Diplazium subsinuatum and Di. tomitaroanum should be Moved to Deparia According to Molecular, Morphological, and Cytological Characters

rbcL sequence data revealed that the putative intergeneric hybrid, Diplazium tomitaroanum Masam. belongs in Deparia, as also does Diplazium subsinuatum (Wall, ex Hook, et Grev.) Tagawa, one of the putative parents. An examination of rachis, scale and spore morphology, and chromosome data provide support for this placement. We propose a new taxonomic treatment of the two Diplazium species as Deparia. Received 7 December 1999/ Accepted in revised form 15 February 2000     

Structure of imidazole glycerol phosphate synthase from Thermus thermophilus HB8: open-closed conformational change and ammonia tunneling

Imidazole glycerol phosphate synthase (IGPs) catalyzes the fifth step in the histidine biosynthetic pathway located at the branch point to de novo purine biosynthesis. IGPs is a multienzyme comprising glutaminase and synthase subunits. The glutaminase activity, which hydrolyzes glutamine to give ammonia, is coupled with substrate binding to the synthase subunit. The three-dimensional structure of the IGPs from Thermus thermophilus HB8 has been determined at 2.3 A resolution, and compared with the previously determined structures for the yeast and Thermotoga maritima enzymes. The structure of each subunit is similar to that of the corresponding domain in the yeast enzyme or subunit in the T. maritima enzyme. However, the overall structure is significantly different from the yeast and T. maritima enzymes, indicating that IGPs may change the relative orientation between the two subunits and close the glutaminase site upon glutamine binding. The putative ammonia tunnel, which carries nascent ammonia from glutaminase to the synthase site, has a closed gate comprising a cyclic salt bridge formed by four charged residues of the synthase subunit. The side chain of Lys100 in the cyclic salt bridge might change its side chain direction to form new interactions with the main chain carbonyl group of glutamine from the synthase subunit and the hydoxyl group of tyrosine from the glutaminase subunit, resulting in the opening of the gate for ammonia transfer.     

Novel assays to monitor gene expression and protein-protein interactions in rice using the bioluminescent protein,NanoLuc

Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.     

Laser micromarking technique in studying the negative gravitropism in pea stem

A laser micromarking technique on plant epidermis was developed to study how a plant can reduce the stress in bending behavior by controlling the growth and morphogenesis. The negative gravitropism in a pea seedling (Pisum sativum L.) was discussed based on the time-dependent displacement of laser marking points which were formed by spatially-selective laser ablation of the cuticle layer that covers the outer surface of a plant. The elongation of the stem in the horizontal direction was remarkable in the first half of the gravitropism. The elongation percentages of the stem length between laser-marking points at around upper surface, middle, and bottom surface were evaluated to be 2.57, 4.87, and 7.70%, respectively. The characteristic feature of the stem bending in gravitropism is the elongation even at the upper surface region, that is, inside of the bending. This is a different feature from cantilever beams for structural materials like metals and polymers, where the compression of the upper surface and elongation of the bottom surface are caused by bending. Another laser micromarking technique was developed to improve the resolution of a dot-matrix pattern by fluorescent material transfer to a plant through a masking film with a micro-hole matrix pattern. Similar time-dependent displacement behavior was observed for a fluorescent dot-marked stem showing a feedback control loop in the mechanical optimization. These results suggested that plants solve the problem of the stress in stem bending through growth. The laser micromarking is an effective method for studying the mechanical optimization in plants.     

Genome-wide quantification of homeolog expression ratio revealed nonstochastic gene regulation in synthetic allopolyploid Arabidopsis

Genome duplication with hybridization, or allopolyploidization, occurs commonly in plants, and is considered to be a strong force for generating new species. However, genome-wide quantification of homeolog expression ratios was technically hindered because of the high homology between homeologous gene pairs. To quantify the homeolog expression ratio using RNA-seq obtained from polyploids, a new method named HomeoRoq was developed, in which the genomic origin of sequencing reads was estimated using mismatches between the read and each parental genome. To verify this method, we first assembled the two diploid parental genomes of Arabidopsis halleri subsp. gemmifera and Arabidopsis lyrata subsp. petraea (Arabidopsis petraea subsp. umbrosa), then generated a synthetic allotetraploid, mimicking the natural allopolyploid Arabidopsis kamchatica. The quantified ratios corresponded well to those obtained by Pyrosequencing. We found that the ratios of homeologs before and after cold stress treatment were highly correlated (r = 0.870). This highlights the presence of nonstochastic polyploid gene regulation despite previous research identifying stochastic variation in expression. Moreover, our new statistical test incorporating overdispersion identified 226 homeologs (1.11% of 20 369 expressed homeologs) with significant ratio changes, many of which were related to stress responses. HomeoRoq would contribute to the study of the genes responsible for polyploid-specific environmental responses.     

Miniature Cell Culture Systems for Use in Field Isolation of Viruses: Cell Stability and Sensitivity to Selected Laboratory-Propagated Viruses During Storage in Miniaturized Environments

Cell cultures were miniaturized in 1-dr vials or in capillary tubes (100 by 3 mm) and examined for their capacity to maintain normal gross morphology and sensitivity to infection by viruses during storage under a variety of conditions. The purpose of these studies was to determine the feasibility of using such cultures in the field during epidemiological investigations. From the results of this work it was concluded that such a feasibility exists.     

Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope

Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning fluorescence microscope and mouse hepatoma Hepa-1c1c7 cells. Five flavonol aglycones showed autofluorescence in the cells under the conditions (Ex. 488 nm to Em. 515–535 nm), whereas three flavonol glycosides and eight compounds belonging to other flavonoid subclasses, i.e., flavones, flavanones, and catechins, did not. The autofluorescence of galangin and kaempferol appeared stronger in the nucleus than cytoplasm, suggesting that they are incorporated into the cells and accumulated in the nucleus. The proposed method provided evidence that flavonol aglycones are incorporated into, and accumulated in the nucleus of, hepatocytes.     

Mechanism of Inhibition by C-terminal α-Helices of the ? Subunit of Escherichia coli FoF1-ATP Synthase

The ϵ subunit of bacterial F_oF₁-ATP synthase (F_oF₁), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal α-helices of ϵ, and the “extended” but not “hairpin-folded” state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of ϵ has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli F_oF₁ (EF_oF₁) mutant in which the ϵ subunit lacked the C-terminal domain (F_oF₁^ϵΔC), and ATP synthesis driven by acid-base transition (ΔpH) and the K⁺-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (F_oF₁^ϵWT). The turnover numbers (k_cat) of F_oF₁^ϵWT were severalfold lower than those of F_oF₁^ϵΔC. F_oF₁^ϵWT showed higher Michaelis constants (K_m). The dependence of the activities of F_oF₁^ϵWT and F_oF₁^ϵΔC on various combinations of ΔpH and ΔΨ was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of ϵ. Solubilized F_oF₁^ϵWT also showed lower k_cat and higher K_m values for ATP hydrolysis than the corresponding values of F_oF₁^ϵΔC. These results suggest that the C-terminal domain of the ϵ subunit of EF_oF₁ slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit.