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First published online February 20, 2008; 10.1104/pp.108.117044 Plant Physiology 146:1759-1772 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Control of Compound Leaf Development by FLORICAULA/LEAFY Ortholog SINGLE LEAFLET1 in Medicago truncatula1,[C],[W],[OA]Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 (H.W., J.C., J.W., M.T., G.L.,Y.L., K.S.M., R.C.); and Institut des Sciences du Vegetale, CNRS, 91198 Gif sur Yvette, France (P.R.)
Molecular genetic studies suggest that FLORICAULA (FLO)/LEAFY (LFY) orthologs function to control compound leaf development in some legume species. However, loss-of-function mutations in the FLO/LFY orthologs result in reduction of leaf complexity to different degrees in Pisum sativum and Lotus japonicus. To further understand the role of FLO/LFY orthologs in compound leaf development in legumes, we studied compound leaf developmental processes and characterized a leaf development mutant, single leaflet1 (sgl1), from the model legume Medicago truncatula. The sgl1 mutants exhibited strong defects in compound leaf development; all adult leaves in sgl1 mutants are simple due to failure in initiating lateral leaflet primordia. In addition, the sgl1 mutants are also defective in floral development, producing inflorescence-like structures. Molecular cloning of SGL1 revealed that it encodes the M. truncatula FLO/LFY ortholog. When properly expressed, LFY rescued both floral and compound leaf defects of sgl1 mutants, indicating that LFY can functionally substitute SGL1 in compound leaf and floral organ development in M. truncatula. We show that SGL1 and LFY differed in their promoter activities. Although the SGL1 genomic sequence completely rescued floral defects of lfy mutants, it failed to alter the simple leaf structure of the Arabidopsis thaliana plants. Collectively, our data strongly suggest that initiation of lateral leaflet primordia required for compound leaf development involves regulatory processes mediated by the SGL1 function in M. truncatula.
Leaves are determinate organs initiated from the periphery of the pluripotent shoot apical meristem (SAM). The class I KNOTTED1-like (KNOX1) homeobox genes are involved in acquisition and maintenance of the meristem activity of SAM (Long et al., 1996  ). Initiation of lateral organs such as leaves requires down-regulation of KNOX1 genes at the incipient leaf primordia, and subsequent acquisition of determinacy in developing organs further requires continued suppression of KNOX1 gene expression (Smith et al., 1992; Sinha et al., 1993; Jackson et al., 1994). Thus, development of lateral organs from the periphery of the SAM requires fine-tuned interactions of multiple regulators in SAM, some maintaining the meristem fate, such as the KNOX1 genes, and others promoting determinacy by suppressing expression of meristem genes, such as ROUGH SHEATH2 (RS2) and ASYMMETRIC LEAVES1 (AS1) in maize (Zea mays) and Arabidopsis (Arabidopsis thaliana), respectively (Timmermans et al., 1999; Tsiantis et al., 1999; Byrne et al., 2000; Ori et al., 2000; Hay et al., 2006).
In compound-leafed species such as tomato (Solanum lycopersicum), the expression of KNOX1 genes is initially excluded from the incipient leaf primordia but subsequently reactivated in developing leaf primordia (Kim et al., 2003
The involvement of KNOX1 genes in compound leaf development is evidenced in a number of compound-leafed species including legumes (Bharathan et al., 2002
Molecular genetic studies indicate that the legume orthologs of the floral meristem (FM) identity gene FLORICAULA (FLO) from snapdragon (Antirrhinum majus) and LEAFY (LFY) from Arabidopsis, and their coregulators FIMBRIATA (FIM) and UNUSUAL FLORAL ORGAN (UFO), respectively, play a role in compound leaf development in compound-leafed legumes (Hofer et al., 1997 Here, we describe compound leaf developmental processes in the model legume species Medicago truncatula and report characterization of a leaf development mutant, single leaflet1 (sgl1) isolated from a tobacco (Nicotiana tabacum) Tnt1 retrotransposon-tagged mutant population of M. truncatula. Our genetic analyses of four loss-of-function alleles of the sgl1 mutant indicate that SGL1 plays a key role in initiation of lateral leaflet primordia during an early stage of leaf development resulting in a complete conversion of compound leaves into simple leaves. We also show that SGL1 controls the development of petioles along the proximodistal axis of compound leaves at a late developmental stage and FM identity during the reproductive phase of growth. Sequence analyses indicate that SGL1 encodes a plant-specific protein related to the snapdragon FLO, Arabidopsis LFY, and pea UNI transcription factors. We discuss possible mechanisms by which FLO/LFY orthologs regulate compound leaf development in legumes.
