209–222www.elsevier.com/locate/gene

Gene 391 (2007)

Identification of PTM5 protein interaction partners, a MADS-box geneinvolved in aspen tree vegetative development

Leland J. Cseke 1, Namritha Ravinder 1, Ajay K. Pandey, Gopi K. Podila ⁎

The University of Alabama in Huntsville, Department of Biological Sciences, Huntsville, AL, 35899, USA

Received 20 September 2006; received in revised form 22 December 2006; accepted 28 December 2006Available online 19 January 2007

Abstract

In a past article, our lab described the identification and characterization of a novel vegetative MADS-box gene from quaking aspen trees,Populus tremuloides MADS-box 5 (PTM5). PTM5 was shown to be a member of the SOC1/TM3 class of MADS-box genes with a seasonalexpression pattern specific to developing vascular tissues including the vascular cambium, the precursor to all woody branches, stems, and roots.Since the proper function of MADS-box proteins is dependent on specific interactions with other regulatory proteins, we further examined PTM5protein–protein interactions as a means to better understand its function. Through yeast two-hybrid analyses, it was demonstrated that, like otherSOC1/TM3 class proteins, PTM5 is capable of interacting with itself as well as other MADS-box proteins from aspen. In addition, yeast two-hybrid library screening revealed that PTM5 interacts with two non-MADS proteins, an actin depolymerizing factor (PtADF) and a novel leucine-rich repeat protein (PtLRR). In situ RNA localization was used to verify the overlapping expression patterns of these genes, and transgenic studiesshowed that over-expression of PTM5 in aspen causes alterations in root vasculature and root biomass development consistent with the cellgrowth and expansion functions of related ADF and LRR genes. These results suggest that the interaction of vegetative MADS-box genes withspecific protein cofactors is a key step in the mechanisms that control woody tissue development in trees.© 2007 Elsevier B.V. All rights reserved.

Keywords: Actin depolymerizing factor (ADF); Cambium; Leucine rich repeat (LRR); Populus; Tree development; Vascular

1. Introduction

MADS-box genes are a class of highly conserved transcrip-tional regulators found in all eukaryotic kingdoms. PlantMADS-box genes play major roles in the development of floraland vegetative meristems, and their structure and function inplants, including tree species, have been reviewed multipletimes (Cseke and Podila, 2004; Kaufmann et al., 2005; De

Abbreviations:AD, Activating Domain; ADF, Actin Depolymerizing Factor;AG, AGAMOUS; AGL, AGAMOUS-like; AP1, APETALA1; BD, BindingDomain; DIG, Digoxigenin; EST, Expressed Sequence Tag; ETL, EucalyptusTM3-like; JGI, Joint Genome Institute; LRR, Leucine Rich Repeat; PTM5, Po-pulus tremuloides MADS-box 5; RT, Reverse Transcription; SEP, SEPALLATA;SQUA, SQUAMOSA; SOC1, Suppressor of Over-expression of CONSTANS 1;TM3, TOMATO MADS 3; UTR, Untranscribed Region.⁎ Corresponding author. Department of Biological Sciences, The University of

Alabama in Huntsville, 142 Wilson Hall, Huntsville, AL 35799, USA. Tel.: +1256 824 6263; fax: +1 256 824 6305.

E-mail address: podilag@uah.edu (G.K. Podila).1 These authors contributed equally to the paper.

0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.gene.2006.12.033

Folter and Angenent, 2006; Leseberg et al., 2006). One of thekey features of MADS-box proteins is that they require in-teraction with members of their own family or with other non-MADS protein cofactors to attain functional protein dimers andprotein complexes. The primary protein domain responsible forspecific protein–protein interactions is the K-domain, a coiled-coil region with a structure similar to that of keratin. However,the I-domain and sections of the C-domain have also beenimplicated in interaction specificity (Cseke and Podila, 2004;Kaufmann et al., 2005).

Currently, the primary method used to identify protein inter-action partners is yeast two-hybrid screening of expressedcDNA libraries. Using this method, a number of MADS-boxprotein interactions with non-MADS proteins have been identi-fied. For example, in Arabidopsis, AGAMOUS was reported tointeract with a non-MADS leucine-rich repeat protein (FLOR1)as well as a carpel-specific acid phosphatase protein complex(Gamboa et al., 2001; Acevedo et al., 2004). It has been shownthat OsMADS18, an AP1/SQUA class MADS-box protein fromrice, interacts with a non-MADS seed-specific histone fold

210 L.J. Cseke et al. / Gene 391 (2007) 209–222

protein called NF-YB (Masiero et al., 2002). In Antirrhinum,the PLE protein, an AG-class MADS-box protein, interacts witha non-MADS leucine zipper protein called MIP1, where MIP1seems to act as part of a ternary complex (Causier et al., 2003).AGL24 in Arabidopsis has been shown to interact with ameristematic receptor-like kinase (MRLK) involved in thetranslocation of AGL24 from cytoplasm to the nucleus (Fujitaet al., 2003). Similarly, MADS-box protein interaction with aprotein having phosphatase activity was identified during theanalysis of transcription factor protein complexes (Remenyiet al., 2004).

Such studies are beginning to shed some light on the diver-sity of MADS-box interactions and their potential functions;however, the study of MADS-box protein interactions betweenmembers of their own family is much more highly developed.For example, a recent comprehensive yeast two-hybrid screenusing the entire collection of Arabidopsis MADS-box proteinsidentified over 260 possible MADS-box homo- and heterodimercombinations (De Folter et al., 2005). Despite such exhaustivecharacterization of interactions in herbaceous species, only onestudy pertaining to tree MADS-box protein interactions hasbeen reported thus far (Sundstrom and Engstrom, 2002). TheAP3/PI class MADS-box proteins DAL11, DAL12, and DAL13from Norway spruce have activities in the development of maleorgan primordia, and like other members of the AP3/PI class,these proteins were shown to form both homo- and heterodimers(Sundstrom and Engstrom, 2002). Thus, despite the fact thattrees have some significant differences in floral and vegetativedevelopment compared to herbaceous species (Taylor, 2002),there remains a significant void in the understanding of MADS-box genes in woody tree species.

Poplar species, such as trembling aspen (Populus tremu-loides), are one of the most wide-spread tree species throughoutthe world, and they are the most highly used species in thelumber industry due to their rapid growth and ability to re-generate from their roots after cutting (Taylor, 2002; Frey et al.,2003). Our lab has isolated a variety of aspen MADS-box genesthat fall within the major phylogenetic classes of MADS-boxgenes. Most of these genes were shown to be important in theprocesses of flower formation (Cseke et al., 2003a, 2005; Csekeand Podila, 2004). However, several MADS-box genes werealso identified from developing vascular tissues including thevascular cambium, the secondary meristem responsible for theincrease in the girth of stems, branches, and roots. PTM5(P. tremuloides MADS-box 5) is seasonally expressed in primaryand secondary vascular tissues including the vascular cambium,differentiating xylem, and the vascular bundles of expandingleaves, petioles, and catkins (Cseke et al., 2003b). Phylogeneticanalysis showed that PTM5 is a novel member of the SOC1/TM3 class MADS-box genes, sharing overall identity withother SOC1/TM3 class proteins but varying significantly in theC-terminal domain (Cseke et al., 2003b). This, along with itsexpression pattern in secondary meristems that extends through-out the tree, suggested that PTM5 may have a function in wholetree development.

To address this possibility, two studies were undertaken thataimed to (1) identify the PTM5 protein interaction partners as a

means to expand upon its function and (2) determine the con-sequences of manipulating PTM5 gene expression in transgenicaspen. In this study, we report that PTM5 is capable of interactingwith itself as well as two other non-MADS proteins, an actindepolymerizing factor and a novel leucine-rich receptor protein.In addition, transgenic studies carried out over the past threeyears indicate that PTM5 plays a role in the development of rootvasculature and root biomass. Through such studies, our labseeks to determine the possible roles of tree vegetative MADS-box genes in the secondary meristematic processes leading towood formation above-ground and root formation below-ground.

