The Sporangiophore of Phycomyces Blakesleeanus: a Tool to Investigate Fungal Gravireception and Graviresponses P

Plant Biology ISSN 1435-8603

REVIEW ARTICLE The sporangiophore of Phycomyces blakesleeanus: a tool to investigate fungal gravireception and graviresponses P. Galland Fachbereich Biologie, Philipps-Universitat€ Marburg, Marburg, Germany

Keywords ABSTRACT Exponential law; gravisusceptors; gravitropism; gravitropism mutants; The giant sporangiophore of the single-celled fungus, Phycomyces blakesleeanus, uti- light-gravity interaction; Phycomyces lises light, gravity and gases (water and ethylene) as environmental cues for spatial blakesleeanus; resultant law; sine law; orientation. Even though gravitropism is ubiquitous in fungi (Naturwissenschaftliche sporangiophore. Rundschau, 1996, 49, 174), the underlying mechanisms of gravireception are far less understood than those operating in plants. The amenability of Phycomyces to classical Correspondence genetics and the availability of its genome sequence makes it essential to fill this P. Galland, Fachbereich Biologie, Philipps- knowledge gap and serve as a paradigm for fungal gravireception. The physiological Universitat€ Marburg, Karl-von-Frisch Str. 8, phenomena describing the gravitropism of plants, foremost adherence to the D-35032 Marburg, Germany. so-called sine law, hold even for Phycomyces. Additional phenomena pertaining to E-mail: [email protected] gravireception, specifically adherence to the novel exponential law and non-adherence to the classical resultant law of gravitropism, were for the first time investigated Editor for Phycomyces. Sporangiophores possess a novel type of gravisusceptor, i.e. lipid K. Palme globules that act by buoyancy rather than sedimentation and that are associated with a network of actin cables (Plant Biology, 2013). Gravitropic bending is associated Received: 4 January 2013; Accepted: 16 with ion currents generated by directed Ca2+ and H+ transport in the growing zone August 2013 (Annals of the New York Academy of Sciences, 2005, 1048, 487; Planta, 2012, 236, 1817). A set of behavioural mutants with specific defects in gravi- and/or photorecep- doi:10.1111/plb.12108 tion allowed dissection of the respective transduction chains. The complex pheno- types of these mutants led to abandoning the concept of simple linear transduction chains in favour of interacting networks with molecular modules of physically interacting proteins.

1992; Chaudhary et al. 2013) are available, all making it INTRODUCTION amenable to modern molecular approaches. In comparison to the classical plant objects of photo- and The Phycomyces literature has been reviewed at regular inter- gravitropism, such as grass coleoptiles and seedlings, the Phyc- vals during the past decades. Older literature, with major omyces sporangiophore is in several ways unique. The sporan- emphasis on blue light reception and the concomitant interac- giophore is a single coenocytic cell, which, for simplicity, can tion with gravitropism (Corrochano & Galland 2006), has been be regarded as a thin, water-filled tube elongating under a critically discussed in previous reviews (Bergman et al. 1969; turgor pressure of about 0.31 MPa at an astonishing rate of Galland & Lipson 1987a; Galland 2001). For physical principles 2–3mmh 1. Light and gravity perception, growth modula- regarding optics, photoreceptor dichroism and excitation pro- tion and phototropism all occur in the small growing zone files, as well as for a critical discussion of problems pertaining extending 2–3 mm below the sporangium (Bergman et al. to adaptation and action spectroscopy, the reader is referred to 1969). The many complexities of signal transduction are thus the reviews of Fukshansky (1993), Galland (2001) and restricted to this fragile transparent cylinder of 2 mm length Corrochano (2007). and 100 lm diameter, whose growth and twist is modulated by unilateral light and gravity in a way that manifests as SPORANGIOPHORE GROWTH tropic bending. During the early decades of the last century it was not The local and time-dependent modulation of growth rate uncommon to investigate Phycomyces tropisms side by side represents a fundamental parameter underlying photo- and with those of higher plants (Banbury 1959, 1962). From gravitropism. Elongation growth and bending is restricted to such comparative investigations it became apparent that the the growing zone, which extends about 2–3 mm below the tip physiological principles underlying the light- and gravirecep- of stage 1 (Fig. 1, left) or below the sporangium in stage 4 tion of Phycomces and higher plants share a similar logical (Fig. 1, right). The growing zone represents the sensitive, and structure (Galland 1990). The many similarities which Phyc- at the same time, also the reactive zone of the sporangiophore. omyces tropisms share with those of plants and other fungi At its upper boundary it is continually formed anew, and at the justifies its use as a model organism, moreover, this organ- lower boundary it is converted into non-reactive material. The ism has been sequenced (http://genome.jgi-psf.org/Phybl2/ growth rate is largely determined by water uptake, transpira- Phybl2.home.html), and detailed genetic maps (Alvarez et al. tion and cell wall extension, which are brought about by the

Fig. 1. Sporangiophores of Phycomyces blakesleeanus. Left: stage 1 sporangiophore. The open arrow points to the complex of lipid globules that contain yellow b-carotene. The central hyaline structure in the lower 360 lm represents the central vacuole. Bar: 100 lm. Photograph: Dr. Irmin Meyer. Middle: close up (interference-contrast micrograph) of the complex of lipid globules. Arrowhead: single lipid globule. Because of interference-contrast other globules appear dark. Small arrows: hyaline zone that houses the lipid globules. Bar: 10 lm. Photograph: Dr. Franz Grolig. Right: dark-ﬁeld photograph of a stage 4 sporangiophore, i.e. with the spore-bearing sporangium. Bar: 100 lm. Photograph: Dr. Irmin Meyer.

allocation of chitosomes (Herrea-Estrella et al. 1982). The tur- Fig. 2. Kinetics of gravitropic bending of stage 4 sporangiophores of Phyc- gor pressure amounts to 0.31 MPa and a growth rate of about omyces. A: Sporangiophores were placed horizontally. Filled circles: wild l 1 33 m min (Ortega et al. 1992). Because of transpiration, the type; open squares: mutant C202 (lacking vacuolar protein crystals); open water uptake of a sporangiophore can exceed seven times the triangles: hypergravitropic mutant C5 containing an excess of vacuolar volumetric growth rate (Ortega et al. 1992). The growth rate is protein crystals. Data from Schimek et al. (1999a). B: Gravitropic bending of not completely steady but rather ﬂuctuates and displays a a single sporangiophore in response to step changes of the centrifugal accel- frequency spectrum with several minor peaks between 0.3 and eration (1.5 g ? 3 g ? 1.5 g; upper solid line). Modiﬁed from Dennison 10 mHz and a maximum at 10 mHz that can be altered in 1961. some of the phototropism mutants (Ensminger & Lipson 1992). A further complication derives from the fact that in gravitational acceleration (Dennison 1961; Dennison & Shrop- stage 4, sporangiophores do not only elongate (2–3mmh 1) shire 1984; Horie et al. 1998; Schimek et al. 1999a), while but rather rotate clockwise, when viewed from above, at a rate mycelial hyphae and zygophores are agravitropic. Cultures of of about 180°h 1 (Oort 1931). Elongation and rotation repre- Phycomyces cultivated for 19.5 days in microgravity in the Rus- sent two growth components that are not evenly distributed in sian biosatellite Cosmos-782 maintained normal vegetative and the growing zone (Cohen & Delbruck€ 1958). The rotation sexual development, but had completely disoriented sporangio- causes a slight complication in gravi- and phototropic bending, phores, forming twists and loops. The normal growth pattern because near the respective thresholds sporangiophores do not of sporangiophores could be recovered on a centrifuge operat- bend exactly in the plane of the stimulus but rather in a plane ing at an acceleration of 1 g (Parfyonov et al. 1979). The effec- that slightly deviates from it, leading to errors in direction tiveness of gravitropism depends to some extent on the (Bergman et al. 1969; Galland et al. 2004). developmental stage, as stage 1 sporangiophores, which lack a sporangium (Fig. 1, left), bend gravitropically more slowly than stage 4 sporangiophores with a sporangium (Schimek et al. GRAVITROPISM 1999a; Grolig et al. 2004). Gravireception is ubiquitous in the fungal kingdom and is It is important to distinguish between two different types of manifested as gravimorphogenesis and gravitropism (reviewed gravitropic response: (i) ‘normal’ gravitropism in response to a in Kern & Hock 1996; Kern 1999; Galland 2001; Corrochano & displacement of the sporangiophore from the vertical (Fig. 2A), Galland 2006). Fruiting bodies usually grow vertically and which persists for as long as the gravitropic stimulus persists, reorient a few hours after displacement from the vertical i.e. it is non-adaptive and slow; and (ii) gravitropism in (Fig. 2A). Sporangiophores of Phycomyces display negative response to sudden step-up or step-down changes of centrifu- gravitropism, i.e. they bend opposite to the Earth’s gal acceleration (Fig. 2B), which is transient and fast.

