Analyses of phylogeny, evolution, conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors

Abstract

Background and aims: The cell cycle is controlled by cyclin-dependent kinases (CDKs), and CDK inhibitors are major regulators of their activities. The ICK/KRP family of CDK inhibitors has been reported in several plants, with seven members in arabidopsis; however, the phylogenetic relationship among members in different species is unknown. Also, there is a need to understand how these genes and proteins are regulated. Furthermore, little information is available on the functional differences among ICK/KRP family members.

Methods: We searched publicly available databases and identified over 120 unique ICK/KRP protein sequences from more than 60 plant species. Phylogenetic analysis was performed using 101 full-length sequences from 40 species and intron-exon organization of ICK/KRP genes in model species. Conserved sequences and motifs were analysed using ICK/KRP protein sequences from arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and poplar (Populus trichocarpa). In addition, gene expression was examined using microarray data from arabidopsis, rice and poplar, and further analysed by RT-PCR for arabidopsis.

Key results and conclusions: Phylogenetic analysis showed that plant ICK/KRP proteins can be grouped into three major classes. Whereas the C-class contains sequences from dicotyledons, monocotyledons and gymnosperms, the A- and B-classes contain only sequences from dicotyledons or monocotyledons, respectively, suggesting that the A- and B-classes might have evolved from the C-class. This classification is also supported by exon-intron organization. Genes in the A- and B- classes have four exons, whereas genes in the C-class have only three exons. Analysis of sequences from arabidopsis, rice and poplar identified conserved sequence motifs, some of which had not been described previously, and putative functional sites. The presence of conserved motifs in different family members is consistent with the classification. In addition, gene expression analysis showed preferential expression of ICK/KRP genes in certain tissues. A model has been proposed for the evolution of this gene family in plants.

Fig. 1.

Phylogenetic tree of the plant ICK/KRP family. The tree was generated by aligning 93 ICK/KRP amino acid sequences by neighbour-joining distance, using clustalw and mega 3·1. ICK/KRPs from arabidopsis, poplar and rice are highlighted (bold). A sub-group of atypical ICK/KRPs (about 90 amino acid residues) is indicated by the shaded box. Classes are indicated to the right. The bar represents 0·1 amino acid substitutions per site in the primary structure. See Supplementary Data Table S2 (available online) for abbreviations.

Fig. 2.

Exon–intron organization of the ICK/KRP genes from arabidopsis, rice and poplar. Exons and introns are represented by boxes and black lines, respectively. White boxes, when full-length cDNA sequences are known, correspond to untranslated regions. The genes are drawn to scale and are aligned by the most conserved exon in all genes (indicated by an arrowhead). Horizontal arrows indicate start codons. Interconnected grey lines correspond to related regions in exons. Sequences are organized according to the phylogenetic classes.

Fig. 3.

Genome localization of ICK/KRP genes in duplicated blocks. Chromosomal distribution of ICK/KRP genes in: (A) arabidopsis, (B) rice and (C) poplar. Chromosomes are presented as vertical bars and identified using roman numerals. In poplar, one unassembled scaffold, Sca130, contains the Poptr;ICK4 gene. Duplicated blocks related to ICK/KRPs are linked by black lines. A scale bar is included in each genome.

Fig. 4.

Putative functional and conserved motifs in plant ICK/KRP proteins. (A) Putative functional motifs are represented by symbols, and conserved motifs are indicated in boxes (with explanations given). Sequences are organized according to the phylogenetic relationship. (B) Sequences of conserved motifs in ICK/KRP proteins of arabidopsis, rice and poplar. Amino acid residues with at least 40 % identity are shaded.

Fig. 4.

Putative functional and conserved motifs in plant ICK/KRP proteins. (A) Putative functional motifs are represented by symbols, and conserved motifs are indicated in boxes (with explanations given). Sequences are organized according to the phylogenetic relationship. (B) Sequences of conserved motifs in ICK/KRP proteins of arabidopsis, rice and poplar. Amino acid residues with at least 40 % identity are shaded.

Fig. 5.

Expression of arabidopsis ICK/KRP genes. (A) ATH Affymetrix expression average values of six arabidopsis ICK/KRP genes from 16 tissues. Codes are: R, roots; S, seedling; L, leaf; Ls, senescing leaf; A, shoot apex; St, stem; Fi, shoot apices after plants were induced to flower (see Schmid et al., 2003); F, flower; Sp, sepal; Pe, petal; Sa, stamen; Ca, carpel; P, pollen; Si, silique; Sd, developing seed in silique; Sg, germinating seed. Bottom bar indicates expression level in mean-normalized values from null (light blue) to high (dark red). (B) RT-PCR expression of the seven arabidopsis ICK/KRP genes in different tissues. Codes are: C, cell suspension; R, root from 13-d seedlings; S, shoot from 13-d seedlings; 3L, leaf from 3-week plants; 5L, leaf from 5-week plants; Ls, senescing leaf from 5-week plants; St, stem from 5-week plants; Pe, petal; F, flower from 5-week plants; 6h and 2d, seeds after 6 h and 2 d of stratification, respectively. Expression of the At4g33380 gene (Czechowski et al., 2005) was used as input control. Pictures were taken under identical conditions (acquisition time and loading volumes) using a UV transilluminator (BioDoc-It System, UVP, www.uvp.com).

Fig. 6.

Expression of rice and poplar ICK/KRP genes. GeneChip expression values for rice and poplar ICK/KRP genes in different tissues and at different developmental stages are used. (A) Rice. Codes are: Sd, seedling; R, root; L, young leaf; ML, mature leaf; Sa, shoot apex; inflorescence stages P1–P6 (P1, up to 3 cm; P2, 3–5 cm; P3, 5–10 cm; P4, 10–15 cm; P5, 15–22 cm; P6, 22–30 cm); seed stages S1–S5 [S1, 0–2 d after pollination (dap); S2, 3–4 dap; S3, 5–10 dap; S4, 11–20 dap; S5, 21–29 dap]. (B) Poplar. Codes are: R, root; LPI1, leaf plastochron index 1; LPI2, leaf plastochron index 2; LPI5, leaf plastochron index 5; St, stem; IN, internode; N, node. The bar at the bottom of each heat map indicates the absolute expression values from null (light blue) to high (dark red).

Fig. 7.

A proposed model for the evolution of the ICK/KRP family. Based on sequence similarity and exon–intron organization, ICK/KRP genes are grouped into three major classes. While genes in the C-class have three exons, genes in the A- and B-classes have four exons. The C-class contains members from dicotyledons, monocotyledons and gymnosperms, suggesting a common ancestral class before the emergence of angiosperm plants. The A- and B- classes have only members from dicotyledons and monocotyledons, respectively, suggesting their split before the speciation of these two groups of plants. The number of family members increased through more recent segmental duplications in the plant genomes. Circles describe dated duplication events according to the literature. Potential origins for the Os01g37740 are marked with dotted lines. See text for more details.