2. Materials and methods

2.1. Plant materials

Terminal bud, young leaf, stem, and root tissues used for yeasttwo-hybrid cDNA library and in situ RNA hybridization, werecollected from young male and female aspen trees propagatedand grown in a greenhouse under controlled conditions (Csekeet al., 2003b). The development of aspen floral tissues waspreviously categorized into 6 distinct stages, and Stage 4 floralbuds were taken from P. tremuloides Michx. populations iden-tified and marked at specific locations in Houghton, Michigan(88.6° latitude/47.1° longitude) (Cseke et al., 2003a,b, 2005).

2.2. Yeast two-hybrid analyses

For identification of protein interaction partners, a combi-nation of LexA- and GAL4-based MATCHMAKER Systems(BD Biosciences/Clontech, CA) was used to detect specificprotein–protein interactions in yeast.

2.2.1. Yeast two-hybrid MADS vs. MADS interaction analysisTo test PTM5 one-on-one interactions with selected MADS-

box genes isolated in our lab, the MATCHMAKER LexATwo-Hybrid System was used as per manufacturer's instructions (BDBiosciences/Clontech, CA). Previous studies have shown thatthe highly conserved MADS domain of MADS-box proteinshas DNA binding activity that can cause non-specific activationof the reporter gene in yeast two-hybrid studies (Moon et al.,1999). Therefore, this domain was not used in the constructionof the binding domain (BD) or activating domain (AD) vectors,and truncated baits containing only the IKC regions of eachgene were constructed by directional cloning of PCR productsinto either the BD containing vector (pLexA) or the AD con-taining vector (pB42AD) (BD Biosciences/Clontech, CA). Thesequence and reading frame of each construct were verifiedusing an ABI 310 Genetic Analyzer (Applied Biosystems, CA)and the DYEnamic ET Terminator Cycle Sequencing Kit (GEHealthcare, NJ). The plasmids obtained were labeled as follows:Binding domain containing plasmids with PTM1, 2, 3, 5 and 6were named 1BD, 2BD, 3BD, 5BD, and 6BD respectivelywhereas activating domain containing plasmids were named1AD, 2AD, 3AD, 5AD, and 6AD respectively.

The reporter yeast strain EGY48 [p8op-lacZ] was co-trans-formed with 5BD and each of the AD vectors (including the

211L.J. Cseke et al. / Gene 391 (2007) 209–222

empty AD vector) as well as the reciprocal experiment with 5ADand each of the BD vectors (including the empty BD vector).Additional control transformations were also performed, includ-ing three negative controls (1) empty BD + empty AD [pLexA +pB42AD]; (2) the kit negative control vectors containing insertsthat do not interact [pLexA-Lam + pB42AD-T]; and (3) our ownnegative control using 5BD + pB42AD-T (the AD vector con-taining an insert that does not interact with PTM5). The kitpositive control [pLexA-53 + pB42AD-T] as well as a control forfalse positive self-activation [pLexA-Pos] was also prepared.Each of the transformants was tested by growth and blue colorformation on each of four different selection media as well asβ-D-galactosidase quantitative assays. We refer to the mediaused as R1 [SD/-His/-Ura/-Trp + X-Gal + BU salts], R2 [SD/-His/-Ura/-Leu/-Trp + X-Gal + BU salts], R3 [SD/Gal/Raf/-His/-Ura/-Trp + X-Gal + BU salts], and R4 [SD/Gal/Raf/-His/-Ura/-Leu/-Trp +X-Gal + BU salts]. Each procedure was carriedout according to the manufacturer's instructions (BD Bios-ciences/Clontech, CA).

2.2.2. Yeast two-hybrid library screeningYeast two-hybrid library construction and screening were

performed in a GAL4-based system using the BD MATCH-MAKER Library Construction and Screening Kit as permanufacturer's instructions (BD Biosciences/Clontech, CA).Construction of the aspen AD vector library was prepared frompooled RNA samples from vegetative tissues including terminalbud, young leaf, young stem, and root. RNA from these tissueswas reverse transcribed using MMLV reverse transcriptase, andthe AD library was constructed by in vivo homologous recom-bination, allowing the cDNAs to be cloned into the AD plasmid(pGADT7-Rec) within the yeast cells. The PTM5 BD baitvector (5BD) was constructed by directional cloning of the PCRproducts into this kit's BD containing vector (pGBKT7).

For screening and analysis of interaction positive clones, thereporter yeast strain AH109 was co-transformed with AD andBD vectors containing the cDNA library and PTM5-pGBKT7bait vector respectively. Approximately 30,000 transformantswere screened on each of the kit's high-stringency drop outselection media. Following such screening, 118 colonies sur-vived the selection conditions, and the AD library inserts wereisolated by colony PCR and sequenced using AD specificprimers (see Section 2.3).

2.3. Analysis and verification of positive interaction clones

The AD cDNA inserts from the yeast colonies showingpositive interactions were isolated by performing colony PCRusing AD vector specific 5′ insert screening amplimer (5′-CTATTCGATGATGAAGATACCCCACCAAACC-3′) and 3′insert screening amplimer (5′-GTGAACTTGCGGGGTTTTT-CAGTATCTACGAT-3′) primers (BD Biosciences, CA). Col-ony PCR was performed under the following conditions: 94 °C(2 min); 35 cycles of 94 °C (30 s), 57 °C (30 s), 72 °C (1 min)and a final extension at 72 °C for 5 min. The PCR products werepurified and sequenced using an ABI 310 Genetic Analyzer(Applied Biosystems, CA) and the DYEnamic ET Terminator

Cycle Sequencing Kit (GE Healthcare, NJ). The sequences thatwere obtained were identified using both the JGI Populusgenome database (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html) as well as NCBI Blast searches. For the clonesthat appeared more than one time in the screening process, yeastAD plasmid DNA recovery was performed by treatingovernight yeast cultures with lyticase enzyme and then usingthe lysed culture as the starting material for extracting plasmidDNA using a QIAGEN plasmid isolation kit (QIAGEN, CA).These AD clones were subsequently transformed into TOP-10chemically competent E. coli cells (Invitrogen, CA).

To verify the results obtained fromY2H library screening, therecovered AD plasmid DNAwas used to perform Y2H one-on-one assays. The reporter yeast strain AH109 was co-transformedwith the PTM5pGBKT7 BD vector and each of the AD vectors(including the empty AD vector). Additional control transfor-mations were also performed, including three negative controls(1) empty BD + empty AD [pGBKT7 + pGADT7-Rec]; (2) thekit negative control vectors containing inserts that do not interact[pGBKT7-Lam + pGADT7-RecT]; and (3) our own negativecontrol using PTM5-pGBKT7 + pGADT7-RecT (the AD vectorcontaining an insert that does not interact with PTM5). The kitpositive control [pGBKT7-53 + pGADT7RecT] was also pre-pared. Each of the transformants was tested by growth on each ofthree different selection media as well as β-D-galactosidasequantitative assays. We refer to the media used as S1 [SD/-Leu/-Trp], S2 [SD/-His/-Leu/-Trp], and S3 [SD/-Ade/-His/-Leu/-Trp].

