Gravitropism of basidiomycetous fungi — On Earth and in microgravity

Pergamon www.elsevier.nlflocate/asr

Adv. Space Res. Vol.24, No. 6, pp. 697-706. 1999 0 1999COSPAR.Published by Elsevier Science Ltd. All rights reserved

Printed in Great Britain 0273-l 177/99 $20.00 + 0.00 PII: SO273-1177(99)00401-9

GRAVITROPISM OF BASIDIOMYCETOUS FUNGI ON EARTH AND IN MICROGRAVITY V. D. Kern

Department of Plant Biologv, Ohio State University, 1735 Neil Ave., Columbus, OH, USA.

ABSTRACT In order to achieve perfect positioning of their lamellae for spore dispersal, fruiting bodies of higher fungi rely on the omnipresent force gravity. Only accurate negatively gravitropic orientation of the fruiting body cap will guarantee successful reproduction. A spaceflight experiment during the STS-55 Spacelab mission in 1993 confirmed that the factor gravity is employed for spatial orientation. Most likely every hypha in the transition zone between the stipe and the cap region is capable of sensing gravity. Sensing presumably involves slight sedimentation of nuclei which subsequently causes deformation of the net-like arrangement of F-actin filament strands. Hyphal elongation is probably driven by hormone-controlled activation and redistribution of vesicle traffic and vesicle incorporation into the vacuoles and cell walls to subsequently cause increased water uptake and turgor pressure. Stipe bending is achieved by way of differential growth of the flanks of the upper-most stipe region. After reorientation to a horizontal position, elongation of the upper flank hyphae decreases 40% while elongation of the lower flank slightly increases. On the cellular level gravity-stimulated vesicle accumulation was observed in hyphae of the 0 1999 COSPAR. Published by Elsevier Science Ltd. lower flank.

INTRODUCTION For centuries gravitational biology focused on specimens from the animal and plant kingdoms. Fungi, finally recognized as an individual kingdom (Whittaker, 1969) but nevertheless often confused with plants even today, ‘suffered’ from reduced effort to analyze their gravitropic behavior (Moore et al., 1996). This review will summarize the current knowledge about graviresponses, cellular mechanisms and events, differential elongation growth and sensing of the gravistimulus through the use of the agaric Fiammulina veZutipesas a model species for the gravitropism of basidiomycetous fungi. Scientists including Schmitz (1842) and Hofmeister (1860) performed initial experiments to define the negative gravitropism of plant shoots and mushroom fruiting bodies. Until the development of rocket engines in the middle of the 20th century, experiments under conditions of reduced gravity were not feasible. Since then, ftmgal material has frequently been included as part of the biological payload flying to space. If early high altitude exposure experiments employing balloons are included, almost 100 experiments can 697

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be listed (Table 1; Kern and Hock, 1993) that used fungal material to evaluate either the effects of spaceflight, the elevated cosmic radiation doses in space or the influence of spaceflight on the circadian rhythm of lower fungi. Only a few of these experiments however, had the intent to evaluate the gravimorphogenesis of basidiomycete fruiting bodies. Initial experiments during the Cosmos 690 flight in 1974 and on board of the orbital stations Salyut 5 and 6 did not answer the proposed questions, because in darkness titing bodies of Polyporus brumalis did not properly develop during 17 days of exposure to microgravity, In parallel experiments in light no hymenium was formed (Kasatkina et al., 1980): Table 1. Total spaceflight missions utilizing fungal material and list of selected experiments transport

system

number of flights

balloon rocket earth-orbiting satellite

40 19 24

automatic lunar station orbital station US space shuttle

1 2 10

experiment

(example)

