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Citrus unshiu Marc. cv. Miyagawa-wase is the most widely cultivated citrus variety in Korea. To determine whether the C. unshiu genome used in this study shows genetic variation compared to the published reference genome (C. unshiu Marc. Miyagawa-wase), we conducted genome re-sequencing of two C. unshiu cultivars (Miyagawa-1 and Miyagawa-2) cultivated on Jeju in Korea. Compared with the reference genome, 1,198,650 and 1,207,084 single-nucleotide polymorphisms (SNPs) and 172,259 and 172,391 insertion/deletion polymorphisms (InDels) were detected in the Miyagawa-1 and -2 genomes, respectively. In SNP and InDel classifications by genome annotation, 367,591 and 369,068 SNPs and 45,362 and 45,464 InDels were located in the genic regions of Miyagawa-1 and -2, respectively. Among the SNPs of Miyagawa-1 and -2, transitions were more frequent than transversions. The majority of InDels was distributed in 1-bp InDels in both cultivars. The comparative number of total SNPs between Miyagawa-1 and -2 was smaller than the number of SNPs between the reference genome and Miyagawa-1 or -2. Gene ontology (GO) analysis showed that 23,164 and 23,049 genes with SNPs and 16,830 and 16,774 genes with InDels were annotated in the GO database. Taken together, Miyagawa-1 and -2 show genome-wide variation, including SNPs and InDels, compared to the published C. unshiu Marc. cv. Miyagawa-wase genome. This study suggests it would be more accurate to use the Miyagawa-1 and -2 genome sequences as a reference when conducting research using C. unshiu cultivated in Korea.

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.

A large part of the rice genome is composed of transposons. Since active excision/reintegration of these mobile elements may result in harmful genetic changes, many transposons are maintained in a genetically or epigenetically inactivated state. However, some non-autonomous DNA transposons of the nDart1-3 subgroup, including nDart1-0, actively transpose in specific rice lines, such as pyl-v which carries an active autonomous element, aDart1-27, on chromosome 6. Although nDart1-3 subgroup elements show considerable sequence identity, they display different excision frequencies. The most active element, nDart1-0, had a low cytosine methylation status. The aDart1-27 sequence showed conservation between pyl-stb (pyl-v derivative line) and Nipponbare, which both lack autonomous activity for transposition of nDart1-3 subgroup elements. In pyl-v plants, the promoter region of the aDart1-27 transposase gene was more hypomethylated than in other rice lines. Treatment with the methylation inhibitor 5-azacytidine (5-azaC) induced transposition of nDart1-3 subgroup elements in both pyl-stb and Nipponbare plants; the new insertion sites were frequently located in genic regions. 5-AzaC treatment principally induced expression of Dart1-34 transposase rather than the other 38 aDart1-related elements in both pyl-stb and Nipponbare treatment groups. Our observations show that transposition of nDart1-3 subgroup elements in the nDart1/aDart1 tagging system is correlated with the level of DNA methylation. Our system does not cause somaclonal variation due to an absence of transformed plants, offers the possibility of large-scale screening in the field and can identify dominant mutants. We therefore propose that this tagging system provides a valuable addition to the tools available for rice functional genomics.

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.

