LATERAL ORGAN BOUNDARIES defines a new family of DNA-binding transcription factors and can interact with specific bHLH proteins

Abstract

Conserved in a variety of evolutionarily divergent plant species, LOB DOMAIN (LBD) genes define a large, plant-specific family of largely unknown function. LBD genes have been implicated in a variety of developmental processes in plants, although to date, relatively few members have been assigned functions. LBD proteins have previously been predicted to be transcription factors, however supporting evidence has only been circumstantial. To address the biochemical function of LBD proteins, we identified a 6-bp consensus motif recognized by a wide cross-section of LBD proteins, and showed that LATERAL ORGAN BOUNDARIES (LOB), the founding member of the family, is a transcriptional activator in yeast. Thus, the LBD genes encode a novel class of DNA-binding transcription factors. Post-translational regulation of transcription factors is often crucial for control of gene expression. In our study, we demonstrate that members of the basic helix-loop-helix (bHLH) family of transcription factors are capable of interacting with LOB. The expression patterns of bHLH048 and LOB overlap at lateral organ boundaries. Interestingly, the interaction of bHLH048 with LOB results in reduced affinity of LOB for the consensus DNA motif. Thus, our studies suggest that bHLH048 post-translationally regulates the function of LOB at lateral organ boundaries.

Figure 1.

Subcellular localization of LOB and results of selection and amplification binding (SAAB) assay. (A) Root tip of transgenic Arabidopsis expressing either 35S:GFP or 35S:GFP–LOB. The GFP–LOB translational fusion localized to the nucleus, whereas GFP alone was observed in both the cytoplasm and the nucleus. (B) Summary of the consensus sequence obtained from analysis of 24 unique oligonucleotides identified in a SAAB assay. The LOB domain of LOB recognized the hexamer GCGGCG, with some degeneracy permitted at the 5′ and 3′ nucleotides.

Figure 2.

LOB and other LBD proteins specifically bind the LBD motif. (A) DNA-binding assays show that the LD binds specifically to a probe containing the wild-type GCGGCG motif and not to a probe containing the mutated sequence GCAACG (lanes 3, 4 versus 6, 7). Incubation with antibody against the T7 epitope on the LD resulted in the appearance of a super-shifted band (lane 4). Binding was specific, as demonstrated by competition with increasing amounts of unlabeled wild-type (W) or mutant (M) DNA at 50-, 250- or 1250-fold excess over labeled probe (lanes 10–16). (B) AS2 and LBD4, two other LBD proteins, also bound specifically to the wild-type motif (lanes 3 versus 7; lanes 10 versus 14). A 500-fold excess of unlabeled wild-type (w) DNA competed this interaction while mutant (m) DNA did not (lanes 3 versus 5; lanes 10 versus 12).

Figure 3.

LOB binds the LBD motif as a dimer and is a transcriptional activator in yeast. (A) LD and LOB proteins were pre-incubated at ratios of 60 ng:0 ng, 45 ng:15 ng, 30 ng:30 ng, 15 ng:45 ng and 0 ng:60 ng (lanes 2–6, respectively), then added to a DNA-binding reaction containing wild-type probe. LD and LOB alone bound to the LBD motif but displayed differences in mobility in EMSAs. The presence of both proteins in the DNA-binding reaction resulted in the formation of a DNA–protein complex of intermediate mobility (lanes 3–5). Free probe (lane 1) was run off the gel to achieve maximal separation of the proteins. (B) LOB fused to the Gal4 DNA-binding domain (LOB–BD) and the empty vector (empty–BD) were transformed separately into yeast strain AH109 and plated on -Trp media to select for transformants. LOB–BD transformants showed growth when plated on 3 mM or 5 mM 3-AT -Trp -His selective plates (bottom row), while clones carrying the empty–BD did not (top row), demonstrating that LOB can function as a transcriptional activator in yeast.

Figure 4.

LOB interacts with members of the bHLH family of transcription factors. (A) Yeast two-hybrid assay: LOB fused to the Gal4 DNA-binding domain (LOB–BD) interacts with bHLH048 fused to the Gal4 activation domain (bHLH048–AD) to permit growth on minimal media lacking histidine and adenine, but does not interact with the activation domain alone (empty–AD). A yeast strain coexpressing AS2–BD and AS1–AD was included as a positive control, as AS1 and AS2 have been previously shown to interact (34,35). (B) Pull-down assay with different recombinant GST-tagged bHLH proteins from the bHLH048 subclade and full-length in vitro transcribed/translated ³⁵S-labeled LOB protein (upper panel). The strongest interactions were between LOB and bHLH048 or the closely related bHLH060. bHLH048 and bHLH060 also interact with the LD alone (lower panel). In vitro synthesized ³⁵S-labeled input protein is shown in the far right lanes.

Figure 5.

bHLH048 specifically reduces the binding affinity of the LD for the LBD motif. (A) Pre-incubation of 25, 50 or 100 ng of bHLH048 (lanes 3–5) with 25 ng of LD increasingly disrupted DNA binding. The LD–T7–antibody complex showed a similar reduction in DNA-binding affinity (lanes 6–9). (B) bHLH048 did not bind wild-type (w) or mutant (m) labeled probe (lanes 12, 13 and 17, 18). Pre-incubation with 50 ng of BP or BSA did not reduce the DNA-binding affinity of the LD (lanes 14 versus 15 and 16). LD–bHLH048 complexes did not recognize the mutant probe (lane 19).

Figure 6.

bHLH048 and LOB expression patterns are distinct, but share regions of overlap. (A, C and E) pLOB:GUS drives expression in the boundaries of all lateral organs throughout plant development, as previously described (1). (B, D and F) Transgenic plants expressing pbHLH048:GUS exhibited GUS activity in the vasculature of leaves and throughout the shoot apex (B), the vasculature of the primary root and incipient lateral roots (D), the base of floral organs (F) and in the stigma, style and ovules of developing flowers (F).