"Journal of Scientific & Industrial Research
Vol.58, September 1999, pp 671-677
Fungal Cells Thrgor Pressure:
Theoretical Approach and Measurement
Patrick Gervais· , Christophe Abadie and Paul Molin
Laboratorie de Genie des Pro cedes Alimentaires et Biotechnologiques
ENSBANA -I , Esplanade Erasme 21000, Dijon
Received: 16 October 1998, accepted: 04 February 1999
Hyph al growth has been examined from a mechanistic standpoint to understand the role of turgor pressure on hyphal extension rate.
From a rigourous momentum bal ance it is show n that turgor pressure can be expressed as a sum of two components : (i) a stati c
contribut ion, related to the membrane e lastic properties and resolved by capillary probe measurements ; and (i i) a dynami c part (M),
responsible for the hyphal extension rate and proportional to it. Bot h contributions add up and explai n why some researchers have found
hyphae growing at finite rates when the capillary probe measures no pressure difference at all; it is simply because the dynamic part of th e
turgor pressure cannot be resolved by such type of technique. The linear relationship between turgor pressure and hypha I extension rat e
means that two measurements need to be taken in order to completely characterize the microscopic parameters involved in this phenomenum.
An experimental setup is proposed based on qui ck osmotic shocks upon the hyphae - to stop tip growth without inducing plasmolysi s
- to accomplish such a di scrimin ati on. This approach has been applied to Aspergillus orizae and an increasing relation between th e
dynamic part of turgor pressure (/'iP) and hyphal extension rate has been found .
Introduction
Growth of microorganisms is very sensi ti ve to env ironmental parameters such as temperature I, water potentia l or water activity2. In the cell ; there is no act ive water
transport and it moves according to the water potentia l
gradient between the intra- and extracellular medium . In
he'terogeneous media, water potential or water activity is
rel ated to the solute concentration , cap illary forces and
absorpt ion propert ies of the insoluble so lid substrate.
Numerou,> workers have demonstrated th e sensitivity.
of hyphal ex tension 3-5, fungal spores germination and
aroma prodllction6.7 to water stress, and a variety of experiments have shown that a turgor pressure exis ts in
hyphal tips.
This work provides a th eoretical :.1pproach for definin g turgor pressure and validates a method for determination of pres ure balance ill growin g fungi.
Eqililibriul1I StOfes and Cel! Waf! Tell sion
Cell turgor press ure is defin ed as the hyd ros tati c pressure due to [he difference between the intracellular and
extracellular osm otic preSSl!re at a steady state, characterized by a steady cell vo lu me . Turgor pressure exists
*Awh or for correspondence
in all types of cell s: vegetal, microorganism and animal
cells. In all these cases, cell turgor pressure corresponds
to an overpressure which allows the cell morphology,
elongaton, division and hence the biomass evolution.
From this it comes that
PI
= · n - oe
I
where P j is the turgor pressure; OJ , the intrace llular osmotic pressure; and ne is the external osmotic pressure.
The turgor pressure value is balanced by the cel l wall
resistance of the organisms and high er the wall res istance, higher the turgor pressures. This can be ex plained
as fo ll ows: since the cell wall must be in equilibrium, it
has to be under tension so th at each surface element can
balance the intra- and ex tracellular osmotic pressures.
T hi s translates mec hani cally int o a compensating pressure, g iven by the ratio of a surface tension oYer;-} curV(]ture rad ius, that is exactly as in the case of a sOap bubble.
It fo llo ws that for a g iven organism:
p I = Pr
' " (I)
where P, has a con stant value unless wall geometry or
properties Li re modified.
672
J SCI IND RES VOL.58 SEPTEMBER 1999
The growth of individual cells is discontinuous and
can be separated into two phases: a slow evolution phase
and a rapid div is ion phase, corresponding to mitosis or
budding. The turgor pressure was found to be constant
for a cell at a specific physiological stage and the budding phenomenon in yeasts was found to be re lated to a
specific and localized hydrolys is of the cell wall which
induces an important decrease in wall resistance and leads
to a volumetric expansion of the cell wall (a pass ive flux
of water into the cell ).
