Phalaenopsis hybrids constitute a major ornamental crop. An important parent species for many of these hybrids is Phalaenopsis amabilis. We developed a convenient method for the genetic modification of P. amabilis using Agrobacterium tumefaciens. The transformed intact protocorms, which are young orchid seedlings of P. amabilis, regenerated plants under the same conditions that showed the highest frequency of shooting. A kanamycin resistance gene under the control of the 35S promoter can be used as a selective marker. In addition, T-DNA vectors containing the Arabidopsis class 1 KNOX gene, BP/KNAT1, were successfully introduced into protocorms. Shoots were generated with an abnormal leaf shape that was easily distinguished from that of normal shoots, indicating that BP/KNAT1 can be used as a visible marker gene. Furthermore, the protocorms transformed with BP/KNAT1 produced multiple shoots. Both the presence and expression of the transgene in transformed plants were confirmed by molecular analysis.
Polyamines (putrescine, spermidine and spermine) have been shown to be important in stress tolerance. Thus, the present study was undertaken with the aim to enhance stress tolerance in eggplant by introduction of a key polyamine biosynthetic gene arginine decarboxylase, adc under the control of a constitutive promoter of cauliflower mosaic virus, CaMV35S through Agrobacterium-mediated transformation. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis, respectively. These transgenic plants have shown an enhanced level of polyamines due to the increase in ADC enzyme activity. The diamine oxidase (an enzyme involved in putrescine and spermidine degradation) activity was also increased in these transgenic plants. Polyamine-accumulating transgenic plants exhibited an increased tolerance levels to multiple abiotic stresses such as salinity, drought, low and high temperature, and heavy-metal and resistance against fungal wilt disease caused by Fusarium oxysporium.
The effects of different culture temperatures, culture media and saccharides on the anther culture efficiency in two salt tolerant rice (Oryza sativa ssp. indica) cultivars, Pokkali and Nona Bokra, were examined. Comparison of two culture temperatures (constant temperature at 25°C and alternating temperatures at 30°C/20°C) indicated that alternating temperatures showed remarkable effects not only on the callus induction but also on the plant regeneration. Among the basal media examined, higher callus productivity was observed in SK-I medium and higher shoot productivity was obtained in SK-II medium than other basal media. When alternating temperatures and revised N6 medium including maltose were used throughout anther culture, callus productivity and green shoot productivity showed the highest score in the salt tolerant two cultivars used in this study. Maltose and alternating temperatures increases the anther culture efficiency of recalcitrant indica rice cultivars. This is the first report suggesting high production of haploids or doubled haploids from anther culture of these salt tolerant indica cultivars.
To induce somaclonal variations related to plant yield, adventitious buds were directly regenerated from immature flowerheads of a selected line A of the ‘Aichi Wase Fuki’ of Japanese butterbur (Petasites japonicus). Among the 280 independent regenerants obtained from 300 immature flowerheads, about 2/3 were regenerated within 90 d of culture. The total yield of the randomly selected 50 lines among the early regenerants ranged from 13.8 to 21.6 kg m−2 (average: 18.2 kg m−2) in the field, and a high correlation (r=−0.869) was observed between the total yield and the period required for the third leaf initiation in flowerhead culture. A high correlation (r=0.923) was also observed between the yield in the field and the fresh weight of rhizomes at 3 months of culture in the greenhouse. Since the yield of the original line A was 16.4 kg m−2, direct adventitious bud regeneration is a useful technique for inducing high yielding variants in Japanese butterbur, which might be efficiently selected by monitoring the period required for third leaf initiation and the weight of rhizomes.
Virus vectors have been used to study plant gene function by transiently overexpressing specific gene products, or conversely, silencing specific endogenous gene functions. Previously, we reported the establishment of tobamovirus vectors and its applications. In this study, we observed that tobamovirus vector could drive ectopic expression of green fluorescent protein (GFP) in roots after infecting leaves, indicating successful invasion into underground tissues. Subsequently, we attempted to apply the tobamovirus vector system to suppress the expression of putrescine N-methyltransferase (PMT), which is the first committed key enzyme for the biosynthesis of nicotine and is active in the underground parts of plants. Two or three weeks after inoculation with the vector harboring a partial fragment of PMT cDNA, we observed a reduction in the PMT mRNA level in the root and in the nicotine content of the aerial parts of the plant Nicotina benthamiana. The possibilities and limitations of the virus vector for the analysis of metabolic pathways are discussed.
