INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The shoot apical meristem (SAM) is a group of cells at the growing tip of a plant that is formed during embryogenesis and is maintained throughout its life. The SAM is organized into a central zone composed of slowly dividing stem cells and a peripheral zone containing more rapidly dividing cells that become incorporated into organ primordia. Thus, the SAM serves as the source of cells for all initiating lateral organs of the shoot. Organs initiate in a specific pattern that depends on the positioning of founder cells in the peripheral zone. This pattern is controlled by a combination of genetic and environmental factors (Steeves and Sussex, 1989). Maintenance of the SAM requires a balance between the pool of central stem cells and the flanking peripheral zone cells. A number of genes required for SAM initiation and maintenance have been identified. Proper meristem function requires the competing action of the CLAVATA (CLV) signaling pathway and the transcription factor WUSCHEL (WUS) (for review, see Clark, 2001). The CLV pathway is required to limit the number and position of stem cells in the meristem by restricting the domain of WUS expression. In contrast, WUS is required for stem cell maintenance and is thought to act on the CLV pathway by positively regulating expression of the putative ligand encoded by CLV3. The interaction between CLV and WUS is thought to function as a feedback loop to limit meristem size (Brand et al., 2000; Schoof et al., 2000).

The class 1 KNOX homeobox genes are also important for SAM function. Class 1 KNOX genes are specifically expressed in the SAM and are down-regulated in lateral organ anlage in a number of plant species (Jackson et al., 1994; Long et al., 1996; Nishimura et al., 1999; Sentoku et al., 1999). Loss-of-function mutations in the Arabidopsis SHOOT MERISTEMLESS (STM) or maize (Zea mays) Knotted1 genes demonstrate that class 1 KNOX genes are important for SAM formation and maintenance (Long et al., 1996; Vollbrecht et al., 2000). One apparent function of STM is to negatively regulate expression of the ASYMMETRIC LEAVES1 (AS1) gene in the meristem (Byrne et al., 2000). AS1 encodes an MYB domain transcription factor that is a homolog of the Antirrhinum PHANTASTICA and maize ROUGH SHEATH2 genes. These genes all show expression in lateral organ primordia (Waites et al., 1998; Timmermans et al., 1999; Tsiantis et al., 1999; Byrne et al., 2000). as1 mutants are epistatic to stm, such that as1 stm double mutants form a vegetative meristem. These observations suggest that the loss of a meristem in stm mutants is due to expression of AS1 in the meristem (Byrne et al., 2000). In turn, AS1 activity is needed to repress KNOX gene expression in the leaf (Byrne et al., 2000; Ori et al., 2000).

Formation of a proper SAM is closely tied to boundary formation and organ separation, as stm mutants show limited fusion at the cotyledon base (Barton and Poethig, 1993). The cup-shaped cotyledon (cuc) mutants also lack a SAM and show extensive cotyledon fusion (Aida et al., 1997). The CUC genes are expressed at the boundary between the SAM and cotyledon primordia, and their activity is required for STM expression (Aida et al., 1999; Takada et al., 2001). We have identified a novel gene that is expressed at the adaxial base of initiating lateral organs. The LATERAL ORGAN BOUNDARIES (LOB) gene encodes a plant-specific protein of unknown function. The LOB protein contains a conserved approximately 100-amino acid domain that is found in 42 other Arabidopsis proteins. Although the function of LOB is unknown, its expression indicates a potential role in organ separation or other aspects of lateral organ development.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

-Glucuronidase (GUS) Expression in the Transposant Line ET22

In a screen for gene-trap expression patterns in the shoot apex of Arabidopsis seedlings (P. Springer and R. Martienssen, unpublished data), an enhancer trap line (ET22) was identified that showed GUS reporter gene activity in defined regions around the SAM. We examined GUS expression in ET22 plants throughout development. GUS activity in ET22 embryos was first detected at the torpedo stage, and was localized throughout the embryo (Fig. 1A). GUS activity became progressively localized to the shoot and root apices during later stages of embryogenesis. In mature embryos, GUS activity was confined to the shoot apex and root tip (Fig. 1B). Following germination, GUS activity was detected in a band of cells at the base of the cotyledons and leaf primordia (Fig. 1, C and D). Longitudinal and transverse sections through the shoot apex revealed that GUS activity was confined to an adaxial domain that was three to five cells deep (Fig. 1D and data not shown). GUS expression persisted at the base of expanded and mature leaves (data not shown). A similar expression pattern was seen at the base of all lateral organs formed from vegetative, inflorescence, and floral meristems (Fig. 1E). GUS activity was also detected in the anthers of the flower (Fig. 1E). In the root, GUS activity was detected at the junction between the primary root and lateral root primordia, in a ring of cells at the base of the lateral root (Fig. 1, F and G). Expression was maintained at the base of fully developed lateral roots.

View larger version (92K): [in this window] [in a new window]   Figure 1.   Analysis of GUS activity in ET22 enhancer trap line and pLOB5.0::GUS transformants. ET22 (A-G). pLOB5.0::GUS transformants (H-J). A, Torpedo-stage embryo. B, Mature embryo. C, Four-day-old seedling; arrow marks cells at base of cotyledons, and arrowhead marks cells at base of leaf primordia. D, Transverse section through 9-d-old seedling apex showing GUS staining on the adaxial side of leaf bases. S, SAM; lp, leaf primordium. E, Inflorescence. F, Lateral root. G, Longitudinal section through lateral root primordium. H, Seven-day-old seedling. I, Inflorescence. J, Lateral root. The tissue in G was counter-stained with Safranin-O after sectioning. Scale bar = 50 µm in A and B; = 100 µm in C, D, F-H, and J; and = 1 mm in E and I.

