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Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput in vitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the in vitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation.

Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.

Gene targeting (GT) is a technique that alter the structure of the specific genes at their original loci in the genome by homologous recombination (HR). It plays an important role in functional genomics because it enables precise modification of the endogenous genes into desired forms such as knockout, knock-in, introduction of point mutations, as well as generation of fusion genes. Also, site-directed mutagenesis by GT can also be applied as an excellent technique for molecular breeding and gene therapy, because it can directly reflect the knowledge acquired from functional genomics. In this section, we introduce well-established GT procedure in rice in combination with positive-negative-selection (PNS) strategy.

Gene targeting (GT) is a technique that alter the structure of the specific genes at their original loci in the genome by homologous recombination (HR). It plays an important role in functional genomics because it enables precise modification of the endogenous genes into desired forms such as knockout, knock-in, introduction of point mutations, as well as generation of fusion genes. Also, site-directed mutagenesis by GT can also be applied as an excellent technique for molecular breeding and gene therapy, because it can directly reflect the knowledge acquired from functional genomics. In this section, we introduce well-established GT procedure in rice in combination with positive-negative-selection (PNS) strategy.

When cultivated rice seed fall into fields, they may overwinter and spontaneously germinate the next spring. Such germinated plants are termed “volunteer rice.” Volunteer grains originating from feed rice varieties may differ in certain traits, such as quality and taste, as compared with those of rice cultivated for human consumption, which may reduce the overall quality of the final harvested grain. Many rice varieties show resistance to benzobicyclon (BBC), a beta-triketone herbicide (bTH) that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Recently, the rice gene HIS1 (HPPD INHIBITOR SENSITIVE 1) conferring resistance to BBC and other bTHs was identified. In this study, to suppress the occurrence of volunteer rice infestation, we attempted to generate a BBC-sensitive rice strain via the knockout of the HIS1 gene using genome editing techniques. The production of a his1 knockout line was carried out by the start-codon substitution or stop-codon creation using CRISPR-Cas9 cytidine deaminase fusion, which is useful as a novel amino acid sequence is not generated due to the shifting of the reading frame. The mutation frequencies of independent transgenic plants were 3.6, 13.5, 13.8, and 21.2% at four gRNAs for start-codon substitution and three stop-codon creations. The his1 knockout lines were conferred with sensitivity to BBC, re-confirming by genome editing that this is indeed the gene responsible for BBC resistance/sensitivity. The his1 knockout lines also exhibited a sensitive phenotype to other bTHs, including sulcotrione, mesotrione, tembotrione, and tefuryltrione, compared with the wild-type variety ‘Nipponbare.' These results demonstrate the potential of herbicide-sensitive rice produced by genome editing technology as a material to control volunteer feed rice using pre-labeled herbicides for varieties consumed by humans.

Acetolactate synthase (ALS) catalyzes the initial step in the biosynthesis of branched-chain amino acids, and is highly conserved from bacteria to higher plants. ALS is encoded by a single copy gene in rice genome and is a target enzyme of several classes of herbicides. Although ALS mutations conferring herbicide-resistance property to plants are well documented, effect of Imazamox (IMZ) on rice and the mutations in ALS correlated with IMZ tolerance were unclear. In this article, the effect of IMZ on rice calli and seedlings in tissue culture conditions were evaluated. Also, the ALS A96V mutation was confirmed to improve IMZ tolerance of rice calli. Based on these results, ALS-assisted multiplex targeted base editing in rice was demonstrated in combination with Target-AID, a CRISPR/Cas9-cytidine deaminase fusion system [1], [2].

The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.

The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.

Targeted editing of single base pairs is achieved in monocot rice and dicot tomato using Target-AID (Cas9 activation-induced cytidine deaminase fusion). We applied a fusion of CRISPR-Cas9 and activation-induced cytidine deaminase (Target-AID) for point mutagenesis at genomic regions specified by single guide RNAs (sgRNAs) in two crop plants. In rice, we induced multiple herbicide-resistance point mutations by multiplexed editing using herbicide selection, while in tomato we generated marker-free plants with homozygous heritable DNA substitutions, demonstrating the feasibility of base editing for crop improvement.

