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.

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.

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.

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.

Actin organization during eucalyptus root hair development and its response to fungal hypaphorine.

The fungus Pisolithus microcarpus establishes an ectomycorrhiza with Eucalyptus globulus. This symbiosis involves a fungal synthesis and secretion of hypaphorine, an indolic compound. Previous studies have shown that hypaphorine induces an alteration in the actin cytoskeleton of elongating root hairs and inhibits hair elongation. Using an alternative approved method, we analyzed the effects of hypaphorine on the E. globulus root hair cyto-architecture and actin configuration in more detail and provide new results. One mM hypaphorine stops root hair elongation within 20 min, and changes the hair cyto-architecture. Semi-quantitative analysis of the actin cytoskeleton before and after treatment with hypaphorine shows that hypaphorine induces a shift from fine F-actin to F-actin bundles in the sub-apex of the hair, which occurs first in the mid-plane of the cell. This creates a sub-apical cell centre free of filamentous actin, an actin configuration that differs from that during developmental growth arrest. The mechanism of action of hypaphorine is discussed.

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.

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.

A dynamical model for plant cell wall architecture formation.

We discuss a dynamical mathematical model to explain cell wall architecture in plant cells. The highly regular textures observed in cell walls reflect the spatial organisation of the cellulose microfibrils (CMFs), the most important structural component of cell walls. Based on a geometrical theory proposed earlier [A. M. C. Emons, Plant, Cell and Environment 17, 3-14 (1994)], the present model describes the space-time evolution of the density of the so-called rosettes, the CMF synthesizing complexes. The motion of these rosettes in the plasma membrane is assumed to be governed by an optimal packing constraint on the CMFs plus adherent matrix material, that couples the direction of motion, and hence the orientation of the CMF being deposited, to the local density of rosettes. The rosettes are created inside the cell in the endoplasmatic reticulum and reach the cell-membrane via vesicles derived from Golgi-bodies. After being inserted into the plasma membrane they are assumed to be operative for a fixed, finite lifetime. The plasma membrane domains within which rosettes are activated are themselves also supposed to be mobile. We propose a feedback mechanism that precludes the density of rosettes to rise beyond a maximum dictated by the geometry of the cell. The above ingredients lead to a quasi-linear first order PDE for the rosette-density. Using the method of characteristics this equation can be cast into a set of first order ODEs, one of which is retarded. We discuss the analytic solutions of the model that give rise to helicoidal, crossed polylamellate, helical, axial and random textures, since all cell walls are composed of (or combinations of) these textures.

Lipochito-oligosaccharide nodulation factors stimulate cytoplasmic polarity with longitudinal endoplasmic reticulum and vesicles at the tip in vetch root hairs.

Vetch root hair development has four stages: bulge, growing, growth terminating, and full-grown hair. In the assay we used, the nodulation factor induced swellings and outgrowths in growth-terminating hairs. Bulges, swellings, and full-grown hairs have transverse endoplasmic reticulum (ER) and no tip-accumulated vesicles. Growing hairs and outgrowths show vesicle accumulation in the tip and longitudinal subapical ER. Bulge walls and walls of swellings appear mottled.

Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas.

Self-incompatibility (SI) is a genetically controlled process used to prevent self-pollination. In Papaver rhoeas, the induction of SI is triggered by a Ca(2)+-dependent signaling pathway that results in the rapid and S allele-specific inhibition of pollen tube tip growth. Tip growth of cells is dependent on a functioning actin cytoskeleton. We have investigated the effect of self-incompatibility (S) proteins on the actin cytoskeleton in poppy pollen tubes. Here, we report that the actin cytoskeleton of incompatible pollen tubes is rapidly and dramatically rearranged during the SI response, not only in our in vitro SI system but also in vivo. We demonstrate that nonspecific inhibition of growth does not result in similar actin rearrangements. Because the SI-induced alterations are not observed if growth stops, this clearly demonstrates that these alterations are triggered by the SI signaling cascade rather than merely resulting from the consequent inhibition of growth. We establish a detailed time course of events and discuss the mechanisms that might be involved. Our data strongly implicate a role for the actin cytoskeleton as a target for signaling pathways involved in the SI response of P. rhoeas.

Spectrin-like proteins in plant nuclei.

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220-240 kDa, typical for human erythrocyte spectrin, and a prominent 60 kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.

The cytoskeleton in plant and fungal cell tip growth.

Tip-growing cells have a particular lifestyle that is characterized by the following features: (1) the cells grow in one direction, forming a cylindrical tube; (2) tip-growing cells are able to penetrate their growth environment, thus having to withstand considerable external forces; (3) the growth velocity of tip-growing cells is among the fastest in biological systems. Tip-growing cells therefore appear to be a system well suited to investigating growth processes. The cytoskeleton plays an important role in cell growth in general, which is why tip-growing cells provide an excellent model system for studying this aspect. The cytoskeletal system comprises structural elements, such as actin filaments and microtubules, as well as proteins that link these elements, control their configuration or are responsible for transport processes using the structural elements as tracks. Common aspects as well as differences in configuration and function of the cytoskeleton in various types of tip-growing cells reveal the general principles that govern the relationship between the cytoskeleton and cell growth.

How the deposition of cellulose microfibrils builds cell wall architecture.

