Glycosylation plays an important role for the stabilization, enhancement of water solubility and detoxification of natural products. It also contributes to the highly diverse nature of plant secondary metabolites. Family 1 glycosyltransferases, often referred to as UDP-glycosyltransferases (UGTs), catalyze the transfer of sugar moieties to a wide range of acceptor molecules including plant hormones, phenylpropanoids, flavonoids, betalains, coumarins, terpenoids, steroids and glucosinolates in planta. Completion of the sequencing of the Arabidopsis genome revealed the presence of 107 UGT genes and also provided novel approaches to determine the functions of UGTs using genomics, transcriptomics and metabolomics. This review describes recent developments in functional genomics efforts on UGTs for plant natural products.
The availability of complete plant genome sequences has opened a new era of plant molecular research using approaches such as comprehensive expression analysis, ectopic expression and gene knockdown. For such purposes, new tools realizing easy and rapid construction of recombinant plasmids are required. Here, we describe Gateway cloning compatible binary vectors for post-genomic research in plant genetic engineering. The vectors comprise a variety of reporters, epitope tags and selective markers that should make them useful for construction of plasmids for Agrobacterium-mediated transformation of plants. We also describe vectors for promoter swapping using Gateway cloning technology.
Cell suspension cultures of Phytolacca americana L. (pokeweed) are capable of glycosylating capsaicinoids that have several biomedical applications. To identify the capsaicinoid glucosyltransferase involved in this biotransformation, we isolated three full-length cDNAs (PaGTs) encoding homologs of plant secondary product glycosyltransferases from cultured cells of P. americana L. These glycosyltransferase cDNAs were heterologously expressed in Escherichia coli cells and the expressed products were functionally characterized. Although all of these glycosyltransferases displayed broad glucosyl-acceptor specificities toward phenolics, capsaicinoid glucosyltransferase activity was found only for one of the cloned enzymes, PaGT3. Phylogenetic analysis showed that PaGT3 is the most closely related to betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis, and in fact, it displayed a weak betanidin 5-O-glucosyltransferase activity. Transcription analyses showed that the expression of PaGT3 in P. americana L. was strongly induced by exposure of the cells to capsaicin (0.65 mM). These results show that PaGT3 should be, at least in part, responsible for the capsaicinoid glucosyltransferase activity of this plant.
Many genes are temporally and spatially regulated during embryogenesis in higher plants. Although many studies have examined transcriptional factors relating to gene regulation during embryogenesis, the molecular mechanisms relating to the initiation of embryogenesis are still unclear. In animals, it was reported that gene regulation by chromatin remodeling contributes to embryogenesis. In contrast, the relationship between chromatin remodeling and the initiation of embryogenesis in higher plants remains to be determined. LEAFY COTYLEDON1 (LEC1) is an important factor in early embryogenesis and is ectopically expressed in the pkl1-1 mutant, which is deficient in chromatin remodeling factor. Therefore, there is a high probability that chromatin remodeling regulates the expression of LEC1. To confirm this possibility, the histone methylation level, which is involved in chromatin remodeling, was examined for the genomic region of LEC1 by chromatin immunoprecipitation analysis. In the promoter region of LEC1, methylation of histone H3 lysine 4 in somatic embryos was higher in rosette leaves. SET domain-containing proteins are an important factor in histone methylation. To isolate the SET domain-containing protein genes (SET gene) involved in Arabidopsis thaliana embryogenesis, expression analyses using RT-PCR were performed. Among 37 SET genes, seven were found to have a high probability of involvement in embryogenesis.