The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus. Inheritance of the introduced mutations, as well as the removal of transgenes, are key issues that must be addressed in order to fully develop Target-AID as a plant breeding technique. Here we report the transmission of such mutations from the callus to regenerants and their progenies, leading to a generation of selectable marker-free (SMF) herbicide tolerant rice plants with simultaneous multiplex nucleotide substitutions. These findings demonstrate that Target-AID can be developed into novel plant breeding technology which enables improvement of multiplex traits at one time in combination with sophisticated targeted base editing with the simplicity and versatility of CRISPR/Cas9 system.

OsRac1 is a member of the plant small GTPase Rac/Rop family and plays a key role in rice immunity. The constitutively active (CA) G19V mutation of OsRac1 was previously shown to induce reactive oxygen species production, phytoalexin synthesis and defense gene activation, leading to resistance to rice blast infection. To study further the effect of the G19V mutation in disease resistance, we introduced a single base substitution by gene targeting and removed the selectable marker using Cre-loxP site-specific recombination. The CA-OsRac1 gene generated by gene targeting was termed CA-gOsRac1. The G19V mutation was transferred from a targeting vector to the OsRac1 locus and stably transmitted to the next generation. In the leaf blade of homozygous CA-gOsRac1 plants, mutant transcript levels were much lower than in those of wild-type plants. In contrast, mutant transcripts in roots, leaf sheaths and panicles were more abundant than those in leaf blades. However, upon chitin treatment, the expression of defense-related genes PAL1 and PBZ1 in the cell culture was greater in the mutants compared with wild-type plants. Furthermore, induction of hypersensitive response (HR)-like cell death was observed in the leaf sheaths of mutant plants infected with a compatible race of rice blast fungus. In the CA-gOsRac1 plants, a number of genes previously shown to be induced by Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo) infection were induced in the leaf sheath without pathogen infection. These results suggest that gene targeting will provide mutations useful for gene function studies and crop improvement.

INTRODUCTION To combat invading pathogens, cells develop an adaptive immune response by changing their own genetic information. In vertebrates, the generation of genetic variation (somatic hypermutation) is an essential process for diversification and affinity maturation of antibodies that function to detect and sequester various foreign biomolecules. The activation-induced cytidine deaminase (AID) carries out hypermutation by modifying deoxycytidine bases in the variable region of the immunoglobulin locus that produces antibody. AID-generated deoxyuridine in DNA is mutagenic as it can be miss-recognized as deoxythymine, resulting in C to T mutations. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) is a prokaryotic adaptive immune system that records and degrades invasive foreign DNA or RNA. The CRISPR/Cas system cleaves and incorporates foreign DNA/RNA segments into the genomic region called the CRISPR array. The CRISPR array is transcribed to produce crispr-RNA that serves as guide RNA (gRNA) for recognition of the complementary foreign DNA/RNA in a ribonucleoprotein complex with Cas proteins, which degrade the target. The CRISPR/Cas system has been repurposed as a powerful genome editing tool, because it can be programmed to cleave specific DNA sequence by providing custom gRNAs. RATIONALE Although the precise mechanism by which AID specifically mutates the immunoglobulin locus remains elusive, targeting of AID activity is facilitated by the formation of a single-stranded DNA region, such as a transcriptional RNA/DNA hybrid (R-loop). The CRISPR/Cas system can be engineered to be nuclease-inactive. The nuclease-inactive form is capable of unfolding the DNA double strand in a protospacer adjacent motif (PAM) sequence-dependent manner so that the gRNA binds to complementary target DNA strand and forms an R-loop. The nuclease-deficient CRISPR/Cas system may serve as a suitable DNA-targeting module for AID to catalyze site-specific mutagenesis. RESULTS To determine whether AID activity can be specifically targeted by the CRISPR/Cas system, we combined dCas9 (a nuclease-deficient mutant of Cas9) from Streptococcus pyogenes and an AID ortholog, PmCDA1 from sea lamprey, to form a synthetic complex (Target-AID) by either engineering a fusion between the two proteins or attaching a SH3 (Src 3 homology) domain to the C terminus of dCas9 and a SHL (SH3 interaction ligand) to the C terminus of PmCDA1. Both of these complexes performed highly efficient site-directed mutagenesis. The mutational spectrum was analyzed in yeast and demonstrated that point mutations were dominantly induced at cytosines within the range of three to five bases surrounding the –18 position upstream of the PAM sequence on the noncomplementary strand to gRNA. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced in the Target-AID complexes. Combination of PmCDA1 with the nickase Cas9(D10A) mutant, which retains cleavage activity for noncomplementary single-stranded DNA, was more efficient in yeast but also induced deletions as well as point mutations in mammalian cells. Addition of the uracil DNA glycosylase inhibitor protein, which blocks the initial step of the uracil base excision repair pathway, suppressed collateral deletions and further improved targeting efficiency. Potential off-target effects were assessed by whole-genome sequencing of yeast as well as deep sequencing of mammalian cells for regions that contain mismatched target sequences. These results showed that off-target effects were comparable to those of conventional CRISPR/Cas systems, with a reduced risk of indel formation. CONCLUSION By expanding the genome editing potential of the CRISPR/Cas9 system by deaminase-mediated hypermutation, Target-AID demonstrated a very narrow range of targeted nucleotide substitution without the use of template DNA. Nickase Cas9 and uracil DNA glycosylase inhibitor protein can be used to boost the targeting efficiency. The reduced cytotoxicity will be beneficial for use in cells that are sensitive to artificial nucleases. Use of other types of nucleotide-modifying enzymes and/or other CRISPR-related systems with different PAM requirements will expand our genome-editing repertoire and capacity.

