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Surface tip-to-base Ca2+ and H+ ionic

fluxes are involved in apical growth and


graviperception of the Phycomyces stage I
sporangiophore

Branka D. Živanović

Planta
An International Journal of Plant
Biology

ISSN 0032-0935

Planta
DOI 10.1007/s00425-012-1738-3

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DOI 10.1007/s00425-012-1738-3

ORIGINAL ARTICLE

Surface tip-to-base Ca2+ and H+ ionic fluxes are involved in apical


growth and graviperception of the Phycomyces stage I
sporangiophore
Branka D. Živanović

Received: 9 July 2012 / Accepted: 3 August 2012


Ó Springer-Verlag 2012

Abstract Net fluxes of Ca2? and H? ions were measured Keywords Gravitropic bending  Growth rate 
non-invasively close to the surface of Phycomyces bla- Ion-selective microelectrodes  Ion fluxes  Tip growth
kesleeanus sporangiophores stage I using ion-selective
vibrating microelectrodes. The measurements were per- Abbreviations
formed on a wild type (Wt) and a gravitropic mutant A909 APW Artificial pond water
kept in either vertical or tilted orientation. Microelectrodes ASET Automated Scanning Electrode Techniques
were positioned 4 lm from the surface of sporangiophore, A909 Gravitropically defective mutant
and ion fluxes were recorded from the apical (0–20 lm) Wt Wild type
and subapical (50–100 lm) regions. The magnitude and
direction of ionic fluxes measured were dependent on the
distance from the tip along the growing zone of sporan-
giophore. Vertically oriented sporangiophores displayed Introduction
characteristic tip-to-base ion fluxes patterns. Ca2? and H?
fluxes recorded from apical region of Wt sporangiophores Although a considerable bulk of experimental evidence
were inward-directed, while ion fluxes from subapical demonstrates that fungal hyphae possess a general feature to
locations occurred in both directions. In contrast to Wt, obey the rules of apical growth, the underlying mechanisms
mutant A909 showed opposite (outward) direction of Ca2? that control this process are poorly understood. This apical
fluxes and reduced H? influxes in the apical region. Fol- growth is an example of extremely polarized form of cell
lowing gravistimulation, the magnitude and direction of growth that establishes a narrow tubular extension of the cell.
ionic fluxes were altered. Wt sporangiophore exhibited The filamentous cell morphology is also present in algae and
oppositely directed fluxes on the lower (influx) and the higher plants (e.g., rhizoids and protonemata in algae; pollen
upper (efflux) sides of the cell, while mutant A909 did not tubes and root hairs in higher plants) (for an overview, see
show such patterns. A variable elongation growth in ver- Geitmann et al. 2001). Tip growth of the fungal hyphae is a
tical position and reduced growth rate upon gravistimula- multicomponent dynamical process that requires coordi-
tion were observed in both strains. The data show that nated activities of the endomembrane and cytoskeletal sys-
tip-growing sporangiophores exhibit a tip-to-base ion flux tems to target growth materials to be inserted into the plasma
pattern which changes characteristically upon gravistimu- membrane and cell wall of the hyphal apex (Heath 1990,
lation in Wt in contrast to the mutant A909 with a strongly 1995; Bartnicki-Garcı́a 2002). Sporangiophores are verti-
reduced gravitropic response. cally oriented aerial vegetative reproductive structures of
unicellular zygomycete fungus Phycomyces blakesleeanus.
These vertically growing unbranched hyphae are able to
B. D. Živanović (&) respond to light, wind, barriers, touch, and gravity (Bergman
Department for Life Sciences, Institute for Multidisciplinary
et al. 1969). Zonal distribution of organelles within the
Research, University of Belgrade, Kneza Višeslava 1,
11030 Belgrade, Serbia growing region of a sporangiophore was earlier found by
e-mail: vunduk@imsi.rs means of light and electron microscopy (Thornton 1968).