Senno (Lychnis senno, Caryophyllaceae), a traditional ornamental plant in Japan, was introduced from China and all the strains of Senno found in Japan are triploid with 2n=3x=36. Hand-pollination was conducted between triploid Senno and allied Lychnis taxa, L. kiusiana (2n=24), L. miqueliana (2n=24), L. miqueliana f. albescens (2n=24), L. chalcedonica (2n=24), L. wilfordii (2n=24), L. sieboldii (2n=24), and L. coronata (2n=24). Immature seeds 17–42 days after pollination were cultured on half-strength Murashige and Skoog media with or without 6-benzyladenine. Although immature seeds were obtained in most cross combinations, seed germination was observed only in three cross combinations, L. senno×L. kiusiana, L. kiusiana×L. senno, and L. senno×L. sieboldii. Seedlings derived from reciprocal crosses between L. senno and L. kiusiana showed an abnormal morphology or a chlorophyll-deficiency, xantha. Seedlings derived from L. senno×L. sieboldii also showed an abnormal morphology or chlorophyll-deficiency, but one seedling grew into a green plantlet. The hybridity of these seedlings was confirmed by random amplified polymorphic DNA analysis. The chromosome number of the interspecific hybrid plantlet between L. senno and L. sieboldii was determined to be 2n=32. The present study shows the possibility of using triploid Senno for breeding by interspecific hybridization, and the effectiveness of immature seed culture for producing interspecific hybrids in the genus Lychnis.
Calli and suspension cell culture were established from Aquilaria species whose resinous portion was called agarwood and used as medicine and incense. Four different strains of calli were analyzed for fragrant compounds such as sesquiterpenoids and chromone derivatives which were the major components of agarwood. Main sesquiterpenoids detected from calli were α-guaiene, α-humulene and δ-guaiene, and those of chromone derivatives were phenylethylchromones (AH3, AH4, AH5, AH6). Amount of these compounds differed among the four strains, indicating that Aquilaria plants may have variation in capacity for fragrant compound production. Incubation temperature analysis was also done from 20°C to 40°C and resulted that cell growth was the best at 25°C, whereas the amount of fragrant compounds was largest at 20°C. Salicylic acid (SA) and methyl jasmonate (MJ) were added to calli and suspension cell culture respectively in order to induce production of fragrant compounds. Both SA and MJ apparently induced production of three sesquiterpenoids, α-guaiene, α-humulene, and δ-guaiene at early stage of treatment of SA or MJ, but did not induce that of chromone derivatives directly. Further studies of time course of chromone production and cell viability suggested that cell death may take part in chromone production, and that phenylethylchromones would be produced via oxydoagarochromones (OACs). These results indicate that sesquiterpenoids are synthesized in living cells, but chromone derivatives may be produced from debris of dying cells.
Autophagy functions in bulk degradation of proteins and organelles for nutrient recycling. Recent evidence suggests that a set of autophagy-related (ATG) genes are induced under starvation, senescence and various abiotic stresses. To understand how these genes function in plants, expression profiles of soybean ATG homologs, GmATG8s, GmATG4, GmATG9, GmATG12 and GmATG18a, were examined using seedlings of Glycine max (L.) Merr. cv. Fukuyutaka) subjected to various nutrient conditions. Among them, GmATG8i mRNA was induced at higher level than any other ATGs under starvation. Immuoblot with a specific antibody raised against GmAtg8i indicated that endogenous GmAtg8i associates to microsomes of soybean seedling extracts. In the presence of vacuolar protease inhibitors, significant accumulation of anti-GmAtg8i was observed and mRNA level of GmATG8i increased in higher level than in the absence of the inhibitors. These results suggest that in soybean gene expression of ATG8i is regulated by both environmental nutrient conditions and intracellular nutrient recycling via proteolysis in vacuole.
The gene TERMINAL FLOWER1 (TFL1) regulates the floral phases and the inflorescence architecture of Arabidopsis. A TFL1 homolog designated as RsTFL1 was isolated from radish (Raphanus sativus). The deduced amino acid sequence had 96% identity with TFL1—higher than the values obtained for other reported TFL1-like proteins. The genomic organization of RsTFL1 is very similar to that of TFL1 and consists of 4 exons and 3 introns. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that RsTFL1, like TFL1, is continuously expressed in both vegetative and reproductive tissues. RsTFL1 was expressed throughout seed development until seed desiccation. RsTFL1 mRNA was detected after seed imbibition, suggesting that the gene expression is initiated during germination. In situ hybridization analysis revealed that RsTFL1 was expressed in the inflorescence meristem but not in the floral meristems. The expression was not limited to the inner cells of the inflorescence meristem, unlike the expression of TFL1 in Arabidopsis. This expression pattern of RsTFL1 may make it possible that radish maintains its indeterminate inflorescence.
