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Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Nagoya University

Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.

The Selaginella genome identifies genetic changes associated with the evolution of vascular plants.

We developed two types of artificial platforms, T-junction and crossroad microchannel devices, and obtained guidance response ratio of pollen tubes to the female tissue as 56-57%. The crossroad device was also able to collect the attracted pollen tubes with high purity, which is useful for future omics analysis.

Quantification of pollen tube attraction in response to guidance by female gametophyte tissue using artificial microscale pathway.

Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure-property relationship study has revealed that the dialkylamino group at the 2-position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double-stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2-position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser-scanning microscope.

Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn-On DNA Probes.

Plant cells are covered with rigid cell walls, yet tip-growing cells can elongate by providing new cell wall material to their apical regions. Studies of the mechanical properties of tip-growing plant cells typically involve measurement of the turgor pressure and stiffness of the cells' apical regions. These experiments, however, do not address how living tip-growing cells react when they encounter physical obstacles that are not substantially altered by turgor pressure. To investigate this issue, we constructed microfabricated platforms with a series of artificial gaps as small as 1 μm, and examined the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to penetrate into these gaps. The cells were grown inside microfluidic chambers and guided towards the gaps using microdevices customized for each cell type. All types of tip-growing cells could grow through the microgaps with their organelles intact, even though the gaps were much smaller than the cylindrical cell diameter. Our findings reveal the dramatic physiological and developmental flexibility of tip-growing plant cells. The microfluidic platforms designed in this study provide novel tools for the elucidation of the mechanical properties of tip-growing plant cells in extremely small spaces.

Capability of tip-growing plant cells to penetrate into extremely narrow gaps.

Under certain circumstances differentiated cells can be reprogrammed to form stem cells in land plants, but only a portion of the cells reprograms successfully. A long-distance inhibitory signal from reprogrammed cells to surrounding cells has been reported in some ferns. Here we show the existence of anisotropic inhibitory signal to regulate stem cell formation in the moss Physcomitrella patens. When single cells were isolated from a gametophore leaf, over 90% of them were reprogrammed to stem cells with characteristic nuclear expansion. By contrast, when two adjacent cells were isolated, the nuclei of both cells expanded, but successful reprogramming of both cells occurred only in approximately one fifth of the pairs. When three aligned cells were isolated, the reprogramming rate of both edge cells decreased with a living middle cell but did not with a dead middle cell. Furthermore, unequal conversion into stem cells was more prominent in cell pairs aligned parallel to the proximal-distal leaf axis than in those perpendicular to the axis. This study gives an insight into the role of the inhibitory signal in development and evolution as well as the efficient stem cell induction from differentiated cells.

Cells reprogramming to stem cells inhibit the reprogramming of adjacent cells in the moss Physcomitrella patens.

Nanographene, a small piece of graphene, has attracted unprecedented interest across diverse scientific disciplines particularly in organic electronics. The biological applications of nanographenes, such as bioimaging, cancer therapies and drug delivery, provide significant opportunities for breakthroughs in the field. However, the intrinsic aggregation behavior and low solubility of nanographenes, which stem from their flat structures, hamper their development for bioapplications. Herein, we report a water-soluble warped nanographene (WNG) that can be easily synthesized by sequential regioselective C-H borylation and cross-coupling reactions of the saddle-shaped WNG core structure. The saddle-shaped structure and hydrophilic tetraethylene glycol chains impart high water solubility to the WNG. The water-soluble WNG possesses a range of promising properties including good photostability and low cytotoxicity. Moreover, the water-soluble WNG was successfully internalized into HeLa cells and promoted photoinduced cell death.

Nagisa Sugimoto

Institute of Transformative Bio-Molecules (ITbM)

Nagoya University

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces