Comparative analysis of the receptor-like kinase family in Arabidopsis and rice

Abstract

Receptor-like kinases (RLKs) belong to the large RLK/Pelle gene family, and it is known that the Arabidopsis thaliana genome contains >600 such members, which play important roles in plant growth, development, and defense responses. Surprisingly, we found that rice (Oryza sativa) has nearly twice as many RLK/Pelle members as Arabidopsis does, and it is not simply a consequence of a larger predicted gene number in rice. From the inferred phylogeny of all Arabidopsis and rice RLK/Pelle members, we estimated that the common ancestor of Arabidopsis and rice had >440 RLK/Pelles and that large-scale expansions of certain RLK/Pelle members and fusions of novel domains have occurred in both the Arabidopsis and rice lineages since their divergence. In addition, the extracellular domains have higher nonsynonymous substitution rates than the intracellular domains, consistent with the role of extracellular domains in sensing diverse signals. The lineage-specific expansions in Arabidopsis can be attributed to both tandem and large-scale duplications, whereas tandem duplication seems to be the major mechanism for recent expansions in rice. Interestingly, although the RLKs that are involved in development seem to have rarely been duplicated after the Arabidopsis-rice split, those that are involved in defense/disease resistance apparently have undergone many duplication events. These findings led us to hypothesize that most of the recent expansions of the RLK/Pelle family have involved defense/resistance-related genes.

Figure 1.

Comparison of Rice and Arabidopsis Protein Kinase Families. The bars indicate the sizes of kinase families from Arabidopsis (open) and rice (closed). The ratios of sizes of kinase families between these two plants (denoted At and Os) are shown at the right. No consistent size deviation can be seen for either organism. In the cases where the sizes or ratios are larger than the scale used, their values are shown. Asterisks indicate families with only rice members.

Figure 2.

The Kinase Domain Phylogeny and Domain Configurations of RLK/Pelle Members from Rice and Arabidopsis. (A) Phylogeny of RLK/Pelles from rice and Arabidopsis. The phylogeny was generated using kinase domain amino acid sequences with sequences from Arabidopsis and rice, which are color-coded orange and blue, respectively. Gray branches represent clusters of truncated sequences. The black circles mark the positions where the bifurcating edges were hypothesized to be because of divergence between monocot and dicot (Figure 3A). The subfamily designations are based on a published classification scheme (Shiu and Bleecker, 2003). The full phylogeny is shown in Supplement C online. (B) Representative domain organizations for RLK/Pelle subfamilies. The RLK/Pelle subfamilies are divided into receptor kinase (RLK) and cytoplasmic kinase (RLCK) categories. Several subfamilies have more than one domain organization shown, representing potential lineage-specific domain fusion events in these subfamilies. Orange and blue arrows represent Arabidopsis- and rice-specific domain organizations, respectively. Arrowheads indicate novel organizations mentioned in the text. The domain organizations of Arabidopsis and rice RLK/Pelles are shown in Supplement C online.

Figure 3.

Expansions of Different RLK Subfamilies Before and After the Arabidopsis–Rice Split. (A) Example phylogenies illustrating the rationale for inferring the presence of an ancestral RLK/Pelle. A tree with five taxa is shown at the left, where two genes are from Arabidopsis (At1a and At1b) and three are from rice (Os1, Os2a, and Os2b). According to the parsimony principle, a bifurcating clade with one branch leading to Arabidopsis gene(s) and the other lead to rice gene(s) indicates the presence of one ancestral RLK/Pelle gene, such as clade 1 shown in the tree at the right. Clade 2 is also regarded as an ancestral unit because it has sister group relationship to clade 1. The absence of an Arabidopsis gene in clade 2 is regarded as a gene-loss event (gray line, shaded gene name in the right panel). (B) Comparison between the numbers of ancestral RLK/Pelle genes (open bar) and those of extant species (closed bar) in different subfamilies. The number of ancestral genes is determined based on the rationale explained in (A). Differential expansion of different subfamilies after the Arabidopsis–rice split is readily detectable in multiple subfamilies, whereas the sizes of others remain relatively constant.