2.4. Phylogenetic analysis and sequence alignments

The phylogenetic analysis was done as described (Csekeet al., 2003a,b) on protein sequences obtained from the Gen-Bank database and sequences obtained from the JGI poplargenome database (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html). Heuristic search analysis using parsimo-ny was done with the PAUP 4.0b10 program on Clustal Wsequence alignments followed by Bootstrap analysis using 1000replicates with amino acids treated as unrooted characters(PAUP 4.0b10, Sinauer Associates, Sunderland, MA). HumanADF/Cofilin (NP_005498) was used to root the ADF tree andArabidopsis FLOR1 (AF136588) was used to root the LRRtree. Accession number for the sequences used in the analysiswas as follows:

[P. tremuloides Michx.] PtADF1, DQ821521; PtLRR1,DQ821522: [Populus alba × Populus tremula] PaPtADF,CF230308: [P. tremula × P. tremuloides] PtPtLRR, BU894996:[Arabidopsis thaliana] AtADF1, NP_190187; AtADF2,NP_566882; AtADF3, NP_851227; AtADF4, NP_851228;AtADF5, NP_565390; AtADF6, NP_565719; AtADF7,NP_194289; AtADF8, NP_567182; AtADF9, NP_195223;AtADF10, NP_171680; AtADF11, NP_190185; AtADF12,NP_568769; NP_174625; NP_197731: [Lycopersicon esculen-tum] Cf-4A, CAA73187; Cf-5, AAC78591; Hcr2-OA,AAC78592; Hcr2-OB, AAC78593; Hcr2-5B, AAC78595;Hcr2-5D, AAC78596; LeSHY, AAR27431: [Lycopersiconpimpinellifolium] Cf-2.1, AAC15779; Cf-2.2, AAC15780; Cf-9, CAA05274; Hcr2-2A, AAC78594; Hcr2-p1, AAX19023;

212 L.J. Cseke et al. / Gene 391 (2007) 209–222

Hcr9-9A, AAT77547; Hcr9-9DC, AF401036; Hcr9-9DC1,AAT77548; Hcr9-9DC2, AAT77549; Hcr9-9DC3, AAT77550:[Petunia hybrida] PhSHY, AF325673.

Sequences were aligned using Clustal W alignments, andpotential sequence motifs were identified using a combinationof web-supported programs as previously described (Csekeet al., 2003b).

2.5. In situ RNA localization

Stage 4 aspen flower buds, terminal buds, Stage 3 youngleaves, Stage 6 young stems, and root tips were used to test thespatial expression patterns of PTM5, PtADF and PtLRR. Tissuesamples were fixed and embedded in paraffin as per proceduresstandardized earlier (Cseke et al., 2003b, 2004). Twelve micro-

Fig. 1. Yeast two-hybrid one-on-one interaction analysis demonstrating the presenceconditions, R1 [SD/-His/-Ura/-Trp + X-Gal + BU salts], R2 [SD/-His/-Ura/-Leu/-Trp +[SD/Gal/Raf/-His/-Ura/-Leu/-Trp + X-Gal + BU salts]. Rows 1 through 6 show PTMPTM1, 2, 3, 5, 6, and the empty AD vector. Rows 12 through 17 show PTM5–AD vec5, 6, and the empty BD vector. Rows 10 and 21 are positive interaction controls supRows 11 and 22 are false positive controls where the yeast cells harbor a vector tha

meter tissue sections were obtained using a microtome and werefixed onto pre-cleaned Superfrost®/Plus microscope slides(Fisher Scientific). The slides were hydrated by treating themwith a series of ethanol and water and made ready for hybri-dization as described (Cseke et al., 2004). Tissue samples werehybridized using DIG-labeled sense (control) and antisensemRNA probes made for the unique regions of PTM5, PtADFand PtLRR following manufacturer's instructions (Roche DIGLabeling Kit; Roche diagnostics, IN). These regions of thePTM5, PtADF and PtLRR genes yield no significant matcheswith any other cloned gene or EST other than their exact matchin NCBI Blast searches or within the poplar genomic sequencedatabase. The DIG Nucleic Acid Detection Kit was used forcolor detection (Roche diagnostics, IN) as previously described(Cseke et al., 2003b). Each of approximately 35 sections per

of growth (+/−) and β-galactosidase activity (+/−) under four different selectionX-Gal + BU salts], R3 [SD/Gal/Raf/-His/-Ura/-Trp + X-Gal + BU salts], and R45–BD vector (5BD) interactions with the various PTM–AD vectors harboringtor (5AD) interactions with the various PTM–BD vectors harboring PTM1, 2, 3,plied by the kit while rows 7, 8, 9, 18, 19, and 20 are various negative controls.t causes auto-activation of the system [pLexA-Pos].

213L.J. Cseke et al. / Gene 391 (2007) 209–222

experiment was visualized using a Nikon dissecting microscopeequipped with a Nikon digital camera system.

2.6. PTM5 binary vector construction and over-expression intransgenic aspen

The binary vector for over-expression of PTM5 (PTM5 sense)was constructed by inserting a double CaMV 35S/AMV RNA4promoter, PTM5 coding region (minus the 5′ and 3′ UTRs), and3′ NOS terminator sequence into the pTCS5 binary vector aspreviously described (Cseke et al., 2005). pTCS5 has a NPTII/GUS fusion gene driven by a 35S-promoter for kanamycinselection and GUS reporter expression. The binary vector wasmobilized into Agrobacterium tumefaciens strain C58/pMP90by a triparental mating method (Cseke et al., 2004). Aspen treeleaf disks were transformed with the PTM5-sense construct aswell as the pTCS5 vector harboring no inserts (used as a control)followingmethods established in our lab (Tsai et al., 1994; Csekeet al., 2005). After kanamycin selection on 100 μg/ml, 21 aspenlines for PTM5 sense and 10 lines for pTCS5 were obtained.Initial integration of each construct was confirmed for each linebased on strong GUS activity in all tissues and construct-specificgenomic PCR. Final conformation of transformation was deter-mined by RNA blot analysis of expressed construct in matureleaf tissue.

3. Results

3.1. PTM5 interacts with itself as well as two non-MADSproteins

Using previously isolated and characterized MADS-boxgenes, a collection of pLexA binding domain (BD) and pB42ADactivating domain (AD) fusion vectors was generated for PTM1,2, 3, 5, and 6. To examine the ability of PTM5 to form associa-tions with theseMADS-box proteins, we compared the results ofthe PTM5–BD vector (5BD) as it interacted with the various ADvectors as well as the results of the PTM5–AD vector (5AD) as

Fig. 2. Liquid β-galactosidase assays demonstrating the relative strength of PTM5 intsimilar to those in Fig. 1 using the 5BD vector in combination with the PTM–AD vectPositive Control is the interaction of pLexA-53 with pB42AD-T, supplied by the kit.Data is expressed as Relative Galactosidase Units, and error bars represent the resul

it interacted with the various BD vectors (Fig. 1). Combinedwith appropriate positive and negative controls, the resultsindicated that, while neither the 5BD or 5AD vectors are self-activating in yeast cells (compare rows 4 and 15 in Fig. 1), eachof the BD vectors harboring PTM1, 2, 3, and 6 was self-activating (compare rows 1, 2, 3, and 5 with rows 12, 13, 14, and16 in Fig. 1). The AD vectors of PTM1, 2, 3, and 6 were notself-activating, and using these vectors, it was clearly shown that5BD can interact with each of the testedMADS-box proteins. Toobtain information on the strength of these interactions, β-D-galactosidase quantitative assays were performed (Fig. 2).PTM5 interaction with the other MADS-box genes (includingitself) showed levels of β-D-galactosidase activity approximate-ly double those of the positive controls, indicating a very strongprotein–protein interaction. These results are similar to resultsseen with the Arabidopsis SOC1/TM3 class MADS-box pro-teins, where some of these proteins are able to interact withthemselves as well as other A- and E-class MADS-box proteins(De Folter et al., 2005). However, only the PTM5–PTM5interaction seems feasible in aspen tree vegetative tissues sincethe other genes have been shown to be floral-specific and are notexpressed in the same tissues as PTM5 (Cseke et al., 2003a,b,2005). Likewise, no other MADS-box factors were isolatedduring yeast two-hybrid vegetative library screening usingPTM5 as bait (see below).

