TY - JOUR
AU - Springer, Patricia S.
AB - Abstract The LATERAL ORGAN BOUNDARIES(LOB) gene in Arabidopsis defines a new conserved protein domain. LOB is expressed in a band of cells at the adaxial base of all lateral organs formed from the shoot apical meristem and at the base of lateral roots. LOB encodes a predicted protein that does not have recognizable functional motifs, but that contains a conserved domain (the LOB domain) that is present in 42 other Arabidopsis proteins and in proteins from a variety of other plant species. Proteins showing similarity to the LOB domain were not found outside of plant databases, indicating that this unique protein may play a role in plant-specific processes. Genes encoding LOB domain proteins are expressed in a variety of temporal- and tissue-specific patterns, suggesting that they may function in diverse processes. Loss-of-function LOB mutants have no detectable phenotype under standard growth conditions, suggesting thatLOB is functionally redundant or required during growth under specific environmental conditions. Ectopic expression ofLOB leads to alterations in the size and shape of leaves and floral organs and causes male and female sterility. The expression of LOB at the base of lateral organs suggests a potential role for LOB in lateral organ development. 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 byCLV3. 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 theASYMMETRIC 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 thatas1 stm double mutants form a vegetative meristem. These observations suggest that the loss of a meristem in stmmutants is due to expression of AS1 in the meristem (Byrne et al., 2000). In turn, AS1 activity is needed to repressKNOX 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 CUCgenes 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 ofLOB is unknown, its expression indicates a potential role in organ separation or other aspects of lateral organ development. RESULTS β-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. Fig. 1. Open in new tabDownload slide 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. Fig. 1. Open in new tabDownload slide 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 theDsE 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 GUSreporter gene in the transposant line, the corresponding gene was namedLOB. The MDC12 sequence has recently been annotated, and theLOB gene corresponds to hypothetical geneAt5g63090 (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. TheDsE insertion was near the 3′ end of LOB and was inserted such that the GUS gene was transcribed opposite toLOB (Fig. 2A). Fig. 2. Open in new tabDownload slide 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. Fig. 2. Open in new tabDownload slide 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::GUSlines and in six of the pLOB5.0::GUSlines (data not shown). These insertions were assumed to be adjacent to strong promoter or enhancer sequences that affected activity of theLOB 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 ofGUS 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::GUStransformants (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 theGUS gene (Fig. 2A). Expression of At5g63100 was not examined. Attempts to detect LOB transcripts using in situ hybridization were unsuccessful, suggesting that LOBtranscripts are present at low abundance. Therefore, we examined the expression pattern of LOB by RT-PCR (Fig.3A). The expression pattern ofLOB shown by RT-PCR was consistent with the GUSexpression 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). Fig. 3. Open in new tabDownload slide Expression of LOB in wild-type tissues. A, RT-PCR analysis of LOB expression. RNA was isolated fromLandsberg 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). Fig. 3. Open in new tabDownload slide Expression of LOB in wild-type tissues. A, RT-PCR analysis of LOB expression. RNA was isolated fromLandsberg 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, theLOB-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 LOBaand 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. TheLOBb 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 LOBdtranscripts 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 LOBaand 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 fromlob::DsE homozygotes, suggesting that the DsE insertion causes a reduction in LOBtranscript 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::DsEhomozygotes (data not shown). Despite the significantly reducedLOB transcript levels, no obvious morphological phenotypes were visible in lob::DsEhomozygotes. 