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In order to investigate nocturnal uptake and assimilation of NO2by C3 and crassulacean acid metabolism (CAM) plants, they were fumigated with 4 µl I-115N-Iabeled nitrogen diox­ide (NO2) for 8 h. The amound of NO2 and assimilation of NO2by plants were determined by mass spectrometry and Kjeldahl-nitrogen based mass spectrometry, respectively. C3 plants such as kenaf (Hibiscus cannabinus), tobacco (Nicotiana tabacum) and ground cherry (Phy- salis alkekengi) showed a high uptake and assimilation during daytime as high as 1100 to 2700 ng N mg-1dry weight. While tobacco and ground cherry strongly reduced uptake and assimilation of NO2during nighttime, kenaf kept high nocturnal uptake and assimilation of NO2as high as about 1500 ng N mg-1dry weight. Stomatal conductance measurements indicated that there were no significant differences to account for the differences in the uptake of NO2by tobacco and kenaf during nighttime. CAM plants such as Sedum sp., Kalanchoe blossfeldiana (kalanchoe) and Aloe arborescens exhibited nocturnal uptake and assimilation of NO2. However, the values of uptake and assimilation of NO2both during daytime and nighttime was very low (at most about 500 ng N mg-1dry weight) as compared with those of above mentioned C3 plants. The present findings indicate that kenaf is an efficient phytoremediator of NO2both during daytime and nighttime.

Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C. 1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The ob­tained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the noil-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 m? TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when they were floated on TNT solution. HPLC analysis showed that the peak due to TNT taken up into plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indi­cates that the expression of bacterial NTR improved the capability of plants to degrade TNT.

Our previous study showed that approximately one-third of the nitrogen of 15N-labeled NO2taken up into plants was converted to a previously unknown organic nitrogen (hereafter designated UN) that was not recoverable by the Kjeldahl method (Morikawa et al., 2004). In this communication, we discuss metabolic and physiological relevance of the UN based on our newest experimental results. All of the 12 plant species were found to form UN derived from NO2(about 10-30% of the total nitrogen derived from NO2). The UN was formed also from nitrate nitrogen in various plant species. Thus, UN is a common metabolite in plants. The amount of UN derived from NO2was greatly increased in the transgenic tobacco clone 271 (Vaucheret et al., 1992) where the activity of nitrite reductase is suppressed less than 5% of that of the wild-type plant. On the other hand, the amount of this UN was significantly decreased by the overexpression of S-nitrosoglutathione reductase (GSNOR). These findings strongly suggest that nitrite and other reactive nitrogen species are involved in the formation of the UN, and that the UN-bearing compounds are metabolizable. A metabolic scheme for the formation of UN-bearing compounds was proposed, in which nitric oxide and peroxynitrite derived from NO2or endogenous nitrogen oxides are involved for nitrosation and/or nitration of organic compounds in the cells to form nitroso and nitro compounds, including N-nitroso and S-nitroso ones. Participation of non-symbiotic haemoglobin bearing peroxidase-like activity (Sakamoto et al., 2004) and GSNOR (Sakamoto et al., 2002) in the metabolism of the UN was discussed. The UN-bearing compounds identified to date in the extracts of the leaves of Arabidopsis thaliana fumigated with NO2include a ?2- 1,2,3-thiadiazoline derivative (Miyawaki et al., 2004) and 4-nitro-ß-carotene.

Mesophyll protoplasts from Nicotiana glaucaand Nicotiana langsdorffiiwere fused electrically. From a total of 9 × 104electrically fused protoplasts (4.5 × 104each), we obtained more than 96 green hybrid colonies that grew vigorously on hormone–free medium after 10 weeks of culture. Electrofocusing analysis of Fraction I proteins isolated from hybrid calluses confirmed the hybridity of the calluses. The hybrid isolation yield was about 0.1 percent. All hybrid colonies regenerated shoots after about 4 months of culture and some formed roots.

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.

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.

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 ß-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 ß-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.

A nitrite reductase (NiR) gene was recovered from Arabidopsis thaliana genomic library by the homology with a cDNA of spinach NiR and sequenced. Based on the comparison with the spinach cDNA, the Arabidopsis NiR gene was concluded to contain 4 exons [exon 1 of 376 bp (beginning with ATG start codon), exon 2 of 355 bp, exon 3 of 289 bp and exon 4 of 741 bp (ending at TGA stop codon)] and 3 introns (intron 1 of 196 bp, intron 2 of 81 bp and intron 3 of 77 bp). This conclusion was confirmed by the analysis using the RT-PCR method. The deduced amino acid sequence of the coding region of the Arabidopsis NiR gene had high similarities with those of NiR genes of other plants including spinach.

