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Department of Botany, PO Box 118526, University of Florida, Gainesville, Florida 32611-8526
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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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.
Key words: 1,4-ß-glucans, cellulase-colloidal-gold labeling, hyphal elongation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Wessels 1988
). Recent evidence (López-Franco et al 1994
) for tip growth, in diverse fungi, has shown a pulsatile type of elongation, which suggests discontinuous wall growth.
Cellulose is a crystalline 1,4-ß-glucan, and in the form of microfibrils provides a strong framework for the addition of various matrix components in hyphal cell walls containing this polymer. It represents about 20% (wt/wt) of the mature wall of Achlya (Reiskind and Mullins 1981a
). Colloidal gold is an effective cytochemical marker in electron microscopy due to its versatility in adsorption to macromolecules, electron density, and small size. Enzyme-linked colloidal gold labeling is a nondestructive, direct labeling technique and has been used to localize cellulose on thin sections (Berg et al 1988
). We have used it to localize cellulose on both thin sections with TEM and the hyphal surface with SEM.
The present study was done in recognition of the cyclic mode of growth and under culture conditions that allowed the distinction between elongating and non-elongating hyphae (Shapiro and Mullins 2002
). We present evidence for an unexpected distribution of cellulose in growing and non-growing colonies of Achlya, as part of an overall examination of hyphal tip growth.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
TEM and SEM preparation and labeling –
The colonies were subjected to the same series of fixation and processing as previously described (Shapiro and Mullins 2002
). Colloidal gold of approximately 15 nm in diameter was made via reduction of chloroauric acid by sodium citrate as described by Frens (1973)
. The enzyme cellulase, LS02601, was purchased from Worthington Biochemical Corporation, (Freehold, New Jersey). This preparation is termed chromatographically pure and is isolated from cultures of a selected strain of Trichoderma reesei. To coat the gold with cellulase, the pH of 10 mL of 15 nm colloidal gold was adjusted to 4.5 and 1 mg of enzyme in 0.1 mL distilled water was added. After 5 min of stirring, the complex was further stabilized by the addition of 0.5 mg/mL polyethylene glycol (MW 20 000). Then the solution was poured into a centrifuge tube, and 1.5 mL of 20% glycerol in citrate buffer pH 4.5 was carefully placed on the bottom of the tube. This step allowed the preparation to be stored at -80 C before use. Centrifugation followed at 12 100 rpm for 1 h. Successful coating of the gold was evident by a mobile pellet, which was then resuspended in 0.75 mL of 20% glycerol.
Before treatment with the enzyme-gold complex, sections were blocked for 5 min by floating them face down on citrate buffer containing 0.5% gelatin. The labeling solution was a 1:10 dilution of the enzyme-gold stock solution in citrate buffer, and the grids were floated on it for 30 min. The grids were then floated on citrate buffer alone for 5 min and rinsed twice for 5 min in distilled water. The colonies destined for SEM observations were treated with the same series of solutions, but were completely submerged, rather than floated. For silver enhancement, following labeling, the colonies were placed in a non-diluted mixture (1:1) of reagents from the Aurion Silver Enhancement Kit for 5 min and washed several times with water to stop the reaction (Scopsi et al 1986
). They were then dehydrated in an ethanol series and critical point dried.
Cytochemical and enzyme controls – A number of cytochemical controls were performed with the cellulase-gold complex to demonstrate the specificity of the label. The substrate competition control used a preincubation with 1 mg/mL carboxy-methylcellulose, sodium salt, medium viscosity, Hercules CMC 7MF, for 30 min before labeling. Nonspecific protein binding sites were determined by incubating the sections or colonies with 18 nm Colloidal Gold-AffiniPure Goat Anti-Mouse IgG (H + L), Jackson ImmunoResearch Laboratories, Inc (West Grove, Pennsylvania). Two glucose polymers from Sigma (St. Louis, Missouri) were tested for nonspecific binding: one was laminarin, 10 mg/mL, and the other was re-acetylated glycol chitosan, 11.8 mg/mL. Finally, in order to determine the effect of partial hydrolysis of wall cellulose on subsequent labeling of the preparation, a 30 min preincubation of the sections with the enzyme cellulase, 1mg/mL, was used before their treatment with the cellulase-gold complex.
A second cellulase enzyme was used, endocellulase III, which was provided by Dr. Tim Fowler, Genencore International, Inc. This enzyme lacks a cellulose-binding domain, but shows excellent catalytic activity (Sandgren et al 2001
), and was used to explore the mechanism of enzyme labeling of cellulose. The solutions used for the conjugation of this enzyme were pH 5.5 rather than the 4.5 used for the commercial preparation of cellulase.