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From the apex to the subapical region, five zones were growth due to cell wall flexure (Horie et al. 1998; Schimek
observed in that growing region of the sporangiophore, et al. 1999a, b; Eibel et al. 2000; Grolig et al. 2004). Ionic
which are not separated by membranes: the most apical zone fluxes measured close to the surface of intact sporangio-
(rich of mitochondria and dense bodies), a zone with several phores argue in favor of their involvement in the gravisen-
hundred nuclei, an exclusion zone in the ovoid region with a sing processes (Živanović 2005). Sporangiophores of Wt
cluster of lipid globules, a peripheral zone rich in nuclei, strain display negative gravitropic bending in stage I in the
mitochondria, dense bodies, and autophagic vesicles, and a air. There is only a single reference that states that the spo-
zone with swollen autophagic vesicles just above the central rangiophore of mutant A909 is affected in gravitropism
vacuole (Thornton 1968). Phycomyces sporangiophores also (Campuzano et al. 1994). Mutant A909 is not completely
exhibit a typical apical growth localized in a small region at agravitropic, but exhibits reduced graviresponse compared
the hyphal apex which depends on a complex interaction of with Wt strain. When Wt and mutant A909 sporangiophores
different physiological processes. Fusion of secretory vesi- were placed horizontally in the air for 12 h, Wt bended 85°,
cles (chitosomes) and microvesicles with the plasma mem- while the mutant A909 bended 25° (Campuzano et al. 1994).
brane (Ruiz-Herrera et al. 2003), Ca2? ionic gradients Sporangiophores are also capable of growth (25 lm min-1
directly or indirectly associated with cytoskeleton (Jackson for Wt) and display a clear gravitropic response in liquid
and Heath 1993), transcellular electrical- and ionic currents media, as previously reported (Stifler 1961; Živanović 2005;
(Živanović et al. 2001; Živanović 2005), and coupled ion Galland et al. 2007). The experiments were done with
transport processes across the plasma membrane (Mogus and solutions that had been thoroughly aerated before the
Wolken 1974; Groves and Gamow 1975; Weiss and Wei- experiments such that oxygen did not become limiting
senseel 1990; Živanović et al. 2001) are all observed during (Živanović 2005; Galland et al. 2007). The data from vertical
apical growth of Phycomyces sporangiophores. The under- or gravistimulated growth of the gravitropic mutant A909 in
lying mechanisms that control this polarized growth remain, liquid medium is still incomplete. This feature of sporan-
however, elusive. Almost all reports on taxonomically giophores growing in liquid medium was employed for
diverse fungi, like Oomycetes, Zygomycetes, Ascomycetes, various electrophysiological investigations on membrane
and Basidiomycetes considered hyphal apical growth as transport processes that drive sporangiophore growth
pulsed with sustained oscillations and periodicity of 4–20 s (Živanović et al. 2001; Živić et al. 2005, 2009).
(López-Franco et al. 1994). However, in some reports, the It is well known that cell wall and plasma membrane are
absence of such pulsative hyphal growth was noted, where the sites where the earliest events take place during apical
the growth rates appeared to fluctuate randomly (Money growth and gravisensing of sporangiophores. Therefore,
2001; Sampson et al. 2003). It is likely that pulsed growth focusing on the mechanisms of ion transport processes at the
displays periodic physiological changes taking place during plasma membrane and in the apoplastic space of Phycomyces
the tip growth. It might be assumed that this pulsed growth sporangiophores in relation to the growth, graviperception,
originates from periodic discharges of the secretory vesicles and their signal transduction chains, could be of importance.
exocytotically carrying material and enzymes for cell wall Various electrophysiological methods, such as intracellular
synthesis implicating the possibility of discontinuous cell microelectrodes (Živanović et al. 2001), non-invasive
wall growth. extracellular 3D vibrating probe (Živanović et al. 2001) and
Castle (1940) was the first to have observed this pulsed Ca2? and H? ion-selective microelectrodes (Živanović
growth on Phycomyces during elongation of sporangio- 2005) were used to study apoplastic and plasma membrane
phores. One of the earliest reports that attempted to model compartments involved in elongation growth and graviper-
tip-growth as a non-homogeneous process was from the ception. The negative correlation was observed between the
nineteenth century (Reinhardt 1892). In spite of the com- growth rate of Phycomyces sporangiophores in non-buffered
plexity of this process, growth velocity of tip-growing cells medium, the magnitude of membrane potential, as well as an
is among the fastest in biological systems. outward current (Živanović et al. 2001). Moreover, the main
Phycomyces sporangiophore is also an excellent unicel- ions creating trans-cellular currents in ascomycete Neuros-
lular fungal model cell for investigating the mecha- pora were previously assumed to be protons or calcium ions,
nism(s) that drive gravity sensing, because gravisensing and and their influxes are necessary for the creation of conditions
graviresponse take place within the same cell. Thus, the that allow the hyphal protoplasm to extend (McGillivray and
gravitropic signal transduction chain is not additionally Gow 1986; Takeuchi et al. 1988). These trans-cellular cur-
complicated by signal transmission between sensing and rents originated from different spatial distribution of ion
responding cells. Three main gravireceptor mechanisms are channels at the apex and the proton-ATPase in the subapical
well established in immature sporangiophore cells (stage I), region of the cells (Harold and Caldwell 1990). The exis-
such as the buoyancy of lipid globules, sedimenting vacuolar tence of ion gradients in this tip-growing system has been
octahedral protein crystals (statoliths), and upward bending proposed as a general mechanism for establishment and