Rpf2 (Ribosome Production Factor 2) is involved in ribosome biogenesis, and functions as one of the pre-rRNA processing factors. Although properties of the Rpf2 protein in yeast have been described in detail, there have been no reports on Rpf2 homologues in higher eukaryotes. To investigate the function of Rpf2 homologues in higher plants, we identified genes encoding Rpf2-like proteins from Arabidopsis and rice and designated them AtRpf2 and OsRpf2, respectively. The predicted amino acid sequence revealed that both genes contain the Brix domain, a characteristic domain associated with ribosome biogenesis, and a sigma 70-like RNA binding motif, a eukaryotic RNA binding domain unique to a superfamily of proteins required for ribosome biogenesis. Both gene transcripts were detected in all organs and tissues tested and the expression patterns were consistent with the typical expression pattern of previously described ribosomal protein genes. Transient expression of GFP::AtRpf2 and GFP::OsRpf2 revealed predominant nucleolar localization within the nucleus of both fusion proteins in onion epidermal cells and tobacco BY-2 cells. These results suggest that Rpf2-like proteins in higher plants are also involved in ribosome biogenesis and function as pre-rRNA processing factors.
Mitogen-activated protein kinase (MAPK) is one of the critical mediators of signaling systems by constituting phosphorylation pathways together with upstream kinases. Despite the importance, few direct targets have so far been identified, making it difficult to understand the whole features of so called MAPK cascade. Notably, a novel transcription factor, which was recently identified from tobacco, is activated upon phosphorylation by wound-induced protein kinase (WIPK), a typical MAPK in tobacco plants, and designated as NtWIF (Nicotiana tobacum WIPK interacting factor). To identify genes that are controlled by NtWIF, a microarray screening was performed using NtWIF overexpressing transgenic tobacco, and a set of affected clones were isolated. Approximately 28% of them were related to defense, confirming the critical role of WIPK in stress response. In addition, 15% of the clones were related to differentiation, suggesting pleiotrophic effects of NtWIF. When NtWIF was overexpressed or silenced by RNAi in tobacco plants, abnormal seed development and root growth were observed. Seeds of RNAi lines exhibited immature endosperm, resulting in germination failure. Root growth was retarded in both transgenic seedlings due to abnormal cell numbers in meristem. These results implicated the participation of phytohormones, of which function might be controlled by NtWIF. Since NtWIF possesses a B3-DNA binding motif, which specifically recognizes auxin-responsive elements found in many auxin-responsive genes, the present finding suggested that WIPK/NtWIF functions not only in stress response but also in developmental process through hormonal control.
Novel methods for foreign-gene introduction into specific cells are required for the functional analysis of specific cells. We report a new transfection method for intact plant cells that uses ArF excimer laser-induced shock waves to introduce foreign gene-coated gold particles into plant cells through the cell wall. Foreign gene (sGFP)-coated gold particles were randomly scattered on the surface of Torenia tissue samples; the laser was irradiated toward the gold particles using a hollow optical fiber and a quartz light-condensing tip to introduce the particles into the target cells. Treated plant tissue samples were then cultured for several weeks on the selection medium, and transformation was assessed by fluorescence microscopy and PCR. Irradiation-induced damage to the cells and coated DNA was insignificant, and the transformation efficiency was similar to that of biolistic transformation based on the particle number. Although this method requires a suitable laser setup and is time-consuming, it could be applied in transformation experiments on specific target cells. Moreover, this method can possibly be used to introduce not only DNA but also biologically active substances such as protein regulators or inhibitors, whose introduction into cells is difficult through the cell wall.
As is the case with other organisms, plants respond to genotoxic stresses by expressing DNA repair genes upon DNA damage. To uncover the mechanisms involved in regulated expression of the DNA damage-responsive gene, we investigated the tissue specific expression and DNA damage-responsiveness of the Arabidopsis RAD51 (AtRAD51) gene promoter in Arabidopsis and tobacco. Transgenic Arabidopsis and tobacco plants harboring AtRAD51 promoter- β-glucuronidase (GUS) were used to study the detailed expression pattern of the AtRAD51 gene. A histochemical GUS assay of bleomycin- or UV-treated plants showed that the AtRAD51 promoter in young tissues is actively expressed particularly in meristematic cells of the root and shoot apex of seedlings. In the absence of genotoxic stress, GUS activities were detected only at very low levels in these same organs. In mature plants, the AtRAD51 promoter is mainly expressed in flower bud, sepal, stigma, later anther, pedicel when treating with DNA damaging agent. The expression patterns of reporter assays were consistent with the AtRAD51 mRNA accumulation pattern. These results suggest that the regulated expression of the AtRAD51 gene is controlled mainly at the level of transcription directed primarily by the promoter function of the gene.
The formation of thiamin-binding proteins (TBPs) from sesame (Sesamum indicum L.) seeds (STBP-I, -II and -III) in developing seeds was investigated. Elution profiles of STBPs obtained from seeds at 4 to 6 weeks after flowering from the Q-Sepharose Fast Flow column showed that only one STBP was contained in the seeds at 4 weeks, and two STBPs were contained in the seeds at 5 weeks, and all three STBPs were contained in the seeds at 6 weeks as well as in mature seeds. SDS-PAGE of those STBPs demonstrated that STBP in the seeds at 4 weeks was large STBP termed as STBP-III, the molecular mass of which was higher than STBP-I and -II. In addition, it was demonstrated that small and large STBPs were contained in the seeds at 5 weeks, and STBP-I, -II and -III were contained in the seeds at 6 weeks. On the other hand, Southern blot analysis of digested genomic DNAs from sesame leaves with an STBP cDNA probe indicated the presence of only one copy of STBP in the sesame genome. These results suggested that STBP-III was first produced from a large proprotein precursor in developing sesame seeds, and STBP-I and -II were produced from the same polypeptide chain by different types of post-translational processing.