Kinetics, threshold, sine law and resultant law (Galland et al. 2002) rather than 90°, as predicted from the sine law. For these ‘head over’ situations a modified sine law Sporangiophores that are placed horizontally have a rather (Larsen 1962, 1969) fits better the observed bending angles: irregular gravitropic latency of some 10–30 min; the vertical position is reached in about 10–13 h (Fig. 2A; Dennison & g a sin c Shropshire 1984; Ootaki et al. 1991; Schimek et al. 1999a). As Sgravi ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð2Þ is the case in phototropism (Shropshire 1971), also in gravitro- b2 þ 2b cos c þ 1Þ pism the bending rate is inversely proportional to the diameter of the sporangiophore. In addition, it also increases with the where a and b are adjustable parameters, c is inclination angle absolute growth rate and the differential growth rates at the of the gravitropic organ and g is gravitational acceleration. lower and upper sides of the horizontal growing zone (Ootaki Another ‘gravitropism law’ that purportedly holds for et al. 1995). The absolute threshold, as determined on a clino- plants is the so-called ‘resultant law’ (Resultantengesetz; Metz- 2 stat centrifuge, is near 10 g (Galland et al. 2004). The gravi- ner 1929; Rawitscher 1932). This law refers to situations in tropic bending rates are rather low and vary between 0.1 and which gravitropic organs are centrifuged in an upright posi- 1 0.3° min , depending on the inclination angle of the sporan- tion. The resultant law claims that in such situations the giophore relative to the vertical. gravitropic organ orients itself parallel to the acceleration vec- The gravitropic bending that occurs in response to sudden tor resulting from the centrifugal and gravitational accelera- acceleration changes in a centrifuge is very different from that tion. A rigorous quantitative examination of this ‘law’ of horizontally placed sporangiophores. A twofold increase of showed, however, that it does not provide a precise descrip- centrifugal acceleration elicits transient bending rates that can tion of the bending of centrifuged sporangiophores of Phyc- 1 reach 6° min (Fig. 2B; Dennison 1961). A subsequent step omyces (Gottig€ & Galland 2014; this volume). Centrifuge down of the acceleration causes a return of the sporangiophore experiments done with sporangiophores of Pilobolus crystalli- to the vertical. During the return phase, the sporangiophore nus also showed a substantial deviation from the resultant law displays positive instead of negative gravitropism, even though (Horie et al. 1998). The purported validity of this ‘law’ was to the stimulus vector has not changed in direction (Fig. 2B). This a large extent based on the fact that the plant material had result shows that the sporangiophore reacts to the change in been tested in the past in most cases at centrifugal accelera- stimulus size rather than direction of the stimulus (Dennison tions of 1 g. When plant material is tested over an extended 1961). The fast gravitropic bending can also be elicited by forc- dynamic range it becomes apparent that the ‘resultant law’ ing the sporangiophore mechanically in the same direction as does not correctly describe the bending pattern of centrifuged the acceleration vector, e.g. by placing a unilateral load as low hypocotyls, and in particular roots of Arabidopsis seedlings as 0.5 mg (Dennison 1961). Even though most investigations (M. Dummer€ & P. Galland, in preparation). The ‘resultant concentrate on the slow graviresponse (‘normal’ gravitropism) law’ is a corollary of the sine law, and any possible deviation elicited by changes in inclination angle (Fig. 2A), one needs to from it represents a strong indication that basic assumptions keep in mind that gravitropic organs are subjected in nature to of the classical statolith–receptor paradigm fail to fully explain sudden acceleration changes such as wind and mechanical the complex spectrum of graviresponses (Gottig€ & Galland disturbances that are equivalent to the step changes in the 2014, this volume). Dennison experiment described above (Fig. 2B). Thus the ecophysiologically relevant gravitropism probably entails two Criteria for gravisusceptors response components: the slow and non-transient response, which is usually investigated, and in addition the fast and tran- Gravitropic bending of plants and fungi depends on the pres- sient response, which is elicited by wall stress (see below) rather ence of statoliths (gravisusceptors), heavy cell organelles that than the action of statoliths. sediment upon reorientation and that generate enough poten- Like gravitropic plant organs, sporangiophores of Phycomy- tial energy to exceed the thermal noise of the hypothetical ces also obey the so-called sine rule or sine law (Sachs 1879, gravireceptor. To achieve this, they must be able to either sedi- 1882), which states that the gravitropic stimulus S that operates ment (statoliths) or else float (buoys), such that a particle on an inclined gravitropic organ is proportional to the product gradient is formed inside the receptive cells. To do this, the of terrestrial gravity and the sine of the inclination angle: gravisusceptors need to possess a density different from that of the surrounding cytoplasm, and additionally, a critical mass to S ¼ g sin c ð1Þ overcome the effect of thermal motion, which counteracts the effect of sedimentation or buoyancy and thus the formation of a particle gradient. Whether or not a cell organelle qualifies as where S is the gravitropic stimulus, g terrestrial gravitational a gravisusceptor can be determined with a function that was acceleration (9.8 ms 2), and c is inclination angle (°) of the originally introduced by Einstein, who modified the Boltzmann sporangiophore relative to the vertical (Galland et al. 2002). distribution by taking into account the terrestrial gravitational The effect of the gravitropic stimulus size S is manifested field. The function describes the ratio of sedimenting (or through the gravitropic bending angle reached after a given floating) particles separating along a distance h: duration. The adherence to the sine law is remarkable in view of the fact that the underlying gravisusceptors and bending = ¼ ½ ðq q Þ = ðÞ mechanisms are completely different from those acting in N N0 exp V c gs gh kT 3 plants. Like plants, Phycomyces sporangiophores also deviate for elevated inclination angles from the sine law, because the where N0 and N are the number of particles separated by a – ° most effective bending occurs at inclinations of 120 135 distance h before (N0) and after (N) sedimentation (flotation),