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maintenance of cell polarity (Reiss and Herth 1979; Schie- Regional Research Laboratory (USDA, Peoria, IL, USA),
felbein et al. 1992; Silverman-Gavrila and Lew 2001; Al- and photo-gravitropically defective mutant A909 madJ(–)
cántara-Sánchez et al. 2004). Ca2? ions have been proposed (Bergman et al. 1973; Campuzano et al. 1994, 1995, 1996)
to regulate and coordinate many of the processes involved in were used in this study. Phycomyces mutant A909 (genotype
hyphal tip growth (Jackson and Heath 1993). It is still unclear madJ) is a photo-gravitropic mutant near-UV/blue-light
how Ca2? ions are tip focused. It has been reported that in photoreceptor system of which is affected. Phycomyces
Neurospora membrane ionic fluxes do not play a role in the mutant A909 is not an agravitropic strain, but displays
tip growth and tip-high Ca2? gradient is generated from defective gravitropism and avoidance response, whereas it is
intracellular stores via InsP3-gated channel located in the normal in photocarotenogenesis. It is not clear whether the
plasma membrane and endoplasmatic reticulum (Silverman- A909 mutant is a double mutant with one mutation affecting
Gavrila and Lew 2001). However, in the fungus Saprolegnia the photoreceptor, and the other affecting the output of the
ferax, tip growth is associated with Ca2? influx at the apical transduction chain of gravitropism and avoidance response
plasma membrane (Lew 1999) through stretch-activated (Campuzano et al. 1994).
Ca2?-permeable channels and via an actin-dependent The experiments were carried out with intact Phycomyces
mechanism (Garrill et al. 1993). In spite of the observed Wt strain and A909 mutant grown in small glass vials, as
surface tip-to-base Ca2? and H? ion flux fluxes across the previously described (Živanović et al. 2001; Živanović
Phycomyces sporangiophore, no clear relationship was 2005). In brief, small glass vials (1-cm diameter and 3.5-cm
detected between the direction of ion fluxes and the exten- height) were filled with PDA medium (Potato Dextrose Agar,
sion growth in vertical or tilted position (Živanović 2005). Life Technologies, Paisley, Scotland) enriched with ca-
Gravitropic stimulation of Phycomyces sporangiophores seinhydrolysate (15 g mL-1, Merck, Darmstadt, Germany)
generated bending growth and different ion flux patterns on up to 2.5 cm. Each vial was inoculated with 20 lL of diluted
the upper and lower sides of the tilted cell (Živanović 2005). spore suspension containing 5–10 spores that had been heat-
A better understanding of transport processes at the shocked for 10 min at 49 °C. The vials were covered with a
sporangiophore plasma membrane was provided by patch- piece of perforated parafilm, placed in transparent plastic
clamp studies of Ca2? and K? ion channels in the Phyc- boxes, and stored in a growth cabinet with continuous
omyces plasma membrane (Edwards 1991; Mitiku and overhead white fluorescent light of 10 W m-2, at a tem-
Edwards 1991; Garrill et al. 1993). The existence of two K? perature of 21 °C and ca. 95 % relative humidity. Sporan-
channels (43 pS and 74 pS) and one ORAC (Outwardly giophores emerged from the holes in parafilm 2–2.5 days
Rectifying Anionic Channel) in plasma membranes of after inoculation, and single sporangiophore was used for ion
cytoplasmic droplets isolated from the growing zone of flux measurements at the time of reaching stage I.
Phycomyces implicates a possible functional role of these
ionic channels (Živić et al. 2005, 2009), together with Ion flux measurements
recorded H? and Ca2? ion fluxes (Živanović 2005), in pro-
cesses that drive apical growth of sporangiophore. Glass shell vial with sporangiophore was submerged into a
The aim of this study was to explore whether surface tip- measuring chamber filled with 15 mL of Artificial Pond
to-base Ca2? and H? ionic fluxes are involved in apical Water (APW: 1.0 mM NaCl, 0.1 mM KCl, and 0.1 mM
growth and graviperception of the Phycomyces stage I CaCl2, non-buffered, pH 6.0). APW was exposed to
sporangiophore of Wt and gravitropic mutant A909. The intensive aeration overnight and adjusted to pH 6.0 before
data suggest that these ionic fluxes might be required for use. A measuring chamber was designed to enable easy
elongation growth of vertically oriented Wt sporangio- positioning of sporangiophore in either vertical or tilted
phore, and also for its gravistimulated bending growth. orientation. The position of sporangiophore in relation to
Gravitropic mutant A909 exhibited inconsistent relation- microelectrode was adjusted using a microscope under cool
ship between surface ionic fluxes and growth both in ver- white light provided by the light guide (KL 1500 LCD,
tical and tilted sporangiophore orientation. Schott, Mainz, Germany) from 150 W halogen lamp
(175 W m-2, Phillips) and a video camera. Images of
sporangiophores were obtained and recorded every 5 min
Materials and methods to observe the growth rate of sporangiophore. Net fluxes of
Ca2? and H? ions were measured non-invasively using
Strains and growth conditions ion-selective microelectrodes, as previously described
(Arend et al. 2004; Živanović 2005). The electrode blanks
The wild-type (Wt) strain of the UV- and blue-light-sen- were pulled with a vertical electrode puller (model PU2V;
sitive fungus Phycomyces blakesleeanus (Burgeff) APS GmbH, Bad Homburg, Germany) from borosilicate
NRRL1555(–) originally obtained from the Northern glass capillaries (1.5–1.8 mm outer diameter; Kimble Glass

