Diffusion and bulk flow in phloem loading - a theoretical analysis of the polymer trap mechanism in plants

Plants create sugar in the mesophyll cells of their leaves by photosynthesis. This sugar, mostly sucrose, has to be loaded via the bundle sheath into the phloem vascular system (the sieve elements), where it is distributed to growing parts of the plant. We analyze the feasibility of a particular loading mechanism, active symplasmic loading, also called the polymer trap mechanism, where sucrose is transformed into heavier sugars, such as raffinose and stachyose, in the intermediary-type companion cells bordering the sieve elements in the minor veins of the phloem. Keeping the heavier sugars from diffusing back requires that the plasmodesmata connecting the bundle sheath with the intermediary cell act as extremely precise filters, which are able to distinguish between molecules that differ by less than 20% in size. In our modeling, we take into account the coupled water and sugar movement across the relevant interfaces, without explicitly considering the chemical reactions transforming the sucrose into the heavier sugars. Based on the available data for plasmodesmata geometry, sugar concentrations and flux rates, we conclude that this mechanism can in principle function. We find that the water flow through the plasmodesmata, which has not been quantified before, contributes only 10-20% to the sucrose flux into the intermediary cells, while the main part is transported by diffusion. On the other hand, the subsequent sugar translocation into the sieve elements would very likely be carried predominantly by bulk water flow through the plasmodesmata. Thus, in contrast to apoplasmic loaders, all the necessary water for phloem translocation would be supplied in this way with no need for additional water uptake across the plasma membranes of the phloem.Comment: 29 pages with 5 figure

Modeling the parameters for plasmodesmal sugar filtering in active symplasmic phloem loaders

Plasmodesmata (PD) play a key role in loading of sugars into the phloem. In plant species that employ the so-called active symplasmic loading strategy, sucrose that diffuses into their unique intermediary cells (ICs) is converted into sugar oligomers. According to the prevalent hypothesis, the oligomers are too large to pass back through PD on the bundle sheath side, but can pass on into the sieve element to be transported in the phloem. Here, we investigate if the PD at the bundle sheath-IC interface can indeed fulfill the function of blocking transport of sugar oligomers while still enabling efficient diffusion of sucrose. Hindrance factors are derived via theoretical modeling for different PD substructure configurations: sub-nano channels, slit, and hydrogel. The results suggest that a strong discrimination could only be realized when the PD opening is almost as small as the sugar oligomers. In order to find model parameters that match the in vivo situation, we measured the effective diffusion coefficient across the interface in question in Cucurbita pepo with 3D-photoactivation microscopy. Calculations indicate that a PD substructure of several sub-nano channels with a radius around 7 Å, a 10.4 Å-wide slit or a hydrogel with 49% polymer fraction would be compatible with the effective diffusion coefficient. If these configurations can accommodate sufficient flux of sucrose into the IC, while blocking raffinose and stachyose movement was assessed using literature data. While the slit-configuration would efficiently prevent the sugar oligomers from “leaking” from the IC, none of the configurations could enable a diffusion-driven sucrose flux that matches the reported rates at a physiologically relevant concentration potential. The presented data provides a first insight on how the substructure of PD could enable selective transport, but indicates that additional factors are involved in efficient phloem loading in active symplasmic loading species

Super-resolution imaging with Pontamine Fast Scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cells

BACKGROUND: In plants, a complex cell wall protects cells and defines their shape. Cellulose fibrils form a multilayered network inside the cell-wall matrix that plays a direct role in controlling cell expansion. Resolving the structure of this network will allow us to comprehend the relationship of cellulose fibril orientation and growth. The fluorescent dye Pontamine Fast Scarlet 4BS (PFS) was shown to stain cellulose with high specificity and could be used to visualize cellulose bundles in cell walls of Arabidopsis root epidermal cells with confocal microscopy. The resolution limit of confocal microscopy of some 200 nm in xy and 550 nm in z for green light, restricts the direct visualization of cellulose to relatively large bundles, whereas the structure of cellulose microfibrils with their diameter below 10 nm remains unresolved. Over the last decade, several so-called super-resolution microscopy approaches have been developed; in this paper we explore the potential of such approaches for the direct visualization of cellulose. RESULTS: To ensure optimal imaging we determined the spectral properties of PFS-stained tissue. PFS was found not to affect cell viability in the onion bulb scale epidermis. We present the first super-resolution images of cellulose bundles in the plant cell wall produced by direct stochastic optical reconstruction microscopy (dSTORM) in combination with total internal reflection fluorescence (TIRF) microscopy. Since TIRF limits observation to the cell surface, we tested as alternatives 3D-structured illumination microscopy (3D-SIM) and confocal microscopy, combined with image deconvolution. Both methods offer lower resolution than STORM, but enable 3D imaging. While 3D-SIM produced strong artifacts, deconvolution gave good results. The resolution was improved over conventional confocal microscopy and the approach could be used to demonstrate differences in fibril orientation in different layers of the cell wall as well as particular cellulose fortifications around plasmodesmata. CONCLUSIONS: Super-resolution light microscopy of PFS-stained cellulose fibrils is possible and the increased resolution over conventional approaches makes it a valuable tool for the investigation of the cell-wall structure. This is one step in method developments that will close the gap to more invasive techniques, such as atomic force and electron microscopy

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays

Spatio-temporal protease activity and cell death induced by Natural Killer cells and soluble CD95L

In the CD95 signaling cascade molecular processes take place, which rely on protein multimerization for activity. In this work, I aimed at measuring the initial events in CD95-mediated apoptosis, in particular the kinetics of the molecular events ligand binding, receptor oligomerization, FADD recruitment and caspase-8 activation to understand which of those processes involve critical kinetic steps. For this, I used the model cell HeLa. I have succeeded in showing and comparing the kinetics of ligand-binding, FADD recruitment and caspase-8 activation by using confocal microscopy. I have found that a critical step before caspase- 8 activation is the binding of FADD to the activated receptor. Furthermore, I found that the activation of the receptor, presumably by its oligomerization, is a limiting step in CD95-mediated apoptosis. Moreover, I showed that the internalization and activation of the receptor CD95 at the plasma membrane are competing processes. To arrive at these findings, two forms of soluble CD95L were compared, one naturally occurring form with weak activity (sCD95L) and one recombinant form with high activity (IZsCD95L). Next to biochemical methods, fluorescence-microscopy based techniques were particularly applied and I have been able to confirm that both forms are trimeric. Notably, I developed a new method to evaluate single-molecule photobleaching data suitable to infer the subunit stoichiometry of molecules at picomolar concentration. Secondly, I aimed at understanding how natural killer cells (NK cells) mediate cell death in target cells. In addition to CD95L, NK cells use own proteases to induce the death of certain cells. In this context, I have been able to distinguish the activity of caspase-8 and granzymes in individual HeLa target cells based on the method of Beaudouin et al. 2013 and by using the NK cell line NK92. While granzyme B activity was measured in cells that died early after NK-cell contact, caspase-8 activity was measured in those which died late upon NK-cell contact. Noteworthy was the observation that granzyme B was mainly active near the plasma membrane, and in lesser amounts inside the nucleus. This was consistent with my results showing that the granzyme B inhibitor serpinB9 is localized at the plasma membrane. In contrast, granzyme A was probably not active in the cytosol, but in the nucleus of the cell