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.

Exploded wood lignin (EXWL) was extracted from moist steam hydrolysed aspen (Populustremuloides) wood using methanol. The lignin was examined by elemental analysis, gel permeation chromatography, infrared spectroscopy, proton and 13C nmr. The observed nmr spectra were compared with those of aspen milled wood lignin (MWL). Typical composition of the material is C9H9.2O2.7(OCH3)1.1Assignment of all the signals in the nmr spectrum led to the conclusion that the explosion process causes cleavage of the β-aryl-ether bond. The weight average molecular weight was found to be 1700–1900 and the polydispersity 2.6. The finely divided exploded wood was soluble in 90% methanol/water mixtures to well beyond 20% by weight at room temperature.

Abstract: Root explants ofArabidopsis thaliana ecotype C24 were bombarded with the plasmid pCH harboring the hygromycin phosphotransferase gene (hpt). A selection condition with post-bombardment culture of 3 days followed by culture with 20 mgl−1 hygromycin gave the highest yield of transformants. More than 44% of explant clumps formed transformant shoots.

Abstract: Chloroplast expression plasmids pTRBCL-GUS (tobaccorbcL promoter-gusA-tobaccorbcL terminator) and pHHU3004 (spinach ‘x gene’ promoter-gusA-spinachrbcL terminator) and a control nuclear expression plasmid pBI221 (CaMV 35S promoter-gusA-NOS terminator) were introduced separately into cultured cells and tissues of tobacco andArabidopsis thaliana, as well as into cultured cells of the lower land plants liverwort and hornwort by a pneumatic particle gun. The pTRBCL-GUS and pHHU3004 plasmids produced many blue spots in the BY-2 cells and the roots ofArabidopsis thaliana, but not in any of the green cells or tissues. The results suggest that the pTRBCL-GUS and pHHU3004 plasmids are expressed more in proplastids and amyloplasts than in chloroplasts. GUS activities of the BY-2 cells bombarded with pTRBCL-GUS and pHHU3004 were insensitive to α-amanitin treatment (10 and 50 μg/ml), while that of the cells with pBI221 greatly decreased by the same treatment. Hence, it is likely that the pTRBCL-GUS and pHHU3004 plasmids were substantially expressed in the proplastids.

Abstract: In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.

Abstract: Significance of bombardment-mediated transformation in plant research is discussed. Apart from general subjects related to this transformation study, a particular emphasis has been placed on the pollen and organelle transformation and on integration mechanism. Over 70 literatures related to this field are cited here.

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×106 cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.

A plasmid pARK 22 harbouring thebar gene encoding phosphinothricin acetyltransferase (PAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator was constructed and introduced into root sections ofArabidopsis thaliana using the pneumatic particle gun. The root sections that had been bombarded with this plasmid gave four to eight times higher yield of drug-resistant calluses than those sections bombarded with pCaMVNEO or pCH, which respectively contain the neomycin phosphotransferase and hygromycin phosphotransferase genes. Among a number of primary transformant (T0) plants obtained from independent bialaphos-resistant calluses, three were studied by Southern blot hybridization and PAT enzyme activity analyses, confirming the stable integration of the foreign gene into theArabidopsis genome and its expression in plants. The progeny analysis showed transmission of the foreign gene and its expression in up to the T2 generation. Some of the T1 progeny showed morphological abnormalities. Thus, thebar gene can be used effectively to allow selection of transgenicA. thalianna plants.

Transformed roots ofCucumis sativus were obtained from cotyledon tissues that had been bombarded with gold particles coated with plasmid pE7.4 using a pneumatic particle gun. This plasmid containsrolA, rolB, rolC genes and ORF 13 of the 7.4 kbEco RI fragment of T-DNA of pRi 1724 isolated fromAgrobacterium rhizogenes MAF 03-01724. The nature of the tissue and the composition of the culture media used greatly influenced the recovery of transformed roots. The transgenic nature of the derived roots was confirmed by the vigorous. highly-branched growth seen on a phytohormone-free medium. The stable integration ofrol genes into the cucumber genome was confirmed by Southern blot analysis.