respectively, V is particle volume, qc and qgs are specific densi- sporangiophores could nevertheless influence gravitropism in ties (kgm 3) of the cytoplasm and the gravisusceptor, respec- nature, as shown by the fact that a laterally applied force of tively, g is the constant of gravitational acceleration (9.8 m only 0.5 mg elicits a relatively fast, transient bending response s 2), k is the Boltzmann constant (1.38 9 10 23 JK 1), and T that subsides after about 6 min (Dennison 1961). Similarly, is absolute temperature (K) (Markl 1982). A value of N/N0 = 1 when a load of 5 mg is applied symmetrically to the growing indicates that the particles do not separate, so that no gradient zone by pulling the sporangium of an inverted sporangiophore is generated; values <1 indicate effective separation. It is appar- downwards, transient growth responses are elicited: lifting the ent from such a plot that particle separation by sedimentation load causes a positive response, while adding to the load causes or by flotation over a distance h of 10 lm occurs at 1 g only for a negative response (Dennison & Roth 1967). Such wall stress- particles with diameters above 0.8 lm (Grolig et al. 2004). At induced bending and growth responses are usually neglected in 0.2 g, particles of diameter >2 lm would separate. At 0.02 g, gravitropism studies, even though they are highly relevant in however, which represents the gravitropic threshold of Phyc- nature, where mechanical stress often adds to the ‘normal’ omyces (Galland et al. 2004), only particles of diameter >4 lm gravitropic bending. will form a substantial gradient. That central vacuoles of the sporangiophore could play a When gravisusceptors sediment or float, they generate a role as buoyant gravisusceptors, as proposed by Dennison & force, F, which can be calculated as: Shropshire (1984). These authors observed that the cytoplasm sediments in horizontal sporangiophores, whereas the central ¼ ðq q ÞðÞ vacuoles are displaced slightly upwards. The central vacuoles of F g V n c gs 4 Phycomyces contain octahedral protein crystals of high density 3 (1.27 gcm ) that rapidly sediment upon reorientation of the where g is the Earth’s gravitational acceleration (9.8 ms 2), V sporangiophore, and must participate in gravisusception the volume of a gravisusceptor, n the number of gravisuscep- because mutants lacking these crystals are gravitropically tors, q the density of the cytoplasm, and q the density of the c gs moderately defective (Schimek et al. 1999a,b) and display an gravisusceptor. elevated threshold (Galland et al. 2004). The octahedral crys- The potential energy, E, of a sedimenting or floating gravi- tals, which had been known for several decades (Thornton susceptor is given by: 1969; Wolken 1969, 1972; Ootaki & Wolken 1973), are made up of three proteins that are associated with pterin- and flavin- ¼ ð Þ E F d 5 like pigments (Eibel et al. 2000; Fries et al. 2002). There is strong circumstantial evidence that the vacuolar crystals also where F is the static force (Newton) and d the distance (m) operate as gravisusceptors in other zygomycete fungi. Sporan- over which the gravisusceptors are displaced. The potential giophores of Pilobolus crystallinus are negatively gravitropic in energy of a gravisusceptor needs to exceed the thermal noise stage 4, during which crystals are present, while they are agravi- 21 (3/2 k T = 6.21 9 10 J at 300 K). For example, at the gravitropic in stage 1, which is characterised by their absence (Horie 2 tropic threshold of Phycomyces, which is near 2 9 10 g et al. 1998). Sporangiophores of Mucor mucedo, which are (Galland et al. 2004), the potential energy generated by floating agravitropic, do not possess vacuolar crystals throughout their 18 lipid globules would amount to 10 J, which is still 360 times development (Horie et al. 1998). more than the thermal noise. The estimated potential energies Beside the protein crystals, apical lipid globules are also are also sufficiently high to explain the adherence of Phycomy- involved in the gravisusception of Phycomyces. The relative ces to the so-called sine law of gravitropism (Galland et al. contributions of protein crystals and lipid globules are depen- 2002). For small inclination angles of the sporangiophore of dent of the developmental stage. In stage 1 sporangiophores, 1–2°, the gravitropic stimuli are, according to the sine law, the vacuoles contain relatively few crystals but more lipid glob- 2 1.7–3.4 9 10 g, which is just above the absolute gravitropic ules; the situation is reversed in stage 4 (with sporangium; threshold, and thus above the thermal noise (see above). Schimek et al. 1999b). In stage 1 sporangiophores, the low- 16 6 Energy of about 10 J could be sufficient to open 10 density lipid globules (0.79 gcm 3) are clustered in a ‘special 2+ mechanosensitive Ca channels (Howard et al. 1988). Such organelle’, a complex of lipid globules (CLG) that resides considerations appear relevant, particularly in view of the 100–200 lm below the apex (Fig. 1, left and centre). The CLG observation that gravireception of the sporangiophore corre- is not surrounded by a membrane, but rather represents an lates with specific ion fluxes in the growing zone (Zivanovic aggregate of lipid globules that are loosely held together in a 2005, 2012). cage made of an actin mesh (Grolig et al. 2014; this volume). Sporangiophores lacking the CLG display a greatly reduced gravitropic response (Grolig et al. 2004). The sedimentation of Gravisusceptors of Phycomyces the octahedral crystals and the buoyancy of the lipid globules The search for fungal gravisusceptors, although a century old, generate a potential energy that exceeds the thermal noise by has only very recently received novel input. Unfortunately, the three to four orders of magnitude (Schimek et al. 1999a; Grolig gravisusceptors of Phycomyces went unnoticed for decades even et al. 2004). The unusually large lipid globules (diameter though it was clear that they must be cell inclusions, as evident 1–4 lm; Grolig et al. 2004) are in continual non-Brownian by the observation that submerged sporangiophores retain motion; the requisite motor molecules are presently unknown, their negative gravitropic bending (Dennison 1961; Galland but the fact that they occur within a dense actin mesh cage et al. 2007). Submerging sporangiophores eliminates ‘flexure’ indicates that they are powered by an actomyosin system (Gro- caused by the weight of the sporangiophore, and particularly lig et al. 2014; this volume). Electron microscopy shows that the sporangium. That flexure present in horizontally placed the globules have different degrees of electron density. Only the

‘pale’ globules of lower electron density can be covered with (Polaino Orts et al. 2013) that confers a hypergravitropic phe- hexagonal matrices of proteins (Thornton 1968), which have notype (Gottig€ & Galland 2014; this volume). The tips of sub- been identified as ferritin with a density of 1.82 gcm 3 (Peat & merged sporangiophores display strong outwardly directed ion Banbury 1968; David & Easterbrook 1971). Because of the large currents at pH 7. Inhibitor studies indicated the presence of an size of the lipid globules the heavy ferritin arrays cannot affect electrogenic H+-ATPase in the plasma membrane, a result of their buoyancy. The lipid globules also contain b-carotene and biological relevance because the growth rate of submerged thus appear deep yellow (Fig. 1, left). In addition, they carry sporangiophores correlates with the membrane potential and pterin- and flavin-like pigments that emit blue and green fluo- the outward current density (Zivanovi c et al. 2001). Gravitrop- rescence upon excitation (Ogorodnikova et al. 2002; Grolig ic stimulation at the tips of sporangiophores elicit Ca2+ and H+ et al. 2004). Micro-irradiation of the lipid globules elicits light fluxes near the surface of sporangiophores (Zivanovi c 2005). In growth responses that are larger than those after irradiation of the apical growth region, vertically oriented stage 1 sporangio- cytoplasmic areas without globules, an observation providing phores display characteristic inwardly directed fluxes of Ca2+ information about the photoreceptor localisation (Ogorodnik- and H+; in the subapical region, however, the currents were ova et al. 2002). bidirectional. The ion fluxes correlate with gravitropic bending Oil droplets are widespread in fungi, and it appears likely because the flux directions are altered after gravitropic stimula- that gravireception mediated by buoyancy might represent a tion. The observed reactions must be specific for gravitropism mechanism that is widespread in the fungal kingdom (Grolig because they were altered in a gravitropism mutant with a et al. 2006). Such a scenario would make perfect sense, because defective madJ gene (Zivanovi c 2012). The combined results fungi mainly utilise lipids for energy storage, while plants use suggest that growth and gravitropic curvature require partici- starch. The redesign of storage organelles for gravisusception pation of the cytoskeleton, and entail a redistribution of Ca2+ thus emerges as a convergent phylogenetic trend that was inde- and calmodulin as well as protons. pendently realised in fungi and plants. Upon reorientation of the sporangiophore, the slowly ensuing bending response of Phycomyces is preceded by very fast molecular events that can be monitored spectroscopically. A gravitropic Cytoskeleton, ion fluxes and gravity-induced absorbance stimulus elicits so-called gravity-induced absorbance changes changes (GIACs) (GIACs) that occur within milliseconds. These events are spe- The paramount role of the actin cytoskeleton in the gravitro- cific for early events of the transduction chain because they are pism of plants (e.g. Pozhvanov et al. 2013) seems also to be altered in a gravitropism mutant of genotype madJ (Schmidt & realised in growth and gravitropism of the Phycomyces Galland 2000, 2004; Schmidt 2006). The fact that GIACs occur sporangiophore, in which actin, myosin, spectrin and integrin not only in Phycomyces sporangiophores but also in seedlings of have been detected after immunolabelling (Doucette et al. Arabidopsis and coleoptiles of Zea mays supports the notion that 1994). Injection of stage 4 sporangiophores with cytochalasin they are also common in plants and that they represent suitable D (inhibitor of actin polymerisation) causes a 4-h delay in tools to spectroscopically dissect their primary responses of gravitropic bending, whereas rhodamin-phalloidin (inhibitor gravireception (Schmidt 2010, 2011, 2012). of actin depolymerisation) enhances the gravitropic bending rate, and sometimes also the bending angle (Edwards et al. Gravitropism mutants 1997). The apical lipid globules of stage 1 sporangiophores (Fig. 1) are encased within a dense mesh of actin filaments that Strains with defective gravitropism were obtained in several fills the dome-like structure of the apex (Grolig et al. 2014; this independent screens for photropism mutants. These so-called volume), a property that makes it a good candidate for a mad mutants (named after Max Delbruck)€ possess abnormal tensegrity-mediated mechanism (Ingber 2003; Yamaoka et al. tropisms in response to light, gravity and chemical stimuli 2012) of gravireception. The advantages of a tensegrity mecha- (Bergman et al. 1973; Alvarez et al. 1983, 1989; Campuzano nism, i.e. its capacity to integrate over a number of diverse et al. 1994). The gravitropism mutants of Phycomyces are thus stimuli, such as differences in local ion concentrations, meta- by-products of the long-term project to genetically dissect the bolic activities or number of statoliths, has been pointed out by light transduction pathway. Mutants of Phycomyces with several authors (e.g. Hasenstein 2009). defects in the genes madD, E, F, G and J are gravitropically par- Gadolinium chloride, an inhibitor of plant gravitropism and tially defective. They are highly pleiotropic, because they show stretch-activated ion channels, delays the onset of gravitropism damped light growth responses, reduced phototropism and an and reduces gravitropic curvature. Asymmetric application of avoidance response (Bergman et al. 1973). Surprisingly, even gadolinium, Ca2+ chelators or compound 48/80 (inhibiting the phototropic action spectra are affected in this class of calmodulin) to the growing zone of the sporangiophore elicits mutant. The observation was unexpected because the gravitro- curvature toward the side to which the inhibitors were applied pism mutants had been interpreted as ‘late’ mutants that affect (Stecker et al. 1990; Edwards 1991). Sporangiophores that are the output of the transduction chain shared by gravi- and kept in Ca2+ buffer display substantially enhanced gravitropism phototropism (Campuzano et al. 1996). The various complex at 10 4 MCa2+; interestingly, the effect ensues only in mutants features indicate, however, that (i) the photoreceptor system is that lack the vacuolar protein crystals (Galland et al. 2007). affected in these gravitropism mutants, (ii) photo- and gravi- The membrane potential inside the growing zone of sub- perception interact at early steps of the transduction chain, and merged sporangiophores is about 131 mV at pH 7 (Zivanovi c (iii) the madD, E, F and G gene products interact with those of et al. 2001). Removal of Ca2+ ions elicited hyperpolarisation in the madA, B and C gene products. the wild type but depolarisation in a madC mutant, from which Another class of mutants with defects in the gene madH we now know that it encodes a Ras GTPase activating protein show enhanced gravitropic and phototropic bending, and also

