Naoki Shinohara

Functional Analysis of Synthetic Substructures of Polytheonamide B: A Transmembrane Channel-Forming Peptide†

1 couldserve as a minimum-structure platform for investigatingrequirements for ion-channel functions and designing noveltransmembrane channels. The structure–function relation-ships of various analogues of 1 should shed light on thespecificconstituentaminoacidsandsequencesthatencodeitsunique channel function.

Isolation of monoclonal antibodies recognizing rare and dominant epitopes in plant vascular cell walls by phage display subtraction

A combination of phage display antibody technology and a subtraction method provides a powerful tool for the isolation of novel biomarkers. However, the dilemma that high stringency screening often reduces the diversity in the subtracted phage antibody repertoires and that it is difficult to isolate phage antibody against rare epitopes remain. Therefore, we carefully monitored the occupancy of differentiation-specific clones in a phage antibody library through an enrichment process, and succeeded in isolating monoclonal antibodies against rare and dominant epitopes in plant vascular cell walls. We also report that clones with stop and frameshift mutations significantly survived the enrichment process, owing to noncanonical translation mechanisms.

Selective modification of the N-terminal structure of polytheonamide B significantly changes its cytotoxicity and activity as an ion channel.

Chemical point mutation: Polytheonamide B is a naturally occurring polypeptide containing 48 amino acids. It both displays potent cytotoxicity and acts as a monovalent cation channel in vitro. Chemoselective methods to modify the 44th, N-, and C-terminal residues of the natural product have been developed, and evaluation of the resultant derivatives suggests that the intrinsic activities of the peptide can only be altered by switching its N-terminal substitution.

Higher extracellular pH suppresses tracheary element differentiation by affecting auxin uptake

In an optimized liquid medium containing auxin and cytokinin, mesophyll cells isolated from Zinnia elegans L. seedlings can be induced to differentiate into tracheary elements (TEs) at high frequency. However, it is known that buffering the medium at neutral pH severely suppresses TE differentiation. In the process of modifying the medium, we found that excessive administration of auxin restored the suppression. Based on this finding, we physiologically characterized auxin actions involved in TE differentiation by focusing on the influence of extracellular pH. First, dose/response relationships between auxin [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] concentrations and differentiated cell ratios were determined under various extracellular pH conditions. Secondly, intracellular concentrations of free forms and metabolites of auxin species were determined by analyzing extracts from cells cultured with radiolabeled NAA and 2,4-D under different extracellular pH conditions with liquid scintillation counting and thin-layer chromatography autoradiograms. Higher extracellular pH was found to reduce both the auxin potency for inducing TE differentiation and intracellular auxin accumulation. Reduction levels correlatively varied depending on the auxin species. These results suggest that the weakening in auxin potency at higher extracellular pH is ascribed to lower auxin uptake, which leads to decreased intracellular perception of the auxin signal. A model to predict auxin action that considers membrane transport, metabolism, and the perception of auxin is also presented.

Evidence for a Role of ANAC082 as a Ribosomal Stress Response Mediator Leading to Growth Defects and Developmental Alterations in Arabidopsis

The existence of a ribosomal stress response in plant cells is revealed by molecular genetic studies that implicate ANAC082 as a crucial mediator of this pathway. Ribosome-related mutants in Arabidopsis thaliana share several notable characteristics regarding growth and development, which implies the existence of a common pathway that responds to disorders in ribosome biogenesis. As a first step to explore this pathway genetically, we screened a mutagenized population of root initiation defective2 (rid2), a temperature-sensitive mutant that is impaired in pre-rRNA processing, and isolated suppressor of root initiation defective two1 (sriw1), a suppressor mutant in which the defects of cell proliferation observed in rid2 at the restrictive temperature was markedly rescued. sriw1 was identified as a missense mutation of the NAC transcription factor gene ANAC082. The sriw1 mutation greatly alleviated the developmental abnormalities of rid2 and four other tested ribosome-related mutants, including rid3. However, the impaired pre-rRNA processing in rid2 and rid3 was not relieved by sriw1. Expression of ANAC082 was localized to regions where phenotypic effects of ribosome-related mutations are readily evident and was elevated in rid2 and rid3 compared with the wild type. These findings suggest that ANAC082 acts downstream of perturbation of biogenesis of the ribosome and may mediate a set of stress responses leading to developmental alterations and cell proliferation defects.

