Evolutionary relationships and expression analysis of EUL domain proteins in rice (Oryza sativa)

Abstract

Background: Lectins, defined as 'Proteins that can recognize and bind specific carbohydrate structures', are widespread among all kingdoms of life and play an important role in various biological processes in the cell. Most plant lectins are involved in stress signaling and/or defense. The family of Euonymus-related lectins (EULs) represents a group of stress-related lectins composed of one or two EUL domains. The latter protein domain is unique in that it is ubiquitous in land plants, suggesting an important role for these proteins.

Results: Despite the availability of multiple completely sequenced rice genomes, little is known on the occurrence of lectins in rice. We identified 329 putative lectin genes in the genome of Oryza sativa subsp. japonica belonging to nine out of 12 plant lectin families. In this paper, an in-depth molecular characterization of the EUL family of rice was performed. In addition, analyses of the promoter sequences and investigation of the transcript levels for these EUL genes enabled retrieval of important information related to the function and stress responsiveness of these lectins. Finally, a comparative analysis between rice cultivars and several monocot and dicot species revealed a high degree of sequence conservation within the EUL domain as well as in the domain organization of these lectins.

Conclusions: The presence of EULs throughout the plant kingdom and the high degree of sequence conservation in the EUL domain suggest that these proteins serve an important function in the plant cell. Analysis of the promoter region of the rice EUL genes revealed a diversity of stress responsive elements. Furthermore analysis of the expression profiles of the EUL genes confirmed that they are differentially regulated in response to several types of stress. These data suggest a potential role for the EULs in plant stress signaling and defense.

Keywords: Carbohydrate; Domain architecture; EUL; Lectin; Phylogeny; Rice.

Fig. 1

Comparison of DNA sequences encoding EUL homologs from O. sativa. a. Distribution and duplication of EUL genes in the Oryza sativa subsp. japonica genome. Sequences for the different EULs were mapped on the chromosomes based on their start position and annotated with their locus number. Blocks containing EUL genes resulting from segmental duplication events are indicated in gray and connected by lines. Tandem duplications are indicated by an asterisk. The chromosome map was prepared using MapChart and drawn to scale. b. Intron-exon structure of the EUL genes. Shown in white are the 5′ and 3′ UTRs, exons are shown in purple. Introns are represented by grey bars between the exons. SNPs identified by SNP-seek are indicated in yellow.c. Distribution of SNP alleles in different rice varieties. The percentage of the Nipponbare reference allele in the different subvarieties is indicated by the heat map. The dendrogram is generated by DendroUPGMA (http://genomes.urv.cat/UPGMA/) (Garcia-Vallve et al. 1999)

Fig. 2

Sequence comparison of EUL homologs from O. sativa at protein level. a. Domain organization of the rice EULs. The EUL domains are represented by a purple bar. b. Sequence alignment of the different EUL domains. c. Phylogenetic relationship of EUL domains. A phylogenetic tree was generated by RaxML based on the sequence alignment shown in panel (b). Branch labels show bootstrapping values

Fig. 3

Ribbon diagrams of the β-trefoil lectin domains of O. sativa EULs. a-h. Ribbon diagrams of the separate EUL domains of OsEULS2 (a), OsEULS3 (b), OsEULD1A_1 (c), OsEULD1A_2 (d), OsEULD1B_1 (e), OsEULD1B_2 (f), OsEULD2_1 (g), and OsEULD2_2 (h). Beta-sheets, stretches of α-helices and loops are colored magenta, orange and green, respectively. The bundles of β-sheet are numbered I, II and III, respectively. The carbohydrate-binding site of the EUL domain is located at the bundle III. i-j. Ribbon structure of the two-β-trefoil domain OsEULD1A (i) and OsEULD1B (j), showing the respective arrangement of the two β-trefoil domains on both sides of the central linker region (L). The arrow indicates the carbohydrate-binding site in subdomain III. N and C correspond to the N- and C-terminal ends of the polypeptide chain of OsEULD1A and OsEULD1B, respectively

Fig. 4

Binding models of selected ligands in the carbohydrate-binding site of OsEULS2 and OsEULD1A. A-C. Side-view of the carbohydrate-binding site of OsEULS2 showing the docking of Man1,2Man (a), Man (b) or LacNAc (c) to the carbohydrate-binding site. d-i. Side-view of the carbohydrate-binding site of OsEULD1A showing the docking of Man1,2Man (d-e), Man (f-g) or LacNAc (h-i) to the carbohydrate-binding site of the first (d, f and h) or second (e, g and i) EUL domain. The H-bond distances are indicated (Å). A stacking interaction occurs between the first Man ring (a, d and e), Man (b, f and g) or Gal (c, h and i) and the aromatic residues F118 and W136 (colored orange) located in the vicinity of the active site

Fig. 5

Expression profiles of the rice EUL genes upon plant stress represented as fold change. The false discovery rate is represented with asterisks to denote statistical significance. One star (*) is used if the value is less than 0.05, two stars (**) represent values less than 0.05 but higher or equal to 0.001, and three stars (***) show a value below 0.001. a. Drought stress. b. Osmotic stress. c. ABA treatment. d. JA treatment

Fig. 6

Cis-regulatory elements in the EUL promoter sequences. Elements identified in the OsEULS2 (a), OsEULS3 (b) and OsEULD1B (c) promoter by the different analyses performed in the integrated approach: motifs in open chromatin, motifs in conserved sequences and motifs enriched in OsEUL regulons. The motifs enriched in the OsEUL regulons are annotated according to the associated transcription factor or motif name. Gray bars represent the 2.000 bp upstream from the ATG codon of the different OsEULs

Fig. 7

Sequence logo for the EUL domains of all species under study. Amino acid residues forming the active triad of the carbohydrate-binding site are indicated with an asterisk (*). Aromatic residues involved in stacking interactions with the sugars are indicated by a hash (#)

Fig. 8

Phylogenetic tree for all EUL domains retrieved from 9 monocot and 8 dicot species. EUL domains are annotated as S-type (red) or D-type EUL domains. The D-type domains are subdivided in domain 1 (N-terminal EUL domain) (blue) and domain 2 (C-terminal EUL domain) (green). Branch labels show bootstrapping values