ORIGINAL PAPER

Alternative respiration of fungus Phycomyces blakesleeanus

M. Zivic Æ J. Zakrzewska Æ M. Stanic ÆT. Cvetic Æ B. Zivanovic

Received: 6 August 2008 / Accepted: 22 December 2008 / Published online: 6 January 2009

� Springer Science+Business Media B.V. 2009

Abstract Respiratory characteristics of germinating

spores, developing mycelium and mitochondria of the

fungus Phycomyces blakesleeanus were investigated

by means of oxygen Clark-type electrode. The effects

of respiratory inhibitors and metabolic compounds on

oxygen consumption were tested. It was demon-

strated that P. blakesleeanus apart of cyanide-

sensitive respiration, CSR, possess alternative respi-

ration, (cyanide-resistant respiration, CRR) which is

constitutive and whose capacity decreases during

development. Maximum is observed for activated

spores where CRR capacity is significantly greater

than CSR. After treatment with antimycin A, a third

type of respiration insensitive to antimycin A and low

concentration of SHAM (sufficient for inhibition of

CRR), but sensitive to cyanide and high concentra-

tion of SHAM, has been expressed.

Keywords Alternative oxidase �Cyanide resistant respiration � Development �Phycomyces blakesleeanus

Introduction

Complexity of respiratory system progressively

increases from animals to plants and fungi. While

animals posses only cytochrome respiratory pathway

(cyanide-sensitive respiration, CSR), performed by

the single terminal CytC oxidase (COX), most fungi,

apart of classical cytochrome respiratory pathway, just

like plants, also posses alternative respiration (cya-

nide-resistant respiration, CRR) sensitive to SHAM,

and performed by alternative oxidase (AOX) which is

located in the inner mitochondrial membrane (Joseph-

Horne et al. 2001). Unlike plants, where CRR is found

in all species investigated to date, it is absent in fungi

capable of aerobic fermentation (Veiga et al. 2000). In

the rest of fungi the AOX expression and activity are

very complex and highly diverse among different

species. In some like Aspergillus niger (Kirimura et al.

1987), Gaeumannomyces graminis (Joseph-Horne

et al. 1998) and Candida parapsilosis (Milani et al.

2001) AOX is both constitutively present and active

component of the electron transport chain, in others

like Allomyces macrogynus (Heldt-Hansen et al.

M. Zivic (&) � M. Stanic

Department of Biology, State University of Novi Pazar,

Vuka Karadzica bb, 36300 Novi Pazar, Serbia

e-mail: ivanas@bf.bio.bg.ac.yu

J. Zakrzewska

Institute of General and Physical Chemistry,

Studentski trg 12-16, 11000 Belgrade, Serbia

T. Cvetic

Institute of Botany, Faculty of Biology,

University of Belgrade, Takovska 43,

11000 Belgrade, Serbia

B. Zivanovic

Institute for Multidisciplinary Research,

Kneza Viseslava 1, 11030 Belgrade, Serbia

123

Antonie van Leeuwenhoek (2009) 95:207–217

DOI 10.1007/s10482-008-9304-5

1983a) and Ustilago maydis (Juarez et al. 2006) it

becomes active only after inhibition of CSR, while in

fungi like Neurospora crassa, (Tanton et al. 2003) and

Magnaporthe grisea, (Yukioka et al. 1998) inhibition

of CSR, or some other environmental stimuli, is

necessary for induction of AOX expression.

There are also differences in proposed function of

CRR between plants and fungi. While the increasing

body of evidence shows that AOX in plants plays a role

in lowering mitochondrial reactive oxygen species

(ROS) formation (Maxwell et al. 1999; Yip and

Vanlerberghe 2001), in fungi this function could not

be confirmed (Veiga et al. 2003a). Even more, in

Podospora anserina mutant overexpression of AOX

considerably increased ROS production (Lorin et al.