Isolation of the LOB Gene

DNA gel-blot hybridization was used to determine that the ET22 transposant line possessed a single DsE element (data not shown). Thermal asymmetric interlaced (TAIL)-PCR (Liu et al., 1995; Tsugeki et al., 1996) was used to amplify genomic DNA flanking the DsE element. Sequence of the TAIL-PCR product matched that of P1 clone MDC12 on chromosome 5. Genomic DNA fragments from the region around the insertion site were amplified and used as probes to screen a cDNA library derived from floral buds (Weigel et al., 1992). Two overlapping cDNA clones were isolated and sequenced. 5'-RACE-PCR (Frohman et al., 1988) was used to identify a full-length cDNA sequence (data not shown). Based on the expression pattern of the GUS reporter gene in the transposant line, the corresponding gene was named LOB. The MDC12 sequence has recently been annotated, and the LOB gene corresponds to hypothetical gene At5g63090 (MDC12.5). At5g63090 is identical to LOB throughout the coding region, but does not contain 5'- and 3'-untranslated regions (UTRs) that were defined by the cDNA sequence. Comparison of the LOB cDNA and genomic DNA sequences showed the presence of one intron in the 5'-UTR, with an open reading frame completely contained within the last exon. The DsE insertion was near the 3' end of LOB and was inserted such that the GUS gene was transcribed opposite to LOB (Fig. 2A).

View larger version (28K): [in this window] [in a new window]   Figure 2.   ET22 genomic structure and sequence of LOB. A, Structure of genomic DNA near the DsE insertion in ET22. Boxes represent exons and arrows show the direction of transcription. B, Amino acid sequence of LOB. The LOB domain is highlighted in gray, and conserved C and GAS blocks are underlined with solid and dashed lines, respectively. The double underline marks the predicted coiled coil. Invariant Cys and Pro residues are shown with dots. , The site of insertion of the T-DNA in lob-2. , The site of insertion of DsE in ET22.

The LOB gene encodes a deduced polypeptide of 186 amino acids (Fig. 2B) with a predicted molecular mass of 20.2 kD. Database searches did not identify similarity to known proteins in any species or to any known functional motifs. However, a number of hypothetical or unknown proteins in the Arabidopsis genome that were similar to LOB were identified. We have named this region of similarity, which spans approximately 100 amino acid residues, the LOB domain (Fig. 2B). Expressed sequence tag (EST) sequences corresponding to related genes from soybean (Glycine max), maize, rice (Oryza sativa), tomato (Lycopersicon esculentum), Lotus japonicus, Medicago truncatula, pine (Pinus sylvestris), aspen (Populus spp.), wheat (Triticum aestivum), and potato (Solanum tuberosum) were also identified. Similar genes were not identified in other species, indicating that the LOB domain proteins are unique to plants.

Expression of LOB

To confirm that GUS activity in the ET22 transposant line accurately reports LOB expression, we constructed two different LOB-promoter::reporter fusion constructs. pLOB2.8::GUS contains the 5'-UTR and 1.1 kb of genomic DNA upstream of the putative transcription start site fused to the uidA gene. pLOB5.0::GUS contains the 5'-UTR and 3.3 kb of genomic DNA upstream of the putative transcription start site fused to the uidA gene. These constructs were introduced into Arabidopsis ecotype Landsberg erecta. GUS expression patterns were examined in seven independent transgenic lines containing pLOB2.8::GUS and in 24 independent transgenic lines containing pLOB5.0::GUS. GUS activity was nearly ubiquitous in two of the pLOB2.8::GUS lines and in six of the pLOB5.0::GUS lines (data not shown). These insertions were assumed to be adjacent to strong promoter or enhancer sequences that affected activity of the LOB promoter. In the remaining transformants for each construct, GUS activity generally mimicked the activity of the ET22 transposant line. However, the pLOB2.8::GUS transformants typically showed weaker and more variable GUS expression than the transposant line (data not shown). In addition, the onset of GUS expression in floral buds was later than in the ET22 line, and GUS activity was occasionally detected in the leaf blade (data not shown). GUS activity in the remainder of the pLOB5.0::GUS transformants resembled the pattern of the transposant line in the shoot apex (Fig. 1H), the inflorescence (Fig. 1I), and the root (Fig. 1J). However, GUS activity was not detected in the anthers of pLOB5.0::GUS transformants (Fig. 1I), suggesting that some regulatory elements were missing from this promoter sequence.

To investigate the possibility that GUS activity in the transposant line was influenced by neighboring genes, expression of the adjacent gene At5g63080 (MDC12.4) was examined. At5g63080 is 3' to LOB and is transcribed in the same orientation, such that the 5' end of At5g63080 is 6.5 kb from the start of transcription of the GUS gene in ET22 (Fig. 2A). At5g63080 encodes an unknown protein. An EST sequence corresponding to At5g63080 was identified from a developing seed cDNA library (White et al., 2000). We could not detect expression of At5g63080 using reverse transcriptase (RT)-PCR in any vegetative or floral tissues (data not shown). The neighboring gene on the 5' side of LOB, At5g63100 (MDC12.6), is 5.6 kb from the site of insertion, placing it approximately 11.6 kb from the start of transcription of the GUS gene (Fig. 2A). Expression of At5g63100 was not examined.

