A genome-wide compilation of the two-component systems in Lotus japonicus

Abstract

The two-component systems (TCS), or histidine-to-aspartate phosphorelays, are evolutionarily conserved common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes including plants. Among higher plants, legumes including Lotus japonicus have a unique ability to engage in beneficial symbiosis with nitrogen-fixing bacteria. We previously presented a genome-wide compiled list of TCS-associated components of Mesorhizobium loti, which is a symbiont specific to L. japonicus (Hagiwara et al. 2004, DNA Res., 11, 57-65). To gain both general and specific insights into TCS of this currently attractive model legume, here we compiled TCS-associated components as many as possible from a genome-wide viewpoint by taking advantage that the efforts of whole genome sequencing of L. japonicus are almost at final stage. In the current database (http://www.kazusa.or.jp/lotus/index.html), it was found that L. japonicus has, at least, 14 genes each encoding a histidine kinase, 7 histidine-containing phosphotransmitter-related genes, 7 type-A response regulator (RR)-related genes, 11 type-B RR-related genes, and also 5 circadian clock-associated pseudo-RR genes. These results suggested that most of the L. japonicus TCS-associated genes have already been uncovered in this genome-wide analysis, if not all. Here, characteristics of these TCS-associated components of L. japonicus were inspected, one by one, in comparison with those of Arabidopsis thaliana. In addition, some critical experiments were also done to gain further insights into the functions of L. japonicus TCS-associated genes with special reference to cytokinin-mediated signal transduction and circadian clock.

Figure 1

A schematic representation of the structural features of plant TCS-associated signal transduction components. HKs (e.g. cytokinin receptors) consists of three domains; N-terminal cytokinin-binding domain, central HK domain containing an invariant phospho-accepting histidine (H) residue, and C-terminal receiver domain containing an invariant aspartate (D) residue, which is capable of accepting a phosphoric group from a phospho-histidine. The phosphoryl group on the receiver domain is transferred to a histidine containing phospho-transmitter (HPt or HP), which subsequently serves as a phospho-donor toward RR. Other details are given in the text.

Figure 2

A phylogenetic tree of the HK family in some representative plants. Amino acid sequences of A. thaliana HKs were characterized previously, whereas those of L. japonicus were done in this study. In addition to these, a set of putative HK amino acid sequences was analyzed by adopting the databases of some other plants; a grape tree (Vitis vinifera, Vvi; http://www.genoscope.cns.fr/spip/Vitis-vinifera-e.html), a popular (Populus truchocarpa, Ptr; http://genome.jgi-psf.org/Poptr1/Poptr1.home.html), and a rice (Oryza sativa, Osa; http://rapdb.dna.affrc.go.jp/). Using these inferred amino acid sequences, a neighbor-joining phylogenetic tree was constructed by the program of ClustalW. The HKs of A. thaliana and L. japonicus were highlighted by red and blue colors, respectively. Ethylene receptor HKs were not integrated extensively for clarity of this tree. Positions of AtHK1 orthologs of L. japonicus were not certain because their inferred entire amino acid sequences are ambiguous (named HK1a and HK1b, Table 1).

Figure 3

Cytokinin responses of L. japonicus. Cytokinin-induced inhibition of root elongation of L. japonicus seedlings. Seedlings were grown on vertically oriented MS agar-plates containing cytokinin (BA, 6-benzylaminopurine) at varied concentrations, as indicated. After being incubated for 9 days, the average lengths of their primary roots (n ≥ 20) were measured. Photographs were taken for each representative (upper panel), and the resulting lengths of root were plotted against the cytokinin concentrations tested. As a reference, essentially the same experiments were carried out with A. thaliana seedlings.

Figure 4

Quantitative confirmation of cytokinin-induced expression of type-A RR genes in L. japonicus seedlings. (A) Cytokinin-induced expression of type-A RR genes in L. japonicus seedlings. Seedlings were grown MS gellan gum-plates under constant light conditions for 17 days, and then these seedlings were sprayed with cytokinin (20 µM t-zeatin in 0.02% DMSO). The seedlings were harvested immediately before and 0.5, 1.0, 3.0 h after the treatment and subjected to RNA preparation. A set of indicated RR transcripts were analyzed by means of semi-quantitative RT–PCR to measure the amounts of each transcript. The UBC transcript encoding ubiquitin-conjugating enzyme (chr1.LjT04O06.40, a homologue of Arabidopsis At5g25760) was used as an internal control, the type-B RRb4 transcript was a negative control, and the CKX3 encoding cytokinin oxidase enzyme (LjT02N03.150, a homologue of Arabidopsis CKX4) was a positive control. The results of reference samples treated with 0.02% DMSO for 1.0 h were also presented at the most right-hand side. The primer set, used for these PCR analyses, was listed in Supplementary Table S2. (B) The essentially the same experiments were repeated in a more quantitative manner by means of real-time quantitative RT–PCR. The experiments were replicated three times to obtain mean values with SD.

Figure 5

A phylogenetic tree of the PRR family in some representative plants. Amino acid sequences of A. thaliana PRRs were characterized previously, whereas those of L. japonicus were done in this study. In addition to these, a set of putative PRR amino acid sequences were analyzed by employing the databases for some other plants, as noted in Fig. 2. In addition, we included a set of putative PRR sequences from Physcomitrella patens (Ppa, a moss) (http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.html). Other details were essentially the same as those in Fig. 2.

Figure 6

Characterization of free-running circadian rhythms of putative clock-associated genes of L. japonicus. (A) A schematic representation of the currently consistent model of central oscillator. This proposed model for A. thaliana means that the morning clock genes CCA1 and LHY act repressors for the evening clock genes TOC1 and LUX, in turn, these evening genes somehow activate the expression of CCA1/LHY. In addition, PRR9, PRR7, and PRR5 are the daytime clock genes, which are essential for the clock function. On the basis of this well-established clock model for A. thaliana, a set of putative clock-associated genes of L. japonicus was analyzed with regard to their free-running expression profiles. For this purpose, L. japonicus plants were grown in the 12 h light/12 h dark cycles for 17 days, and then they were released into the continuous light. RNA samples were prepared at intervals (every 3 h), as schematically shown in the panels (the shaded duration corresponds to the final dark period). The resulting expression profiles were analyzed with regard to the putative LHY and TOC1 genes (B) and the putative LHY and LUX genes (C), respectively, by means of both real-time and semi-quantitative RT–PCR (upper and lower parts, respectively). The primer set, used for these PCR analyses, was presented in Supplementary Table S2.

Figure 7

Further characterization of free-running circadian rhythms of putative PRR clock-associated genes of L. japonicus. To extend the results of Fig. 6, other putative clock genes (PRR9, PRR7, and PRR5) were also analyzed with regard to their circadian rhythms. Other details were the same as those given in Fig. 6.