SEP-class genes in Populus tremuloides and their likely role in reproductive survival of poplar trees

Gene 358 (2005) 1 – 16 www.elsevier.com/locate/gene

SEP-class genes in Populus tremuloides and their likely role in reproductive survival of poplar trees Leland J. Cseke a, Sarah Beth Cseke a, Namritha Ravinder a, Lorelei C. Taylor b, Anupama Shankar b, Banalata Sen c, Ramesh Thakur c, David F. Karnosky c, Gopi K. Podila a,* a

The University of Alabama in Huntsville, Department of Biological Sciences, Huntsville, AL 35899, USA b Michigan Technological University, Department of Biological Sciences, Houghton, MI 49931, USA c Michigan Technological University, School of Forestry, Houghton, MI 49931, USA Received 11 October 2004; received in revised form 27 April 2005; accepted 17 May 2005 Available online 25 July 2005 Received by G. Theissen

Abstract One of the most important processes to the survival of a species is its ability to reproduce. In plants, SEPALLATA-class MADS-box genes have been found to control the development of the inner whorls of flowers. However, while much is known about floral development in herbaceous plants, similar systems in woody trees remain poorly understood. Populus tremuloides (trembling aspen) is a widespread North American tree having important economic value, and its floral development differs from that of well-studied species in that the flowers have only two whorls and are truly unisexual. Sequence based analyses indicate that PTM3 (Populus tremuloides MADS-box 3), and a duplicate gene PTM4, are related to the SEPALLATA1-and 2-class of MADS-box genes. Another gene, PTM6, is related to SEP3, and each of these genes has a counterpart in the poplar genomic database along with additional members of the A, B, C, D, and E-classes of MADS-box genes. PTM3/4 and 6 are expressed in all stages of male and female aspen floral development. However, PTM3/4 is also expressed in the terminal buds, young leaves, and young stems. In situ RNA localization identified PTM3/4 and 6 transcripts predominantly in the inner, sexual whorl, within developing ovules of female flowers and anther primordia of male flowers. Tree researchers often use heterologous systems to help study tree floral development due to the long juvenile periods found in most trees. We found that the participation of PTM3/4 in floral development is supported by transgenic experiments in both P. tremuloides and heterologous systems such as tobacco and Arabidopsis. However, phenotypic artifacts were observed in the heterologous systems. Together the results suggest a role for poplar SEP-class genes in reproductive viability. D 2005 Elsevier B.V. All rights reserved. Keywords: Floral; Flower; MADS-box; Ovule; Pollen; SEPALLATA

1. Introduction 1.1. SEP-class genes: key regulatory MADS-box proteins

Abbreviations: AG, AGAMOUS; AGL, AGAMOUS-like; AP1, APETALA1; AP3, APETALA3; DIG, Digoxigenin; EST, Expressed Sequence Tag; FBP, FLORAL BINDING PROTEIN; IR, Inverted Repeat; JGI, Joint Genome Institute; NR, non-redundant; PI, PISTILLATA; PTM, Populus tremuloides MADS-box; PTGS, Post-transcriptional Gene Silencing; RT, Reverse Transcription; SEP, SEPALLATA; SHP, SHATTER-PROOF; STK, SEEDSTICK. * Corresponding author. Tel.: +1 256 824 6263; fax: +1 256 824 6305. E-mail address: [email protected] (G.K. Podila). 0378-1119/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.05.035

From a biological standpoint, one of the most important processes during the life of a plant is its ability to sexually reproduce. In angiosperm plants, this ability is dependent on the proper development of flowers which is controlled by a large array of both structural and regulatory genes whose coordinated activity works to ensure the proper development of sexual organs at an appropriate time during the plant_s life. MADS-box genes are perhaps the bestcharacterized family of developmental genes that regulate

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the timing and formation of flowers. Their structure and action in floral development has been reviewed in many species including trees (Becker and Theissen, 2003; Cseke and Podila, 2004; Kaufmann et al., 2005; Theissen, 2001). Recent studies suggest that models used to explain MADS-box gene activity based on the A-, B-, and Cfunction organ identity genes are insufficient in explaining the diversity of MADS-box gene functions. The B- and Cfunction organ-identity genes in Arabidopsis, for example, require four closely related and functionally redundant MADS-box genes. These genes, SEPELATTA 1, 2, 3 and 4 (previously named AGL2, AGL4, AGL9 and AGL3), are required not only for the development of the inner three whorls consisting of petals, stamens, and carpels but also for ovules and the outer whorl of sepals as demonstrated by SEP quadruple mutants (Ditta et al., 2004; Favaro et al., 2003; Pelaz et al., 2000). While ectopic expression of SEPclass genes is reported to stimulate early flowering in Arabidopsis, Petunia, tobacco, lilly, silver birch, and rice (Ferrario et al., 2003; Jang et al., 1999, 2002; Lemmetyinen et al., 2004; Moon et al., 1999; Pelaz et al, 2001; Tzeng et al., 2003), suppression of SEP-class genes, such as SEP1/2/ 3/4, FBP2/5, and TM5/29, produces flowers with continuous whorls of sepals or leaf-like structures, one inside of the other, often with indeterminate growth of the floral meristem (Ampomah-Dwamena, et al., 2002; Angenent, et al., 1994; Ditta et al., 2004; Pelaz et al., 2000; Pnueli, et al., 1994; Vandenbussche et al., 2003). However, in addition to their activity in flowering time and formation, SEP-class genes from some plant species have been reported to play a role in fruit development (e.g. LeMADS-RIN and TAGL2 in tomato and SEP1/2 homologs in apple trees) as well as in vegetative plant growth (e.g. over-expressing lines of NsMADS3 and NtMADS4 in tobacco plants; OsMADS1/ LHS1 when expressed in tobacco, and LMADS3 from lily when expressed in Arabidopsis) (Busi et al., 2003; Chung et al., 1994; Jang et al., 1999, 2002; Tzeng et al., 2003; Vrebalov et al., 2001). Evidence that SEP-class proteins can form higher order MADS-box interactions suggests that differences in complex formation is one way these transcription factors obtain their functions (Busi et al., 2003; Favaro et al., 2003; Ferrario et al., 2003; Honma and Goto, 2001; Immink et al., 2002; Jang et al., 2002; Kaufmann et al., 2005; Lee et al., 2003; Shchennikova et al., 2004; Theissen and Saedler, 2001). For example, SEP3 may be able to interact with combinations of AP1, PI/AP3, and AG as well as ovulespecific D-function genes such as SHP and STK to promote the formation of differing regulatory complexes in the various floral organs (Favaro et al., 2003; Honma and Goto, 2001; Pelaz et al., 2001). Such complexes have been shown to occur in Petunia where a family of SEP-class genes (FBP2, 4, 5, 9, and 23) have been shown to act similarly to those found in Arabidopsis. FBP2 (the SEP3 homolog) was seen to interact in ternary and even quaternary complexes with other B- and C-

