LOV KELCH PROTEIN 2 (LKP2) belongs to a group of blue light receptors in Arabidopsis thaliana that possess 1 light, oxygen, or voltage (LOV) domain, 1 F-box motif, and 1 kelch repeat domain. Members of this group regulate both the circadian clock and photoperiodic flowering. Transgenic Arabidopsis plants overexpressing a fusion of the genes for green fluorescent protein (GFP) and LKP2 exhibited long hypocotyls and arrhythmicity under constant light and late flowering under long-day conditions, as previously reported for LKP2-overexpressing Arabidopsis plants. The GFP-LKP2-overexpressing plants with elongated hypocotyls had more cortical cells than control plants but the same number of epidermal cells. Further, the cells in the hypocotyls of GFP-LKP2-overexpressing plants, especially in the middle regions of the epidermis and cortex, were longer and possessed larger nuclei, with increased DNA content. Therefore, LKP2 overexpression induces cell elongation and increases in cell number and ploidy in the Arabidopsis hypocotyls.
Forsythia spp. are perennial woody plants containing abundant amounts of beneficial lignans, and the transgenic metabolic engineering of Forsythia is expected to produce plants with a more efficient production of specific lignans on demand. However, the transgenic methods for Forsythia have yet to be fully established. In this study, we have investigated the optimal conditions for the regeneration, growth, and antibiotic-based selection of Forsythia suspense (Fs), F. koreana (Fk), and F. intermedia (Fi), and compared the possibility of the construction of transgenic plants among the three species. Fk, Fi, and Fs explants regenerated more than 100, 36, and 4 shoots per leaf, respectively, revealing that Fk is especially endowed with potent regeneration ability. Fi initiated shoot formation 6 days earlier, but required 6 extra days for the initiation of rooting than the two Forsythia spp. Moreover, Fs, Fk, and Fi displayed different preferences for the components of the MS macro elements in the regeneration media. Fk and Fi calli grew to 10-cm long plants for approximately 120 day. We also found that Fk and Fi exhibited prominent dose-dependent sensitivity to hygromycin, but not to kanamycin. We constructed transgenic hygromycin-resistant Fk and Fi via agrobacterium-based transformation with a hygromycin-resistant gene, hptII. Polymerase chain reaction analyses confirmed the introduction and expression of hptII in the transgenic Fk and Fi. Altogether, these data showed the establishment of the methods for Fk and Fi transgenic plants, and revealed multiple different propensities among Forsythia species.
The sweet taste-modifying protein miraculin has been successfully expressed in the tomato, a high-yield commercial plant (Sun et al. 2007). Previously proposed methods of purifying untagged miraculin from transgenic tomato fruit are laborious and time consuming and do not allow the separation of undenatured and denatured miraculin. Therefore, it is necessary to develop an efficient method of purifying undenatured miraculin from transgenic tomato fruit. In this study, we have demonstrated that the combined use of nickel-immobilized affinity chromatography and ion-exchange chromatography readily provides highly purified undenatured miraculin, as demonstrated by the observation of both a single peak from reverse phase high performance liquid chromatography and a single band from SDS-PAGE.
Lignans are phenylpropanoid dimers in which the monomers are linked by the central carbon (C8) atoms. Because many lignans have physiological effects, including antioxidant activity, they are now in demand as components in health foods. However, the lignan biosynthetic pathways in plants are only now being understood. Recently, lariciresinol was detected in Arabidopsis thaliana. This observation indicated the existence of common lignan biosynthetic pathways in A. thaliana, despite a low amount of lignans other than lariciresinol glycosides. In this study, we established a highly sensitive analytical method that enables quantification of both glycoside and aglycone forms of lignans in A. thaliana simultaneously using capillary liquid chromatography-electrospray ionization-ion trap mass spectrometry. Some lignans not previously detected in A. thaliana were quantified in extracts of both roots and shoots. Our method can be used for the comprehensive analysis of lignans in small samples from mutants and transformants. This method will be utilized to elucidate the metabolic pathways and physiological roles of lignans as well as the regulation of their biosynthesis in plants.