Attempts to detect LOB transcripts using in situ hybridization were unsuccessful, suggesting that LOB transcripts are present at low abundance. Therefore, we examined the expression pattern of LOB by RT-PCR (Fig. 3A). The expression pattern of LOB shown by RT-PCR was consistent with the GUS expression pattern in the trap line. Amplified fragments were detected in RNA isolated from 6-d-old seedlings, inflorescence stems, roots, buds, and open flowers (Fig. 3A). Amplification of a faint band was detected from RNA isolated from rosette and cauline leaves (which included the leaf base). LOB expression was not detected in an RNA sample isolated from the apical one-half of rosette leaves (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 3.   Expression of LOB in wild-type tissues. A, RT-PCR analysis of LOB expression. RNA was isolated from Landsberg erecta 6-d-old seedlings (S), rosette leaves (RL), cauline leaves (CL), stem (ST), root (RT), flower buds (B), and open flowers (FL). The four products are indicated by arrows. The lower panel shows RT-PCR using primers to the ACT2 gene as a control. B, Schematic showing the four LOB transcripts that are produced due to alternative splicing in the 5'-UTR. Boxes represent exons, and lines represent introns. The pair of arrows indicates the location of the primers used in the PCR reactions. Exon positions in the individual transcripts are 1..296, 1673..2402 (LOBa); 1..300, 1673..2402 (LOBb); 1..296, 1046..1104, 1673..2402 (LOBc); 1..296, 1046..1175, 1673..2402 (LOBd). Position 1 indicates the putative start of transcription and corresponds to position 19,609 of MDC12 (AB008265); position 2,402 corresponds to position 17,208. Accession numbers for each transcript are AF447897 (LOBa), AF447898 (LOBb), AF447899 (LOBc), and AF447900 (LOBd).

Based on the sequence of the largest cDNA clone characterized, the LOB-specific primers were expected to amplify a 245-bp PCR product. Several amplified products were detected (Fig. 3A), including one of the expected size. Sequencing of the RT-PCR products demonstrated that the multiple PCR products were derived from alternatively spliced LOB transcripts. Four different splice variants were identified in the 5'-UTR (Fig. 3B). The LOBa and LOBb transcripts differed by four nucleotides at the splice donor site. LOBa, which was identical to the original cDNA sequences, used a non-consensus GC at the splice donor site. The LOBb transcript used a consensus GU splice donor site four nucleotides downstream of the LOBa site (Fig. 3B). Use of a 5'-GC is unusual; however, 1% of Arabidopsis introns have a GC in the 5' position (Brown et al., 1996). The remainder of the nucleotides at the splice site conform to the consensus sequence. The RT-PCR products derived from the LOBa and LOBb transcripts could not be resolved on agarose gels, but cloning and sequencing of 16 clones suggested that the two transcripts were present at similar levels in seedlings. The LOBc and LOBd transcripts used the LOBa splice donor site and included an additional exon that differed at its 3' end (Fig. 3B). These larger transcripts appeared to be present at lower levels than LOBa and LOBb, based on band intensities of the RT-PCR products.

All four 5'-UTR splice variants are predicted to encode an identical protein, as the predicted open reading frame is not affected. However, the additional exon introduces out of frame AUG codons upstream of the translation start site in both of the larger transcripts. If these upstream AUGs were used, they could perhaps affect translation initiation of the downstream open reading frame. It is not clear if translation would initiate at any of the out of frame AUGs, however, as none of them occurs in a consensus context (Joshi et al., 1997).

Alterations in Expression of LOB

The transposant line ET22 contains a DsE insertion in the 3' end of the LOB gene, corresponding to the non-conserved C terminus of the LOB protein (Fig. 2, A and B). To determine whether the insertion affected LOB transcript accumulation, RT-PCR was used to examine LOB expression in seedlings that were homozygous for the DsE insertion. After 30 PCR cycles, a LOB-specific PCR product could be readily amplified from cDNA derived from 6-d-old wild-type seedlings (Fig. 3A). In contrast, only a faint band could occasionally be detected after amplification of cDNA derived from lob::DsE homozygotes, suggesting that the DsE insertion causes a reduction in LOB transcript levels (data not shown). Reconstruction experiments using 20 cycles of PCR, followed by blotting and hybridization, demonstrated that LOB transcript abundance is reduced 20- to 50-fold compared with wild type in lob::DsE homozygotes (data not shown). Despite the significantly reduced LOB transcript levels, no obvious morphological phenotypes were visible in lob::DsE homozygotes.

To identify additional loss-of-function lob mutations, we screened the Arabidopsis Knock-Out Facility's T-DNA insertion collection (Krysan et al., 1999) and identified a T-DNA insertion in the conserved LOB domain (Fig. 2B). This allele was designated lob-2. RT-PCR showed that full-length LOB transcripts did not accumulate in plants homozygous for the T-DNA insertion (data not shown). Examination of lob-2 homozygotes again revealed no obvious visible phenotypes in plants grown under standard growth conditions.

To determine the effect of expression of LOB outside of its normal expression domain, the LOB coding sequence was fused to the cauliflower mosaic virus 35S promoter and introduced into wild-type Arabidopsis plants. Thirty-seven independent transformants were recovered, and 25 of them showed a similar phenotype (Fig. 4), whereas the remaining nine transformants resembled wild-type plants. Fewer transformants were recovered than in control experiments using empty vector or other transgenes (data not shown), suggesting that high levels of LOB expression may be detrimental. RNA-blot analysis was performed on individual transformants to verify LOB overexpression (Fig. 4M). Plants overexpressing LOB were much smaller than wild type at all stages of development (Fig. 4, B and C). 35S::LOB rosette leaves had short petioles and were more rounded than wild type (Fig. 4, B-E). Leaves were often curled upward (Fig. 4, D and E). After flowering, the inflorescence stem did not elongate appreciably, resulting in a tightly packed cluster of flowers (Fig. 4D). 35S::LOB plants produced abnormal flowers that contained reduced floral organs and were infertile. Organs in the outer three whorls failed to elongate, resulting in exposed gynoecia (Fig. 4, G and H). Anthers only rarely produced pollen grains. Although 35S::LOB carpels elongated and the stigma occasionally appeared to develop normally, pollination with wild-type pollen did not result in the production of seeds, suggesting that 35S::LOB plants are female sterile. We examined 35S::LOB leaves by clearing and viewing with DIC optics. Cell size and shape were similar to that of wild type (Fig. 4, I-L). In addition, 35S::LOB leaves appeared to have a normal arrangement of cells in transverse section (data not shown).

