Hyphal tip growth in Achlya bisexualis. II. Distribution of cellulose in elongating and non-elongating regions of the wall

Cellulose has been localized in the hyphal wall of elongating and non-elongating hyphae of Achlya bisexualis using a direct enzyme-colloidal-gold method. A number of controls, including several different types of fixation, support the idea that this labeling is specific for cellulose. Both TEM and SEM were used and they gave similar results. The apical area of an elongating hypha lacks cellulose, but the same area of a non-elongating hypha contains cellulose. We have used specific culture media and light microscopic measurements to ensure that we could distinguish between elongating and non-elongating hyphae. The lack of cellulose at the apex of elongating hyphae seems to require a reevaluation of the current concepts of hyphal tip growth in Achlya and related genera. A major question now is to determine whether or not the lack of a microfibrillar component is a universal pattern among all organisms having tip growth.     

Changes in Pectin Structure during Epidermal Cell Elongation in Pea (Pisum sativum) and Its Implications for Cell Wall Architecture

Changes in cell wall architecture during elongation of epidermalcells in pea epicotyls were visualized by rapid-freezing anddeep-etching (RFDE) techniques. The abundant network structurecomposed of the association of granular substances disappearedfrom the cell wall during elongation. The granular substanceswere demonstrated to be pectic polysaccharides by their disappearanceupon EDTA treatment and by chemical analysis of the EDTA-ex-tractablesubstances. Labeling with the monoclonal antibody JIM5, whichrecognizes unesterified pectins, was much more extensive inthe cell walls of the non-elongating region than in those ofthe elongating region. The pore size of the cell wall was largerin the non-elongating region than in the elongating region.These observations suggest that the formation of the pecticgel itself is not involved in the control of the wall porosity.We proposed that the association of the granular substancesis involved in the swelling of the cell walls in the elongatingregion. (Received May 18, 1998; Accepted September 25, 1998)     

Orientation of Wall Microfibrils in Avena Coleoptiles and Mesocotyls and in Pisum Epicotyls

Microfibrils (MFs) on the inner surface of the walls of Avenacoleoptile and mesocotyl cells and of Pisum epicotyl cells wereexamined by a replica method. In the elongating epidermis ofthese three organs, cells having MFs that were transverse, obliqueor longitudinal to the elongation axis were intermingled. Inthe elongating parenchymal tissues, all cells deposited MFstransversely. In non-elongating cells of Avena coleoptiles andPisum epicotyls, the orientation of MFs on the inner wall surfaceof both epidermal and parenchymal cells was more longitudinalthan in elongating cells. These observations on the orientationsof MFs are compatible with those our previously reported observationson the orientations of microtubules (MT) (Iwata and Hogetsu1988). Disruption of MTs of Avena coleoptiles by treatment withamiprophosmethyl caused changes in the orientation of depositionof MFs. These results support the idea that MFs are usuallyco-aligned with MTs in organ cells and that the orientationof MFs is controlled by MTs. The averaged direction of MFs, visualized under polarized light,showed a clear difference between the epidermal and inner-tissuecell walls in the elongating regions of the three organs. Inalmost all elongating and non-elongating epidermal cells, theaveraged direction of MFs was longitudinal, while it was transversein all inner-tissue cells. (Received December 16, 1988; Accepted April 28, 1989)     

Solute production and net wall synthesis in the growing and non-growing cells of gravistimulated sunflower hypocotyls

Solute generation and cell wall synthesis were examined in sunflower hypocotyl peripheral layers, the growth rate of which had been altered by gravistimulation. Measurements of both the concentrations of the major solutes and the osmotic potential showed that although upper cells stopped growing, the solute levels in these cells continued to increase at rates comparable to those in lower cells. This indicated that altered growth rates, generated during gravicurvature, are not based on solute generation but must result from cell wall changes. Gravimetric and precursor incorporation studies showed that net wall synthesis continued in upper cells despite their lack of growth. An ultrastructural study of the epidermal cells on the uppermost (non-elongating) and lowermost (elongating) surfaces of horizontal cucumber hypocotyls showed that the relative amounts of the various membrane fractions were similar in upper and lower cells despite their very different growth rates.     