2001). Another well established hypothesis is that

AOX, due to its not-protonmotive nature, can function

as ‘‘overflow’’ for excess electrons under conditions of

saturated or limited cytochrome pathway, allowing the

functioning of tricarboxylic acid cycle with minimal

ATP synthesis (Lambers 1985; Simons and Lambers

1999). In fungi this function could not be regarded as

ubiquitous, since there are evidences that in several

fungal species AOX has equally important role in

maintaining mitochondrial membrane potential and

ATP synthesis as cytochrome pathway (Mizutani et al.

1996; Veiga et al. 2003a; Joseph-Horne et al. 1998,

2001).

Moreover, some fungi possess additional terminal

oxidases. Thus in Kluyveromyces lactis (Ferrero et al.

1981) and in Schwanniomyces castellii (Claisse et al.

1991) a cyanide- and SHAM-insensitive, but azide-

sensitive respiration, was brought up. A second

respiratory chain, insensitive to antimycin A, but

inhibited by amital, SHAM, myxothiazol and cyanide,

was found in C. parapsilosis and Candida albicans

(Guerin and Camougrand 1994; Ruy et al. 2006). Two

terminal oxidases insensitive to both SHAM and azide

have been found in Debaryomyces hansenii (Shel-

emekh et al. 2006), and in G. graminis terminal oxidase

sensitive to antimycin A, but insensitive to cyanide

was observed (Joseph-Horne and Hollomon 2000).

In spite of great diversity, fungal respiration is less

investigated than that of plants. One of the least

investigated areas of fungal respiration is CRR

capacity during development. Most of the investiga-

tion in this area was qualitative in its nature. In Botrytis

cinerea, respiration in 24 h-old cultures was solely

sensitive to inhibitors of the core pathway (Tamura

et al. 1999), while in 48 h cultures it became sensitive

to AOX inhibitors, but insensitive to inhibitors of

Complex III (Joseph-Horne et al. 2001). In Pichia

membranifaciens (Veiga et al. 2003b) and Yarrowia

lipolytica (Medentsev et al. 1999) CRR has been found

in transition between exponential and stationary phase

of culture. In D. hansenii CRR was absent only in very

early exponential phase (Veiga et al. 2003b), while in

Debarymyces occidentalis (Zimmer et al. 1997),

C. parapsylosis (Milani et al., 2001) and C. albicans

(Shepherd et al. 1978) it was detected under all

conditions tested. There are only two detailed, quan-

titative investigations of AOX activity during growth.

In U. maydis, a sharp increase in AOX activity at the

beginning of exponential growth phase was seen in

cells cultured at 34�C, reaching a maximum value at

about 24 h, and decreasing slowly afterwards (Juarez

et al. 2006). In A. niger it was shown that CRR

increases during development, especially at the late-

log phase (Kirimura et al. 1987). To the best of our

knowledge germinating spores is the least investigated

developmental stage concerning AOX activity. There

is only one report on Mucor rouxii germinating spores

where CRR capacity was greater than that of CSR

(Salcedo-Hernandez et al. 1994).

Clarifying the specific details of fungal respiration

pathways is a challenging task due to its large

diversity within fungal kingdom. Investigation of

lower fungi group like Zygomycota might be partic-

ularly helpful, as Zygomycota lie in the basis of

fungal tree of life (Lutzoni et al. 2004) and the single

known AOX of Zygomycota is basal to the rest of the

fungal AOXs (McDonald and Vanlerberghe 2006). In

spite of this, respiration pathways of Zygomycota are

the least investigated among fungi so far. Alternative

respiration was investigated in only two species of

Zygomycota, M. rouxii (Cano-Canchola et al. 1988;

Salcedo-Hernandez et al. 1994) and A. macrogynus

(Heldt-Hansen et al. 1983a, b).