Recent methylome analyses of the entire Arabidopsis thaliana genome using various mutants have provided detailed information about the DNA methylation pattern and its function. However, information about DNA methylation in other plants is limited, partly because of the lack of mutants. To study DNA methylation in rice (Oryza sativa) we applied homologous recombination-mediated gene targeting to generate targeted disruptants of OsDRM2, a rice orthologue of DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2), which encode DNA methyltransferases responsible for de novo and non-CG methylation in Arabidopsis. Whereas Arabidopsis drm1 drm2 double mutants showed no morphological alterations, targeted disruptants of rice OsDRM2 displayed pleiotropic developmental phenotypes in both vegetative and reproductive stages, including growth defects, semi-dwarfed stature, reductions in tiller number, delayed heading or no heading, abnormal panicle and spikelet morphology, and complete sterility. In these osdrm2 disruptants, a 13.9% decrease in 5-methylcytosine was observed by HPLC analysis. The CG and non-CG methylation levels were reduced in RIRE7/CRR1 retrotransposons, and in 5S rDNA repeats. Associated transcriptional activation was detected in RIRE7/CRR1. Furthermore, de novo methylation by an RNA-directed DNA methylation (RdDM) process involving transgene-derived exogenous small interfering RNA (siRNA) was deficient in osdrm2-disrupted cells. Impaired growth and abnormal DNA methylation of osdrm2 disruptants were restored by the complementation of wild-type OsDRM2 cDNA. Our results suggest that OsDRM2 is responsible for de novo, CG and non-CG methylation in rice genomic sequences, and that DNA methylation regulated by OsDRM2 is essential for proper rice development in both vegetative and reproductive stages.

Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice.

