A.M.C. Emons

Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells

The rate of insertion and lifetime of cellulose-synthesizing complexes at the plasma membrane is dependent on the organization of the actin cytoskeleton. The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.

Differential Regulation of Cellulose Orientation at the Inner and Outer Face of Epidermal Cells in the Arabidopsis Hypocotyl

Whereas microtubules and cellulose synthase trajectories are transversely oriented on the outer epidermal surface of Arabidopsis hypocotyl cells only for part of the growth cycle, microtubules and microfibrils at the inner surface remain transverse throughout growth. The inner face of the epidermis is thus established as a regulator of growth anisotropy in the hypocotyl. It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.

Rates of exocytosis and endocytosis in Arabidopsis root hairs and pollen tubes

Exocytosis and endocytosis are pivotal in many biological processes, but remain difficult to quantify. Here we combine a new algorithm for estimating vesicle size with a detailed morphological analysis of tip‐growing cells, in which exocytosis is highly localized and therefore more readily quantified. Cell preservation was rendered as life‐like as possible by rapid freezing. This allowed us to produce the first estimates of exocytosis rates in the root hairs and pollen tubes of the model plant Arabidopsis. To quantify exocytosis and endocytosis rates during cell growth, we measured the diameter of vesicles located in the tips of Arabidopsis root hairs and pollen tubes and the widths of cell walls and the cell lumen in longitudinal thin transmission electron microscopic sections. In addition, we measured growth velocities of Arabidopsis root hairs and pollen tubes, using video microscopy. The number of exocytotic vesicles required for cell wall expansion, and the amount of excess membrane inserted into the plasma membrane to be internalized, were estimated from the values that were obtained. The amount of excess membrane that is inserted into the plasma membrane during cell growth was estimated as 86.7% in root hairs and 79% in pollen tubes. This membrane has to be recycled by endocytosis. From counting of the total number of vesicles that is present in thin EM sections through the pollen tube tip, we estimated the average number of vesicles that is present in the tip of pollen tubes. By calculating the total amount of membrane and cell wall material that is required for continued cell growth, assuming that all vesicles are exocytotic, we estimated that pollen tubes continue to grow for 33 s when delivery of vesicles to the tip is inhibited. We arrested vesicle delivery to the tip by application of cytochalasin D. After cytochalasin D application, pollen tubes continued to grow for 30–40 s, which is in the same range as the estimated value of 33 s and shows that in this time frame, the availability of exocytotic vesicles is not a limiting factor.

EXO70A1-Mediated Vesicle Trafficking Is Critical for Tracheary Element Development in Arabidopsis

Genes encoding for EXO70, a component of the exocyst complex, are highly expanded in plant genomes, with reasons unknown. EXO70A1 expressed primarily in tracheary elements regulates vesicle trafficking during xylem formation, suggesting that individual EXO70 members in plants may act in cell type– or cargo-specific exocytosis. Exocysts are highly conserved octameric complexes that play an essential role in the tethering of Golgi-derived vesicles to target membranes in eukaryotic organisms. Genes encoding the EXO70 subunit are highly duplicated in plants. Based on expression analyses, we proposed previously that individual EXO70 members may provide the exocyst with functional specificity to regulate cell type– or cargo-specific exocytosis, although direct evidence is not available. Here, we show that, as a gene expressed primarily during tracheary element (TE) development, EXO70A1 regulates vesicle trafficking in TE differentiation in Arabidopsis thaliana. Mutations of EXO70A1 led to aberrant xylem development, producing dwarfed and nearly sterile plants with very low fertility, reduced cell expansion, and decreased water potential and hydraulic transport. Grafting of a mutant shoot onto wild-type rootstock rescued most of these aboveground phenotypes, while grafting of a wild-type shoot to the mutant rootstock did not rescue the short root hair phenotype, consistent with the role of TEs in hydraulic transport from roots to shoots. Histological analyses revealed an altered pattern of secondary cell wall thickening and accumulation of large membrane-bound compartments specifically in developing TEs of the mutant. We thus propose that EXO70A1 functions in vesicle trafficking in TEs to regulate patterned secondary cell wall thickening.