Compound Leaf Development in M. truncatula
In pea, a legume species forming odd-pinnate compound leaves with distal tendrils, development of leaflet primordia is acropetal during early stages of leaf development. However, development of distal tendrils follows a basipetal pattern, giving rise to the unique compound leaf structure of pea plants (Hofer and Ellis, 1998
Morphological changes during compound leaf primordial development can be divided into 10 distinct stages in M. truncatula. At Stage 0 (S0), cells along the periphery of SAM were recruited and became an incipient leaf primordium, albeit no signs of outgrowth were visible at this stage (Fig. 1B). At S1, a common leaf primordium formed as a strip of cells outgrew along the periphery of SAM (Fig. 1B). At the subsequent S2, a pair of stipule primordia emerged as small bumps of cells that grew out of the proximal end of the common leaf primordium (Fig. 1C). At S3, a pair of lateral leaflet primordia emerged between the stipule and common leaf primordia (Fig. 1D). Because no additional primordia developed after this stage, the common leaf primordium differentiated into a terminal leaflet primordium. Subsequently, at S4, the stipule and lateral leaflet primordia were separated away from each other so that boundaries were established between the stipule and lateral leaflet primordia (Fig. 1E). Adaxial-abaxial specification of the terminal leaflet primordium was apparent at this stage, because trichomes initiated as spherical outgrowth from the abaxial surface of the terminal leaflet primordium, indicating that the adaxial-abaxial polarity first established in the terminal leaflet primordium. Following S4, the lateral and terminal leaflet primordia grew away from each other, and boundaries formed between them at S5 (Fig. 1F). At this stage, it was apparent that trichomes differentiated further as tubular trichomes elongated from the abaxial surface of the terminal leaflet primordium (Fig. 1F). At S6, trichomes developed from the abaxial surface of the stipule and lateral leaflet primordia. Furthermore, at this stage, the abaxial surface of the terminal leaflet primordium outgrew the adaxial surface such that the terminal leaflet primordium became folded (Fig. 1G). At the subsequent stage (S7), the region between stipule and lateral leaflet primordia expanded to become a petiole as a result of cell division and cell expansion (Fig.1, H and I). At S8, differentiation of petioles was apparent as trichomes initiated from the adaxial surface of petioles. Furthermore, at this stage, the region between lateral and terminal leaflet primordia expanded to form a rachis (Fig. 1, J–M). And, at the last stage (S9), the proximal regions of lateral and terminal leaflet primordia expanded to form petiolules (Fig. 1, L and M; data not shown).
We screened and isolated a leaf development mutant with four alleles from a M. truncatula mutant collection generated by tobacco Tnt1 retrotransposon insertion mutagenesis (d'Erfurth et al., 2003
To characterize leaf development defects in sgl1 mutants, SEM analyses of leaf development were carried out. These analyses indicated that in sgl1 mutants, leaf development was initially normal until it reached S3, in which the pair of lateral leaflet primordia failed to initiate between the stipule and common leaf primordia (Fig. 2, D–F). All four alleles of the sgl1 mutant exhibited identical defects. The defect in the initiation of lateral leaflet primordia was persistent throughout subsequent developmental stages, resulting in the formation of simple adult leaves in sgl1 mutants (Fig. 2, A–G). We also examined development of petioles in sgl1 mutants and found that the length of petioles on newly developed leaves at node 10 was not significantly different between sgl1 mutants and the wild-type plants (Fig. 2H). However, petioles on older leaves were significantly shorter in the sgl1 mutants than in the wild-type plants (Fig. 2H), indicating that SGL1 also plays a role in petiole development.