An endogenous 0.6-kb rice DNA transposon, nDart1-0, was found as an active nonautonomous element in a mutable virescent line, pyl-v, displaying leaf variegations. Here, we demonstrated that the active autonomous element aDart in pyl-v corresponds to Dart1-27 on chromosome 6 in Nipponbare, which carries no active aDart elements, and that aDart and Dart1-27 are identical in their sequences and chromosomal locations, indicating that Dart1-27 is epigenetically silenced in Nipponbare. The identification of aDart in pyl-v was first performed by map-based cloning and by detection of the accumulated transposase transcripts. Subsequently, various transposition activities of the cloned Dart1-27 element from Nipponbare were demonstrated in Arabidopsis. Dart1-27 in Arabidopsis was able to excise nDart1-0 and Dart1-27 from cloned sites, generating footprints, and to integrate into new sites, generating 8-bp target site duplications. In addition to Dart1-27, Nipponbare contains 37 putative autonomous Dart1 elements because their putative transposase genes carry no apparent nonsense or frameshift mutations. Of these, at least four elements were shown to become active aDart elements in transgenic Arabidopsis plants, even though considerable sequence divergence arose among their transposases. Thus, these four Dart1 elements and Dart1-27 in Nipponbare must be potential autonomous elements silenced epigenetically. The regulatory and evolutionary implications of the autonomous Dart1 elements and the development of an efficient transposon-tagging system in rice are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s00438-008-0410-x) contains supplementary material, which is available to authorized users.

Floral induction is a crucial developmental step in higher plants. Florigen, a mobile floral activator that is synthesized in the leaf and transported to the shoot apex, was recently identified as a protein encoded by FLOWERING LOCUS T (FT) and its orthologs; the rice florigen is Heading date 3a (Hd3a) protein. The 14-3-3 proteins mediate the interaction of Hd3a with the transcription factor OsFD1 to form a ternary structure called the florigen activation complex on the promoter of OsMADS15, a rice APETALA1 ortholog. However, crucial information, including the spatiotemporal overlap among FT-like proteins and the components of florigen activation complex and downstream genes, remains unclear. Here, we confirm that Hd3a coexists, in the same regions of the rice shoot apex, with the other components of the florigen activation complex and its transcriptional targets. Unexpectedly, however, RNA-sequencing analysis of shoot apex from wild-type and RNA-interference plants depleted of florigen activity revealed that 4,379 transposable elements (TEs; 58% of all classifiable rice TEs) were expressed collectively in the vegetative and reproductive shoot apex. Furthermore, in the reproductive shoot apex, 214 TEs were silenced by florigen. Our results suggest a link between floral induction and regulation of TEs.

Gene targeting (GT) refers to the designed modification of genomic sequence(s) through homologous recombination (HR). GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ) occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive-negative selection (PNS) is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice—a major crop worldwide—reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology (NPBT) based on PNS are discussed.

Recent methylome analyses of the entire Arabidopsis thaliana genome using various mutants have provided detailed information about the DNA methylation pattern and its function. However, information about DNA methylation in other plants is limited, partly because of the lack of mutants. To study DNA methylation in rice (Oryza sativa) we applied homologous recombination-mediated gene targeting to generate targeted disruptants of OsDRM2, a rice orthologue of DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2), which encode DNA methyltransferases responsible for de novo and non-CG methylation in Arabidopsis. Whereas Arabidopsis drm1 drm2 double mutants showed no morphological alterations, targeted disruptants of rice OsDRM2 displayed pleiotropic developmental phenotypes in both vegetative and reproductive stages, including growth defects, semi-dwarfed stature, reductions in tiller number, delayed heading or no heading, abnormal panicle and spikelet morphology, and complete sterility. In these osdrm2 disruptants, a 13.9% decrease in 5-methylcytosine was observed by HPLC analysis. The CG and non-CG methylation levels were reduced in RIRE7/CRR1 retrotransposons, and in 5S rDNA repeats. Associated transcriptional activation was detected in RIRE7/CRR1. Furthermore, de novo methylation by an RNA-directed DNA methylation (RdDM) process involving transgene-derived exogenous small interfering RNA (siRNA) was deficient in osdrm2-disrupted cells. Impaired growth and abnormal DNA methylation of osdrm2 disruptants were restored by the complementation of wild-type OsDRM2 cDNA. Our results suggest that OsDRM2 is responsible for de novo, CG and non-CG methylation in rice genomic sequences, and that DNA methylation regulated by OsDRM2 is essential for proper rice development in both vegetative and reproductive stages.

