Epigenetic regulation is defined as mechanisms that control gene expression without altering base sequences. Cytosine methylation, chromatin remodeling, and modifications at the N-termini of core histones are key factors in this regard. Epigenetic modifications are found throughout the eukaryotes, suggesting that they developed at an early stage in biological evolution, although actual molecular mechanisms show considerable variation among species. In particular, plants are unique in establishment and maintenance of epigenetic states, as exemplified by species-specific enzymes that catalyze DNA methylation. Since the function and diversity of DNA methyltransferases in individual species are not fully understood, I here summarize recent findings in plant epigenetics, focusing on DNA methyltransferases classified into three major groups. Their possible biological functions are also discussed with reference to histone modification and chromatin remodeling.
Millets are small seeded grasses grown for food, feed or forage and cultivated mostly in less developed countries in poor soil and dry conditions. There are at least 10 genera and 14 species of millets belonging to the Poaceae (Gramineae) family. Tissue culture and plant regeneration occurring through different morphogenic pathways have been reported in great detail in millets. Gene transfer has been attempted using various methods, but so far transgenic plants have been developed only in Pearl millet and Bahiagrass. Not much work has been done on transgenesis in other millets. This is primarily because they have less economic value and are cultivated in poor countries, where research and development are also poor. In the present review we have attempted to provide available information on millet tissue culture and genetic transformation. We have underlined the importance of transgenesis in millet improvement and the role that biotechnology can play in the improvement of these crops grown in a variety of harsh conditions.
We compared three transgenic rice plants transformed with pIJ2C9, pIES2C9, or pIJU2C9, which express the human CYP2C9 gene under the control of the CaMV 35S, chimeric CaMV 35S, or maize polyubiquitin 1 promoter, respectively. The plants, especially those transformed with pIJU2C9, showed high tolerance to the sulfonylurea herbicides chlorsulfuron, imazosulfuron, and triasulfuron, owing to metabolic detoxification promoted by the introduced CYP2C9. The levels of expression of CYP2C9 were highly related to the tolerance to sulfonylurea herbicides. In this study, we achieved to produce transgenic rice plants with high expression of CYP2C9 by use of a maize polyubiquitin 1 promoter. Imazosulfuron is used in rice, but chlorsulfuron and triasulfuron are not, because of the sensitivity of rice plants to them. Transgenic rice plants expressing CYP2C9 will be useful for introducing tolerance to chlorsulfuron and triasulfuron into rice lines.
We examined the applicability of small and branching varieties of sunflower (Helianthus annuus L. cv. Sonja, Valentine, and Pacino) for plant regeneration and gene introduction. Some small and branching sunflower varieties have advantages as transformation materials. The frequency of shoot regeneration in MS+0.1 mg l−1 BA medium was high in Sonja and Valentine and many shoots formed on each explant. Roots formed easily from regenerated shoots in MS+0.1 mg l−1 NAA medium and the regenerated plants grew normally and formed flowers and seeds in Pacino. In addition, the introduction of genes into branching sunflowers was shown to be straightforward. About 20–50% of regenerated shoots displayed GUS expression over wide areas of tissue in all three varieties. In Pacino, some uniformly transformed shoots were observed after simple infection with Agrobacterium. All of the steps required to obtain transformants of branching sunflowers are simple and straightforward. These small and easily transformed sunflower varieties are therefore useful subjects for molecular genetic experiments.
Tobacco BY-2 cells were successfully cryopreserved by a simple slow prefreezing (equilibrium freezing) method using an encapsulation technique. After the cells were immobilized in alginate gel beads, the beads were treated with cryoprotectant solution (2 M glycerol, 0.4 M sucrose) for 45 min. The beads were then transferred to a laboratory freezer at −30°C, stored for 2 h, and then immersed in liquid nitrogen. To initiate the regrowth of cells, the beads were warmed in a water bath. Following dilution of cryoprotectant solution, the beads were suspended and cultured in normal medium. With this method, suspension cell cultures were regrown within 7 days. There were no differences in the morphology or growth profiles between cryopreserved cell cultures and the original cell cultures.