For the identification of potential vegetative interacting part-ners, a yeast two-hybrid vegetative library was screened usingPTM5 fused to the GAL4 binding domain of the BD vector(pGBKT7). Out of approximately 30,000 transformants, only118 colonies survived the high-stringency selection conditions(see Section 2.3). Each of these clones was identified, throughcolony PCR and sequencing, using both the JGI poplar genomedatabase as well as NCBI Blast searches. Only 8 of the cloneswere shown to be reproducible, being identified more than onetime from the same library (Table 1). Of these 8 clones, four ofthem had very weak β-D-galactosidase activity, likely being theresult of abundant transcripts found in the yeast library. Theother 4 clones were considered to be true potential interaction

eraction with PTM1, 2, 3, 5, and 6. Positive and negative interactions were set upors to avoid auto-activation from their presence in the BD vector. The InteractionThe Gal Positive Control is the auto-activation control generated by pLexA-Pos.ts of triplicate experiments.

Table 1Replicate clones resulting from yeast two-hybrid library screening

Potential interacting clone Poplar database hit Number ofreplicates

Actin depolymerizing factor eugene3.00102018 4Leucine-rich repeat protein eugene3.00100641 4Calmodulin related protein(EF-Hand superfamily)

estExt_fgenesh1_pg_v1.C_LG_III0972

2

Rapid alkalinization factor grail3.0019005101 2Mitochondrial carnitine-acylcarnitine carrier protein

fgenesh1_pg.C_LG_IV000498 8

60S ribosomal protein L31 estExt_Genewise1_v1.C_LG_IX3224

4

40S ribosomal protein S26 eugene3.00190574 240S ribosomal protein S19 eugene3.00820032 2

Approximately 30,000 colonies were initially screened, and 118 blue coloniessurvived the drop out media selection process. Sequencing of each of theseclones indicated that 8 clones had replicate results. Of these clones, the potentialinteraction clones were re-isolated and further analyzed using Y2H one-on-oneinteraction analysis (see Fig. 3).

214 L.J. Cseke et al. / Gene 391 (2007) 209–222

partners and include (1) an actin depolymerizing factor(PtADF); (2) a leucine-rich repeat protein (PtLRR); (3) acalmodulin related protein (EF-Hand superfamily) (PtCRP);and (4) a rapid alkalinization factor (PtRAF). These clones werere-isolated in E. coli and then re-introduced into the GAL4-based Y2H one-on-one system to verify the results of the Y2Hlibrary screening (Fig. 3). β-galactosidase quantitative assayswere also used to obtain information on the strength of theirinteraction with PTM5–BD. While PtADF and PtLRR showedcomparable results to the interaction positive controls includinggrowth on S2 and S3 media, PtCRP and PtRAF showed veryweak interaction with PTM5 and did not grow on S2 and S3,indicating that only PtADF and PtLRR were true PTM5 in-teraction partners in vitro (Fig. 3).

Fig. 3. Yeast two-hybrid one-on-one interaction analysis demonstrating the presence oS1 [SD/-Leu/-Trp], S2 [SD/-His/-Leu/-Trp], and S3 [SD/-Ade/-His/-Leu/-Trp]. Rowpotential Y2H interaction AD clones as well as the empty AD vector. Additional contrthe AD vector containing an SV40 large T antigen insert that does not interact with Pinteract (row 8); and (4) the kit positive control vectors (row 9). The liquid β-galacPositive Control is the interaction of pGBKT7-53 with pGADT7-RecT, supplied by ththe results of quadruplicate experiments.

3.2. PtADF is a Group-I ADF protein while PtLRR appears tobe a novel member of the LRR superfamily

To better understand the relationships of PtADF and PtLRRto related sequences in other species, phylogenetic analysis wasperformed using amino acid sequences obtained from searchesof the JGI poplar genome database as well as the GenBankdatabase (Fig. 4). PtADF was found to be an almost exact match(99% identical) to the Populus trichocarpa sequence identifiedas eugene3.00102018 as well as to the P. alba × P. tremulacambial zone EST sequence which we are calling PaPtADF(identified in Dejardin et al., 2004). Using each of the 12members of the Arabidopsis ADF gene family described byFeng et al. (2006), 11 ADF family genes were identified in thegenome of P. trichocarpa that correlate with the Arabidopsissequences. PtADF along with 5 additional ADF sequences fromP. trichocarpa fell within the Group-I branch of ADF proteinsthat includes the Arabidopsis sequences of AtADF1, 2, 3, 4, and11 (Mun et al., 2000; Maciver and Hussey, 2002; Feng et al.,2006). Using sequence alignments, it was also observed thatthere is a considerable amount of amino acid conservationwithin this group of ADF proteins, where there is approximately83% identity between PtADF and the Arabidopsis sequences(Fig. 5).

Unlike the relatively small ADF gene family, the LRR su-perfamily is considerably larger (Baumberger et al., 2003;Forsthoefel et al., 2005). Consequently, to examine the phylo-genetic relationships of PtLRR, we performed the analysis usingthe Arabidopsis, Petunia, and tomato sequences shown to havethe best E-value scores duringGenBank searches (Fig. 4). PtLRRis an almost exact match (99% identical) to the P. trichocarpasequence identified as eugene3.0010061 and is related to theP. tremula × P. tremuloideswood library ESTsequence which we

f growth (+/−) and β-galactosidase activity under different selection conditions,s 1 through 5 show GAL4-based PTM5–BD vector (5BD) interactions with theol transformations were (1) empty BD + empty AD (row 6); (2) 5BD vector withTM5 (row 7); (3) the kit negative control vectors containing inserts that do nottosidase assays were performed under S1 media conditions, and the Interactione kit. Data is expressed as Relative Galactosidase Units, and error bars represent

Fig. 4. Phylogenetic analysis of PtADF and PtLRR using the PAUP 4.0b10program. (A) Analysis of PtADF compared to the amino acid sequences of theArabidopsis ADF gene family (Feng et al., 2006) as well as sequences obtainedfrom data-mining of the JGI poplar genome database. (B) Analysis of PtLRRcompared to Arabidopsis, Petunia, and tomato amino acid sequences selectedbased on NCBI blast search results as well as poplar sequences identified in theJGI poplar genome database. This is a comparison of only a small number ofprotein sequences from various species, and thus the purpose of this analysis isonly to identify the general class in which PtLRR falls. Bootstrap percentagesrefer to the percentage of replicates that support the branch. See Section 2.4 foraccession numbers.

215L.J. Cseke et al. / Gene 391 (2007) 209–222

named PtPtLRR. The most closely related Arabidopsis sequencecomes from accession number NP_197731 which shares only26% amino acid identity (Fig. 5). While PtLRR has somesimilarity to many of the R genes shown to be involved inresistance responses in tomato (Kruijt et al., 2004; Caicedo andSchaal, 2004), it is more closely related to the much shortersequences of PhSHY and LeSHY shown to be involved in thegrowth of pollen tubes (Guyon et al., 2004; Tang et al., 2004).

Sequence alignments indicated that PhSHY and LeSHY havemore sequence identity to PtLRR in the N-terminal half of theprotein (having approximately 41% identity), with the otherpoplar sequences having more sequence identity in the C-terminal half of the protein (Fig. 5). This suggested that PtLRRmay be one of a group of LRR genes in poplar trees that haveevolved a novel function through the addition of extra copies ofthe leucine rich repeats.