Turgor pressusre can be measured in three ways9. First,
osmometry can be used to determine the osmotic potential difference between cytoplasm and growth medium
and so turgor pressure. Internal osmotic potential is difficult to determ ine accurately since the cytoplasm mu st
be extracted by freezing and thawing lo. 12 • Second, by
exposing the cell to varying solute concentrations, the
onset of plasmolysis can be determined. Third, pressure
probe micropipettes can be inserted into a cell and the
pressure th at mu st be applied to prevent a low-v iscosity
oil dropl et from being forced back up the pipette can be
measured 9.1.1.
Nonequilibriwn States: Fungi Cell Gro wth
For fungi species, growth occurs continuously at the
apex of numerous hyphae of a colony and at constant
growth rate under fixed conditions of the medium (i.e.
pH, temperature and water activityfl4, and the maintenance of the turgor pressure cannot be sufficient to explain the growth phenomena l5. 17
During hyphal elongation, synthesis of cell material s
such as cytoplasmic organcelles or membrane components mu st continu ously occur to keep the cell wall mechanical resistance constant in spite of the continuous
deformation induced by the turgor pressure. The most
numerous components in the apical zone are the apical
vesicles 18 that originate in the Spitzenkorper (or Vesicule
Supply Centre) : a central and dense component of the
apex consisting of accumulated vesicles and presumably
anchored to the apex walIs l8. Hence, cell growth is the
result of a dynamic balance between the maintenance of
an intracellular turgor pressure via solute synthesis and
water input, and a synthesis of macromol ecules which
causes the membrane to provide a constant res istance
despite elongation phenomenon l9 ; we give furth er detail s below.
In the case of continuously growing cells such as
filam entou s fungal tips, the steady state conditions men-
tioned in the preceding section [see Eq. ( I)] can no longer
be applied . So the evaluation of intrace llular turgo r pressure must take into accou nt the type of system under consideration : it will be, different between non-ex. tendincrb
cells (such as bacteria, yeasts or vegetal cells) for which
both- cell volume V and intrace llular osmotic pressure
TC j could be consid,ered as constant, and extending nonequilibrium cells (such as fungi). In order to study the
case of fungi more precisely, let us consider the exampl e
of fungal hypae growth during a time interval dt . During
this interval of dt, dn, mol es of a solute j are synthesized or incorporated in the internal medium which induces a dn j increase in os motic pressure, as H could be
.
'
conslde(ed
for diluted medi a as equal to :
/I
RT
I
j =
I
C.
J
... (2)
with R is the ideal gas constant; ~j' the molar concentration of intrace llular solute } ; and T is the
temperature. Such an increase in osmotic pressure added
to a continuous apex wall elongation (through vesicle
incorporation) will simultaneou sly provoke a thermodynamic water flow into the cell which will decrease the
Cj concentration and so will simultaneously reduce the
osmotic pressure level of -dn j as shown in the previous sentence. Obviously, all these phenomena occur simultaneously, which correspond to a continuous hyphal
extension. Cell growth could then be attributed to a constant increase in the osmolytes concentration near the
apical part which is continuou sly balanced through a
water flow input therein . So in the case of filamentous
fungi, growth regarded as volume variation (essentially
due to to water input) sho'uld occur under a constant intracellular osmotic pressure (H).
The increase in cell volume'is in tum limited by the
mechanical properties of cell wall, as dis cussed previously. Assuming that the tens ion developed by the cell
wall is constant in the apical part during hyphal growth
(i.e. the continuously synthesized wall e xhibits steady
mechanical properties), it could then be possible to calculate the cell pressure balance for extending fungi . In
this case, the sum of the exerted pressure is no more equal
to zero [as previously shown for non-continuously extending cells in Eq. (I)] but equals to a pressure loss value
(!1P) which is the origin of the continuous apical extension (that is, water inflow to the cell) so at a time t it
could be written:
673
GERVAIS el al. : FUNGAL CELLS TURGOR PRESSURE
P.