View larger version (102K): [in this window] [in a new window]   Figure 4.   Phenotypes of transgenic plants that ectopically express LOB. A, Wild-type 19-d-old Landsberg erecta plant. B and C, Two independent transgenic 35S::LOB plants (19-d-old). D, Thirty-two-day-old 35S::LOB plant. E, Scanning electron microscopy of 35S::LOB rosette leaf. F, Wild-type Landsberg erecta flower. G, 35S::LOB flower. H, Scanning electron microscopy of 35S::LOB flower. I through L, Differential interference contrast (DIC) images of wild-type (I and K) and 35S::LOB (J and L) cleared rosette leaves. The images show the epidermis (I and J) and mesophyll (K and L). M, Northern-blot analysis of LOB expression in wild-type and five different 35S::LOB transgenic plants. Ten micrograms of total RNA was loaded in each lane. The filter was probed with the LOB cDNA (top) or 18S rDNA as a loading control (bottom). Scale bar in A through C = 5 mm; in I through L = 50 µm.

The LOB Domain Gene Family

The Arabidopsis genome database was searched to identify all Arabidopsis genes related to LOB. Searches were performed using TBLASTN with the entire LOB amino acid sequence as a query. A total of 42 genes was identified in the Arabidopsis genome that showed similarity to LOB (Table I). All 42 predicted proteins share varying degrees of similarity in the LOB domain (Fig. 5, A and B). No genes were identified that were similar to the carboxy-terminal 75 residues of LOB, suggesting that this region of the LOB protein is unique.

View larger version (115K): [in this window] [in a new window]   Figure 5.   A, Alignment of the LOB domains of class I protein sequences. LBD34 is not included in the alignment because the annotation is not certain. B, Alignment of LOB with the class II protein sequences. The alignments were produced by the Alignment program of Vector NTI, which uses the Clustal W algorithm. Conserved amino acids are highlighted in black, and similar amino acids are highlighted in gray. The conserved blocks and invariant residues are shown above the alignments.

EST sequences were available for 13 of the LBD genes (Table I). cDNA clones corresponding to ESTs for LBD6, 13, 15, 18, 25, 29, 30, 37, 41, and 42 were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus), Kazusa DNA Research Institute (Chiba, Japan), or Genome Systems Inc. (St. Louis) and were fully sequenced. We also isolated and sequenced a cDNA clone corresponding to LBD16. In most cases, the cDNA sequences agreed with the annotated gene models and included 5'- and 3'-UTRs. In the case of LBD13, an additional intron was present relative to the annotated gene model, resulting in a change in the first four residues in the amino terminus of the deduced protein. The LBD18 cDNA sequence differed from the predicted gene model at a splice acceptor site. This change resulted in an insertion of five amino acids in the deduced protein sequence, which allowed a better alignment with the other LBD protein sequences (Fig. 5A). Although a cDNA clone was not available for LBD31, examination of the gene model revealed a similar situation to LBD18, and movement of the position of a splice acceptor site also resulted in an insertion of five amino acids, allowing better alignment to the consensus. The LBD25 cDNA sequence extended the 5' end of the first exon relative to the gene model. This extended the open reading frame, adding 31 amino acids to the amino terminus of the deduced protein sequence. The cDNA sequences have been deposited in GenBank and accession numbers are shown in Table I.

Genes encoding LBD proteins fall into two classes. Members of class I include 36 Arabidopsis genes that are predicted to encode proteins that are similar to LOB (25%-82% identity) throughout the LOB domain (Fig. 5A). Class II consists of six Arabidopsis genes that encode deduced proteins that are less similar to LOB (28%-33% identity) and the other class I proteins (Fig. 5B). Class II proteins share a conserved amino terminus (62%-93% identity in pair-wise comparisons). The class II proteins share limited sequence conservation outside of the LOB domain as well. Signature sequences that define class I and class II proteins were identified (see below).

To identify potential functionally important domains within the class I and class II proteins, blocks were generated with Block Maker (Henikoff et al., 1995). These analyses defined two conserved blocks in the class I proteins (Fig. 5A). The C block is 22 amino acids in length and contains four absolutely conserved Cys residues in a CX₂CX₆CX₃C motif. LBD3 deviates from this motif slightly, containing four amino acids between the third and fourth Cys residues (Fig. 5A). The GAS block is 49 amino acids in length, beginning with a FX₂VH motif and ending with a DP(V/I) YG motif (Fig. 5A). The Pro residue in the DP(V/I) YG signature is present in all class I proteins.

Three conserved blocks were detected in the class II proteins that together span the entire length of the LOB domain. These blocks are in close proximity to each other and therefore will be considered as one large block (Fig. 5B). The class II block contains a Cys motif similar to the class I proteins. Spacing between the four Cys residues is the same in both classes, but the intervening amino acids differ. The class I consensus sequence is CAACKFLRRKCX₃C, whereas the class II consensus sequence is CNGCRVLRKGCSE(D/N)C. The class II block contains an invariant Pro residue that is also found in the DP(V/I) YG signature in the class I LOB domains. One distinguishing feature of the class II proteins is that they are more Cys rich than the class I proteins, containing from nine to 13 total Cys residues, whereas the class I proteins contain four to seven cysteines.