function MADS-box proteins (Ferrario et al., 2003; Vandenbussche et al., 2003). The diversity and impact of such interactions supports the idea that SEP-class genes belong to an independent class of MADS-box genes termed the Eclass (Theissen, 2001). 1.2. Floral development in aspen: a different story Poplar species are becoming one of the most important model organisms for the study of tree processes primarily due to the recently completed genome sequence of Populus trichocarpa (Taylor 2002; http://genome.jgi-psf.org/Poptr1/ Poptr1.home.html). Our lab is interested in a close relative, Populus tremuloides (trembling aspen), because it is an essential component of paper, plywood, oriented stand board, and many other engineered wood products (Isebrands and Karnosky, 2002). It is also one of the most widely distributed tree species in North America, making it a valuable forest resource and an important ecological species whose reproduction deserves attention (Taylor 2002). Poplar trees have several important differences in the floral developmental process that set them apart from herbaceous plants. They typically have an 8 to 10 year juvenile period that has greatly inhibited selective breeding programs and the study of floral development. Once the tree reaches maturity, inflorescences are borne only on axillary buds approximately one year prior to anthesis, which occurs the following spring after a winter dormancy period. Each inflorescence or catkin contains many individual flower primordia developing in unison, and male and female flowers are produced on separate trees (Boes and Strauss, 1994; Cseke et al., 2003a). It is important to note that many tree species, including aspen, are dioecious while most well-studied herbaceous plants are monoecious. Dioecy occurs in only 4% of all flowering plants and is achieved by several different means (Ainsworth et al., 1998). Usually both male and female organs are initiated but then the development of one or the other sex organ is blocked. In poplar species, however, only organs of a single sex are initiated and the floral meristems are thus truly unisexual (Boes and Strauss, 1994; Sheppard et al., 2000). Thus, instead of four concentric whorls of floral organs, each aspen flower has only two whorls consisting of a highly reduced perianth cup enclosing either stamens or one pistil (Sheppard et al., 2000). Normally, in other flowers, the perianth is the floral envelope that contains either the calyx (sepals) or the corolla (petals) or both (Kempin et al., 1993). In aspen, however, it is unknown if the perianth cup is derived from the equivalent of sepals and/or petals. Populus species along with willows (Salix species) are members of the Salicaceae family which is in the order Malipighiales. Most of the families in this order have flowers with four whorls, and comparisons within the fossil record, as well as genetic level studies, indicate that the genus Populus is of relatively recent origin most likely

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derived from an ancestor with typical four-whorled flowers (Eckenwalder, 1996). We have previously characterized AP1-class genes such as PTM1 and PTM2 in floral development as well as TM3/SOC1-class genes such as PTM5 in floral and vegetative development (Cseke et al., 2003a; 2003c). Considering the differences in poplar floral structure resulting from the evolution of highly reduced, wind-pollinated flowers, we undertook the study of P. tremuloides SEP-class MADS-box genes to determine if similar mechanisms are conserved in trees with two-whorled flowers as compared to other species.

2. Materials and methods 2.1. Plant material Male and Female Populus tremuloides Michx. trees were identified and marked at specific locations in Houghton, Michigan (47.1-N Latitude / 88.6-W Longitude). The development of aspen floral tissues was previously divided into 6 distinct stages, from stage 1 catkins, where the sex of the primordia was not yet identifiable, through stage 6 catkins having mature floral organs (Cseke et al., 2003a,c). These tissues were collected at pre- and post-winter time points and used for RNA blots, protein blots, and in situ RNA hybridizations. Growing terminal buds, young leaf, stem, and root tissues used for the analyses were taken from young male and female aspen trees propagated and grown in a greenhouse under controlled conditions. 2.2. cDNA cloning of aspen SEP-class genes Total RNA, free from contaminating genomic DNA, was isolated from developing male and female floral buds as described (Cseke et al., 2003b). Reverse transcription was carried out in 20 Al reactions following protocols supplied with SUPERSCRIPTi II reverse transcriptase (Gibco BRL, Gaitherburg, MD). A degenerate primer based on the conserved regions near the start codon of plant MADSbox sequences in the Genbank database was previously used to obtain male and female aspen MADS-box homologs (Cseke et al., 2003a,c). The cDNAs thus obtained from several independent reactions lacked the 5V ends of the genes including the regions spanning the first ten amino acids. Consequently, male and female flower cDNA library lysates, constructed in the k-triplex vector according to the Smart cDNA Library Construction Kiti protocol (Clontech, Palo Alto, CA), were used as templates to obtain the 5V ends of PTM3 and PTM4. Full-length clones were then generated using protocols contained in the Marathon cDNA Cloning Kit (Clontech, CA) and primers designed for the 5V and 3V ends of the cDNAs. PTM6 was isolated during the amplification of fulllength PTM3 clones using RT –PCR performed on cDNAs from stage 4 floral tissues. 5V-UTR and 3V-UTR PTM3

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specific primers generated a 1120bp PTM6 product in addition to the PTM3 product. This second product was determined to be a new SEP-class clone. Note: the 5V and 3V PTM6 sequences were confirmed using internal sequencespecific 5V and 3V RACE generated products from cDNAs obtained from stage 4 floral tissue as detailed in the 5V/3V RACE Kit (Roche, Indianapolis, IN). 2.3. Sequence alignments and phylogenetic analysis The sequence of each cDNA was obtained by double strand sequencing using an ABI 310 Genetic Analyzer (Applied Biosystems, CA) and the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham-General Electric, NJ). Sequencing of both strands of each clone was accomplished using sequence specific primers, and the final sequence was obtained by contig assembly of all overlapping sequences. Sequences were analyzed using NCBI Blast searches and Clustal W alignments. Potential sequence motifs were identified using a combination of websupported programs as previously described (Cseke et al., 2003c). The phylogenetic analysis was done as described (Cseke et al., 2003a,c) on protein sequences obtained from Genbank (see accession numbers below) and sequences obtained from the poplar genomic database (http:// genome.jgi-psf.org/Poptr1/Poptr1.home.html). Heuristic search analysis using parsimony was done with the PAUP 4.0b10 program on Clustal W sequence alignments followed by Bootstrap analysis using 1000 replicates with amino acids treated as unrooted characters (PAUP 4.0b10, Sinauer Associates, Sunderland, MA). AGL38 and 41 were used to root the tree because they are more closely related to the ancestral MADS-box genes found in plants, animal, and fungi (Alvarez-Buylla et al., 2000). [Populus tremuloides Michx.] PTM1,AF034093; PTM2,AF034094; PTM3,AF185574; PTM4,AF034095; PTM5,AF377868; PTM6,AY235222: [Populus balsamifera subsp. trichocarpa] PtAG1,AAC06237; PtAG2,AAC06238; PtAP11,AAT39554; PtAP1-2,AAT39556; PtD,AAC13695: [Betula pendula] BpMADS1,CAB95648: [Arabidopsis thaliana] AG,P17839; SEP1/AGL2,T51409; SEP4/ AGL3,S57793; SEP2/AGL4,D39534; SHP2/ AGL5,E39534; AP1/AGL7,P35631; FUL/AGL8,S71208; S E P 3 / A G L 9 , T 0 0 6 5 6 ; S T K / A G L 11 , T 0 4 0 0 0 ; AGL14,T09347; AGL19,AAG37901; SOC1/AGL20, AAG16297; AGL38,AAC27144; AGL41,AC005168; AGL42,BAB10179; AP3,A42095; CAL,AAA64789; PI,A53839: [Eucalyptus grandis] EGM1,AAC78282; EGM3,AAC78284: [Gerbera hybrid cultivar] GRCD1,CAC13148: [Lycopersicon esculentum] LeMADS-RIN,AAM15775; TAG1,T07185; TM3, S23729; TM4,S23730; TM5,CAA43170; TM6,S23731; TM29,CAC83066: [Malus domestica] MdMADS1,AAC25922; MdMADS2,AAC83170; MdMADS3, AAD51422; MdMADS4,AAD51423; MdMADS5,

