Pavement cells distinguish touch from letting go.

A micro-cantilever technique applied to individual leaf epidermis cells of intact Arabidopsis thaliana and Nicotiana tabacum synthesizing genetically encoded calcium indicators (R-GECO1 and GCaMP3) revealed that compressive forces induced local calcium peaks that preceded delayed, slowly moving calcium waves. Releasing the force evoked significantly faster calcium waves. Slow waves were also triggered by increased turgor and fast waves by turgor drops in pressure probe tests. The distinct characteristics of the wave types suggest different underlying mechanisms and an ability of plants to distinguish touch from letting go.

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness.

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein-protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.

The diffusive injection micropipette (DIMP).

The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern cell biology. Microinjection techniques include the penetration of the plasma membrane and, if present, the cell wall with micropipettes, and the application of pressure or electrical currents to drive the micropipette contents into the cell. These procedures interfere with cellular functions and therefore may induce artifacts. We designed the diffusive injection micropipette (DIMP) that avoids most of the possible artifacts due to the drastically reduced volume of its fluid contents and the utilization of diffusion for cargo delivery into the target cell. DIMPs were successfully tested in plant, fungal, and animal cells. Using the continuity of cytoplasmic dynamics over ten minutes after impalement of Nicotiana trichome cells as a criterion for non-invasiveness, we found DIMPs significantly less disruptive than conventional pressure microinjection. The design of DIMPs abolishes major sources of artifacts that cannot be avoided by other microinjection techniques. Moreover, DIMPs are inexpensive, easy to produce, and can be applied without specific equipment other than a micromanipulator. With these features, DIMPs may become the tool of choice for studies that require the least invasive delivery possible of materials into live cells.

Phloem unloading in Arabidopsis roots is convective and regulated by the phloem-pole pericycle.

In plants, a complex mixture of solutes and macromolecules is transported by the phloem. Here, we examined how solutes and macromolecules are separated when they exit the phloem during the unloading process. We used a combination of approaches (non-invasive imaging, 3D-electron microscopy, and mathematical modelling) to show that phloem unloading of solutes in Arabidopsis roots occurs through plasmodesmata by a combination of mass flow and diffusion (convective phloem unloading). During unloading, solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of 'funnel plasmodesmata'. While solutes are unloaded without restriction, large proteins are released through funnel plasmodesmata in discrete pulses, a phenomenon we refer to as 'batch unloading'. Unlike solutes, these proteins remain restricted to the phloem-pole pericycle. Our data demonstrate a major role for the phloem-pole pericycle in regulating phloem unloading in roots.