Va
lime I
... (3)
This very simple equation allows us to describe the fungal growth; thus turgor pressure in extending cells is no
longer counterbalanced only by the membrane pressure
resistance (P r) but also by the apical deformation (due to
6.P) .
Previous workers l7 ,2o have not considered this last parameter (6.P) in the turgor pressure evaluation or measurement in continuously extending cells.
Now it is possible to relate the volume variation dV
during the time dt to this constant pressure loss 6.P
over time (as
and Pr are constant) and to
,
i
physical characteristics of the fungal apex through the
application of an equation 21 describing laminar water
flow driven by pressure gradients between intra- and extracellular media [Eq .(4)].
---'-I~
lime I + dl
or
J
dV
dt
=
AL
!1P
I'
...
Integration of Eq . (4) yields Eq. (5) :
=
(A Lp
I1P) t
+ Vo
)
dV
Va
1/
Figure 1- Representation of the apex vo lume vari ation during the
time cll (the cell act ive volume V, remains constant )
(4)
where A = exchange surface where water flow takes
place (m2 ) d VI dt = hydraulic permeabi lity of the apex
wall (m .s' l . Pa· l )
V
;7L
\\
n ne
l dV
- - =L, M or
Adt
/
... (5)
In obtaining Eg. (5) it is assumed that the sUlface A
is constant versus time. Indeed the parameter A is
relative to the surface of the apex where the water flow
takes pl ace, i.e. where the press ure gradient occurs. This
apex wall surface corresponds to the cell active vo lume
(V) whi ch can be defined as the hyphal apex vo lume
which is osmo ticall y balanced or equ ilibrated through
both co ntinu ous incorporat ion or sy nthes is of solutes and
water input. Indeed it was shown through 14C labeling
of g lucose 22 . 24 , that glucose in corporation was about ten
times hi gher at the apex than in the subapical zone of
Sapro legnia mon oica. Such a local increase in so lute
incorporation (mainly sugars and ions) would immediately tri gger a fast water input to th e fungi, thi s is known
to be many times fa ster than so lute diffu sion towards th e
rear of the apex 25 .
This confirms the view that the rate of hyphal growth
wi ll be only controlled by the water input in the apical
part, as Eq. (5) estimates.
As the apex was extending during the time dt, the active volume Va was increased by dV, but was also decreased by dV due to the advance of the apex which is
supposed to be the only place of the osmotic perturbation (i.e ., solute incorporation or sy nthesi s), as proposed
in Fig . I . The surface A could then be considered as a
constant during fungal extension . Two hypothesis could
be proposed to exp lain thi s behav ior, (i) cell wa ll s corresponding to the dV volume in Eq. ( I ) have evo lved
toward s a weaker permeability to external so lu tes, and
(ii) the cytoplasmic vo lume correspondin g to dV has lost
its ab ility to sy nthes ize compatibl e so lutes (g lycero l,
manitol, ..... ).
With these assumpti ons, the fungal growth expansion
could be represented by Eg . (5) . As the hypha I growth
rate is unidirectional (x-axi s), the vo lume vari ation dV
during the time dt could be written as dV = dx.S where
dx is the ax ial extension and S is the secti on of th e hyphal
tube.
So Eq. (4) becomes Eq. (6):
dx
dt
ALI' Ap
L.l.
or M =
S
S dx
ALI' . dt
... (6)
674
] SCI IND RES VOL.58 SEPTEMBER 1999
These equations imply that the hyphal growth rate
dxldt is constant for defined medium conditions (A. L p"
11P, S are constant), a well known fact since 1957 2 .
The expression of the turgor pressure could be easily
obtained from Eg . (6). Substituting I1P by its value
(PI - P,) given by Eq. (3) in Eq. (6) g ives:
with
K --
... (7)
A comparison of Eq. (7) with Eq. (I) leads us to conclude th at in the case of g rowing tips, the turgo r pressure
considered as a pote nti a l pressure, is greater than the
equi libriu m value PI = P, (case of non-extending cells).
Equation (7) triviall y reduces to Eq . ( I ) when cell extension van ishes.