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 designatedlob-2. RT-PCR showed that full-length LOBtranscripts 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 ofLOB expression may be detrimental. RNA-blot analysis was performed on individual transformants to verify LOBoverexpression (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). Fig. 4. Open in new tabDownload slide Phenotypes of transgenic plants that ectopically express LOB. A, Wild-type 19-d-old Landsberg erecta plant. B and C, Two independent transgenic35S::LOB plants (19-d-old). D, Thirty-two-day-old 35S::LOB plant. E, Scanning electron microscopy of35S::LOB rosette leaf. F, Wild-typeLandsberg 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. Fig. 4. Open in new tabDownload slide Phenotypes of transgenic plants that ectopically express LOB. A, Wild-type 19-d-old Landsberg erecta plant. B and C, Two independent transgenic35S::LOB plants (19-d-old). D, Thirty-two-day-old 35S::LOB plant. E, Scanning electron microscopy of35S::LOB rosette leaf. F, Wild-typeLandsberg 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 (TableI). 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. Table I. Arabidopsis genes encoding LOB domain proteins Gene . Bacterial Artificial Chromosome/P1 Locus . Genbank Protein ID . Chromosome Locus1-a . Accession No. . Clone Representing EST Sequence1-b,1-c . Class I  LOB MDC12.5 10177290 At5g63090 BAB10551  LBD1 F24B9.1 8439879 At1g07900 AAF75065  LBD2 F9P14.14 8844133 At1g06280 AAF80225  LBD3 F3O9.33 4966373 At1g16530 AAD34704  LBD4 T19E23.11 6692123 At1g31320 AAF24588  LBD5 T22A15.8 12324709 At1g36000 AAG52312  LBD6 F5I14.15 2190548 At1g65620 AAB60912 VBVYB03  LBD7 F3N23.18 5903087 At1g72980 AAD55645  LBD8 F3P11.11 4191781 At2g19510 AAD10150  LBD9 F6F22.15 3687236 At2g19820 AAC62134  LBD10 F27L4.15 3152616 At2g23660 AAC17095  LBD11 T17D12.6 4510398 At2g28500 AAD21485  LBD12 T27E13.13 3150407 At2g30130 AAC16959  LBD13 T9D9.15 2347197 At2g30340 AAC16936 RZ93g04,RZ123h01  LBD14 F16D14.15 4432826 At2g31310 AAD20676  LBD15 T2P4.18 2651309 At2g40470 AAB87589 241G2,701514676, 600038330  LBD16 MHK10.15 4567314 At2g42430 AAD23725  LBD17 MHK10.16 4567315 At2g42440 AAD23726  LBD18 F4L23.7 2583113 At2g45420 AAB82622 M13A6, M22C7  LBD19 F4L23.8 2583114 At2g45410 AAB82623  LBD20 F20H23.21 6006865 At3g03760 AAF00641  LBD21 F9F8.10 6016686 At3g11090 AAF01513  LBD22 MCP4.8 9294609 At3g13850 BAB02910  LBD23 MLJ15.2 At3g26620  LBD24 MLJ15.6 At3g26660  LBD25 MGF10.6 9294470 At3g27650 BAB02689 701546372  LBD26 K24A2.3 9294310 At3g27940 BAB01481  LBD27 T23J7.200 4741204 At3g47870 CAB41870  LBD28 T20E23.110 6561991 At3g50510 CAB62480  LBD29 F9D24.100 6735331 At3g58190 CAB68157 RZ58d08  LBD30 F6N15.4 3193318 At4g00220 AAC19300 232A14, 701500638, SQ185e01  LBD31 F6N15.25 3193312 At4g00210 AAC19294  LBD32 T12H17.90 2827547 At4g22700 CAA16555  LBD33 K16F4.4 8978344 At5g06080 BAA98197  LBD34 F2G14 Not annotated1-d  LBD35 MIK22.21 10176709 At5g35900 BAB09931  LBD36 MUD21.13 9758137 At5g66870 BAB08629 Class II  LBD37 K8K14.16 9758441 At5g67420 BAB09027 AA07F04, AB01G02, APD23d08, APD43f11, APZ06a08, OBO52, RZ108b01,RZ126f11, 47B5, 65DI, 179D12, 217P3, 240N2, 701551301  LBD38 F3A4.20 6522915 At3g49940 CAB62102 147D14T7, VBVQC02  LBD39 F19F18.30 4468979 At4g37540 CAB38293 701500839, 165E11T7, RZ23c09  LBD40 F5A8.2 4204277 At1g67100 AAD10658 M11G10, M13C9, M15B4, M22A4, M53F11  LBD41 F16B3.18 6957718 At3g02550 AAF32462 RZ101f04, SQ182b06, SQ193f07, 111E10, 195F24, 701667942  LBD42 T26J14.8 12324879 At1g68510 AAG52389 SQ124h04 Gene . Bacterial Artificial Chromosome/P1 Locus . Genbank