2, 6-dichlorobenzonitrile Causes Multiple Effects on Pollen Tube Growth beyond Altering Cellulose Synthesis in Pinus bungeana Zucc

Cellulose is an important component of cell wall, yet its location and function in pollen tubes remain speculative. In this paper, we studied the role of cellulose synthesis in pollen tube elongation in Pinus bungeana Zucc. by using the specific inhibitor, 2, 6-dichlorobenzonitrile (DCB). In the presence of DCB, the growth rate and morphology of pollen tubes were distinctly changed. The organization of cytoskeleton and vesicle trafficking were also disturbed. Ultrastructure of pollen tubes treated with DCB was characterized by the loose tube wall and damaged organelles. DCB treatment induced distinct changes in tube wall components. Fluorescence labeling results showed that callose, and acidic pectin accumulated in the tip regions, whereas there was less cellulose when treated with DCB. These results were confirmed by FTIR microspectroscopic analysis. In summary, our findings showed that inhibition of cellulose synthesis by DCB affected the organization of cytoskeleton and vesicle trafficking in pollen tubes, and induced changes in the tube wall chemical composition in a dose-dependent manner. These results confirm that cellulose is involved in the establishment of growth direction of pollen tubes, and plays important role in the cell wall construction during pollen tube development despite its lower quantity.     

MicroFilament Analyzer,an image analysis tool for quantifying fibrillar orientation,reveals changes in microtubule organization during gravitropism

Image acquisition is an important step in the study of cytoskeleton organization. As visual interpretations and manual measurements of digital images are prone to errors and require a great amount of time, a freely available software package named MicroFilament Analyzer (MFA) was developed. The goal was to provide a tool that facilitates high‐throughput analysis to determine the orientation of filamentous structures on digital images in a more standardized, objective and repeatable way. Here, the rationale and applicability of the program is demonstrated by analyzing the microtubule patterns in epidermal cells of control and gravi‐stimulated Arabidopsis thaliana roots. Differential expansion of cells on either side of the root results in downward bending of the root tip. As cell expansion depends on the properties of the cell wall, this may imply a differential orientation of cellulose microfibrils. As cellulose deposition is orchestrated by cortical microtubules, the microtubule patterns were analyzed. The MFA program detects the filamentous structures on the image and identifies the main orientation(s) within individual cells. This revealed four distinguishable microtubule patterns in root epidermal cells. The analysis indicated that gravitropic stimulation and developmental age are both significant factors that determine microtubule orientation. Moreover, the data show that an altered microtubule pattern does not precede differential expansion. Other possible applications are also illustrated, including field emission scanning electron micrographs of cellulose microfibrils in plant cell walls and images of fluorescent actin.     

Cellulose microfibril alignment recovers from DCB-induced disruption despite microtubule disorganization

Cellulose microfibril deposition patterns define the direction of plant cell expansion. To better understand how microfibril alignment is controlled, we examined microfibril orientation during cortical microtubule disruption using the temperature-sensitive mutant of Arabidopsis thaliana, mor1-1. In a previous study, it was shown that at restrictive temperature for mor1-1, cortical microtubules lose transverse orientation and cells lose growth anisotropy without any change in the parallel arrangement of cellulose microfibrils. In this study, we investigated whether a pre-existing template of well-ordered microfibrils or the presence of well-organized cortical microtubules was essential for the cell to resume deposition of parallel microfibrils. We first transiently disrupted the parallel order of microfibrils in mor1-1 using a brief treatment with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). We then analysed the alignment of recently deposited cellulose microfibrils (by field emission scanning electron microscopy) as cellulose synthesis recovered and microtubules remained disrupted at the mor1-1 mutant's non-permissive culture temperature. Despite the disordered cortical microtubules and an initially randomized wall texture, new cellulose microfibrils were deposited with parallel, transverse orientation. These results show that transverse cellulose microfibril deposition requires neither accurately transverse cortical microtubules nor a pre-existing template of well-ordered microfibrils. We also demonstrated that DCB treatments reduced the ability of cortical microtubules to form transverse arrays, supporting a role for cellulose microfibrils in influencing cortical microtubule organization.     

