Sulfur is one of the essential macronutrients required for plant growth. Since the expression of several sulfur-assimilatory genes is stimulated under the condition of sulfur deficiency (−S), transcriptional regulation of these genes is considered to be critical for the control of sulfur assimilation. In the last several years, the author and coworkers have been investigated molecular mechanisms of −S-inducible expression of high-affinity sulfate transporters, SULTR1;1 and SULTR1;2 in Arabidopsis thaliana. SULTR1;1 and SULTR1;2 facilitate sulfate uptake in roots. This review summarizes the recent progress about the transcriptional regulation of these sulfate transporters by focusing on three major topics. 1) Identification of a cis-acting element involved in the −S-inducible expression of SULTR1;1. 2) Cytokinin-dependent repression of SULTR expression and sulfate uptake. 3) Central transcription regulator SLIM1 controlling −S responsive gene expression including sulfur assimilation and metabolism.
The gene encoding the taste-modifying protein miraculin was introduced under the control of the 35S or El2 promoter into strawberry (Fragaria x ananassa) by Agrobacterium-mediated transformation to produce transgenic plants. Although miraculin was detected in the leaves and fruits of the transgenic plants, the level of accumulation among the transgenic lines, which ranged from 0.5 to 2.0 μg g−1 fresh fruit, was not significantly different and was lower than that in miracle fruits (145 μg g−1 fresh fruit). High levels of miraculin accumulation were detected in the mature fruits. The transgenic lines were subsequently propagated via the runners for three vegetative generations, and miraculin was detected at equal levels in the leaves and fruits of the plants from each generation. In conclusion, although the level of accumulation was not high, miraculin was stably expressed and accumulated in the vegetative progeny of the transgenic strawberry plants.
Sugars are important regulators of germination and seedling development, and sugar signaling is thought to be involved in the repression of embryonic characteristics after germination. In contrast, chromatin remodeling induced by histone modification plays an important role in the control of plant development. We found that HDA6 and HDA19 redundantly repress embryo-specific gene expression during germination in Arabidopsis. We analyzed the effects of sucrose-containing or sucrose-free medium on post-germination growth arrest in an HDA6/19 double repression line (HDA6/19:RNAi). Although HDA6/19:RNAi seeds showed post-germination growth arrest when grown in sucrose-containing medium, sucrose-free medium relieved growth arrest, indicating the suppression of embryo-specific genes. Thus, sugar signaling may prevent the HDAC-mediated repression of embryonic characteristics after germination. Based on expression analyses of VSP2 in HDA6/19:RNAi seedlings, the repression of HDA6 and HDA19 may not affect sugar signaling. During germination, HDA6 and HDA19 repress embryonic properties via regulation of embryo-specific transcription factors. Sugar signaling may also contribute to this repression of embryonic properties.
In vitro culture from seeds to full maturity of Cypripedium macranthos var. rebunense, one of the most endangered species in Japan, was studied. Immature seeds from wild plants germinated efficiently in Tsutsumi-Tomita medium and 1/3 strength Murashige-Skoog medium both supplemented with 1% sucrose. Formed protocorms were transferred onto the same Murashige-Skoog medium developed into seedlings, which were subjected to a cold-treatment. The seedlings were further successively cultured in artificial soils (Florialite and then Cryptomoss) under artificially controlled photoperiod and temperature conditions. After 7 years, fully matured plants successfully flowered. The genetic diversity of the wild colony of this orchid in Rebun Island was examined. Leaf samples randomly collected were subjected to random amplified polymorphic DNA analysis. Initially examined 31 samples were classified into independent 5 groups, indicating that the colony consists of genetically different individuals. This suggested that plants in the habitat are heterogenous as to the genetic structure. Introduction of artificially propagated plants with a similar genetic background to this habitat would match the genetic diversity of the existing population. The results of this study may be helpful in planning the rescue of other endangered plant species.
The hairy root culture system has several desirable features as an experimental model for functional analyses of the genes involved in root metabolism and/or physiology. We developed a binary vector set for efficient target gene overexpression and RNA interference (RNAi) in transformed (hairy) roots. The vectors, pHR-OX and pHR-RNAi, contain a cluster of rol (rooting locus) genes, together with the single GATEWAYTM conversion cassette (in pHR-OX vectors) or inverted repeats of the GATEWAYTM conversion cassette separated by an intron sequence (in pHR-RNAi vectors), flanked by a dual CaMV 35S promoter and nopaline synthase polyadenylation signal, on the same T-DNA. Transformation experiments with pHR-OX vectors using Arabidopsis, potato, and tobacco as model plants revealed that by inoculating Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants in a short period of time, and subsequent establishment of root culture lines was possible. A model experiment that focused on the sterol biosynthetic pathway in Arabidopsis validated the utility of pHR-RNAi vectors for gene functional analysis in cultured roots. Use of our vector system may facilitate identification of regulatory or biosynthetic genes for the production of valuable secondary metabolites in plant roots.
