IC4R005-Metabolomics-2010-20085895

Project Title

Metabolomic Screening Applied to Rice FOX Arabidopsis Lines Leads to the Identification of a Gene-Changing Nitrogen Metabolis

The Background of This Project

• The construction of the rice full-length (FL) cDNA collection (Oryza sativa L. ssp. japonica ‘Nipponbare’) led to the develop- ment of the FOX hunting system (full-length cDNA overexpressor gene hunting system) (Ichikawa et al., 2006), which allows the ectopic expression of essentially any plant-cDNA library in even heterologous plant systems. Using this approach, a rice cDNA-expression library consisting of approximately 13 000 in- dependent FL cDNA clones was introduced into the dicot model plant Arabidopsis (Arabidopsis thaliana), creating a high-throughput screening system for novel rice genes (Kondou et al., 2009).
  

  Alignment of the LBD/AS2s of the LBD37/ASL39 protein sequences of Arabidopsis (At5g67420) and rice (Os03g0445700).
• Metabolomic data can connect the transcriptional and translational states of a cell to its physiological responses, which are manifested in the morphological and biochemical characteristics of the plant. In addition to applications in functional genomics (Fukushima et al., 2009; Joshi et al., 2006; Tohge et al., 2005), metabolite-profiling technology has been used for assessing the impact of environmental stimuli (Cook et al., 2004; Kaplan et al., 2004), in plant-breeding studies (Schauer et al., 2008), and for diagnostic analyses (Sauter et al., 1991). However, few reports describing the application of metabolomics in rice exist (Oikawa et al., 2008; Sato et al., 2004; Tarpley et al., 2005).
• In this project, the researchers used a novel approach combining metabolite-profiling techniques with the FOX-hunting system to perform functional genomic analysis of rice. Here, the researchers report the study of one exemplary rice FOX Arabidopsis line that overexpressed the FL cDNA of the rice LBD37/ASL39 (Os-LBD37/ASL39) gene. Integrated metabolomic and transcriptomic characterization of Os-LBD37/ASL39-overexpressing Arabi- dopsis and rice lines were described in this project.

Phylogenetic Analysis of Class II LOB/AS2-Protein Sequences from Arabidopsis and Rice.

Plant Materials

• The researchers used wt plants (Oryza sativa L. ssp. japonica ‘Nipponbare’), the control line FOX3, and transgenic lines transformed using the high-speed Agrobacterium-mediate transformation system (Toki et al., 2006). qRT–PCR analysis of the T0 generation of 19 independent transformant lines revealed high LBD37/ASL39 expression in the RK16331–4 and RK16331–13 transformant lines; therefore, these lines were selected for further analysis.
• The samples for the metabolite- and transcript-profiling experiments and the qRT–PCR experiments were prepared using the following procedure. The husks were separated from the seeds, and the brown rice seeds were sterilized by treating them with 70% ethanol for approximately 20 s. Subsequently, the seeds were stirred in 10 ml of sterilizing solution for 20 min by using an agitator (Rotator RT-50; Taitech Co., Ltd, Japan). After sterilization, the seeds were washed four times in 10 ml of distilled water by using the agitator. The sterilized seeds were inoculated on a solid half-strength MS medium containing 3% sucrose and 50 mg l 1 hygromycin (pH 5.8). After a 4-d incubation under dark conditions at room temperature, the plates were transferred to the growth chamber (MLR-350h/MLR-350 H; Sanyo, Japan).

Research Findings

• The FOX-hunting system in Arabidopsis can also be used to easily recover and sequence heterologous FL cDNAs conferring phenotypic variations or metabolite changes, as described in this study. Sequencing of the FL cDNA inserts from the overex- pressor lines K16331, and from K19624, which harbors the same rice FL cDNA fragment as K16331(accession no. AK071624), revealed that the rice cDNA shows high sequence similarity to the LOB/AS2-domain of the Arabidopsis LBD37/ ASL39 gene (Supplemental Figure 1).

• LBD37/ASL39 belongs to class II, and its function is unclear. However, previous investigations in Arabidopsis (Scheible et al., 2004; Wang et al., 2003) have demonstrated that the addition of nitrate to the liquid culture medium concom- itantly induces LBD37/ASL39 along with LBD38/ASL40 and LBD39/ASL41. Furthermore, LBD37/ASL39, LBD38/ASL40, and LBD39/ASL41 showed similar structures in Arabidopsis and rice (Figure 2), indicating a functional overlap between these three genes.

Labs working on this Project

• RIKEN Plant Science Center, Tsurumi-ku, Yokohama 230-0045, Japan
• National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan
• Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan

Corresponding Author

• Kazuki Saito (E-mail: ksaito@psc.riken.jp)