To determine if the cellulase enzyme retained enzyme activity when conjugated to gold, the following experiment was done. The samples were combined with 1 mL of gold-cellulase complex (1:10 dilution) and incubated at room temperature. A control for each sample was prepared with substrate and 0.05 M sodium citrate buffer pH 4.5. The citrate buffer alone was used as a blank. After 30 min and 3 h, the enzyme activity was checked by a reducing sugar determination (Somogyi 1952
). The substrates tested were CMC, an isolated whole wall fraction, and a small colony of Achlya.
Some preliminary experiments were done in order to determine the effect of 1,3-ß-glucanase treatment on elongating and non-elongating colonies. Either chemically fixed or live colonies were incubated at room temperature with 0.05 mg/mL Zymolyase 100 T (Arthrobacter luteus) (Seikagaku America, Inc, Ijamsville, Maryland) in 66 mM sodium phosphate pH 7.5. The hydrolysis was monitored with light microscopy to determine the condition of hyphal integrity, especially the hyphal tips. After 24 h of treatment the colonies were fixed, labeled with the cellulase-gold complex, and processed for SEM.
To determine the effect of the cellulose biosynthesis inhibitor dichlorobenzonitrile (DCB), flasks containing 250 mL of PYG and ten small colonies were grown for 12 h, after which appropriate aliquots of a stock solution of DCB in 100% DMSO were added to obtain final concentrations of 10, 20, 30, 40, 50, 60, 100, and 200 µM. Colony growth was monitored with light microscopy, and after 36 h of incubation, colonies were chemically fixed, labeled with cellulase-gold complex, and processed for SEM. Control colonies grown in PYG, including the same treatment as above with DMSO, without DCB, were processed in the same way. In addition, the ability of spores to germinate in PYG plus DCB was followed by light microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) gave the same result as the chemically fixed preparation (data not shown).
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), before being subjected to fixation and labeling. Cellulase-gold affinity labeling of a cross section in the mature area of an elongating hypha localizes cellulose to the cell wall exclusively (Fig. 5 ). The distribution of the gold particles in the wall is even, and the level of nonspecific labeling is very low. In longitudinal sections of elongating hyphae the label is present in mature and subapical regions, but is significantly reduced in apical regions (Fig. 6 ).
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In living colonies, under growing conditions, treatment with Zymolyase resulted in most of the hyphal apices' remaining intact. A small number had broken apices, and the remaining cell wall was labeled with the cellulase-gold complex (Figs. 8–9 ). In the growing colonies that were chemically fixed first and then hydrolyzed, all the hyphae had intact apices. Most of the apices were labeled, but some were not (Fig. 10 ).
The non-growing colonies from glucose-only medium treated with Zymolyase, either living or chemically fixed before treatment, gave the same result. All the hyphae were intact and the tips were labeled as intensively as the rest of the hyphae (data not shown).
The addition of DCB to the growth medium had no affect on the growth of Achlya. The average rate of elongation was 3.6 µm/min and the hyphal morphology was not changed. Spores germinated equally well in PYG medium with or without DCB. No difference in the expected labeling pattern of cellulose, with the cellulase-gold reagent, was found for mycelium grown with or without DCB (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and in the present study. The cellulose-binding domains are required for efficient hydrolysis of crystalline but not for soluble substrates (Teeri 1997
), suggesting that in the carboxy-methylcellulose control binding to the soluble ß-glucans occurs primarily through the catalytic domain. Differences in fixation techniques had no affect on the labeling pattern. Thus this labeling technique provides a specific and reliable method for localizing cellulose on thin sections and hyphal surfaces.
The fact that cellulose labeling was found on the hyphal surface (Figs. 1–4
), may contradict a general assumption that microfibrillar polysaccharides are accumulated mostly in the inner layers of the cell walls, while amorphous components are more abundant in the external layers (Hunsley and Burnett 1970
, Reiskind and Mullins 1981b
). The unique structure of the cellulase enzyme complex may contribute to an explanation for the presence of cellulose labeling on the hyphal surface. These enzymes contain a small, highly homologous 36-residue region called the A domain, connected to the globular catalytic domain by a threonine- and serine-rich sequence. The A domain lacks catalytic activity and is thought to have a cellulose-binding function (Rouveinen et al 1990
). Thus apparent surface labeling may result from the small cellulose-binding domain penetrating the wall, while the catalytic domain conjugated to gold remains on the surface. The lack of cellulose labeling with EG III enzyme (Fig. 7
) supports this view, since it is a genetically modified enzyme that lacks a cellulose-binding domain. The conjugation of this enzyme with gold was successful, based on the raspberry red color of the enzyme gold complex and the presence of a mobile pellet. Negative staining of the enzyme-gold complex also confirmed successful conjugation (data not shown). The lack of enzyme activity of the cellulase-gold complex against insoluble substrates, and very low activity against soluble substrates, also provides support for this view.