In this study, the unicellular zygomycetous fungus

P. blakesleeanus was chosen for investigation of the

main respiratory characteristics throughout mycelial

development. P. blakesleeanus is a strictly aerobic

Zygomycota with very short life cycle and it is widely

used in studies of phototropic and geotropic responses

(Cerda-Olmedo and Lipson 1987). In addition, P. bla-

kesleeanus genome sequencing is at its final phase

(www.es.embnet.org/*genus/phycomyces.html). Our

earlier results showed that there is a difference

208 Antonie van Leeuwenhoek (2009) 95:207–217

123

between effects of cyanide and anoxia on 31P NMR

spectra (Zakrzewska et al. 2005), which indicated the

existence of some alternative respiratory pathway in

P. blakesleeanus. Previous investigation showed that

P. blakesleeanus has all components of cytochrome

respiratory pathway (Cerda-Olmedo and Lipson

1987). The data presented in this study provide evi-

dence for existence of alternative type of respiration in

P. blakesleeanus apart of cytochrome respiratory

pathway.

Materials and methods

Strains and growth conditions

The wild type strain of the fungus P. blakesleeanus

(Burgeff) (NRRL 1555(-)) was used in this work.

One milliliter of spore suspension containing about

106 spores was transferred to modified liquid standard

minimal medium (Sutter 1975) containing (in mM):

36.7 KH2PO4, 2 MgSO4�7H2O, 0.376 CaCl2, and (in

lM): 3 thiamine 9 HCl, 1 citric acid 9 H2O, 3.7

Fe(NO3)3�9H2O, 3.5 ZnSO4�7H2O, 1.8 MnSO4�H2O,

0.2 CuSO4�5H2O, 0.2 NaMoO4�2H2O, supplemented

with (in mM): 220 glucose and 26.2 L-asparagine to

ensure adequate supply of carbon and nitrogen sources

for optimal growth of the mycelium. The pH of this

liquid nutrient medium was 4.5, since the growth of

mycelium is optimal at this pH value (Martinez-

Cadena et al. 1995). P. blakesleeanus spores are

endogenously dormant and they need to be activated to

germinate. Prior to inoculation, the spore suspension

was heat shocked for 10 min at 49�C. The mycelium

was grown in Petri dishes placed in transparent plastic

boxes and stored in the growth cabinet with contin-

uous overhead white fluorescent light of 10 W/m2, at

temperature of 20�C, and ca. 95% relative humidity.

Under these experimental conditions, the development

of vegetative mycelium was detected up to 40 h when

the mycelium started to develop sporangiophores.

Germination of spores and mycelium development

were checked under microscope.

Mitochondria isolation and enzyme assays

Mitochondria were isolated from 28 h old mycelium

according to the procedure described previously

(Salcedo-Hernandez et al. 1994). Mycelium (10 g

wet wt per 100 ml) resuspended in 10 mM TRIS/HCl

buffer, pH 7.5, containing 2 mM EDTA, 1 mM

CaCl2, 0.7 M sucrose and 1.1 M glycerol (medium

A) was homogenized 3 9 5 s with Polytron homog-

enizer (Kinematica GmbH). The suspension was

centrifuged at 3,3009g for 5 min and the supernatant

was recovered and centrifuged for 10 min at

20,0009g. Sediment was carefully removed in Tris

buffer containing EDTA, CaCl2 and sucrose as above,

but no glycerol (medium B) and the suspension was

centrifuged for 5 min at 3,3009g. Mitochondria in the

supernatant were sedimented by centrifugation at

13,3009g for 10 min and recovered in a minimal

volume of medium B. Purity of mitochondrial fraction

was assayed by measuring hydroxypyruvate reductase

(Titus et al. 1983) to establish peroxisomal contam-

ination, NAD(P)H cytochrome c reductase (Terry and

Williams 2002) as a marker for endoplasmic reticu-

lum and cytochrome c oxidase (Tolbert 1974) as a

mitochondrial marker enzyme. All enzyme assays

were performed in the presence of 0.015% Triton X-

100. Protein concentration was estimated according to

Bradford (1976) with BSA as a standard. Marker

enzyme assays indicated that the final pellet is

mitochondria-enriched fraction of substantial purity.

