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In order to screen for the best species for mitigating nitrogen dioxide (NO2) by plants at urban levels, we investigated assimilation of nitrogen dioxide by 70 taxa of woody plants that are mostly utilized as roadside trees. They were fumigated with 15N-labeled NO2 at 0.1 microl l(-1) for 8h, and the amount of reduced nitrogen derived from NO2 (in mg Ng(-1) dry weight) in the leaves (designated NO2 assimilation capability hereafter) were determined. Data were analyzed in the comparison with the previously reported ones obtained at 4 microl l(-1) NO2. Among the 70 taxa, the value of NO2 assimilation capability differed by a factor of 122 between the highest (Prunus yedoensis; 0.061) and the lowest (Cryptomeria japonica; 0.0005). Based on the analysis of NO2 assimilation capability values at 0.1 and 4 micro l(-1) NO2, the 70 taxa of woody plants appeared to be classified into four types; those of high NO2 assimilation and high NO2 resistance, those of high NO2 assimilation but low NO2 resistance, those of low NO2 assimilation and low NO2 resistance, and those of low NO2 assimilation but high NO2 resistance. The first, second, third and fourth types include 13, 11, 35 and 11 taxa, respectively. The broad-leaf deciduous trees may have advantages of high biomass and fast growth as compared with woody plants of other habits. Thus, four broad-leaf deciduous species, Robinia pseudo-acacia, Sophora japonica, Populus nigra and Prunus lannesiana, were concluded here to be the best phytoremediators for the urban air.

In this short review, basic processes for phytoremediation and plant enzymes that are potentially useful in phytoremediation are briefly summarized. The remaining part describes the applications of the basic processes to decontaminate pollutants in the environments that have been done in our laboratory. Our work includes (1) analysis of the capability of various naturally occurring plants to decontaminate atmospheric nitrogen dioxide and selection of nitrogen-dioxide-philic plants that grow with nitrogen dioxide as the sole nitrogen source, (2) production of transgenic plants to improve plants' capability to decontaminate atmospheric nitrogen dioxide, and (3) analysis of the denitrification process in plants to innovate a gas--gas-converting plants that convert nitrogen dioxide to nitrogen gas.

We succeeded expressing the streptomyces cholesterol oxidase gene (choA), a potent insecticidal protein active against boll weevil larvae, in tobacco cells. A plasmid, pBC4, containing the choA gene under the control of the 35S promoter of cauliflower mosaic virus, was introduced into tobacco cells (BY-2) using a pneumatic particle gun. Stable integration of the choA gene in the genome of the transformed callus line was confirmed by Southern hybridization analysis. Northern blot analysis revealed material corresponding in size to the choA transcript in the transformed cells. Cholesterol oxidase activity in transformed callus was about eightfold higher than that in control cell line.

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10(6) per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.

Phylogenetic relationships between four Phaseolus (P. angularis, P. calcaratus, P. coccineus and P. vulgaris (including six cultivars)) and two Vigna (V. sesquipedalis and V. sinensis) species were studied by restriction fragment length polymorphism (RFLP) analysis using 15 restriction enzymes and two ribosomal DNA fragments from rice and mung bean as probes. Based on the RFLP data, phylogenetic trees were constructed by the distance matrix and maximum parsimony methods. The two trees constructed by the distance matrix method and one tree constructed by the maximum parsimony method had congruent topology and recognized three clusters: one containing V. sesquipedalis and V. sinensis; a second containing P. angularis; and a third containing P. calcaratus, P. coccineus and six cultivars of P. vulgaris. Grouping of the six species based on the molecular taxonomic data obtained is compared with grouping based on previously reported cytological data.

Assimilatory nitrite reductase (aNiR) reduces nitrite ions (NO(2)(-)) to ammonium ions (NH(4)(+)), whereas assimilatory sulfite reductase reduces sulfite (SO(3)(2-)) to hydrogen sulfide (HS(-)). Although aNiR can also reduce SO(3)(2-), its activity is much lower than when NO(2)(-) is reduced as the substrate. To increase the SO(3)(2-)-reduction activity of aNiR, we performed a N226K mutation of Nii3, a representative aNiR. The resulting Nii3-N226K variant could bind non-native targets, SO(3)(2-), and HCO(3)(-), in addition to its native target, i.e., NO(2)(-). We have determined the high-resolution structure of Nii3-N226K in its apo-state and in complex with SO(3)(2-), NO(2)(-), and HCO(3)(-). This analysis revealed conformational changes of Lys226 and the adjacent Lys224 upon binding of SO(3)(2-), but not NO(2)(-)In contrast, HCO(3)(-) binding induced a conformational change at Arg179. After replacing Asn226 with a positively charged Lys, aNiR showed affinity for several anions. A comparison of all ligand-bound structures for Nii3-N226K revealed that structural changes in the active site depend on the size of the substrate.