62 Plant Biology 16 (Suppl. 1) (2014) 58–68 © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands Galland Gravireception of Phycomyces blakesleeanus an enhanced avoidance response (Lipson et al. 1983; Lopez- A B Dıaz & Lipson 1983). Similarly, the geo-10 mutation also confers enhanced gravitropism, which manifests in a shorter latency and elevated differential bending rates (Ootaki et al. 1991, 1995; Grolig et al. 2000). Recently it was found that the madC mutation also confers hypergravitropism (Gottig€ & Galland 2014; this volume). In the past, the madC gene had been associated exclusively with photoreception, because the mutation confers night blindness (106-fold increased phototropic threshold) and also abnormal phototropic action spectra C (Galland & Lipson 1985). The recent discovery that madC codes for a Ras GTPase-activating protein (Polaino Orts et al. 2013) indicates that (i) the complex phenotype of madC mutants is caused by elevation of a small G-protein in its activated state (GTP bound) and (ii) the small G-protein is an element that integrates gravi- and photo-responses. It is noteworthy that a number of ergosterol-deﬁcient mutants are unaffected in gravitropism even though ergosterol and its derivatives are thought to affect membranes, i.e. the sites where presumed elements of the gravitropic transduction chain reside (Barrero et al. 2002). Sporangiophores of mutants lacking the vacuolar protein crystals bend less effectively than the wild type (Fig. 2A; Schimek et al. 1999a,b) and also have a raised gravitropic threshold, as determined on a clinostat centrifuge (Galland et al. 2004). The fact they still retain residual gravitropism (Fig. 2A) shows that the protein crystals are not absolutely necessary for gravitropism.

INTERACTION BETWEEN LIGHT AND GRAVITROPISM In nature, gravitropism operates to a large extent in the presence of light, while phototropism practically never occurs with- Fig. 3. A: Standard phototropism experiment. Unilateral collimated light impinges perpendicularly on the sporangiophore. Within about 8 h, a photo- out interference of gravitropism, it can act independently of gravitropic equilibrium angle a is established. B: Experimental set up for phototropism. It is thus essential to study the two tropisms determining the fluence rate Icomp that exactly compensates a given gravi- independently of each other and subsequently clarify their tropic stimulus S (equation 1) of a tilted sporangiophore. At time zero the mode of interaction. sporangiophore and the light source are tilted by an inclination angle c.At the compensating fluence rate the sporangiophore does not bend, i.e. the Photogravitropic equilibrium and light growth response gravitropic and the phototropic stimuli neutralise each other. The optical conditions do not change at Icomp, i.e. the light is always impinging perpen- When exposed for 6–8 h to unilateral light (Fig. 3A), sporan- dicularly on the sporangiophore. C: The relative compensating fluence rates giophores bend in response to near-UV and blue light of flu- as a function of the inclination angle of the sporangiophores irradiated for 9 2 2 ence rates between 10 and 10 W m (Bergman et al. 1969; 8 h. The relative compensating fluence rate is defined as Icomp/I0, where I0 is Galland & Lipson 1987a,b; Campuzano et al. 1996). In such the threshold fluence rate obtained from phototropism experiments done as long-term irradiation experiments, the sporangiophores reach in Fig. 3A. Open symbols: Mutant C2 geo-3 lacking vacuolar protein crystals a photogravitropic equilibrium, in which the bending angles (statoliths). The smooth curve is a fit to equation 6, with kk = 0.56. Filled are determined by the fluence rate and by gravity. The corre- symbols: wild type C171 containing vacuolar protein crystals. Data replotted sponding stimulus–response curves are modelled as exponen- from Galland et al. (2004). tial rising functions. The contribution of gravity can be easily seen when the fluence rate–response curves for photogravitrop- madJ) have been physiologically characterised (reviewed in ic equilibrium are generated in the presence of clinostatisation, Galland 2001) and genetically mapped (Alvarez et al. 1992). which causes an increase in the slope of these curves and also Apart from the photoreceptor genes madA and madB, all other an increase in the maximum bending angles at saturating mad genes have some effect on gravitropism. In the future, a irradiances (Grolig et al. 2000). major role will probably be played by madC, which until very The flavin-containing blue light photoreceptor eliciting recently was exclusively understood as an early phototropism phototropism is a molecular complex of MADA (similar to the mutant conferring a 106-fold elevated threshold. It turned out, zinc-finger protein WC-1 (white collar) of Neurospora; Idnurm however, that madC is in addition hypergravitropic (Gottig€ & et al. 2006) and MADB (similar to the zinc-finger protein Galland 2014; this volume). This discovery happily coincided WC-2 of Neurospora; Sanz et al. 2009). The MADA–MADB with the finding that madC encodes a Ras GTPase-activating complex acts as a light-regulated transcription factor eliciting protein (Polaino Orts et al. 2013; see below). phototropism and other responses pertaining to photo-differ- When dark-adapted sporangiophores are irradiated with entiation. The numerous phototropism mutants (madA to unilateral light they respond not only with bending (Fig. 3A)