Protein ID . Chromosome Locus1-a . Accession No. . Clone Representing EST Sequence1-b,1-c . Class I  LOB MDC12.5 10177290 At5g63090 BAB10551  LBD1 F24B9.1 8439879 At1g07900 AAF75065  LBD2 F9P14.14 8844133 At1g06280 AAF80225  LBD3 F3O9.33 4966373 At1g16530 AAD34704  LBD4 T19E23.11 6692123 At1g31320 AAF24588  LBD5 T22A15.8 12324709 At1g36000 AAG52312  LBD6 F5I14.15 2190548 At1g65620 AAB60912 VBVYB03  LBD7 F3N23.18 5903087 At1g72980 AAD55645  LBD8 F3P11.11 4191781 At2g19510 AAD10150  LBD9 F6F22.15 3687236 At2g19820 AAC62134  LBD10 F27L4.15 3152616 At2g23660 AAC17095  LBD11 T17D12.6 4510398 At2g28500 AAD21485  LBD12 T27E13.13 3150407 At2g30130 AAC16959  LBD13 T9D9.15 2347197 At2g30340 AAC16936 RZ93g04,RZ123h01  LBD14 F16D14.15 4432826 At2g31310 AAD20676  LBD15 T2P4.18 2651309 At2g40470 AAB87589 241G2,701514676, 600038330  LBD16 MHK10.15 4567314 At2g42430 AAD23725  LBD17 MHK10.16 4567315 At2g42440 AAD23726  LBD18 F4L23.7 2583113 At2g45420 AAB82622 M13A6, M22C7  LBD19 F4L23.8 2583114 At2g45410 AAB82623  LBD20 F20H23.21 6006865 At3g03760 AAF00641  LBD21 F9F8.10 6016686 At3g11090 AAF01513  LBD22 MCP4.8 9294609 At3g13850 BAB02910  LBD23 MLJ15.2 At3g26620  LBD24 MLJ15.6 At3g26660  LBD25 MGF10.6 9294470 At3g27650 BAB02689 701546372  LBD26 K24A2.3 9294310 At3g27940 BAB01481  LBD27 T23J7.200 4741204 At3g47870 CAB41870  LBD28 T20E23.110 6561991 At3g50510 CAB62480  LBD29 F9D24.100 6735331 At3g58190 CAB68157 RZ58d08  LBD30 F6N15.4 3193318 At4g00220 AAC19300 232A14, 701500638, SQ185e01  LBD31 F6N15.25 3193312 At4g00210 AAC19294  LBD32 T12H17.90 2827547 At4g22700 CAA16555  LBD33 K16F4.4 8978344 At5g06080 BAA98197  LBD34 F2G14 Not annotated1-d  LBD35 MIK22.21 10176709 At5g35900 BAB09931  LBD36 MUD21.13 9758137 At5g66870 BAB08629 Class II  LBD37 K8K14.16 9758441 At5g67420 BAB09027 AA07F04, AB01G02, APD23d08, APD43f11, APZ06a08, OBO52, RZ108b01,RZ126f11, 47B5, 65DI, 179D12, 217P3, 240N2, 701551301  LBD38 F3A4.20 6522915 At3g49940 CAB62102 147D14T7, VBVQC02  LBD39 F19F18.30 4468979 At4g37540 CAB38293 701500839, 165E11T7, RZ23c09  LBD40 F5A8.2 4204277 At1g67100 AAD10658 M11G10, M13C9, M15B4, M22A4, M53F11  LBD41 F16B3.18 6957718 At3g02550 AAF32462 RZ101f04, SQ182b06, SQ193f07, 111E10, 195F24, 701667942  LBD42 T26J14.8 12324879 At1g68510 AAG52389 SQ124h04 F1-a  Designation from Munich Information Center for Protein Sequences Arabidopsis thaliana Database (MATDB) (www.mips.biochem.mpg.de/proj/thal/db/index. html). F1-b  Clones shown in bold were fully sequenced. F1-c  EST accession nos.: Z33806, Z25656, AF447887 (clone VBVYB03);AV554524 (clone RZ93g04); AV551296, AV538901, AF447888(clone RZ123h01); N97300 (clone 241G2); AI993799, AF447889(clone 701514676); BE529105 (clone 600038330); BE520513, BE520514,AF447891 (clone M13A6); BE521897, BE521898 (clone M22C7);AI998629, AF447892 (clone 701546372); AV553261, AV544763,AF447893 (clone RZ58d08); AI994962 (clone 701500638);AV563330 (clone SQ185e01); N65781, AF432232 (clone 232A14);BE038006 (clone AA07F04); BE038601 (clone AB01G02); AV525400 (clone APD23d08); AV518911 (clone APD43f11); AV526154, AV520071 (clone APZ06a08); F14269, F14441 (clone OBO52); AV550089 (clone RZ108b01);AV539081, AV551431, AF447894 (clone RZ126f11); T14105 (clone 47B5); T41721 (clone 65D1); H36818 (clone 179D12); N38449 (clone 217P3); N65652 (clone 240N2); AI996949 (clone 701551301); T76164 (clone 147D14); Z29130, F13856 (clone VBVQC02); AI994989 (clone 701500839);R65200 (clone 165E11); AV552189 (clone RZ23c09); BE520344 (clone M11G10); BE520541 (clone M13C9); BE520808, BE520809, BE520810 (clone M15B4); BE521846, BE521847 (clone M22A4); AV537704, AV549715,AF447895 (clone RZ101f04); AV563144 (clone SQ182b0);AV563797 (clone SQ193f07); AI100279, T42227 (clone 111E10); H76116(195F24); AI996685 (clone 701667942); AV559860, AF447896(clone SQ124h04); AF447890 (LBD16). Accession nos. shown in italics represent the complete sequence of the corresponding clone. F1-d  Complement (AL391146.1:60133.60555). Open in new tab Table I. Arabidopsis genes encoding LOB domain proteins Gene . Bacterial Artificial Chromosome/P1 Locus . Genbank Protein ID . Chromosome Locus1-a . Accession No. . Clone