Phylogenetic relationships among eight taxa of seven species of Phaseolus and Vigna (Phaseolus angularis, P. aureus, P. calcaratus, P. coccineus, P. vulgaris, Vigna sesquipedalis and V. sinensis; 2n = 22 each) were studied by the fluorescent chromosome banding technique. Preparations of somatic metaphase chromosomes of each taxon were sequentially stained with Giemsa, GC-specific fluorochrome chromomycin A3 (CMA) and AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). On the basis of the fluorescent banding patterns of the 22 chromosomes of each taxon, P. angularis, P. coccineus (from China and Korea) and P. vulgaris were grouped into one group ("Phaseolus group"), P. aureus and two Vigna species were grouped into another ("Vigna group") and P. calcaratus was grouped in an independent group.

Transient expression of the β-glucuronidase (GUS) gene in suspension-cultured cells of tobacco was obtained after gene delivery with a pneumatic particle gun device driven by compressed air. Both the pneumatic pressure used to accelerate the particles as well as the distance between the point of release of the particles from the device and the location of the target cells could be varied. Efficiency of gene delivery was tested by accelerating pressure from 63 to 200 kg/cm(2) and distances from 5 to 15 cm. At 150 kg/cm(2) and 10cm, 618±49 blue spots of GUS-expressing cells were evenly distributed over the surface of the cell layer (3.5 cm diameter). The frequency of transient gene expression was estimated to be more than 1.2×10(-3), which is comparable to, or even higher than, that obtained by the gunpowder-driven device.

Xanthine dehydrogenase (XDH) is a ubiquitous enzyme involved in purine metabolism which catalyzes the oxidation of hypoxanthine and xanthine to uric acid. Although the essential role of XDH is well documented in the nitrogen-fixing nodules of leguminous plants, the physiological importance of this enzyme remains uncertain in non-leguminous species such as Arabidopsis. To evaluate the impact of an XDH deficiency on whole-plant physiology and development in Arabidopsis, RNA interference (RNAi) was used to generate transgenic lines of this species in which AtXDH1 and AtXDH2, the two paralogous genes for XDH in this plant, were silenced simultaneously. The nearly complete reduction in the total XDH protein levels caused by this gene silencing resulted in the dramatic overaccumulation of xanthine and a retarded growth phenotype in which fruit development and seed fertility were also affected. A less severe silencing of XDH did not cause these growth abnormalities. The impaired growth phenotype was mimicked by treating wild-type plants with the XDH inhibitor allopurinol, and was reversed in the RNAi transgenic lines by exogenous supplementation of uric acid. Inactivation of XDH is also associated with precocious senescence in mature leaves displaying accelerated chlorophyll breakdown and by the early induction of senescence-related genes and enzyme markers. In contrast, the XDH protein levels increase with the aging of the wild-type leaves, supporting the physiological relevance of the function of this enzyme in leaf senescence. Our current results thus indicate that XDH functions in various aspects of plant growth and development.

To clarify the molecular structure of the integration sites of transgenes, we used particle bombardment to examine the DNA sequences of transgene loci. Three transgenic Arabidopsis lines gave a single Southern hybridization band with a selectable gene as the probe. Junction regions flanked by the transgenes were cloned by the inverse polymerase chain reaction method, and the characteristics of the DNA sequences of the 10 junction regions were investigated. All but two of these were AT-rich sequences bearing motifs characteristic of a scaffold/matrix-attachment region (S/MAR). Calculations showed that seven of them should have a propensity for curvature. An assay of in-vitro binding to tobacco nuclear matrices showed that all the junction regions bound to nuclear matrices and that the two input DNAs did not bind. The 12 chromosome/transgene (CT) junctions in these three transgene loci were investigated. Cleavage sites for topoisomerase I were found at 10 of the 12, near the junction point. The other two junctions had sites within 6 bp of the junction point. The sequence near one terminal of the transgene in the transgene loci was compared with that near the other terminal. Short, direct repeats consisting of 4–6 bp were present within 10 bp of the junction points in the sequence. We speculate that the transgene introduced by particle bombardment is delivered on AT-rich S/MAR that has a propensity for curvature, and then a nucleotide near the short, direct repeat on the transgene is joined near the cleavage sites on the genome for topoisomerase I.

A ferredoxin-nitrite reductase (EC 1.7.7.1) cDNA was isolated and sequenced from a lambda gt 11 cDNA library constructed from nitrate-induced greening shoots of rice (Oryza sativa L.) seedlings. The nucleotide sequence of the cDNA clone contains an open reading frame of 1788 nucleotides. There exists a strong bias for the third codon usage of G/C (95.5%) as in the case of the maize enzyme. The deduced amino acid sequence shows an overall homology to the maize (81%) and the dicot enzymes (70-74%), suggesting that the primary structure of ferredoxin-nitrite reductase is highly conserved in higher plants.

Gold particles coated with beta-glucuronidase (GUS) mRNA with a 5' cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.