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CAA04321; MdMADS6,CAA04322; MdMADS7, CAA04323; MdMADS8,CAA04919; MdMADS9, CAA04920; MdMADS10,CAA04324; MdPI,CAC28022: [Nicotiana sylvestris] NsMADS3,AAD39034: [Nicotiana t a b a c u m ] N t A G 1 , Q 4 3 5 8 5 ; N t D E F, C A A 6 5 2 8 8 ; NtGLO,Q03416; NtMADS1,S46526; NtMADS4, AAF76381; NtMADS5,AAD39035: [Petunia hybrida] FBP2,AAA86854; FBP4,AAK21247; FBP5,AAK21248; FBP9,AAK21249; FBP21,AAK21252; FBP23, AAK21254; FBP28,AAK21257: [Pinus radiata] PrAG, AAD09342; PrMADS1,T09569. Gene IDs from the P. trichocarpa genomic database that we have named according to their respective Arabidopsis homologues: PtFUL-1, grail3.0042013901; PtFUL-2, grail3.0155001801; PtPI, grail3.0002017601; PtSEP1, grail3.0139000303; PtSEP2, eugene3.01550021; PtSEP31, grail3.0008011201; PtSEP3-2, grail3.0047013501; PtSEP4, gw1.VIII.1808.1; PtSTK, eugene3.00190689. 2.4. DNA blot analysis Total genomic DNA was extracted from young male and female aspen leaves using a CTAB method as described (Cseke et al., 2003b). The resulting genomic DNA was digested with BamHI, HindIII, SacI and XbaI. Fourteen Ag of each digested DNA was run on a 0.8% agarose gel and Southern blotted onto charged nylon membranes (Hybond N+, Amersham-General Electric, NJ) using an alkaline capillary transfer method. The membranes were probed at 65 -C using the Church hybridization method and [a-32P] dCTP labeled 3V end of PTM3 or PTM6 as probes (Cseke et al., 2003b). These probe regions were determined to be specific to PTM3/4 and PTM6 respectively using both genbank searches on the NR and EST databases and JGI searches of the poplar genomic sequence. Exposure was performed for four days on a phosphoimaging screen (BioRad Technologies, Hercules, CA). Both male and female DNA resulted in the same patterns. 2.5. Binary vector construction and plant transformation The binary vector for over-expression of PTM3 (PTM3sense) was constructed by inserting a double CaMV 35S/ AMV RNA4 promoter, PTM3 coding region, and 3V NOS terminator sequence into the pTCS5 binary vector (Datla et al., 1993). pTCS5 has a NPTII-GUS fusion gene driven by a 35S-promoter for kanamycin selection and GUS reporter expression. The binary vector for suppression of PTM3/4 related genes through post-transcriptional gene silencing (PTM3-IR) was constructed as above using an inverted repeat region created from PTM3 specific sequence. The 417bp inverted repeat region spanned parts of the K-, C-and 3V-UTR (bp 453 to 870). The remaining span of the 3V-UTR (bp 870 to 1020) was designed as the loop region of the repeat required for the construct to be stable in bacterial cells (Waterhouse et al., 2001).

The binary vectors were mobilized into Agrobacterium tumefaciens strain C58/pMP90 by a triparental mating method (Cseke et al., 2003b). Aspen tree leaf disks were transformed with the sense and inverted repeat PTM3 constructs as well as the pTCS5 vector harboring no inserts (used as a control) following methods established in our lab (Tsai et al., 1994). After kanamycin selection at 100 Ag/ml, we obtained 13 aspen lines for PTM3-sense, 11 lines for PTM3-IR, and 10 lines for pTCS5. These plants were maintained on soil under controlled conditions at 25 -C in a growth chamber with a 16 h photoperiod. Integration of each construct was confirmed for each line based on strong GUS activity in all tissues and PTM3 cDNA specific PCR on isolated genomic DNA. All lines were determined not to be chimeric, and final conformation of transformation was determined by RNA blot analysis of expressed construct in older leaf tissue. For the analysis of flower formation, we were able to force some of the PTM3-sense and pTCS5 lines to flower in 2.5 years instead of the typical 8 to 10 years. This was done by keeping the trees root bound in small pots under continuously warm greenhouse conditions that shorten each dormancy period. The protocol established by Horsch et al. (1985) was used to introduce constructs into tobacco (Nicotiana tabacum cv SR1) with minor modifications as described in (Cseke et al., 2003c). For each of the PTM3-sense, PTM3-IR and pTCS5, 43, 19, and 37 independent transgenic lines respectively were selected on 100 Ag/ml kanamycin. Analysis of the kanamycin resistant/sensitive ratios of the T1 and T2 generations suggested that only one copy of the insert was obtained in almost all lines. For the single copy, GUS positive lines, 11 PTM3-sense, 11 PTM3IR and 7 pTCS5 lines were chosen for analysis using PTM3 specific PCR on genomic DNA and RNA blot analysis for expressed constructs. All rooted plants were maintained on soil under controlled conditions at 25 -C in a growth chamber with a 16 h photoperiod. Transformation of Arabidopsis was performed by a vacuum infiltration method (Bechtold and Pelletier, 1998) and analysis of the T1 and T2 generations was performed as for tobacco. For each of the PTM3-sense, PTM3-IR and pTCS5, 34, 35, and 28 independent transgenic lines respectively were selected on 40 Ag/ml kanamycin. For the single copy, GUS positive lines, 12 lines of each of PTM3-sense, PTM3-IR and pTCS5 lines were chosen for analysis using PTM3 specific PCR on genomic DNA and RNA blot analysis for expressed constructs. All rooted plants were maintained on soil under controlled conditions at 22 -C in a growth chamber with a 16 h photoperiod. To avoid phenotypic differences stimulated by selection on media containing kanamycin, we also performed soil experiments on populations of the seeds collected from T1 and T2 generation lines that had GUS positive, single copy insertions and were proven to have expression of the insertion via RNA blots. Phenotypic analysis of the 3/4 of the plants in each line having GUS activity and grown under