Representing EST Sequence1-b,1-c . Class I  LOB MDC12.5 10177290 At5g63090 BAB10551  LBD1 F24B9.1 8439879 At1g07900 AAF75065  LBD2 F9P14.14 8844133 At1g06280 AAF80225  LBD3 F3O9.33 4966373 At1g16530 AAD34704  LBD4 T19E23.11 6692123 At1g31320 AAF24588  LBD5 T22A15.8 12324709 At1g36000 AAG52312  LBD6 F5I14.15 2190548 At1g65620 AAB60912 VBVYB03  LBD7 F3N23.18 5903087 At1g72980 AAD55645  LBD8 F3P11.11 4191781 At2g19510 AAD10150  LBD9 F6F22.15 3687236 At2g19820 AAC62134  LBD10 F27L4.15 3152616 At2g23660 AAC17095  LBD11 T17D12.6 4510398 At2g28500 AAD21485  LBD12 T27E13.13 3150407 At2g30130 AAC16959  LBD13 T9D9.15 2347197 At2g30340 AAC16936 RZ93g04,RZ123h01  LBD14 F16D14.15 4432826 At2g31310 AAD20676  LBD15 T2P4.18 2651309 At2g40470 AAB87589 241G2,701514676, 600038330  LBD16 MHK10.15 4567314 At2g42430 AAD23725  LBD17 MHK10.16 4567315 At2g42440 AAD23726  LBD18 F4L23.7 2583113 At2g45420 AAB82622 M13A6, M22C7  LBD19 F4L23.8 2583114 At2g45410 AAB82623  LBD20 F20H23.21 6006865 At3g03760 AAF00641  LBD21 F9F8.10 6016686 At3g11090 AAF01513  LBD22 MCP4.8 9294609 At3g13850 BAB02910  LBD23 MLJ15.2 At3g26620  LBD24 MLJ15.6 At3g26660  LBD25 MGF10.6 9294470 At3g27650 BAB02689 701546372  LBD26 K24A2.3 9294310 At3g27940 BAB01481  LBD27 T23J7.200 4741204 At3g47870 CAB41870  LBD28 T20E23.110 6561991 At3g50510 CAB62480  LBD29 F9D24.100 6735331 At3g58190 CAB68157 RZ58d08  LBD30 F6N15.4 3193318 At4g00220 AAC19300 232A14, 701500638, SQ185e01  LBD31 F6N15.25 3193312 At4g00210 AAC19294  LBD32 T12H17.90 2827547 At4g22700 CAA16555  LBD33 K16F4.4 8978344 At5g06080 BAA98197  LBD34 F2G14 Not annotated1-d  LBD35 MIK22.21 10176709 At5g35900 BAB09931  LBD36 MUD21.13 9758137 At5g66870 BAB08629 Class II  LBD37 K8K14.16 9758441 At5g67420 BAB09027 AA07F04, AB01G02, APD23d08, APD43f11, APZ06a08, OBO52, RZ108b01,RZ126f11, 47B5, 65DI, 179D12, 217P3, 240N2, 701551301  LBD38 F3A4.20 6522915 At3g49940 CAB62102 147D14T7, VBVQC02  LBD39 F19F18.30 4468979 At4g37540 CAB38293 701500839, 165E11T7, RZ23c09  LBD40 F5A8.2 4204277 At1g67100 AAD10658 M11G10, M13C9, M15B4, M22A4, M53F11  LBD41 F16B3.18 6957718 At3g02550 AAF32462 RZ101f04, SQ182b06, SQ193f07, 111E10, 195F24, 701667942  LBD42 T26J14.8 12324879 At1g68510 AAG52389 SQ124h04 Gene . Bacterial Artificial Chromosome/P1 Locus . Genbank Protein ID . Chromosome Locus1-a . Accession No. . Clone Representing EST Sequence1-b,1-c . Class I  LOB MDC12.5 10177290 At5g63090 BAB10551  LBD1 F24B9.1 8439879 At1g07900 AAF75065  LBD2 F9P14.14 8844133 At1g06280 AAF80225  LBD3 F3O9.33 4966373 At1g16530 AAD34704  LBD4 T19E23.11 6692123 At1g31320 AAF24588  LBD5 T22A15.8 12324709 At1g36000 AAG52312  LBD6 F5I14.15 2190548 At1g65620 AAB60912 VBVYB03  LBD7 F3N23.18 5903087 At1g72980 AAD55645  LBD8 F3P11.11 4191781 At2g19510 AAD10150  LBD9 F6F22.15 3687236 At2g19820 AAC62134  LBD10 F27L4.15 3152616 At2g23660 AAC17095  LBD11 T17D12.6 4510398 At2g28500 AAD21485  LBD12 T27E13.13 3150407 At2g30130 AAC16959  LBD13 T9D9.15 2347197 At2g30340 AAC16936 RZ93g04,RZ123h01  LBD14 F16D14.15 4432826 At2g31310 AAD20676  LBD15 T2P4.18 2651309 At2g40470 AAB87589 241G2,701514676, 600038330  LBD16 MHK10.15 4567314 At2g42430 AAD23725  LBD17 MHK10.16 4567315 At2g42440 AAD23726  LBD18 F4L23.7 2583113 At2g45420 AAB82622 M13A6, M22C7  LBD19 F4L23.8 2583114 At2g45410 AAB82623  LBD20 F20H23.21 6006865 At3g03760 AAF00641  LBD21 F9F8.10 6016686 At3g11090 AAF01513  LBD22 MCP4.8 9294609 At3g13850 BAB02910  LBD23 MLJ15.2 At3g26620  LBD24 MLJ15.6 At3g26660  LBD25 MGF10.6 9294470 At3g27650 BAB02689 701546372  LBD26 K24A2.3 9294310 At3g27940 BAB01481  LBD27 T23J7.200 4741204 At3g47870 CAB41870  LBD28 T20E23.110 6561991 At3g50510 CAB62480  LBD29 F9D24.100 6735331 At3g58190 CAB68157 RZ58d08  LBD30 F6N15.4 3193318 At4g00220 AAC19300 232A14, 701500638, SQ185e01  LBD31 F6N15.25 3193312 At4g00210 AAC19294  LBD32 T12H17.90 2827547 At4g22700 CAA16555  LBD33 K16F4.4 8978344 At5g06080 BAA98197  LBD34 F2G14 Not annotated1-d  LBD35 MIK22.21 10176709 At5g35900 BAB09931  LBD36 MUD21.13 9758137 At5g66870 BAB08629 Class II  LBD37 K8K14.16 9758441 At5g67420 BAB09027 AA07F04, AB01G02, APD23d08, APD43f11, APZ06a08, OBO52, RZ108b01,RZ126f11, 47B5, 