This difference between non-extending cells and growing tips is eas ily ul1lJerstanuable. because in the case of
continuous hyphal e longali oll, a part [K.dx Idt ] of the
turgor pressure continuously dri ves th e vo lume expan~ I o n and so cannot be measu red ju st th iOugh the memhrane pressure res istance P,. The experimental details
, re desc ribed below.
·'.,;fajor COllsequences
Recent work s usin g capi ll ary probe technique have
' '1easured suc h a low turgo r pressure le ve l in growing
lips that the question of ho w g rowth is poss ibl e without
u. rgor prc~ ,'ure has bee n put fo rward 17 Thi s co ntroversy
;..; c l:u ifi ed by our theory just presented . In fac t, such a
probe in corporated as the hyp hae is sti ll growing and will
,n easure a hydrostat ic pressure gradient be tween intraan d extracell ular medium, but without taking int o
accXOllllt the pressure drop which continuously drives
the vo lume e xpansion . i.e I1P value in Eq. (3).
Whithin the framework de veloped here, such mea surements wi ll only give th e value P't = ni' -He = P,
'md not the total value which is Hi' - n t. = P, + I1P,
i.e. PI = P, + I1P
So, a main an alyti cal concillsion of thi s theoretical development is that intc:l11ai fun ga l osmotic pressure as well
.. 'ungal tu rgor pre. ~ 1I;-l: were underestimated when the
~. illary I "obe techlOl q e WaS used (or when any othe r
" " ic ml'~",u reme t j <; don e;.
Experimental Considerations
Previous theoretical considerations allowed us to understand the cell turgor pressure, particul arly in mobile
microorganisms. For such cells, an important part of the
turgor pressure was continuously diverted into volume
expansion and so cannot be measured through an intracellu lar pressure measurement. Equation (7) also provides
evidence of a linear relationship between the turgor pressure and the hyphal extension rate . Neverthel ess, suc h a
linear relation ship w ill only be valid for standard cu lture
condition s (tempera ture, pH, osmotic pressure) which
co rres pond to constant me mbrane characteristics.
In o rder to relate ex perimental method s to the previous consideration s about the underestimation of turgor
pressure in growing tips, we propose a m ethod intended
to evaluate only that part of turgor pressure in growing
filamentous fung i cont inuou sly used for vo lume expansion . This method is based on the appli cat ion of different osmotic step increases to the e xternal culture medium
in order to evaluate the intensity of the spec ific osmotic
shift which would stop the hypha l ex ten sion rate without
any plasmolysis, i.e. without any decrease of the hyph al
vo lume. The osmotic shift must be as fa st as possibl e so
as to preve nt any active os moregulation mechanism from
the fungi. The perturbation corresponded to a sudden
increase of the rI,. value from n<1 (growing fungu s at a
c onstant hyphal growth rate : dx I dt ) to nco (stopped
hyphae, i. e. dx I dt =0). Then, Eq. (8) follows- from Eq .
(7).
n
e2
=
n
CI
= K. dxldt
... (8)
Hence, the differe nce between th e two e xternal levels
in osmoti c pressure g ives the part of the tu rgo r pressure
wh ic h is continuously used for vo lume e xpansion, i.e.
the I1P value of Eq. (3) .
Total turgor pressure value could the n be experimentally determined by the sum of th e previous experime ntal I1P va lue and of the P, value g ive n by the capillary
probe techniques or by a direct measurment of the osmotic step increase !eading to the incipi ent plasmolysis 9 .
Materials and Methods
Microorganism and Culture M edia
Aspergillus orizae CBS 81772 var. Bruneus was furnished by the Central Bureau voa r chi mm elcultures (T he
Netherlands). The fungu . was u \t !\,jjt..?( on potato-dext 'ose agar (PDA, Institute Pasteu r PI' (~,:c ti on , Paris) con-
GERVAIS e/ al. : FUNGAL CELLS TURGOR PRESSURE
3.0 , -- - -- -- - -- -- --
c
·E
:;;
-
-
-.,
2.5
a.