Cell wall hydroxyproline-polysaccharide associations in Lupinus hypocotyls

Extraction of lupin hypocotyl cell walls with guanidine thiocynate, both before and after dilute acid treatment does not dissolve the hydroxyproline indicating that compounds containing this amino acid are probably covalently linked to insoluble wall constituents other than through acid labile arabinofuranose-hydroxyproline links. Dilute alkali does extract all of the wall hydroxyproline largely as non-dialysable material. Sequential extraction of cell walls with alkali at two temperatures (2° and 22–25°) removes most of the hemicellulose at the lower temperature but only dissolves the hydroxyproline at the higher temperature. Other studies show that the hydroxyproline containing polymer is co-precipitated with hemicellulose-B arabino-xylan. When cell walls from elongating and non-elongating hypocotyl sections are compared using this sequential extraction, the hemicellulose-B arabino-xylan containing hydroxyproline from the non-elongating wall has a much higher proportion of arabinoseand galactose than the same polymer from the elongating wall. Much more of the hydroxyproline from the elongating wall is dialysable. These results indicate more bonding of the hydroxyproline-containing glycoprotein within the wall of non-elongating tissue consistent with its suggested role in stopping cell elongation. It is suggested that the glycoprotein is linked to insoluble wall constituents such as cellulose through galactose or by direct protein to cellulose links.     

Evidence that actin reinforces the extensible hyphal apex of the oomyceteSaprolegnia ferax

Summary Filamentous actin in the apices of growing hyphae of the oomyceteSaprolegnia ferax is distributed such that it could compensate for weakness in the expanding apical cell wall and thus play a role in morphogenesis of the tip. The tapered extensible portion of the hyphal tip where the cell wall is plastic contains a cap of actin which differs in organization from the actin in subapical, inextensible regions of the hypha. Rapidly growing hyphae which are expected to have a longer plastic cell wall region contain longer actin caps. Furthermore, the weakest point in the hyphal apex, demonstrated by osmotic shock-induced bursting, was within the taper where the wall is plastic but never in the extreme apex where actin was most densely packed and presumably the strongest. Treatment of hyphae with cytochalasin E/dimethyl sulphoxide induced rapid changes in actin caps. Cap disruption was accompanied by transient growth rate increases, subsequent rounding and swelling of apices and a shift of osmotically induced burst points closer to the apex. These correlated changes are consistent with a role for the actin cap in tip morphogenesis. The association between regions of plasticity in the apical cell wall, the extent of the actin cap, the location of the weakest point in the apex and the effects of damage to the actin cap suggest that the cap functions to support the apex in regions where the cell wall is weak.Abbrevations CE cytochalasin E - DMSO dimethyl sulphoxide - RP rhodamine phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

Autolytic enzymes in hyphae of Aspergillus nidulans: their action on old and newly formed walls.

Walls, purified from hyphae of the ascomycete Aspergillus nidulans, autolyzed on incubation and liberated glucose, mannose, galactose, N-acetylglucosamine, and soluble oligosaccharides. Digestion proceeded at linear rates until approximately 3% of the wall polymers had been hydrolyzed and then slowed markedly. The change in the rate of autolysis was not due to loss of enzyme activity but was caused by the disappearance of a fraction of the wall which was highly susceptible to digestion. Radioactive labeling showed that this fraction was the newly formed wall. The new wall was highly susceptible to enzyme action both when it was deposited at the apex in growing hyphae or when deposited laterally in hyphae treated with cycloheximide. The relations between wall modification and apical growth are discussed.     

Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls

Plant cell walls contain a glycoprotein component rich in the otherwise rare amino acid hydroxyproline. We examined the synthesis and accumulation of wall hydroxyproline during different states of elongation growth in pea epicotyls. Light-grown peas contained more wall hydroxyproline than their taller, dark-grown counterparts. When elongation was studied by marking growing stems in situ, there was a marked accumulation of wall hydroxyproline coincident with the cessation of elongation. Dividing and elongating regions of the epicotyl showed less wall hydroxyproline than did regions where elongation was no longer occurring.Hydroxyproline biosynthesis was examined by incubation of excised sections of tissues in various growth states in ¹⁴C-proline. The extent of conversion of these residues to ¹⁴C-hydroxyproline served as a measure of the rate of hydroxyproline synthesis. This rate was highest in tissues which had ceased elongation. The low rate of hydroxyproline synthesis in dividing and elongating cells was probably not due to the inability to hydroxylate peptidyl proline or to secrete proteins.These data show a positive correlation between the synthesis and accumulation of cell wall hydroxyproline and the cessation of cell elongation in pea epicotyls.     

