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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.
Highlights
Pollen tubes are one of the fastest, if not the fastest, growing cellular systems with in vivo growth speeds reaching around 2.7 μm/s in maize and only rivaled in the natural world by specially cultured neuronal cells [1]
The basic functional unit of the LoC, called the unit cell (Fig 1b), consists of three sections: (i) a large circular fluidic chamber that serves as the inlet for loading a pollen-growth medium, (ii) a grain reservoir connected to the inlet via a tapering neck, and (iii) dozens of collinear microchannels emerging from the grain reservoir
Our two-height microfluidic chip is fabricated via two-step photo-lithography, followed by soft-replica molding of polydimethylsiloxane (PDMS), which allows for a higher height for the pollen grain chambers with the tubes growing in a narrower channel (Fig 1c)
Summary
Pollen tubes are one of the fastest, if not the fastest, growing cellular systems with in vivo growth speeds reaching around 2.7 μm/s in maize and only rivaled in the natural world by specially cultured neuronal cells [1]. The maize pollen starts to germinate within 5 minutes after contact with the stigma [2] and can grow 300 mm long in the style to fertilize the ovary, PLOS ONE | DOI:10.1371/journal.pone.0168138. Lab-on-a-Chip: Pollen Tube Phenotyping & Mechanics the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section
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