M. truncatula flowers have pentamerous organs in the outermost four whorls (sepals, petals, and outer and inner stamens) and a single carpel in the center. The development of M. truncatula flowers has been previously described (Benlloch et al., 2003
The floral homeotic defects of the sgl1 mutants resemble that of uni and stp mutants. PCR amplification of M. truncatula STP genomic sequence from sgl1 mutants and wild-type plants yielded identical products, indicating that the M. truncatula STP gene is not interrupted in the sgl1 mutants (data not shown). On the contrary, PCR amplification of M. truncatula FLO/LFY/UNI genomic sequence from the sgl1 mutants and the wild-type plants exhibited a difference of 5.3 kb, suggesting that each of the four sgl1 alleles carried a single Tnt1 insert (Fig. 4A ; data not shown).
Flanking sequence analyses indicated that Tnt1 was inserted in the corresponding SGL1 gene in the first exon at positions 198, 314, and 333 bp, and in the third exon at the position 831 bp downstream from the translation initiation codon ATG in sgl1-1 to sgl1-4 alleles, respectively (Fig. 4B). Furthermore, the orientation of Tnt1 in sgl1-1 and sgl1-4 was opposite from that in sgl1-2 and sgl1-3 alleles (Fig. 4B).
All four sgl1 alleles were infertile. Segregation analysis of an F2 population of the sgl1-1 allele indicated that 34 out of a total of 139 individuals were homozygous for Tnt1 insertion and exhibited both simple leaf and floral homeotic defects, suggesting that Tnt1 insertion in the corresponding SGL1 gene cosegregated with the mutant phenotype (Supplemental Table S1). To confirm that we have cloned the SGL1 gene, we carried out a genetic complementation test in sgl1-1 mutant using Agrobacterium tumefaciens-mediated stable transformation with a wild-type SGL1 genomic sequence. Phenotypic analysis of transgenic plants indicated that three lines were completely rescued, exhibiting both wild-type-like compound leaves and flowers (Figs. 4C; data not shown). The other two lines were only rescued for the compound leaf phenotype (data not shown), the floral phenotype of which was only partially rescued in one line but not at all in the other line (Fig. 4D). We examined the expression level of the introduced SGL1 gene in these stable transgenic lines using reverse transcription (RT)-PCR. The results indicated that the introduced SGL1 gene was expressed in all five transgenic lines (Fig. 4E). However, the expression level was much higher in lines that exhibited fully and partially rescued flowers than in the line with nonrescued flowers (Fig. 4E). Thus, the interruption of the same gene in four independent lines together with the genetic complementation data unambiguously confirmed that the SGL1 gene is the M. truncatula FLO/LFY/UNI ortholog.
Comparison of the genomic and full-length complementary DNA (cDNA) sequences of SGL1 indicate that SGL1 consists of three exons and two introns (Fig. 4B). Furthermore, the intron and exon structure of SGL1 was very similar to that of FLO, LFY, and UNI. Phylogenetic analysis of selected FLO/LFY orthologs from a diverse group of species placed SGL1 in close proximity to UNI from pea and PFM from L. japonicus (Fig. 4F). Together, they formed a cluster that is distantly related to FLO from snapdragon (Coen et al., 1990
RNA in situ hybridization data revealed that SGL1 was expressed in the SAM and emerging leaf primordia (Fig. 5A
). The highest expression was detected in the distal region of leaf primordia (Fig. 5A). During the reproductive phase of growth, transcripts of SGL1 were detected in the I2 (Fig. 5A). SGL1 transcripts were also detected in the peripheral region of FMs (data not shown). During S7 of floral development (Benlloch et al., 2003
To gain a better spatial and temporal resolution, we fused the SGL1 promoter to the Escherichia coli uidA gene encoding GUS and introduced the resulting SGL1:GUS construct into M. truncatula wild-type plants (R108 ecotype). Out of four independent transgenic lines obtained, three showed strong and consistent GUS staining patterns, and the remaining one did not show any detectable GUS activity. We found that GUS staining from the GUS positive lines was restricted to the SAM and emerging leaf anlagen during early stages of leaf development (Fig. 5D, inset), consistent with our RNA in situ hybridization data. Interestingly, we observed that strong GUS staining was in the entire young leaflets (Fig. 5D, inset). In older leaves, GUS staining was detected at the proximal region of leaflets and regions in rachis where leaflets were attached (Figs. 5, D–F). GUS staining in leaves was gradually reduced and eventually disappeared when leaves aged (Fig. 5G). In petioles and rachis, GUS staining was mainly detected in the vascular tissues (Figs. 5, D–F).