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Inc., Vineland, NJ, USA), dried overnight in an oven, and concentration difference (concentration calculated from
silanized with 100 lL N,N-Dimethyl-trimethylsilylamine measured voltages) observed at these two positions of
(Fluka 41720). Cooled microelectrodes were backfilled microelectrode using Fick’s first law of diffusion. All
with solutions (50 mM KCl ? 25 mM K2HPO4 ? 25 mM measurements were performed in a Faraday cage resting on
KH2PO4 for H?; 100 mM CaCl2 for Ca2?). Commercially a heavy, vibration-damped table with sporangiophores
available ionophore cocktails (H? 95297; Ca2? 21048, submersed in APW.
Fluka, Taufkirchen, Germany) were used for filling the
electrode tips. The base of the micropipette was inserted Measurement of sporangiophore growth
into an electrolyte-filled Ag/AgCl pellet electrode holder
(World Precision Instruments, Berlin, Germany). Ion- Growing sporangiophores were recorded with a video-
selective microelectrodes were calibrated in a set of stan- imagining system. Digital images of sporangiophores were
dard solutions before use. The prepared microelectrodes taken every 5 min, stored on hard drive and analyzed for
were mounted on three-dimensional motorized microma- growth by PC package ‘‘Image tool’’. Elongation growth of
nipulator (computerized motion control system CMC4; both strains was monitored during ion flux measurements
Applicable Electronics, Forestdale, MA, USA). The refer- both in vertical and tilted orientation. Elongation growth
ence electrode consisted of an Ag/AgCl electrode inserted was expressed as growth rate in lm per min. Ca2? and H?
into a 1 mL Eppendorf pipette tip filled with 100 mM KCl ion fluxes were tested for correlation in PC package Excel
and plugged with a ceramic stopper to minimize leakage of (Microsoft).
the electrolyte while allowing electrical contact with the
bathing solution. Signals from the electrodes were mea- Data analysis
sured by a high impedance amplifier (model IP-2; Appli-
cable Electronics) and fed to a computer. The position and Initial calculations of ion fluxes were performed in PC
movement of electrodes were controlled by ASET software package Excel (Microsoft). The ion fluxes obtained from
(Automated Scanning Electrode Techniques, Science individual sporangiophores were averaged. Data are pre-
Wares, East Falmouth, MA, USA). The electrodes were sented as mean ± SE and tested for significance by using
placed 4 lm from the surface of sporangiophores and Student’s t test.
moved between 4 lm (position 1) and 14 lm (position 2)
from the sporangiophore surface by a computer-controlled
stepper motor at a sampling rate of 1,000 samples s-1 Results
during measurements. One measurement cycle lasted 4.5 s.
Sporangiophore was viewed from the front through a Ca2? and H? fluxes from vertical and tilted
horizontal microscope with attached CCD video camera sporangiophores
(TELI, Tokyo, Japan), which was connected to a TV
monitor. The electrode position was constantly adjusted to Net Ca2? and H? ion fluxes were recorded at different
maintain approximately the same distance from the grow- positions (apex, 20–100 lm from the apex) 4 lm from the
ing apex. Net ion fluxes were recorded at different posi- surface of intact Phycomyces sporangiophores (stage I)
tions along the growing zone of Phycomyces submerged in electrophysiological solution (APW) and kept
sporangiophore starting from the apex, and subsequently to in vertical or tilted (45°) orientation. Extracellular Ca2? and
20, 50, and 100 lm from the apex kept in vertical- or tilted H? ionic fluxes were recorded at the tips of individual spo-
orientation by 45°. Custom-made chamber enabled easy rangiophores of Wt and gravitropic mutant A909. Direction
tilting by 45°, and the position of microelectrode was of ionic fluxes indicated a net influx of Ca2? and H? ions into
adjusted to the appropriate measuring point 4 lm from the apical part of growing sporangiophores (Figs. 1a, 4a,
surface of sporangiophore while being observed with a 6d).The fluxes were of opposite (outward) direction near the
microscope. The data acquisition was stopped for 5 min slow-growing sporangiophores (Fig. 6e). As ion flux mea-
during sporangiophore displacement from vertical position surements were performed on living sporangiophores, they
and then resumed again. For each position (apex, 20, 50, were passing through normal developmental processes and
and 100 lm) of ion-selective microelectrode along the exhibited variable pattern of ion fluxes and growth at dif-
sporangiophore, appropriate sample rules were chosen. The ferent developmental stages. The values and directions of
ion fluxes were recorded for 5 min, and in most cases, Ca2? and H? ion fluxes depended on sporangiophore
measurements were repeated 2–3 times on all screened developmental stage, distance from the tip along the surface
points. Data recorded by ASET program was saved as of sporangiophore, and orientation (vertical or tilted) of
ASCII file and imported into Excel program to calculate sporangiophore. Ca2? fluxes recorded from the apical region
the ion fluxes. Ion fluxes were calculated from (apex and 20 lm from the apex) of Wt sporangiophores were

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a 6 fluxes displayed high correlation with the distance from the


Wt
20-V tip both in Wt (r = 0.83) and mutant A909 (r = -0.82)
A909
4 sporangiophores. Ca2? fluxes were recorded initially from
50-V
Ca2+ flux (pmol·cm-2·s-1)

apex-V the vertical sporangiophore, followed by tilting; measure-


2 ments were then performed on tilted sporangiophore. The
100-V
direction and magnitude of Ca2? fluxes were altered fol-
0 lowing such a gravistimulation. Thus, Ca2? fluxes at the apex
changed direction to efflux or significantly reduced magni-
-2 tude of influx, and sporangiophore exhibited obvious oppo-
site direction of Ca2? fluxes on lower and upper side of tilted
-4 Wt sporangiophore (Figs. 1b, 2a–c). Ca2? fluxes recorded
from lower and upper sides of Wt sporangiophore (Fig. 2a–
-6
c) were significantly different and negatively correlated
(r = -0.52). Ca2? influxes recorded on lower side of Wt
b 6 sporangiophore corresponded to extending side of cell. The
upper side of Wt sporangiophore displayed outward direc-
4 tion of Ca2? fluxes (Figs. 1b, 2a–c). These opposite (‘‘up-
Ca2+ flux (pmol·cm-2·s-1)

