Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade

Abstract

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.

Figure 1

Position of the 11 Arabidopsis LRX genes on the five Arabidopsis chromosomes. The large chromosomal duplications harboring the LRX genes are represented by shaded boxes with identical pattern, and paralogs are linked by arrows. AtLRX5, 6, and 7 are single genes, although they are located on duplicated segments. Centromeres are indicated by constrictions in the chromosome schematic representation. The chromosomal duplications are deduced from Blanc et al. (2000) and Vision et al. (2000). Gene positions on the chromosomes are approximated according to the mapping of the BAC clones available at The Arabidopsis Information Research Web site (http://www.arabidopsis.org).

Figure 2

Domain organization of the LRX proteins. A, Schematic representation of the conserved domain organization of the LRX proteins. The different domains are drawn to scale and their identity is indicated (SP, predicted signal peptide; and α, position of an α-helix). The N-terminal domain contains two predicted α-helices (gray oblique stripes) that flank a hypervariable region (light-gray box). The region of the N-terminal domain beginning with the second predicted α-helix is strongly conserved (stippled box). The C terminus (black oblique stripes) is conserved between several of the LRX proteins. B, Consensus amino acid sequence of each conserved domain. Capital letters, Residues that are conserved in at least 80% of the Arabidopsis, rice, maize, and tomato LRX genes; x, Non-conserved residues; and a, Any aliphatic amino acids. The 10 LRRs are aligned to the plant extracellular LRR consensus sequence, and positions matching the consensus are indicated in bold letters. The predicted β-strand/β-turn structural motif is framed. Lower script numbers indicate the number of non-conserved residues between two consensus amino acids. C, N-terminal domain sequences of the LRX proteins. The α-helix regions and the hypervariable regions are indicated at the top. Conserved residues are in bold, and regions of particular homology are framed. D, C terminus of the LRX proteins. Conserved residues are indicated in bold, and sequences sharing a same consensus are grouped together. The consensus motifs are shown on the right of the sequences. Capital letters, Residues conserved in 50% or more of the sequences; minuscule letters, residues common in less than 50% of the sequences.

Figure 3

Northern-blot analysis of the Arabidopsis LRX transcript levels. Total RNA was extracted from roots (Rt), young developing leaves and cotyledons (yL), rosette mature leaves (rL), cauline leaves (cL), floral stems (St), flower buds and opened flowers (Fl), stamens (S), carpels (Ca), pollen (Po), and pollinated carpels (PCa). Roots and young developing leaves were harvested from 14-d-old Columbia seedlings grown vertically on solidified Murashige and Skoog medium. All the other material was harvested from 35- to 40-d-old Columbia plants. Carpels were harvested from unopened flowers with young stamens to reduce pollen contamination. For northern analysis, 5 μg (2 μg for pollen) of total RNA was hybridized with ³²P-labeled gene-specific probes amplified by PCR from genomic DNA. Ribosomal RNAs were used as loading control (lower). No signal was obtained with the LRX7 probe and the result is not shown. AtLRX2 data will be presented elsewhere (N. Baumberger, M. Steiner, U. Ryser, B. Keller, and C. Ringli, unpublished data).

Figure 4

Histochemical localization of LRX expression in LRX promoter::uidA transgenic plants. A and B, pPEX3::uidA expression. C, pLRX6::uidA expression. D through F, pLRX5::uidA expression. G through I, pLRX4::uidA expression. A, Open flower. Mature pollen grains show a strong expression of pPEX3::uidA. B, Pollen germinating on a stigma. C, Emerging lateral roots, showing strong expression of pLRX6::uidA in the lateral root meristem, as well as in the vascular tissue of the mature primary root. D, Root of a 7-d-old seedling; GUS activity is restricted to the emerging lateral roots. E, Fourteen-day-old seedlings; pLRX5::uidA is expressed in the leaf primordia and stipules (inset image) at the basis of the rosette leaves. A moderate activity persisted during further expansion of the leaves, preferentially in petioles. F, Opened flowers and flower buds. GUS activity is detected in the carpels and faintly in the pedicels. Carpel expression seems higher in the style directly under the stigma and at the base of the organ and the junction with the receptacle (abscission zone). G, Primary mature roots; the vascular bundle is strongly stained. H, Fourteen-day-old seedlings; the inset picture shows a leaf stained for a shorter time to reveal the stronger expression in vascular tissue. I, Open flower; GUS activity is detected in sepals, mostly in the veins. Bars = 1 mm (A, F, and I), 50 μm (B–D), 2 mm (E and H), 350 μm (E, inset image), and 200 μm (G).

Figure 5

Phylogenetic analysis of the LRX family. The strict consensus of the two most parsimonious amino acid based phylogenetic trees is reproduced, and the jackknife support values mapped above the branches. The number of steps was 1,721, the ensemble consistency index of the most parsimonious trees was 0.65 (excluding uninformative characters), and the ensemble retention index was 0.64. Monocot and dicot LRX proteins cluster in a vegetatively expressed LRX and a reproductive-expressed LRX clade.