Immature pollen fromNicotiana rustica was bombarded with gold particles coated with plasmid DNA encoding neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS) genes which, respectively, are under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator in the plasmid. Kanamycin-resistant pollen embryoids were selected from the bombarded pollen cells and two independent lines of transgenic plants were regenerated. Enzyme assays showed that one has both NPTII and GUS activities and the other only weak NPTII activity. Southern blot analyses indicated that the former has a DNA fragment corresponding to the intact expression cassettes for both genes in its genome; whereas the latter lacks intact expression cassettes for both genes and has only the intactnptII coding sequence in its genome. The transgenic plants of both lines have 24 chromosomes, confirming haploidy, and they are infertile. These results indicate that transgenic haploid plants can be produced directly by the bombardment-mediated transformation of immature pollen.

The solid state properties of poly-β-hydroxybutyrate (PHB) were investigated for samples with degrees of polymerization from 4 to 994. The observed melting points ranged from 47 °C to 180 °C. Electron diffractograms on carefully prepared single crystals of a high molecular weight sample provided data which confirmed the reported aand bparameters from X-ray fiber diffraction and provided clear justification for the P212121space group. The observed intensities in the electron diffractogram, corresponding to (hk0) reflections, were compared with the predicted intensities for two proposed structures in the literature. The observed and calculated structure factors for both structures were in good agreement. Small angle X-ray diffraction of the meridional maximum for annealed "cold drawn" and "hot drawn" fibers showed a distinctly different dependence on temperature than the maximum for stacks of lamellar single crystals. For oligomers a long spacing was observed which was about twice the length of the sample multiplied by the crystalline advance per monomer.

Pyrolysis products from bacterial polyesters, poly-β-hydroxybutyrate (PHB), and from a heteropolyester (β-hydroxyvalerate and β-hydroxybutyrate) were identified. Different physical forms of PHB were studied: PHB purified by dissolution in chloroform, native granules of PHB, and PHB in bacterial cells. The products were characterized by gc, ms, and nmr analysis. The yield of crotonic acid obtained by the pyrolysis of purified PHB was 60 to 65%. Pyrolysis of PHB native granules yielded crotonic acid as well as oligomers of PHB with a terminal crotonate. Upon direct pyrolysis of dry bacterial cells, the yield of crotonic acid was 20 to 25% of the PHB in the cells. From the heteropolymer, 2-pentenoic acid and terminally unsaturated oligomers of polyhydroxyvalerate were obtained. The usefulness of the pyrolysis method for obtaining vinyl compounds from bacterial cultures containing polyalkanoates is discussed.

Gold particles coated with ß-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.

The plastid gene rpoBencodes a plastid‐specific, DNA‐dependent RNA polymerase. A study of a barley mutant, albostrians, indicated that rpoBitself is transcribed by a nuclear‐encoded RNA polymerase. However, the molecular nature of the nuclear‐encoded RNA polymerase and mechanisms of the transcriptional regulation of rpoBexpression have not been elucidated. Using an in vivotransient assay system for gene expression in plastids, the 5′‐flanking region of rpoBfrom Arabidopsis thalianain plastids of cultured BY‐2 tobacco cells was analyzed. A series of deletion analyses of the 5′‐flanking region indicated that there were two positive and one negative regulatory regions for rpoBexpression. Comparison of the 5′‐flanking sequences of rpoBamong several plant species revealed the existence of three conserved motifs in these regions.

Successful transient expression of genes (luciferase and ß-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.

ABSTRACTFoliar uptake of nitrogen dioxide (NO2) is governed by its reactive absorption mechanism, by which NO2molecules diffuse through cell wall layers and simultaneously react with apoplastic ascorbate to form nitrous acid, which freely diffuses across plasmalemma. However, whether free diffusion of nitrous acid is the sole mechanism of foliar uptake of NO2remains unknown. The involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO2, as reported in nitrite transport in Arabidopsis roots, is also unknown. In this study, we treated Arabidopsis thalianaleaves with methionine sulfoximine (MSX) to inhibit incorporation of ammonia into glutamate and exposed them to 4 ppm 15N-labeled NO2for 4 h in light followed by quantification of total nitrogen, reduced nitrogen, and ammonia nitrogen derived from NO2using mass spectrometry and capillary electrophoresis. The total nitrogen derived from NO2in leaves without MSX treatment was 587.0 nmol NO2/g fresh weight, of which more than 65% was recovered as reduced nitrogen. In comparison, MSX treatment decreased the total nitrogen and reduced nitrogen derived from NO2by half. Thus, half of the foliar uptake of NO2is not attributable to passive diffusion of nitrous acid but to ammonia-inhibitable nitrite transport. Foliar uptake of NO2is mediated by a dual mechanism in A. thaliana: nitrous acid-free diffusion and nitrite transporter-mediated transport.