50-down down’’) Ca2? fluxes are in an agreement with general pattern


apex-T
2 of differential growth of sporangiophore because of the
20-down
100-down
bending growth upon gravitropic stimulation. This distinct
0 ‘‘up-down’’ direction of Ca2? fluxes was not observed in
20-up 50-up 100-up
sporangiophores of the mutant A909 (Figs. 1b, 2d–f). Ca2?
-2 fluxes observed from lower and upper side of mutant A909
sporangiophore were not significantly different at P \ 0.05.
-4 Moreover, Ca2? fluxes exhibited oscillatory properties in
both strains of sporangiophore regardless of their orientation
-6 (Fig. 2a–f). The amplitude of oscillations of Ca2? fluxes
measured in the vertical oriented sporangiophores varied
Fig. 1 Average net Ca2? ion fluxes recorded at different positions
(apex, 20, 50, 100 lm from the apex) of Ca2?-selective microelec-
from 7.0 to 11.5 pmol cm-2 s-1 and 1.9–2.4 pmol
trodes near (4 lm) the surface of Phycomyces blakesleeanus sporan- cm-2 s-1 in Wt and mutant A909, respectively (Fig. 3a).
giophores of Wt and mutant A909 immersed in experimental Artificial The amplitudes of Ca2? oscillations in Wt increased
Pond Water (APW) solution (1 mM NaCl, 0.1 mM KCl and 0.1 mM (12–21 pmol cm-2 s-1), while in mutant A909 the ampli-
CaCl2, unbuffered, pH 6.0) kept in vertical orientation (a), and
subsequently tilted by 45° (b). The displacement of the experimental
tudes were not affected by the gravistimulation (Fig. 3b). H?
chamber with the sporangiophore from the vertical position was fluxes displayed inward direction from apical region (apex
performed manually, data acquisition being temporarily (for 5 min) and 20 lm from the apex) of Wt sporangiophores (Fig. 4a).
stopped during the repositioning of the microelectrode. Positive and In the case of subapical locations, outward (50 lm from the
negative values of ion fluxes (pmol cm-2 s-1) correspond to efflux
and influx, respectively; A909-gravitropical mutant; 20, 50, and 100
apex)- and inward (100 lm from the apex)-directed H?
marks denote distance from the apex in lm; up, down denotes upper, fluxes were recorded (Fig. 4a). Characteristic tip-to-base H?
lower surface of tilted sporangiophore; T—tilted position; V— ion flux patterns were detected near Wt sporangiophores,
vertical position; Wt—wild-type strain. Data are mean values ± SE while mutant A909 sporangiophores lacked consistent fluxes
of seven (Wt) and five (A909) different sporangiophores
(Fig. 4a).Upon gravitropical stimulation, the magnitudes
and direction of H? fluxes were significantly altered in both
Wt and mutant A909 sporangiophores, but some differences
inward-directed (negative, influxes), while from subapical were observed between the two strains (Fig. 4b). H? fluxes
locations (50–100 lm from the apex) outward-directed were significantly changed and their opposite (‘‘up-down’’)
(positive, effluxes) (Fig. 1a). In contrast to Wt sporangio- distribution was detected in Wt strain, when compared to the
phores, A909 mutant showed an opposite direction of Ca2? readings of the vertical orientation (Figs. 4b, 5a–c), Thus,
fluxes along the sporangiophores and from all but one H? influxes were reduced 74 % in Wt, while in mutant A909
(100 lm from the apex) locations Ca2? fluxes were outward- H? fluxes changed direction to the efflux upon gravitropic
directed (Fig. 1a).The magnitude of Ca2? fluxes was stimulation, and fluxes recorded near the opposite sides of
dependent on the distance from the tip along the growing mutant A909 sporangiophore cell were not significantly
zone of sporangiophore kept in vertical orientation. Ca2? different at P \ 0.05 (Figs. 4b, 5d–f).

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a 40 d 6
20 up 20 up

Ca2+ flux (pmol·cm-2·s-1)


Ca2+ flux (pmol·cm-2·s-1)

20 down 20 down
20 4

0 2

-20 0

-40 -2

-60 -4
0 1 2 3 4 5 0 1 2 3 4 5

b 50 e 6
50 up 50 up
Ca2+ flux (pmol·cm-2·s-1)

Ca2+ flux (pmol·cm-2·s-1)


50 down 50 down
30 4

10 2

-10 0

-30 -2

-50 -4
0 1 2 3 4 5 0 1 2 3 4 5

c 40 f 6
100 up 100 up
Ca2+ flux (pmol·cm-2·s-1)

Ca2+ flux (pmol·cm-2·s-1)

100 down 100 down


20 4

0 2

-20 0

-40 -2

-60 -4
0 1 2 3 4 5 0 1 2 3 4 5
Time (min) Time (min)

Fig. 2 Five-min tracings of original measurements of Ca2? ion fluxes 100 lm (f) kept in tilted orientation. Up, down denotes upper, lower
(pmol cm-2 s-1) recorded on three measuring positions of Ca2?- surface of the tilted sporangiophore. Opposite Ca2? fluxes were
selective microelectrodes along the surface of the Wt Phycomyces recorded from lower (influx) and upper (efflux) sides of Wt
sporangiophore, 20 lm from the apex (a), 50 lm (b), and 100 lm (c), sporangiophore cells (a–c), whereas such a difference was not
and the mutant A909, 20 lm from the apex (d), 50 lm (e), and observed in the case of the mutant A909 (d–f)

Relationship between growth and ion fluxes fast and slow rates in the form of cycles with more or less
regular intervals (Fig. 6a). Phycomyces sporangiophores
As measurements of ion fluxes by Ca2? and H? ion- stage I had the ability to grow longitudinally in the bathing
selective microelectrodes were non-invasive and performed solution, either in vertical or tilted position, but growth rate
on intact sporangiophores, it was also possible to monitor of vertically oriented sporangiophores was higher
growth dynamics of these cells simultaneously. During ion (11–13 lm min-1) as compared to tilted ones (5–7 lm
flux measurements living sporangiophores were passing min-1) in both strains (Fig. 6b). Growth rate of Wt spo-
through different developmental stages, which was moni- rangiophores was significantly different from the growth
tored under light microscope connected to a video imaging rate of mutant A909 ones, both in vertical and tilted ori-
system. Images were taken every 5 min and were subse- entation (Fig. 6b). The pattern of Ca2? and H? fluxes was
quently processed after experiments (Fig. 7). Sporangio- related to growth of sporangiophores. Thus, growing spo-
phores exhibited variable growth rate depending on rangiophores exhibited mainly influxes (negative) of these
developmental stage and sporangiophore orientation. Thus, ions in the apical region (Fig. 6d), while slow-growing
growth rate of sporangiophores fluctuated with periods of sporangiophores displayed their effluxes (positive) in the