Cell walls, the extracytoplasmic matrices of plant cells, consist of an ordered array of cellulose microfibrils embedded in a matrix of polysaccharides and glycoproteins. This construction is reminiscent of steel rods in reinforced concrete. How a cell organizes these ordered textures around itself, creating its own desirable environment, is a fascinating question. We believe that nature adopted an economical solution to this design problem: it exploits the geometrical constraints imposed by the shape of the cell and the limited space in which microfibrils are deposited, enabling the wall textures essentially to 'build themselves'. This does not imply that the cell cannot control its wall texture. On the contrary, the cell has ample regulatory mechanisms to control wall texture formation by controlling the insertion of synthases and the distance between individual microfibrils within a wall lamella.

Spectrin-like proteins in green algae (Desmidiaceae).

Immunochemical detection of actin as well as spectrin-like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43 kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross-reactivity with desmid proteins is found at about the molecular mass of 220 kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120 kDa and 70 kDa, which are probably breakdown products. An antibody against chicken alpha-actinin, a small protein of the spectrin superfamily, recognizes bands at 90 kDa, where it is expected, and 70 kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI 4.6 with antibodies against spectrin. Immunogold labelling of spectrin and alpha-actinin antigens on high-pressure frozen, freeze-substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin-like proteins in desmid cells which are probably functional in exocytosis.

The making of the architecture of the plant cell wall: how cells exploit geometry.

Cell wall deposition is a key process in the formation, growth, and differentiation of plant cells. The most important structural components of the wall are long cellulose microfibrils, which are synthesized by synthases embedded in the plasma membrane. A fundamental question is how the microfibrils become oriented during deposition at the plasma membrane. The current textbook explanation for the orientation mechanism is a guidance system mediated by cortical microtubules. However, too many contraindications are known in secondary cell walls for this to be a universal mechanism, particularly in the case of helicoidal arrangements, which occur in many situations. An additional construction mechanism involves liquid crystalline self-assembly [A. C. Neville (1993) Biology of Fibrous Composites: Development Beyond the Cell Membrane (Cambridge Univ. Press, Cambridge, U.K.)], but the required amount of bulk material that is able to equilibrate thermally is not normally present at any stage of the wall deposition process. Therefore, we have asked whether the complex ordered texture of helicoidal cell walls can be formed in the absence of direct cellular guidance mechanisms. We propose that they can be formed by a mechanism that is based on geometrical considerations. It explains the genesis of the complicated helicoidal texture and shows that the cell has intrinsic, versatile tools for creating a variety of textures. A compelling feature of the model is that local rules generate global order, a typical phenomenon of life.

The ipiO gene of Phytophthora infestans is highly expressed in invading hyphae during infection.

The expression of the in planta-induced gene ipiO of the potato late blight pathogen Phytophthora infestans was analyzed during various developmental stages of its life cycle. ipiO mRNA was detected in zoospores, cysts, germinating cysts, and young mycelia, but not in sporangia or in old mycelia grown in vitro. ipiO is not only expressed in stages prior to infection but also during colonization of potato and tomato leaves. In disease lesions, ipiO mRNA was detected in the water-soaked area and the healthy-looking plant tissue surrounding it. In contrast, ipiO mRNA was not found in necrotized tissue or in sporulating areas of a lesion. To determine more precisely the location and time of ipiO gene expression in planta, cytological assays were performed using a P. infestans transformant expressing a transcriptional fusion between the ipiO1 promoter and the beta-glucuronidase (GUS) reporter gene. GUS staining was found specifically in the subapical and vacuolated area of tips of invading hyphae. The histochemical GUS assays demonstrate that ipiO is expressed during biotrophic stages of the disease cycle.

Ethylene provides positional information on cortical cell division but is not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction.

Nod factors secreted by Rhizobium leguminosarum bv. viciae induce root hair deformation, involving a reinitiation of tip growth, and the formation of nodule primordia in Vicia sativa (vetch). Ethylene is a potent inhibitor of cortical cell division, an effect that can be counteracted by applying silver ions (Ag+) or aminoethoxy-vinylglycine (AVG). In contrast to the inhibitory effect on cortical cell division, ethylene promotes the formation of root hairs (which involves tip growth) in the root epidermis of Arabidopsis. We investigate the possible paradox concerning the action of ethylene, putatively promoting Nod factor induced tip growth whilst, at the same time, inhibiting cortical cell division. We show, by using the ethylene inhibitors AVG and Ag+, that ethylene has no role in the reinitiation of root hair tip growth induced by Nod factors (root hair deformation) in vetch. However, root hair formation is controlled, at least in part, by ethylene. Furthermore, we show that ACC oxidase, which catalizes the last step in ethylene biosynthesis, is expressed in the cell layers opposite the phloem in that part of the root where nodule primordia are induced upon inoculation with Rhizobium. Therefore, we test whether endogenously produced ethylene provides positional information controlling the site where nodule primordia are formed by determining the position of nodules formed on pea roots grown in the presence of AVG or Ag+.

Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells.

An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.

Helicoidal cell-wall texture in root hairs.

It is shown that root hairs of most aquatic plants have a helicoidal cell-wall texture. Cell walls of root hairs of the aquatic/marshland plant Ranunculus lingua, however, have an axial microfibril alignment. The occurrence of a helicoidal wall texture is not limited to root hairs of aquatic plants: the terrestrial plant Zebrina purpusii has a helicoidal root-hair wall texture, too. With the exception of the grasses, the occurrence of root hairs with helicoidal cell walls pertains to species with predetermined root-hair-forming cells, trichoblasts. The rotation mode of the helicoid is species-specific. The average angle between fibrils of adjacent lamellae varies from 23° to 40°. In Hydrocharis morsus-ranae, cortical microtubules have a net-axial orientation and thus do not parallel nascent microfibrils. The deposition of the helicoidal cell wall is discussed.