Misa Takahashi

Three distinct Arabidopsis hemoglobins exhibit peroxidase-like activity and differentially mediate nitrite-dependent protein nitration

All plants examined to date possess non‐symbiotic hemoglobin whose physiological role remains unclear. The present study explored the catalytic function of three representative classes of the plant hemoglobin from Arabidopsis thaliana: AtGLB1, AtGLB2, and AtGLB3. Purified recombinant proteins of these hemoglobins displayed hydrogen peroxide‐dependent oxidation of several peroxidase substrates that was sensitive to cyanide, revealing intrinsic peroxidase‐like activity. In the presence of nitrite and hydrogen peroxide, AtGLB1 was the most efficient at mediating tyrosine nitration of its own and other proteins via the formation of reactive nitrogen species as a result of nitrite oxidation. AtGLB1 mRNA significantly accumulated in Arabidopsis seedlings exposed to nitrite, supporting the physiological relevance of its function to nitrite and nitrite‐derived reactive nitrogen species.

Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: junction regions can bind to nuclear matrices.

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 6bp 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 4-6bp were present within 10bp 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.

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism.

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO2) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from 15N-labeled NO2 taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with 15NO2. Secondly, tobacco (Nicotiana tabacum L.) plants fed with 15N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.

Molecular characterization of atmospheric NO2-responsive germin-like proteins in azalea leaves.

Atmospheric nitrogen dioxide (NO(2)) is an environmental oxidant that is removed through direct uptake by foliage, but plant responses to this highly reactive gas are not well understood at the molecular level. From NO(2)-exposed leaves of a woody azalea (Rhododendron mucronatum), we cloned two cDNAs (RmGLP1 and RmGLP2) for germin-like proteins (GLPs), a group of ubiquitous plant proteins that have been implicated in various plant physiological and developmental processes. Quantitative analysis of mRNA expression, together with immunoblotting data, showed that foliar exposure to NO(2) caused a robust induction of these GLP-encoding genes. When produced in tobacco cell culture, recombinant RmGLP2 was secreted into the apoplast, where it exhibited superoxide dismutase activity. RmGLP1 and RmGLP2 represent the first examples of plant genes that are responsive to airborne NO(2). These enzymes might have a potential role in extracellular defense mechanisms through attenuation of interactions between reactive nitrogen and oxygen species.

Dual selective nitration in Arabidopsis: Almost exclusive nitration of PsbO and PsbP, and highly susceptible nitration of four non‐PSII proteins, including peroxiredoxin II E

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti‐3‐nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non‐exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non‐PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5∼6.6). Tyr9 of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non‐PSII proteins in Arabidopsis.

Stable transformation of Eustoma grandiflorum by particle bombardment

Abstract Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome.

High frequency stable transformation ofArabidopsis thaliana by particle bombardment

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.

The reductive reaction mechanism of tobacco nitrite reductase derived from a combination of crystal structures and ultraviolet–visible microspectroscopy

Assimilatory nitrite reductase (aNiR) reduces nitrite to an ammonium ion and has siroheme and a [Fe4S4] cluster as prosthetic groups. A reaction mechanism for Nii3, an aNiR from tobacco, is proposed based on high resolution X‐ray structures and UV–Vis (ultraviolet–visible) microspectroscopy of Nii3‐ligand complexes. Analysis of UV–Vis spectral changes in Nii3 crystals with increasing X‐ray exposure showed prosthetic group reductions. In Nii3‐NO  2− structures, X‐ray irradiation enhanced the progress of the reduction reaction, and cleavage of the NO bond was observed when X‐ray doses were increased. Crystal structures of Nii3 with other bound ligands, such as Nii3‐NO and Nii3‐NH2OH, were also determined. Further, by combining information from these Nii3 ligand‐bound structures, including that of Nii3‐NO  2− , with UV–Vis microspectral data obtained using different X‐ray doses, a reaction mechanism for aNiR was suggested. Cleavage of the two NO bonds of nitrite was envisaged as a two‐step process: first, the NO bond close to Lys224 was cleaved, followed by cleavage of the NO bond close to Arg109. X‐ray structures also indicated that aNiR‐catalyzed nitrite reduction proceeded without the need for conformation changes in active site residues. Geometrical changes in the ligand molecules and the placement of neighboring water molecules appeared to be important to the stability of the active site residue interactions (Arg109, Arg179, and Lys224) and the ligand molecule. These interactions may contribute to the efficiency of aNiR reduction reactions. Proteins 2012;

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

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.

Structure-function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high-resolution structures

Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf‐type (Nii1 and Nii3), and root‐type (Nii2 and Nii4). The high‐resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe–4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3‐M175G, Nii3‐M175E, and Nii3‐M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3‐Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild‐type (Nii3‐WT) and the Met175 variants showed that the hydrophobic side‐chain of Met175 facilitated enzyme efficiency (kcat/Km). The Nii4‐WT has Lys449 at the equivalent position of Gln448 in Nii3‐WT. The enzyme activity assay revealed that the turnover number (kcat) and Michaelis constant (Km) of Nii4‐WT were lower than those of Nii3‐WT. However, the kcat/Km of Nii4‐WT was about 1.4 times higher than that of Nii3‐WT. A comparison of the kinetics of the Nii3‐Q448K and Nii4‐K449Q variants revealed that the change in kcat/Km was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low‐affinity and Nii4 is the high‐affinity aNiR.