In the grass species Brachypodium distachyon,the production of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) occurs in the Golgi apparatus

Mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase‐like F or H families of proteins, with CSLF6 being the best‐characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno‐localization analyses with MLG‐specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post‐Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)‐β‐glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.     

Aluminium effects on mechanical properties of cell wall analogues

Aluminium (Al) toxicity adversely impacts plant productivity in acid soils by restricting root growth and although several mechanisms are involved the physiological basis of decreased root elongation remains unclear. Understanding the primary mechanisms of Al rhizotoxicity is hindered due to the rapid effects of soluble Al on root growth and the close proximity of many cellular components within the cell wall, plasma membrane, cytosol and nucleus with which Al may react. To overcome some of these difficulties, we report on a novel method for investigating Al interactions with Komagataeibacter xylinus bacterial cellulose (BC)‐pectin composites as cell wall analogues. The growth of K. xylinus in the presence of various plant cell wall polysaccharides, such as pectin, has provided a unique in vitro model system with which to investigate the interactions of Al with plant cell wall polysaccharides. The BC‐pectin composites reacted in a similar way with Al as do plant cell walls, providing insights into the effects of Al on the mechanical properties of the BC‐pectin composites as cell wall analogues. Our findings indicated that there were no significant effects of Al (4–160 μM) on the tensile stress, tensile strain or Young's modulus of the composites. This finding was consistent with cellulose, not pectin, being the major load bearing component in BC‐pectin composites, as is also the case in plant cell walls.     

Cell wall synthesis during growth and maturation of Nitella internodal cells

An improved ¹³C-density-labeling method was used to study cell wall synthesis in rapidly expanding, slowly expanding and recently mature internodes of Nitella translucens var axillaris (A.Br.) R.D.W. As cells matured, the rate of wall synthesis slowed and the deposition of cellulose microfibrils changed from a predominantly transverse direction in the primary wall of rapidly expanding internodes to a helicoidal array in the secondary wall of mature internodes. The secondary wall was characterized by relatively higher rates of cellulose synthesis and lower rates of pectin synthesis than the primary wall. The synthesis of xyloglucan also decreased markedly at the transition to secondary wall synthesis, while the synthesis of mannose-rich hemicellulose increased. Even though structural differences were striking between the primary and secondary walls of Nitella, compositional differences between the two types of wall were quantitative rather than qualitative. The authors appreciate the assistance of Martin Yousef with the electron microscopy.     

Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy

We used atomic force microscopy (AFM), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis – a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5‐nm wide cellulose microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high‐resolution AFM images of the newly deposited wall surface showed that single microfibrils merge into and out of short regions of microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose microfibrils when imaged by FESEM, but not by AFM. The AFM thus preferentially detects cellulose microfibrils by probing through the soft matrix in these hydrated walls. AFM‐based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near‐native state, with implications for microfibrils motions in different lamellae during uniaxial and biaxial extensions.     

Phospholipases may play multiple roles in anisotropic plant cell growth

Both the cortical microtubule cytoskeleton and cellulose microfibrils are important for the anisotropic growth of plant cells. Although the two systems interact, the details of this interaction are far from clear. It has been shown the inhibitors of phospholipase D, phospholipase A₂ and phospholipase C all cause disorganisation of the microtubule cytoskeleton. Since the phospholipases act on the plasma membrane, which links cortical microtubules to cellulose microfibrils in the cell wall, they may play a key role in the communication between the two structures. This communication may take various forms. Microtubule-linked phospholipase activity may cause the organisation of underlying cellulose microfibril liquid crystals. Alternatively, phospholipases may co-operate in the regulation of plasma membrane fluidity, affecting the movement of cellulose synthase complexes in the underlying plasma membrane. GPI-anchored proteins in the plasma membrane, which are cleaved by phospholipases, may possibly play a role.     