Intrusive growth of flax phloem fibers is of intercalary type.

Flax (Linum usitatissimum L.) phloem fibers elongate considerably during their development and intrude between existing cells. We questioned whether fiber elongation is caused by cell tip growth or intercalary growth. Cells with tip growth are characterized by having two specific zones of cytoplasm in the cell tip, one with vesicles and no large organelles at the very tip and one with various organelles amongst others longitudinally arranged cortical microtubules in the subapex. Such zones were not observed in elongating flax fibers. Instead, organelles moved into the very tip region, and cortical microtubules showed transversal and helical configurations as known for cells growing in intercalary way. In addition, pulse-chase experiments with Calcofluor White resulted in a spotted fluorescence in the cell wall all over the length of the fiber. Therefore, it is concluded that fiber elongation is not achieved by tip growth but by intercalary growth. The intrusively growing fiber is a coenocytic cell that has no plasmodesmata, making the fibers a symplastically isolated domain within the stem.

Hydrodynamic flow in the cytoplasm of plant cells

Plant cells show myosin‐driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor‐driven organelle transport? Our recent simulation study ( Houtman et al., 2007 ) shows that active transport of organelles gives rise to a drag in the cytosol, setting up a hydrodynamic flow, which contributes to a fast distribution of proteins and nutrients in plant cells. Here, we show experimentally that actively transported organelles produce hydrodynamic flow that significantly contributes to the movement of the molecules in the cytosol. We have used fluorescence recovery after photobleaching and show that in tobacco Bright Yellow 2 (BY‐2) suspension cells constitutively expressing cytoplasmic green fluorescent protein (GFP), free GFP molecules move faster in cells with active transport of organelles than in cells where this transport has been inhibited with the general myosin inhibitor BDM (2,3‐butanedione monoxime). Furthermore, we show that the direction of the GFP movement in the cells with active transport is the same as that of the organelle movement and that the speed of the GFP in the cytosol is proportional to the speed of the organelle movement. In large BY‐2 cells with fast cytoplasmic streaming, a GFP molecule reaches the other side of the cell approximately in the similar time frame (about 16 s) as in small BY‐2 cells that have slow cytoplasmic streaming. With this, we suggest that hydrodynamic flow is important for efficient transport of cytosolic molecules in large cells. Hydrodynamic flow might also contribute to the movement of larger structures than molecules in the cytoplasm. We show that synthetic lipid (DOPG) vesicles and ‘stealth’ vesicles with PEG phospholipids moved in the cytoplasm.

Cellulose microfibril deposition: coordinated activity at the plant plasma membrane.

Plant cell wall production is a membrane‐bound process. Cell walls are composed of cellulose microfibrils, embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose synthase complexes into the plasma membrane. These complexes, the nanomachines that produce the cellulose microfibrils, move inside the plasma membrane leaving the cellulose microfibrils in their wake. Cellulose microfibril angle is an important determinant of cell development and of tissue properties and as such relevant for the industrial use of plant material. Here, we provide an integrated view of the events taking place in the not more than 100 nm deep area in and around the plasma membrane, correlating recent results provided by the distinct field of plant cell biology. We discuss the coordinated activities of exocytosis, endocytosis, and movement of cellulose synthase complexes while producing cellulose microfibrils and the link of these processes to the cortical microtubules.

Dissection of Nod factor signalling in legumes: cell biology, mutants and pharmacological approaches

Nodulation factors (NFs) are lipochito‐oligosaccharide signal molecules excreted by soil‐living rhizobia. These molecules elicit a range of responses in the legume roots, with which the bacteria can live in symbiosis. In this review we focus on the genetic, pharmacological and cell biological approaches that have been, and are being, undertaken to decipher the signalling pathways that lead to the symbiotic responses in the plant.