§,
2.0
Q)
'@
§
1.5
675
A CCD camera (Cohu, model 6710, Japan) was set
on an inverted light microscope and this sent a numerical
signal to the image analyser (series 151, Imaging Technology Inc., USA) . The image produced consisted of
pixels which represented diferent gray intensity leve ls
varying from 0 to 225 .
·iii
c:
Q)
X
Q)
1.0·
n;
-a>.
I
0.5
0.0 +---+---+--
o
---+-
- - -- - +- - --
5
10
----1
15
Time af1er an osmotic shock (min)
Figure 2 - Evolution of th.e hyphal extension rate for different
step decreases in water potenti al :
A : -3.1 MPa
: -3.5 MPa
+ : -3 .9 MPa
(i nitial water potential va lue was 2.8 Mpa)
tai nin g 20 gIL glucose, 15 giL agar and 4gIL potato extract. Xylitol , a compatible so lute, was added to the PDA
medium to obtain different water activity values. Media
were steri lized at 12 1"C for 20 min . Water activity values indicating the water potential were checked before
and after cultivation , us in g a dew point osmometer
Aqualab CX2 (Decagon Devices Inc., USA) . Four different water potential (\jf ) levels of the medium were
tested in order to measure th e e volution of the turgor pressure (in MPa ) as a function of the external water potential (-0. 3, -2 .8, -4.2 ; -7.0). Petri dishes were partially
filled with 4 .5 mL of PDA along a band as shown in
Fig. 2 and in oculated with 24 hours old mycel ia Dishes
were stored at 27"C in bo xes containing 200 mL of water-x ylitol sol uti on to co ntrol the relative humidi ty of the
atmosphere. The water potential of the water-xylitol soluti on was the same as that of the dishes, so the surrou nding environment was in equ ilibrium with the so lid media. Measure ments were then made afte r 24 hours cu ltivati on on the band .
Microscopic Measurement
The hypft al growth rate of Aspergillus orizae was
measured after 24 h of cultivation when the radical extension rate had reached a constant value for a given a w .
Th e hypha I growth rate ( VI) was measured at the periphery of th e co lony by analyzi ng images of individual hyphae ; three hyphae, chosen at random , were tracked for
IS min taking images every three minutes}('. At the colony
front, th e observed hyphae were always in contact with
the gelose medium 27 .2x .
Experimental Procedure for I1P Determination
To estimate the I1P value of the fungus, differents plates
were subjected to slightly decreasing step changes of
water potential (i .e. increasing step changes of osmotic
pressure levels ) unti I a step change stopped hyphal
growth, but without any volume decrease . Different levels of step changes for water potential were then used in
order to estimate I1P for different water potential culture lev~ l s (see Table I) .
Practical realization of previous step changes was made
through the instantaneous injection ofaxylitol-water
solution at the defi ned water potenti al in the plate in order to totally and instantaneou sly submerge the fungus
cultivation. Micro scopical observation of the hypha l
growth rate was going on during and after thi s immersion . It has been verified that immers ion of filam entous
fungi (with a so lution at the same water potential as the
cultivation med ium for periods of up to 45 minutes) does
not modi fy the ex tension rate
Results and Discussion
Hypha! Ettension Rate
Table I gives the different hyphal ex tension rates for different water potenti a l values of the culture medium.
These results confi rm previous findings 2 and show th at
hypha I extension rate is constant for a given water activity in the culture medium. Nevertheless , there are significant variation s as the water potenti al of the medium
is modi fied . The hyph al extension rate corresponding to
the optical water potential value (about -4MPa) was
about twice the va lues found for higher water potenti al
values ( -0.3 MPa) or for lower water potenti al values
(- 7MPa) ..