Measurement of oxygen consumption

Oxygen consumption was measured with a Clark-type

electrode (Hansatech Instruments, Norfolk, England).

Spore or mycelial suspensions were diluted in fresh

liquid nutrient medium to appropriate concentration

and aerated for 10 min prior to the measurement of

oxygen uptake. Initial concentration of the oxygen in

the reaction buffer was 254 nmol ml-1. One milliliter

of mycelial suspension was transferred to 2.5 ml

electrode chamber and kept at constant temperature of

25�C. All measurements were performed at pH 4.5.

Mitochondrial respiration was measured according to

a procedure described by Salcedo-Hernandez et al.

1994. Mitochondrial aliquots, resuspended in medium

B, were mixed with a solution containing 120 mM

KCl, 5 mM MOPS/K2HPO4, 5 mM MgCl2, and

2 mM EDTA.

The inhibitors of appropriate concentrations were

added directly to the electrode chamber containing

mycelial suspension, and O2 uptake was subsequently

Antonie van Leeuwenhoek (2009) 95:207–217 209

123

followed for 5–15 min. Estimation of CSR and CRR

intensity was performed by using KCN and SHAM,

respectively. KCN and SHAM were added to reach

final concentrations of 1–10 and 2–10 mM, respec-

tively. The extent of engagement of CRR, determined

as percentage of total respiration blocked by SHAM

corresponds to CRR activity, while residual respiration

measured after addition of KCN to CRR capacity. The

extent of engagement of alternative pathway was

investigated at different stages of mycelial develop-

ment (1–40 h-old mycelium). The respiratory

inhibitor, antimycin A (20 lM), was used for enhance-

ment of the CRR. Cyclohexymide, as translation

inhibitor, actinomycin D, as transcription inhibitor,

and CCCP, as an uncoupler and inhibitor of energy

dependent import of polypeptides into mitochondria,

were added at 0.24, 16, and 20 lM, respectively. After

measurements, the biomass of the sample was recov-

ered by vacuum filtration and dried at 90�C in order to

determine dry weight.

Chemicals

KCN, SHAM, CCCP, NADH, antimycin A, actino-

mycin D and cyclohexymide were from Sigma–

Aldrich (St Louis, MO, USA). All other reagents and

chemicals were of analytical grade. All stock solu-

tions were prepared using glass-distilled deionized

water or 96% ethanol.

Results

Effects of respiratory inhibitors

The effects of inhibitors, KCN and SHAM

on respiration of 28 h old mycelium of fungus

P. blakesleeanus are shown in Fig. 1a. Addition of

1 mM KCN inhibited 73 ± 2% (n = 3) of total

respiration. The rest of the respiration was inhibited

by 2 mM SHAM. When inhibitors were added in

reverse order, 2 mM SHAM decreased respiration by

27 ± 3% (n = 5), while subsequent addition of

1 mM KCN inhibited the rest of respiration. Thus,

the presence of both inhibitors totally inhibited

mycelial respiration, regardless of the sequence of

their addition (Fig. 1). In order to confirm that the

effect of SHAM is a consequence of CRR inhibition,

the experiments were performed on mitochondria

isolated from 28 h old mycelium. NADH (1 mM)

was added as external respiration substrate. Obtained

results were similar to those on mycelium, 1 mM

KCN inhibited 74% of respiration, while 2 mM

SHAM, when added first, inhibited 24% of respira-

tion and in both cases addition of second inhibitor

blocked respiration completely (Fig. 1b).