Rice is an important staple food for more than half the world’s population, and it has become the first crop plant to have its 389-Mb genome sequenced (IRGSP 2005). Even though various functional genomic tools for elucidating the function of rice genes are available (Hirochika et al. 2004; Leung and An 2004; Sasaki et al. 2005; Upadhyaya 2007), developing new methods for characterizing genes of interest by both forward and reverse genetic approaches has become particularly important. This chapter contains a description of the current state of gene targeting mediated by homologous recombination (HR) and gene tagging promoted by a nonautonomous DNA-based active rice transposon, nDart, for rice functional genomics, both of which are being developed (Terada et al. 2002, 2007; Tsugane et al. 2006; Takagi et al. 2007). Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome and, often, to

Two evolutionarily distant plant species, rice (Oryza sativa L.), a short-day (SD) plant, and Arabidopsis thaliana, a long-day plant, share a conserved genetic network controlling photoperiodic flowering. The orthologous floral regulators—rice Heading date 1 (Hd1) and Arabidopsis CONSTANS (CO)—integrate circadian clock and external light signals into mRNA expression of the FLOWERING LOCUS T (FT) group floral inducer. Here, we report that the rice Early heading date 1 (Ehd1) gene, which confers SD promotion of flowering in the absence of a functional allele of Hd1, encodes a B-type response regulator that might not have an ortholog in the Arabidopsis genome. Ehd1 mRNA was induced by 1-wk SD treatment, and Ehd1 may promote flowering by inducing FT-like gene expression only under SD conditions. Microarray analysis further revealed a few MADS box genes downstream of Ehd1. Our results indicate that a novel two-component signaling cascade is integrated into the conserved pathway in the photoperiodic control of flowering in rice.

We used an 8987-EST collection to construct a cDNA microarray system with various genomics information (full-length cDNA, expression profile, high accuracy genome sequence, phenotype, genetic map, and physical map) in rice. This array was used as a probe to hybridize target RNAs prepared from normally grown callus of rice and from callus treated for 6 hr or 3 days with the hormones abscisic acid (ABA) or gibberellin (GA). We identified 509 clones, including many clones that had never been annotated as ABA-or GA-responsive. These genes included not only ABA- or GA-responsive genes but also genes responsive to other physiological conditions such as pathogen infection, heat shock, and metal ion stress. Comparison of ABA- and GA-responsive genes revealed antagonistic regulation for these genes by both hormones except for one defense-related gene, thionin. The gene for thionin was up-regulated by both hormone treatments for 3 days. The upstream regions of all the genes that were regulated by both hormones had cis-elements for ABA and GA response. We performed a clustering analysis of genes regulated by both hormones and various expression profiles that showed three notable clusters (seed tissues, low temperature and sugar starvation, and thionin-gene related). A comparison of the cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response or for expression of amylase gene as Arabidopsis gene-specific or rice gene-specific, respectively.

We collected and completely sequenced 32,127 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. “Nipponbare.” Mapping of these clones to genomic DNA revealed ∼20,500 transcriptional units (TUs) in the rice genome. For each TU, we selected 60-mers using an algorithm that took into account some DNA conditions such as base composition and sequence complexity. Using in situ synthesis technology, we constructed oligonucleotide arrays with these TUs on glass slides. We targeted RNAs prepared from normally grown rice callus and from callus treated with abscisic acid (ABA) or gibberellin (GA). We identified 200 ABA-responsive and 301 GA-responsive genes, many of which had never before been annotated as ABA or GA responsive in other expression analysis. Comparison of these genes revealed antagonistic regulation of almost all by both hormones; these had previously been annotated as being responsible for protein storage and defense against pathogens. Comparison of the cis-elements of genes responsive to one or antagonistic to both hormones revealed that the antagonistic genes had cis-elements related to ABA and GA responses. The genes responsive to only one hormone were rich in cis-elements that supported ABA and GA responses. In a search for the phenotypes of mutants in which a retrotransposon was inserted in these hormone-responsive genes, we identified phenotypes related to seed formation or plant height, including sterility, vivipary, and dwarfism. In comparison of cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response as Arabidopsis specific and for protein storage as rice specific.