Coptis japonica is a perennial medicinal plant grown in Asia, and its rhizome is used as crude drug, in which berberine is highly accumulated as the main alkaloid. In this study, a transformation method for C. japonica using Rhizobium has been established. Young petioles were infected with Rhizobium radiobactor Gv3101 (pMP90) harboring a plasmid with β-glucuronidase (gus) driven by cauliflower mosaic virus 35S promoter and hygromycin phosphotransferase (hpt) gene as the selection marker. GUS assay showed that 46% of the calli grown on hygromycin plates clearly expressed the gus gene. As an application of the transformation method, an endogenous cDNA encoding an ABC protein, CjMDR1, which is involved in berberine transport, was introduced in sense orientation. Cjmdr1 transgenic plant (Sense-2) regenerated from hygromycin resistant callus line had a single copy of integrated hpt gene in its genomic DNA. Northern analyses of the Sense-2 plant showed that Cjmdr1 mRNA levels were lower in all organs examined than those of wild-type plant, which suggested that co-suppression of Cjmdr1 expression occurred. Indeed, the berberine content in Sense-2 was also suppressed. This is the first report of stable transformation of Coptis spp. and also of the alteration of a secondary metabolite by transport engineering.
A modified 1/2MS medium supplemented with 3 µM 2,4-D was effective for callus induction from bamboo shoots of Phyllostachys nigra Munro var. Henonis. In the first phase (phase 1), some parts of the explants enlarged and gave rise to whitish-yellow calli after 2–3 weeks of culture. During maintenance subcultures, almost all explants and the initially formed calli turned brown and these calli gradually lost their proliferation capacity (phase 2). Removal of the necrotic potions of explants, and frequent subcultures at phase 2 was essential. Secondary proliferated calli were subsequently produced on the surface of brown tissues (phase 3). These calli could be maintained on both solid and liquid media. The liquid suspension cells had a blue to pale blue autofluorescence in the cell walls. These cells fluoresced strongly when stained with Calcofluor White M2R and Aniline Blue, indicating the presence of callose (β-1,3-glucan) in a cellulosic wall. Endogenous free amino acids analyses indicated that glutamine, γ-aminobutyric acid, and alanine were the major amino acids in callus tissues whereas asparagine and tyrosine were abundant in the regenerated bamboo shoots.
Reactive oxygen species (ROS) are proposed to function as diffusible signaling molecules in plant immune response. Rice respiratory burst oxidase homologs (Osrboh genes) are proposed to play a role in ROS generation. We examined a role in rice immune responses of four Osrboh homologs, OsrbohA, OsrbohB, OsrbohD, and OsrbohE. OsrbohA and OsrbohD transcripts were induced after inoculation with an incompatible N1141 strain of Acidovorax avenae, whereas OsrbohaB and OsrbohE mRNA levels did not obviously change even after inoculation with the incompatible strain. We examined the function of the Osrboh genes in ROS generation and in the plant immune response using RNAi-based knockdown in rice cells. OsrbohA and OsrbohE knockdown lines showed that rapid H2O2 generation is caused by OsrbohA, whereas OsrbohE is involved in late H2O2 production during the immune response. Hypersensitive cell death was decreased only in the OsrbohA knockdown line. We further demonstrated that among immune related genes, the induction of EL2 and LOX genes is controlled by ROS generated by OsrbohE, whereas expression of Cht-1 gene is regulated by both OsrbohA and OsrbohE. These results indicate that the ROS molecules generated by OsrbohA and OsrbohE regulate different signaling pathways in the plant immune response.
A 507-bp nuclear matrix attachment region (MAR) sequence (designated TJ1) was flanked at both at 5′ and 3′ ends of the expression cassette for a modified green fluorescent protein (GFP) gene of the 35 Ω-sGFP(S65T) in four different orientations. A plasmid p507TH bearing a 507-bp non-MAR sequence from yeast at both ends of the cassette also was constructed. These five plasmids and 35 Ω-sGFP(S65T) were separately delivered into cultured tobacco BY-2 cells by particle bombardment. The yield of stable transformants that showed a uniform green fluorescence was determined. Southern blot analysis confirmed the integration of the GFP gene in tobacco genome. Transformant yields of the two TJ1-lacking plasmids were not significant. Transformant yields appeared to be increased by more than 2 times by the presence of TJ1-MAR sequences. The orientation of TJ1-MAR sequences did not influence the transformant yield.