We hypothesized that root nodule formation would be promoted by lower expression of a β-1,3-glucanase gene (designated as LjGlu1), because expression of this gene is increased in transgenic Lotus japonicus that shows reduced nodulation. In order to suppress the expression of this gene, we introduced the antisense LjGlu1 gene into L. japonicus via Agrobacterium rhizogenes. Although there was no significant difference in shoot and root growth between L. japonicus possessing the antisense construct and plants containing an empty vector, the number of root nodules 28 days after inoculation with Mesorhizobium loti MAFF303099 increased in the antisense plant compared with the control plant. Moreover, the nitrogen fixation activity of the antisense plant was drastically enhanced. The mechanism of enhanced nitrogen fixation by suppression of the expression of LjGlu1 is unknown, but this phenomenon should be an important breeding target for leguminous plants. This is the first report of strong enhancement of nitrogen fixation activity by manipulation of a gene of the leguminous host plant.
The cyanide-insensitive alternative oxidase (AOX) respiratory pathway can reduce the excessive production of reactive oxygen species. Here we showed that transgenic rice seedlings transformed with OsAOX1a for rice alternative oxidase under the control of ubiquitin promoter exhibited thermotolerance after acute exposure, 41–45°C for 10 min, or chronic exposure, 37°C for up to 8 days. In contrast, these high temperature stresses resulted in significant growth inhibition in wild-type and transgenic plants with antisense OsAOX1a. The enhanced tolerance was significant in shoot growth, suggesting that the increased levels of AOX protein would dissipate the excess reductants produced in the chloroplasts suffering from oxidative damage due to high temperature stress.
ANGUSTIFOLIA (AN), the first gene encoding a putative C-terminal binding protein (CtBP) identified in plants, controls leaf cell width in a polarity-dependent manner by regulating the arrangement of cortical microtubules. However, several differences in the sequence of AN have been detected in its conserved CtBP domains, which are transcriptional co-repressors in animals; thus, it is unclear whether AN is a transcriptional co-repressor in plants and how it may function. In this study, we found that AN possesses incomplete D2-HDH and GxGxxG(17x)D motifs, which confer CtBP dehydrogenase activity and NAD-binding for interaction with PxDLS motifs, respectively. In addition, full-length AN was unable to couple the reduction of pyruvate to lactic acid with the oxidation of NADH to NAD+, suggesting that it might not have dehydrogenase activity. Moreover, we found that AN has an additional short open reading frame (ORF), which was identified as an upstream ORF (uORF), in its 5′-untranslated region that overlaps with the start codon of the AN gene. Transcriptional analysis revealed that the uORF and AN ORF are transcribed as a single molecule, indicating that the uORF might influence AN transcription during leaf development.
Four 1-O-hydroxycinnamoyl-β-D-glucoses (HCA-Glcs), sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-glucoses, were synthesized using a recombinant protein of sinapate glucosyltransferase from Gomphrena globosa coupled with a recycling system of UDP-glucose by sucrose synthase from Arabidopsis thaliana. The substrate preference of HCA-Glc-dependent acyltransferase activity was examined in a protein extract prepared from anthocyanin-producing cultured cells of Daucus carota and Glehnia littoralis. The main anthocyanin molecule of the aglycon and the sugar moiety produced and accumulated in both cultured cells were exactly the same; the only difference was found in modification with sinapoyl moiety in D. carota and with feruloyl moiety in G. littoralis. The protein extracts from both D. carota and G. littoralis cultured cells showed higher activity with feruloyl-Glc than with sinapoyl-Glc. The major HCA-Glcs that accumulated in cultured cells of D. carota and G. littoralis were sinapoyl-Glc and feruloyl-Glc, respectively. These results suggested that the specificity of HCA moieties of major anthocyanin molecules in cultured cells of D. carota and G. littoralis might be dominated by produced and accumulated acyl donor molecules in vivo rather than by the substrate specificity of acyltransferase enzymes.
Most plant metabolites are uncharacterized, even in well-analyzed plant species. High-accuracy measurement of mass values by state-of-the-art mass spectrometers such as Fourier transform ion cyclotron resonance mass spectrometers allows prediction of possible molecular formulas for each metabolite. As a first step in comprehensive metabolite identification from mass spectrometry data, here we have developed a computational tool for high-throughput prediction of molecular formulas and identification of isotopic peaks. The program generates all possible formulas for each mass value under given parameters. To reduce calculation time, monophosphate, diphosphate, triphosphate and bisulfate groups are regarded as monovalent units during formula generation. Prediction of isotopic peaks associated with each metabolite also facilitates reduction in the number of possible formulas. The tool implements these procedures for all mass values from a set of mass spectrometry data. The tool facilitates subsequent annotation of metabolites, which can be integrated with metabolome databases.