A number of A/T-rich sequences and a CATGCAT/A sequence are contained in the 5'-upstream regions of the genes encoding concanavalin A (Con A) and canavalin, two major seed storage proteins of Canavalia gladiata D.C. To study the role of these sequences in the seed-specific gene expression, we constructed 5'-deletion mutants and examined the transient expression of beta-glucuronidase reporter gene by particle bombardment and the stable expression by Agrobacterium-mediated transformation of tobacco plants. Positive regulatory elements were located in the -894/-602 and -602/-74 regions of the Con A gene, and in the -428/-376, -281/-155 and -155/-50 regions of the canavalin gene. In addition, the results suggested that the A/T-rich sequences in the 5'-upstream region of the Con A gene play a role in transcriptional activation, but that those of the canavalin gene have little effect on the gene expression. The CATGCAT/A sequence was not sufficient by itself for high levels of expression of both the Con A and canavalin genes. The canavalin polypeptide amounted to about 1% of the total extractable protein in the transgenic tobacco seeds, but the Con A polypeptide was not detected in the extractable protein.

Successful transient expression of beta-glucuronidase (GUS) in Arabidopsis thaliana leaves and roots and Brassica napus stems was obtained after gene delivery with a pneumatic particle gun driven by compressed air. Effects of the pneumatic pressure used to accelerate the particles (accelerating pressure; 85 to 200 kg/cm2) and of preculture periods of plant tissues (0 to 6 days) on the efficiency of gene delivery were studied. In A. thaliana leaves, best results were obtained at 115 kg/cm2 of accelerating pressure and 3 days of preculture. In A. thaliana roots, the optimum was at 200 kg/cm2 of accelerating pressure and 3 days of preculture. These results indicate that both preculture period and accelerating pressure are vital factors that determine the efficiency of gene delivery by particle gun.

Stable transformation of suspension-cultured cells of tobacco with plasmid DNA (pCaMV-NEO) harbouring the neomycin phosphotransferase II (npt-II) gene was achieved using a previously described gas-pressure-driven particle acceleration device. The cells were bombarded with DNA-coated gold particles accelerated by the device, and callus containing the introduced gene was selected in the presence of geneticin disulphate. The geneticin-resistant callus exhibited npt-II enzyme activity, and Southern analysis confirmed the stable integration of the foreign gene into the tobacco genome. Transformants were obtained at a rate of more than 1.9 × 10−4.

When stirred with powdered samarium and iodine in methanol at ice-cold temperature, ethyl 1,2,3-thiadiazole-4-carboxylate 5 underwent unusual reduction involving the dimeric ring enlargement with a sulfur addition, giving dimethyl 1,2,5-trithiepan-4,6-dicarboxylates 7a,b as a cis/trans-isomeric mixture in acceptable yield. The 1,2,3-thiadiazole ring of 5 proved to resist reduction by ordinary reducing agents, where the only choice in most cases was either recovery of the unchanged substrate or decomposition to an intractable mixture of unidentified products and tar.

We obtained complete hybrid plants by electrofusion of mesophyll protoplasts from Nicotiana glauca and N. langsdorffii. Electrofocusing analysis of Fraction I proteins isolated from the leaves of these plants confirmed their hybridity. Cytological analysis indicated that the chromosome number (2n) of these plants is between 60 to 66, suggesting that they are the products of triple fusion. All plants were fertile and set viable seeds after self pollination. As we did not use an AC field for electrofusion, the present results indicate that an AC field is not essential for obtaining hybrid plants with electrofusion.

It was found that plasmid DNA (YEp13) can be introduced into intact yeast cells by electric field pulses. The frequency of transformation by this electroinjection method depend upon the initial electric field intensity, the capacitance of the electric discharge capacitor, and the number of pulses applied. A maximum number of transformants (90±20/μgDNA) was obtained by three successive pulses with an initial intensity of 5 KV/cm and with a capacitance of 1 μF. The present electroinjection method is simple, and transformants can be obtained within 2 to 3 days after transformation treatment.

Successful transient expression of genes (luciferase and β-glucuronidase) in cultured tobacco cells and adventitious shoots of egg-plant hypocotyls can be obtained by use of a simple biolistic device. This device is driven by controlled gas pressure from a cylinder of nitrogen gas. It has the advantages of being free of explosive heat and of avoiding cell damage caused by expanding gas, due to the “self-sealing” effect of the projectile.

Cultured cells of Lavandula vera were entrapped with photosensitive synthetic resin prepolymers (PVA-SbQ). PVA-SbQ-entrapped cells grew well inside gel matrices and synthesized de novo blue pigments in the presence of l-cysteine as an inducer. The entrapped cells were superior to calcium alginate-entrapped cells judging from cell growth and total pigment productivity. Release of the pigments, which were almost insoluble in water, from the gels was markedly enhanced by the increase in hydrophilicity of the cell-entrapping gels. The entrapped cells could be used repeatedly for the pigment production.