Wall architecture in the cellulose-deficientrsw1 mutant ofArabidopsis thaliana: Microfibrils but not microtubules lose their transverse alignment before microfibrils become unrecognizable in the mitotic and elongation zones of roots

Summary Thersw1 mutant ofArabidopsis thaliana has a single amino acid substitution in a putative glycosyl transferase that causes a temperature-dependent reduction in cellulose production. We used recently described methods to examine root growth by surface marker particles, cell wall structure by field emission scanning electron microscopy and microtubule alignment by immunofluorescence after the mutant is transferred to its restrictive temperature. We find that raising the temperature quickly accelerates root elongation in both wild type and mutant, presumably as a result of general metabolic stimulation, but that in the mutant, the rate declines within 7–8 h and elongation almost ceases after 24 h. Radial swelling begins at about 6 h in the mutant and root diameter continues to increase until about 24 h. The normal transverse alignment of microfibrils is severely impaired in the mutant after 8 h, and chemical inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile causes a similar loss of orientation. After 24 h, microfibrils are not clearly visible in the walls of cells that would have been in the mitotic and early-elongation zone of wild-type roots. Changes in older cells are less marked; loss of transverse microfibril orientation occurs without disruption to the transverse orientation of cortical microtubules. The wild type shows none of the changes except for acceleration of elongation, which in its case is sustained. We conclude that microfibril alignment requires the normal functioning of RSW1 and that, in view of the effects of dichlorobenzonitrile, there may be a more general linkage between the rate of cellulose production and its proper alignment.Abbreviations DCB 2,6-dichlorobenzonitrile - REGR relative elemental growth rate Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Inhibition and Recovery of Cell Wall Formation in Root Hairs of Ceratopteris thalictroides

The processes occurring in root hairs of Ceratopteris thalictroidesduring recovery after 10 min incubation in cellulose crystallizationinhibitors (Congo Red and Calcofluor White) and in cellulosesynthesis inhibitors (Coumarin and Dichlorobenzonitrile) werestudied using light and electron microscopy. All these drugscause reversible cessation of growth of the root hairs. AfterCR and CW treatment the nuclei proceed in their normal directionof movement, whereas after Cm and DCB treatment the directionof movement is reversed immediately. After CR and CW treatment the organization of the cytoplasmin the root hair tip is temporarily altered. Cell wall synthesiscontinues, although the resulting wall has a different appearance.After Cm and DCB treatment, the cytoplasm in the root hair tiphas disintegrated and cell wall synthesis is stopped. Recoveryof cell wall synthesis results in a new cell wall between thedisintegrated cytoplasm and the remaining cytoplasm. The effects of the drugs on cytoplasmic organization are discussedin relation to their specificity in inhibiting cell wall synthesis. Key words: Ceratopteris thalictroides, root hairs, cellulose synthesis, inhibition of cell wall formation     

A computational approach for inferring the cell wall properties that govern guard cell dynamics

Guard cells dynamically adjust their shape in order to regulate photosynthetic gas exchange, respiration rates and defend against pathogen entry. Cell shape changes are determined by the interplay of cell wall material properties and turgor pressure. To investigate this relationship between turgor pressure, cell wall properties and cell shape, we focused on kidney‐shaped stomata and developed a biomechanical model of a guard cell pair. Treating the cell wall as a composite of the pectin‐rich cell wall matrix embedded with cellulose microfibrils, we show that strong, circumferentially oriented fibres are critical for opening. We find that the opening dynamics are dictated by the mechanical stress response of the cell wall matrix, and as the turgor rises, the pectinaceous matrix stiffens. We validate these predictions with stomatal opening experiments in selected Arabidopsis cell wall mutants. Thus, using a computational framework that combines a 3D biomechanical model with parameter optimization, we demonstrate how to exploit subtle shape changes to infer cell wall material properties. Our findings reveal that proper stomatal dynamics are built on two key properties of the cell wall, namely anisotropy in the form of hoop reinforcement and strain stiffening.     

IAA stimulates pollen tube growth and mediates the modification of its wall composition and structure in Torenia fournieri