Examination of the LOB protein sequence for possible secondary structure revealed a predicted coiled coil of 30 amino acids in length at the end of the LOB domain. The predicted coiled coil contains four leucines in a LX₆LX₃LX₆L spacing that is reminiscent of a Leu-zipper (Landschultz et al., 1988). To determine whether this potential structural domain is conserved, the LBD protein sequences were examined for predicted coiled-coil structures. Among the class I proteins, 33 of the 36 proteins were predicted to form a coiled coil at the end of the LOB domain with >90% probability. LBD2, 26, and 34 were not predicted to form coiled-coils. None of the class II proteins were predicted to form coiled-coil structures.

Expression of LBD Genes

Twenty-nine of the 42 LBD genes were hypothetical in that they were predicted from genomic sequence but had not experimentally been shown to be expressed. We performed RT-PCR to examine the patterns of expression of 30 different LBD genes in a variety of Arabidopsis tissues. In all cases, primers spanning predicted introns were used to distinguish between amplification of genomic DNA and amplification of cDNA. Expression was detected for 24 LBD genes (Fig. 6). No expression was detected for LBD5, 8, 9, 23, 24, and 42 in any of the tissues tested. Only one of these genes, LBD42, is represented by an EST sequence. At this time, we do not know if LBD5, 8, 9, 23, and 24 are expressed at levels that were undetectable under the conditions used, or are expressed in tissues that were not tested. It is also possible that these genes are pseudogenes.

View larger version (70K): [in this window] [in a new window]   Figure 6.   RT-PCR analysis of the expression profiles of 24 different LBD genes. SH, Twelve-day-old shoot tissue; RL, rosette leaves; CL, cauline leaves; ST, inflorescence stem; RT, root; BD, floral buds; FL, open flowers.

LBD gene expression patterns were quite variable, with many genes showing tissue or developmental stage-specific patterns (Fig. 6). Transcripts from LBD1, 3, 4, 6, 15, 25, 37, 38, 39, and 41 were detected in all tissues examined, although at variable levels. LBD11 transcripts were detected in all tissues except root. LBD17 transcripts were detected in all tissues except 12-d-old shoots. Transcripts from LBD14, 29, and 33 were detected only in roots, whereas transcripts from LBD16 were primarily detected in roots, but a faint band was also amplified in shoots. In vegetative tissues, LBD12 was also expressed predominantly in roots; low levels were detected in shoots and floral stems. LBD12 transcripts were also detected in open flowers, but not flower buds. LBD19 transcripts were detected in shoots, roots, and floral tissues, but not in stems or leaves. LBD13 transcripts were detected in shoots and roots but not in rosette or cauline leaves or inflorescence stems. Low levels were also detected in floral buds and open flowers. Transcripts from LBD20 and 40 were detected in roots and floral tissues, although at different levels. Transcripts from LBD18 and 30 were not detected in shoots or rosette leaves, but were present in all other tissues tested. LBD31 transcripts were detected in roots, stems, and floral tissues.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The LOB gene was identified based on the expression pattern of an enhancer trap insertion. Although we were not able to visualize LOB transcript localization, a LOB promoter::GUS fusion largely recapitulated the expression pattern of GUS in the ET22 line. The pLOB5.0::GUS fusion differed from the transposant line in that it did not drive expression in anthers. This raises the possibility that sequences within the LOB coding region or 3' to the gene contribute to its expression. Another possible explanation is that the anther staining in ET22 plants does not reflect expression of LOB. The DsE insertion in LOB is oriented so that the GUS gene is transcribed opposite to LOB. This could place GUS under the control of 3' regulatory elements or cryptic enhancers that do not normally function. Differences between the expression patterns conferred by the two pLOB::GUS constructs indicate the presence of enhancer elements in the region of the promoter unique to pLOB5.0::GUS.

LOB is expressed at the base of lateral organs in the shoot and the root (Fig. 1). No obvious morphological characteristics distinguish LOB-expressing cells from adjacent cells that do not express LOB. One possible function of genes expressed in such a pattern is to define a boundary between the initiating organ primordia and the stem cells they are derived from. As lateral organs initiate in the shoot and the root, founder cells from the SAM and pericycle, respectively, are recruited into forming lateral organs (Steeves and Sussex, 1989; Laskowski et al., 1995). The establishment of a boundary between a primordium and its progenitor cells is likely important for maintaining the integrity of the stem cells and the initiating organ primordium.

A number of plant genes that are expressed in the vegetative shoot apex in a pattern similar to LOB have been described, including UNUSUAL FLORAL ORGANS (UFO), NO APICAL MERISTEM, CYP78A5, and the CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 genes (Souer et al., 1996; Aida et al., 1997; Lee et al., 1997; Zondlo and Irish, 1999; Takada et al., 2001). Analyses of loss-of-function mutations support the idea that some of these genes are important for the establishment of a boundary between organs. Mutations in no apical meristem cause a loss of the SAM and fusion of the cotyledons. CUC1 and CUC2 are functionally redundant and cuc1 cuc2 double mutants have fused cotyledons and do not form a SAM. ufo mutants have aberrant floral organs, but no vegetative phenotypes, suggesting that UFO acts redundantly in the vegetative shoot apex. Based on GUS activity in the transposant line, LOB expression appears to commence later in leaf initiation than that of UFO or CUC2. Although it is possible that LOB is expressed earlier, but at levels that are not detectable using the GUS reporter, these observations may indicate that LOB functions in the later stages of leaf development. Other possible functions for a gene expressed in such a domain are involvement in control of cell division or differentiation at the leaf base, establishment of adaxial cell fates, or functions in abscission. The Arabidopsis HAESA Leu-rich repeat receptor kinase, which is required for proper abscission of floral organs, is expressed in a pattern similar to LOB (Jinn et al., 2000). HAE expression appears to initiate later than that of LOB however.