Plant Biology 16 (Suppl. 1) (2014) 58–68 © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands 63 Gravireception of Phycomyces blakesleeanus Galland but also with a transient acceleration of the growth rate, which The fact that such unrelated organs as sporangiophores, subsides within 40 min and which does not interfere with the coleoptiles and hypocotyls have acquired via different phyloge- phototropic bending (Galland et al. 1985). Usually, light netic routes the same algorithm enabling them to balance grav- growth responses, however, were studied with symmetrically ity and light stimuli indicates that adherence to the exponential irradiated sporangiophores (Foster & Lipson 1973; Russo law provides an intrinsic advantage for spatial orientation, and 1980). Typically, the growth rate increases after a latency of thus for survival. 3–4 min and may display a transient deceleration before the Gravitropic bending is substantially affected by symmetric normal growth rate is resumed. A step-down or pulse-down of (tonic) irradiation (Dennison 1964). The dependence on irra- the fluence rate elicits a dark growth response, i.e. a transient diance and wavelength is complex and has not been studied decrease in growth rate. The duration depends on the exact pre- extensively. Surprisingly, red light, which is phototropically stimulus and stimulus conditions. As a rule, the response ceases ineffective, accelerates gravitropism, although the underlying after 30–40 min (Foster & Lipson 1973; Russo 1980). Because photoreceptor is currently unknown (Galland et al. 2007). turgor remains constant during a light growth response, the growth rate changes must be explained in terms of altered cell PHYCOMYCES IN THE CONTEXT OF FUNGAL AND wall mechanical properties (Ortega et al. 1988a,b) and chitin PLANT GRAVIRESEARCH synthase activity (Herrera-Estrella & Ruiz-Herrera 1983). Pres- ently, it remains unclear whether or not a strong gravistimulus The major focus during the past decades on molecular is likewise able to elicit a transient growth response. approaches may have partially obscured the realisation that quite a few basic problems pertaining to classical physiology of gravireception have remained unaddressed and are presently Exponential law – quantitative interaction between gravi- and far from being resolved. In the given historical context, Phyc- phototropism omyces may be instrumental in highlighting such unresolved When sporangiophores are unilaterally irradiated for several questions and help in catalysing appropriate solutions. hours, as shown in Fig. 3A, a photogravitropic equilibrium 1 One of the most pressing questions in fungal gravitropism angle is established that results from two antagonistic research is rooted in the very fact that fungi possess the phy- responses, i.e. phototropism and gravitropism (Varju et al. tohormone auxin, indole-3-acetic acid. It may thus come as 1961; Ootaki et al. 1991). Thus, the organism requires an algo- a surprise to learn that we still do not know whether fungi rithm that allows it to calculate the fluence rate required to require auxin(s) for tropic bending in a similar way to balance the gravitropic stimulus: would the relationship plants. It is probably related to the small size of the research between gravi- and photostimulus be linear or rather exponen- community dedicated to fungal gravitropism that this most tial? The standard phototropism experiment (Fig. 3A) does not obvious question has remained unanswered, or even worse, help to fully solve this problem because the effective fluence remained largely ignored. Phycomyces, alas, has not helped rate at the photoreceptor site changes dynamically during in the past to improve this lamentable state, even though bending because of changes in optical properties (e.g. reflection the routes of biosynthesis of auxin, its precursors and the losses, path lengths). It is thus essential to set up an experiment requisite enzymes were elucidated for Phycomyces during in such a way that optics of the irradiation do not change. This the 1960s to 1990s (Hilgenberg 1965; Hofmann & Hilgen- is achieved with a set up in which the sporangiophore and also berg 1978; Hilgenberg et al. 1980; Schramm et al. 1987a,b). the light source are tilted relative to the vertical by a variable As in plants, auxin biosynthesis starts in Phycomyces with inclination angle c (Fig. 3B). One then needs to determine the tryptophan and proceeds via tryptamine and indole-3- fluence rate Icomp that exactly compensates the gravitropic acetaldehyde to auxin (Ludwig-Muller€ et al. 1990, 1991). It stimulus, which is given by the sine law (equation 1). When is noteworthy that auxin appears during development with the fluence rate is too high, positive phototropism prevails and the initiation of sporangiophores (Hilgenberg 1965), which the sporangiophore bends downward, when the fluence rate is contain an about ten times higher concentration than too low, negative gravitropism prevails and the sporangiophore mycelia (Hilgenberg et al. 1980). Despite the elevated auxin bends upward (Fig. 3B). The irradiance required to compen- concentration in sporangiophores (6 nmolg 1 DW; Hof- sate for the ensuing gravitropic response is described in a novel mann & Hilgenberg 1982), it remains a not fully resolved exponential law that holds for Phycomyces (Grolig et al. 2000; question whether or not it plays a role in gravi- or photot- Galland et al. 2004), Avena coleoptiles (Galland 2002) and also ropism. Early attempts to demonstrate a possible role for hypocotyls of Arabidopsis (Feil & Galland, unpublished): this important hormone in tropic bending through unilateral application of auxin droplets or pastes were unsuccess- Icomp ¼ I0expðkk g sin cÞð6Þ ful (Banbury 1952; Maas 1958; Thimann & Gruen 1960), and have not since been repeated. There is nevertheless sub- where Icomp is the irradiance of the unilateral light that com- stantial evidence for the existence of apical growth-promot- pensates the gravitropic response elicited at an inclination ing substances that are stored in the spore mass of the 9 2 angle c,I0 the absolute threshold irradiance (10 Wm , sporangium (Fig. 1, right) and that are required for the 1 2 450 nm), kk (m s ) is a wavelength-dependent constant, g maintenance of elongation growth. When the sporangium terrestrial gravitational acceleration, and c inclination angle of is removed from a stage 4 sporangiophore, growth ceases the sporangiophore. immediately and resumes when the sporangium or a mass The exponential law states that the fluence rate that compen- of spores is replaced on the columella, i.e. the internal struc- sates a gravitropic stimulus needs to be raised exponentially ture that carries the sporangium (Goodell 1971). Even if the when the gravitropic stimulus (g sin c) rises linearly (Fig. 3C). growth-promoting substances were unrelated to auxin, it

would still be very worthwhile to identify them and test major key elements of gravireception and graviresponses whether they are involved in gravi- or phototropism. (Bastien et al. 2013). The fact that one needs to distinguish 2 The classical concept that statoliths act through the capabil- between the slow, non-adaptive gravitropic bending ity to exert a pressure force on a yet unidentified gravirecep- response (‘normal’ gravitropism, Fig. 2A) and the fast, tran- tor has rarely been questioned, and represents the current sient adaptive gravitropic response triggered by mechanical state of thinking. One model proposed by Braun and associ- load or acceleration changes (Fig. 2B) encourages a read- ates on the basis of investigations of rhizoids of Chara justment of current gravitropism models by taking into presents a quite unconventional view (Limbach et al. 2005). account this dual nature of gravistimulation. It is very likely In this model, the statoliths (crystals of BaSO4) do not act that both graviresponses operate in nature, and it is thus through physical contact and pressure but rather through pertinent to elucidate their interrelationship and their indi- the release of diffusible molecules that they contain. Stato- vidual contribution to spatial orientation in still as well as lith sedimentation would thus represent a prerequisite to in turbulent environments, which would generate changing build up a gradient of effector molecules. We postulated a (rhythmic) stimuli. similar mode of action for the vacuolar protein crystals of Apart from the above considerations there is a second Phycomyces sporangiophores that are associated with pterin- strong motivation to study gravitropism with changing and flavin-like molecules (Eibel et al. 2000). stimuli. This motivation derives from the realisation that 3 The actin cytoskeleton of the sporangiophore of Phycomyces the gravitropism literature does not, alas, provide the slight- stands out among those of other fungi in that its size est insight on the fundamental parameter ‘discrimination surpasses that of other fungal hyphae and of plant cells by threshold’, a classical parameter in the field of sensory phys- several orders of magnitude (Grolig et al. 2014; this iology. While this parameter was recognised early in photot- volume). The growing zone of stage 1 sporangiophores ropism research, it has never appeared to play a role in possesses different types of actin filaments (filaments, graviresearch. Spatial orientation with respect to light strands and cables), a situation that offers the possibility to requires an innate ability to evaluate and react to differences investigate, in the context of tensegrity models (Ingber of opposing light stimuli. For phototropism, the spatial or 2003), the functions of the different actin filaments and even temporal difference thresholds are 1–5% (reviewed in their requisite roles in contributing to bending. Studies of Galland 1989). A corresponding question has never been basidiomycete fungi such as Flammulina (Kern 1999) and raised in the area of graviresearch in fungi and plants. The Coprinus have indicated a prominent role for the actin cyto- graviphysiology of the past and present has typically relied skeleton in gravitropism, particularly in combination with on experimental designs in which statoliths exert pressure nuclei as potential statoliths and redistribution of microvac- on only one side of an inclined organ, such that inside a uoles (Moore et al. 1996) and microvesicles (Kern et al. single cell, e.g. a sporangiophore, the stimulus difference 1997) after gravistimulation. It appears necessary to investi- between the lower and upper sides becomes maximal. In gate in greater detail whether the nuclei of Phycomyces, which nature the situation can, however, be far more complex. are embedded in the apical actin network (Grolig et al. Consider, for example, tilted fungal or plant organs that are 2014), could similarly contribute to gravitropic bending. exposed to wind and that are thus subjected to rhythmic 4 Even though physiologists have been aware for a long time motion. In such situations statoliths, and also flexure, that flexure and wall stress both evoke bending responses would act in a complex manner on opposing sides of the (see above), these phenomena have not always been consid- graviresponsive organs. It would, therefore, be worthwhile ered to be intrinsic parts of gravitropism. Mainstream to test, for example, whether the time-honoured sine law gravitropism research was largely centred on the statolith– would hold even under such dynamic stimulus conditions receptor paradigm, and wall stress was viewed at best as an that are more akin to the natural environment than epi-phenomenon. As a result, gravitropism experiments ‘wind-free’ experiments done under laboratory conditions. were generally done in ‘undisturbed’ environments so as to 5 An important topic of classical graviresearch concerns the avoid wall stress elicited by wind or mechanical movement. so-called ‘resultant law’ of gravitropism (see above). An With the benefit of hindsight, the self-imposed restriction inspection of the older literature shows, however, that the on the statolith paradigm is actually hard to comprehend, data basis supporting this law is quite small and thus some- because in nature gravitropic organisms are continually what questionable. Our own on-going investigations with exposed to internal (statolith) as well as external (flexure, sporangiophores of Phycomyces (Gottig€ & Galland 2014, this wall stress) stimuli. The experiment of Dennison (see volume), coleoptiles of Avena and hypocotyls and roots of above) could serve as a guidepost for future fungal gravitro- seedlings of Arabidopsis show that the law holds reasonably pism studies that scrutinise more closely the relationship well only for coleoptiles and hypocotyls (Dummer€ & between statolith- and wall stress-mediated bending. Also Galland, in preparation), not, however, for Phycomyces spo- relevant in this context is the finding that some of the rangiophores and roots of Arabidopsis seedlings. The very so-called ‘stiff” mutants of Phycomyces with reduced gravi- apparent violations of the resultant law are at variance with tropic bending possess an increased turgor pressure but the strict adherence of the sine law; they indicate instead reduced rates of cell wall deformation (Ortega et al. 2012). that the gravistimuli acting longitudinally (parallel to the An increased focus on the nanomechanics would very likely longitudinal axis) are of paramount importance for gravi- lead to integrative gravitropism concepts that have already tropic bending. At first glance, the resultant law might been better explored for plants (reviewed in Baluska & appear of somewhat academic interest, because centrifugal Volkmann 2011). Such integrative concepts should include accelerations may be thought absent in nature, a view that phenotypic plasticity (Niklas 2009) and proprioreception as is very misleading. Even superficial observation of natural