Using tobacco BY-2 cells transformed with a promoter-luciferase gene fusion, we developed a non-destructive and sensitive in vivo assay system for monitoring defense gene expression in higher plant cells. A promoter fragment of a tobacco salicylic acid (SA) inducible pathogenesis-related gene, PR-1a isolated from the genomic DNA of tobacco BY-2 cells, was fused to the luciferase reporter gene and introduced into plant cells by Agrobacterium-mediated transformation. To detect the PR-1a promoter expression as a luciferase activity, transformed cells were mixed with luciferin solution and the bioluminescence levels were monitored in vivo using a conventional luminometer. Because the PR-1a promoter expression levels of the BY-2 cells are relatively high, the induction of the luciferase activities by the treatment with SA was barely detectable under log phase growth conditions. However, we could observe concentration dependent induction of the luciferase activities following SA application under stationary phase. Treatment with benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) and methyl-2,6-dichloroisonicotinic acid (INA) also resulted in a drastic increase in luciferase activities of transgenic cells in a dose dependent manner. On the other hand, treatment with an inactive SA analog 4-hydroxybenzoic acid (4-HBA) showed no influence on luciferase activities. The sensitivity of the assay system was higher than the previously reported techniques for the detection of induction by SA or BTH. These results indicate that this rapid, inexpensive and versatile assay system would be useful for the identification and characterization of chemicals capable of inducing defense gene expression in higher plant cells.
Cold stress on crops results in severe yield losses through growth retardation and irreversible damage. Recently, Inducer of CBF Expression 1 (ICE1) was identified as the master regulator inducing dehydration responsive element binding protein/C-repeat binding factor (DREB/CBF)-type transcriptional factors involved in the signaling of cold and osmotic stress in Arabidopsis. To examine whether rice ICE homologs function in cold acclimation via regulation of rice DREB homologs in response to cold stress, we assessed a polypeptide epitope containing an ICE-specific motif. Chilling stress on rice seedlings induced two ICE-related proteins with molecular masses of approximately 55 and 40 kDa. These sizes are consistent with those predicted for OsICE1 and OsICE2, respectively. In contrast to the proteins, cold stress had little or no effect on the expression of OsICE1 and OsICE2. Semi-quantitative RT-PCR indicated that both were constantly expressed, but that cold stress sequentially upregulated OsDREB1B, rice heat shock factor A3 (OsHsfA3), and trehalose-6-phosphate phosphatase (OsTPP1). Trehalose treatment enhanced the cold tolerance of seedlings. These results suggest that OsICE homologs function in transcriptional regulation at upstream of a cold-stress-induced transcription factor cascade involving OsDREB1B and OsHsfA3, leading to cold acclimation, possibly involving trehalose synthesis.
Recently, we characterized an acyl-glucose : anthocyanin 5-O-glucosyltransferase (DcAA5GT) in Dianthus caryophyllus that has novel characteristics. We also showed that a homologous enzyme, acyl-glucose:anthocyanin 7-O-glucosyltransferase (DgAA7GT), is present in Delphinium grandiflorum. These enzymes are both classified as members of glycoside hydrolase family 1 (GH1). Here, we searched the Arabidopsis thaliana database for genes with homology to DcAA5GT and DgAA7GT and identified 11 β-glucosidases (BGLU1–11) of unknown function. In a co-expression analysis, using genes for enzymes involved in anthocyanin modification as baits, we found that expression of AtBGLU6 and AtBGLU9 appeared to be correlated to that of the baits. Real-time RT-PCR showed that AtBGLU1, 6, 7 and 9 were expressed at their highest levels in anthocyanin-inducing medium, although AtBGLU6 was a pseudogene in which there was a stop codon on the middle of its cDNA sequence. These results suggest that AtBGLU1, 7 and 9 might be candidate glucosyltransferases for anthocyanin/flavonoid modification or biosynthesis.
The objective of this study was to determine a possible application of hairy roots as biotechnological producers of furanocoumarins, by establishing root clones of Glehnia littoralis after infection from Agrobacterium rhizogenes. None of the root clones grew under hormone-free conditions, but when cultured in the presence of indole-3-butyric acid, growth varied substantially across clones. Analysis of the insertion and expression of pathogenic rol (A, BTL, BTR, and C) genes for 10 clones showed that the stable expression of rolC and irregular expression of rolB were detected in 2 clones, which exhibited active growth and higher furanocoumarin induction. Following ascorbic acid treatment, the productivity of xanthotoxin and bergapten in a root clone harbouring a pathogenic gene was 2.8 and 3.7 times greater than that recorded in wild-type roots, respectively. These results indicate that root cultures possessing pathogenic rol genes offer a potential means to produce furanocoumarins.