Studies on the Structure and Elongating Growth of the Gynophore of Peanut plant

The epigeal portion of the gynophore has a typical herbaceous stem structure. It is found that the young gynophore is composed of epidermis, cortex and vascular cylinder. Periderm, lenticelle and secondary vascular tissue in the gynophore are produced due to its secondary growth. The region of cell division in the gynophore is located at 0.9–1.9 mm from the peg apex, and that of cell elongation at 2.0–4.5 mm. These two regions are found to overlap at 2.0–2.5 mm from peg apex. The results of the experiment exhibit clearly that the growing region of the gynophore grows toward the gravitional direction of the earth when the gynophore is placed either in a vertical position or in a horizontal position, thus the elongating gynophore shows a positive geotropic response. The distributed position of starch grains in the pith parenchyma sedimented to the side of cell wall near the earth surface. All this shows that there is a close relation between the positive geotropic growth of the peanut gynophore and the distribution of starch statoliths in the pith of the gynophore under the influence of gravity.     

An architectural analysis of the elongation of field-grown sunflower root systems. Elements for modelling the effects of temperature and intercepted radiation

The effects of photosynthetic photon flux density (PPFD) andsoil temperature on root system elongation rate have been analysedby using an architectural framework. Root elongation rate wasanalysed by considering three terms, (i) the branch appearancerate, (ii) the individual elongation rates of the taproot andbranches and (iii) the proportion of branches which stop elongating.Large ranges ofPPFD and soil temperature were obtained in aseries of field and growth chamber experiments. In the field,the growth of root systems experiencing day-to-day natural fluctuationof PPFD and temperature was followed, and some of the plantsunder study were shaded. In the growth chamber, plants experiencedcontrasting and constant PPFDs and root temperatures. The directeffect of apex temperature on individual root elongation ratewas surprisingly low in the range 13–25C, except forthe first days after germination. Root elongation rate was essentiallyrelated to intercepted PPFD and to distance to the source, bothin the field and in the growth chamber. Branch appearance ratesubstantially varied among days and environmental conditions.It was essentially linked to taproot elongation rate, as theprofile of branch density along the taproot was quite stable.The length of the taproot segment carrying newly appeared brancheson a given day was equal to taproot elongation on this day,plus a 'buffering term' which transiently increased if taprootelongation rate slowed down. The proportion of branches whichstopped elongating a short distance from the taproot rangedfrom 50–80% and was, therefore, a major architecturalvariable, although it is not taken into account in current architecturalmodels. A set of equations accounting for the variabilitiesin elongation rate, branch appearance rate and proportion ofbranches which stop elongating, as a function of interceptedPPFD and apex temperature is proposed. These equations applyfor both field and growth chamber experiments. Key words: Sunflower, root system, model, temperature, radiation     

Microtubule orientation in pea stem cells: a change in orientation follows the initiation of growth rate decline

The orientation of microtubules in cells of redlight-grown pea plants (Pisum sativum L.) was examined by means of immunofluorescence. Microtubules (MTs) in rapidly elongating, subepidermal cells commonly form multiple, parallel strands that run transverse to the cell's axis of elongation. By contrast, MTs in nonelongating subepidermal cells form steeply pitched helical arrays; MTs in non-elongating epidermal cells are oriented parallel to the axis of elongation. This change in orientation occurs during the time interval in which growth rate is declining. The transition is abrupt rather than gradual and occurs in both epidermal and subepidermal cells at the same time. Plants irradiated for 2 h with a growth-inhibiting fluence of blue light did not undergo the same transition, indicating that factors other than changing elongation rates must be responsible for triggering the reorganization of MT arrays.     