Previous studies have shown that FLO or LFY encodes a plant-specific transcription factor that controls FM identity and plant architecture (Coen et al., 1990
Second, homozygous lfy mutant plants were transformed with the full-length SGL1 cDNA driven by the LFY promoter. Sixteen out of 17 transgenic lines expressed the SGL1 gene and rescued the lfy floral defects (Figs. 7, A–C, E and F; Supplemental Table S2). The remaining line did not express the SGL1 gene to a detectable level and did not rescue the lfy phenotype (data not shown). However, both rosette and cauline leaves of lfy LFY:SGL1 transgenic lines remained to be simple (Fig. 7C ; data not shown). These data indicate that SGL1 and LFY are also functionally conserved in control of FM identity and that expression of SGL1 driven by the LFY promoter was not sufficient to alter the simple leaf structure of the Arabidopsis plants.
M. truncatula SGL1 Genomic Sequence Functionally Rescued lfy Floral Defects But Failed to Alter the Simple Leaf Structure of the Arabidopsis Plants
Even though LFY was expressed at the periphery of vegetative SAM, its expression was largely excluded from developing leaves and pedicels (Weigel et al., 1992 To address the question whether differences in the expression patterns between LFY and SGL1 underlie development of different forms of leaves in Arabidopsis and M. truncatula, we generated homozygous lfy transgenic lines transformed with the SGL1 genomic sequence. Out of 17 transgenic lines obtained, all properly expressed the SGL1 gene and rescued the lfy floral defects (Fig. 7, D and G; Supplemental Table S2), indicating that the SGL1 genomic sequence is sufficient to rescue the lfy floral defects, despite differences in the promoter activities between SGL1 and LFY. However, both rosette and cauline leaves in transgenic lfy SGL1:SGL1 lines remained to be simple (Fig. 7D; data not shown). These data strongly suggest that expression of the M. truncatula SGL1 gene driven by the SGL1 promoter, which is active in the initiating leaf primordia and developing leaves, was not sufficient to alter the simple leaf structure in Arabidopsis.