EXPLORER II, 1935; spores of 7 fungal species DISCOVERER 18, 1960; Neurospora crassa (spores) VOSTOK 2, 1961; several Saccharomyces species GEMINI 12, 1966; Penicillium roqueforti APOLLO 16, 1972; 4 different species ZOND 8, 1970; Candida tropicalis SK-4 SALYUT 5 and 6, 1976178; Polyporus brumalis STS-9 Spacelab 1, 1983; Neurospora crassa STS-6 1A D- 1, 1985; Physarum polycephalum STS-32, 1990; Neurospora crassa STS-42 IML- 1, 1992; Physarum, Saccharomyces STS-47 Spacelab J, 1992; Neurospora crassa STS-55 D-2, 1993; Flammulina velutipes, Saccharomyces STS-49+57 EURECA, 1992193; AspergilIus ochraceus STS-65 IML-2, 1994; Physarum, Saccharomyces STS-8 1 SMM-5, 1997; Sordaria macrospora STS-87 CUE, 1997; Phytophthora soiae

Gravity Causes Fruiting, Bodies to Bend Up Very early Schmitz (1842) analyzed the upward growth of Agaricus fruiting bodies. He was convinced that gravity was responsible for this orientation and created the term “negative geotropism” (now gravitropism). If a fruiting body is placed horizontally, it will bend up in 3-4 h (Coprinus; Kher et al., 1992) or more slowly (24 h in Flammulina) to reach the vertical position. This is an extremely accurate reorientation. The titing bodies reach the perfectly upright position, exactly 180” opposite to the gravitational vector (Kern, 1994). Nonetheless, it took until 1993 to document that gravity alone is required for this orientation, because of the inability to eliminate gravity as a variable in any experiment on Earth. The FUNGI experiment during the STS-55 spacelab mission was the first launch to space of basidiomycete fruiting body primordia designed to document their development when exposed to microgravity. During this mission 6 cultures were either incubated for 10 days in microgravity or on a lg-reference centrifuge. Fruiting body orientation was photo-documented (Figure 1) and samples were harvested and chemically fixed in microgravity for subsequent electron microscopical analysis after landing (Kern and Hock, 1994). The absolute requirement of gravitational force for gravimorphogenesis was demonstrated by comparing centrifuged (Figure 1C) with microgravity samples (D). Figure IE is a plot of the fmal cap orientation in circular histograms that shows negative gravitropism in lg versus random orientation in pg. Additionally, a substrate avoidance reaction (probably negative hydrotropism) could be detected and documented in the

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Fig. 1. Gravitropism of Flammulina fruiting bodies on earth (A) and during spaceflight (C, D). Bar in D = 2cm for A, C, D. A Four days old culture with negatively gravitropic fruiting bodies in lg on earth. B Set-up during the STS-55 flight (details in Kern and Hock, 1994). Four sample containers (left) were placed in front The centrifuge was of the BOTEX lg-reference centrifuge that accommodated 2 additional containers. rotating (black arrow) with 90rpm thus simulating exactly lg. The BOTEX incubator as part of the Biolabor Spacelab rack allowed incubation of 6 Flammulina cultures for 10 days in darkness at 24’C. C Culture on lgreference centrifuge (50h old). All fruiting bodies reacted negatively gravitropic (arrow indicates acceleration vector) thus demonstrating that gravimorphogenesis does require a gravitational vector. D Cultures incubated in microgravity for 7 days. The fruiting bodies displayed random orientation and additionally a negatively hydrotropic substrate avoidance reaction. E Circular histograms indicating final fruiting body cap orientation in lg (upper-most histogram), lg on the centrifuge during spaceflight (middle) and pg (lower-most histogram).

pg cultures. The morphology of fruiting bodies in space-grown cultures did not differ from lg samples. The hymenium showed normal development in pg with spores maturing on the basidia (Kern, 1994). Successful Reproduction

Requires Negatively Gravitropic Orientation of the Fruiting Body

Early this century Buller (1905) demonstrated that young fruiting bodies of Lenlinus lepidus exhibit gravitropic reactions only at the maturation of spores. Furthermore, Buller (1909) showed that when the fiuiting body lame&e are incorrectly oriented a significant amount of spores will not fall out (down; Figure 2D) and cannot be dispersed by wind. If the lamellae are tilted 5” off the vertical orientation, up to 50% of the total spore mass will stick to neighboring lamellae and be lost. An incorrect orientation of 30” most likely causes a total loss (Figure 2D). These investigations underline the importance of appropriate fruiting body orientation for successful reproduction, and as discussed earlier gravity is employed for spatial orientation. Some species like Agaricus bisporus do not solely rely on the negatively gravitropic orientation of their fruiting bodies, but also actively orient their lamellae positively gravitropic (Buller, 1909). This could not be demonstrated for Flammulina velutipes (data not shown). Recently, it was confirmed for Flammulina that a graviresponse is seen only when the basidia in the hymenium are fully differentiated

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D. Kern

Flammulina velutipes. Bars = lmm.