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identical growing conditions (i.e. day-length, temperature, humidity) helped to determine what phenotypic differences were consistent with transgene expression and not due to culturing conditions. 2.6. RNA blot analysis Total RNA was extracted from 20 different tissues in both male and female aspen including floral bud stages 1 to 6 as well as terminal buds, young leaves, young stems, and roots as described (Cseke et al., 2003b). RNA quantitation was performed using Ribogreen dye (Molecular Probes, OR) and a flourometer (Turner, PicoFluor), and 12 Ag of RNA were run on a 1% denaturing agarose gels. The RNA from each experiment was then transferred onto a charged nylon membrane (Hybond N+, AmershamPharmacia, New Jersey) and hybridized at 65 -C using the Church hybridization method and the same [32P] dCTP labeled PTM6-specific probe as described in DNA blot analysis (Cseke et al., 2003b). After stripping the membranes, they were then hybridized with the PTM3specific probe (described above), and following one more round of stripping, they were probed with an aspen 26S rRNA probe as a loading control. 2.7. Antibody production and protein blot analysis The PCR amplified IKC-region of the PTM3 cDNA was cloned into the pET 22b+ His-Tag expression vector (Novagen, Madison, WI) and mobilized into BL-21 cells. Protein expression was induced using 0.5mM IPTG as described (Cseke et al., 2003b), and purification of the recombinant protein fragment was accomplished as described in the HisIBind Purification Kit manual (Novagen, Madison, WI). 1.2 mg of the pure 22 kDa protein was used to generate polyclonal antibodies in rabbits (Alpha Diagnostic International, Inc., San Antonio, TX). Antibodies were determined to be specific to the PTM3/4 protein with no signal resulting from preimmune serum when using pure protein and protein extracts described below. Protein extracts from both male and female floral bud stages 1– 6, terminal buds, young and mature leaves, young stems, as well as xylem and phloem tissues were prepared from 300 –400 mg each of tissue as described (Cseke et al., 2003b) using a modified extraction buffer (10 mM HEPES pH 7.9, 50 mMMgCl2, 10 mMKCl, 0.5 mM DTT, 1.0 mM PMSF, 0.2 mM pepstatin A, serine protease inhibitor cocktail (Roche, Indianapolis, IN), and 9 Ag/ml DNase). The amount of total protein was determined using the Bradford method and bovine serum albumin as standards. Protein blot analysis was then accomplished by subjecting 15 Ag of each protein sample to SDS-PAGE on 13% SDS-PAGE gels containing 2 M urea. Purified recombinant protein was used as a positive control, and the proteins were transferred onto PVDF membranes (Millipore Corporation, Bedford, MA) (Cseke et al.,

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2003b). Anti-PTM3 antibodies were used at a 1 : 2000 dilution followed by a 1 : 30,000 dilution of the secondary Goat anti-rabbit IgG conjugated with horseradish peroxidase (Pierce Biochem, Rockford, IL). Signal was visualized following protocols in the SuperSignal\ West Pico Chemiluminescent Substrate kit (Pierce Biochem, Rockford, IL). The blot was exposed to film for 2 min. 2.8. In situ hybridization Localization of PTM3/4 and PTM6 transcripts within 12 Am thick developing floral sections was performed using in situ RNA hybridizations as previously described (Cseke et al., 2003c). DIG-labeled sense and antisense PTM3/4 and PTM6 probes were prepared from their respective 3V-UTRs following the manufacturer’s instructions (Roche DIG labeling kit, Indianapolis, IN). These regions of the PTM3/4 and PTM6 clones yield no significant matches with any other cloned gene or EST other than their exact match in NCBI Blast searches or within the poplar genomic sequence database. Each of approximately 35 sections per experiment was visualized using a Nikon dissecting microscope equipped with a Sony digital imaging system and software. In situ localization was also done using a similar number of vegetative tissue sections including young leaf and stem tissues, but no RNA signal was detected in nonfloral tissues (data not shown).

3. Results 3.1. The poplar genome contains a SEP-class family of genes PTM3 and PTM4 were originally isolated from RNA obtained from stage 4 floral catkins using a degenerate primer based RT – PCR method (see Materials and Methods). The full-length PTM3 cDNA sequence is 1190 bp in length containing a 231bp 5V UTR, a 224 bp 3V UTR, and a 28.1 kDa deduced amino acid sequence of 245 residues (Fig. 1). PTM4 is 926 bp in length and was repeatedly cloned as a second copy of PTM3. It shares 99% nucleic acid sequence identity with PTM3 with only 3 amino acid differences in the deduced amino acid sequence and possesses a 3V UTR that is 99% identical to PTM3. The deduced amino acid sequences were aligned with other known MADS proteins revealing that PTM3/4 share 72% overall identity with SEP1 and 74% identity with SEP2 (Fig. 1). Each gene codes for a protein having all of the hallmark features of typical plant MADS-box transcription factors, including the MADS-box region with internal nuclear localization motif, a coiledcoil structure characteristic of the K-domain that is responsible for protein-protein interactions, and various predicted motifs for phosphorylation, myristilation, and glycosylation.

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Fig. 1. PTM3, 4, and 6 deduced amino acid sequences aligned with SEPALLATA genes from Arabidopsis using Clustal W analysis. Also shown is BpMADS1, a SEP3 homolog from silver birch. Each gene is compared with respect to the PTM3 sequence. Black boxes refer to residues that are identical to those found in PTM3. Grey boxes identify similar residues, and dashes indicate gaps in the sequences used to optimize the alignments. Within the PTM3 sequence is the typical nuclear localization motif (double overscore) as well as predicted phosphorylation sites (triangles), myristilation sites (archs), and glycosylation sites (stars).

In order to check the copy number and authenticity of PTM3/4, high stringency male and female genomic DNA hybridizations were performed (Fig. 2). In both male and female DNA, BamHI resulted in two bands, while HindIII, SacI, and XbaI resulted in three primary bands when probed with a 3V PTM3-specific sequence. While none of these enzymes cut within the coding region that spans the PTM3 probe used, analysis of the homologous sequences contained within the P. trichocarpa genome database, which is 99% identical to P. tremuloides sequences, suggest that HindIII, SacI, and XbaI cut within the intron regions that span this segment (see the genomic sequence for what we call PtSEP1). Thus, there appear to be two copies of PTM3/ 4 in the P. tremuloides genome. A third MADS-box cDNA, PTM6, is 1,120 bp in length including an open reading frame that codes for a 27.5 kDa, 242 amino acid long protein sharing 64% amino acid identity with PTM3/4 (Fig. 1). PTM6 shares only 59% overall identity with SEP1 and SEP2, but it has 74% identity with SEP3. As above, high stringency male and female genomic DNA hybridizations were performed using a 3V PTM6-specific sequence (Fig. 2). For PTM6, BamHI and HindIII resulted in two bands while SacI and XbaI resulted in one band each. As with PTM3/4 none of these enzymes cut within the coding region that spans the probe used. However, analysis of the homologous sequences within the P. trichocarpa genome database suggests that HindIII cuts in an intron region that spans the segment.

Thus, PTM6 is most likely a single copy gene in aspen. However, P. trichocarpa has an additional SEP3 homologue (which we call PtSEP3-2), and the PTM6 Southern blot does have faint bands that may be representative of another homologous gene. To better understand the relationships of these three genes to the major classes of MADS-box genes in other species, phylogenetic analysis was performed using deduced amino acid sequences (Fig. 3). Comparison with similar genes from four-whorled flower species, such as Arabidopsis thaliana, Lycopersicon esculentum, Nicotiana sylvestris, Nicotiana tabacum, Petunia hybrida, Eucalyptus grandis, and Malus domestica, showed that PTM3/4 fall within the SEP1/2-subclass of MADS-box proteins while PTM6 is related to the SEP3-subclass (Fig. 3). The analysis also compares sequences obtained from the P. trichocarpa genome database. These sequences not only indicate that the P. trichocarpa genome contains genes falling within all of the A, B, C, D, and E-class MADSbox genes but also indicate that PTM3 and PTM4 match in sequence and copy number with what we call PtSEP1 and PtSEP2 from P. trichocarpa (Fig. 3). PTM6 matches two sequences that we name PtSEP3-1 and PtSEP3-2, and we identified a fourth P. trichocarpa SEP-like sequence that we call PtSEP4. Therefore, poplar species (two-whorled species) maintain families of MADS-box genes, including SEP-class genes, similar to those found in four-whorled herbacious species.