Two different lob mutations were identified, and we examined homozygous mutant plants for abnormal morphology. Plants that contained a disrupted LOB gene made reduced levels of LOB transcript in the case of lob::DSE or a truncated transcript in the case of lob-2. In both cases, homozygotes were viable and had normal morphology under standard growth conditions. These data may indicate that LOB is functionally redundant, or required under a particular growth condition that we did not examine. Further support for functional redundancy comes from the fact that the LOB gene lies within a duplicated region of the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000). The corresponding region lies on chromosome 3 and contains the LBD27 gene. However, LBD27 is only 41% identical to LOB in the LOB domain. Another LOB domain protein, LBD25, also encoded by a gene on chromosome 3, is 83% identical to LOB in the LOB domain. Phylogenetic analyses also place LOB and LBD25 in the same clade and LBD 27 in a different clade (B. Shuai and P. Springer, unpublished data). For this reason, LBD25 may be more likely to have functions that overlap with LOB. Analyses of LBD25 transcript distribution by RT-PCR revealed that the LBD25 and LOB expression domains overlap, although LBD25 expression appears to be broader than LOB (Figs. 3A and 6). Functionally redundant genes with expression patterns that are not identical have been described; for example, the CUC1 expression domain is broader than that of CUC2 (Takada et al., 2001). Mutations in LBD25 will need to be identified to determine if LOB and LBD25 are functionally redundant.

Ectopic expression of LOB outside of its normal domain caused pleiotropic defects, making it difficult to attribute a specific role in plant development to LOB. 35S::LOB plants made generally smaller organs. The effects on organ size appeared to be largely due to differences in cell numbers, as we could not detect significant differences in cell size (Fig. 4, I-L). This may suggest that LOB functions to limit cell division at the base of lateral organs. An alternative possibility is that the effect on cell division is a pleiotropic stress response.

The deduced LOB protein is not similar to any previously described proteins in plants or animals, and does not contain defined functional domains. However, the amino terminal one-half of the LOB protein contains a conserved domain that is present in a large group of plant proteins that have been identified by EST and genomic sequencing. A search of the Arabidopsis genome sequence revealed 42 other genes that are predicted to encode LOB domain proteins (Table I). The LBD genes fall into two distinct classes based on sequence similarity to LOB in the LOB domain (Fig. 5). Examination of LBD expression profiles revealed that LBD genes are expressed in a variety of different patterns, with some genes being expressed in all tissues tested, whereas other genes were expressed in a more limited fashion (Fig. 6). These data may indicate diverse roles for the LBD genes.

The LOB domain contains conserved blocks of amino acids that identify the LOB domain gene family. In particular, a conserved CX₂CX₆CX₃C motif, which is the defining feature of the LOB domain, is present in all LBD proteins. It is possible that this motif forms a zinc finger, although the spacing between the cysteines is not typical of a C₂/C₂ type zinc finger (Takatsuji, 1998). LOB and many of the class I LOB domain proteins are predicted to form a coiled-coil motif that may function in protein-protein interactions. The lack of a predicted coiled coil in the class II proteins suggests that their function may be distinct from the class I LOB domain proteins.

LOB is expressed at low levels, is not present in EST databases, and is apparently functionally redundant, suggesting that LOB is unlikely to have been identified by conventional forward mutagenesis or differential expression approaches. The use of a gene trap approach allowed the identification of LOB, a gene encoding a novel, plant-specific protein of unknown function. The fact that LOB is plant specific could suggest its involvement in processes that are unique to plants. Further characterization of LOB and related LBD genes will be needed for the role of LOB in plant development to be understood.

A major goal in plant biology in the coming years will be to determine the function of every plant gene. Analyses of the annotated regions of the Arabidopsis genome suggest that approximately 30% of the 25,498 Arabidopsis genes are predicted to encode proteins that cannot be classified into functional groups based on sequence (The Arabidopsis Genome Initiative, 2000). Determining the function of genes in this category will be especially challenging. The analysis of members of multigene families can be particularly difficult, as these genes may be functionally redundant. In these instances, information about a gene's expression pattern can often provide important information regarding a potential biological role.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Growth Conditions

Seedlings were grown on germination media as previously described (Springer et al., 2000). Soil-grown plants were grown in Sunshine Mix No. 1 (SunGro, Bellevue, WA) supplemented with fertilizer and insecticide as previously described (Springer et al., 2000). Plants were grown in a 16-h light:8-h dark cycle (180 microeinsteins m² s¹).

Histochemical Localization of GUS Activity and Microscopy

Plant tissues were stained for GUS activity in 5-bromo-4-chloro-3-indolyl--glucuronic acid and were cleared in 70% (v/v) ethanol as previously described (Sundaresan et al., 1995). Stained tissue was processed for sectioning as previously described (Springer et al., 2000) and was viewed with a stereomicroscope or mounted on slides and viewed with DIC optics. Leaves were cleared as described (Berleth and Jürgens, 1993) and were viewed with DIC.

