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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.

Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.

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.

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.

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.