65DI, 179D12, 217P3, 240N2, 701551301  LBD38 F3A4.20 6522915 At3g49940 CAB62102 147D14T7, VBVQC02  LBD39 F19F18.30 4468979 At4g37540 CAB38293 701500839, 165E11T7, RZ23c09  LBD40 F5A8.2 4204277 At1g67100 AAD10658 M11G10, M13C9, M15B4, M22A4, M53F11  LBD41 F16B3.18 6957718 At3g02550 AAF32462 RZ101f04, SQ182b06, SQ193f07, 111E10, 195F24, 701667942  LBD42 T26J14.8 12324879 At1g68510 AAG52389 SQ124h04 F1-a  Designation from Munich Information Center for Protein Sequences Arabidopsis thaliana Database (MATDB) (www.mips.biochem.mpg.de/proj/thal/db/index. html). F1-b  Clones shown in bold were fully sequenced. F1-c  EST accession nos.: Z33806, Z25656, AF447887 (clone VBVYB03);AV554524 (clone RZ93g04); AV551296, AV538901, AF447888(clone RZ123h01); N97300 (clone 241G2); AI993799, AF447889(clone 701514676); BE529105 (clone 600038330); BE520513, BE520514,AF447891 (clone M13A6); BE521897, BE521898 (clone M22C7);AI998629, AF447892 (clone 701546372); AV553261, AV544763,AF447893 (clone RZ58d08); AI994962 (clone 701500638);AV563330 (clone SQ185e01); N65781, AF432232 (clone 232A14);BE038006 (clone AA07F04); BE038601 (clone AB01G02); AV525400 (clone APD23d08); AV518911 (clone APD43f11); AV526154, AV520071 (clone APZ06a08); F14269, F14441 (clone OBO52); AV550089 (clone RZ108b01);AV539081, AV551431, AF447894 (clone RZ126f11); T14105 (clone 47B5); T41721 (clone 65D1); H36818 (clone 179D12); N38449 (clone 217P3); N65652 (clone 240N2); AI996949 (clone 701551301); T76164 (clone 147D14); Z29130, F13856 (clone VBVQC02); AI994989 (clone 701500839);R65200 (clone 165E11); AV552189 (clone RZ23c09); BE520344 (clone M11G10); BE520541 (clone M13C9); BE520808, BE520809, BE520810 (clone M15B4); BE521846, BE521847 (clone M22A4); AV537704, AV549715,AF447895 (clone RZ101f04); AV563144 (clone SQ182b0);AV563797 (clone SQ193f07); AI100279, T42227 (clone 111E10); H76116(195F24); AI996685 (clone 701667942); AV559860, AF447896(clone SQ124h04); AF447890 (LBD16). Accession nos. shown in italics represent the complete sequence of the corresponding clone. F1-d  Complement (AL391146.1:60133.60555). Open in new tab Fig. 5. Open in new tabDownload slide 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. Fig. 5. Open in new tabDownload slide 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 (TableI). cDNA clones corresponding to ESTs for LBD6,13, 15, 18, 25,29, 30, 37, 41, and42 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. TheLBD18 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 isCAACKFLRRKCX3 C, whereas the class II consensus sequence isCNGCRVLRKGCSE(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 24LBD 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 ifLBD5, 8, 9, 23, and24 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. Fig. 6. Open in new tabDownload slide 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. Fig. 6. Open in new tabDownload slide 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. LBD11transcripts were detected in all tissues except root. LBD17transcripts were detected in all tissues except 12-d-old shoots. Transcripts from LBD14, 29, and 33were detected only in roots, whereas transcripts from LBD16were 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 fromLBD20 and 40 were detected in roots and floral tissues, although at different levels. Transcripts fromLBD18 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 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 LOBpromoter::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 inLOB is oriented so that the GUS gene is transcribed opposite to LOB. This could place GUSunder the control of 3′ regulatory elements or cryptic enhancers that do not normally function. Differences between the expression patterns conferred by the two pLOB::GUSconstructs 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, includingUNUSUAL 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 meristemcause a loss of the SAM and fusion of the cotyledons. CUC1and CUC2 are functionally redundant and cuc1 cuc2double 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 ofLOB 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 