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Fig. 2. Confirmation of the presence of PTM3/4 and 6 within the aspen genome using high stringency genomic DNA blot analysis probed at 65 -C with either a PTM3/4-specific fragment or a PTM6-specific fragment made from the C-domain and 3V-UTR regions. While none of the enzymes cut in the coding regions that span the probes used, evidence from the poplar sequence database suggests that some of these enzymes cut within intron regions.

3.2. Alteration in PTM3/4 expression in aspen does not affect vegetative development but alters catkin and flower development We generated 13 independent PTM3-sense transgenic lines of aspen showing resistance to kanamycin, positive GUS reporter gene assays, and appropriate PTM3 fragments during PTM3-specific PCR on genomic DNA (data not shown). Of these 13 lines, 8 were shown to have strong PTM3 expression in older leaves, where expression is not normally observed, while the remaining had very low levels (Fig. 4). This evidence confirms the transgenic nature of the trees; however, upon comparison with 10 control lines, confirmed to harbor the empty pTCS5 binary vector insert, no phenotypic differences were seen in any vegetative tissues during development. As described above, aspen trees require 8 to 10 year to begin flowering under unforced conditions. However, we were able to force some of the PTM3-sense and pTSC5 lines to flower in only 2.5 years. Interestingly, the PTM3-sense lines that flowered were the lines that showed very low levels of PTM3-sense transcript. Moreover, the flowers of these lines had the unusual

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phenotype of having catkins divided into three developmental regions including shoot-like growth at the base followed by normal appearing individual flowers that turned to leaf-like structures near the ends of the catkins (Fig. 4panel F). While it is possible that this phenotype is a result of co-suppression, these are preliminary results, and the full consequences of PTM3 manipulation in aspen will require more study over the next few years. For PTM3/4 experiments using post-transcriptional gene silencing (PTGS) to target the suppression of homologous genes in aspen, we generated 11 independent PTM3-IR transgenic lines of aspen showing resistance to kanamycin, positive GUS reporter gene assays, and appropriate PTM3 fragments during PTM3-specific PCR on genomic DNA (data not shown). Of these lines, none showed RNA production of the 1300 bp inverted repeat product (inverted repeat plus loop plus NOS terminator). However, all showed a ladder of hybridizing degradation products when probed with a PTM3-specific 3V probe, although only one line (G11) had band intensities comparable to the PTM3 over-expressing lines (Fig. 4). Upon comparison with 10 control lines, no phenotypic differences were seen in any vegetative tissues during aspen tree development. To date, we have been unable to force any of these lines to flower, but whether this is due to the inserted construct is unknown. Tree researchers often use heterologous systems to help study tree floral development due to the long juvenile periods found in most trees (Kotoda et al., 2002; Lemmetyinen et al., 2001, 2004; Rottmann et al., 2000; Rutledge et al., 1998; Southerton et al., 1998; Sundstrom and Engstrom, 2002; Sung et al., 1999). To make initial comparisons between woody and herbaceous species and to test if heterologous systems can yield similar results to those that we obtained from aspen, we performed parallel experiments in tobacco and Arabidopsis. For tobacco, 43 independent PTM3-sense lines were generated. Following analysis for kanamycin resistant ratios and GUS activity in the T1 and T2 generations (data not shown), 11 lines were selected for further analysis of PTM3 expression and transgenic phenotypes (Fig. 5). Of these lines, all showed strong PTM3 ectopic expression compared to control plants harboring the pTCS5 binary vector alone (Fig. 5-panel C). When these lines were grown under culture conditions with selection on kanamycin, they showed the typical and significant early flowering phenotype seen during the over-expression of other SEP-class genes in various species (Ferrario et al., 2003; Jang et al., 1999, 2002; Lemmetyinen et al., 2004; Moon et al., 1999; Pelaz et al., 2001; Tzeng et al., 2003). However, we also performed screening experiments by planting T1 and T2 generation seeds on soil without selection. None of the members of the lines under soil conditions displayed the early flowering phenotype despite confirmation that approximately 3/4 of each line were transgenic. Thus, we concluded that the early flowering phenotype had more to do with the culture conditions

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Fig. 4. Phenotypes of transgenic aspen harboring PTM3 over-expression and inverted repeat suppression constructs. All of the trees shown were grown side-byside under identical conditions and proven to have functional incorporated construct. Panel A: control trees harboring the empty pTCS5 vector. Panel B: transgenic aspen harboring the PTM3-sense construct and shown to have ectopic expression of PTM3. Panels C: transgenic aspen harboring the PTM3-IR inverted repeat construct. No phenotypic differences in vegetative growth were seen in any of the 24 transgenic lines tested as compared to controls or wildtype trees. Panel D: PTM3 RNA analysis from mature leaves of selected aspen transgenic lines showing no expression in the control plants, high expression in most of the over-expression plants, and degradation resulting from the suppression construct. The probe used was identical to that used in Fig. 2, the ethidium bromide stained gel is provided as a loading control. The lane labeled YL is an RNA sample from aspen young leaf showing the typical signal in this tissue. Panel E: a normal late stage floral catkin. Panel F: a late stage catkin displaying conversion of normal floral development to more shoot and leaf-like development generated by aspen harboring the PTM3-sense yet expressing low levels of transcript (lines P2 and P3). Panel G: a normal lateral shoot generated by aspen harboring the PTM3-IR construct and identical to control and wild-type trees.

rather than the effect of PTM3 on development. However, as was seen with the over-expression of OsMADS1/LHS1, NsMADS3, or NtMADS4 (all SEP homologs) in tobacco as well as LMADS3 from lily when expressed in Arabidopsis, one consistent PTM3 over-expression phenotype in both culture and on soil was reduced apical dominance that

triggered growth of the lateral meristems which in turn developed new floral meristems (Fig. 5-panels A and B) (Chung et al., 1994; Jang et al., 1999, 2002; Tzeng et al., 2003; Vrebelov et al., 2001). For Arabidopsis, 34 independent PTM3-sense lines were generated. Following analysis for kanamycin resistant

Fig. 3. Phylogenetic analysis of predicted amino acid sequences of PTM3, 4 and 6 compared to selected herbaceous and tree MADS-box sequences (including others from P. tremuloides and P. trichocarpa) using the PAUP 4.0b10 program. This is a comparison of only a small number of MADS-box proteins known to exist, and thus the purpose of this analysis is only to identify the general class of MADS-box proteins in which PTM3, 4 and 6 fall in comparison to sequences found in the poplar database. Vertical bars indicate the primary classes of MADS box genes. Bootstrap percentages refer to the percentage of replicates that support the branch. AGL38 and AGL41 were used to root the tree because it is believed that they are more closely related to the ancestral-type MADS-box genes (Alvarez-Buylla et al., 2000).