ABSTRACTIt is known that when plant leaves are exposed to exogenously applied nitrogen dioxide (NO2), nitrogen derived from NO2is reduced to amino acid nitrogen. However, whether this is the sole metabolic fate of exogenously applied NO2is unclear. In this study, Arabidopsisleaves were exposed to 4 ppm 15N-labeled NO2for 4 h in light, followed by capillary ion analysis and elemental analysis–mass spectrometry with an elemental analyzer connected directly to a mass spectrometer. We found that leaf cells exposed to 15N-labeled NO2accumulated a large amount of 15N-labeled nitrate. Neither 15N-labeled nitrite nor endogenous nitrite was present in exposed leaves. It is likely that exogenously applied NO2is first converted to nitrite, and that nitrite is oxidized to nitrate in Arabidopsisleaf cells. The complete disappearance of nitrite derived from exogenously applied NO2and endogenous nitrite supports this mechanism.

ABSTRACTExposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in the selective nitration of specific proteins, such as PsbO1. The 9th tyrosine residue (9Tyr) of PsbO1 has been identified as the nitration site. This nitration is triggered by light and inhibited by photosynthetic electron transport inhibitors. During protein nitration, tyrosyl and NO2radicals are formed concurrently and combine rapidly to form 3-nitrotyrosine. A selective oxidation mechanism for 9Tyr of PsbO1 is required. We postulated that, similar to 161Tyr of D1, 9Tyr of PsbO1 is selectively photo-oxidized by photosynthetic electron transport in response to illumination to a tyrosyl radical. In corroboration, after reappraising our oxygen evolution analysis, the nitration of PsbO1 proved responsible for decreased oxygen evolution from the thylakoid membranes. NO2is reportedly taken into cells as nitrous acid, which dissociates to form NO2−. NO2−may be oxidized into NO2by the oxygen-evolving complex. Light may synchronize this reaction with tyrosyl radical formation. These findings suggest a novel role for PsbO1 in photosynthetic electron transport.

ABSTRACTExposure of intact Arabidopsis leaves to 40 ppm nitrogen dioxide (NO2) in light resulted almost exclusively in nitration of PsbO1, PsbO2, and PsbP1 of photosystem II (PSII), with minor nitration of four non-PS II proteins, including peroxiredoxin II E, as reported previously. Our previous findings that light-triggered selective nitration of PsbO1 decreased oxygen evolution and that inhibition of photoelectric electron transport inhibited nitration of PsbO1 implied that the nitratable tyrosine residue of PsbO1 is redox-active. However, whether the nitratable tyrosine residues of PsbO2 and PsbP1 are redox-active is unknown. In this study, we determined the oxygen evolution and maximal photochemical efficiency of PSII in intact Arabidopsis leaves following exposure to 40 ppm NO2in light and found that these parameters were decreased to 60 and 70% of the non-exposed control, respectively. Because PsbO1, PsbO2, and PsbP1 accounted for > 80% of anti-3-nitrotyrosine antibody signal intensities, observed decreases in the oxygen evolution and maximal photochemical efficiency of PSII were mainly attributable to nitration of the tyrosine residues of these PSII proteins. Thus, it is postulated that nitratable tyrosine residues of PsbO2 and PsbP1 are redox-active, as in the case of PsbO1. A new hypothetical model is proposed.