Cloning of LOB

Genomic DNA was isolated from pooled F₃ seedlings as previously described (Springer et al., 1995). TAIL-PCR (Liu et al., 1995) was performed as described (Tsugeki et al., 1996). TAIL-PCR products were cloned using the pGEM-T Easy vector system (Promega, Madison, WI) and were sequenced at the University of Maine DNA sequencing facility (Orono, ME). For cDNA library screening, PCR primers were designed to amplify genomic DNA fragments in the vicinity of the DsE insertion in ET22. Primers MDC5, 5'-GGCATTCAAGCAGGTTTACG-3'; MDC6, 5'-AGCTAATGCTGACTTGGCAC-3'; MDC7, 5'-AAGATTTTGTGGACGTTGGC-3'; and MDC8, 5'-TTGGAAGCGAAATTCAAAGG-3' were used. MDC5 and MDC6 amplified a 1.5-kb fragment, and MDC 7 and MDC8 amplified a 1.6-kb fragment. Both fragments were labeled using a random-primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis) and were used together to screen a cDNA library made from Arabidopsis flower buds (Weigel et al., 1992). The library, CD4-6, was obtained from the Arabidopsis Biological Resource Center. Approximately 300,000 clones were screened, and two clones were identified that hybridized to both fragments. To clone the 5' end of the LOB cDNA, 5'-RACE-PCR was performed as previously described (Frohman et al., 1988) with the following modifications. First strand cDNA synthesis was performed with the primer 5'-RACE_O, cDNAs were tailed with terminal transferase, and first round amplification was done with primers Q_O, Q_T, and 5'-RACE_O. First round PCR products were reamplified using Q_I and nested gene-specific primer 5'-RACE_I. Conditions for the second round PCR amplification were as follows: 45 s at 94°C, 1 min at 55°C, and 1 min at 72°C for three cycles; 45 s at 94°C, 1 min at 60°C, and 1 min at 72°C for 10 cycles; and 45 s at 94°C, 1 min at 55°C, and 1 min at 72°C for 10 cycles. The LOB gene-specific primers were 5'-RACE_O, 5'-TTTCTTCCTCTTTCAAGGGC-3' and 5'-RACE_I, 5'-AGGGATCCTTACCCTTTGAATTTCGC-3'. Q_T, Q_O, and Q_I primer sequences have previously been described (Frohman et al., 1988).

Constructs and Generation of Transgenic Plants

The LOB promoter fragments were amplified from genomic DNA using primers pET22-3': 5'-CATGCCATGG ACGACGCCATTTGTTTTTCTT-3' and pET22-5'a: 5'-CCGCTCGAGTTCCCACCACTAACCACCAT-3' (pLOB2.8) or pET22-5'b: 5'-TCCCCCGGGTTGCTTGGTCATCGTGTCTT-3' (pLOB5.0). The primers contained introduced restriction sites to facilitate cloning. The amplified pLOB2.8 fragment was cloned into SLJ4D4, which contains a uidA gene fused to the octopine synthase transcription terminator (Jones et al., 1992). The resulting promoter::GUS fusion was cloned into the binary vector pPZP111 (Hajdukiewicz et al., 1994) to create the pLOB2.8::GUS plasmid. The amplified pLOB5.0 fragment was fused to the uidA gene and was cloned into the binary vector pCAMBIA3200 (Center for the Application of Molecular Biology to International Agriculture, personal communication) using the SmaI and PstI sites to create the pLOB5.0::GUS plasmid. A construct for ectopic expression of LOB was made by introducing the LOB coding region into pPS119 (P. Springer and R. Martienssen, unpublished data), which contains the 35S cauliflower mosaic virus promoter (Odell et al., 1985) and a 3' octopine synthase transcription terminator (DeGreve et al., 1983) interrupted by multiple cloning sites in a pPZP111 backbone (Hajdukiewicz et al., 1994). The single exon containing the LOB coding region was amplified from genomic DNA using PFU polymerase (Stratagene, La Jolla, CA) and primers SET22-5, 5'-CCGCTCGAGATGGCGTCGTCATCAAACTC-3' and SET22-3, 5'-GCTCTAGACTCACATGTTACCTCCTTGC-3'. Both primers contain introduced restriction sites for cloning. The PCR product was cloned into pBS SK⁺ (Stratagene), sequenced to verify its integrity, and subsequently subcloned into pPS119 to create the 35S::LOB construct. Binary vectors were introduced into wild-type Landsberg erecta Arabidopsis plants by floral dip (Clough and Bent, 1998).

Scanning Electron Microscopy

Thirty-two-day-old 35S::LOB transgenic plants were fixed in 3% (v/v) glutaraldehyde (EM Sciences, Fort Washington, PA) in 1× phosphate-buffered saline at 4°C overnight. Plants were rinsed with 1× phosphate-buffered saline and dehydrated through an ethanol series at 4°C. Dehydrated tissue was critical point-dried in liquid carbon dioxide. Individual leaves were mounted on scanning electron microscope stubs, coated, and observed in a scanning electron microscope (XL30-FEG; Philips, Eindhoven, The Netherlands) at an accelerating voltage of 20 kV.

Screening for T-DNA Insertions in LOB

Primers were designed based on the recommendations of the Arabidopsis Knockout Facility (http://www. biotech.wisc.edu/Arabidopsis/). Primers used in the screening were: ET22-4, 5'-CACTTTGTCTTTTGCTCTTTCTCCTTCCT-3' and ET22-5, 5'-AAGCAGAGACCTTCAATTATTAGCACCCT-3' in pair-wise combination with T-DNA left border primer JL-202. After identification of a pool containing a T-DNA insertion in the LOB coding region, seeds from subpools were obtained from the Arabidopsis Biological Resource Center. PCR reactions on single plants were used to identify plants homozygous for the T-DNA insertion.