Fig. 2. Scanning electron microscopy images of the fruiting body morphology and the hymenium. A, B Mature fruiting bodies of

C Four basidiospores mature on each basidium (bar = 2Spm). Fruiting bodies were fixed in 3% (v/v) glutardialdehyde in 75mM potassium phosphate buffer (pH 7.2) for 2h and postfixed with 2% Os04 for lh. The specimens were dehydrated in an acetone dilution series with increasing concentrations, critical point dried with COz, sputtered with a palladium/ gold complex (10:90) and viewed on a Stereoscan 360 SEM (Cambridge Instruments Ltd.) at the Department of Physics (TU Mtinchen, Weihenstephan). D Cartoon model of fruiting body lamellae (basidia and spores are Perfect negatively superimposed). orientation gravitropic guarantees spore release (the arrows show the trajectory of the spores) and dispersal by wind (W, left). If the fruiting body cap is shifted 25” from its vertical position, spores fall down and stick to neighboring lamellae and therefore are lost (right).

(Monzer et al., 1994). Figure 2 shows SEM details of fruiting body morphology the hymenium with basidia and mature spores. Gravity is Sensed in the Upper-Most

of Flammulina including

Stipe Region

While the basidiomycete mycelium does not show any spatial orientation with respect to gravity and simply grows along and throughout its medium (Kern and Hock, 1994) fruiting bodies display obvious negatively gravitropic orientation (Figures 1A and 3). Intensive studies with Flammulina fruiting body explants revealed that only the region between upper-most stipe and cap (3-5mm) is sensitive to gravity (Monzer et al., 1994; Haindl and Monzer, 1994). Microscopical analyses revealed that this region has a distinct morphology, in that it contains the transition zone from cap to stipe where the strictly paralleloriented hyphae of the stipe randomly spread into the umbrella-like cap region of the fruiting body (Kern, 1994). It was demonstrated that if the transition zone was cut into halves, quarters or 8 equal pieces, each of those segments sensed gravity and oriented negatively gravitropic after horizontal displacement (Figure 3B). This and early detailed experiments conducted by Hofmeister (1860) lead to the assumption that each individual hypha carries the potential to sense and to react to gravity. In Flammulina, the fruiting body cap and the lamellae apparently are not involved in gravisensing (Figure 3). Instead, the cap region plays a role as source for a growth promoting hormone-like factor (reviewed by Gruen, 1982 and Moore, 1991). After decapitation, fruiting bodies will demonstrate normal gravitropism initially including stipe elongation (residual growth). After about 3 days elongation terminates and the fruiting body deteriorates (Kern, 1994). Presumably, at that time all of the growth-promoting factor that could reach its receptors in the upper-most stipe is used, and no further substance can be transported from the missing cap.

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show significant graviresponses. Note that, likely due to incomplete removal of the transition zone, the lower-most stipe displays some gravitropism. B Graviresponse of a split stipe. Each individual segment (8 segments) gets reoriented negatively gravitropic after displacement. horizontal C Dependence of the graviresponse on specimen size. Gravitropic curvature of 5mm, lOmm, decapitated 25mm-long explants and an intact fruiting body. Image A from Monzer et al. (1994); images B and C from Hamdl and Monzer (1994).

types

Fig. 3. The transition zone between cap region the graviperceptive zone in Flammulina fruiting C Bar = Smm. A Surgically treated specimen Except for the lower-most gravistimulation.

and the upper-most stipe is bodies. A, B Bar = 10mm; types after 24h of lateral stipe specimen, all sample