Antimycin A and cyanide had similar effects on

O2 uptake of mycelium; all cyanide insensitive

respiration was insensitive to antimycin A and vice

versa (data are not presented). The results indicated

B

0

-31.3

-21.0

0

-37.4

-9.6

1mMKCN

1mMKCN

2mMSHAM

2mMSHAM

A

20nmol O2

1 min

1mMKCN

2mMSHAM

1mMNADH

-51.9

0

-13.3

10nmol O2

1 min

2mMSHAM

1mMKCN

1mMNADH

0

-38.6

-51.0

Fig. 1 Representative oxygen uptake traces of 28 h-old

mycelium (a) and mitochondria isolated from 28 h old

mycelium (b) of P. blakesleeanus before and after treatment

with 1 mM KCN and 2 mM SHAM, added subsequently. In

the case of mitochondria (0.21 mg ml-1 of protein) oxygen

uptake was measured with external NADH (1 mM) as a

reducing substrate. The numbers at the right of the traces are

respiration rates in lmolO2 min-1 gDW-1 (a) and nmol-

O2 min-1 (mg protein)-1 (b). Arrows indicate the moment of

inhibitors addition, dashed lines indicate expected course of

oxygen uptake in the absence of inhibitors

210 Antonie van Leeuwenhoek (2009) 95:207–217

123

that only cytochrome and CRR pathways are consti-

tutively active in young mycelia.

The engagement of CRR during mycelial

development

To find out whether the engagement of CRR is

changing during development, the effects of inhibitors

were monitored at different growth stages (Fig. 2).

Grey bars represent decrease in respiration after

addition of 2 mM SHAM which corresponds to

CRR activity; black bars represent residual respiration

after addition of 1 mM KCN (CRR capacity). The

results presented in Fig. 2 implicate that alternative

respiration is active at all times during development of

P. blakesleeanus mycelium. However, the extent of

engagement of alternative pathway varies among

different stages of development (grey bars). Data

presented also indicate that not all CRR present is

active under standard growth conditions as its

engagement is significantly greater when KCN is

added first. The only exceptions are at 20 and 28 h old

mycelia when CRR activity and capacity were the

same. CRR activity is almost constant (*20%) up to

24 h-old mycelium, when it reaches statistically

significant minimum of 10 ± 1% (n = 8, P \ 0.05).

In 28 h-old mycelium CRR activity reaches a max-

imum of 27 ± 3% followed by a further decrease to

approximately 17%. On the other hand, CRR capacity

(Fig. 2, black bars) decreases with mycelium growth.

It is maximal for activated spores (123 ± 6%, n = 3,

P \ 0.001), minimal for 20 h-old mycelium (19 ±

2%, n = 6, P \ 0.05), and after 24 h of growth, it

shows constant level of approximately 27%. An

unexpected respiratory increase of 23 ± 6% (n = 3)

after addition of 1 mM KCN (Fig. 3) was observed for

activated spores (1 h after activation). Total respira-

tion was inhibited by subsequent addition of 2 mM

SHAM (Fig. 3). When 2 mM SHAM was added first,

decrease in respiration amounted to a mere 18 ± 1%,

(n = 3), while residual respiration was completely

inhibited by 1 mM cyanide (Fig. 3).

Course of changes of absolute values of residual

respiration during development after addition of 1 mM

KCN (gray circles, Fig. 2) follows that of relative

values with characteristic minimum at 20 h (6.1 ± 0.5

lmolO2 min-1 gDW-1, n = 6, P \ 0.05), except for

36 and 40 h old mycelia, where statistically significant

AGE1h 12h 16h 20h 24h 28h 36h 40h

% A

OX

0

10

20

30

40

50120

130

Res

pir

atio

n r

ate

(µm

olO

2min

-1g

DW

-1)

0

2

4

6

8

10

12

AGE

12h 16h 20h 24h 28h 36h 40h

Res

pir

atio

n r

ate

(µm

olO

2min

-1g

DW

-1)

