Protoplast Isolation and Manipulation in the Unicellular Model Plant Penium margaritaceum.

The unicellular freshwater green alga Penium margaritaceum has become a novel and valuable model organism for elucidating cell wall dynamics in plants. We describe a rapid and simple means for isolating protoplasts using commercial enzymes in a mannitol-based buffer. Protoplasts can be cultured and cell wall recovery can be monitored in sequentially diluted mannitol-based medium. We also describe an optimized protocol to prepare highly pure, organelle-free nuclei fractions from protoplasts using sucrose gradients. This technology provides a new and effective tool in Penium biology that can be used for analysis of cell wall polymer deposition, organelle isolation and characterization, and molecular research including genetic transformation and somatic hybridization.

Arabinogalactan Proteins and the Extracellular Matrix of Charophytes: A Sticky Business.

Charophytes represent the group of green algae whose ancestors invaded land and ultimately gave rise to land plants 450 million years ago. While Zygnematophyceae are believed to be the direct sister lineage to embryophytes, different members of this group (Penium, Spirogyra, Zygnema) and the advanced thallus forming Coleochaete as well as the sarcinoid basal streptophyte Chlorokybus were investigated concerning their vegetative extracellular matrix (ECM) properties. Many taxa exhibit adhesion phenomena that are critical for affixing to a substrate or keeping cells together in a thallus, however, there is a great variety in possible reactions to e.g., wounding. In this study an analysis of adhesion mechanisms revealed that arabinogalactan proteins (AGPs) are most likely key adhesion molecules. Through use of monoclonal antibodies (JIM13) or the Yariv reagent, AGPs were located in cell surface sheaths and cell walls that were parts of the adhesion focal zones on substrates including wound induced rhizoid formation. JIM5, detecting highly methyl-esterfied homoglacturonan and JIM8, an antibody detecting AGP glycan and LM6 detecting arabinans were also tested and a colocalization was found in several examples (e.g., Zygnema) suggesting an interplay between these components. AGPs have been described in this study to perform both, cell to cell adhesion in algae forming thalli and cell to surface adhesion in the filamentous forms. These findings enable a broader evolutionary understanding of the function of AGPs in charophyte green algae.

Imaging the Dynamics of Cell Wall Polymer Deposition in the Unicellular Model Plant, Penium margaritaceum.

The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.

Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in Arabidopsis.

BACKGROUND: The Arabidopsis root hair represents a valuable cell model for elucidating polar expansion mechanisms in plant cells and the overall biology of roots. The deposition and development of the cell wall is central to the root hair expansion apparatus. During this process, incorporation of specific wall polymers into the growing wall architecture constitutes a critical spatio-temporal event that controls hair size and growth rate and one that is closely coordinated with the cell's endomembrane, cytoskeletal and signal transduction apparatuses. RESULTS: In this study, the protocol for live cell labeling of roots with monoclonal antibodies that bind to specific wall polymers is presented. This method allows for rapid assessment of root hair cell wall composition during development and assists in describing changes to cell wall composition in transgenic mutant lines. Enzymatic "unmasking" of specific polymers prior to labeling allows for refined interpretation of cell wall chemistry. Live cell immunofluorescence data may also be correlated with transmission electron microscopy-based immunogold labeling. CONCLUSIONS: Live Arabidopsis root hairs may be labeled with cell wall polymer-specific antibodies. This methodology allows for direct visualization of cell wall dynamics throughout development in stable transgenic plant lines. It also provides an important new tool in the elucidation of the specific interactions occurring between membrane trafficking networks, cytoskeleton and the cell wall deposition/remodeling mechanism.

The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum.

BACKGROUND AND AIMS: Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division. METHODS: Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy. KEY RESULTS: The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division. CONCLUSIONS: The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross wall will form during cytokinesis. This suggests that prior to the evolution of land plants, a dynamic, cortical cytoskeletal array similar to a pre-prophase band had evolved in the charophytes. However, an interesting variation on the cortical band theme is present in Penium, where two satellite microtubule bands are produced at the onset of cell expansion, each of which is destined to become an IMB in the two daughter cells after cytokinesis. These unique cytoskeletal components demonstrate the close temporal control and highly coordinated cytoskeletal dynamics of cellular development in Penium.