ABSTRACTTreatment of isolated Arabidopsis thalianathylakoid membranes with nitrogen dioxide (NO2) induces selective nitration of the tyrosine residue at the ninth amino acid (9Tyr) of PsbO1. This selective nitration is triggered by light and is inhibited by photosynthetic electron transport inhibitors. Therefore, we postulated that, similar to 161Tyr of D1 (YZ), 9Tyr of PsbO1 is redox active and is selectively oxidized by photosynthetic electron transport in response to illumination to a tyrosyl radical that is highly susceptible to nitration. This tyrosyl radical may combine rapidly at diffusion-controlled rates with NO2to form 3-nitrotyrosine. If this postulation is correct, the nitration of 9Tyr of PsbO1 should decrease oxygen evolution activity. We investigated the effects of PsbO1 nitration on oxygen evolution from isolated thylakoid membranes, and found that nitration decreased oxygen evolution to ≥ 0% of the control. Oxygen evolution and nitration were significantly negatively correlated. This finding is consistent with redox active properties of the 9Tyr gene of PsbO1, and suggests that PsbO1 9Tyr acts as an electron relay, such as YZin the photosystem II oxygenic electron transport chain.

ABSTRACTPsbO1 is exclusively nitrated when isolated thylakoid membranes are incubated in a buffer bubbled with nitrogen dioxide (NO2) containing NO2and nitrite. NO2is the primary intermediate for this selective nitration. Isolated thylakoid membranes were incubated in NO2-bubbled buffer at 25°C in the light or dark. Protein analysis confirmed the selective nitration of PsbO1. Illumination was found to be essential in PsbO1 nitration. A nitration mechanism whereby nitratable tyrosine residues of PsbO1 are, prior to nitration, selectively photo-oxidized by photosynthetic electron transport to tyrosyl radicals to combine with NO2to form 3-nitrotyrosine was hypothesized. We tested the electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1- dimethylurea, sodium azide, and 1,5-diphenylcarbazide and found distinct inhibition of nitration of PsbO1. We also propose a possible nitration mechanism.

ABSTRACTExposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in nitration of specific chloroplast proteins. To determine whether NO2itself and/or nitrite derived from NO2can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO2-bubbled or potassium nitrite (KNO2) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO2concentrations in the NO2-bubbled buffer were calculated by numerically solving NO2dissociation kinetic equations. The two buffers were adjusted to have identical nitrite concentrations. Both treatments yielded an NT-immunopositive band that LC/MS identified as PSBO1. The difference in the band intensity between the 2 treatments was designated nitration by NO2. Both NO2and nitrite mediated nitration of proteins, and the nitration ability per unit NO2concentration was ∼100-fold greater than that of nitrite.

ABSTRACTProtein tyrosine nitration is an important post-translational modification. A variety of nitrated proteins are reported in Arabidopsis leaves and seedlings, sunflower hypocotyls, and pea roots. The identities of nitrated proteins are species-/organ-specific, and chloroplast proteins are most nitratable in leaves. However, precise mechanism is unclear. Here, we investigated nitroproteome in tobacco leaves following exposure to nitrogen dioxide. Proteins were extracted, electrophoresed and immunoblotted using an anti-3-nitrotyrosine antibody. Mass spectrometry and FASTA search identified for the first time an exclusive nitration of pathogenesis-related proteins, PR-1, PR-3 and PR-5, which are reportedly located in the apoplast or the vacuole. Furthermore, Tyr36of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.

To elucidate the stimulation of leaf growth by atmospheric nitrogen dioxide (NO2), we performed a kinematic analysis of the eighth leaves of Arabidopsis thaliana(accession C24) plants grown for 17–35 days after sowing in the presence or absence of 50 ppb NO2(designated +NO2plants and –NO2plants, respectively). We found that the peak and mean values of the relative rates of leaf expansion, cell division and cell expansion were always greater in +NO2plants than in –NO2plants. No evidence for prolonged duration was obtained. Thus, NO2treatment increased the rates of both cell proliferation and enlargement to increase leaf size. Furthermore, a fold-change analysis showed that cell proliferation and enlargement differentially regulated NO2-induced leaf expansion.

A negative correlation has consistently been reported between the change in flowering time and the change in leaf number at flowering in response to environmental stimuli, such as the application of exogenous compounds, cold temperature, day length and light quality treatments in Arabidopsis thaliana(Arabidopsis). However, we show here that the application of exogenous nitrogen dioxide (NO2) did not change the number of rosette leaves at flowering, but actually accelerated flowering in Arabidopsis. Furthermore, NO2treatment was found to increase the rate of leaf appearance. Based on these results, reaching the maximum rosette leaf number earlier in response to NO2treatment resulted in earlier flowering relative to controls.