The effects of several hormones on pollen tube growth were compared in Torenia fournieri and it was found that IAA was the most effective, stimulating pollen tube growth and causing the shank part of pollen tubes to be slender and straighter. The role of IAA was investigated by studying the changes in ultrastructure and PM H(+)-ATPase distribution in the pollen tubes and the modification of the tube wall. Using the fluorescent marker FM4-64, together with transmission electron microscopy, it was shown that secretory vesicles and mitochondria increased in IAA-treated tubes. Immunolocalization and fluorescence labelling, together with Fourier-transform infrared analysis, detected that IAA enhanced the level of PM H(+)-ATPase and the synthesis of pectins, and reduced the cellulose density in pollen tubes. Importantly, to observe the orientation of cellulose microfibrils in pollen tubes in situ, atomic force microscopy was used to examine the 'intact' tube wall. Atomic force microscopy images showed that cellulose microfibrils were parallel to each other in the subapical region of IAA-treated tubes, but disorganized in control tubes. All results provided new insights into the functions of cellulose microfibrils in pollen tube growth and direction, and revealed that the IAA-induced changes of pollen tubes were attributed to the increase in secretory vesicles, mitochondria, and PM H(+)-ATPase, and the modification of pectin and cellulose microfibrils in the tube wall.     

The use of radioautography for investigating wall secretion in plant cells

Summary The paper summarises results of simple radioautographic experiments using tritiated glucoses to investigate wall secretion in plant cells. In outer root cap cells, labelled material was first concentrated in the Golgi bodies; it later appeared in vesicles, and was incorporated into the wall immediately under the plasmalemma. It finally collected mainly in the slime layer surrounding the root tip. Biochemical analyses have indicated that this material was pectic in nature. In inner root cap and epidermal cells, labelled material incorporated into the walls and also the cell plates of dividing cells was also apparently mainly derived from Golgi bodies. In meristematic (less differentiated) cells, however, the endoplasmic reticulum was more frequently labelled than the Golgi bodies near walls that had incorporated derivatives of labelled glucose. Considerable incorporation of labelled derivatives into the wall thickenings in coleoptile xylem cells was often detected; nearby elements of the endoplasmic reticulum were again frequently labelled in these cells and less often, Golgi bodies and the cytoplasm in the region occupied by microtubules contained radioactivity. Labelling of starch grains in the plastids was generally observed, but not in cells secreting large amounts of wall materials (outer root cap and older xylem cells); however, addition of larger amounts of exogenous glucose to outer root cap cells, following their incubation in tritiated glucose, promoted such incorporation. The paper finally sets forth some considerations on experimental techniques for radioautography that might be of more general application.     

CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana

Pectins are critical polysaccharides of the cell wall that are involved in key aspects of a plant's life, including cell‐wall stiffness, cell‐to‐cell adhesion, and mechanical strength. Pectins undergo methylesterification, which affects their cellular roles. Pectin methyltransferases are believed to methylesterify pectins in the Golgi, but little is known about their identity. To date, there is only circumstantial evidence to support a role for QUASIMODO2 (QUA2)‐like proteins and an unrelated plant‐specific protein, cotton Golgi‐related 3 (CGR3), in pectin methylesterification. To add to the knowledge of pectin biosynthesis, here we characterized a close homolog of CGR3, named CGR2, and evaluated the effect of loss‐of‐function mutants and over‐expression lines of CGR2 and CGR3 in planta. Our results show that, similar to CGR3, CGR2 is a Golgi protein whose enzyme active site is located in the Golgi lumen where pectin methylesterification occurs. Through phenotypical analyses, we also established that simultaneous loss of CGR2 and CGR3 causes severe defects in plant growth and development, supporting critical but overlapping functional roles of these proteins. Qualitative and quantitative cell‐wall analytical assays of the double knockout mutant demonstrated reduced levels of pectin methylesterification, coupled with decreased microsomal pectin methyltransferase activity. Conversely, CGR2 and CGR3 over‐expression lines have markedly opposite phenotypes to the double knockout mutant, with increased cell‐wall methylesterification levels and microsomal pectin methyltransferase activity. Based on these findings, we propose that CGR2 and CGR3 are critical proteins in plant growth and development that act redundantly in pectin methylesterification in the Golgi apparatus.     