A microtubule-associated protein in maize is expressed during phytochrome-induced cell elongation

Plants can adapt their shape to environmental stimuli. This response is mediated by the reorganization of cortical microtubules, a unique element of the cytoskeleton. However, the molecular base of this response has remained obscure so far. In an attempt to solve this problem, signal-dependent changes in the pattern of microtubule-binding proteins were analysed during coleoptile elongation in maize, that is, under the control of the plant photoreceptor phytochrome. Two putative MAPs of 100 kDa (P₁₀₀) and 50 kDa apparent molecular weights were identified in cytosolic extracts from non-elongating and elongating cells. Both proteins co-assembled with endogenous tubulin, bound to neurotubules and were immunologically related to the neural MAP τ: the P₁₀₀ protein, depending on the physiological situation, was manifest as a double band and was always found to be heat-stable. In contrast, the 50 kDa MAP was heat-stable only for particular tissues and physiological treatments. The P₁₀₀ protein was present in all tissues, however in a reduced amount in elongating coleoptiles. The 50 kDa MAP was expressed exclusively upon induction of phytochrome-dependent cell elongation. As shown by immunofluorescence double-staining, an epitope shared by both proteins colocalized with cortical microtubules in situ, but exclusively in elongating cells. In non-elongating cells, only the nuclei were stained. Partially purified nuclei from elongating cells were enriched in P₁₀₀, whereas the 50 kDa MAP became enriched in a partially purified plasma membrane fraction.     

Mapping the growth of fungal hyphae: orthogonal cell wall expansion during tip growth and the role of turgor

By computer-enhanced videomicroscopy, we mapped the trajectory of external and internal cell surface markers in growing fungal hyphae to determine the pattern of cell wall expansion during apical growth. Carbon particles (India ink) were chosen as external markers for tip expansion of Rhizoctonia solani hyphae. Irregularities in the growing apical walls of R. solani served as internal markers. Marker movement was traced in captured frames from the videotaped sequences. External and internal markers both followed orthogonal trajectories; i.e., they moved perpendicular to the cell surface regardless of their initial position in the hyphal apex. We found no evidence that the tip rotates during elongation. The discovery that the cell wall of a growing hypha expands orthogonally has major repercussions on two fronts: 1) It supports the long-held view that turgor pressure is the main force driving cell wall expansion. 2) It provides crucial information to complete the mathematical derivation of a three-dimensional model of hyphal morphogenesis based on the vesicle supply center concept. In three dimensions, the vesicle gradient generated by the vesicle supply center is insufficient to explain shape; it is also necessary to know the manner in which the existing surface is displaced during wall expansion.     

Auxin regulation and spatial localization of an endo-1,4-β-D-glucanase and a xyloglucan endotransglycosylase in expanding tomato hypocotyls

Xyloglucan, the primary hemicellulosic cell wall polysaccharide in dicotyledons, undergoes substantial modification during auxin-stimulated cell expansion. To identify candidates for mediating xyloglucan turnover, the expression and auxin regulation of tomato Cel7 and LeEXT , genes encoding an endo-1,4-β-glucanase (EGase) and a xyloglucan endotransglycosylase (XET), respectively, were examined. LeEXT mRNA was present primarily in elongating regions of the hypocotyl and was induced to higher levels by hormone treatments that elicited elongation of hypocotyl segments. Cel7 mRNA abundance was very low in both elongating and mature regions of the hypocotyl but was induced to accumulate to high levels in both hypocotyl regions by auxin application. Analysis of the time dependence of expression of Cel7 and LeEXT during auxin treatment suggested that induction of these genes is not required for rapid growth responses but may participate in the cell wall changes involved in sustained cell elongation. Localization of Cel7 and LeEXT mRNA by in situ hybridization revealed that both genes are expressed in outer cell layers of the hypocotyl. In untreated etiolated seedlings, LeEXT mRNA was detected in epidermal cells of the elongating region, a tissue considered to play a key role in auxin-induced elongation. After auxin treatment, Cel7 and LeEXT mRNA showed an overlapping spatial distribution in the epidermis and outer cortical cell layers. We conclude that LeEXT and Cel7 exhibit both unique and overlapping patterns of expression and have the potential to act cooperatively in mediating cell wall disassembly associated with expansive growth.     