Phyllostachys meyeri cultivated in the Fuji Bamboo Garden, Japan showed monocarpic mass flowering from 2004 through 2005. We first examined in vitro germination of the caryopses and found that sterilized water supplemented with 0.1% Plant Preservative MixtureTM was effective for their growth with an efficient germination rate (75%). Seedlings were transferred to a liquid modified half strength Murashige and Skoog (m1/2MS) medium for further growth and were acclimatized in soil mixture under ex vitro condition. Nodal segments which were collected from ca. 1.5 year-old seedlings and 1 year-old tissue cultured clone plants were re-sterilized and then cultured in a liquid m1/2MS medium. Shoot elongation with rooting could be seen within 8 weeks and they were successfully multiplied under ex vitro condition.
Techniques of exogenous gene introduction in plant cells have been actively applied for producing transgenic cultured cells that produce high levels of useful biological substances and are used for the molecular breeding of plants. A novel gene transfer technique has been developed using calcium alginate micro-beads (bioactive beads) to entrap genetic material. Although this technique has several advantages such as high transformation efficiency and the ability to introduce large DNA molecules, the precise optimal application conditions remain to be determined. Here, the optimal conditions for the concomitant use of bioactive beads and electroporation, such as polyethylene glycol (PEG) concentration, alginate concentration, electric field intensity, pulse duration, and pulse numbers, were determined for improved gene transfer efficiency of bioactive bead-mediated plant transformation. Consequently, highly efficient transient transgene expression (up to 4.7%) was achieved by applying electrical pulses (0.75 kV CM−1, 30 μs, 3×) to a protoplast suspension in 12% polyethylene glycol (PEG).
Analysis of GUS expression patterns was previously carried out for 20,000 gene trap lines of Arabidopsis thaliana generated by the Kazusa DNA Research Institute. This analysis indicated that 51 lines showed GUS expression within stamens. In this paper, we further focused on 42 of these lines in order to examine the detailed expression patterns at different anther developmental stages. Seven lines showed GUS expression exclusively in the tapetum during the early stage of anther development, thirty lines in pollen grains at the tricellular pollen stage and five lines in filaments at the tricellular pollen stage. We identified four trapped-genes by sequencing the adjacent fragments to T-DNA insertions. RT-PCR analysis confirmed that the mRNA expression patterns in these four genes were consistent with the expression pattern observed by GUS activity. Our results demonstrated that the gene trap system is an efficient strategy for identifying stamen specific genes in Arabidopsis.
In eukaryotic cells, accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the transcriptional induction of ER-resident molecular chaperones and folding enzymes to maintain cellular homeostasis, termed the ER stress response or the unfolded protein response (UPR). In this study we focused on a putative transcriptional regulator of Arabidopsis thaliana whose transcripts accumulate in response to the ER stress inducer tunicamycin. This gene, designated as TCR1 (tunicamycin-induced CONSTANS-like-related 1), encodes a predicted protein of 195 amino acids with a plant-specific CCT (CONSTANS, CONSTANS-like, TOC1) domain considered to function in transcriptional regulation. In a dual-luciferase assay using Arabidopsis protoplasts, we showed that induction of the TCR1 gene by tunicamycin is independent of a nucleotide sequence similar to the cis-element ER stress response element on its promoter. Our result further indicates that this induction could be mediated by the transcription factor AtbZIP60. This study is a first step to understand the role of TCR1 in the Arabidopsis ER stress response.
We describe a simple cost-effective technique for the transport of binary vector plasmid DNA by mail. Our results demonstrate that common multipurpose printing paper is a satisfactory substrate and superior to the more absorbent 3 MM chromatography paper for the transport of plasmid DNA through the mail. We show that depositing as little as 100 ng of purified plasmid onto multipurpose printing paper is sufficient to allow recovery of the mailed DNA. Also observed was an inverse correlation between vector size and recovery of intact plasmid DNA, thus larger binary vectors require the deposition of more plasmid to ensure successful recovery.
Tobacco (Nicotiana tabacum) was recently shown to detoxify heavy metals by exudation of metals as a metal-substituted calcite (calcium carbonate) through leaf trichomes. In this paper, we describe the applications of the variable-pressure scanning electron microscopy (VP-SEM) system to investigate tobacco trichomes exudates after heavy metal treatment. An energy-dispersive X-ray analysis (EDX) system fitted to VP-SEM revealed that the exudates contain amounts of heavy metals. Overexpression of cysteine synthase confers cadmium (Cd) tolerance to tobacco, and the endogenous concentration of Cd was 20% less in transgenic plants than in wild-type plants. We evaluated the numbers of trichomes on the leaf surfaces of wild-type and transgenic plants using VP-SEM. The numbers of both long and short trichomes in the transgenic plants were 25% higher than in that of wild-type plants, indicating the active excretion of Cd from trichomes in transgenic plants. The VPSEM-EDX system is a powerful tool to investigate plant epidermal structures and functions.