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a 25 10
Wt
a Wt
A909 A909

20
Amplitude (pmol·cm-2·s-1)

H+ flux (pmol·cm-2·s-1)
0

15 50-V
-10
100-V
10

-20
5 apex-V

20-V
0 -30
apex-V 20-V 50-V 100-V
20
b
b 25

H+ flux (pmol·cm-2·s-1)
20 10
50-down
Amplitude (pmol·cm-2·s-1)

20-down
100-down
15
0

20-up
10 apex-T
-10
50-up

5
100-up
-20
0
apex-T 20 down 50 down 100 down Fig. 4 Average net H? ion fluxes (pmol cm-2 s-1) recorded at
different positions (apex, 20, 50, and 100 lm from the apex) of H?-
Fig. 3 Average amplitudes of one-min Ca2? ion flux oscillations selective microelectrodes near (4 lm) the surface of Phycomyces
(pmol cm-2 s-1) observed continuously during Ca2? ion flux mea- sporangiophores of Wt and mutant A909 kept in vertical orientation
surements performed at different positions of Ca2?-selective micro- (a), and subsequently tilted by 45° (b). Data are mean values ± SE of
electrodes along the surface of Phycomyces sporangiophores of Wt five (Wt) and eight (A909) different sporangiophores. For details, see
(gray bar, n = 5–9) and mutant A909 (open bar, n = 5–9) kept in Fig. 1
vertical orientation (apex-, 20, 50, 100V, a), and subsequently tilted
by 45° (apex-T, 20 down, 50 down, 100 down, b); down-lower side of
tilted sporangiophore. T, tilted position; V, vertical position; and
n number of single oscillations. Bars represent SE invasively during tip growth of Phycomyces sporangio-
phore. Tip-focused Ca2? gradients, directly or indirectly
same region of the cell (Fig. 6e). Upon vertical displace- associated with the cytoskeleton, are thought to be char-
ment, sporangiophores responded to gravistimulation by its acteristic of all tip-growing cells such as fungal hyphae,
intracellular reorganization and bending growth, because of algal rhizoids, pollen tubes, and root hairs (Jackson and
differential growth between lower and upper side of the Heath 1993; Leitz et al. 1995; Messerli et al. 1999; Braun
cell (Figs. 6a, 7). Gravistimulation of sporangiophores and Limbach 2006; Braun and Hemmersbach 2008;
induced intensive cytoplasmatic vacuolization just above Monshausen et al. 2008). The data presented in this study
the central vacuole (Fig. 6a). show that extracellular Ca2? and H? ionic fluxes detected
near the surface of intact Phycomyces Wt sporangiophores
are required for normal hyphae elongation in the vertical
Discussion position. A clear correlative relationship emerged between
direction of ion fluxes and elongation growth of sporan-
Tip-to-base ionic fluxes patterns and sporangiophore giophores (Fig. 6b–e). The direction of Ca2? and H? ions
growth is related to sporangiophore developmental stage, distance
from the sporangiophore tip (apical or subapical), and
In this study, ion-selective vibrating microelectrode sporangiophore orientation (vertical or tilted). In most
enabled us to perform measurements of ion fluxes non- cases, inward-directed Ca2? and H? ion fluxes were

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a 3 d
20 up 20 up
-1

H+ flux (pmol·cm-2·s-1)
20 down
H+ flux (pmol·cm-2·s-1)

20 down

1
-5

-1
-9

-3 -13
0 1 2 3 4 5 0 1 2 3 4 5

b 3 e
50 up 50 up
H+ flux (pmol·cm-2·s-1)

H+ flux (pmol·cm-2·s-1)
50 down 5 50 down

1
1

-1
-3

-3
-7
0 1 2 3 4 5
0 1 2 3 4 5

c 3 f
100 up 100 up
5
H+ flux (pmol·cm-2·s-1)

100 down 100 down


H+ flux (pmol·cm-2·s-1)

1
1

-1
-3

-3 -7
0 1 2 3 4 5 0 1 2 3 4 5
Time (min) Time (min)

Fig. 5 Five-minute traces of original measurements of H? ion fluxes down denotes the upper, lower surface of the tilted sporangiophore.
(pmol cm-2 s-1) recorded on three measuring positions along the Opposite directions of H? fluxes recorded from lower (influx) and
surface of the Phycomyces sporangiophore of Wt (20 lm from upper (efflux) side of Wt sporangiophore cell (a–c) were recorded,
the apex) (a), 50 lm (b), 100 lm (c), and mutant A909 (20 lm from whereas such a difference was not observed from mutant A909 one
the apex) (d), 50 lm (e), 100 lm (f) kept in tilted orientation; up, (d–f)

recorded at the tips (apical region) of growing sporangio- mutant A909 of Phycomyces as compared with Wt
phores (stage I) (Figs. 1a, 4a), while slow-growing spo- (Figs. 1a, 4a). Although tip growth is common and domi-
rangiophores (stage II) exhibited mostly outward-directed nant growth mechanism in almost all fungal organisms,
ionic fluxes (Fig. 6e). The magnitude of net Ca2? fluxes in some differences were observed between different fungal
Wt was highly correlated (r = 0.83) with the distance from groups. Saprolegnia ferax exhibited well-defined transport
the sporangiophore tip. This is in contrast to data previ- zones absent in Neurospora crassa (Levina et al. 1995;
ously reported on membrane potential which was inde- Lew 1999). Ca2? fluxes were located within 8 lm of the
pendent on microelectrode measuring position within the growing hyphal tip and the net Ca2? flux was either
growing zone of sporangiophore (Živanović et al. inward, driven by stretch-activated Ca2? channels, or out-
2001).The reason for this disparity might be in different ward by exocytosis (Lew 1999). We have recorded 75 and
compartments (apoplastic space and plasma membrane) 73 % of all measurements on Phycomyces sporangiophores
investigated. We recorded inconsistent and significantly as inward-directed Ca2? and H? fluxes, respectively.
different ionic fluxes in sporangiophores of gravitropic Interestingly, the direction of ionic fluxes was recorded as