To better understand the response of plants to atmospheric nitrogen dioxide (NO2), we investigated biomass accumulation in 3 accessions of Arabidopsis thaliana: C24, Columbia (Col-0), and Landsberg erecta (Ler). Plants were grown in NO2-free air for 1 week after sowing, followed by 3 (Col-0 and Ler) to 4 (C24) weeks in air with or without NO2(10 or 50 ppb). NO2treatment increased the biomass of all 3 accessions to varying extents. Treatment with 10 ppb NO2increased shoot biomass in C24, Col-0, and Ler by 3.2-, 1.4-, and 2.3-fold, respectively, compared with control. Treatment with 50 ppb gave similar increases, except in C24 (2.7-fold). The physiological, evolutionary, and genetic significance of these results are discussed below.

Atmospheric nitric oxide (NO) and nitrogen dioxide (NO2) have long been recognized as either detrimental or beneficial for plant development. Recent research has established that NO is a phytohormone. Our present knowledge of the physiological role of NO2is incomplete. We do know, however, that exogenous NO2positively regulates the vegetative and reproductive growth of plants. We may therefore postulate that NO2is a positive growth regulator for plants. We are now in a position to coherently summarize what is known of NO2physiology; collated information on the topic is presented here.

For Abstract see ChemInform Abstract in Full Text.

Infrared absorption spectra of film specimens of oat coleoptile cell walls, before and after protease treatment and after treatment for removal of lipid materials, pectic substances and hemicellulose, were recorded, and the characteristic bands in the spectrum of the wall assigned. Polarization spectrum measurements on the wall provided evidence indicating that the non-cellulosic polysaccharide matrix as well as cellulose micronbrils has an oriented structure in the wall and that the oriented structure changes during extension growth as well as upon mechanical extension of the walls.

Infrared evidence indicated that the oriented structure of matrix polysaccharides changed on mechanical extension as well as during extension growth of Nitella cell walls. The importance of the ultrastructure of polysaccharide gels in controlling extension growth is discussed.

Polarized infrared absorption spectra of film specimens of the epidermal cell wall of the third internode of pea stems were recorded before and after treatment with endopolygalacturonase (endo-PG) and endo-pectin lyase (endo-PL). The spectra showed that the pectic polysaccharides solubilized with endo-PG were essentially the same as those solubilized with endo-PL. The degree of esterification of the pectic polysaccharides was about 20%, and their major sugar components were uronic acids (32.8%), arabinose (48.1%) and galactose (19.2%). The polarized infrared spectra showed that pectic polysaccharides have an oriented structure in cell walls with their molecular chains oriented preferentially parallel to the direction of cell elongation.

By a direct microinjection through pressurized glass micropipettes, we injected fluorescent Lucifer Yellow, berberine and berberine-bound DNA into protoplasts isolated from cultured Euphorbia millii cells and from tobacco mesophyll cells. The protoplasts to be injected were held by a holding pipette with which the cytoplasm or nucleus of the protoplasts could be oriented with reference to the tip of the injection pipette. Upon microinjection of a berberine-bound DNA, the nucleus and intranuclear organelles, possibly including nucleolus, specifically exhibited yellow fluorescence due to berberine. This provides a direct evidence that DNA molecules can be microinjected into the intranuclear compartment of the protoplasts. The injected protoplasts survived the capillary microinjection.

Protoplasts isolated from cultured cells of Coptis japonica and Euphorbia millii were electrically fused using platinum microelectrodes. The process involved two stages, cellular and vacuolar fusion, which are characterized respectively by transient wrinkling of the membrane and the formation of a dark-red precipitate.

The effects of three physical parameters upon protoplast electrofusion were studied using protoplasts from cultured cells of Coptis japonica and Euphorbia millii. The osmotic potential of the medium did not appreciably affect the AC-field-induced protoplast-pair formation, but significantly influenced the fusion process of the paired protoplasts in response to DC pulses. The optimum osmotic potential was 0.55 to 0.60 Osm/kg H2O in our system. The density of the medium markedly influenced both pair formation and fusion process. The optimum density was 1.13 to 1.14 g/cm3, and at this density the yield of the fused protoplasts increased to more than twice that of the control (1.10 g/cm3). Hydrophilic coating of the bottom surface of the chamber with Gellan gum or polyacrylamide gel was also effective for both pair formation and the fusion process, while coating with hydrophobic silicone was entirely inhibitory. Possible interpretations of the effects of these physical parameters upon protoplast electrofusion are presented.