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Fig. 5. Phenotypes of transgenic tobacco and Arabidopsis harboring PTM3 over-expression and inverted repeat suppression constructs for comparison with experiments done in aspen. The plants shown were grown side-by-side under identical conditions and proven to have incorporated construct. Panels A: late stage T2 generation control tobacco transformed with the empty pTCS5 vector. Panels B: a late stage T2 generation PTM3 over-expressing transgenic line showing reduced apical dominance causing conversion of axillary meristems to floral meristems. Panel C: RNA expression of the PTM3 gene in leaves of tobacco constitutively expressing PTM3 as compared to a control plant. Panel D: RNA expression of the PTM3 in leaves of Arabidopsis constitutively expressing PTM3 as compared to control plants, although none of these lines showed phenotypic differences from control plants (data not shown). Panel E: a late stage T1 generation control Arabidopsis flower harboring the empty pTCS5 vector. Panel F: a late stage T1 generation Arabidopsis flower harboring the PTM3 inverted repeat construct and showing the loss of normal development of the stamen and carpel whorls resulting in sterility. Panel G: RNA expression and degradation of the inverted repeat PTM3 transcript in the leaves of Arabidopsis lines. Typically the 1300 bp IR transcript can be seen as a 900, 300, and 21 bp ladder as it is degraded. The ethidium bromide stained gels in Panels C, D, and G are provided as a loading controls.

ratios and GUS activity in the T1 and T2 generations (data not shown), 12 lines were selected for further analysis. Of these lines, 10 showed PTM3 ectopic expression compared to control plants harboring the pTCS5 binary vector alone, although expression levels varied widely compared to those in tobacco (Fig. 5-panel D). As with tobacco, most of these lines had an early flowering phenotype when selected in culture. However, this phenotype disappeared when seeds were grown on soil alone. No other phenotype was observed for Arabidopsis over-expressing PTM3 vs. control lines. As a means to test the ability of the PTM3-IR construct to cross-suppress SEP-class genes in heterologous systems, we also generated transgenic tobacco and Arabidopsis using our PTM3-IR construct. Prior to these experiments, however, we searched the genome of Arabidopsis as well as the known SEP homologs in tobacco to validate that expression of PTM3-IR would be able to target the genes of interest during PTGS. In Arabidopsis, it is clear that only SEP1 and SEP2 have stretches of sequence with enough identity to be targeted by PTGS using double-strand PTM3 RNA (data not

shown). The results for tobacco were not as clear due to the limited number of sequences. For Arabidopsis, 35 independent PTM3-IR lines were generated. Integration of the PTM3-IR construct was confirmed using kanamycin selection, GUS assays of the NPTII/GUS fusion gene, and PCR reactions on genomic DNA specific for the PTM3 inverted repeat. Twelve transgenic lines were then selected for further analysis, and the ratios of kanamycin resistance to sensitive germinating seeds suggested that each of these lines was the result of single copy insertions (data not shown). All of these lines had the expected ladder of PTM3-IR degradation products consistent with PTGS that results in degradation of the expressed construct (Fig. 5-panel G). As with the PTM3sense construct, we again observed phenotypic differences between plants grown under culture conditions compared to those grown on soil alone. When these lines are grown under culture conditions with selection on kanamycin, they show a severe reduction in plant size and a complete lack of flowering. Similar results have been observed by other researchers

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during manipulation of SEP-class genes in various species (Chung et al., 1994; Jang et al., 1999, 2002; Lemmetyinen et al., 2004; Tzeng et al., 2003). However, when T1 and T2 generation seeds were planted on soil without selection, none of the members of populations of these lines displayed the same phenotypes. One consistent phenotype in both culture and on soil were was highly reduced seed production in flowers that appeared to have no other alterations in floral morphology as compared to control lines grown under identical conditions. This is similar to the phenotype that we observed in SEP1/SEP2 double mutants using seed supplied to us by Dr. Martin F. Yanofsky (data not shown). Another consistent phenotype was observed in 5 of the PTM3-IR lines where neither the stamens nor carpels developed in resulting in sterility. While the sepal and petal whorls (the perianth organs) did develop in these lines, both were elongated compared to control flowers (Fig. 5-panels E and F). Similar results were obtained from T1 and T2 generations of tobacco transgenic lines where 8 out of 19 lines showed premature termination of the development of petals, stamens, and carpels (data not shown). Therefore, the PTM3-IR construct does appear to be able to target the expected floral organs in heterologous systems. However, unlike sep-mutant phenotypes, we did not observe the conversion of inner floral whorls to sepals or leaf-like structures.

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3.3. PTM3/4 and PTM6 expression is consistent with a function in inner whorl, pollen and ovule development Aspen flower development has been previously divided into 6 distinct stages (see Materials and Methods for more details). Northern hybridizations were performed in order to check the expression of PTM3/4 throughout the tree. Total RNA was extracted from stages 1 to 6 as well as vegetative tissues from both male and female aspen, and the 1.2 Kb PTM3/4 transcript was detected in each of the floral stages 1 –6 in both male and female flowers (Fig. 6-panel A). Transcript levels of PTM3/4 are lower in stages 1 and 2 buds of both sexes, which may reflect a reduction in transcript levels prior to winter dormancy (see arrow heads within Fig. 6). In contrast, there is a uniformly strong transcript expression in the after-winter stages 3– 6 in both male and female buds while vegetative tissues show very low levels of expression in the terminal buds, young leaves, and young stems. PTM6 on the other hand, has no detectable levels of transcript in the vegetative tissues, and it has overall much lower levels of expression as compared to PTM3/4. The PTM3/4 protein expression pattern differs slightly from the RNA results (Fig. 6-panel C). Polyclonal antibodies were raised against the E. coli expressed polypeptide including the I-, K-, and C-terminal domain of the protein. The purified 22kDa peptide was used as a positive control

Fig. 6. Expression patterns of PTM3/4 and PTM6 in male and female aspen floral and vegetative tissues. Panel A: Stage 1 through 6 male and female floral RNA was probed under high stringency conditions with PTM3/4, PTM6, and 26S rRNA fragments specific to each gene. 12 Ag of total RNA from each tissue was loaded per lane and exposed for four days on a phosphoimaging screen. Vegetative tissues were used as controls (TB: terminal bud; YL: young leaf; YS: young stem; R: root), and arrowheads indicate where winter dormancy occurs. Panel B: the protein purification scheme for the PTM3/4 peptide. Lane M: marker lane; Lane 1: crude extract from IPTG induced E. coli harboring the PTM3 expression construct; Lane 2: sample from an intermediate Urea purification step; Lane 3: purified PTM3 IKC-region peptide after His-tag purification. This fragment was used to prepare PTM3 specific polyclonal antibodies. Panel C: Western blot analysis of protein extracts from stage 1 through 6 female floral tissues as well as terminal bud (TB) and young leaf (YL) visualized using PTM3/4 specific polyclonal antibodies. Purified E. coli expressed PTM3/4 protein fragment was used as a positive control.