KOBITO1 encodes a novel plasma membrane protein necessary for normal synthesis of cellulose during cell expansion in Arabidopsis

The cell wall is the major limiting factor for plant growth. Wall extension is thought to result from the loosening of its structure. However, it is not known how this is coordinated with wall synthesis. We have identified two novel allelic cellulose-deficient dwarf mutants, kobito1-1 and kobito1-2 (kob1-1 and kob1-2). The cellulose deficiency was confirmed by the direct observation of microfibrils in most recent wall layers of elongating root cells. In contrast to the wild type, which showed transversely oriented parallel microfibrils, kob1 microfibrils were randomized and occluded by a layer of pectic material. No such changes were observed in another dwarf mutant, pom1, suggesting that the cellulose defect in kob1 is not an indirect result of the reduced cell elongation. Interestingly, in the meristematic zone of kob1 roots, microfibrils appeared unaltered compared with the wild type, suggesting a role for KOB1 preferentially in rapidly elongating cells. KOB1 was cloned and encodes a novel, highly conserved, plant-specific protein that is plasma membrane bound, as shown with a green fluorescent protein-KOB1 fusion protein. KOB1 mRNA was present in all organs investigated, and its overexpression did not cause visible phenotypic changes. KOB1 may be part of the cellulose synthesis machinery in elongating cells, or it may play a role in the coordination between cell elongation and cellulose synthesis.     

Actin-Based Domains of the "Cell Periphery Complex" and their Associations with Polarized "Cell Bodies" in Higher Plants

Abstract: Nascent cellulosic cell wall microfibrils and transverse (with respect of cell growth axis) arrays of cortical microtubules (MTs) beneath the plasma membrane (PM) are two well established features of the periphery of higher plant cells. Together with transmembrane synthase complexes, they represent the most characteristic form of a “cell periphery complex” of higher plant cells which determines the orientation of the diffuse (intercalary) type of their cell growth. However, there are some plant cell types having distinct cell cortex domains which are depleted of cortical MTs. These particular cell cortex domains are, instead, typically enriched with components of the actin‐based cytoskeleton. In higher plants, this feature is prominent at extending apices of two cell types displaying tip growth ‐ pollen tubes and root hairs. In the latter cell type, highly dynamic F‐actin meshworks accumulate at extending tips, and they appear to be critical for the apparently motile character of these subcellular domains. Importantly, tip growth of both root hairs and pollen tubes is immediately stopped when the most dynamic F‐actin population is depolymerized with low levels of anti‐F‐actin drugs. Intriguingly, MTs of tip‐growing plant cells are organized in the form of longitudinal arrays, throughout the cytoplasm, which interconnect the extending tips with the subapical nuclei. This suggests that actin‐rich cell cortex domains polarize plant “cell bodies” represented by nucleus‐MTs complexes. A similar polarization of “cell bodies” is typical of mitotic and cytokinetic plant cells. A further type of MT‐depleted and actomyosin‐enriched plant cell cortex domain comprises the plasmodesmata. Primary plasmodesmata are formed during cytokinesis as part of the myosin VIII‐enriched callosic cell plates, representing “juvenile” forms of the plant “cell periphery complex”. In phylogenetic terms the association between F‐actin and the PM may be considered for a more “primitive” form of cellular organization than does the association of cortical MTs with the PM. We hypothesize that the actin cytoskeleton is a natural partner of the PM in all eukaryotic cells. In most plant cells, however, it was replaced by a tubulin‐based “cell periphery apparatus” which regulates, via still unknown mechanisms, the spatial deposition of nascent cellulosic microfibrils synthesized by PM‐associated synthase complexes.     

The exogenous application of AtPGLR,an endo‐polygalacturonase,triggers pollen tube burst and repair

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine‐tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark‐grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo‐PG that preferentially releases non‐methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non‐methylesterified pectin epitopes are detected. Those leaks could either be repaired by new β‐glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.     

Pectin methylesterase modulates aluminium sensitivity in Zea mays and Solanum tuberosum