20

25

30

35Fig. 2 Changes in

alternative respiration

during growth of P.blakesleeanus. Gray barsand white trianglesrepresent decrease in

respiration after addition of

2 mM SHAM (CRR

activity) in relative (%

AOX) and absolute

(lmolO2 min-1 gDW-1)

units, respectively. Blackbars and gray circlesrepresent residual

respiration after addition of

1 mM KCN (CRR capacity)

in relative (AOX %) and

absolute

(lmolO2 min-1 gDW-1)

units, respectively. Insert:absolute values of total

respiration during growth of

P. blakesleeanus. The data

are means ± SE (n = 3–8)

Antonie van Leeuwenhoek (2009) 95:207–217 211

123

decrease of CRR capacity is observed (6.2 ± 0.5

lmolO2 min-1 gDW-1, n = 3, P \0.05 and 6.4 ± 0.6

lmolO2 min-1 gDW-1, n = 5, P \ 0.05, respec-

tively). After addition of 2 mM SHAM (white

triangles, Fig. 2), maximum of CRR activity at 20 h

(7.2 ± 0.4 lmolO2 min-1 gDW-1, n = 4, P \ 0.01)

not present in the case of relative values, is observed.

These discrepancies are a consequence of variation in

absolute values of total respiration, with maxima at

20 h (33.3 ± 1.6) and 28 h (35.9 ± 1.0) of fungal

growth (Fig. 2, insert).

CRR induction

The time course of enhancement of CRR of 24 h old

mycelium of P. blakesleeanus after incubation in

20 lM antimycin A is shown in Fig. 4. In the absence

of respiratory inhibitor (control), CRR increased only

slightly, but in the presence of antimycin A, SHAM

sensitive O2 uptake gradually increased from

2.9 times in the 80th min from the beginning of

incubation to 6.7 times after 6 h.

The regulatory mechanism of CRR induction was

examined by monitoring the influence of metabolic

inhibitors on enhancement and repression of CRR in

antimycin A-incubated 24 h-old mycelium. In the

presence of translation inhibitor, cycloheximide

(0.24 mM) or CCCP (20 lM), the uncoupler that also

acts as inhibitor of energy dependent import of

polypeptides into mitochondria, CRR was completely

repressed (Fig. 4). On the other hand, actinomycin D,

the inhibitor of eukaryotic gene transcription, added in

usual concentration of 1.6 lM (Kirimura et al. 1996)

had only a slight effect on CRR; even an elevated

concentration (16 lM) gave the same result (Fig. 4).

In 24 h old mycelia, 3 h from the start of incuba-

tion in minimal media with 20 lM antimycin A,

addition of 2 mM SHAM only partially suppressed

respiration (Fig. 5), while the residual respiration was

completely inhibited by addition of 1 mM KCN

(Fig. 5). Total respiration was blocked by 10 mM

SHAM. Increase of cyanide concentration from 1 to

10 mM had no significant effects (Fig. 5). The results

indicated that P. blakesleeanus expresses a type of

respiration sensitive to cyanide and high concentra-

tion of SHAM, but insensitive to antimycin A and low

concentration of SHAM, sufficient for inhibition of

CRR. This type of respiration is not constitutive for

24 h old mycelium, since cyanide and antimycin A

have the same effect on the respiration of P. blakes-

leeanus in standard experimental conditions.

Discussion

Results obtained on both mycelium and isolated

mitochondria (Fig. 1) indicate that P. blakesleeanus,

Fig. 3 Representative oxygen uptake traces of P. blakeslee-anus spores 1 h after activation. The numbers at the right of the

traces are O2 uptake rates in nmolO2 min-1 (106 spores)-1.