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20 b Fig. 6 Average growth rate (a–c) and net Ca2? and H? ion fluxes (d,
a e) observed near the surface of intact sporangiophores kept in vertical
vertical (V) and tilted orientation (T). a Variable growth rate (lm min-1) of
Growth rate (µm·min-1)

15 Wt sporangiophores obtained from images taken every 5 min during


ion flux measurements. Sporangiophores were kept in vertical
position first for 30 min and then tilted from the vertical position
by 45° for 85 min. Inset denotes images of the sporangiophore in
10 vertical and tilted orientation. A number of small vacuole-like
structures was observed in the tilted sporangiophore. Data are mean
values ± SE of eight (Wt) different sporangiophores. Bar marker
5 tilted 100 lm. b Average growth rate (lm min-1) of Wt (gray bar) and
mutant A909 (open bar) sporangiophores in vertical (apex-V) and
tilted orientation (apex-T) observed on normal (b) and slow-growing
0 (c) sporangiophore. Growth rate of vertical sporangiophores was
0 20 40 60 80 100 120 significantly different from tilted ones, and the difference between Wt
Time (min) and mutant A909 was significant at P \ 0.05. d Average net Ca2?
and H? ion fluxes (pmol cm-2 s-1) recorded on vertical (Ca2?-V;
16 H?-V) and tilted (Ca2?-T; H?-T) normal (d) and slow-growing
b c Wt (e) sporangiophores. Data are mean values ± SE of five (Wt) and
normal growth
A909 seven (A909) different normal-growing sporangiophores, and three
slow-growing ones. Growth rate of vertical sporangiophores in both
Growth rate (µm·min-1)

12 strains was significantly different from tilted ones at P \ 0.05 both in


normal- and slow-growing sporangiophores (b, c)
slow growth
8
tip-localized inward Ca2? flux during growth of Sapro-
legnia ferax. Some short reports on Phycomyces patch-
4
clamp study detected stretch-activated Ca2? channels in the
growing zone of sporangiophore (Edwards 1991; Mitiku
and Edwards 1991). Moreover, maximal inward H? fluxes
0 were observed in apical region (20 lm from the apex) of
apex-V apex-T apex-V apex-T
Phycomyces sporangiophore similarly as Lew recorded
(1999) on Saprolegnia ferax (10–30 lm from the apex).
10
d normal growth e slow growth Lew (1999) assumed that the location of maximal H?
influxes corresponded to the highest density of mitochon-
dria at the onset of the vacuolization zone. We have also
Ion flux (pmol·cm-2·s-1)

0
Ca2+-V Ca2+-T H+-T observed an intensive vacuolization in the apical and sub-
Ca2+-T H+-V
apical regions of Phycomyces sporangiophores that could
2+
Ca -V +
H -T be associated with gravitropic response and/or with regular
-10
developmental processes occurring during measurements
(Fig. 6a). Beside the well-established gravireceptor-induced
alterations in immature sporangiophore cells (stage I), such
-20
H+-V as buoyancy of lipid globules, sedimenting of vacuolar
octahedral protein crystals and upward bending growth due
to the cell wall flexure (Horie et al. 1998; Schimek et al.
-30
1999a, b; Eibel et al. 2000), intensive cytoplasmatic vacuo-
lization could also be associated with gravisensing. Future
detailed ultrastructural analysis is needed to address this
variable from one to another set of measurements on the issue.
same sporangiophore passing throughout the normal Elongation growth of Phycomyces sporangiophore was
development. These data indicated complex and dynamic recorded as variable; this is in an agreement with previous
transport processes taking place during tip growth of the findings reporting the growth of sporangiophores as pul-
sporangiophore. Our data are in agreement with findings on sative (Castle 1940; Lafay and Matricon 1985). In this
Saprolegnia ferax (Lew 1999) where both inward- and study, growth rate was analyzed every 5 min, so it was not
outward net fluxes were observed in the growing hyphae, possible to recognize growth rate as pulsative. Direction
and both are specifically localized at the extreme tip. and magnitude of ion fluxes were related to the growth
Earlier reports on stretch-activated channels (Garrill et al. rate in Wt sporangiophores. Growing sporangiophores
1993; Levina et al. 1994) predicted an obligatory exhibited ionic influxes, while fluxes measured from

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H+ Ca2+
H+

Ca2+

H+
Ca2+

H+
Ca2+

4.04 pm 4.09 pm 4.19 pm 4.23 pm


Ca2+ Ca2+
H+ H+
Ca2+
H+
H+
Ca2+
Ca2+

H+

Ca2+ H+

H+
Ca2+

4.51 pm 5.28 pm 5.56 pm

Fig. 7 Original images of an intact wild-type strain Phycomyces indicate the direction of Ca2? and H? ion fluxes (outward-efflux as
sporangiophore (stage I) taken during measurements of net ion fluxes positive, and inward-influx as negative values) recorded at different
at different positions of microelectrode near the surface of the cell positions of microelectrodes near the surface of the cell. The length of
kept in vertical orientation (from 4.04–4.23 p.m.), and subsequently the arrows (scaled as indicated on Figs. 1, 4) is representative of the
tilted by 45° (from 4.51–5.56 p.m.). The numbers in the right corner magnitude of the fluxes. Bending growth of the sporangiophore apex
of images denote the real time when the images were taken. Arrows is visible on the last image. Bar marker 50 lm