LOBtranscript 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 theLOB 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 ofCUC2 (Takada et al., 2001). Mutations in LBD25will need to be identified to determine if LOB andLBD25 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 thatLOB 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). TheLBD genes fall into two distinct classes based on sequence similarity to LOB in the LOB domain (Fig. 5). Examination ofLBD 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 thatLOB 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 ofLOB 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 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 m−2 s−1). Histochemical Localization of GUS Activity and Microscopy Plant tissues were stained for GUS activity in 5-bromo-4-chloro-3-indolyl-β-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 theLOB 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′-RACEO, cDNAs were tailed with terminal transferase, and first round amplification was done with primers QO, QT, and 5′-RACEO. First round PCR products were reamplified using QI and nested gene-specific primer 5′-RACEI. 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′-RACEO, 5′-TTTCTTCCTCTTTCAAGGGC-3′ and 5′-RACEI, 5′-AGGGATCCTTACCCTTTGAATTTCGC-3′. QT, QO, and QI 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 uidAgene 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 theuidA gene and was cloned into the binary vector pCAMBIA3200 (Center for the Application of Molecular Biology to International Agriculture, personal communication) using theSmaI 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::LOBconstruct. Binary vectors were introduced into wild-typeLandsberg erecta Arabidopsis plants by floral dip (Clough and Bent, 1998). Scanning Electron Microscopy Thirty-two-day-old35S::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 At5g63080were 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. ACKNOWLEDGMENTS We thank Mary Byrne, Elizabeth Bray, and Linda Walling for comments on the manuscript, Janena Williams and Rob Lennox for assistance with plant growth, Catherine Bushell for help with RT-PCR, and members of the Springer laboratory for helpful discussions. We also thank the Arabidopsis Biological Resource Center and the Kazusa DNA Research Institute for supplying cDNA clones and the Arabidopsis Knock-Out Facility for identifying the lob-2allele. LITERATURE CITED 1 Aida M Ishida T Fukaki H Fujisawa H Tasaka M Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9 1997 841 857 Google Scholar Crossref Search ADS PubMed WorldCat 2 Aida M Ishida T Tasaka M Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes. Development 126 1999 1563 1570 Google Scholar Crossref Search ADS PubMed WorldCat 3 An Y-Q McDowell JM Huang S McKinney EC Chambliss S Meagher RB Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10 1996 107 121 Google Scholar Crossref Search ADS PubMed WorldCat 4 Barton MK Poethig RS Formation of the shoot apical meristem in Arabidopsis thaliana: an analysis of development in the wild type and in the shoot meristemless mutant. Development 119 1993 823 831 Google Scholar Crossref Search ADS WorldCat 5 Berleth T Jürgens G The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo. Development 118 1993 575 587 Google Scholar Crossref Search ADS WorldCat 6 Brand U Fletcher JC Hobe M Meyerowitz EM Simon R Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity. Science 289 2000 617 619 Google Scholar Crossref Search ADS PubMed WorldCat 7 Brown JWS Smith P Simpson CG Arabidopsis consensus intron sequences. Plant Mol Biol 32 1996 531 535 Google Scholar Crossref Search ADS PubMed WorldCat 8 Byrne ME Barley R Curtis M Arroyo JM Dunham M Hudson A Martienssen RA Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature 408 2000 967 971 Google Scholar Crossref Search ADS PubMed WorldCat 9 Clark SE Meristems: start your signaling. Curr Opin Plant Biol 4 2001 28 32 Google Scholar Crossref Search ADS PubMed WorldCat 10 Clough SJ Bent AJ Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16 1998 735 743 