Carotenoid isomerase (CRTISO) catalyzes the isomerization of prolycopene to all-trans-lycopene in the carotenoid biosynthetic pathway. We isolated a promoter region of CuCRTISO from Citrus unshiu. We determined whether the promoter encoded organ-specific or developmental-specific expression and identified possible cis-acting promoter elements. The promoter and two truncated versions were fused to the β-glucuronidase (GUS) gene and transformed into Arabidopsis thaliana. Transgenic lines expressing the 2501-bp promoter (pCiso-Prom1) and truncated promoters (pCiso-Prom2 and pCiso-Prom3) showed the same developmental and organ-specific activity. GUS expression was detected in the cotyledon and root at 5 and 10 days after germination, mature leaf, and anther. The CuCRTISO promoter contained several cis-acting elements involved in hormonal and environmental stress. Drought stress or abscisic acid treatment did not induce GUS expression in any transgenic lines. Heat stress induced GUS expression in the pCiso-Prom1 line; this promoter construct contains the heat stress-responsive element. Ethylene and cold stress treatments induced GUS expression only in the pCiso-Prom3 line, although all transgenic lines contained the same cis-acting ethylene and low-temperature response elements, which could indicate the existence of unknown repressor element(s) in the CuCRTISO promoter. The CuCRTISO expression of Citrus unshiu during fruit maturation showed that in the green stage, it was highly expressed and then subsequently decreased after the stage. These studies indicate that CuCRTISO promoter activity is regulated in a developmental and organ-specific manner that responds to heat, cold, and ethylene. These results provide new insights into the role of cis-acting element(s) in CuCRTISO promoter activity.

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.

In carrot (Daucus carota L.) somatic embryos, desiccation tolerance is induced by treatment with abscisic acid (ABA). Six cDNA clones that showed ABA-enhanced expression were isolated from carrot by differential screening with ABA-treated and ABA-untreated somatic embryos, and they were named the CAISE (carrot ABA-induced in somatic embryos) genes. Five of the clones encode late embryogenesis abundant (LEA) proteins, and the other clone encodes a glucose and ribitol dehydrogenase. The expression of the CAISE genes was detected in maturing seeds, embryogenic cells, and ABA-treated somatic embryos in which exhibit desiccation tolerance induced by endogenous or exogenous ABA. These results indicate that ABA-induced desiccation tolerance in carrot somatic embryos may be induced by the LEA proteins and the glucose and ribitol dehydrogenase encoded by these ABA-inducible genes.

Expression of carrot embryogenic cell specific protein genes (DcECPs) that are late-embryogenesis abundant proteins is ABA-inducible and embryo-specific. The expression of some DcECPs is controlled by an embryo-specific transcription factor C-ABI3, a carrot homologue of Arabidopsis ABI3. In order to understand the molecular mechanisms regulating ABA-inducible and embryo-specific expression of DcECP genes, we carried out a detailed analysis of DcECP31 promoter with deletion analysis, base-substitution mutagenesis and electrophoretic mobility shift assay (EMSA). We identified two important elements in the promoter of DcECP31, motif X (CACACGTGGG) and motif Y (CACACGTATC), which are sufficient for the embryo-specific and ABA-inducible promoter activity. By precise EMSA analysis, it was demonstrated that a nuclear protein which has sequence specific-binding ability binds to the flanking ACGT core motifs.

Expression of carrot ECP (DcECP) genes encoding late-embryogenesis abundant proteins is embryo-specific and ABA-inducible. The expression is regulated by C-ABI3, a carrot homologue of Arabidopsis ABI3. To understand the molecular mechanisms controlling ABA-inducible and embryo-specific gene expression, we compared and analyzed the promoter regions of some ECP genes. The accumulation of DcECPs mRNA in response to ABA was not inhibited by an inhibitor of protein synthesis, cycloheximide. These results indicate that the ABA-inducible expression of the DcECPs did not require de novo protein synthesis. Sequence comparison among some ECP gene promoters revealed that the promoters contained several conserved motifs including ABRE (ABA responsive element)-like ACGT core motifs, and an Sph box (CATGCATG), which has been identified as a motif mediating gene activation of maize antocyanin regulatory gene C1. To investigate the promoter activity of DcECP31 promoter region, we carried out a deletion analysis with a transient assay system using protoplasts of embryogenic cells or with a transformation system using embryogenic cells of carrot. We found that the -250bp upstream region of the DcECP31 promoter is sufficient for embryo-specific and ABA-inducible promoter activity. Following deletion analysis of DcECP40 promoter, we clarified that the distal (-670∼-390) and proximal regions (-140∼-50), are essential for the ABA-inducible expression.

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