Tomoya Asano

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells. In the present study, we used high-speed atomic force microscopy (HS-AFM) to image the molecular dynamics of living bacterial cell surfaces. HS-AFM visualizes the dynamic molecular processes of isolated proteins at sub-molecular resolution without the need for complicated sample preparation. In the present study, magnetotactic bacterial cells were anchored in liquid medium on substrate modified by poly-L-lysine and glutaraldehyde. High-resolution HS-AFM images of live cell surfaces showed that the bacterial outer membrane was covered with a net-like structure comprising holes and the hole rims framing them. Furthermore, HS-AFM captured the dynamic movement of the surface ultrastructure, showing that the holes in the net-like structure slowly diffused in the cell surface. Nano-dissection revealed that porin trimers constitute the net-like structure. Here, we report for the first time the direct observation of dynamic molecular architectures on a live cell surface using HS-AFM.

Plant Cells Without Detectable Plastids are Generated in the crumpled leaf Mutant of Arabidopsis thaliana

Plastids are maintained in cells by proliferating prior to cell division and being partitioned to each daughter cell during cell division. It is unclear, however, whether cells without plastids are generated when plastid division is suppressed. The crumpled leaf (crl) mutant of Arabidopsis thaliana is a plastid division mutant that displays severe abnormalities in plastid division and plant development. We show that the crl mutant contains cells lacking detectable plastids; this situation probably results from an unequal partitioning of plastids to each daughter cell. Our results suggest that crl has a partial defect in plastid expansion, which is suggested to be important in the partitioning of plastids to daughter cells when plastid division is suppressed. The absence of cells without detectable plastids in the accumulation and replication of chloroplasts 6 (arc6) mutant, another plastid division mutant of A. thaliana having no significant defects in plant morphology, suggests that the generation of cells without detectable plastids is one of the causes of the developmental abnormalities seen in crl plants. We also demonstrate that plastids with trace or undetectable amounts of chlorophyll are generated from enlarged plastids by a non-binary fission mode of plastid replication in both crl and arc6.

Gene expression analysis of wounding-induced root-to-shoot communication in Arabidopsis thaliana.

Root-to-shoot communication plays an important role in the adaptation to environmental stress. In this study, we established a model system for root-to-shoot signalling to observe global gene expression in Arabidopsis thaliana. The roots of Arabidopsis seedlings were wounded and the expression in the shoots of 68 and 5 genes was up-regulated threefold at 30 min and 6 h post-injury, respectively. These genes were designated early and late Root-to-Shoot responsive (RtS) genes, respectively. Many of the early RtS genes were found to encode transcription factors such as AtERFs, whereas others were associated with jasmonic acid (JA) and ethylene (ET). Some of the late RtS genes were shown to be regulated by 12-oxo-phytodienoic acid (OPDA). In fact, elevated levels of JA and OPDA were detected in the shoots of seedlings 30 min and 6 h, respectively, after wounding of the roots. A mutant analysis revealed that JA and ET are involved in the expression of the early RtS genes. Thus, root-to-shoot communication for many RtS genes is associated with the systemic production of JA, OPDA and possibly ET.

The secreted antifungal protein thionin 2.4 in Arabidopsis thaliana suppresses the toxicity of a fungal fruit body lectin from Fusarium graminearum.

Plants possess active defense systems and can protect themselves from pathogenic invasion by secretion of a variety of small antimicrobial or antifungal proteins such as thionins. The antibacterial and antifungal properties of thionins are derived from their ability to induce open pore formation on cell membranes of phytopathogens, resulting in release of potassium and calcium ions from the cell. Wheat thionin also accumulates in the cell walls of Fusarium-inoculated plants, suggesting that it may have a role in blocking pathogen infection at the plant cell walls. Here we developed an anti-thionin 2.4 (Thi2.4) antibody and used it to show that Thi2.4 is localized in the cell walls of Arabidopsis and cell membranes of F. graminearum, when flowers are inoculated with F. graminearum. The Thi2.4 protein had an antifungal effect on F. graminearum. Next, we purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST) and coupled the proteins to an NHS-activated column. Total protein from F. graminearum was applied to GST-Thi2.4 or Thi2.4-binding columns, and the fungal fruit body lectin (FFBL) of F. graminearum was identified as a Thi2.4-interacting protein. This interaction was confirmed by a yeast two-hybrid analysis. To investigate the biological function of FFBL, we infiltrated the lectin into Arabidopsis leaves and observed that it induced cell death in the leaves. Application of FFBL at the same time as inoculation with F. graminearum significantly enhanced the virulence of the pathogen. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that overexpressed Thi2.4. We found that a 15 kD Thi2.4 protein was specifically expressed in flowers and flower buds and suggest that it acts not only as an antifungal peptide, but also as a suppressor of the FFBL toxicity. Secreted thionin proteins are involved in this dual defense mechanism against pathogen invasion at the plant-pathogen interface.