Basic helix-loop-helix (bHLH) proteins constitute one of the most abundant types of transcription factor in both plants and animals. They are considered to be localized in the nucleus, but recent surveys have revealed two exceptions, NtWIN4 from tobacco and AtPTF1 from Arabidopsis, both reported to localize to plastids. Their specific subcellular localization was suggested to be the result of structural change, whose analysis might provide clues to understanding protein evolution. Consequently we performed comparative analyses on localization features of both NtWIN4 and AtPTF1 using GFP-tagged fusion proteins. While epifluorescence from NtWIN4-GFP was clearly observed in plastids, that from AtPTF1-GFP was unexpectedly seen only in nuclei. Subsequent transcription assays using a dual-luciferase system indicated that AtPTF1 possesses clear nuclear transcriptional repression activity. It is concluded that, in contrast to the previous paradigm, AtPTF1 is a nuclear transcriptional repressor, and therefore that NtWIN4 is the only non-nucleus resident bHLH protein.
Cytosines in eukaryotic DNA is often methylated to yield 5-methylcytosines (m5C), which play an important role in controlling gene expression. This is referred as DNA methylation, and its status dynamically changes during plant growth by active methylation and demethylation. DNA glycosylases have been known to possess base excision DNA repair activity, and an Arabidopsis enzyme, ROS1 (Repressor of Silencing 1), was recently shown to exhibit m5C DNA glycosylase activity. We isolated and characterized four genes encoding ROS1-like proteins from tobacco plants (Nicotiana tabacum), and designated as NtROS1, NtROS2a, NtROS2b and NtROS3, each respectively encoding a polypeptide with 1796, 1673, 1673 and 1662 amino acids. Purified NtROS1 and NtROS2a proteins expressed in Sf9 insect cells clearly exhibited activity of m5C removal from tobacco genomic DNA in vitro. GFP fusion assay showed that NtROS1 and NtROS2a were localized in nucleus. Transcripts of NtROS1, NtROS2a and NtROS3 were induced by abiotic stresses, including aluminium, salt and reactive oxygen species. These observations suggested that NtROS proteins function in demethylating process of genomic DNA during plant stress response, thereby maintaining the balance of gene expression in combination with DNA methyltransferases.
It is well known that DNA methylation is involved in the control of transposable elements in eukaryotic cells. Recent studies indicate that demethylation of DNA in a mutant of Chlamydomonas and Arabidopsis causes transcriptional activation and mobilization of transposons. In this report, transposition of a retrotransposon was investigated in the phytopathogenic fungus Fusarium oxysporum treated with 5-azacytidine (5azaC), a reagent that causes reduction in the DNA methylation level. The results showed elevated transposition frequency in 5azaC-treated isolates when they were incubated for a long time. However, increase of retrotransposon transcripts was not observed, suggesting that the retrotransposon was mobilized by a mechanism other than its transcriptional activation.
Carbon-ion beam is a new irradiation source for inducing mutations in plant breeding effectively. In order to apply this new technique in banana breeding program, we studied the critical doses for in vitro irradiation and the genetic variability for black Sigatoka in the regenerated plants. Carbon-ion beam was irradiated to in vitro plantlets of banana cultivars ‘Cavendish Enano’ and ‘Williams’ with the dose of 0, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 Gy. Biological effects on survival rate were recorded and 8 Gy was supposed to be the best dose. Survived plantlets were propagated in vitro to evaluate resistance to black Sigatoka. Six plants from ‘Williams’ population and two plants from ‘Cavendish Enano’ population were selected as candidates for resistant plants to black Sigatoka in the field, suggesting that carbon-ion beam could be useful for mutation breeding in banana.
Boron (B) is an essential nutrient for plants but is toxic at high levels. We performed transcriptome analysis under conditions of both low and high B in Arabidopsis thaliana. There is a positive correlation between the levels of gene induction by low and high B, suggesting that common pathways operate during these responses. Induction by high B was also confirmed for nine genes in an independent experiment. Transcripts of At1g03770 (transcription factor) and At5g57340 were found to be elevated in roots, whereas At2g04040, At2g04050, At2g04070 (all encoding multidrug and toxic compound extrusion transporters), At1g32870 (transcription factor), At5g51440 (heat shock protein-like), At2g41730 and At2g21640 are induced in the shoots. The accumulation of both At2g04050 and At5g51440 transcripts was further found to be elevated by more than 100-fold after two days of high B treatment. Our findings thus represent the first identification of high B-induced genes.