Ion-beam irradiation is attracting increasing attention as a new mutagen. Here, we describe for the first time the dose response and mutagenic effects of ion-beam irradiation in soybean. We irradiated the hilum side of dried mature soybean seeds with 320-MeV carbon ions within a 0.25–20-Gy range. The growth or seed production of the irradiated plants was profoundly affected. In particular, the number of plants that survived until seed-set decreased with the increase of the irradiation dose and was very low in plants irradiated at doses higher than 5.0 Gy, whereas the frequency distribution of the number of seeds produced by each seed-setting plant was not affected by lower doses of irradiation. Based on these results, we produced plant populations irradiated at 2.5 Gy and 5.0 Gy on a large scale to obtain M2 seeds. Despite the duplicate composition of the soybean genome, which originated from tetraploids, chlorophyll-deficient mutants were detected with a frequency of 0.47% in the M2 generation of plants irradiated at 5.0 Gy. These results demonstrate that irradiation of the hilum side of dried soybean seeds with carbon-ion beams at a dose range around 2.5–5.0 Gy induces genetic changes while also allowing the production of a considerable number of seed-setting plants, suggesting that these irradiation conditions are suitable for producing a mutant population potentially useful for breeding and/or identifying gene function.
In a previous study, we reported the production of the exogenous lignan, sesamin, using the Forsythia koreana transgenic cells (CPi-Fk cells) in which an exogenous sesamin-synthase CYP81Q1 is stably expressed while an endogenous pinoresinol-lariciresinol reductase is suppressed by RNA interference. Here, we present the effects of light on the production of sesamin and an endogenous lignan pinoresinol which is a precursor of sesamin in CPi-Fk cells. CPi-Fk cells showed a 2.3-fold, 2.7-fold, or 1.6-fold increase in sesamin production after two-week irradiation with white fluorescent, blue LED, or red LED light, respectively, compared with the level obtained under the dark condition. Likewise, CPi-Fk cells showed an approximately 1.5 to 3.0-fold increase in pinoresinol (aglycone and glucosides) production. Furthermore, expression of the pinoresinol-glucosylating enzyme UGT71A18 was suppressed in CPi-Fk cells under blue or red light. Considering that white fluorescent light contains the blue wavelength and that CYP81Q1 fails to convert pinoresinol glucosides to sesamin, it is concluded that blue light plays a major role in the up-regulation of the production of sesamin by CPi-Fk via an enhancement of the production of pinoresinol aglycone and a reduction of UGT71A18. This is the first report on the elevation of lignan biosynthesis by light.
Multiple meristematic nodules were induced on leaf segments from the Japanese persimmon (Diospyros kaki Thunb.) ‘Fuyu’ that were cultured on a solidified half-strength Murashige and Skoog's medium containing 2% (w/v) sucrose and 1 μM thidiazuron (TDZ). The nodules 1 to 3 mm in diameter were formed in the cut ends and abaxial side of the leaf segments two weeks after inoculation. The nodule changed from pale green to dense green and multiplied to form many daughter nodules. Adventitious buds were thereafter formed on the nodules, suggesting that the meristematic nodule is an intermediate structure for bud differentiation. The adventitious buds grew poorly on the medium with TDZ, but resumed growth and developed into shoots after transfer to the medium containing 2% (w/v) sucrose and 10 μM zeatin. The meristematic nodules survived more than one year on the medium containing 2% (w/v) sucrose and 1 μM TDZ without transfer, and did not lose their ability to form adventitious buds for more than 5 years when transferred regularly to a fresh medium. These results suggest that the meristematic nodule is a promising material for propagation and long-term conservation of this plant.
Estragole (EG) is biosynthesized in herbs including anise, basil, bay, tarragon, fennel and marjoram, and is thought to be a useful biomass for the food and health industries. Moreover, the metabolites from estragole are useful intermediates in organic synthetic chemistry. However, estragole has been transformed only by chemical methods, and no biocatalysts have been reported. In this paper, we report the biotransformation of estragole using the plant cells of Caragana chamlagu gave 4-methoxycinnamaldehyde (MCAL), 4-methoxycinnamyl alcohol (MCA) and 4-methoxybenzaldehyde (MBAL). In addition, we propose a reaction mechanism in the biotransformation of estragole using Caragana chamlagu. Since estragole generates malignant liver tumors in the rat, it is necessary to reduce exposure. The present study reveals the transformation of harmful estragole. Furthermore, we succeeded in biotransforming estragole as biomass using plant cells into useful compounds.