environments shows that fungi and plants are continually gravitropism experiments that exploit fast gravi-induced exposed to wind, and as a consequence, the terrestrial absorption changes of Zea coleoptiles have taken up and organs are continually subjected to swaying, rhythmic reinforced this train of thought (Schmidt 2012). The inner motions that generate centrifugal acceleration. To simulate and the outer cell walls (and the associated gravireceptors) such motions under controlled laboratory conditions is that are parallel to the longitudinal axis should thus possess technically feasible and would fill a gap in knowledge in the different sensitivities to the gravisusceptors, a conclusion classical physiology of gravitropism. that again reinforces the demand for investigations that 6 Last, but not least, I like to consider a question that, regret- clarify the question of the difference threshold of gravitro- tably, does not enjoy full attention from the modern reader- pism. As a single-celled organism, the sporangiophore of ship, and of which the research community has lost sight. Phycomyces can be used as a model to determine the differ- Tropisms require differential stimulation at the flanks of the ence threshold for a situation in which opposing cell walls bending organ. In plant phototropism, the opaqueness of and their associated gravireceptors must possess equal the organ provides the differential stimulus by generating gravisensitivity. The phylogenetic progression from uni- to an internal light gradient; in Phycomyces, the differential multicellularity, e.g. stipe of macromycete fungi or hypoco- stimulation is achieved through the lens effect of the trans- tyls, cannot be simply explained as the additive action of parent sporangiophore. In the case of gravitropism of numerous such single-celled systems. One rather needs to multicellular organs, e.g. horizontal coleoptiles or hypoco- rely on models that provide integrative functions that are tyls, the situation is more complex, if not a true paradox, beyond the scope of the single-celled sporangiophore of because the gravisusceptors of the lower and upper flanks Phycomyces. should actually generate identical gravistimuli. To explain the gravitropism of such organs, early investigators thus ACKNOWLEDGEMENT rightly concluded that there must be a physiological asym- metry of the outer and inner cell walls of the gravisensitive I thank Dr. Franz Grolig and Dr. Irmin Meyer for providing cells (Rawitscher 1932 and literature therein). Recent the photographs shown in Fig. 1.

REFERENCES Bergman K., Burke P.W., Cerda-Olmedo E., David Dennison D.S. (1964) The effect of light on the geotro- C.N., Delbruck€ M., Foster K.W., Goodell E.W., Hei- pic responses of Phycomyces sporangiophore. Journal Alvarez M.I., Ootaki T., Eslava A.P. (1983) Mutants of senberg M., Meissner G., Zalokar M., Dennison of General Physiology, 47, 651–665. Phycomyces with abnormal phototropism induced D.S., Shropshire W. Jr (1969) Phycomyces. Bacterio- Dennison D.S., Roth C.C. (1967) Phycomyces sporan- by ICR-170. Molecular and General Genetics, 191, logical Reviews, 33,99–157. giophores: fungal stretch receptors. Science, 156, 507–511. Bergman K., Eslava A.P., Cerda-Olmedo E. (1973) 1386–1388. Alvarez M.I., Eslava A.P., Lipson E.D. (1989) Phototro- Mutants of Phycomyces with abnormal phototro- Dennison D.S., Shropshire W. Jr (1984) The gravirecep- pism mutants of Phycomyces blakesleeanus isolated at pism. Molecular and General Genetics, 123,1–16. tor of Phycomyces: its development following gravity low light intensity. Experimental Mycology, 13,38–48. Campuzano V., Galland P., Senger H., Alvarez M.I., exposure. Journal of General Physiology, 84, 845–859. Alvarez M.I., Benito E.P., Campuzano V., Eslava A.P. Eslava A.P. (1994) Isolation and characterization of Doucette A., Voorthuis R., Havrilla M. (1994) Cyto- (1992) Genetic loci of Phycomyces blakesleeanus. In: phototropism mutants of Phycomyces insensitive to skeletal and associated components and calcium O’Brien S.J. (Ed.), Genetic maps. Locus maps of ultraviolet light. Current Genetics, 26,49–53. binding proteins of Phycomyces gravity sensitive complex genomes, 6th edition. Cold Spring Harbor Campuzano V., Galland P., Alvarez M.I., Eslava A.P. sporangiophores. American Society for Gravitational Laboratory Press, Cold Spring Harbor, NY, USA, (1996) Blue-light receptor requirement for gravitro- and Space Biology, 8,82–88. pp 3.120–3.126. pism, autochemotropism and ethylene response in Edwards K.L. (1991) The gravitational response of Baluska F., Volkmann D. (2011) Mechanical aspects of Phycomyces. Photchemistry and Photobiology, 63, Phycomyces with respect to gadolinium and factors gravity-controlled growth, development and mor- 686–694. affecting calcium function. NASA Space/Gravita- phogenesis. In: Wojtaszek P. (Ed.), Mechanical inte- Chaudhary S., Polaino S., Shakya V.P.S., Idnurm A. tional Biology Accomplishments 1989–1990. NASA gration of plant cells and plants. Springer, Berlin, (2013) A new genetic linkage map of the zygomycete Technical Memorandum, 4258,32–33. Germany, pp 195–223. fungus Phycomyces blakesleeanus. PLoS ONE, 8, Edwards K.L., Chew J., Harris S., di Muzio E. (1997) Banbury G.H. (1952) Physiological studies in the e58931. Effects of microinjected cytoskeletal inhibitors on Mucorales. Part II. Some observations on growth Cohen R., Delbruck€ M. (1958) Distribution of stretch the gravitropic response of the Phycomyces sporan- regulation in the sporangiophore of Phycomyces. and twist along the growing zone of the sporangio- giophore. American Society for Gravitational and Journal of Experimental Botany, 3,86–94. phore of Phycomyces and the distribution of Space Biology Bulletin, 11, 47. Banbury G.H. (1959) Phototropisms of lower plants. response to a periodic illumination programme. Eibel P., Schimek C., Fries V., Grolig F., Schapat T., In: Ruhland W. (Ed.) Encyclopedia of plant physiol- Journal of Cellular and Comparative Physiology, 52, Schmidt W., Schneckenburger H., Ootaki T., Gal- ogy, physiology of movements, Vol. 17/1. Springer, 361–388. land P. (2000) Statoliths in Phycomyces: characteriza- Berlin, Germany, pp 530–578. Corrochano L.M. (2007) Fungal photoreceptors: sen- tion of octahedral protein crystals. Fungal Genetics Banbury G.H. (1962) Geotropism of lower plants. In: sory molecules for fungal development and behav- and Biology, 29, 211–220. Ruhland W. (Ed.) Encyclopedia of plant physiology, iour. Photochemical and Photobiological Sciences, 6, Ensminger P.A., Lipson E.D. (1992) Growth rate ﬂuc- physiology of movements, Vol. 17/2. Springer, Berlin, 725–736. tuations in Phycomyces sporangiophores. Plant Phys- Germany, pp 344–377. Corrochano L.M., Galland P. (2006) Photomorpho- iology, 99, 1376–1380. Barrero A.F., Oltra J.E., Robinson J., Burke P.V., genesis and gravitropism in fungi. In: Kues U., Foster K.W., Lipson E.D. (1973) The light growth Jimenez D., Oliver E. (2002) Sterols in erg mutants Fischer R. (Eds), The Mycota I, growth differentiation response of Phycomyces. Journal of General Physiol- of Phycomyces: metabolic pathways and physiological and sexuality. Springer, Heidelberg, Germany, pp ogy, 62, 590–617. effects. Steroids, 67, 403–409. 231–257. Fries V., Krockert T., Grolig F., Galland P. (2002) Bastien R., Bohr T., Moulia B., Douady S. (2013) Uni- David C.H., Easterbrook K. (1971) Ferritin in the fun- Statoliths in Phycomyces: spectroﬂuorometric char- fying models of shoot gravitropism reveals proprio- gus Phycomyces. Journal of Cell Biology, 48,15–28. acterization of octahedral protein crystals. Journal of ception as a central feature of posture control in Dennison D.S. (1961) Tropic responses of Phycomyces Plant Physiology, 159,39–47. plants. Proceedings of the National Academy of sporangiophores to gravitational and centrifugal Fukshansky L. (1993) Intracellular processing of a Sciences USA, 110, 755–760. stimuli. Journal of General Physiology, 45,23–38. spatially non-uniform stimulus: case study of