The Role of FLO/LFY Orthologs in Compound Leaf Development
In most compound-leafed species, activation of KNOX1 gene expression in initiating leaf primordia is correlated with development of compound leaves. In simple-leafed species, however, expression of the KNOX1 genes is permanently down-regulated in the initiating leaf primordia in a process that requires MYB domain transcription repressors RS2 and AS1 in maize and Arabidopsis, respectively, and involves hormonal signaling (Smith et al., 1992
In compound-leafed legume species such as pea and alfalfa, in which the KNOX1 genes are excluded from the leaf primordia and thus are not likely correlated with compound leaf development in these plants, the FLO/LFY orthologs appear to function in place of the KNOX1 genes in control of compound leaf development (Champagne et al., 2007
By contrast, M. truncatula sgl1 mutants exhibit strong compound leaf defects with all adult leaves changed to simple ones (Fig. 2), indicating that the M. truncatula FLO/LFY ortholog SGL1 plays a significant role in the initiation of lateral leaflet primordia required for compound leaf development (Figs. 1 and 2). Similar to sgl1 mutants, pea uni mutants also exhibit strong compound leaf defects, i.e. the number of leaflets is greatly reduced to one to three, and the terminal tendrils are completely missing. Genetic analyses of uni, afila (af), and tendril-less (tl) pea mutants lead to a hypothesis that UNI functions as a determinant of a central-fated shoot-like structure (Hofer and Ellis, 1998
It is well established that FLO/LFY plays a key role in vegetative to reproductive transition, leading to flower development (Blazquez et al., 1997
The fact that proper expression of the Arabidopsis LFY rescued compound leaf and floral defects of the sgl1 mutants and proper expression of the M. truncatula SGL1 rescued the floral defects but did not alter the simple leaf structure of the lfy mutants unambiguously demonstrates that LFY and SGL1 are functional orthologs and that differences in leaf forms between the simple-leafed Arabidopsis and compound-leafed M. truncatula are not due to functional differences between LFY and SGL1. Rather, our results support a hypothesis that regulatory processes mediated by the SGL1 function are involved in initiation of leaflet primordia and development of compound leaves in M. truncatula.
Alterations in cis-regulatory sequences, e.g. 5'-upstream sequences, are also implicated in morphological variations during plant evolution (Busch and Gleissberg, 2003
In contrast to its role in compound leaf development being prominent only in legume species, particularly in the IRLC studied to date, the role of FLO/LFY in inflorescence and FM identity control has been shown to be highly conserved in flowering plants. In loss-of-function mutants of FLO/LFY orthologs, flowers exhibit homeotic defects, including lack of petals and stamens, and FMs being converted into inflorescence-like structures (Coen et al., 1990
Plant Materials and Growth Condition
Medicago truncatula ecotypes R108 and ‘Jemalong’ A17 were used in this study. NF740 (sgl1-1), NF1240 (sgl1-2), NF2138 (sgl1-3), and NF2703 (sgl1-4) alleles were isolated from a tobacco (Nicotiana tabacum) Tnt1 retrotransposon tagged mutant collection of M. truncatula (d'Erfurth et al., 2003
Shoot apical and FMs were collected from shoot apices of wild-type and mutant plants 2 to 4 weeks and 2 to 3 months postgermination, respectively. Plant tissues were fixed by vacuum infiltration with 3.0% glutaraldehyde in 25 mM phosphate buffer, pH 7.0, for 1 h and then incubated in 4°C overnight. Plant tissues were further fixed with 1.0% osmium tetroxide in the same phosphate buffer overnight and then dehydrated in a graded ethanol series. Before electron scanning microscopic observations, tissues were critical-point dried in liquid CO2, mounted on aluminum stubs, dissected, and sputter-coated with gold. Specimens were then examined under a Zeiss DSM-960A SEM (Carl Zeiss) at an accelerating voltage of 5 kV. Digital photographs were collected and assembled using Photoshop.
To clone full-length M. truncatula SGL1 (MtUNI) and MtSTP genes, we first searched all available M. truncatula sequences in GenBank against UNI and STP. The genomic sequence was available for SGL1 in the database. Therefore, sequence alignments of the SGL1 genomic sequence with UNI/LFY/FLO coding sequences allowed us to define the open reading frame of SGL1. In the case of MtSTP, no sequences were available in the database. Therefore, oligonucleotide primers were designed based on consensus sequences derived from UFO, FIM, and STP sequences. Total RNA was prepared from shoot apices of 2-week-old wild-type plants (‘Jemalong’ A17). RT-PCR was carried out by following the manufacturer's instructions (Invitrogen) using the following oligonucleotide primers: SGL1-forward: 5'-GCTTACCATGGATCCCGACGCATT-3'; SGL1-reverse: 5'-TAACTTAAAAAGGAAGGTGAGCAGTTC-3'; MtSTP-forward: 5'-ATGGAAGGTTTTCACCCATCTATGA-3'; and MtSTP-reverse: 5'-CATAAGCAAAACCATGCAACTCAAAC-3'. PCR products were cloned into pGEM-T Easy vector (Promega) and sequences were confirmed by sequencing and deposited into the GenBank (AY928184 and AY932821). We also PCR amplified and sequenced the SGL1 genomic sequence from M. truncatula R108 ecotype. The corresponding sequence was also deposited into the GenBank (DQ672589).