3.3. PtADF and PtLRR have overlapping vascular-specificexpression patterns with PTM5

To determine if the spatial localization of PtADF and PtLRRgene expression matches that of PTM5 in tissues known toexpress PTM5, RNA in situ hybridization analysis was per-formed in Stage 4 aspen flower buds, terminal buds, Stage 3young leaves, Stage 6 young stems, and root tips (all stageshave been described in Cseke et al., 2003a,b, 2005). For bothPTM5 and PtADF, independent analyses showed expressionin the elongating vascular tissues and outer carpel wall of floraltissues as compared to controls (Fig. 6, panels 1A through 1C).The same expression pattern was observed for PtLRR, althoughthe relative level of signal was lower (Fig. 6, panel 1D). Interminal buds, all three genes were preferentially expressed inthe procambium and developing primary xylem and phloem celllayers (Fig. 6, panels 2A through 2D). Overlapping expressionpatterns were also observed in young leaves, where all threegenes were expressed in the vasculature of the leaf midrib aswell as within the expanding leaf mesophyll (Fig. 6, panels 3Athrough 3D).

Within developing stems, expression of all three genes wasobserved in the developing cambium and xylem ray parenchy-ma stemming from the cambium (Fig. 6, panels 4A through4D). However, the relative signal of PtLRR was reduced ascompared to PTM5. In addition, while PTM5 is expressed in thedeveloping xylem ray parenchyma stemming from the pith, nosuch signal was seen for either PtADF or PtLRR. In roots,PTM5 and PtADF have identical expression patterns, whereeach is expressed in the root central vascular cylinder as well asthe root meristem (Fig. 6, panels 5A through 5D and 6A through6D). PtLRR, on the other hand, is not expressed in either ofthese tissues. Instead, it appears to be activated during lateralroot formation (Fig. 6, panel 5D) as well as in a thin layer at theoutermost edge of the root vascular cylinder. Especially withrespect to PtADF, these data clearly show the overlappingvascular-specific expression patterns of all three genes through-out the tree.

3.4. PTM5 over-expression causes alterations in root vasculardevelopment consistent with a function in cell growth andexpansion

As a means to examine the function of PTM5, we generated21 P. tremuloides transgenic lines over-expressing the PTM5coding region and tracked the results of the trees (along withappropriate controls) growing under floral inducing conditionsas described in Cseke et al. (2005). It has been shown that the

Fig. 5. PtADF and PtLRR deduced amino acid sequence alignments compared to their most closely related sequences. (A) PtADF compared with PaPtAD (a hybridpoplar cambial zone EST), a closely related P. trichocarpa ADF sequence as well as AtADF1 and AtADF2 (two members of the Arabidopsis Group-I ADF genefamily). (B) PtLRR compared with closely related P. trichocarpa LRR sequences, the most closely related sequence from Arabidopsis (NP_197731), and the SHYsequences from Petunia and tomato. Black boxes refer to residues that are identical to those found in PtADF or PtLRR. Grey boxes identify similar residues, anddashes indicate gaps in the sequences used to optimize the alignments. Over-scored regions represent the involucrin repeat and actin depolymerizing regions of PtADFas well as each of the leucine rich repeats of PtLRR as predicted by Pfam.

216 L.J. Cseke et al. / Gene 391 (2007) 209–222

over-expression of SOC1-related genes in a variety of plantspecies causes early flowering, and it was one of our initialhypotheses that PTM5may have a similar role in flowering time(Kim et al., 2003; Cseke and Podila, 2004; Lee et al., 2004).However, after tracking the transgenic trees for three years,none of the PTM5 transgenic lines flowered early despite the

fact that other transgenic lines harboring MADS-box genes witha function in floral development (PTM3 and PTM6) did flowerearly (Cseke et al., 2005). Similar results for later stage trans-genics were obtained from other researchers studying the over-expression of the PTM5 ortholog in transgenic hybrid aspen(personal communications with Dr. Cetin Yuceer, Mississippi

217L.J. Cseke et al. / Gene 391 (2007) 209–222

State University and Dr. David Karnosky, Michigan Techno-logical University).

Instead, a drooping phenotype was observed in the branchesof several of the later stage PTM5 transgenics (2–3 years old)

Fig. 6. In situmRNA localization of PTM5, PtADF and PtLRR in (1) Stage 4 aspen fl(5) cross-sections of the roots near the root tip, and (6) root tips. Panels A representSense probes for PtADF and PtLRR were also used in the analysis and appeared idenlabeled PTM5-specific antisense probe showing expression of PTM5 in developing pwith DIG-labeled PtADF-specific antisense probe showing identical expression pattPanels D represent each tissue incubated with the DIG-labeled PtLRR-specific antisbuds and young leaves, reduced expression levels in floral and stem vascular tissues aPTM5. Arrows indicate the tissues where expression is most abundant, and size barsperianth cup; Re, receptacle. Panel 2A: PC, procambium. Panel 3A: Me, mesophyll; MCo, cortex; En, endodermis; Ep, epidermal layer; VC, vascular cylinder. Panel 6A:

compared to control lines harboring empty expression vectorsand wild-type trees of the same age (Fig. 7, panels 2A and 2B).While this phenotype did not have an identifiable cause uponexamination, the more obvious phenotypic difference seen in

ower buds, (2) terminal buds, (3) Stage 3 young leaves, (4) Stage 6 young stems,each tissue incubated with DIG-labeled PTM5-sense probe, acting as controls.tical to the PTM5 controls. Panels B represent each tissue incubated with DIG-rimary and secondary vascular tissues. Panels C represent each tissue incubatederns to those of PTM5 with the exception of a lack of expression in pith tissue.ense probe showing identical expression patterns to those of PTM5 in terminals compared to PTM5, and a different expression pattern in the roots compared toindicate 1 mm. Tissue labels are as follows: Panel 1A: Br, bract; Cp, carpel; Pe,R, midrib. Panel 4A: Ca, cambium; Ph, phloem; Pi, pith; Xy, xylem. Panel 5A:

RM, root meristem, VC, vascular cylinder.

Fig. 6 (continued ).

218 L.J. Cseke et al. / Gene 391 (2007) 209–222

the PTM5 over-expressing lines was observed in early stages ofdevelopment. Here the roots from 14 transgenic lines over-expressing PTM5 showed significant thickening as compared tocontrol lines or wild-type roots (Fig. 7, panels 3A and 3B).Upon further observation of root sections∼2.0 cm from the roottips, the morphology of these fat roots at identical magnifica-tions appears to be due to alterations including (1) changes inthe number of vascular cylinders, (2) an increase in the numberof cells in the root central vascular cylinder, and (3) a dramaticincrease in the size of root cortex cells derived from the rootvascular cambium (Fig. 7, panels 4A, 4B, 5A and 5B). Thesechanges are consistent with a function in cell growth and

expansion that is linked to the functions of known ADF andLRR genes.

4. Discussion

4.1. Exploration of PTM5 interactions

We have shown that PTM5 has the ability to form homodi-mers with itself as well as heterodimers with other AP1/SQUAand SEP class MADS-box proteins (Figs. 1 and 2). However,with the exception of PTM5 itself, the expression patterns ofPTM1, 2, 3 and 6 do not correlate with the vegetative expression

Fig. 7. Phenotypes of transgenic aspen over-expressing PTM5. Panel 1A shows Northern hybridization results obtained from RNA samples acquired from the leaves ofselected control lines harboring the empty pTSC5 vector and selected PTM5 transgenic plants harboring the PTM5 over-expression construct. The upper bandcorresponds to the normal levels of native PTM5 in young leaves and the lower bands represent the over-expressing PTM5 transcript. Panel 1B shows the ethidiumbromide stained gel as a control for RNA loading in each lane. Panel 2A shows the normal development of the control lines in comparison to Panel 2B, showing thedrooping phenotype seen in several of the over-expression lines grown under identical conditions. Panel 3A demonstrates the development of normal roots in thecontrol lines, and panel 3B shows the formation of the thick root phenotype in the transgenic aspen. Cross-sections of the roots from the control and transgenic aspenplants under the same magnification are shown in Panels 4A and 4B respectively, including the micrometer used to approximate cell size and number. Highermagnification views (20×) of the same tissues are shown in Panels 5A and 5B respectively to give a better representation of the changes taking place in different rootcell layers (a, epidermal layers; b, cortex; c, endodermis; d, phloem tissues; e, xylem tissues).

