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Massively Parallelized Pollen Tube Guidance and Mechanical Measurements on a Lab-on-a-Chip Platform.

ABSTRACT: Pollen tubes are used as a model in the study of plant morphogenesis, cellular differentiation, cell wall biochemistry, biomechanics, and intra- and intercellular signaling. For a "systems-understanding" of the bio-chemo-mechanics of tip-polarized growth in pollen tubes, the need for a versatile, experimental assay platform for quantitative data collection and analysis is critical. We introduce a Lab-on-a-Chip (LoC) concept for high-throughput pollen germination and pollen tube guidance for parallelized optical and mechanical measurements. The LoC localizes a large number of growing pollen tubes on a single plane of focus with unidirectional tip-growth, enabling high-resolution quantitative microscopy. This species-independent LoC platform can be integrated with micro-/nano-indentation systems, such as the cellular force microscope (CFM) or the atomic force microscope (AFM), allowing for rapid measurements of cell wall stiffness of growing tubes. As a demonstrative example, we show the growth and directional guidance of hundreds of lily (Lilium longiflorum) and Arabidopsis (Arabidopsis thaliana) pollen tubes on a single LoC microscopy slide. Combining the LoC with the CFM, we characterized the cell wall stiffness of lily pollen tubes. Using the stiffness statistics and finite-element-method (FEM)-based approaches, we computed an effective range of the linear elastic moduli of the cell wall spanning the variability space of physiological parameters including internal turgor, cell wall thickness, and tube diameter. We propose the LoC device as a versatile and high-throughput phenomics platform for plant reproductive and development biology using the pollen tube as a model.

SUBMITTER: Shamsudhin N

PROVIDER: S-EPMC5158026 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Massively Parallelized Pollen Tube Guidance and Mechanical Measurements on a Lab-on-a-Chip Platform.

Shamsudhin Naveen N Laeubli Nino N Atakan Huseyin Baris HB Vogler Hannes H Hu Chengzhi C Haeberle Walter W Sebastian Abu A Grossniklaus Ueli U Nelson Bradley J BJ

PloS one 20161215 12

Pollen tubes are used as a model in the study of plant morphogenesis, cellular differentiation, cell wall biochemistry, biomechanics, and intra- and intercellular signaling. For a "systems-understanding" of the bio-chemo-mechanics of tip-polarized growth in pollen tubes, the need for a versatile, experimental assay platform for quantitative data collection and analysis is critical. We introduce a Lab-on-a-Chip (LoC) concept for high-throughput pollen germination and pollen tube guidance for para ...[more]

PMID: 27977748

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Project description:BackgroundAs in animals, cell-cell communication plays a pivotal role in male-female recognition during plant sexual reproduction. Prelaid peptides secreted from the female reproductive tissues guide pollen tubes towards ovules for fertilization. However, the elaborate mechanisms for this dialogue have remained elusive, particularly from the male perspective.ResultsWe performed genome-wide quantitative liquid chromatography-tandem mass spectrometry analysis of a pistil-stimulated pollen tube secretome and identified 801 pollen tube-secreted proteins. Interestingly, in silico analysis reveals that the pollen tube secretome is dominated by proteins that are secreted unconventionally, representing 57 % of the total secretome. In support, we show that an unconventionally secreted protein, translationally controlled tumor protein, is secreted to the apoplast. Remarkably, we discovered that this protein could be secreted by infiltrating through the initial phases of the conventional secretory pathway and could reach the apoplast via exosomes, as demonstrated by co-localization with Oleisin1 exosome marker. We demonstrate that translationally controlled tumor protein-knockdown Arabidopsis thaliana plants produce pollen tubes that navigate poorly to the target ovule and that the mutant allele is poorly transmitted through the male. Further, we show that regulators of the endoplasmic reticulum-trans-Golgi network protein secretory pathway control secretion of Nicotiana tabacum Pollen tube-secreted cysteine-rich protein 2 and Lorelei-like GPI-anchor protein 3 and that a regulator of endoplasmic reticulum-trans-Golgi protein translocation is essential for pollen tube growth, pollen tube guidance and ovule-targeting competence.ConclusionsThis work, the first study on the pollen tube secretome, identifies novel genome-wide pollen tube-secreted proteins with potential functions in pollen tube guidance towards ovules for sexual reproduction. Functional analysis highlights a potential mechanism for unconventional secretion of pollen tube proteins and reveals likely regulators of conventional pollen tube protein secretion. The association of pollen tube-secreted proteins with marker proteins shown to be secreted via exosomes in other species suggests exosome secretion is a possible mechanism for cell-cell communication between the pollen tube and female reproductive cells.

Dataset Information

Massively Parallelized Pollen Tube Guidance and Mechanical Measurements on a Lab-on-a-Chip Platform.

Publications

Massively Parallelized Pollen Tube Guidance and Mechanical Measurements on a Lab-on-a-Chip Platform.

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