Genomic DNA samples were prepared from wild-type M. truncatula (R108), and sgl1-1, sgl1-2, sgl1-3, and sgl1-4 alleles using a standard protocol. PCR amplification was carried out using either SGL1 or MtSTP primers listed above. PCR amplification gave rise to a product 5.3 kb larger from sgl1-1 to -4 alleles than from the wild type when SGL1 primers were used. We PCR amplified the Tnt1 flanking sequences from sgl1-1 to sgl1-4 alleles using a combination of SGL1 primers (see above) and Tnt1 primers (Tnt1-upstream, 5'-CTCCAGACATTTTTATTTTTCACCAAG-3'; Tnt1-downstream, 5'-GCATTCAAACTAGAAGACAGTGCTACC-3'). These PCR products were fully sequenced.
To generate the SGL1 genomic clone for functional complementation of M. truncatula sgl1 mutants, we first PCR amplified SGL1 promoter sequence from wild-type M. truncatula (‘Jemalong’ A17) with the following primers: (SGL1 promoter-forward) 5'-AATTGAATTCAAAAATGGTGTACCAAACATGAGGTAGAA-3' (an EcoRI site was introduced as underlined) and (SGL1 promoter-reverse) 5'-CGTCGGGATCCATGGTAAGCAATG- 3' (a unique BamHI site was highlighted). The PCR product was digested by EcoRI and BamHI and cloned into the EcoRI-BamHI sites of pCAMBIA3300 binary vector (the resulting construct was labeled as pCAMBIA3300-pSGL1). Similarly, we PCR amplified the SGL1 coding sequence and 3' untranslated region from the wild type with the following primers: (SGL1-forward) 5'-AGTTTCATTGCTTACCATGGATCC-3' (a unique BamHI site was highlighted) and (SGL1-reverse) 5'-AATTCTGCAGAAAAATTAGCCTTTCCCATGCTAAACTTC-3' (a PstI site was introduced as underlined). The PCR product was digested by BamHI and PstI and cloned into the BamHI-PstI sites of the pCAMBIA3300-pSGL1 construct. The resulting construct pCAMBIA3300-SGL1 was sequenced and confirmed to contain no errors and was then introduced into disarmed Agrobacterium tumefaciens EHA105 and AGL1 strains via electroporation.
To generate the Arabidopsis LFY clone for complementation of M. truncatula sgl1 mutants, we first recloned the SGL1 promoter into the EcoRI-BamHI sites of pCAMBIA3301. We then PCR amplified the full-length LFY cDNA from pDW123 (Blazquez et al., 1997 We generated pCAMBIA3301-pSGL1::GUS construct by restriction enzyme digestion of the pCAMBIA3301-pSGL1 with NcoI and followed by religation, which removed cauliflower mosaic virus 35S promoter between SGL1 promoter and GUS gene from the vector. The resulting construct CAMBIA3301-pSGL1::GUS was introduced into A. tumefaciens EHA105 and AGL1 strains. To clone the full-length SGL1 cDNA for genetic complementation of Arabidopsis lfy mutants, we amplified the Arabidopsis LFY promoter from Columbia-0 wild-type plants by PCR using the following primers: (LFY promoter-forward) 5'-TATAGAATTCGTAATGGGCTGACCGAGAAGATATAAA-3' (an EcoRI site was introduced as underlined) and (LFY promoter-reverse) 5'-CGTGAAACCTTCAGGATCCATAATCTA-3'(a unique BamHI site was highlighted). The PCR product was digested with EcoRI and BamHI and ligated into the EcoRI-BamHI sites of pCAMBIA3301, resulting in pCAMBIA3301-pLFY. The full-length SGL1 cDNA was reamplified using the following primers: (SGL1-forward) 5'-AGTTTCATTGCTTACCATGGATCC-3' (a unique BamHI site was highlighted) and (SGL1-reverse2) 5'-AATTGGTCACCTAACTTAAAAAGGAAGGTGAGCAGTTC-3' (a BstEII site was introduced as underlined). The PCR product was digested by BamHI and BstEII and ligated into the BamHI-BstEII sites of pCAMBIA3301-pLFY. The resulting construct, pCAMBIA3301-pLFY:SGL1, was confirmed by sequencing and introduced into A. tumefaciens GV3101 strain.