219L.J. Cseke et al. / Gene 391 (2007) 209–222

patterns of PTM5, as they are predominantly floral tissue-specific (Cseke et al., 2003a,b, 2005). Therefore, while theformation of PTM5 homodimers is probable, it is not likely thatPTM5 interacts with the other proteins in vivo. Likewise, noother MADS-box factors were isolated during yeast two-hybridvegetative library screening using PTM5 as bait.

It is interesting to note that we were not able to isolate PTM5as an interaction partner with itself when screening the yeasttwo-hybrid library. This may be due in part to the fact that theexpression levels of PTM5 are quite low (Cseke et al., 2003b),and one would anticipate that PTM5 transcripts in the yeasttwo-hybrid library would be quite rare. In addition, it issometimes difficult to identify homodimers with the yeast two-hybrid system as expressed AD–AD or BD–BD interactionscan mask the desired BD–AD interactions. This may be one

reason that the relative strength of the PTM5–PTM5 interac-tions in the β-galactosidase quantitative assays was lowercompared to the other tested MADS-box proteins (Fig. 2).However, yeast two-hybrid library screening did identify twoadditional non-MADS interaction partners, an actin depolymer-izing factor (PtADF) and a leucine-rich repeat protein (PtLRR)(Table 1 and Fig. 3).

4.1.1. Interaction with actin depolymerizing factorActin is a fundamental element involved in the formation of

the cytoskeleton in all eukaryotes, and its polymerization anddepolymerization are key events in the development of varioustissues throughout the plant. Actin depolymerizing factors orcofilins have been classified into four Groups (I–IV) and areessential regulators in plant growth and development as their

220 L.J. Cseke et al. / Gene 391 (2007) 209–222

activity modulates the depolymerization of actin filaments ne-cessary for cell expansion (Mun et al., 2000; Hussey et al.,2002; Maciver and Hussey, 2002). In trees, ADF activity in thesecondary meristematic tissues of the stem and root vascularcambium is thought to be especially important in the deve-lopment of phloem and xylem tissues throughout the tree. Weshow in this study that PTM5 and PtADF have overlappingvascular-specific expression patterns that extend throughout thetree including expression in the stem and root vascular cambium(Fig. 6). Likewise, the PtADF EST homolog (PaPtADF) isexpressed in the cambial zone of P. alba × P. tremula (Dejardinet al., 2004).

Through comparison of the 11 ADF family members thatwere identified in the P. trichocarpa genome database with the12 ADF family members found in the genome of Arabidopsis(Feng et al., 2006), we show that PtADF is a Group-I ADFprotein closely related to the Arabidopsis genes AtADF1, 2, 3,4, and 11 (Figs. 4 and 5). AtADF1 is specifically expressed inthe developing vascular tissues of all Arabidopsis vegetativeorgans and has been shown in vivo to be a key regulator in celland organ expansion (Dong et al., 2001a,b). Similar results werefound with PhADF1, the Petunia ortholog (Mun et al., 2000).In fact, the expression patterns of both AtADF1 and PhADF1are an almost exact match with those of PTM5 and PtADF(Fig. 6; Dong et al., 2001a; Mun et al., 2002). The fact thatPTM5 and PtADF interact in vitro, along with their overlappingexpression patterns in developmental tissues, suggests that theirassociation in vivo is important to vegetative development inthese tissues. While we can only speculate at this time as to theimportance of the PTM5/PtADF interaction, it seems likely thatPTM5 may be participating in PtADF gene expression. This islikely when considering the changes that we saw in rootdevelopment as a result of PTM5 over-expression and dimerformation, including the increase in the number of cells withinthe vascular cylinder as well as the increase in cell size withinthe cortex tissues (Fig. 7). The interaction of PTM5 with PtADFmay simply be part of a feedback loop that helps to control theamount of PtADF gene expression.

4.1.2. Interaction with a leucine-rich repeat proteinLeucine-rich repeat (LRR) proteins constitute a large and

functionally diverse superfamily of proteins composed of atleast six subfamilies. Their functions range from cell signalingand signal transduction to defense against pathogens, thedocking of specific proteins with various pathway components,the growth of pollen tubes, as well as the development of roots(Baumberger et al., 2003; Caicedo and Schaal, 2004; Guyonet al., 2004; Suzuki et al., 2004; Tang et al., 2004; Forsthoefelet al., 2005). At the heart of their activity is the LRR domain,composed of tandemly repeated leucine-rich motifs that mediatespecific protein–protein interactions. The binding specificityand subsequent function of each of these proteins are deter-mined in part by the sequence characteristics of the leucine-richmotifs that make up the LRR domain (Forsthoefel et al., 2005).

We have shown that PTM5 interacts in vitro with PtLRR andthey share overlapping expression patterns in all tissues with theexception of roots, where PtLRR seems to be activated during

lateral root initiation (Fig. 6 panel 5D). The overall expressionpattern of PtLRR is similar to the expression pattern of some ofthe LRR-extensins including LRX proteins (Baumberger et al.,2003). Despite this, the PtLRR sequence does not fall within oneof the distinct subfamilies of LRR proteins during phylogeneticanalysis. While the PtLRR protein is related to pathogen resistantgenes (R genes) such as the Cf-type receptor proteins, it is moreclosely related to as yet uncharacterized LRR sequencesidentified from both poplar and Arabidopsis (Figs. 4 and 5).Even in these cases, the overall percent identity between theproteins is low. Interestingly, our sequence analysis shows thatthe N-terminal half of the PtLRR protein is more closely relatedto PhSHY and LeSHY, proteins thought to control signal trans-duction events that mediate pollen tube growth (Fig. 5) (Guyonet al., 2004; Tang et al., 2004). This suggests that the PtLRRmaybe one of a group of LRR genes in poplar trees that have evolveda novel functions through the addition of extra copies of theleucine rich repeats. Such domain swapping (resulting fromunequal recombination events) and divergence to form newgenes are not uncommon in the evolution of LRR-type genes(Wulff et al., 2001; Caicedo and Schaal, 2004; Kruijt et al., 2004).

It should also be noted that this is not the first report of aMADS-box protein having an LRR protein interaction partner.AGAMOUS (AG) interacts with a non-MADS intracellularLRR protein called FLOR1, which may be mediating the in-teraction of AG with an acid phosphatase protein complexduring the development of floral organs (Gamboa et al., 2001;Acevedo et al., 2004). Considering that a common theme in theactivity of many LRR proteins is to act as a mediator of specificprotein–protein interactions, we can speculate that function ofthe PTM5/PtLRR interaction may be to mediate additionalprotein–protein interactions in the PTM5 pathway. In addition,the fact that PTM5 and PtLRR do not have the same spatialexpression patterns in roots suggests that PtLRR may be faci-litating differential protein interactions in above-ground andbelow-ground tissues, possibly allowing the regulation of dif-ferent target genes in stems versus roots.