phototropism in Phycomyces. Journal of Photochemis- omyces blakesleeanus and synthesis of chitin microfi- fungus Phycomyces blakesleeanus Bgff Life Science try and Photobiology B: Biology, 19, 161–186. brils. Experimental Mycology, 6, 385–388. Advances. Plant Physiology, 10,45–49. Galland P. (1989) Photosensory adaptation in plants. Herrera-Estrella L., Ruiz-Herrera J. (1983) Light Maas W. (1958) Zur Frage einer Beteiligung von Indol- Botanica Acta, 102,11–20. response in Phycomyces blakesleeanus: evidence from ylessigs€aure beim Wachstum und beim Phototropis- Galland P. (1990) Phototropism of the Phycomyces roles in chitin biosynthesis and breakdown. Experi- mus von Phycomyces. Archives of Microbiology, 30, sporangiophore: a comparison with higher plants. mental Mycology, 7,362–369. 73–90. Photochemistry and Photobiology, 52, 233–248. Hilgenberg W. (1965) Wuchsstoffe aus verschiedenen Markl H. (1982) Geo-Biophsik: mediendruck, Galland P. (2001) Phototropism in Phycomyces. In: Entwicklungsstadien von Phycomyces blakesleeanus Schwerefeld, Magnetfeld und Organismen. In: H€ader D.P., Lebert M. (Eds), Photomovement. Com- (Bgff.). Beitr€age der Biologie der Pflanze, 41,69– Hoppe W., Lohmann W., Markl H., Ziegler H. prehensive series in photosciences, Vol. 1. Elsevier, 122. (Eds), Biophysik. Springer, Berlin, Germany, pp Amsterdam, the Netherlands., pp 621–657. Hilgenberg W., Sandmann G., Hofmann F. (1980) The 805–816. Galland P. (2002) Tropisms of Avena coleoptiles: sine influence of indole-3-acetic acid on the growth of Metzner P. (1929) Uber€ die Wirkung der L€angskraft law for gravitropism, exponential law for photo- Phycomyces blakesleeanus and its quantitative deter- beim Geotropismus. Jahrbuch der wissenschaftlichen gravitropic equilibrium. Planta, 215, 779–784. mination in the fungus. Journal of Plant Physiology, Botanik, 71, 325–385. Galland P., Lipson E.D. (1985) Modified action spectra 97,89–94. Moore D., Hock B., Greening J.P., Kern V.D., Frazer of photogeotropic equilibrium in Phycomyces blakes- Hofmann F., Hilgenberg W. (1978) Aminooxidasen in L.N., Monzer J. (1996) Gravimorphogenesis in aga- leeanus with defects in genes madA, madB, madC, Phycomyces blakesleeanus und ihre Bedeutung fur€ die rics. Mycological Research, 100, 257–273. and madH. Photochemistry and Photobiology, 41, Indol-3-Essigs€aurebiosynthese des Pilzes. Berichte Niklas K.J. (2009) Functional adaptation and pheno- 331–335. der Deutschen Botanischen Gesellschaft, 91, 611–622. typic plasticity at the cellular and whole plant level. Galland P., Lipson E.D. (1987a) Light physiology of Hofmann F., Hilgenberg W. (1982) On the role of try- Journal of Biosciences, 34, 613–620. Phycomyces. In: Cerda-Olmedo E., Lipson E. D. ptophol in Phycomyces. Plant Physiology(supple- Ogorodnikova U., Sineshchekov O., Galland P. (2002) (Eds), Phycomyces. Cold Spring Harbour Laboratory ment), 69, Abstract 63. Blue-light perception in stage-1 sporangiophores of Press, Cold Spring Harbour, NY, USA, pp 49–92. Horie T., Schimek C., Fukui J., Miyazaki A., Mihara Phycomyces: a role for apical lipid globules? Journal Galland P., Lipson E.D. (1987b) Blue-light reception H., Tsuru T., Maki K., Ootaki T. (1998) Develop- of Plant Physiology, 159, 205–209. im Phycomyces phototropism: evidence for two mental stage-dependent response of Pilobolus crystal- Oort A.J.P. (1931) The spiral-growth of Phycomyces. photosystems operating in low- and high-intensity linus sporangiophores to gravitative and centrifugal Verhandelingen der koninklijke Akademie van ranges. Proceedings of the National Academy of stimulation. Mycoscience, 39, 463–470. Wetenschapen Amsterdam, 34,564–575. Sciences USA, 84, 104–108. Howard J., Roberts W.M., Hudspeth A.J. (1988) Ootaki T., Wolken J.J. (1973) Octahedral crystals in Galland P., Palit A., Lipson E.D. (1985) Phycomyces: Mechanoelectrical transduction by hair cells. Annual Phycomyces II. Journal of Cell Biology, 57, 278–288. phototropism and light-growth response to pulse Reviews of Biophysical Chemistry, 17,99–124. Ootaki T., Ishikawa N., Miyazaki A., Tsuru T. (1991) stimuli. Planta, 165, 538–547. Idnurm A., Rodrıguez-Romero J., Corrochano L.M., The determination of the maximal bending angle in Galland P., Wallacher Y., Finger H., Hannappel M., Sanz C., Itturiaga E., Eslava A.P., Heitman J. (2006) Phycomyces sporangiophores. Experimental Mycol- Troster€ S., Bold E., Grolig F. (2002) Tropisms in The Phycomyces madA gene encodes a blue-light ogy, 15, 336–345. Phycomyces: sine law for gravitropism, exponential photoreceptor for phototropism and other light Ootaki T., Ito K., Abe M., Lazarova G., Miyazaki A., law for photogravitropic equilibrium. Planta, 214, responses. Proceedings of the National Academy of Tsuru T. (1995) Parameters governing gravitropic 931–938. Sciences USA, 103, 4546–4551. response of sporangiophores in Phycomyces blakes- Galland P., Finger H., Wallacher Y. (2004) Gravitro- Ingber D.E. (2003) Tensegrity II. How structural net- leeanus. Mycoscience, 36, 263–270. pism in Phycomyces: threshold determination on a works influence cellular information processing Ortega J.K.E., Keanini R.G., Manica K.J. (1988a) Pres- clinostat centrifuge. Journal of Plant Physiology, 161, networks. Journal of Cell Science, 116, 1397–1408. sure probe technique to study transpiration in Phyc- 733–739. Kern V.D. (1999) Gravitropism of basidiomycetous omyces sporangiophores. Plant Physiology, 87,11–14. Galland P., Fries V., Grolig F., Schmidt W. (2007) fungi – on earth and in microgravity. Advances in Ortega J.K.E., Manica K.J., Keanini R.G. (1988b) Phyc- Gravireception in Phycomyces blakesleeanus. Space Research, 24, 697–706. omyces: turgor pressure behavior during the light Advances in Space Research, 39, 1134–1139. Kern V.D., Hock B. (1996) Gravitropismus bei Pilzen. and avoidance growth response. Photochemistry and Goodell E.W. (1971) ‘Apical dominance’ in the sporan- Naturwissenschaftliche Rundschau, 49, 174–180. Photobiology, 48, 697–703. giophore of the fungus Phycomyces. Planta, 98, Kern V.D., Mentgen K., Hock B. (1997) Flammulina as Ortega J.K.E., Bell S.A., Erazo A.J. (1992) Pressure 63–75. a model system for fungal graviresponses. Planta, clamp method to measure transpiration in growing Gottig€ M., Galland P. (2014) Gravitropism in Phyc- 203, S23–S32. single plant cells. Demonstration with sporangio- omyces: violation of the so-called resultant law – Larsen P. (1962) Orthogeotropism in roots. In: Ruh- phores of Phycomyces. Plant Physiology, 100, 1036– evidence for two response components. Plant Biol- land W. (Ed.) Encyclopaedia of plant physiology, Vol. 1041. ogy, 16 (Suppl. 1), 158–166. 17. Springer, Berlin, Germany, pp 153–199. Ortega J.K.E., Munoz C.M., Blakley S.E., Truong J.T., Grolig F., Eibel P., Schimek C., Schapat T., Dennison Larsen P. (1969) The optimum angle of geotropic stim- Ortega E.L. (2012) Stiff mutant genes of Phycomyces D.S., Galland P. (2000) Interaction between photot- ulation and its relation to the starch statolith affect turgor pressure and wall mechanical properties ropism and gravitropism in the sporangiophore of hypothesis. Physiologia Plantarum, 22,469–488. to regulate elongation growth rate. Frontiers in Plant Phycomyces. Plant Physiology, 123, 765–776. Limbach C., Hauslage J., Sch€afer C., Braun M. (2005) Science, 3, 99. Grolig F., Herkenrath H., Pumm T., Gross A., Galland How to activate a plant gravireceptor: early mecha- Parfyonov G.P., Platanova R.N., Tairbekov M.G., Bele- P. (2004) Gravity susception by buoyancy: floating nisms of gravity sensing studied in Charean rhizoids nev Y.N., Olkhovenko V.P., Rostopshina A.V., lipid globules in sporangiophores of Phycomyces. during parabola flights. Plant Physiology, 139, 1030– Oigenblick E.A. (1979) Biological investigations Planta, 218, 658–667. 1040. aboard biosatellite Cosmos-782. Acta Astronautica, Grolig F., Doring€ M., Galland P. (2006) Gravisuscep- Lipson E.D., Lopez-D ıaz I., Pollock J.A. (1983) 6, 1235–1238. tion by buoyancy: a mechanism ubiquitous among Mutants of Phycomyces with enhanced tropism. Peat A., Banbury G.H. (1968) Occurrence of ferritin- fungi? Protoplasma, 229, 117–123. Experimental Mycology, 7, 241–252. like particles in a fungus. Planta, 79, 268–270. Grolig F., Moch J., Schneider A., Galland P. (2014) Lopez-D ıaz I., Lipson E.D. (1983) Genetic analysis of Polaino Orts S., Chaudhary S., Shakya V., Miralles- Actin cytoskeleton and organelle movement in the hypertropic mutants of Phycomyces. Molecular and Duran A., Corrochano L., Idnurm A. (2013) Map- sporangiophore of the zygomycete Phycomyces bla- General Genetics, 190, 318–325. based identification of the mad photosensing genes kesleeanus. Plant Biology, 16 (Suppl. 1), 167–178. Ludwig-Muller€ J., Schramm P., Hilgenberg W. (1990) of Phycomyces blakesleeanus. Fungal Genetics Reports, Hasenstein K.H. (2009) Plant responses to gravity – Indole-3-acetaldehyde reductase in Phycomyces 60 (Suppl.), Abstract # 211. insights and extrapolations from ground studies. blakesleeanus. Characterization of the enzyme. Physi- Pozhvanov G.A., Suslov D.V., Medvedev S.S. (2013) Gravitational and Space Biology, 22,21–32. ologia Plantarum, 80, 472–478. Actin cytoskeleton rearrangements during the Herrea-Estrella L., Chavez B., Ruiz-Herrera J. (1982) Ludwig-Muller€ J., Schramm P., Hilgenberg W. (1991) gravitropic response of Arabidopsis roots. Cell and Presence of chitosomes in the cytoplasm of Phyc- Indole-3-acetaldehyde dehydrogenase activity in the Tissue Biology, 7, 185–191.