slow-growing ones were outward-directed (Fig. 6b–e). The 1997; Monshausen et al. 2008). Growth rate of root hairs
gravitropic mutant of Phycomyces A909 displayed an oscillated at the same frequency as the apical cytoplasmatic
inconsistent relationship between ion fluxes and growth Ca2? levels, but growth preceded Ca2? increases about 5 s.
rate (Fig. 6b–e). The sporangiophore growth rate was sig- Further studies are needed to investigate the association
nificantly reduced upon gravistimulation in both strains between Ca2? oscillations and sporangiophore growth rate
(Fig. 6a, b). Significant reduction in sporangiophore in a higher temporal resolution.
growth rate upon vertical displacement might be a conse-
quence of both, developmental processes (transition to Ca2? and H? ion fluxes may participate in gravity
stage II) and upward growth. Previous findings on Phyc- signal transduction
omyces reported that tip-focused Ca2? fluxes are provided
from stretch-activated Ca2? channels and from internal Previous reports (Schmidt and Galland 2000, 2004;
stores by fusion of secretory vesicles (chitosomes) and Schmidt 2007) demonstrated that gravity-induced absorp-
microvesicles with the plasma membrane (Ruiz-Herrera tion changes occur rapidly and most likely represent the
et al. 2003). Therefore the net ion fluxes we observed could primary responses of gravireception in the fungus Phyc-
be a consequence of a number of distinct tip-localized omyces blakesleeanus. These absorption changes occur
plasma membrane transport processes, as also Lew (1999) very fast in comparison to the gravitropically induced
concluded for Saprolegnia ferax. Additional pharmaco- redistribution of protons, calcium, auxin and bending,
logical investigation are needed to determine the origin of which require minutes to hours. Altered ionic fluxes are the
Ca2? fluxes necessary for sporangiophore tip growth more next to absorption changes on the list of events associated
precisely. with the input of the gravitropic transduction chain. Ca2?
Although we are not able to consider the growth rate of and H? ion flux changes probably localized at the plasma
the sporangiophore as pulsative, owing to an inadequate membrane have been also proposed to be early conse-
temporal resolution, we observed that Ca2? fluxes oscil- quences of gravity sensing in tip-growing Characean rhi-
lated with a frequency of six to eight peaks per minute. zoids and protonemata (Limbach et al. 2005). The data
These ‘‘Ca2? signatures’’ observed close to the surface of presented in the present study is comparable to data
the intact Phycomyces sporangiophore implicate that reported for Characean protonemata (Braun and Limbach
elongation growth and gravisensing might be controlled by 2006). In gravistimulated protonemata, an asymmetric
an oscillatory mechanism as was reported for pollen tubes distribution of the calcium gradient could originate from
and root hairs (Pierson et al. 1996; Holdaway-Clarke et al. statolith-induced repositioning of calcium channels or,

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more likely, might be caused by differential activation and/ gravistimulation. The fact that the direction of Ca2? and
or inhibition of apical calcium channels (Braun and Lim- H? ion fluxes is altered upon tilting suggests that ion
bach 2006). This asymmetric distribution of Ca2? ions is channels activities change leading to an asymmetric
also observed near the surface of the gravistimulated spo- intracellular ionic distribution, and subsequently, differen-
rangiophore which could mediate the change in the growth tial growth of the opposite subapical flanks of the sporan-
pattern. Statoliths are also detected in Phycomyces spo- giophore. The existence of these characteristic apoplastic
rangiophore as gravisusceptors (Schimek et al. 1999b), and tip-to-base ionic fluxes patterns seem to reflect a tip-high
it is likely that they affect the direction of Ca2? ions via Ca2? gradient which was found in many tip-growing cell
plasma membrane ionic channels upon gravistimulation. types including the growing sporangiophore and reveals
These ionic fluxes recorded from the sporangiophore sur- their fundamental importance for establishing optimal
face could be transferred via plasma membrane channels hyphal tip growth. The direction of ion fluxes varied during
and/or transporters in both directions (inward or outward) sporangiophore growth, so that a clear relationship between
depending on the side of the tilted sporangiophore cell. The the direction of ion fluxes and elongation growth is still
lower side of the gravistimulated sporangiophore is likely missing. Additional pharmacological and biochemical
to scuttle ion fluxes inside the cell, while the upper side experimentation are required to delineate this complex
operates in opposite direction. It is still incompletely mechanism underlying tip growth and gravitropical reac-
explored whether such channels operate in the plasma tion of Phycomyces sporangiophore.
membrane of the sporangiophore upon gravitropic stimu-
lation. Calcium might be the major key player in the Acknowledgments This study was performed in Botanisches In-
stitutder Universität Karlsruhe (TH), Germany, through a DAAD
gravitropic signaling pathways. The data previously scholarship and partial support by Grant 173040 from the Serbian
reported on Phycomyces (Galland et al. 2007) confirm the Ministry of Education and Science. The author is grateful to Prof. Dr.
importance of calcium for Wt sporangiophore bending M.H. Weisenseel for helpful discussions, Dr. G.Monshausen for help
growth upon gravistimulation. Thus, Wt sporangiophore in in setting up measuring line and processing data, and Ms. B. Schlicke
and Mr. W. Müller for their excellent technical assistance. The author
liquid medium without calcium bended 50°, while in the is also grateful to Prof. Dr. Ž. Vučinić and Prof. Dr. P. Galland for
presence of 0.1 mM Ca2? in the solution bending increased their critical reading and useful suggestions during preparation of the
twice as much. Although there are no reports on the manuscript, and to M.Sc. D. Mutavdžić for help in statistical data
gravitropic mutant A909, one can assume that the mutant analysis.
A909 would behave gravidefective even under the same
experimental conditions in liquid medium. The fact that
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