The lateral organ boundaries gene defines a novel, plant-specific gene family

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 that LOB is functionally redundant or required during growth under specific environmental conditions. Ectopic expression of LOB 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.

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.

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.

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).

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.

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.

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.