Functional divergence in the Arabidopsis LOB-domain gene family

Abstract

The Arabidopsis LOB-domain (LBD) gene family is composed by 43 members divided in two classes based on amino acid conservation within the LOB-domain. The LOB domain is known to be responsible for DNA binding and protein-protein interactions. There is very little functional information available for most genes in the LBD family and many lbd single mutants do not exhibit conspicuous phenotypes. One plausible explanation for the limited loss-of-function phenotypes observed in this family is that LBD genes exhibit significant functional redundancy. Here we discuss an example of one phylogenetic subgroup of the LBD family, in which genes that are closely related based on phylogeny exhibit distinctly different expression patterns and do not have overlapping functions. We discuss the challenges of using phylogenetic analyses to predict redundancy in gene families.

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes, also known as AS2-like (ASL), encode a plant-specific family of transcriptional regulators.^1,2 LBD/ASL proteins are characterized by the presence of a highly conserved N-terminal DNA-binding domain termed the LOB domain.^1,3 The LOB domain also mediates protein-protein interactions, likely through a predicted leucine-zipper/coiled-coil domain.^1,3 There are 43 LBD genes in Arabidopsis. Differences in conserved residues in the LOB domain divide this family in two classes. Whereas the LOB domain is highly conserved, little conservation is found in the C-termini of LBD proteins.^1,2,4 Phylogenetic analyses have therefore only been possible using alignment of the LOB domain region of the proteins.^2,5-7 Lack of conservation in the C-terminus of LBD proteins may complicate identification of orthologs in other plant species.

A number of Arabidopsis LBD genes have been molecularly and genetically characterized; however the function of LBD proteins remains poorly understood.^7-12 Few loss-of-function mutations in Arabidopsis LBD genes lead to conspicuous phenotypes and many of the available functional data are derived from gain-of-function experiments.^8,10,13-22 The limited phenotypes in loss-of-functions mutants suggest that redundancy plays a significant role in LBD function, at least in Arabidopsis. Interestingly, a number of LBD genes in maize and rice have been identified based on their loss-of-function phenotypes,^5,6,23-26 indicating that redundancy may be less significant in these species.

LATERAL ORGAN BOUNDARIES (LOB), the founding member of the LBD family, was identified through an enhancer trap screen due to its appealing expression pattern in the boundaries of lateral organs.¹lob mutants do not present a conspicuous vegetative phenotype. A closely related LBD gene, ASYMMETRIC LEAVES2 (AS2), functions to repress KNOX gene expression in the leaf.^13,27 Based on misexpression phenotypes and its expression pattern, AS2 has also been implicated in specification of adaxial cell fate in lateral organs.^2,14,28,29 Loss-of-function as2 mutations do not result in significant adaxial-abaxial polarity phenotypes however, suggesting that AS2 may function redundantly with other proteins to specify adaxial cell fate.^13,30 Since the lob and as2 mutants do not appear to reflect true loss-of-function phenotypes, we were interested in characterizing other closely related LBD genes. Phylogenetic analyses place LOB and AS2 in a subgroup that also includes LBD25/DDA1, LBD10 and LBD36.^2,5-7 The latter three genes were therefore considered candidates to have redundant functions with LOB and/or AS2. With the exception of AS2,¹³ mutations in genes in the LOB/AS2 subgroup result in either no observable phenotype (data not shown and ref. 16) or quite subtle phenotypes (Bell et al., unpublished results).

To test the possibility that LBD genes in the LOB/AS2 subgroup have functional overlap, we examined expression patterns and generated double and triple mutant combinations between all genes in the subgroup. No double or triple mutant combination resulted in additional phenotypes (data not shown), indicating that these genes are not likely to have overlapping functions. We did not observe enhancement of as2 in as2 lbd36 double mutants as previously reported,¹⁶ perhaps due to the presence of genetic modifiers in the mixed background used in that study. Furthermore, the examination of individual expression profiles demonstrated that the five genes within this subgroup are expressed in dissimilar fashion.³¹

To further examine their expression patterns, we generated promoter:GUS fusions for each of the five genes. Analysis of promoter:GUS lines revealed that the expression patterns were quite distinct. Expression of LOB is restricted to the lateral organ boundaries during vegetative and reproductive development as shown previously.¹ AS2 expression is restricted to the adaxial side of leaves and cotyledons and was also observed in root tips.^28,29 DDA1 is expressed in the vasculature throughout the plant, at the base of flowers and siliques, in the stigma, and in pollen grains.¹⁰ LBD36 is expressed in the vasculature of the leaf as well as in the trichomes. In the flower, its expression is regulated during development and in the silique, expression was observed in developing seeds. pLBD36:GUS expression was also observed in the branch points of the inflorescence.¹⁶ Expression of LBD36 is somewhat overlapping with DDA1 and LOB,^1,10,16 although double and triple mutant combinations with these genes did not present any obvious phenotypes (data not shown). Finally, expression of LBD10 was restricted to the pollen grains (Fig. 1).

Figure 1.LBD10 expression is restricted to pollen. Whole mount images of LBD10_pro:GUS transgenic Arabidopsis plants showing GUS activity in (A) the inflorescence; (B) a flower; (C) pollen grains and pollen tubes on the stigma and style. The LBD10 promoter region, from –1895 bp upstream of the ATG to +63, was cloned into the BamHI site of pCB308⁴³ to create an in-frame translational fusion of the first 21 amino acids of LBD10 to GUS. Images were captured using a stereo microscope (A,B) or compound microscope with Nomarski optics (C). Bars = 2 mm (A), 500 μm (B), 100 μm (C).

Phylogenetic analyses have been successfully used to dissect redundancy for several Arabidopsis gene families. There are many examples of genes with overlapping functions encoding highly similar proteins. For example, mutations in the YABBY,³² KANADI,³³ and HD-ZIPIII^34,35 families resulted in phenotypes only in double or triple mutant combinations. Similarly, the paralogous PENNYWISE and POUNDFOOLISH genes function redundantly to regulate internode patterning and the double mutant revealed a function in the vegetative to reproductive transition that was not apparent in the single mutants.³⁶ The CUP-SHAPED COTYLEDON³⁷ and LATERAL ORGAN FUSION³⁸ genes function redundantly to regulate lateral organ separation whereas the BLADE-ON-PETIOLE genes regulate growth and development of lateral organs³⁹ and the SEPALLATA MADS box genes function to specify floral organ identity.⁴⁰ Thus there are many examples where phylogenetic analyses have successfully been used to inform a search for functionally redundant components of a process.

In order for two genes to have overlapping function, they must be expressed in the same location. In the case of the LOB/AS2 subgroup of the Arabidopsis LBD family, which includes LOB, AS2, DDA1, LBD36 and LBD10, the expression pattern of each gene is quite distinct (Fig. One and refs. 1,10,16,29). Thus, for this subgroup of the family, the coding regions are conserved, whereas the regulatory regions are not. In a genome-wide study of the expression patterns of duplicated genes in Arabidopsis, divergence of expression patterns was commonly observed, suggesting that this is a common phenomenon.⁴¹ As transcriptional regulators, the location of LBD proteins is critical for function due to the presence of partners that will together with LBD proteins form transcriptional complexes that might regulate myriad developmental processes. The diversification of regulatory regions can lead to rapid sub-functionalization or neo-functionalization.⁴²

Additionally, domain swap experiments revealed that amino acid differences in the LOB domain are responsible for specific functions of these proteins, as the LOB domains of the four other members of LOB/AS2 subgroup were unable to complement the as2 phenotype.⁴ Thus, changes in both regulatory and coding sequences contribute to the evolution of new LBD gene functions.