M. truncatula sgl1-1 (R108 ecotype) and R108 wild type were transformed with A. tumefaciens EHA105 or AGL1 strain harboring the various binary constructs described, using a transformation-regeneration protocol previously described (Chabaud et al., 1996
For Arabidopsis transformation, heterozygous (lfy +/–) plants (SALK_057202) were transformed by A. tumefaciens GV3101 strain harboring various binary constructs described, using the floral dipping method as previously described (Clough and Bent, 1998
Total RNA was prepared using Tri Reagent (Molecular Research Center). Genomic DNA was removed using a DNA-free kit (Ambion). cDNA synthesis was performed using SuperScript II reverse transcriptase (Invitrogen) starting with 2 µg of total RNA in a 20-µL reaction with oligo(dT)15 primers (Promega) at 42°C for 1 h. Primers 5'-AGACGCCTTGATGAAGAGGAAATTAA-3' and 5'-TAGCAATTGCTTGAACCTGAATCAAG-3' were designed to amplify 424 bp of the internal coding region of SGL1. Primers 5'-TCTTACTCTCAAGTACCCCATTGAGC-3' and 5'-GTGGGAGTGCATAACCTTCATAGATT-3' were designed to amplify 329 bp M. truncatula Actin as an internal loading control. Primers 5'-CAGTGTCTGGATCGGAGGAT-3' and 5'-TGAACAATCGATGGACCTGA-3' were designed to amplify the Arabidopsis Actin gene (At5G09810) as an internal loading control. The PCR was performed as follows: 2 min at 95°C, 25 cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C, and 5 min at 72°C. Amplified products were separated by electrophoresis according to their molecular weights.
Phylogenetic analysis was performed using PAUP version 4 software (paup.csit.fsu.edu/). A maximum parsimony criterion was used to generate the single most parsimonious tree, with bootstrap values from 1,000 replicates and greater than 50% shown above the relevant branches.
RNA in situ hybridization was essentially carried out as previously described (Coen et al., 1990
GUS staining was carried out as previously described (Shin et al., 2005 Sequence data from this article can be found in the EMBL/GenBank data libraries under accession numbers AY928184, AY932821, and DQ672589.
The following materials are available in the online version of this article.
We thank members of the Chen laboratory for helpful discussions, Kelly Craven and Neelima Sinha for comments on a previous version of the manuscript, Detlef Weigel for providing plasmids, Zeng-yu Wang and Elaine Wright for advice on M. truncatula transformation, Bill Chisso and Preston Larson for assistance with SEM, Kelly Craven and Nikki Charlton for phylogenetic analysis, the Arabidopsis Stock Center at The Ohio State University for providing Arabidopsis lines, and Kuihua Zhang, Xirong Xiao, and Shuirong Zhang for technical assistance. Received January 28, 2008; accepted February 14, 2008; published February 20, 2008.
1 This work was supported by The Samuel Roberts Noble Foundation and by the European Union (EU FP6–GLIP project). 
2 These authors contributed equally to the article.
3 Present address: Department of Microbiology and Molecular Genetics, Oklahoma State University, 307 Life Science East, Stillwater, OK 74078. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Rujin Chen (rchen{at}noble.org).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.117044 * Corresponding author; e-mail rchen{at}noble.org.
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