Differential Elongation Allows Gravitropic Bendii In contrast to tip-growing fungal mycelium, the fruiting body stem grows by diffuse elongation of its api-

cal region (Wessels, 1993). Differential growth of the upper-most stipe allows gravitropic bending. In this region of the stipe all hyphae (-1.2 million throughout the diameter) are arranged strictly parallel to each other (Figure 4A, B). In contrast to other genera with direct hyphal connections (anastomoses) such as Armillaria, in Flammulina individual hyphae do not have direct contact to neighboring cells (plectenchymous arrangement without plasmodesmal cell communication). Differential growth of gravistimulated fruiting bodies of Flammulina (Figure 4C, D, E) has been investigated in great detail (Monzer et al., 1994; Kern et al., 1997, 1998). About 12h after horizontal displacement fruiting bodies bent up negatively gravitropic (Figure 4C) by differentially elongating the apical region of the stipe. Note that marks of white paint were stretched and eventually torn (arrows) in regions of growth. Balloon model studies supported the proposed mechanism of gravitropic bending of a non-tissue cellular arrangement (Kern et al., 1997). Differential inflation of balloons (simulating differential elongation of individual hyphae and hyphal bundles) that were partially glued together or restricted by unilaterally applied tape will lead to upward bending. Detailed measurements of gravistimulated fruiting body elongation revealed that the elongation growth of hyphae of the upper flank was inhibited to about 40% of that in vertically growing controls (Figure 4D). The lower flank showed elongation with a slightly increased rate, Active shrinking of upper flank surface cells such as that observed in stems of the plant Xanthium strumarium (Sliwinski and Salisbury, 1984) could not be demonstrated. Greening et al. (1997) recently showed that during the bending reaction of gravireacting fruiting body stipes of Coprinus cinereus the hyphae of the physically lower flank increased their length up to a ratio of 5:l when compared to upper flank hyphae. However, the packing density of neighboring hyphae did not differ between flanks. Ultrastructural Responses During Gravitropic Bending Early cellular responses to gravistimulation were revealed in ultrastructural analyses of l.tg-grown and lg reference samples (Kern et al., 1997). Both differential elongation and diffise growth depend on the incorporation of extracellular and membrane components throughout the total length of the hyphae (Craig et al.,

Fig. 4.

Gravitropic reorientation of horizontally displaced fruiting bodies. A, B The apical stipe region of fruiting bodies contains up to 1.2 million of individual hyphae that are arranged strictly parallel. SEM image of sectioned upper stipe (A); cross section of the same region (B). Bars = 500pm (A), 50pm (B). C Negatively gravitropic bending of an intact fruiting body. White paint marks (intact marks on the left) stretched and torn (arrows) on the lower flank of the stipe apex (48h after replacement, bar = 5mm). D Graphic analysis of elongation rate (BE) of negatively gravitropic fruiting bodies (upright vertical control) and lower versus upper flanks of horizontally displaced fruiting bodies. While the elongation rate of the lower side of gravistimulated apical stipes only slightly increased, the growth of the physically upper flank was reduced (inhibited) to 40% of comparable vertical growth rates (from Monzer et al., 1994). E Cartoon model showing differential growth in the apical stipe region (from Kern et al., 1997).

Fhmmulina

1977). Cell wall extension is assumed to be triggered by exocytosis. Individual vesicles (Figure 5A, arrowheads) and multi-vesicular aggregations (5A, arrows) have been observed (Kern and Hock, 1994) which are suspected to fuse with the plasma membrane to export wall-building materials like D-D-glucan chains and chitin-microfibrils, as well as new membrane. Multi-vesicular aggregations, previously suspected to be fixation artifacts, were observed after employing high-pressure-freezing technology (Kern et al., 1997). Vesicles are most likely synthesized at the endoplasmatic reticulum and the Golgi-like comThese vesicles are guided to the plasma membrane by F-actin plexes near the nuclei of the hyphae. strands (Heath and Kaminskij, 1989). Additionally, it has been shown that the driving force for hyphal elongation is an increase in turgor pressure due to water uptake and vacuolar extension (reviewed by Wessels, 1993). This was shown by incubating stipes in hypertonic solutions (decreasing turgor pressure) which lead to a decrease in gravitropic bending response (Kern et al., 1998). Volume increase of the central vacuoles of apical stipe hyphae is achieved by incorporation of microvesicles into the vacuolar membrane (Figure 5B, arrowheads). In vertically growing apical stipes microvesicles were found most abundantly in the hyphae at the center of the stipe. By 30-60min after horizontal displacement and gmvistimulation a significant vesicle accumulation could be found in peripheral hyphae of the lower flank (Figure SC). The vesicle concentration in upper side hyphae decreased significantly during that timeframe.