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for the Western blot analyses and showed a sharp signal with the anti-PTM3 IgG antibodies while the pre-immune serum showed no signal (Fig. 6-panel C and data not shown). PTM3/4 protein was detected in floral stages 1 – 5,

but these levels seem to be lower in pre-winter tissues with strongest signal in stage 3, just after the breaking of winter dormancy. Overall, this correlates with the RNA results; however, PTM3/4 protein was not detected in mature male or female organs. In addition, high signal was detected in young leaves, and a small amount of protein signal was detected in terminal buds. To identify the spatial localization of PTM3/4 and PTM6 transcripts within the developing floral organs, RNA in situ hybridization analysis was performed in stage 2 and 4 male and female catkins using the respective 3V-UTR DIG-labeled sense (control) and antisense RNA probes. In three independent analyses, strong PTM3/4 transcript expression was observed in both stages using the antisense probe (Fig. 7-panels A, C, and E), and no color development was observed using an equal amount of the sense control probe and equal color development (Fig. 7-panels B, D, and F). In stage 2 male and female catkins, the transcript is visualized within the anther primordia and the ovule primordia, respectively (Fig. 7-panels A, B, C, and D). This expression continued and expanded in the inner whorl of stage 4 female floral catkins where it encompassed developing ovules and the outer wall of the carpel (Fig. 7-panels E and F). Similar results were obtained for PTM6 where transcripts were localized to developing ovules, the outer wall of the carpel, and the stigma (Fig. 7-panels G and H). No expression of either PTM3/4 or PTM6 was seen in the perianth cups of any stage (approximately 100 sections for each stage), nor was expression visualized in young leaves or stems (data not shown). These results are consistent with a function in the development of inner whorl organs including ovules and pollen.

4. Discussion 4.1. Floral MADS-box genes in poplar species: the implications of SEP-class genes for wind-pollinated flower morphology Fig. 7. In situ mRNA localization of PTM3/4 and PTM6 in developing stage 2 and 4 floral tissues. Panels A, C, and E are tissues incubated with the DIGlabeled PTM3 antisense probe made from the 3V UTR showing the expression of PTM3/4 in the developing anther primordia (Ap) of stage 2 male floral buds (Panel A), stage 2 female developing ovule primordia (Op) (Panel C), and stage 4 female ovules and carpel walls (Panel E). Arrows indicate the tissues where PTM3/4 expression is most abundant. Panels B, D, and F are negative controls showing the same tissues incubated with the same amount of DIG-labeled PTM3 sense probe made from the 3_ UTR and undergoing an identical amount of color development. Panel G is tissue incubated with the DIG-labeled PTM6 antisense probe made from the 3_ UTR showing the expression of PTM6 in stage 4 female ovules, carpel walls, and stigma. Panel H is a negative control showing the same tissue incubated with the same amount of DIG-labeled PTM6 sense probe made from the 3V UTR and undergoing an identical amount of color development. ‘‘St’’ indicates the location of the stigma, ‘‘o’’ indicates the location of an ovule, and ‘‘p’’ indicates the location of the perianth cup while ‘‘s’’ indicates the whorl holding the sexual organs in these two whorl flowers. The size bars indicate 200 Am.

In an effort to address the sparse understanding behind the two-whorled floral development of poplar trees, our lab has identified, cloned, and characterized, from P. tremuloides, several distinct cDNA clones that are homologous to MADS-box genes that control flower formation in many other plant species (Cseke and Podila 2004; Cseke et al., 2003a,c). Among these are a group of SEP-class MADSbox homologues that are required for the proper development of the inner three whorls of four-whorled species such as Arabidopsis, Petunia, tobacco, tomato, apple trees and Eucalyptus trees (Pelaz et al., 2000; Angenent et al., 1994; Ampomah-Dwamena et al., 2002; Jang et al., 1999; Pnueli et al., 1994; Southerton et al., 1998; Sung et al., 2000). However, recent evidence from Arabidopsis SEP4 indicates that SEP-class genes work together in specifying the development of ovules, meristems, and all four floral whorls

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including sepals (Ditta et al., 2004). These diverse functions are thought to result from the differential formation of protein complexes with other MADS-box factors (reviewed in Kaufmann et al., 2005). We show that PTM3 and PTM4 are duplicate genes within P. tremuloides and are closely related to the SEP1/2subclass of MADS-box proteins (Figs. 1, 2 and 3). PTM6, appears to be a single copy gene more closely related to the SEP3-subclass (Figs. 1, 2 and 3). Similar results are found with homologous sequences in the poplar genome database where P. trichocarpa has two copies related to PTM3/4 (PtSEP1 and PtSEP2), two copies related to PTM6 (PtSEP3-1 and PtSEP3-2), and one copy of a sequence related to SEP4 from Arabidopsis. In addition, the P. trichocarpa genome contains genes falling within all primary classes of floral MADS-box genes including a group of A-class genes, PtAP1-1 and PtAP1-2 (related to PTM1 and PTM2 from P. tremuloides) as well as PtFUL-1 and PtFUL-2 (Fig. 3). Likewise, there are genes that fall into the B-class (PtD and PtPI), C-class (PtAG1 and PtAG2), and D-class (PtSTK) (Brunner et al., 2000; Sheppard et al., 2000). Therefore, poplar species (twowhorled species) maintain families of MADS-box genes, including SEP-class genes, similar to those found in fourwhorled herbacious species. As described in the introduction, Populus species are thought to be recently derived from a four-whorled ancestor, and this would explain the presence of A, B, C, D, and Eclass MADS-box genes within the genome (Eckenwalder, 1996). It is sometimes assumed that the perianth cup of the flowers is a result of the reduction of four-whorled perianth organs (sepals and petals) to a more simplified structure, although the true identity of the perianth cup remains unknown. In addition, the stamen and carpel whorls in poplars have been reduced through a mechanism of dioecy that initiates only one type of sexual organ, making the flowers truly unisexual (Boes and Strauss, 1994; Sheppard et al., 2000). Such control must occur at a very early stage of development, and these changes in floral development put into question how the behavior of the primary classes of MADS-box genes has changed in poplars. To explore the role of poplar SEP genes in floral development, we conducted experiments to localize the expression of PTM3/4 and PTM6. As with other SEP-class genes, RNA expression of PTM3/4 and 6 was detected in all stages of flower development; however, lower levels of PTM3/4 were also detected in terminal buds, young leaves, and young stems (Fig. 6). The largely uniform expression pattern of PTM3/4 and PTM6 in the flowers appears to be due to transcripts localized to the inner, sexual whorl starting in ovule and anther primordia and expanding to other tissues of the inner whorl at later stages (Fig. 7). However, unlike other SEP-class genes in four-whorled species, which are expressed in at lease one of the perianth organs (sepals or petals), PTM3/4 and 6 are excluded from the outer whorl (the perianth cup). As noted above, the poplar B and C-function

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genes are also expressed in the inner, sexual whorl but are excluded from the perianth cup (Brunner et al., 2000; Sheppard et al., 2000). However, if the poplar perianth cup is homologous to petals and develops through a mechanism similar to four-whorled species, then one would expect to find both B and E-function genes expressed in the tissue (reviewed in Cseke and Podila, 2004; Kaufmann et al., 2005). Likewise, if the perianth cup is homologous to sepals, then one would expect to find the expression of at least some E-class genes. Therefore, while the expression patterns of B, C, and E-class genes, combined with the rather unique mechanism of dioecy in poplars, matches what would be expected from four-whorl species, the PTM3/4 and 6 results suggest that the perianth cup of poplar trees may not be homologous to perianth organs. Perhaps the poplar perianth cup is derived from receptacle tissues; however, in situ data from the remaining poplar SEP-class genes would be very helpful in making final conclusions, and while our lab has performed initial characterization of A-class genes such as PTM1 and PTM2 from P. tremuloides (Cseke et al. 2003a), nothing seems to have been published on the tissue-specific expression patterns of A-function genes from other poplars. In addition, we found that PTM3/4 protein was not detected in mature flowers despite RNA expression, and there were significant levels of protein expressed in terminal buds and young leaves despite low levels of RNA expression (Fig. 6). Thus, there may be post-transcriptional mechanisms that help control the spatial and temporal activity of each gene product (Angenent et al., 1994; Sung et al., 1999; Sung et al., 2000). Since protein –protein interactions are key to the true function of MADS-box proteins, information on the in situ protein localization and protein interaction patterns of the poplar A, B, C, D, and E-function MADS-box genes will be essential to the explanation of why poplar flowers develop differently than four-whorled flowers. 4.2. Are heterologous systems reliable for testing the function of tree MADS-box genes? The high level of homology between MADS-box proteins makes it difficult to assign a function based solely on sequences. The role of such genes is better studied by using transgenic plants and observing the consequences of over-expression and/or suppression of the gene of interest. Unfortunately, poplar trees have juvenile periods lasting from 8 to 10 years, making it very difficult to analyze transgenic trees for alterations in floral phenotypes. Consequently, many tree researchers have used heterologous systems for the analysis of transgenic alterations of tree MADS-box genes (Kotoda et al., 2002; Lemmetyinen et al., 2001; 2004; Rottmann et al., 2000; Rutledge et al., 1998; Southerton et al., 1998; Sundstrom and Engstrom, 2002; Sung et al., 1999). Non-tree researchers have also made use of heterologous systems to test SEP-class gene function (Ferrario et al., 2003; Shchennikova et al., 2004; Tzeng et