Involvement of rRNA biosynthesis in the regulation of CUC1 gene expression and pre-meristematic cell mound formation during shoot regeneration

At an early stage of shoot regeneration from calli of Arabidopsis, pre-meristematic cell mounds develop in association with localized strong expression of CUP-SHAPED COTYLEDON (CUC) genes. Previous characterization of root initiation-defective 3 (rid3), an Arabidopsis mutant originally isolated as being temperature-sensitive for adventitious root formation, with respect to shoot regeneration implicated RID3 in the negative regulation of CUC1 expression and the restriction of cell division in pre-meristematic cell mounds. Positional cloning has identified RID3 as a WD40 repeat protein gene whose molecular function was not investigated before. Here we performed in silico analysis of RID3 and found that RID3 is orthologous to IPI3, which mediates pre-rRNA processing in Saccharomyces cerevisiae. In the rid3 mutant, rRNA precursors accumulated to a very high level in a temperature-dependent manner. This result indicates that RID3 is required for pre-rRNA processing as is IPI3. We compared rid3 with rid2, a temperature-sensitive mutant that is mutated in a putative RNA methyltransferase gene and is impaired in pre-rRNA processing, for seedling morphology, shoot regeneration, and CUC1 expression. The rid2 and rid3 seedlings shared various developmental alterations, such as a pointed-leaf phenotype, which is often observed in ribosome-related mutants. In tissue culture for the induction of shoot regeneration, both rid2 and rid3 mutations perturbed cell-mound formation and elevated CUC1 expression. Together, our findings suggest that rRNA biosynthesis may be involved in the regulation of CUC1 gene expression and pre-meristematic cell-mound formation during shoot regeneration.

Isolation of Monoclonal Antibodies Recognizing Xylem Cell Wall Components by Using a Phage Display Subtraction Method

ABSTRACT Monoclonal antibodies (mAbs) recognizing differentiated cell-specific wall components are useful in distinguishing types of cells and dissecting developmental processes. We tried to generate such mAbs using a strategy consisting of (i) isolation of cell walls from synchronously differentiating cells of an in vitro Zinnia culture system, which reflects vascular differentiation in plants (ii) construction of phage display library of recombinant antibody against the cell walls (iii) screening via biopannings with subtractive procedures using non-differentiating cell walls. The advantage of this strategy is that purposive mAbs can be easily screened without purifying antigens of interest. Moreover, this approach can be applied to search for novel subpopulations of cells that are difficult to address by conventional methods because of small subpopulations in heterogeneous mixtures. As a result, we succeeded in isolation of three mAbs, designated CN 8, XD 3, and XD 27. Immunolocalization analyses in Zinnia plants revealed that CN 8 epitope localized in walls of immature tracheary elements (TEs) and xylem parenchyma cells, XD 3 epitope localized in walls of TE precursors and immature fiber cells, and XD 27 epitope localized only in walls of immature TEs. In the Zinnia culture system, these three mAbs distinguished subpopulations of cells in different developmental stages. These results demonstrate that some wall components change dynamically in association with xylem cell differentiation, as cell-surface antigens of animal cells. These mAbs, therefore, are useful as molecular markers to dissect the vascular developmental stage and also as tools to isolate specific xylem cells using a cell sorter.

Monoclonal antibody-based analysis of cell wall remodeling during xylogenesis

Xylogenesis, a process by which woody tissues are formed, entails qualitative and quantitative changes in the cell wall. However, the molecular events that underlie these changes are not completely understood. Previously, we have isolated two monoclonal antibodies, referred to as XD3 and XD27, by subtractive screening of a phage-display library of antibodies raised against a wall fraction of Zinnia elegans xylogenic culture cells. Here we report the biochemical and immunohistochemical characterization of those antibodies. The antibody XD3 recognized (1→4)-β-d-galactan in pectin fraction. During xylogenesis, the XD3 epitope was localized to the primary wall of tracheary-element precursor cells, which undergo substantial cell elongation, and was absent from mature tracheary elements. XD27 recognized an arabinogalactan protein that was bound strongly to a germin-like protein. The XD27 epitope was localized to pre-lignified secondary walls of tracheary elements. Thus these cell-wall-directed monoclonal antibodies revealed two molecular events during xylogenesis. The biological significance of these events is discussed in relation to current views of the plant cell wall.

Erratum: Total synthesis of the large non-ribosomal peptide polytheonamide B

Nature Chemistry doi: 10.1038/nchem.554 (2010); published online: 21 February 2010; corrected online 23 February 2010. In the version of this Article originally published online, an in-house error led to the incorrect representation of stereochemistry in Figs 1, 2 and 6. These have now been corrected in all versions of the Article.