All the hyphae from non-growing colonies were evenly labeled for cellulose from apex to base (Fig. 4
). In contrast, hyphae from growing colonies revealed three patterns of labeling at the apex: a very small number (about 5%) were labeled; most had a sharply decreasing label toward the apex; and some were unlabeled. We have previously shown that a small number of hyphae under growing conditions are not elongating (Shapiro and Mullins 2001
), and this may account for the small number of hyphae with labeled apices. Thus in a non-elongating hypha cellulose is evenly distributed along the hyphal wall including the apex, whereas in an elongating hypha there is either no cellulose at the apex or there is a gradual decrease in the amount of cellulose toward the apex. It has generally been accepted that among the oomycetes and the eufungi the qualitative composition of the elongating apex wall and the mature wall is the same (Hunsley and Burnett 1970
, Wessels 1990
, Kaminskyj and Heath 1996
, Heath and Steinberg 1999
).
The results of the experiments with Zymolyase hydrolysis generally support the conclusion that in growing colonies there is no cellulose in the apical cell wall (Figs. 8–10
). In the colonies that were treated with Zymolyase prior to fixation, some hyphae show broken apices, which could result from the lack of cellulose at the apex and the hydrolysis of 1,3-ß-glucans, shown to be present at the apex of both elongating and non-elongating hyphae (Shapiro and Mullins 2002
). This enzyme preparation also contains a protease activity, which could hydrolyze the structural protein in the wall (Reiskind and Mullins 1981a
). In the colonies that were chemically fixed prior to treatment with Zymolyase, elongating hyphae have intact unlabeled apices. Chemical fixation may produce cross-links among the wall components, especially proteins, and protect them from hydrolysis. As would be expected, treated non-elongating hyphae, with or without fixation, have intact apices with uniform cellulose labeling of the hyphal wall. Additional data is needed to show that most apical regions of elongating hyphae can be broken with 1,3-ß-glucanase, but it is not apparent what conditions will allow this result to be obtained.
The experiments with DCB gave an unexpected result. In these experiments it was the intention to use a different approach to show the absence of cellulose in the apical wall of elongating hyphae. DCB is a classic inhibitor of cellulose biosynthesis in higher plants (Delmer 1999
). It was expected that the hyphae would continue to elongate by synthesizing 1,3-ß-glucans, perhaps producing large regions of apical wall without a tubular form, because of a lack of the structural support of cellulose. This inhibitor, however, had no effect on growth or hyphal morphology when examined by light microscopy, TEM, or SEM (data not shown). Hyphal elongation rates in colonies incubated with or without DCB were the same. Cellulose labeling of cross sections or hyphal surfaces revealed no differences. Similar results for the lack of an inhibitory effect of DCB were found in the cellular slime mold Dictyostelium (Blanton 1997
, Blanton pers comm). Actually, none of the three major inhibitors used in higher plants, DCB, Isoxaben, or phthoxazolin, had an effect in this organism.
These results of cellulose localization suggest that in a non-elongating hypha, cellulose is evenly distributed along the wall including the apex. In contrast, an elongating hypha lacks cellulose at the apex, or there is a gradual decrease of the amount of cellulose toward the apex. The major matrix component of the wall, 1,3-ß-glucan, is evenly distributed over the entire wall, including the apex, in both elongating and non-elongating hyphae (Shapiro and Mullins 2001
). This contradicts an idea shared by the major theories of hyphal tip growth, namely that all wall components are present in the elongation zone (Hunsley and Burnett 1970
, Wessels 1988
). In a major contribution to the structure of fungal walls, Hunsley and Burnett (1970)
rejected the evidence of Marchant (1966)
and Strunk (1968)
that hyphal tips either lack microfibrillar components or when found the components are artifacts of chemical treatment. They found not only microfibrils, but also matrix materials in the apical region, and suggested that to account for hyphal tip growth microfibrils must be formed at the apex together with protein and 1,3-ß-glucans. A careful reading of their culture methods, however, reveals that it is very unlikely that they were studying elongating hyphae. Based on the evidence that we have presented here, it seems likely that elongating hyphae of Phytophthora would lack cellulose at the apex.
The relatively small area lacking cellulose deposition suggests that cellulose may be required to maintain the tubular cell shape and the integrity of the tip. The recent evidence that activity of the secreted endocellulase (endo-1,4-ß-D-glucanase), first described by Thomas and Mullins (1967), correlates with the tensile strength of the apical hyphal wall supports this idea (Money and Hill 1997
). In higher plants a similar enzyme plays a central role in the assembly of the cellulose-matrix network in expanding cell walls (Nicol et al 1998
).