Anthers of transgenic rice plants with two foreign genes were cultured to obtain doubled haploid plants that possessed the introduced foreign genes homozygously. Anther culture response varied with the transgenic line. Green plantlets were regenerated from the microspore-derived calli, and 5.9–88.9% of the regenerated plants set numerous seeds in a greenhouse. The haploid or doubled haploid green plants were confirmed to have the foreign genes by Southern hybridization analysis. They had both or no foreign genes, and no plants with heterozygosity in transgenes were observed. The ratio of the plants with the transgenes homozygously to those without the transgenes varied with the donor transgenic lines.
In an attempt to increase the amount of Rubisco in a green alga, the Chlamydomonas reinhardtiirbcL gene connected to the aadA cassette was integrated into chloroplast inverted repeats in this organism to generate a transformant with three copies of rbcL (3L). Synthesis and accumulation of the rbcL transcript in 3L were 2.3 and 1.4 times those of the control, respectively. Although synthesis of the large subunit (LSU) in 3L was 2.6 times that in the control, accumulation of the LSU was only 1.1 times that in the control. Synthesis of rbcS transcript and the small subunit (SSU) in 3L was also upregulated to 1.7 and 1.3 times those in the control, respectively, while their accumulation was unaffected. These results suggest that an increase in rbcL gene number upregulates transcription of rbcL from chloroplast genomes and that of rbcS from a nuclear genome, but does not affect the accumulation of LSU and SSU.
To investigate the usefulness of multi-color luciferase technology as reporter genes in higher plants, we assayed the transient expression of click beetle luciferase genes introduced into plant cells by microprojectile bombardment. Although their expression levels were relatively low, luminescence from green and red luciferases were separable under the CCD camera equipped with interference filters. Results of time-course experiments and the inducible promoter assay suggest that the multi-color luciferase system optimized primarily for mammalian cells is also applicable to monitor reporter activities in plant cells.
Tobacco genes regulated during the hypersensitive response (HR) to infection by tobacco mosaic viruses were screened by differential display and one particular clone with a rapid expression profile was selected and further characterized. The obtained cDNA encoded a polypeptide of 205 amino acids with an apparent molecular mass of 23 kDa and a pI of 10.37 and production of a GFP fusion protein revealed localization in the cytoplasm. Transcripts of the clone were strongly induced in leaves 3 h after HR onset, and 10 min after mechanical wounding, as well as by treatment with jasmonic and salicylic acids. The induction profile satisfies the definition of pathogenesis-related (PR), and the protein was designated as NtPRp23. Database searches indicated the presence of similar proteins in Arabidopsis and rice, but with functions yet to be assigned. Our results point to the existence of a novel PR protein family in tobacco plants.
Tomato genome sequencing projects have started to become an internationally coordinated program. To accelerate tomato functional genomics studies in coordination with the complete sequencing of the tomato genome, we prepared a full-length cDNA library from the miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom, which has attracted attention as a laboratory-grown model plant. Total RNA from maturing fruits was subjected to a vector-capping protocol for full-length cDNA synthesis. We generated 8,046 expressed sequence tags (ESTs), which comprised 3,484 contigs. We calculated that 80.7% of the cDNA clones in the library met the criteria for full-length clones, and 1,920 non-redundant full-length clones were identified. As a pilot experiment, we chose seven clones, whose encoded proteins shared low homology with Arabidopsis proteins, for full sequencing. Of these, three genes had no or very low homology with Arabidopsis genes, indicating the usefulness of the library for analyses of “not-found-in-Arabidopsis” genes.
We screened about 50 species of plants collected from local abandoned rice fields for capability to decolorize Remzol Brillinant Blue R (RBBR), an anthraquinone dye. We chose Rumex crispus L. subsp. japonicus (Houtt.) Kitamura (a curly dock; “Gishi-gishi” in Japanese) and rice, and investigated the capability of these plants to remove bisphenol A (BPA), an endocrine disruptor, from the culture medium. Aseptically-grown curly dock appeared to completely remove BPA added in the culture medium (1000 mg BPA kg−1 fresh plants, 40 mg l−1) by 15 days after treatment (DAT). At 7 DAT methanol-extractable BPA was present in curly dock. However, no such BPA was detectable at 14 DAT. Curly dock was found to remove completely BPA at an environmental pollution level (1 µg l−1) by 15 DAT. Rice showed a similar or less capability to clean up BPA from the culture medium.