In vitro regeneration protocol was developed from hypocotyl-derived callus of Psoralea corylifolia L. Green compact nodular calli were induced from 3 day-old hypocotyl explants on Phillips and Collins (L2) medium containing 25 g l−1 sucrose, 7 g l−1 agar and supplemented with 10 μM NAA and 2 μM TDZ. Higher shoot regeneration (89.5 ± 1.18) was achieved in enriched L2 medium supplemented with 2 μM BA, 4 μM TDZ and 50 mg l−1 BVN. Regenerated shoots elongated on 1/2-enriched L2 medium containing 10 g l−1 sucrose, 7 g l−1 agar and 2 μM 2iP. Elongated shoots (45–55 mm in length) were exposed simultaneously 15.0 roots per shoot as well as hardened in moistened (1/8-L2 basal salt solution with 5 mM IBA and 100 mg l−1 BVN) soil mixture and vermiculite (3 : 1 v/v). The plants were subsequently established in the field. The higher survival percentage (100%) was achieved in winter season (September–December, 25–28°C). This system would be useful for mass propagation and germplasm conservation of P. corylifolia.
For research and development of plants suitable for forage and biofuel production, the development of a high-throughput system of lignin determination, especially for herbaceous species, is important. However, currently available methods for lignin determination are not suitable for high-throughput analysis of herbaceous samples. In this paper, we describe a straightforward, high-throughput method for lignin determination as thioglycolic acid lignin from rice straw using disposable plastic microtubes, TissueLyser, and a UV microplate reader. We successfully plotted calibration curves derived from the readings of a spectrophotometer and a microplate reader using thioglycolic acid lignin prepared from bamboo milled wood lignin as a lignin standard. Based on the calibration curve, we could determine the lignin content in various organs from dried rice straw and rice seedlings. We confirmed that 20 mg of dried material from rice plants is adequate for stable determination of the lignin content. Using this method, 100 independent samples from rice straw can be analyzed in three days per person.
Concern about the use of bacterial antibiotic resistance genes as selectable markers is considered to be one of the factors in public resistance towards genetically modified crops. We had previously shown that the Arabidopsis AtHOL1 protein had high S-adenosyl-L-methionine-dependent methyltransferase activity toward the thiocyanate ion (NCS−), which is toxic to plants beyond certain concentrations. Contrary to our expectations regarding the usability of AtHOL1 as a selectable marker, our initial trial screenings for AtHOL1-overexpressing Arabidopsis on 1/2 MS agar medium containing various concentrations of potassium thiocyanate under normal growth conditions had been unsuccessful. In order to explore the possibility of using AtHOL1 in Arabidopsis transformation, the screening conditions for AtHOL1-overexpressing Arabidopsis were further examined. We found that the transgenic seedlings could be screened when the seeds were germinated and grown under conditions of 7-day darkness followed by a 12-h light/12-h dark cycle for 3–7 days on a medium containing 3.0–5.0 mM potassium thiocyanate. Analyses of transgene insertions into the genomes and AtHOL1 mRNA accumulation in the screened seedlings were also performed. Neither escaped seedlings nor altered growth was observed with the seedlings screened under the conditions reported here.
To develop a screening system for plant defense activators, which are novel substances that protect plants by enhancing their inherent disease-resistance mechanisms, we utilized a GUS histochemical staining assay using promoters of the defense-related genes, PR-1 and PDF1.2. We can perform about 1,000 screenings per week per person by this high-throughput screening method. This GUS assay for plant defense activator candidates was evaluated by QRT-PCR analysis to elucidate the functions of the plant defense activators in detail. In the present preliminary screening, we evaluated two hundred chemicals chosen at random. Some chemicals induced GUS activity in a PR-1 promoter::GUS transformant, i.e., abietic acid, allose, glycine, and thymol. The induction of PR-1 expression by the treatments with these chemicals was confirmed using QRT-PCR. The foliar treatment with abietic acid 1 d prior to inoculation with the fungal pathogen Colletotrichum higginsianum led to a significant reduction of necrotic surface area compared with distilled water treated controls, as observed 6 d after inoculation. These results suggest that this GUS histochemical staining assay is an effective and available screening system for plant defense activators.