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After 60min the ratio of vesicle number is 7:l in favor of hyphae of the lower flank (Kern et al., 1997). Vacuole size was also increased in these hyphae (Kern, unpublished results).

Fig. 5. Vesicle incorporation into the vacuoles and the cell wall causes an increase of turgor pressure and drives cell wall elongation (diffusive growth). A Individual (arrowheads) and multi-vesicular aggregations supposedly fuse (arrows) with the plasma membrane to supply cell wall elongation with wall and membrane-building material. Bar = 0.25pm. B As a prerequisite, the turgor pressure of the hyphae increases by incorporating electron-lucient vesicles into the vacuoles (arrowheads) and subsequent water uptake. Bar = lpm. C After 60min of horizontal displacement significant vesicle accumulation are observed in hyphae of the lower flank. Bar = lpm. cw = cell wall, m = mitochondria, v = vacuole. TEM techniques as described in Kern et al., 1997.

Hypotheses

for Gravisensinq

Gravisensing in fruiting bodies is still an unanswered question though several models and hypotheses have been proposed (Dennison and Shropshire, 1984; Moore et al., 1996; Monzer, 1996; Kern et al., 1997, 1998; Schimek, 1998). As shown, gravisensing requires the transition zone of the Fhmmulinu fruiting body. A detailed analysis of the hyphal ultrastructure (Kern, 1994) revealed a lack of organelles that sediment in response to gravity. Dennison and Shropshire (1984) suggested that the lightest component in Phycomyces sporangiophore cell, the vacuoles, might float in the more dense cytoplasmic framework and allow gravisensing through pressure on the upper plasma membrane. In FZummuZinu,however, floating of cell components could not be observed in gravistimulated hyphae (Kern, 1994). Another more recent model (Schimek, 1998) proposed that heavy protein crystals in vacuoles of Phycomyces sporangiophore cells could be involved in gravisensing. This also does not seem to be applicable for the multihyphal t?uiting bodies of FZummuZinu,because large crystals were distributed very infrequently throughout the stipe, cap region and lamellae of old fruiting bodies, and sedimentation of these structures could not be documented (Kern, 1994). Furthermore, protein crystals were not observed in young fruiting bodies that were fully capable of gravisensing. A hypothetical model for graviscnsing in multihyphal fruiting bodies was drafted for FZummuZinu. Monzer (1996) compared density values for different hyphal organelles and determined that only the nuclei with a density of 1.22g cm-3 could be considered as valid candidates for gravity-related sedimentation (statoliths). Hyphae of the upper-most stipe of FZummuZinufruiting bodies are multinuclear and up to 10 individual nuclei (-2l.trn in diameter) were observed in hyphal compartments. Considering equations

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for thermal energy and the range of displacement necessary to be detectable against the thermal motion of the cytoplasm (‘ minimal condition’; Bj&krnan, 1988), nuclei of Fhmmulirzu clearly fulfill these criteria (Monzer, 1996). After horizontal displacement, slight but significant gravity-dependent sedimentation of transition zone nuclei was documented (Figure 6C). Furthermore, it was shown that these nuclei were encased by a network of F-actin filaments (Monzer, 1995). Immunofluorecence revealed spindle-shaped Factin aggregates that were connected to the plasma membrane in the hyphal periphery and closely encased the nuclei (Figure 6A, B). When treated with the actin-depolymerizing drug cytochalasin D, the gravitropic response decreased significantly (Monzer, 1995). Inhibition of microtubules did not influence the gravitropic response. Summarizing these observations, a model for gravisensing in the upper-most FZummulina stipes has been formulated (Figure 6D, E): Under normal growth conditions the nuclei that are ‘hanging’ in a network of actin filaments cause specific actin strands to be stretched and others to be relaxed (Figure 6D). When gravitropically stimulated (Figure 6E) slight sedimentation of the nuclei towards the gravity vector most likely causes a change in the pattern of which strands are stretched or relaxed. mulinu transition zones. The nuclei were stained with the vital stain SYTO 11. Elapsed times after reorientation and the g-vector are indicated. Bar = 5p.m. D Cartoon model of a typical transition zone hypha during vertical growth. The nuclei are encased by actin filaments that in addi-