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide, PBZ) is a chemical inducer of resistance against plant pathogens and has long been practically used for protecting rice plants against a blast fungus, Magnaporthe grisea. We have been investigating the expression profiles of rice genes in response to PBZ using cDNA microarray technique. An array composed of 1265 rice expressed sequenced tag (EST) clones from various source including shoots, calli etc. was targeted by Cy5 labeled mRNAs from PBZ treated and untreated rice plants, respectively. As a result, ten cDNA clones exhibited greater than three-fold over-expression in the PBZ treated plants compared to untreated controls. Some of these clones have identities with disease resistance, pathogenesis-related and cell maintenance/development-related genes. The enhanced gene expression was confirmed for nine out of ten clones by using Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Several clones were further tested for Northern blot analysis using total RNA from blast fungus infected rice. Under the conditions used, three out of seven clones showed enhanced level of mRNA accumulation in infected plants. This may suggest that the enhanced expression of some genes by PBZ may contribute to the disease resistance in host plants. Furthermore cDNA library was constructed using mRNAs from leaves of PBZ treated rice plants and a microarray of the randomly selected 2880 clones from the library was used for choosing clones whose expression was changed in response to PBZ. Thirty nine clones were found to change their expression levels by a factor of 3 folds after PBZ treatment. Characterization including sequencing is now underway for these clones.

In order to understand the molecular mechanisms by which two types of phytohormones, brassinosteroids (BRs) and gibberellins (GAs), regulate plant growth and development, a cDNA microarray was made containing 4000 clones randomly selected from a rice cDNA library constructed from RNA isolated from seedlings treated with GA(3) and brassinolide (BL). The array was then probed to identify differences in expression using labelled cDNAs prepared from treated and control seedlings. Nine and 29 unique cDNA clones were up-regulated, while 32 and 42 unique cDNA clones were down-regulated by BL and GA(3), respectively. The predicted products of these BL- and GA-regulated genes fall into such functional categories as signal transduction, transcription, metabolism, cellular organization, and defense or anti-stress responses. Northern analysis of the selected BL- and GA(3)-regulated genes not only confirmed their BL or GA(3) dose dependent expression, but also revealed significant differences in the degree of expression in different organs and in response to other phytohormones. These results demonstrate that BR and GA influence growth and development by coordinately regulating the expression of specific groups of genes.