Glycosylated Porphyra-334 and Palythine-Threonine from the Terrestrial Cyanobacterium Nostoc commune

Mycosporine-like amino acids (MAAs) are water-soluble UV-absorbing pigments, and structurally different MAAs have been identified in eukaryotic algae and cyanobacteria. In this study novel glycosylated MAAs were found in the terrestrial cyanobacterium Nostoc commune (N. commune). An MAA with an absorption maximum at 334 nm was identified as a hexose-bound porphyra-334 derivative with a molecular mass of 508 Da. Another MAA with an absorption maximum at 322 nm was identified as a two hexose-bound palythine-threonine derivative with a molecular mass of 612 Da. These purified MAAs have radical scavenging activities in vitro, which suggests multifunctional roles as sunscreens and antioxidants. The 612-Da MAA accounted for approximately 60% of the total MAAs and contributed approximately 20% of the total radical scavenging activities in a water extract, indicating that it is the major water-soluble UV-protectant and radical scavenger component. The hexose-bound porphyra-334 derivative and the glycosylated palythine-threonine derivatives were found in a specific genotype of N. commune, suggesting that glycosylated MAA patterns could be a chemotaxonomic marker for the characterization of the morphologically indistinguishable N. commune. The glycosylation of porphyra-334 and palythine-threonine in N. commune suggests a unique adaptation for terrestrial environments that are drastically fluctuating in comparison to stable aquatic environments.

The defense response in Arabidopsis thaliana against Fusarium sporotrichioides

BackgroundCertain graminaceous plants such as Zea mays and Triticum aestivum serve as hosts for Fusarium sporotrichioides; however, molecular interactions between the host plants and F. sporotrichioides remain unknown. It is also not known whether any interaction between Arabidopsis thaliana and F. sporotrichioides can occur. To understand these interactions, we performed proteomic analysis.ResultsArabidopsis leaves and flowers were inoculated with F. sporotrichioides. Accumulation of PLANT DEFENSIN1.2 (PDF1.2) and PATHOGENESIS RELATED1 (PR1) mRNA in Arabidopsis were increased by inoculation of F. sporotrichioides. Furthermore, mitogen-activated protein kinase 3 (MPK3) and mitogen-activated protein kinase 6 (MPK6), which represent MAP kinases in Arabidopsis, were activated by inoculation of F. sporotrichioides. Proteomic analysis revealed that some defense-related proteins were upregulated, while the expression of photosynthesis- and metabolism-related proteins was down regulated, by inoculation with F. sporotrichioides. We carried out the proteomic analysis about upregulated proteins by inoculation with Fusarium graminearum. The glutathione S-transferases (GSTs), such as GSTF4 and GSTF7 were upregulated, by inoculation with F. graminearum-infected Arabidopsis leaves. On the other hand, GSTF3 and GSTF9 were uniquely upregulated, by inoculation with F. sporotrichioides.ConclusionsThese results indicate that Arabidopsis is a host plant for F. sporotrichioides. We revealed that defense response of Arabidopsis is initiated by infection with F. sporotrichioides.

Characterization of the chemical diversity of glycosylated mycosporine-like amino acids in the terrestrial cyanobacterium Nostoc commune.

Mycosporine-like amino acids (MAAs) are UV-absorbing pigments, and structurally unique glycosylated MAAs are found in the terrestrial cyanobacterium Nostoc commune. In this study, we examined two genotypes of N.commune colonies with different water extract UV-absorption spectra. We found structurally distinct MAAs in each genotype. The water extract from genotype A showed a UV-absorbing spectrum with an absorption maximum at 335nm. The extract contained the following compounds: 7-O-(β-arabinopyranosyl)-porphyra-334 (478Da), pentose-bound shinorine (464Da), hexose-bound porphyra-334 (508Da) and porphyra-334 (346Da). The water extract from genotype B showed a characteristic UV-absorbing spectrum with double absorption maxima at 312 and 340nm. The extract contained hybrid MAAs (1050Da and 880Da) with two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen linked to 2-O-(β-xylopyranosyl)-β-galactopyranoside. A novel 273-Da MAA with an absorption maximum at 310nm was also identified in genotype B. The MAA consisted of a 3-aminocyclohexen-1-one linked to a γ-aminobutyric acid chain. These MAAs had potent radical scavenging activities in vitro and the results confirmed that the MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune. The two genotypes of N. commune exclusively produced their own characteristic glycosylated MAAs, which supports that MAA composition could be a chemotaxonomic marker for the classification of N. commune.

Comparative Analysis of Phosphoprotein Expression Using 2D-DIGE

Two-dimensional gel electrophoresis (2-DE) has often been used to compare protein expression pattern between different samples. This method is also useful to compare protein expression between wild-type and RNAi plants. 2D-DIGE (difference gel electrophoresis) was developed to perform quantitative proteomics of two or more samples. In this method, proteins are fluorescently labeled by Cy2, Cy3, or Cy5 and are mixed and subjected to electrophoresis on the same gel, thereby gel-to-gel variations are eliminated. We perform phosphoproteomics between Arabidopsis wild-type and a stress-activated MAPK (mitogen-activated protein kinase) mutant after the phytotoxin treatment. For this purpose, proteins are extracted from both samples, and then phosphorylated proteins are purified by phosphoprotein enrichment columns. Purified proteins are fluorescently labeled by different CyDyes using the minimum labeling method. The labeled protein samples are separated on the same gel by 2-DE, and gels are scanned by a variable mode laser imager. Then, high-resolution images are analyzed by 2D analysis software. Thus, we identified several phosphoprotein spots that were differentially accumulated between wild-type and mapk mutant.

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1-0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.

Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels

Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl-ch) or without chloroplasts (crl-no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl-no ch were approximately 65-70% those of crl-ch and approximately 50-60% those of wild type. The weakened stomatal opening in crl-no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K(+) accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl-no ch compared with crl-ch and wild type. Determination of the relative ATP levels in individual cells showed that crl-no ch guard cells contained considerably lower levels of ATP than did crl-ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H(+) extrusion by guard cells.