whole-mount-clearing-staining-arabidopsis-flower-organs

Whole-mount Clearing and Staining of <em>Arabidopsis</em> Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

University of Zurich

Expansion des cellules v&eacute;g&eacute;tales est contr&ocirc;l&eacute;e par un &eacute;quilibre finement &eacute;labor&eacute; entre la pression osmotique intracellulaire, le rel&acirc;chement de la paroi cellulaire et la biosynth&egrave;se de la paroi cellulaire. Pour comprendre ces processus, il est important d&#39;acqu&eacute;rir une connaissance approfondie de la m&eacute;canique de la paroi cellulaire. Les tubes polliniques sont productrices de pointe cellules qui fournissent un syst&egrave;me id&eacute;al pour &eacute;tudier les propri&eacute;t&eacute;s m&eacute;caniques au niveau de la cellule unique. Avec les approches disponibles, il n&#39;&eacute;tait pas facile de mesurer des param&egrave;tres m&eacute;caniques importants des tubes polliniques, tels que l&#39;&eacute;lasticit&eacute; de la paroi cellulaire. Nous avons utilis&eacute; un microscope &agrave; force cellulaire (CFM) pour mesurer la rigidit&eacute; apparente des tubes polliniques de lily. En combinaison avec un mod&egrave;le m&eacute;canique bas&eacute; sur la m&eacute;thode des &eacute;l&eacute;ments finis (MEF), cela nous a permis de calculer la pression de turgescence et &eacute;lasticit&eacute; de la paroi de la cellule, que nous avons trouv&eacute; &agrave; environ 0,3 MPa et 20-90 MPa, respectivement. En outre et contrairement aux rapports pr&eacute;c&eacute;dents, nous avons montr&eacute; que la diff&eacute;rence de rigidit&eacute; entre l&#39;extr&eacute;mit&eacute; du tube pollinique et la tige peut s&#39;expliquer uniquement par la g&eacute;om&eacute;trie du tube pollinique. CFM, en combinaison avec un mod&egrave;le MEF, fournit une m&eacute;thode puissante pour &eacute;valuer les param&egrave;tres m&eacute;caniques importants d&#39;unique, les cellules en croissance. Nos r&eacute;sultats indiquent que la paroi cellulaire des tubes polliniques de plus en plus a des propri&eacute;t&eacute;s m&eacute;caniques similaires au caoutchouc. Ceci sugg&egrave;re qu&#39;un tube pollinique enti&egrave;rement turgescents est une cellule relativement raide, mais souple qui peut r&eacute;agir tr&egrave;s rapidement &agrave; des obstacles ou des substances attractives en ajustant la direction de la croissance sur son chemin &agrave; travers la femelle transmettant des tissus.

Le tube pollinique : une carapace molle avec un noyau dur.

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.

The pollen tube: a soft shell with a hard core.

Espansione delle cellule vegetali &egrave; controllato da un perfetto equilibrio tra turgescenza intracellulare, allentamento della parete cellulare e la parete delle cellule biosintesi. Per capire questi processi, &egrave; importante acquisire una conoscenza approfondita della meccanica della parete cellulare. Tubi pollinici sono punta-crescita delle cellule che forniscono un sistema ideale per studiare le propriet&agrave; meccaniche a livello di singola cellula. Con gli approcci disponibili non &egrave; stato facile misurare importanti parametri meccanici di tubi pollinici, ad esempio l&#39;elasticit&agrave; della parete delle cellule. Abbiamo utilizzato un microscopio a forza cellulare (CFM) per misurare l&#39;apparente rigidit&agrave; dei tubi pollinici giglio. In combinazione con un modello meccanico basato sul metodo degli elementi finiti (FEM), questo ci ha permesso di calcolare il turgore e l&#39;elasticit&agrave; della parete delle cellule, che abbiamo trovato a circa 0,3 MPa e 20-90 MPa, rispettivamente. Inoltre e contrariamente a precedenti relazioni, abbiamo dimostrato che la differenza di rigidezza tra la punta del tubo pollinico e la tibia pu&ograve; essere spiegata unicamente dalla geometria del tubo di polline. CFM, in combinazione con un modello basato su FEM, fornisce un metodo potente per valutare importanti parametri meccanici del singolo, le cellule in crescita. I nostri risultati indicano che la parete cellulare dei tubi pollinici in crescita ha propriet&agrave; meccaniche simili alla gomma. Questo suggerisce che un tubo pollinico completamente turgido &egrave; una cella relativamente rigida, ma flessibile che pu&ograve; reagire molto rapidamente a ostacoli o attrattivi regolando la direzione della crescita nel suo cammino attraverso la femmina trasmissione di tessuto.