Expression Analyses

RNA was isolated from various tissues from wild-type plants, and RNA gel-blot hybridizations were performed as previously described (Martienssen et al., 1989). For RT-PCR analysis, cDNA was synthesized from 2 µg of total RNA using an oligo-(dT) primer and M-MLV RNase H minus reverse transcriptase (Promega). One-twentieth volume of each cDNA sample was used as the template for PCR amplification. Primers MDC7 and MDC8 (Fig. 3B, see above), which flanked an intron in the 5'-UTR, were used for amplification of LOB under the following conditions: denaturation at 94°C for 3 min, followed by 30 cycles of 45 s at 94°C, 45 s at 57°C, and 1 min at 72°C. Control reactions using primers to the ACT2 gene (An et al., 1996; Li et al., 2001) were performed on the same cDNA samples. The gene-specific primers used were: LBD1, 5'-GGAATCCCAAATCATTGCTC-3' and 5'-TTAGTCCATGTGCTGCTTGC-3'; LBD3, 5'-ACAAAAGGGTCACAGACACG-3' and 5'-AAGACCAAAGGAAGTCTCCG-3'; LBD4, 5'-CGTTTTCTCGCCGTATTTTC-3' and 5'-ACTCTCCCAAACTGGCTTCA-3'; LBD5, 5'-CCTGGAGTTCACGGAGGTAG-3' and 5'-CCTCTAGGAAACCGTCGTCC-3'; LBD6, 5'-ATTTCCCCTCTGAGCAACAG-3' and 5'-AAGACGGATCAACAGTACGG-3'; LBD8, 5'-TCGTCCTTGCTGCGTATGTA-3' and 5'-TCCACATGATCTTTTGCACC-3'; LBD9, 5'-TGCGTAATTCAATTTGCCAC-3' and 5'-TCAATGTTAAACGTGCTCCTTG-3'; LBD11, 5'-TTTGGCACCGTACTTTCCTC-3' and 5'-ATGTCCAAAGAGGATCCCAC-3'; LBD12, 5'-GATCCTCACAAATTCGCCAT-3' and 5'-TAAGAGGGTCTTGCATTTGC-3'; LBD13, 5'-TGGGAATCAGGAGACATGTG-3' and 5'-GTGGCGTAGGATTTCCGTAC-3'; LBD14, 5'-TTTTGCAGCCATTCACAAAG-3' and 5'-CAGACCAAGGAAAATTGACC-3'; LBD15, 5'-GAATGTCCCTTTTCGCCATA-3' and 5'-TCTCACTTTCAATGTTGCCG-3'; LBD16, 5'-TCGCAGCTATTCACAAGGTG-3' and 5'-CCTCCGGTTTGATGATGAGT-3'; LBD17, 5'-AAAAGGATGTGTGTTTGCCC-3' and 5'-ATCAGATTATTGCCGCCATG-3'; LBD18, 5'-AGGTCCGATGCTGTCGTAAC-3' and 5'-ACATAGTTCGAGACGGCGAG-3'; LBD19, 5'-TGAGATTGCCTCTGCACAAG-3' and 5'-AAGTGCAAGCCGGAAGTTTG-3'; LBD20, 5'-CATGGTGAAGCTGTTCATGG-3' and 5'-TTTTGGGTCAGACCAAGGAG-3'; LBD23, 5'-GAATCCAAAAAGATGTGCAGC -3' and 5'-TGGCCTCTTGATTATGAGTCTG-3'; LBD24, 5'-GCTAATGGCCTCTTGATTATGATT-3' and 5'-GAATCCAAAAAGATGTGCAGC-3'; LBD25, 5'-AAGGACCTTTTCTTGTTGCG-3' and 5'-CGCCGCTAATTTTCTCAAAG-3'; LBD29, 5'-TGAGGAGGTTTCGTTGTGGT-3' and 5'-CGCTGTGAAGCCGCTATTA-3'; LBD30, 5'-TGCGTCTCTCACATCGTCTC-3' and 5'-ACTGACGAGGCAGAACCACT-3'; LBD31, 5'-CTTACGAGGCATTGGCTAGG-3' and 5'-GAAGATGGTCGGTATTTGCC-3'; LBD33, 5'-GGTCGTGGCCATAGTCATCT-3' and 5'-CTAAGGAGGAAATGCAACCG-3'; LBD37, 5'-AGATGGTTGGTCTTCCGATG-3' and 5'-CCGTCTTCGTCGCTAAATTC-3'; LBD38, 5'-CGTGCCGGTTTAATGTCTTT-3' and 5'-ACGAAGGTTGTTGTTCCGAC-3'; LBD39, 5'-GTGGATCTGGAGGTGGAGAA-3' and 5'-CCTCCGTACCTGAACTCCAA-3'; LBD40, 5'-TACGAAAAGGCTGCAGTGAA-3' and 5'-GGTACCACCACGTGATTTCC-3'; LBD41, 5'-TCCTTCATGAGCAGCCACTA -3' and 5'-AAACCAAAGATGCGGATGAG -3'; and LBD42, 5'-AATGGATCAAATCCGCAGAC-3' and 5'-GAACTTGGGAGTGCCACAT-3'. Primers to At5g63080 were MDC12.4-1, 5'-GCCATTGGAGGAGAAGCATC-3' and MDC12.4-2, 5'- TTTCCAGCCATCGTGTCATA-3'.

Sequence Alignment and Block Analysis

Database searches were performed using TBLASTN (http://www.ncbi.nlm.nih.gov/BLAST/). Protein sequences from each gene were aligned using AlignX program from Vector NTI suite (InforMax, Bethesda, MD). Alignments were done using the LOB sequence as the selected profile with a gap opening penalty of 10 and a gap extension penalty of 0.1. The aligned sequences were shaded using MacBoxshade (http://www.isrec.isb-sib.ch/sib-isrec/boxshade/MacBoxshade/) in an Encapsulated PostScript output. Conserved blocks were predicted by BlockMaker (http://www.blocks.fhcrc.org/) using the Motif algorithm and all class I or class II sequences as input. Secondary structure predictions were performed with NNPredict (http://www.cmpharm.ucsf.edu/~nomi/nnpredict; Kneller et al., 1990) and COILS (http://www.ch.embnet. org/software/COILS_form.html; Lupas et al., 1991) programs. COILS parameters used the MTIDK matrix and a 2.5-fold weighting of positions a and d. A coiled coil of 30 amino acids in length was predicted (>95% probability) in LOB with window sizes of 14, 21, and 28.