As a major provision for more than half of the world’s population, rice (Oryza sativa L.) is one of the most important plant species and has great economic importance. Also, rice has been focused on as an excellent model plant for cereal genomics studies due to its following features; 1) the smallest genome size (389 Mb) among the cereal grasses, 2) syntenic relation with other agronomically important Poaceae species such as maize, barley and wheat, 3) available varied resources including a large number of genetic markers, genomic libraries, many mutant lines and retrotransposon tagged lines, 4) there exists well developed technologies for rice genome manipulation such as Agrobacterium tumefaciens-mediated gene transformation and gene targeting with homologous recombination. Consequently, fine quality map-based genome sequencing of rice was completed in 2005. One of the most challenging goals for the plant-research community going forward is to identify the function and regulation of rice genes. Thus, both forward and reverse genetic approaches have been developed to elucidate these functions. However, because the tissue culture process is a necessary step in most of the currently available procedures used in rice genome research, somaclonal variations, which refer to genetic and epigenetic mutations induced by tissue culture, can hamper these approaches. Therefore, a lot of attention has been given to recently identified endogenous DNA transposons that are active under natural growth conditions, a character that is quite useful to development of more efficient rice transposon tagging as a functional genomics tool free from somaclonal variation. One such DNA transposon, nDart1-0 (non-autonomous DNA-based active rice transposon one-zero) in the hAT superfamily, had been identified as a causative element of spontaneous leaf variegation shown in the mutant line pyl-v (pale-yellow-leaf variegated). This mutable phenotype is caused by the disruption and restoration of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to the insertion and subsequent excision of nDart1-0. As a typical non-autonomous transposon in the hATsuperfamily, nDart1-0 can transpose only when the trans-acting transposase is supplied from an autonomous element, aDart (active autonomous Dart). On the other hand, an indicator line, pyl-stb (pyl-stable) shows uniform pale-yellow leaves with no nDart1-0 excision due to a lack of an aDart. The result of test crosses between pyl-v and pyl-stb lines indicated that the pyl-v line carries an aDart element in its genome. In the published genomic sequence of the cultivar Nipponbare, there are 38 candidate autonomous Dart elements that have putative transposase genes with no apparent nonsense or frameshift mutations. However, from the result of test crosses with the pyl -stb line, it was shown that Nipponbare carries no aDart elements in its genome. Meanwhile, the excision of some endogenous nDart1 elements in Nipponbare and pyl- stb was induced by treatment with a DNA methylation inhibitor, 5-azacytidine. Hence, these lines were predicted to carry epigenetically silenced autonomous elements, iDarts (inactive autonomous Darts). The first aims of this study were identifying the aDart element in the pyl-v line and demonstrating its molecular criteria as an autonomous element. To this end, I performed map-based cloning and revealed that the aDart element in the pyl-v line coincides with one of the 38 candidate autonomous elements, iDart1-27, residing on chromosome 6 in Nipponbare. Also, I have found that all of the examined transcripts of the Dart transposase gene were derived from Dart1-27in the pyl-v line. These results strongly suggested that Dart1-27in pyl-v acts on nDart1-0 as an active aDart element. Then, I demonstrated that iDart1-27 cloned from the Nipponbare genome can be converted to an active aDart element in Arabidopsis thaliana plants when its methylation status was eliminated during the cloning process; Dart1-27 excised nDart1-0 as well as itself from the introduced vectors and integrated into various sites of the A. thalianagenome. These results clearly indicated that Dart1-27 is a functional autonomous element, and it is active as an aDart element in the pyl-v line whereas epigenetically silenced as iDart1- 27 in Nipponbare. Furthermore, I showed other Dart elements, Dart1-1, Dart1-20, Dart1-28 and Dart1-52 are also functional autonomous elements, but they are epigenetically silenced as iDarts in Nipponbare. Next, in order to study if there are any regulatory mechanisms that control the activity of the Dart/nDart system in the pyl-stb line, I introduced Dart1-27 derivatives into the pyl-stb line and evaluated their activity. As a prerequisite for this transgenic approach, I carefully confirmed that during each step of the A. tumefaciens-mediated transformation process the endogenous iDart elements in the pyl-stb genome are almost never activated (0.1%). Based on this confirmation, I introduced Dart1-27 derivatives into pyl-stb and demonstrated that they can mobilize nDart1-0 elements from the OsClpP5 gene as well as from an introduced GUSPlus gene at a high frequency in transgenic pyl-stb plants. This result reconfirmed that Dart1-27 is a functional autonomous element able to act on nDart elements when its methylation status is eliminated, as shown in A. thalianaplants. From the results of phenotypic analysis of transgenic pyl-stb plants, it was suggested that there is a development-dependent regulation of Dartactivity in regenerated pyl-stb plants; most of the transgenic pyl-stb plants introduced with Dart1-27 derivatives were the pyl-stb phenotype at their 4-6 leaves stage, but almost all of them became the pyl-v phenotype at their 7-10 leaves stage. In this manuscript, I have unambiguously demonstrated that the active autonomous element in the pyl-v line is Dart1-27 on chromosome 6 and that the rice genome contains multiple potential autonomous Dart elements silenced epigenetically. From analysis of transgenic pyl-stb plants, I have also indicated a development-dependent regulation that could be a key to further elucidating Dart/nDart regulation mechanisms in the rice genome. I believe these results will facilitate an effective gene tagging system using the Dart/nDart elements in rice.