The current results may implicate an expanded role for 1,3-ß-glucans in concert with the cytoskeleton during apical elongation. Microtubules, polymers of the tubulin protein, are generally held to be responsible for the orientation of cellulose microfibrils within plant cell walls (Nogales 2000
). These microfibrils then provide a scaffold for the assembly of other wall components, and influence the orientation of cell growth (Shaw et al 2000
). The use of specific inhibitors of F-actin or microtubules suggest that hyphal tip growth in Saprolegnia, a close relative of Achlya, is independent of microtubules, but obligatorily requires F-actin caps (Heath et al 2000
). A somewhat different result was found in the hyphal tip cells of Allomyces, a member of the Chytridiomycota, where vesicle and mitochondrion transport and positioning were microtubule-based and the actin cytoskeleton played uncertain roles (McDaniel and Roberson 2000
). The uniform distribution of 1,3-ß-glucans, the major component of the Achlya cell wall, suggests that in the elongation zone the matrix is synthesized before cellulose (Shapiro and Mullins 2002
). The challenge now will be to integrate these new findings on the distribution of the two major wall components of Achlya into the complex picture of hyphal tip growth. A major issue will be to determine if the lack of a microfibrillar component at the elongating apex is unique to Achlya or is also the pattern for true fungi. Among the wide range of tip growing cells, pollen tubes lack cellulose and callose at the tip, and cellulose is first detected about 5–15 µm behind the growing apex (Ferguson et al 1998
). It was suggested that the control of wall plasticity at the growing tube tip does not involve the interaction between cellulose microfibrils, or between cellulose and other non-cellulosic polysaccharides, as occurs in the elongating walls of other cell types. Thus Achlya may be more similar to pollen tubes than to eufungi or other cell types of higher plants.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication August 7, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Blanton RL., 1997 Cellulose biogenesis in Dictyostelium discoideum In: Maeda Y, Inouye K, Takeuchi K, eds. Dictyostelium, A model system for cell and developmental biology. Frontiers Science Series No. 21. Tokyo: Universal Academy Press. p 379–391
Delmer DP., 1999 Cellulose biosynthesis: exciting times for a difficult field of study Annu Rev Plant Physiol Plant Mol Biol 50:245-276
Ferguson C, Teeri TT, Siika-aho M, Read SM, Bacic A., 1998 Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum Planta 206:452-460
Frens G., 1973 Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions Nature Phys Sci 241:20-22
Heath IB, Steinberg G., 1999 Mechanisms of hyphal tip growth: tube dwelling amebae revisited Fungal Genet Biol 28:79-93[Medline]
Heath IB, Gupta G, Bai S., 2000 Plasma membrane-adjacent actin filaments, but not microtubules, are essential for both polarization and hyphal tip morphogenesis in Saprolegnia ferax and Neurospora crassa Fungal Genet Biol 30:45-62[Medline]
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Kaminskyj SGW, Heath IB., 1996 Studies on Saprolegnia ferax suggest the general importance of the cytoplasm in determining hyphal morphology Mycologia 88:20-37
López-Franco R, Bartnicki-Garcia S, Bracker CE., 1994 Pulsed growth of fungal hyphal tips Proc Natl Acad Sci USA 91:12228-12232
Marchant R., 1966 Wall structure and spore germination in Fusarium culmorum Annals Bot 30:821-830
McDaniel DP, Roberson RW., 2000 Microtubules are required for motility and positioning of vesicles and mitochondria in hyphal tip cells of Allomyces macrogynus Fungal Genet Biol 31:233-244[Medline]
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Nicol F, His I, Jauneau A, Vernhettes S, Canut H, Hofte H., 1998 A plasma membrane-bound putative endo-1,4-ß-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis EMBO J 17:5563-5576[Medline]
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Reiskind JB, Mullins JT., 1981a Molecular architecture of the hyphal wall of Achlya ambisexualis Raper. I. Chemical analyses Can J Microbiol 27:1092-1099
———. 1981b Molecular architecture of the hyphal wall of Achlya ambisexualis Raper. II. Ultrastructural analyses and a proposed model Can J Microbiol 27:1100-1105
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Sandgren M, Shaw A, Ropp TH, Wu S, Bott R, Cameron AD, Ståhlberg J, Mitchinson C, Jones TA., 2001 The x-ray crystal structure of the Trichoderma reesei family 12 endoglucanase, Cel12A, at 1.9 Å resolution J Mol Biol 308:295-310[Medline]
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———. 1990 Role of cell wall architecture in fungal tip growth generation In: Heath IB. ed. Tip growth in plant and fungal cells. Toronto and New York: Academic Press. p 1–29
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