Levels of a cysteine endopeptidase, EP-Cl, and its mRNA increased slightly in cotyledons of French bean detached from embryonic axes. A transient expression assay by a particle gun indicated that the downstream region from position –340 of the EP-Cl gene was sufficient for gene expression in cotyledons of germinating seeds.

Replacement of calcium ion with magnesium ion in the cell wall of the pea epicotyl makes the wall more extensible. A possible role of this differential effect of Ca2+ and Mg2− in regulating cell elongation in pea epicotyl is discussed. The ratio of the content of calcium ion to that of magnesium ion (Ca2+/Mg2+) in the walls decreased markedly in the order of the first > the second > the third internodes of the pea epicotyl. The capacity of the walls for cation exchange increased in the same order, whereas the calcium-magnesium ion exchange selectivity of the walls was virtually constant. Our results indicate that the changes in the Ca2+/Mg2+ ratio among the internodes is not attributable to the ion exchange properties of the walk per se, but is due to other physiological conditions which regulate the activities of free calcium and magnesium ions in the environment with which the walls are in equilibrium.

The increase in the dichroism of the ester-C=O and COO− bands in response to mechanical extension in oat coleoptile cell walls was much enhanced by IAA pretreatment while the decrease in that of the C-O-C band was not enhanced or even suppressed. The results indicate the importance of the ultrastructure of non-cellulosic polysaccharides in controlling auxin-induced cell elongation.

Infrared absorption spectra of film specimens of the epidermal and parenchyma cell walls of the third internode of pea stem, before and after protease treatment and after treatment for removal of lipid materials, pectic substances and hemicellulose, were recorded, and characteristic bands in the spectrum of the wall were assigned. Polarization spectrum measurements of the wall provided evidence indicating that the non-cellulosic polysaccharide matrix as well as cellulose microfibrils has an oriented structure in the wall which changes during extension growth as well as upon mechanical extension of the walls.

The electrophysiological properties of the membrane of Nicotiana tabacum var. Sarnsun cultured cells were determined using a microelectrode technique in standard medium containing 1 mM KC1, 1 mM NaCl and 1 mu CaCl2 at pH 7. Tobacco callus was derived from the pith (Em=−104.4%16.2 mV). The membrane potentials of the callus cells did not show a symmetrical Gaussian distribution but were scattered over a wide range. The percentage of high membrane potential cells increased as the subculture was continued until about 11 months and then decreased. The response of the membrane potential to electric stimulus, ionic composition, metabolic inhibitors, sugars and amino acids was characteristic of high (Em=−{small tilde}−160 220 mV; H-cells) and low (Em=−80{small tilde}−90 mV; L-cells) membrane potential cells. The membrane potential of H-cells was largely depolarized by addition of CN−, carbonium cyanide m-chlorophenylhydrazone, decyclohexylcarbodiimide, and triphenyltin chloride and transiently depolarized by addition of glucose, galactose, mannose or sucrose, and D-alanine, L-alanine or Llysine, but the membrane potential of L-cells was not.

Protoplasts isolated from suspension-cultured cells of high-berberine-producing Coptis japonica (Polycarpiidae) and of high anthocyanin-producing Euphorbia millii (Sapindaifloriidae) were electrically fused. The “inter-infraclass” heterokaryons were manually selected with a glass micropipette, using the dark red vesicles which had formed in them as markers, and cultured in a microculture chamber in the presence of nurse cells. Various culture conditions were examined. Among a total of 2,830 heterokaryons, three showed cell division and one developed into a small cell colony of 10 to 20 cells after 260 days of culture.

Permeability coefficients for the cell membrane of Nitella jlexilis have been determined for some organic substances by measuring their transcellular transport with the double chamber method. Benzyl alcohol is rapidly taken up in the vacuole of the cell. In the double chamber method benzyl alcohol taken up in the vacuole at one end of the cell is not as rapidly transported to the other end as expected from the rate of protoplasmic streaming in the vacuole. This can be explained by assuming the presence of a “non-streaming part” in the vacuole. The uptake of urea into the streaming part of the cell is stimulated by directly illuminating the part where the cell is immersed in urea solution.