4.2. Exploration into the function of PTM5

SOC1 (Suppressor of Over-expression of CONSTANS fromArabidopsis) is active in floral induction through the long-day, autonomous, vernalization, and gibberellin (GA)-depen-dent pathways (Kaufmann et al., 2005). It has been shown thatthe over-expression of SOC1 homologs in a variety of plantspecies causes early flowering that is sometimes so extreme thatit occurs in callus stages of transformation (Kim et al., 2003;Cseke and Podila, 2004; Lee et al., 2004; Schonrock et al.,2006). To test such a possibility for PTM5 and to examinepossible links with known ADF and LRR genes, we examinedthe over-expression phenotypes of PTM5 in transgenic aspenfor a period of three years under floral induction conditions.None of the 21 generated transgenic lines showed any dif-ference in flowering time as compared to controls. Instead, apronounced thickening of the roots in 14 of the transgenic linesas well as a drooping phenotype in the branches of some ofthese lines was observed.

221L.J. Cseke et al. / Gene 391 (2007) 209–222

Comparison of cross-sections of transgenic and control rootsindicated that the “fat” root phenotype was due to (1) changes inthe number of vascular cylinders, (2) an increase in the numberof cells in the root central vascular cylinder, and (3) a dramaticincrease in the size of root cortex cells derived from the rootvascular cambium (Fig. 7). Such phenotypes are reminiscent tochanges observed in cell and organ development during themanipulation of AtADF1 gene expression in Arabidopsis (Donget al., 2001b). Presumably these changes were a result of over-expressing PTM5 in cell types where it is not normally ex-pressed. Likewise, since we have shown that PTM5 interactsvery strongly with itself, over-expression likely resulted in theabundant and expanded expression of PTM5 dimers which mayhave altered early-stage vegetative processes. Therefore, whileover-expression of PTM5 did not alter flowering time as seenwith other SOC1-related genes, it did have an impact onvegetative development including the proper development ofroots, perhaps through the regulation of PtADF cell expansionactivity.

Using comparisons between poplar and Arabidopsis se-quences, PTM5 was previously shown to be a member of theSOC1/TM3 class MADS-box genes including SOC1, AGL14and AGL19 (Cseke et al., 2003b). We have also identified fiveSOC1/TM3 class genes in the genome of poplar species, in-cluding the PTM5 ortholog. A recent paper by Leseberg et al.(2006) supports the presence of four of these genes, includingPtMADS1, 9, 12, and 41 where PtMADS41 is the PTM5 ortho-log (Leseberg et al., 2006). PTM5 and one other gene appear tobe co-orthologs to SOC1, while the remaining genes belong tothe AGL14/AGL19 subgroup that is sister to the SOC1 sub-group. AGL14 and AGL19 were both originally described asroot-specific MADS-box genes, likely involved in root de-velopment (Rounsley et al., 1995; Alvarez-Buylla et al., 2000;Feng et al., 2006). However, it was recently shown that AGL19is also expressed in shoot tissues, is involved in vernalizationprocesses, and also promotes flowering similar to SOC1(Schonrock et al., 2006). Similarly, ETL from Eucalyptusglobulus was shown to be a tree AGL14/AGL19 homolog ex-pressed predominantly in the vasculature of young roots and inthe root meristems but also in organ primordia, shoot meristems,and reproductive organs (Decroocq et al., 1999; De OliveiraDias et al., 2005). However, unlike PTM5, ETL transcripts werenot found in wood formation.

Suggesting differences in protein–protein interactions, theK-domain of PTM5 was previously predicted to have alternatefolding that is more prone to the formation of a trimeric coiled-coil rather than the dimeric coil predicted for genes such asSOC1, AGL14, AGL19, and ETL (Cseke et al., 2003b).Likewise, we have isolated and characterized the promoterregion of PTM5 (Acc #: AY345123), and while it has somesequence motifs and regulatory elements related to the promoterregion of AGL14, its sequence is significantly different from thepromoter of SOC1 (data not shown). A similar study found thatthe promoter of the poplar SOC1 co-ortholog, which was calledPMADS5, has retained more motifs in common with SOC1,whereas the PTM5 promoter has diverged, suggesting neofunc-tionalization (De Bodt et al., 2006). This sets up the possibility

that the PTM5 gene may be responding to different regulatoryevents through a divergent promoter, and the PTM5 protein mayhave functionally diverged from other members of the SOC1/TM3 class, developing a novel tree-specific set of protein in-teractions, such as those seen with PtADF and PtLRR, throughan altered K-domain.

It is interesting to note that when Gan et al. (2005) examinedthe expression pattern of SOC1 in conjunction with root MADS-box genes like ANR1, AGL14, and AGL19, they observed thateach of these genes was differentially regulated in response todiffering levels of nitrate. SOC1 expression was additionallyfound to respond to changes in the phosphorus and sulfur sup-ply, suggesting a more general and additional role of these genesin the response to nutrient stresses (Gan et al., 2005). Clearly, thestory behind the function of SOC1/TM3 class MADS-box genesrequires further attention. While we have shown here that PTM5is functional in root development and cell expansion with atransgenic phenotype consistent with the predicted function ofits potential interactions partners, PtADF and PtLRR, the pos-sibility of there being gene redundancy and the alternativeprotein complex formation within the SOC1/TM3 class MADS-box genes is highly likely. It may soon be found that genes,including PTM5, have a more broad range function in plantresponses to environmental conditions that inevitably controlwhole plant development.

Acknowledgments

Part of this work is supported by a subcontract from theOffice of Science (BER), U.S. Department of Energy GrantNo. DEFG02-04ER63792, to GKP. We would like to thank Dr.Karnosky for providing stock material for aspen transformation.

References

Acevedo, F.G., Gamboa, A., Paez-Valencia, J., Jimenez-Garcia, L.F., Izaguirre-Sierra,M., Alvarez-Buylla, E.R., 2004. FLOR1, a putative interaction partnerof the floral homeotic protein AGAMOUS, is a plant-specific intracellularLRR. Plant Sci. 167, 225–231.

Alvarez-Buylla, E.R., et al., 2000. MADS-box gene evolution beyond flowers:expression in pollen, endosperm, guard cells, roots and trichomes. Plant J. 24,457–466.

Baumberger, N., et al., 2003. Whole genome comparison of leucine richrepeat extensins in Arabidopsis and rice. A conserved family of cell wallproteins form a vegetative and a reproductive clade. Plant Physiol. 131,1313–1326.

Caicedo, A.L., Schaal, B.A., 2004. Heterogeneous evolutionary processes affectR gene diversity in natural populations of Solanum pimpinellifolium. Proc.Natl. Acad. Sci. U. S. A. 101, 17444–17449.

Causier, B., Cook, H., Davies, B., 2003. An Antirrhinum ternary complex factorspecifically interacts with C-function and SEPALLATA-like MADS-boxfactors. Plant Mol. Biol. 52, 1051–1062.

Cseke, L.J., Podila, G.K., 2004. MADS-box genes in dioecious aspen II: areview of MADS-box genes from trees and their potential in forestbiotechnology. Physiol. Mol. Biol. Plants 10, 7–28.

Cseke, L.J., Sen, B., Ravinder, N., Karnosky, D.F., Podila, G.K., 2003a. MADS-box genes in dioecious aspen I: characterization of PTM1 and PTM2 floralMADS-box genes. Physiol. Mol. Biol. Plants 9, 187–196.

Cseke, L.J., Zheng, J., Podila, G.K., 2003b. Characterization of PTM5 in aspentrees: a MADS-box gene expressed during woody vascular development.Gene 318, 55–67.

222 L.J. Cseke et al. / Gene 391 (2007) 209–222

Cseke, L.J., Kaufman, P.B., Podila, G.K., Tsai, C.-J., 2004. Handbook ofMolecular and Cellular Methods in Biology and Medicine, 2nd ed. CRCPress, Boca Raton, Florida.