Texture of cellulose microfibrils of root hair cell walls of Arabidopsis thaliana, Medicago truncatula, and Vicia sativa

Cellulose is the most abundant biopolymer on earth, and has qualities that make it suitable for biofuel. There are new tools for the visualisation of the cellulose synthase complexes in living cells, but those do not show their product, the cellulose microfibrils (CMFs). In this study we report the characteristics of cell wall textures, i.e. the architectures of the CMFs in the wall, of root hairs of Arabidopsis thaliana, Medicago truncatula and Vicia sativa and compare the different techniques we used to study them. Root hairs of these species have a random primary cell wall deposited at the root hair tip, which covers the outside of the growing and fully grown hair. The secondary wall starts between 10 (Arabidopsis) and 40 (Vicia) μm from the hair tip and the CMFs make a small angle, Z as well as S direction, with the long axis of the root hair. CMFs are 3–4 nm wide in thin sections, indicating that single cellulose synthase complexes make them. Thin sections after extraction of cell wall matrix, leaving only the CMFs, reveal the type of wall texture and the orientation and width of CMFs, but CMF density within a lamella cannot be quantified, and CMF length is always underestimated by this technique. Field emission scanning electron microscopy and surface preparations for transmission electron microscopy reveal the type of wall texture and the orientation of individual CMFs. Only when the orientation of CMFs in subsequent deposited lamellae is different, their density per lamella can be determined. It is impossible to measure CMF length with any of the EM techniques.

Microtubule dynamics during preprophase band formation and the role of endoplasmic microtubules during root hair elongation.

Plant cells possess a number of distinct microtubule cytoskeleton configurations that are successively being formed, remodeled and broken down during the life of a cell: the interphase cortical microtubules and those encaging the nucleus, the cortical preprophase band, the spindle and the phragmoplast. In order to understand whether and how dynamic instability of microtubules contributes to the transition from a cortical array to the preprophase band, we studied the dynamic instability of individual microtubules in BY2 cells expressing a GFP-microtubule binding domain (GFP-MBD) fusion protein. The step from cortical array to preprophase band was also studied at the level of the microtubule population dynamics. The results showed that microtubule dynamic instability is altered during preprophase band formation and that the process is biphasic. Interphase plant cells have not been reported to possess endoplasmic microtubules, except around the nucleus. However, using both a GFP-MBD fusion protein and immuno-cytochemistry after freeze fixation/ freeze substitution, we found endoplasmic microtubules in the sub-apical area of root hairs of the legume Medicago truncatula. Drug experiments showed that these microtubules are less stable than the cortical microtubules. Real-time 4D confocal microscopy revealed that the endoplasmic microtubules in the subapical region are very dynamic, constantly changing between phases of growth and shrinkage. Moreover, single microtubules were observed to grow from the sub-apical region into the very tip of the root hair. The endoplasmic sub-apical microtubules contribute to the organization of the cell, i.e. the cell architecture, which includes keeping the nucleus at a certain distance from the growing root hair tip. After depolymerization of the endoplasmic microtubules only, the distance between the growing root hair tip and the nucleus became larger, but the nucleus still followed the expanding tip, albeit at a larger distance than before depolymerization of the endoplasmic microtubules. Depolymerization of sub-apical endoplasmic microtubules, alone or along with the cortical microtubules, retarded, but did not stop, hair elongation. This novel microtubule array has not been reported for Arabidopsis root hairs. Further study should show whether it is specific for legumes and related to their interaction with Rhizobium bacteria. * Corresponding author. Tel.: +31-317-484329; fax: +31-317-485005. E-mail address: annemie.emons@pcb.dpw.wag-ur.nl (A.M.C. Emons). Cell Biology International 27 (2003) 295 Cell Biology International