Google Scholar Crossref Search ADS PubMed WorldCat 11 DeGreve H Dhaese P Seurinck J Lemmers S van Montagu M Schell J Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet 1 1983 499 511 Google Scholar OpenURL Placeholder Text WorldCat 12 Frohman MA Dush MK Martin GR Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85 1988 8998 9002 Google Scholar Crossref Search ADS PubMed WorldCat 13 Hajdukiewicz P Svab Z Maliga P The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25 1994 989 994 Google Scholar Crossref Search ADS PubMed WorldCat 14 Henikoff S Henikoff JG Alford WJ Pietrokovski S Automated construction and graphical presentation of protein blocks from unaligned sequences. Gene 163 1995 17 26 Google Scholar Crossref Search ADS WorldCat 15 Jackson D Veit B Hake S Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Development 120 1994 405 413 Google Scholar Crossref Search ADS WorldCat 16 Jinn T-L Stone JM Walker JC HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission. Genes Dev 14 2000 108 117 Google Scholar PubMed OpenURL Placeholder Text WorldCat 17 Jones JDG Shlumukov L Carland F English J Scofield SR Bishop GJ Harrison K Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants. Transgenic Res 1 1992 285 297 Google Scholar Crossref Search ADS PubMed WorldCat 18 Joshi CP Zhou H Huang X Chiang VL Context sequences of translation initiation codon in plants. Plant Mol Biol 35 1997 993 1001 Google Scholar Crossref Search ADS PubMed WorldCat 19 Kneller DG Cohen FE Langridge R Improvements in protein secondary structure prediction by an enhanced neural network. J Mol Biol 214 1990 171 182 Google Scholar Crossref Search ADS PubMed WorldCat 20 Krysan PJ Young JC Sussman MR T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11 1999 2283 2290 Google Scholar Crossref Search ADS PubMed WorldCat 21 Landschultz WH Johnson PF McKnight SL The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240 1988 1759 1764 Google Scholar Crossref Search ADS PubMed WorldCat 22 Laskowski MJ Williams ME Nusbaum HC Sussex IM Formation of lateral root meristems is a two-stage process. Development 121 1995 3303 3310 Google Scholar Crossref Search ADS PubMed WorldCat 23 Lee I Wolfe DS Nilsson O Weigel D A LEAFY co-regulator encoded by UNUSUAL FLORAL ORGANS. Curr Biol 7 1997 95 104 Google Scholar Crossref Search ADS PubMed WorldCat 24 Li H Shen J-J Zheng Z-L Lin Y Yang Z The Rop GTPase switch controls multiple developmental processes in Arabidopsis. Plant Physiol 126 2001 670 684 Google Scholar Crossref Search ADS PubMed WorldCat 25 Liu Y-G Mitsukawa N Oosumi T Whittier RF Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8 1995 457 463 Google Scholar Crossref Search ADS PubMed WorldCat 26 Long JA Moan EI Medford JI Barton MK A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379 1996 66 69 Google Scholar Crossref Search ADS PubMed WorldCat 27 Lupas A Van Dyke M Stock J Predicting coiled coils from protein sequence. Science 252 1991 1162 1164 Google Scholar Crossref Search ADS PubMed WorldCat 28 Martienssen RA Barkan A Freeling M Taylor WC Molecular cloning of a maize gene involved in photosynthetic membrane organization that is regulated by Robertson's Mutator. EMBO J 8 1989 1633 1639 Google Scholar Crossref Search ADS PubMed WorldCat 29 Nishimura A Tamaoki M Sato Y Matsuoka M The expression of tobacco knotted1-type class 1 homeobox genes correspond to regions predicted by the cytohistological zonation model. Plant J 18 1999 337 347 Google Scholar Crossref Search ADS PubMed WorldCat 30 Odell JT Nagy F Chua N-H Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313 1985 810 812 Google Scholar Crossref Search ADS PubMed WorldCat 31 Ori N Eshed Y Chuck G Bowman JL Hake S Mechanisms that control knox gene expression in the Arabidopsis shoot. Development 127 2000 5523 5532 Google Scholar Crossref Search ADS PubMed WorldCat 32 Schoof H Lenhard M Haecker A Mayer KFX Jürgens G Laux T The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100 2000 635 644 Google Scholar Crossref Search ADS PubMed WorldCat 33 Sentoku N Sato Y Kurata N Ito Y Kitano H Matsuoka M Regional expression of the