Il tubo pollinico: un guscio morbido con un nocciolo duro.

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.

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

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).

Measuring the Mechanical Properties of Plant Cell Walls.

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.

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

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.

Starch Turnover and Metabolism during Flower and Early Embryo Development.

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.

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

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.

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

Pollen tubes are tip-growing plant cells that deliver the sperm cells to the ovules for double fertilization of the egg cell and the endosperm. Various directional cues can trigger the reorientation of pollen tube growth direction on their passage through the female tissues. Among the external stimuli, protons serve an important, regulatory role in the control of pollen tube growth. The generation of local guidance cues has been challenging when investigating the mechanisms of perception and processing of such directional triggers in pollen tubes. Here, we developed and characterized a microelectrode device to generate a local proton gradient and proton flux through water electrolysis. We confirmed that the cytoplasmic pH of pollen tubes varied with environmental pH change. Depending on the position of the pollen tube tip relative to the proton gradient, we observed alterations in the growth behavior, such as bursting at the tip, change in growth direction, or complete growth arrest. Bursting and growth arrest support the hypothesis that changes in the extracellular Hconcentration may interfere with cell wall integrity and actin polymerization at the growing tip. A change in growth direction for some pollen tubes implies that they can perceive the local proton gradient and respond to it. We also showed that the growth rate is directly correlated with the extracellular pH in the tip region. Our microelectrode approach provides a simple method to generate protons and investigate their effect on plant cell growth.

High precision, localized proton gradients and fluxes generated by a microelectrode device induce differential growth behaviors of pollen tubes.

In this introductory chapter, we describe male germline development in plants taking Arabidopsis thaliana as a reference species. We first describe the transition from sporophytic to germline development, then microsporogenesis including meiosis, followed by male gametophyte development prior to pollination, and finally the progamic phase culminating in double fertilization, which leads to the formation of the embryo and the endosperm. For detailed information on some of these processes or on the molecular underpinning of certain fate transitions, we refer the reader to recent reviews. An important but often neglected aspect of male gametophyte development is the formation of the unique pollen cell wall. In contrast to that of other plant cells, the pollen cell wall is composed of two principal layers, the intine and exine. While the intine, the inner pecto-cellulosic cell wall layer, is biochemically and structurally similar to a "classical" plant cell wall, the exine is a unique composite with sporopollenin as its main component. Biosynthesis of the cell wall is remarkably similar between the spores of mosses and ferns, and pollen of seed plants, although slight differences exist, even between closely related species (reviewed in Wallace et al., AoB Plants 2011:plr027, 2011). In the latter sections of this chapter, we will present a brief overview of cell wall development in Arabidopsis pollen, where this aspect has been intensively studied.

An Introduction to Male Germline Development.

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-expressedin Arabidopsis (). Mutations in multiple pollen-expressedgenes cause severe defects in pollen germination and pollen tube growth, resulting in a reduced seed set. Physiological experiments demonstrate that manipulating Caavailability partially suppresses the pollen tube growth defects, suggesting that LRX proteins influence Ca-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.

Article	Year
The pollen tube: a soft shell with a hard core. The Plant journal : for cell and molecular biology\| PubMed ID: 23106269	2013
A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery. Developmental cell\| PubMed ID: 24814317	2014
Measuring the Mechanical Properties of Plant Cell Walls. Plants (Basel, Switzerland)\| PubMed ID: 27135321	2015
Maybe she's NOT the boss: male-female crosstalk during sexual plant reproduction. Genome biology\| PubMed ID: 27159978	2016
Starch Turnover and Metabolism during Flower and Early Embryo Development. Plant physiology\| PubMed ID: 27794100	2016
Characterization of size-dependent mechanical properties of tip-growing cells using a lab-on-chip device. Lab on a chip\| PubMed ID: 27883138	2016
Massively Parallelized Pollen Tube Guidance and Mechanical Measurements on a Lab-on-a-Chip Platform. PloS one\| PubMed ID: 27977748	2016
High precision, localized proton gradients and fluxes generated by a microelectrode device induce differential growth behaviors of pollen tubes. Lab on a chip\| PubMed ID: 28098283	2017
An Introduction to Male Germline Development. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28936645	2017
LRX Proteins Play a Crucial Role in Pollen Grain and Pollen Tube Cell Wall Development. Plant physiology\| PubMed ID: 29247121	2018

Hannes Vogler

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