Salinity-induced inhibition of leaf elongation in maize is not mediated by changes in cell wall acidification capacity

The physiological mechanisms underlying leaf growth inhibition under salt stress are not fully understood. Apoplastic pH is considered to play an important role in cell wall loosening and tissue growth and was demonstrated to be altered by several growth-limiting environmental conditions. In this study we have evaluated the possibility that inhibition of maize (Zea mays) leaf elongation by salinity is mediated by changes in growing cell wall acidification capacity. The kinetics of extended apoplast pH changes by leaf tissue of known expansion rates and extent of growth reduction under stress was investigated (in vivo) and was found similar for non-stressed and salt-stressed tissues at all examined apoplast salinity levels (0.1, 5, 10, or 25 mM NaCl). A similar rate of spontaneous acidification for the salt and control treatments was demonstrated also in in situ experiments. Unlike growing cells that acidified the external medium, mature nongrowing cells caused medium alkalinization. The kinetics of pH changes by mature tissue was also unchanged by salt stress. Fusicoccin, an enhancer of plasmalemma H(+)-ATPase activity level, greatly stimulated elongation growth and acidification rate to a similar extent in the control and salt treatments. That the ability of the growing tissue to acidify the apoplast did not change under same salt stress conditions that induced inhibition of tissue elongation rate suggests that salinity does not inhibit cell growth by impairing the acidification process or reducing the inherent capacity for cell wall acidification.     

A possible role of hydroxyproline-containing proteins in the cessation of cell elongation

Hydroxyproline-containing proteins increase markedly in the cell walls of the Alaska pea epicotyl during the transition of rapidly elongating tissue into non-elongating, mature tissue. The increase in these proteins may be a factor in the cessation of cell elongation.     

Cell wall proteome in the maize primary root elongation zone. II. Region-specific changes in water soluble and lightly ionically bound proteins under water deficit

Previous work on the adaptation of maize (Zea mays) primary roots to water deficit showed that cell elongation is maintained preferentially toward the apex, and that this response involves modification of cell wall extension properties. To gain a comprehensive understanding of how cell wall protein (CWP) composition changes in association with the differential growth responses to water deficit in different regions of the elongation zone, a proteomics approach was used to examine water soluble and loosely ionically bound CWPs. The results revealed major and predominantly region-specific changes in protein profiles between well-watered and water-stressed roots. In total, 152 water deficit-responsive proteins were identified and categorized into five groups based on their potential function in the cell wall: reactive oxygen species (ROS) metabolism, defense and detoxification, hydrolases, carbohydrate metabolism, and other/unknown. The results indicate that stress-induced changes in CWPs involve multiple processes that are likely to regulate the response of cell elongation. In particular, the changes in protein abundance related to ROS metabolism predicted an increase in apoplastic ROS production in the apical region of the elongation zone of water-stressed roots. This was verified by quantification of hydrogen peroxide content in extracted apoplastic fluid and by in situ imaging of apoplastic ROS levels. This response could contribute directly to the enhancement of wall loosening in this region. This large-scale proteomic analysis provides novel insights into the complexity of mechanisms that regulate root growth under water deficit conditions and highlights the spatial differences in CWP composition in the root elongation zone.     

Mycelial differentiation and spore formation by Streptomyces brasiliensis in submerged culture.

Streptomyces brasiliensis ATCC 23727 showed extensive sporulation when cultured in a liquid medium containing galactose and glutamic acid as carbon and nitrogen sources. Sporogenic hyphae formed under these conditions were morphologically similar and developmentally equivalent to aerial hyphae and metamorphosed into chains of spores by following a sequence of ultrastructural changes similar to that observed during growth on solid media. In addition, our electron microscopy study revealed two previously unrecognized aspects of hyphal development in streptomycetes: the formation of sporogenic hyphae was always preceded by changes in the structure of the nucleoid, and the sheath that characteristically covered these hyphae was not deposited coincidently with wall formation in the apical growing portion of the hypha.