I1P Measurements
Prev ious considerations (see Theoretical considerati on
secti on) have all owed us to propose a method for I1P
evaluation (I1P = K .dxld t , with K = SiAL )
I'
As an exampl e of I1P determination, Fig. 2 shows th e
evolution of the hyphal ex tension rate for an init ia l water
Table I -
Evaluation of I1P val ues for Aspergillus orizae cultivated at
different levels of water potential
Water potential of the culture medium (MPa)
Hyphal extension rate
( ~m/min )
Water potential of the so lu tions
used to stop growth (MPa)
I1P values (MPa)
K = I1PIV"
1.60 (0.34)*
1.94 (0.30)
3.08 (0.60)
1.43 (0.41)
-0.5
- 1.0
-3.9
-3. 1
-3 .5
-5 .6
-4.9
-5 . 1
-7. 3
0.2<P, <0.7
0.7<P, <1.1
0.9<P, <1.4
O<P, <0.3
0 .1 3-0.44
0.36-0.56
0.29-0.45
0-0.21
• Valu es within the parentheses represent confidence intervals at 95%
potential value of -2.8 MPa and for three different step
decreases in water potential : -3.1 MPa, -3.5 MPa, and
- 3.9 MPa, respectively. From the plots, the minimum
level of water potenti al required in order to stop growth
without modificati on of the hyphal volume would be
between the step decrease va lues -3.5 MPa and -3.9
MPa. So I1P will be bounded by (+ 3.5 -2.8) < P, <
(+ 3.9 -2.8) and so 0.7 < PI < 1.1 (in MPa).
The procedure has been repeated for three other initia l water potentials and the results are presented in Table I .
Analy~is of the fungu s I1P values proposed in Table
! shows that Aspergillus orizae exhibits relatively high
!\P values, which depend on the water potential value of
the media. Such high va lu c~ for I1P are in contradiction
""ith previous theoreti cal co nsiderations which have conc luded that a minuscule difference in water potential suffices to account fo r water inflow during hypha I growth
and that this does not significantl y influence turgor pressures as measured by a press ure probe2~. Similar values
have been previously reported JII ·30 • Maximal I1P values
(;lbout 1.3 MPa) were found for water potenti al values
vf the external med ium of -4MPa (the one hav ing the
highest VI). For higher as for lower values of external
water potential (-0.3 or -7 MPa) the I1P was found to
decrease drastically until 0.5 MPa and 0.1 MPa, re;;pectively ( like behaviour VIl)'
The measured I1P pal1 of the turgor pressure is
plotted in F ig. 3 against hyphal extension rate. It can be
~:t'en tha higher the !1P, hi gher the ap ical rate. The variabili ty of the I1P measurements does not allow to defini tively c0ncJ ude in a linear relation between the two
3.5
C
- .... - - - - - -.. • - . - . - - - - - -- . - - - -- - - -___ .... _
3.0
'E
[ 2.5 E
.3
~
2.0
II
~
-i 1.5t-_..:=-~--·
Q)
x
j
10
: :: +I-
I
.
12
1.4
I
---+.-~
-<--+----+----+,--+-,
0.0
0 .2
OA
0.6
0.8
10
aP (Pa)
Figure 3 -
Representati on of the hyph al extension rate as a
fun ction of the pressure I1P
parameters as expected from Eg. (6) . Nevertheless, the
relationship between the two parameters is clearly an increasing function . Further experiments will allow us to
expl ore thi s relation in details.
Conclusions
The theory developed here has allowed us a better understanding of turgor pressure in fungi and mobile microorganisms. For such organisms a main part of the
turgor pressure (I1P) was show n to be continuously used
for cell vol ume expansion. On the basis of this theoretical approach, a method allowing the evaluation of the
I1P of mobile microorganisms has been proposed . This
method does not give a precise value but defines a range
for the I1P values. Nevertheless, the u"e of osmotic step
GERVAIS el al. : FUNGAL CELLS TURGOR PRESSURE
changes of weaker intensity would certainly improve the
resolution of the method .
To measure the whole turgor pressure of mobile microorgani sms, it would be necessary to combine the capillary probe technique in order to evaluate Pr , and the
prev ious osmoti c step technique in order to evalute /1P ;
the turgor pressure is then the sum of both values. Another method to reach the whole turgor pressure is to use
the previous osmotic step technique until the incipient
cell pl asmolys is, as proposed prev iousll.
Acknowledgements
This work was supported by fund s from the Burgundy
di strict. The auth ors are indebted to Pr Hector Jorquera
from the Catholic University of Santiago (Chile) for helpful ad vices and di scuss ions.
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