Hannes Vogler

A calcium dialog mediated by the FERONIA Signal transduction pathway controls plant sperm delivery

Sperm delivery for double fertilization of flowering plants relies on interactions between the pollen tube (PT) and two synergids, leading to programmed cell death (PCD) of the PT and one synergid. The mechanisms underlying the communication among these cells during PT reception is unknown. We discovered that the synergids control this process by coordinating their distinct calcium signatures in response to the calcium dynamics and growth behavior of the PT. Induced and spontaneous aberrant calcium responses in the synergids abolish the two coordinated PCD events. Components of the FERONIA (FER) signaling pathway are required for initiating and modulating these calcium responses and for coupling the PCD events. Intriguingly, the calcium signatures are interchangeable between the two synergids, implying that their fates of death and survival are determined by reversible interactions with the PT. Thus, complex intercellular interactions involving a receptor kinase pathway and calcium-mediated signaling control sperm delivery in plants.

The pollen tube: a soft shell with a hard core

Plant cell expansion is controlled by a fine-tuned balance between intracellular turgor pressure, cell wall loosening and cell wall biosynthesis. To understand these processes, it is important to gain in-depth knowledge of cell wall mechanics. Pollen tubes are tip-growing cells that provide an ideal system to study mechanical properties at the single cell level. With the available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used a cellular force microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20-90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM, in combination with an FEM-based model, provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue.

Simple hormones but complex signalling

It has not been easy to make sense of the pleiotropic effects of plant hormones, especially of auxins; but now, it has become possible to study these effects within the framework of what we know about signal transduction in general. Changes in local auxin concentrations, perhaps even actively maintained auxin gradients, signal to networks of transcription factors, which in turn signal to downstream effectors. Transcription factors can also signal back to hormone biosynthetic pathways.

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

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.

Measuring the Mechanical Properties of Plant Cell Walls

The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM) and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM), and its automated successor, real-time CFM (RT-CFM).

Real-time automated characterization of 3D morphology and mechanics of developing plant cells

In this article, we introduce the real-time cellular force microscope (RT-CFM), a high-throughput microrobotic platform for mechanical stimulation and characterization of single cells. We developed computer vision algorithms that fully automate the positioning of target cells and localization of the sensor tip. The control and acquisition architecture dramatically increases the accuracy, speed, and reliability of force measurements. Pollen tubes provide an ideal model system for the study of plant mechanics at the single-cell level. To quantitatively obtain the physical properties of the plant cell wall, we generated topography and stiffness measurements from 3D scans of living, growing pollen tubes. We report techniques for real-time monitoring and analysis of intracellular calcium fluxes during mechanical intervention. Our platform is compatible with various imaging systems and enables a powerful screening technology to facilitate biomechanical and morphological characterization of developing cells.

Characterization of size-dependent mechanical properties of tip-growing cells using a lab-on-chip device

Quantification of mechanical properties of tissues, living cells, and cellular components is crucial for the modeling of plant developmental processes such as mechanotransduction. Pollen tubes are tip-growing cells that provide an ideal system to study the mechanical properties at the single cell level. In this article, a lab-on-a-chip (LOC) device is developed to quantitatively measure the biomechanical properties of lily (Lilium longiflorum) pollen tubes. A single pollen tube is fixed inside the microfluidic chip at a specific orientation and subjected to compression by a soft membrane. By comparing the deformation of the pollen tube at a given external load (compressibility) and the effect of turgor pressure on the tube diameter (stretch ratio) with finite element modeling, its mechanical properties are determined. The turgor pressure and wall stiffness of the pollen tubes are found to decrease considerably with increasing initial diameter of the pollen tubes. This observation supports the hypothesis that tip-growth is regulated by a delicate balance between turgor pressure and wall stiffness. The LOC device is modular and adaptable to a variety of cells that exhibit tip-growth, allowing for the straightforward measurement of mechanical properties.

Starch turnover and metabolism during flower and early embryo development

A systematic characterization of starch turnover in Arabidopsis during reproductive development unravels new starch deposits and sheds light on starch metabolism and transport in reproductive tissues. The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed.

LRX Proteins play a crucial role in pollen grain and pollen tube cell wall development

LRR extensins are extracellular proteins that associate with and influence processes at the plasma membrane that are important for pollen grain germination and pollen tube growth. Leu-rich repeat extensins (LRXs) are chimeric proteins containing an N-terminal Leu-rich repeat (LRR) and a C-terminal extensin domain. LRXs are involved in cell wall formation in vegetative tissues and required for plant growth. However, the nature of their role in these cellular processes remains to be elucidated. Here, we used a combination of molecular techniques, light microscopy, and transmission electron microscopy to characterize mutants of pollen-expressed LRXs in Arabidopsis (Arabidopsis thaliana). Mutations in multiple pollen-expressed lrx genes cause severe defects in pollen germination and pollen tube growth, resulting in a reduced seed set. Physiological experiments demonstrate that manipulating Ca2+ availability partially suppresses the pollen tube growth defects, suggesting that LRX proteins influence Ca2+-related processes. Furthermore, we show that LRX protein localizes to the cell wall, and its LRR-domain (which likely mediates protein-protein interactions) is associated with the plasma membrane. Mechanical analyses by cellular force microscopy and finite element method-based modeling revealed significant changes in the material properties of the cell wall and the fine-tuning of cellular biophysical parameters in the mutants compared to the wild type. The results indicate that LRX proteins might play a role in cell wall-plasma membrane communication, influencing cell wall formation and cellular mechanics.

Maybe she's NOT the boss: male–female crosstalk during sexual plant reproduction

New insights into the molecular dialogue between male and female during sexual plant reproduction show that even plant sex does not work without clear communication.Please see related Research article: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0928-x