1 vacuole i

1._

.______ ____.__ __.-. ___._______

Li

Fig. 6. Gravisensing in hyphae of the transition zone between cap and stipe of Flammulinu - facts and models. A F-actin immunolocalization reveals spindleshaped aggregates that encase the nuclei (non-stained black holes, arrows). B A bisbenzimide staining of nuclei perfectly matches the pattern in A (arrows). A, B Bar = 10pm. C Nuclear motion (arrows) in horizontally displaced Flam-

tion are connected to the plasma membrane. Some of the F-actin filament strands are under more tension than others. E Gravireacting hypha after horizontal reorientation with displacement of the nuclei. Note the new pattern in the Faction arrangement. Some actin strands are stretched while others are relaxed. Vesicle traffic is directed to the lower flank hyphae; vacuoles incorporate vesisubsequently cles which leads to an increase in size and increased turgor pressure by way of water uptake. A, B, from Monzer, 1995, C from Monzer, 1996; D, E modified after Moore et al., 1996.

In a non-tissue cellular arrangement of hyphae that do not possess cytoplasmic intercellular connections, any exchange of information or cell communication must rely on a mobile factor. The existence of mobile growth regulators as an essential component of the signal transduction process is hypothetically involved

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in controlling the differential response of individual hyphae to F-actin net deformation (discussed in detail in Kern et al., 1998). Deformation of the F-a&in filament strands during gmvisensing most likely activates a cascade-like redistribution of an as yet unknown hormone-like growth factor within the stipe. Differential distribution (enrichment or reduction) of a growth regulating substance in hyphae of the lower flank subsequently would induce increased vesicle incorporation into the vacuoles of those hyphae and cause rising turgor pressure. Additionally, increased vesicle traffic that shuttles wall material to the hyphal wall accommodates increased cell elongation. Alternatively, a potential redistribution of growth-inhibiting factors in the upper flank hyphae (Figure 4D shows a 40% growth inhibition) should be considered. Further studies need to be conducted to isolate and identify these growth regulating factors with potential mycohormonal function and quantify their redistribution during the graviresponse. In any case, collectively, differential growth coordinated across a pseudo-tissue arrangement of independent hyphae allows gravitropic bending.

SUMMARY Fruiting bodies of higher fungi orient their cap region in an upright negatively gravitropic position in order to optimize conditions for spore release and spore dispersal by wind. The fruiting body hyphae employ the omnipresent factor gravity to determine their spatial orientation. It appears that each individual hypha can sense and react to gravity, but elongation growth of these non-interconnected cells is coordiitely regulated to cause stipe bending. The actin microfilament system that surrounds cell organelles in a netlike arrangement may be involved in sensing since microfilament-depolymerizing drugs eliminate gmvitropism without causing strong effects on hyphal elongation. We hypothesize that a barely-detectable sedimentation of nuclei deforms the microfilament net. This may initiate a cascade-like redistribution of hormone-analogous growth factors that trigger differential elongation. Still, many aspects of the graviperception and reaction chain are unknown. Nevertheless, the last 5 years of extensive studies in several labs around the world and the opportunity to successfully cultivate basidiomycetous fruiting bodies in microgravity allowed the discovery and description of many more exciting details and permitted significant answers to the question: “How do fruiting bodies grow up?”

ACKNOWLEDGEMENTS I am grateful to Prof. Bertold Hock at the Department of Botany, TU Mtinchen at Weihenstephan, for confidently granting this project into my hands, continuously inspiring new ideas and assisting with helpful discussions. Thanks to Dr. Jeanette Nadeau and Nathan White at Ohio State University for discussion of the manuscript. Supported by a grant of DARA (50 WB 9250) to B.H.

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