Figure 4.

Comparison of the Extents of Lineage-Specific Expansion in Arabidopsis and Rice. (A) The clade sizes indicate the numbers of duplications that occurred after the divergence between Arabidopsis and rice. The relative extent of expansion that occurred after this period is illustrated by comparing the number of Arabidopsis and rice genes in each clade inferred (with the scheme shown in Figure 3A) and are plotted on the x and y axes, respectively. The number of each clade size relationship is counted and plotted on the z axis. Most clades are of similar sizes in Arabidopsis and rice. However, some clades have expanded rather dramatically in a lineage-specific fashion, but very few show similar degrees of expansion in both lineages as the arrow indicates a clade from the LRR-I subfamily. (B) The contribution of lineage-specific expansions on the sizes of the RLK/Pelle family. Note the gradual separation between Arabidopsis (At) and rice (Os) cumulative total between cluster sizes of 5 and 20. The most significant contribution to the large RLK/Pelle family in rice, however, is because of the presence of lineage-specific expanded clades, accounting for ∼80% of the differences between Arabidopsis and rice.

Figure 5.

Large-Scale Duplication in the RLK/Pelle Family. (A) An example phylogeny showing the rationales for determining the relative ages of duplications. Only ancestral units with both an Arabidopsis branch (A clade) and a rice branch (O clade) were analyzed. The node that represents the species divergence is called basal node (black bar). Four nodes represent duplications occurred in the Arabidopsis lineage (A-I through A-IV, white bars), and two occurred in rice (O-I and O-II, gray bars). The Ks value for each node was calculated by taking the arithmetic mean of all left-right branch combinations. For example, Ks for node A-II was determined by the sum of Ks of sequence pairs A1-A3 and A2-A3 divided by the number of pairs. The node Ks was then divided by the basal Ks to obtain its relative age. (B) and (C) The frequency distribution of relative ages of Arabidopsis nodes. In (B), the relative ages of all nodes, regardless of their duplication mechanisms, were plotted. In (C), each tandem cluster was treated as one locus, and nodes were determined by excluding all but the first members in tandem clusters. (D) and (E) The frequency distribution of rice RLK/Pelle relative ages of all nodes or locus-based nodes, respectively.

Figure 6.

The Ka/Ks Ratios for Different Domains in RLKs. (A) A schematic representation of the domains in question. Kinase, kinase domain; signal, signal sequence; TM, transmembrane region. (B) to (D) The Ka/Ks frequency distribution for ECD, ICD, and kinase, respectively. The average Ka/Ks for each domain is indicated by an arrowhead. (E) The Ka of ECD plotted against that of ICD of the same protein. The line indicates a one-to-one relationship between Ka of these two domains. (F) and (G) The relationships between Ka/Ks and Ks of ECDs and ICDs. Regression lines with the highest correlation coefficients are shown. The Ka/Ks values decline sharply as Ks values get larger, a pattern that is more pronounced in ECDs. Because Ks is a proxy for time, this pattern suggests that, for newly duplicated RLKs, the ECDs in general evolved faster than the ICDs of the newly duplicated RLKs.

Figure 7.

Detection of Positively Selected Regions in RLKs. Four LRR RLK pairs differ widely in their Ka/Ks between ECDs and ICDs and are shown as examples. For each pair, the subfamily designation and the Ka/Ks values for ECD and ICD are indicated, followed by the graphical representation of domains and the Ka/Ks values calculated with sliding windows. The signal sequences, if present, are the black boxes at the beginning of each entry. The transmembrane regions are the internal black boxes. Gray boxes indicate motifs in the ECDs; they are LRRs in all examples. The kinase domains are black boxes. Dotted lines define the boundaries between ECDs, transmembrane regions, and ICDs. The window size was 30 amino acids, and the step size was 15. For each window, the Ka value was calculated and divided by the full length Ks value. This was done to reduce false positives because of relatively low Ks values in the windows. The line indicates the Ka/Ks value of 1.