Rawitscher F. (1932) Der Geotropismus der Pflanzen. Bremen (FRG). Microgravity Science and Technology, phore. American Society for Gravitational and Space Gustav Fischer, Jena, Germany. 22,79–85. Biology, 4, 98. Russo V.E.A. (1980) Sensory transduction in Schmidt W. (2011) Gravireception in Phycomyces Thimann K.V., Gruen H.E. (1960) The growth and phototropism: genetic and physiological analysis in blakesleeanus and Arabidopsis thaliana: hysteretic curvature of Phycomyces sporangiophores. Beiheft zu Phycomyces. In: Lenci F., Colombetti G. (Eds), behaviour of primary reactions. Microgravity Science den Zeitschriften des Schweizerischen Forstvereins, 30, Photoreception and sensory transduction in aneural and Technology, 23,355–364. 237–263. organisms. Plenum Press, New York, USA, pp 373– Schmidt W. (2012) Differential gravity induced Thornton R.M. (1968) The fine structure of Phycomy- 395. absorption changes in coleoptiles of Zea mays as ces. II. Organization of the stage I sporangiophore Sachs J. (1879) Uber€ Ausschließung der geotropischen measured with the single wavelength space discrimi- apex. Protoplasma, 66, 269–285. und heliotropischen Krummungen€ w€ahrend des nator (SWSD). Microgravity Science and Technology, Thornton R.M. (1969) Crystalloids of Phycomyces spo- Wachsens. Arbeiten des Botanischen Instituts in 24, 103–112. rangiophores: nature and photosensitive accumula- Wurzburg€ , 2,209–225. Schmidt W., Galland P. (2000) Gravity-induced absor- tion. Plant Physiology, 44, 861–865. Sachs J. (1882) Uber€ orthotrope und plagiotrope bance changes in Phycomyces: a novel method for Varju D., Edgar L., Delbruck€ M. (1961) Interplay Pflanzenteile. Arbeiten des Botanischen Instituts in detecting primary reactions of gravitropism. Planta, between the reactions to light and to gravity in Phyc- Wurzburg€ , 2,226–284. 210, 848–852. omyces. Journal of General Physiology, 45,47–58. Sanz C., Rodrıguez-Romero J., Idnurm A., Christie Schmidt W., Galland P. (2004) Optospectro- Wolken J.J. (1969) Microspectrophotometry and the J.M., Heitman J., Corrochano L.M., Eslava A.P. scopic detection of primary reactions associated photoreceptor of Phycomyces I. Journal of Cell (2009) Phycomyces MADB interacts with MADA to with the graviperception of Phycomyces: effects of Biology, 13, 354–360. form the primary photoreceptor complex for fungal micro- and hypergravity. Plant Physiology, 135, Wolken J.J. (1972) Phycomyces: a model photosensory phototropism. Proceedings of the National Academy 183–192. cell. International Journal of Neuroscience, 3, 135–146. of Sciences USA, 106, 7095–7100. Schramm P., Rausch T., Hilgenberg W. (1987a) Yamaoka H., Matsushita S., Shimada Y., Adachi T. Schimek C., Eibel P., Grolig F., Horie T., Ootaki T., Indole-3-ethanol oxidase in Phycomyces blakeslee- (2012) Multiscale modelling and mechanics of Galland P. (1999a) Gravitropism in Phycomyces:a anus. Is indole-3-ethanol a ‘storage pool’ for IAA? filamentous actin cytoskeleton. Biomechanics and role for sedimenting protein crystals and for floating Physiologia Plantarum, 69,99–104. Modeling in Mechanobiology, 11, 291–302. lipid globules. Planta, 210, 132–142. Schramm P., Rausch T., Hilgenberg W. (1987b) Zivanovi c B. (2005) Ca2+ and H+ ion fluxes near the Schimek C., Eibel P., Horie T., Galland P., Ootaki T. Indole-3-ethanol oxidase in Phycomyces blakeslee- surface of gravitropically stimulated Phycomyces (1999b) Protein crystals in Phycomyces sporangio- anus Bgff Characterization of the enzyme. Plant sporangiophore. Annals of the New York Academy of phores are involved in graviperception. Advances in Physiology, 84, 541–544. Sciences, 1048,487–493. Space Research, 24, 687–696. Shropshire W. Jr (1971) Phototropic bending rate in Zivanovi c B. (2012) Surface tip-to-base Ca2+ and H+ Schmidt W. (2006) Gravity-induced absorption Phycomyces as a function of average growth rate and ionic fluxes are involved in apical growth and gravi- changes in Phycomyces blakesleeanus during para- cell radius. In: Broda E. (Ed.), Biophysics of cells and perception of the Phycomyces stage I sporangio- bolic flights: first spectral approach in the visible. organs. Proceedings of 1st European biophysics con- phore. Planta, 236, 1817–1829. Protoplasma, 229, 125–131. gress. Medizinische Akademie, Vienna, Austria. Vol. Zivanovi c B., Kohler€ K., Galland P., Weisenseel M. Schmidt W. (2010) Gravity induced absorption 5, pp 111. (2001) Membrane potential and endogenous ion changes in Phycomyces blakesleeanus and coleoptiles Stecker T., Joiner W., Edwards K.L. (1990) Inhibitors current of Phycomyces sporangiophores. Electro- and of Zea mays as measured on the drop tower in affecting gravitropism of the Phycomyces sporangio- Magnetobiology, 20, 343–362.