA Purification Kit (Merck KGaA,

Darmstadt,

Figure 4.A phylogenetic tree with topology representative for the Arabidopsis 14-3-3(GF14)protein family.The putative gene prod-ucts of grf13and grf14are designated Grf13and Grf14,respectively.A heuristic search using the maximum parsimony method was done on the alignment of GF14protein sequences using the PAUP 4.0b4software (Sinauer Associates,Inc.Publishers,Sunderland,MA),with gaps treated as missing data.Bootstrap values for 12,000replicates are indicated on each branch.The hypothetical 14-3-3-like protein coded by grf15(Table I;Fig.1)was excluded from the analysis due to strong deviance in both sequence and length.The 82amino acids encoded by grf15show highest homology (44%identity)to GF14psi and upsilon,and correspond to the C terminus of a functional 14-3-3(data not shown).

The Arabidopsis 14-3-3Gene Family

Germany).First-strand synthesis was performed with the First Strand cDNA Synthesis Kit (Boehringer Mannheim,Basel).The same procedures were used on roots harvested after 10weeks of cultivation.Total RNA was extracted according to the method developed by Chang et al.(1993)from a mix of flowers in all stages of anthesis.

Primary RT-PCR was carried out with Taq polymerase (Roche,Basel)and primers designed against the largest exon of the newly identified GF14genes (see Fig.1),as well as for an alternatively spliced grf6.Fragments obtained were run on a 1.5%(w/v)agarose gel and sequenced for identification.RT-PCR subsequently was done with prim-ers corresponding to the full-length genes.Full-length frag-ments were cloned into the TA vector pGEM (Promega Corp.,Madison,WI),and sequenced.Restriction sites were added to full-length primers to facilitate cloning into suit-able vectors.

cDNA Library PCR Screening

A cDNA library from Arabidopsis ecotype Landsberg erecta developing flowers (Weigel et al.,1992)was obtained from the Arabidopsis Biological Resource Center.Conven-tional in vivo excision on the entire library was performed on infected Escherichia coli XL1-Blue with E.coli SOLR (AH Diagnostics,Stockholm).PCR with exon-specific primers was performed on the excised library,for detection pur-pose only.Full-length clones were amplified from cDNA

synthesized from the flower mRNA preparation from Ara-bidopsis (Columbia-0)described above.

ACKNOWLEDGMENT

We thank Dr.Ove Nilsson (Swedish Agricultural Uni-versity,Ume ?,Sweden)for preparation of total RNA from Arabidopsis flowers.

Received April 6,2001;returned for revision April 30,2001;accepted May 18,2001.

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Figure 5.Arabidopsis chromosome map.BAC clones harboring 14-3-3(GF14)genes are indicated with black lines.Gene names,with corresponding gene products written in parentheses,are indicated by each BAC.Centromeres are indicated with white circles.The gray dashed arrow indicates a major duplication event that took place 170million years ago,and the two gray arrows indicate another major duplication event that took place 50million years ago (Vision et al.,2000).Thin dashed and dotted lines connect relatively closely related genes and hence suggest other duplication events.The gene grf14(T16E15.9)is located 1,329bases from grf10(T16E15.8)within the same BAC clone.Given the low homology of the severely truncated 14-3-3gene grf15in F12P23,no connection to other genes is indicated.

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The Arabidopsis14-3-3Gene Family