Cseke, L.J., et al., 2005. SEP-class genes in Populus tremuloides and their likelyrole in reproductive survival of poplar trees. Gene 358, 1–16.

De Bodt, S., Theissen, G., Van de Peer, Y., 2006. Promoter analysis of MADS-box genes in eudicots through phylogenetic footprinting. Mol. Biol. Evol.23, 1293–1303.

Decroocq, V., et al., 1999. A TM3-like MADS-box gene from Eucalyptusexpressed in both vegetative and reproductive tissues. Gene 228, 155–160.

De Folter, S., Angenent, G.C., 2006. Trans meets cis in MADS science. TrendsPlant Sci. 11, 224–231.

De Folter, S., et al., 2005. Comprehensive interaction map of the ArabidopsisMADS box transcription factors. Plant Cell 17, 1424–1433.

Dejardin, A., Leple, J.C., Lesage-Descauses, M.C., Costa, G., Pilate, G., 2004.Expressed sequence tags from poplar wood tissues — a comparativeanalysis from multiple libraries. Plant Biol. 6, 55–64.

De Oliveira Dias, B.F., Simoes-Araujo, J.L., Russo, C.A.M., Margis, R., Alves-Ferreira, M., 2005. Unraveling MADS-box gene family in Eucalyptus spp.:a starting point to an understanding of their developmental role in trees.Genet. Mol. Biol. 28, 501–510.

Dong, C.H., Kost, B., Xia, G., Chua, N.H., 2001a. Molecular identification andcharacterization of the Arabidopsis AtADF1, AtADF5 and AtADF6 genes.Plant Mol. Biol. 45, 517–527.

Dong, C.H., Xia, G.X., Hong, Y., Ramachandran, S., Kost, B., Chua, N.H.,2001b. ADF proteins are involved in the control of flowering and regulate F-actin organization, cell expansion, and organ growth in Arabidopsis. PlantCell 13, 1333–1346.

Feng, Y., Liu, Q., Xue, Q., 2006. Comparative study of rice and Arabidopsisactin-depolymerizing factors gene families. Plant Physiol. 163, 69–79.

Forsthoefel, N.R., Cutler, K., Port, M.D., Yamamoto, T., Vernon, D.M., 2005.PIRLs: a novel class of plant intracellular leucine-rich repeat proteins. PlantCell Physiol. 46, 913–922.

Frey, B.R., Lieffers, V.J., Landhausser, S.M., Comeau, P.G., Greenway, K.J.,2003. An analysis of sucker regeneration of trembling aspen. Can. J. For.Res. 33, 1169–1179.

Fujita, H., Takemura, M., Tani, E., Nemoto, K., Yokota, A., Kohchi, T., 2003.An Arabidopsis MADS-box protein, AGL24, is specifically bound to andphosphorylated by meristematic receptor-like kinase (MRLK). Plant CellPhysiol. 44, 735–742.

Gamboa, A., Paez-Valencia, J., Acevedo, G.F., Vazquez-Moreno, L., Alvarez-Buylla, R.E., 2001. Floral transcription factor AGAMOUS interacts in vitrowith a leucine-rich repeat and an acid phosphatase protein complex. Molec.Cell Biol. Res. Commun. 288, 1018–1026.

Gan, Y., Filleur, S., Rahman, A., Gotensparre, S., Forde, B.G., 2005. Nutritionalregulation of ANR1 and other root-expressed MADS-box genes in Arabi-dopsis thaliana. Planta 222, 730–742.

Guyon, V., et al., 2004. Antisense phenotypes reveal a role for SHY, a pollen-specific leucine-rich repeat protein, in pollen tube growth. Plant J. 39,643–654.

Hussey, P.J., Allwood, E.G., Smertenko, A.P., 2002. Actin-binding proteins inthe Arabidopsis genome database: properties of functionally distinct plantactin-depolymerizing factors/cofilins. Philos. Trans. R. Soc. Lond., B Biol.Sci. 357, 791–798.

Kaufmann, K., Melzer, R., Theissen, G., 2005. MIKC-type MADS-domainproteins: structural modularity, protein interactions and network evolution inland plants. Gene 347, 183–198.

Kim, K.W., et al., 2003. The function of the flowering time gene AGL20 isconserved in Crucifers. Mol. Cells 16, 136–141.

Kruijt, M., Brandwagt, B.F., De Wit, P.J.G.M., 2004. Rearrangements in the Cf-9 disease resistance gene cluster of wild tomato have resulted in three genesthat mediate Avr9 responsiveness. Genetics 168, 1655–1663.

Lee, S., Kim, J., Han, J.J., Han, M.J., An, G., 2004. Functional analyses of theflowering time gene OsMADS50, the putative SUPPRESSOR OF OVER-EXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20) ortholog inrice. Plant J. 38, 754–764.

Leseberg, C.H., Li, A., Kang, H., Duvall, M., Mao, L., 2006. Genome-wideanalysis of the MADS-box gene family in Populus trichocarpa. Gene 378,84–94.

Maciver, S.K., Hussey, P.J., 2002. The ADF/cofilin family: actin-remodelingproteins. Genome Biol. 3 (5), 1–12.

Masiero, S., et al., 2002. Ternary complex formation between MADS-boxtranscription factors and the histone fold protein NF-YB. The J. Biol. Chem.277, 26429–26435.

Moon, Y.H., Jung, J.Y., Kang, H., An, G., 1999. Determination of plant APE-TALA3 homolog by yeast two-hybrid screening. PlantMol. Biol. 40, 167–177.

Mun, J.-H., et al., 2000. Two closely related cDNAs encoding actin-depo-lymerizing factors of petunia are mainly expressed in vegetative tissues.Gene 257, 167–176.

Mun, J.H., et al., 2002. Petunia actin-depolymerizing factor is mainly accu-mulated in vascular tissue and its gene expression is enhanced by the firstintron. Gene 292, 233–243.

Remenyi, A., Scholer, H.R., Wilmanns, M., 2004. Combinatorial control of geneexpression. Nat. Struct. Mol. Biol. 11, 812–815.

Rounsley, S.D., Ditta, G.S., Yanofsky, M.F., 1995. Diverse roles for MADS boxgenes in Arabidopsis development. Plant Cell 7, 1259–1269.

Schonrock, N., et al., 2006. Polycomb-group proteins repress the floral activatorAGL19 in the FLC-independent vernalization pathway. Genes Dev. 20,1667–1678.

Sundstrom, J., Engstrom, P., 2002. Conifer reproductive development involvesB-type MADS-box genes with distinct and different activities in male organprimordia. Plant J. 31, 161–169.

Suzuki, T., et al., 2004. A novel Arabidopsis gene TONSOKU is required forproper cell arrangement in root and shoot apical meristems. Plant J. 38,673–684.

Tang, W., Kelley, D., Ezcurra, I., Cotter, R., McCormick, S., 2004. LeSTIG1, anextracellular binding partner for the pollen receptor kinases LePRK1 andLePRK2, promotes pollen tube growth in vitro. Plant J. 39, 343–353.

Taylor, G., 2002. Populus: Arabidopsis for forestry: do we need a model tree?Ann. Bot. 90, 681–689.

Tsai, C.J., Podila, G.K., Chiang, V.L., 1994. Agrobacterium-mediated trans-formation of quaking aspen (Populus tremuloides) and regeneration oftransgenic plants. Plant Cell Rep. 14, 94–97.

Wulff, B.B.H., Thomas, C.M., Smoker, M., Grant, M., Jones, J.D.G., 2001.Domain swapping and gene shuffling identify sequences required forinduction of an Avr-dependent hypersensitive response by the tomato Cf-4and Cf-9 proteins. The Plant Cell 13, 255–272.