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al., 2003). Therefore, to make initial comparisons between woody and herbaceous species and to test if heterologous systems can yield similar results to those we obtain from aspen, we performed parallel experiments with the PTM3/4 gene in both aspen that were forced to flower and the heterologous systems of tobacco and Arabidopsis. Our transgenic analyses indicated that neither ectopic expression nor suppression of PTM3/4 altered the development of any vegetative tissue in transgenic aspen trees. However, upon forcing aspen harboring the PTM3-sense construct to flower, alterations in normal catkin development were observed, including conversion of sections of the floral catkins to more shoot and leaf-like structures (Fig. 4). The flowers in the center of these catkins appeared normal but turned to leaf-like structures near the ends of the catkins. Interestingly, the aspen lines having the observed phenotype had very low levels of expressed PTM3, and the phenotype is similar to suppression phenotypes of other SEP-class genes, such as SEP1/2/3/4, FBP2/5, and TM5/29, where flowers have continuous whorls of sepals or leaf-like structures, one inside of the other (Ampomah-Dwamena et al., 2002; Angenent et al., 1994; Ditta et al., 2004; Pelaz et al., 2000; Pnueli et al., 1994; Vandenbussche et al., 2003). This suggests that the phenotype is a result of cosuppression in aspen, but we were unable to force flowering of the trees harboring the PTM3-IR suppression construct for comparison. It is also not clear why different regions of the catkins were affected differently, although one possibility is that the SEP-class genes in aspen have differential redundancy in different regions of catkins. Thus, the PTM3 construct indicates a role for PTM3/4 in floral development as apposed to vegetative processes, but the full consequences of manipulating PTM3/4 will require more study over the next few years. In our heterologous experiments, while Arabidopsis showed no change in floral development in response to the PTM3-sense construct other than an early flowering artifact, reduced apical dominance was observed in tobacco over-expressing PTM3/4 (Fig. 5). Early flowering and reduced apical dominance phenotypes have been observed during the ectopic expression of other SEP-class genes including NsMADS3 and NtMADS4 in tobacco, OsMADS1/ LHS1 when expressed in tobacco, LMADS3 from lily when expressed in Arabidopsis, and MdMADS2 (a SQUAMOSAhomolog in apple trees) when over-expressed in tobacco (Chung et al., 1994; Jang et al., 1999, 2002; Tzeng et al., 2003; Sung et al., 1999). However, as evidenced by their ability to form differing complexes in different tissues, one must be very careful when interpreting experiments using over-expressed MADS-box genes, even in the native species, because the products of these genes may influence the normal function of other MADS-box genes resulting in phenotypic artifacts through protein – protein interactions. Thus, the reduced apical dominance phenotype could be one example of this, and more studies would be required to correlate the phenotype with gene function.

In addition, we have found that antisense approaches in aspen are largely unstable, and other reports have suggested the same (Rottmann et al., 2000). However, it has been clearly shown that inverted repeats containing a loop are the most effective constructs at suppressing genes in as much as 99% of plant transformants though the stimulation of PTGS mechanisms (Chuang and Meyerowitz, 2000; Fagard et al., 2000; Waterhouse et al., 2001). Such constructs are known to target genes having as little as 75% overall nucleic acid identity through the production of 21 to 23 bp fragments (Chuang and Meyerowitz, 2000; Waterhouse et al., 2001). These siRNAs are subsequently assembled into active RNA-Induced Silencing Complexes (RISC) that seek out and cleave specific target mRNAs. Through analysis of the Arabidopsis genome sequence, we determined that only SEP1 and SEP2 have sufficient identity to PTM3/4 to be affected by PTGS. When the PTM3-IR construct was expressed in Arabidopsis, as a means to test the ability of the construct to cross-suppress SEP-class genes, most of the lines had significantly less seed production while not necessarily showing alterations in floral morphology. While Arabidopsis mutant for SEP1, 2, and 3 produce flowers with continuous whorls of sepals, double and single mutants have only subtle phenotypes (Pelaz et al., 2000; 2001), and through comparisons conducted in our lab, we observed that double SEP1/SEP2 mutants of Arabidopsis have the same phenotype as our transgenic lines including flowers that develop relatively normally with the exception of having significantly less seed yield. In addition, we observed more severe phenotypes in some Arabidopsis and tobacco PTM3IR lines, including the lack of growth of the inner whorls (causing sterility). This phenotype does not copy known SEP mutant phenotypes, and while the PTM3-IR construct was able to target the expected tissues, this may be another example of differences that are seen when manipulating native vs. heterologous genes in transgenic plants. Finally, it is important to note that the phenotypes from both PTM3/4 constructs, when expressed in heterologous systems, were riddled with phenotypic artifacts. For example, when the plants were grown in culture conditions, both constructs were able to generate additional phenotypes including early flowering by the PTM3-sense construct as well as reduction in plant size and lack of flowering by the PTM3-IR construct. These phenotypes did not manifest when the transgenic lines were germinated on soil without selection pressure. Thus, such phenotypes appear to be artifacts and cause us to question other published results including the early flowering that is often observed during over-expression of SEP genes as well as changes in vegetative morphology seen with some SEP genes. In addition, while our constructs were able to target floral tissues in both native and heterologous systems, the phenotypes in heterologous systems did not clearly match those seen in aspen. Therefore, despite the appeal of using heterologous systems to test gene function, extreme care should be taken when dealing with MADS-

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box genes. If such experiments are to be done, it is probably necessary to test the progeny on non-selective soil media to make sure that the phenotypes are not artifacts due to selection pressure from antibiotics or other tissue culture conditions.

Acknowlegments This work was supported by grants from USDA-FRA (# 97-36200-5275) to DFK and GKP, USDA-NRI grant (# 9835103-6533) to GKP and by the Office of Science (BER), U.S. Department of Energy Grant No. DEFG0204ER63792, to DFK and GKP. We would also like to thank Dr. Martin Yanofsky for providing various SEP double and triple mutant Arabidopsis lines and Dr. Chung-Jui Tsai for the use of her dissecting microscope system.

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