SummaryEctopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis. This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.

Plant metabolomics developed as a powerful tool to examine gene functions and to gain deeper insight into the physiology of the plant cell. In this study, we screened Arabidopsis lines overexpressing rice full-length (FL) cDNAs (rice FOX Arabidopsis lines) using a gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS)-based technique to identify rice genes that caused metabolic changes. This screening system allows fast and reliable identification of candidate lines showing altered metabolite profiles. We performed metabolomic and transcriptomic analysis of a rice FOX Arabidopsis line that harbored the FL cDNA of the rice ortholog of the Lateral Organ Boundaries (LOB) Domain (LBD)/Asymmetric Leaves2-like (ASL) gene of Arabidopsis, At-LBD37/ASL39. The investigated rice FOX Arabidopsis line showed prominent changes in the levels of metabolites related to nitrogen metabolism. The transcriptomic data as well as the results from the metabolite analysis of the Arabidopsis At-LBD37/ASL39-overexpressor plants were consistent with these findings. Furthermore, the metabolomic and transcriptomic analysis of the Os-LBD37/ASL39-overexpressing rice plants indicated that Os-LBD37/ASL39 is associated with processes related to nitrogen metabolism in rice. Thus, the combination of a metabolomics-based screening method and a gain-of-function approach is useful for rapid characterization of novel genes in both Arabidopsis and rice.

Endoreduplication is a type of cell cycle in which DNA replication continues without cell division. We have isolated several dominant mutants from Arabidopsis thaliana activation tagging lines by flow cytometry. One of the mutants, increased level of polyploidy1-1D (ilp1-1D), showed increased polyploidy in both light-and dark-grown hypocotyls. The corresponding gene of ilp1-1D encodes a protein homologous to the C-terminal region of mammalian GC binding factor. We demonstrate that this protein functions as a transcriptional repressor in vivo. The expression of all members of the CYCLINA2 (CYCA2) family was reduced in an ILP1 overexpressing line, and the mouse (Mus musculus) homolog of ILP1 repressed cyclin A2 expression in mouse NIH3T3 cells. T-DNA insertion mutants of ILP1 showed reduced polyploidy and upregulated all CYCA2 expression. Furthermore, loss of CYCA2;1 expression induces an increase in polyploidy in Arabidopsis. We demonstrate that this protein regulates endoreduplication through control of CYCA2 expression in Arabidopsis.

These authors contributed equally to this study. SummaryPlant genomic resources harbouring gain-of-function mutations remain rare, even though this type of mutation is believed to be one of the most useful for elucidating the function of unknown genes that have redundant partners in the genome. An activation-tagging T-DNA was introduced into the genome of Arabidopsis creating 55 431 independent transformed lines. Of these T1 lines, 1262 showed phenotypes different from those of wild-type plants. We called these lines`AT1Ps' (activation T1 putants). The phenotypes observed include abnormalities in morphology, growth rate, plant colour,¯owering time and fertility. Similar phenotypes re-appeared either in dominant or semi-dominant fashion in 17% of 177 AT2P plants tested. Plasmid rescue or an adaptor-PCR method was used to identify 1172 independent genomic loci of T-DNA integration sites in the AT1P plants. Mapping of the integration sites revealed that the chromosomal distribution of these sites is similar to that observed in conventional T-DNA knock-out lines, except that the intragenic type of integration is slightly lower (27%) in the AT1P plants compared to that observed in other random knock-out populations (30±35%). Ten AT2P lines that showed dominant phenotypes were chosen to monitor expression levels of genes adjacent to the T-DNA integration sites by RT-PCR. Activation was observed in 7 out of 17 of the adjacent genes detected. Genes located up to 8.2 kb away from the enhancer sequence were activated. One of the seven activated genes was located close to the left-border sequence of the T-DNA, having an estimated distance of 5.7 kb from the enhancer. Surprisingly, one gene, the ®rst ATG of which is located 12 kb away from the enhancer, showed reduced mRNA accumulation in the tagged line. Application of the database generated to Arabidopsis functional genomics research is discussed. The sequence database of the 1172 loci from the AT1P plants is available (http://pfgweb.gsc.riken.go.jp/ index.html).