rice KN1-type homeobox gene family during embryo, shoot, and flower development. Plant Cell 11 1999 1651 1663 Google Scholar Crossref Search ADS PubMed WorldCat 34 Souer E van Houwelingen A Kloos D Mol J Koes R The No Apical Meristem gene of petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell 85 1996 159 170 Google Scholar Crossref Search ADS PubMed WorldCat 35 Springer PS Holding DR Groover A Yordan C Martienssen RA The essential Mcm7 protein PROLIFERA is localized to the nucleus of dividing cells during the G1 phase and is required maternally for early Arabidopsis development. Development 127 2000 1815 1822 Google Scholar Crossref Search ADS PubMed WorldCat 36 Springer PS McCombie WR Sundaresan V Martienssen RA Gene trap tagging of PROLIFERA, an essential MCM2–3-5-like gene in Arabidopsis. Science 268 1995 877 880 Google Scholar Crossref Search ADS PubMed WorldCat 37 Steeves TA Sussex IM Patterns in Plant Development. 1989 Cambridge University Press Cambridge, UK 38 Sundaresan V Springer P Volpe T Haward S Jones JDG Dean C Ma H Martienssen R Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev 9 1995 1797 1810 Google Scholar Crossref Search ADS PubMed WorldCat 39 Takada S Hibara K-I Ishida T Tasaka M The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation. Development 128 2001 1127 1135 Google Scholar Crossref Search ADS PubMed WorldCat 40 Takatsuji H Zinc-finger transcription factors in plants. Cell Mol Life Sci 54 1998 582 596 Google Scholar Crossref Search ADS PubMed WorldCat 41 The Arabidopsis Genome Initiative Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408 2000 796 815 Crossref Search ADS PubMed WorldCat 42 Timmermans MCP Hudson A Becraft PW Nelson T ROUGH SHEATH2: a Myb protein that represses knox homeobox genes in maize lateral organ primordia. Science 284 1999 151 153 Google Scholar Crossref Search ADS PubMed WorldCat 43 Tsiantis M Schneeberger R Golz JF Freeling M Langdale JA The maize rough sheath2 gene and leaf development programs in monocot and dicot plants. Science 284 1999 154 156 Google Scholar Crossref Search ADS PubMed WorldCat 44 Tsugeki R Kochieva EZ Fedoroff NV A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation. Plant J 10 1996 479 489 Google Scholar Crossref Search ADS PubMed WorldCat 45 Vollbrecht E Reiser L Hake S Shoot meristem size is dependent on inbred background and presence of the maize homeobox gene, knotted1. Development 127 2000 3161 3172 Google Scholar Crossref Search ADS PubMed WorldCat 46 Waites R Selvadurai HRN Oliver IR Hudson A The PHANTASTICA gene encodes a MYB transcription factor involved in growth and dorsoventrality of lateral organs in Antirrhinum. Cell 93 1998 779 789 Google Scholar Crossref Search ADS PubMed WorldCat 47 Weigel D Alvarez J Smyth DR Yanofsky MF Meyerowitz EM LEAFY controls floral meristem identity in Arabidopsis. Cell 69 1992 843 859 Google Scholar Crossref Search ADS PubMed WorldCat 48 White JA Todd J Newman T Focks N Girke T Martı́nez de Ilárduya O Jaworski JG Ohlrogge JB Benning C A new set of Arabidopsis expressed sequence tags from developing seeds: the metabolic pathway from carbohydrates to seed oil. Plant Physiol 124 2000 1582 1594 Google Scholar Crossref Search ADS PubMed WorldCat 49 Zondlo SC Irish VF CYP78A5 encodes a cytochrome P450 that marks the shoot apical meristem boundary in Arabidopsis. Plant J 19 1999 259 268 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the National Science Foundation (grant no. IBN–9875371 to P.S.). 2 Present address: Departamento de Ingenierı́a Genética, CINVESTAV Unidad Irapuato, Irapuato, Gto. CP 36500, Mexico. * Corresponding author; e-mail patricia.springer@ucr.edu; fax 909–787–4437. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010926. Copyright © 2002 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - The Lateral Organ Boundaries Gene Defines a Novel, Plant-Specific Gene Family
JF - Plant Physiology
DO - 10.1104/pp.010926
DA - 2002-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-lateral-organ-boundaries-gene-defines-a-novel-plant-specific-gene-VLfooEToR6
SP - 747
EP - 761
VL - 129
IS - 2
DP - DeepDyve
ER -