Cell suspension cultures of Zeamays L. were adapted to grow under conditions of NaCl stress, which increased the cell‐wall pectin content of these cells by 31% compared with unadapted cells (controls). Both cultures were treated for 5 or 10 min with pectin methylesterase (PME) and afterwards incubated in the presence of Al for 2 h. The different capabilities of the cells to synthesise callose due to pre‐treatment were taken into account by calculating relative Al‐induced callose induction (digitonin=100%). Only in salt‐adapted cells with a degree of methylation of cell‐wall pectin (DM) decreasing from 34% (control) to 13%, did PME treatment enhance total and BaCl₂‐non‐exchangeable Al contents and Al sensitivity as indicated by increased callose formation. In a further step, a wider variation in DM was achieved by subculturing the NaCl‐adapted cells for up to 3 weeks without NaCl supply and adapting them to the cellulose‐synthesis inhibitor 2,6‐dichlorbenzonitrile (DCB). This reduced DM to 26%, while short‐term treatment with pectolyase resulted in the lowest DM (12%). After the 2 h Al treatment, there was a close negative relationship between DM and relative callose formation of Al contents, with the exception of pectolyase‐treated cells. In addition, intact plants of Solanumtuberosum L. genotypes were characterised for their Al sensitivity in hydroponics using root elongation, Al‐induced callose formation and Al contents of root tips as parameters. Based on all three parameters, the transgenic potato mutant overexpressing PME proved to be more Al‐sensitive than the wild type, the Al‐resistant and even the Al‐sensitive potato cultivar. Especially in the root tips (1 cm), Al treatment (2 h, 50 μM) increased the activity of PME more in the Al‐sensitive than in the Al‐resistant genotypes. The presented data emphasise the importance of the DM of the pectin matrix and the activity of PME for the expression of Al toxicity and Al resistance.     

THE SITES OF CELLULOSE SYNTHESIS IN ALGAE: DIVERSITY AND EVOLUTION OF CELLULOSE-SYNTHESIZING ENZYME COMPLEXES

Information on the sites of cellulose synthesis and the diversity and evolution of cellulose-synthesizing enzyme complexes (terminal complexes) in algae is reviewed. There is now ample evidence that cellulose synthesis occurs at the plasma membrane-bound cellulose synthase, with the exception of some algae that produce cellulosic scales in the Golgi apparatus. Freeze-fracture studies of the supramolecular organization of the plasma membrane support the view that the rosettes (a six-subunit complex) in higher plants and both the rosettes and the linear terminal complexes (TCs) in algae are the structures that synthesize cellulose and secrete cellulose microfibrils. In the Zygnemataceae, each single rosette forms a 5-nm or 3-nm single “elementary” microfibril (primary wall), whereas rosettes arranged in rows of hexagonal arrays synthesize criss-crossed bands of parallel cellulose microfibrils (secondary wall). In Spirogyra, it is proposed that each of the six subunits of a rosette might synthesize six β-1,4-glucan chains that cocrystallize into a 36-glucan chain “elementary” microfibril, as is the case in higher plants. One typical feature of the linear terminal complexes in red algae is the periodic arrangement of the particle rows transverse to the longitudinal axis of the TCs. In bangiophyte red algae and in Vaucheria hamata, cellulose microfibrils are thin, ribbon-shaped structures, 1–1.5 nm thick and 5–70 nm wide; details of their synthesis are reviewed. Terminal complexes appear to be made in the endoplasmic reticulum and are transferred to Golgi cisternae, where the cellulose synthases are activated and may be transported to the plasma membrane. In algae with linear TCs, deposition follows a precise pattern directed by the movement and the orientation of the TCs (membrane flow). A principal underlying theme is that the architecture of cellulose microfibrils (size, shape, crystallinity, and intramicrofibrillar associations) is directly related to the geometry of TCs. The effects of inhibitors on the structure of cellulose-synthetizing complexes and the relationship between the deposition of the cellulose microfibrils with cortical microtubules and with the membrane-embedded TCs is reviewed In Porphyra yezoensis, the frequency and distribution of TCs reflect polar tip growth in the apical shoot cell.The evolution of TCs in algae is reviewed. The evidence gathered to date illustrates the utility of terminal complex organization in addressing plant phylogenetic relationships.     

Formation of root hairs by the wheat Triticum Vulgare Host. Trichoblasts.

The process of root hair formation has been studied by light-, transmission-and scanning electron microscopy. In the course of root hair development a break of the outer cell wall is observed by electron microscopy. It apparently occurs after the break of the fibrillar layer. The break of the outer layer of the cell wall is assumed to represent the break of the outer mucilage, the cuticle and the adjoining amorphous matrix with irregularly oriented cellulose microfibrils. The scheme of successive ultrastructural changes in the outer cell wall pattern during root hair formation is presented.