Arrows indicate the moments of inhibitor addition, dashedlines indicate expected course of oxygen uptake in the absence

of inhibitors

Fig. 4 Effects of addition of metabolic inhibitors on cyanide

insensitive respiration 24 h-old mycelium of P. blakesleeanusincubated in 20 lM antimycin A. s Control, d antimycin A

(20 lM), r antimycin A and actinomycin D (16 lM), .

antimycin A and cycloheximide (0.24 mM), h antimycin A

and CCCP (20 lM)

212 Antonie van Leeuwenhoek (2009) 95:207–217

123

beside the cytochrome respiratory pathway also

expresses an alternative type of respiration. The

result was expected, since CRR is absent only in

fungi capable of aerobic fermentation (Veiga et al.

2000). Experiments on isolated mitochondria were

performed in order to confirm that the changes in

respiratory rate of mycelium could be attributed to

effect of KCN and SHAM on CSR and CRR,

respectively. Inhibitors demonstrated the same effect

on mycelium and isolated mitochondria; therefore it

could be concluded that the effect of SHAM is indeed

a consequence of CRR inhibition and that mycelium

is a good model system for further studies of CRR.

Cyanide resistant respiration is found in all plants and

many fungi (Siedow and Umbach 2000; Minagawa

and Yoshimoto 1987). CRR insensitive to cyto-

chrome pathway inhibitors (cyanide and antimycin

A) and sensitive to SHAM, was found to be conferred

by enzyme alternative oxidase or AOX (Veiga et al.

2003a; Siedow and Umbach 2000; Joseph-Horne

et al. 2001; Juarez et al. 2004). Behavior of CRR

found in P. blakesleeanus is identical to that

conferred by AOX, so it can be assumed that AOX

is responsible for cyanide resistant respiration of

P. blakesleeanus.

During mycelial development the respiration of

P. blakesleeanus is sensitive to SHAM (Fig. 2, grey

bars), indicating that AOX is active at all times

during development, which is not always the case in

fungi. For example, in gametangia of zygomycetous

fungus A. macrogynus AOX is not active constitu-

tively, but its activity is gradually induced after

treatment with cyanide (Heldt-Hansen et al. 1983a).

A certain amount of AOX in P. blakesleeanus is

inactive under standard growth conditions, as con-

firmed by the cyanide-induced increase of CRR

engagement (Fig. 2, black bars). Stimulation of the

alternative pathway by cyanide was also reported

for Y. lipolytica (Medentsev and Akimenko 1999),

P. membranifaciens and D. hansenii (Veiga et al.

2003b). In Y. lipolytica it was accompanied by a

marked decrease in ATP and an increase in ADP and

AMP, both of which stimulate CRR in fungi (Sakajo

et al. 1997; Milani et al. 2001; Medentsev and

Akimenko 1999; Umbach and Seidow 2000; Lambers

1982; Vanderleyden et al. 1980). Medentsev et al.

1999 interpreted the effect of cyanide as due to

activation of AOX mediated by an increase in AMP

concentration. Also, 31P NMR spectra of P. blakes-

leeanus showed that the concentration of ATP

decreases by about 40% after addition of KCN (Zivic

2005). Alternatively, since the activation of CRR by

cyanide is very fast, Veiga et al. 2003b suggested that

the increase in oxygen consumption through the

alternative pathway may be caused by an overall

increase in metabolic flux, as if mitochondria were

counterbalancing the rapid switch to this relatively

low-yielding proton motive force pathway.

Unlike most fungi, in which CRR capacity

increases during development (Kirimura et al. 1987;

Juarez et al. 2006), in P. blakesleeanus it decreases

with the most pronounced drop in first few hours after

activation of the spores. It could be explained by the

fact that in P. blakesleeanus, during the first 8 h of

germination, amount of cytochromes increase mark-

edly (Keyhani et al. 1972). Minimum of CRR capacity

in both absolute and relative values is attained at 20 h

of growth which is accompanied by a marked increase

of total respiration. Since P. blakesleeanus is strictly

aerobic fungus, the finding could be explained by a

gradual favorable environmental change from low-

oxygen water medium to high-oxygen medium at the

surface which induced both further increase in

cytochrome respiration and decrease in alternative

-12.8

0

0-6.7

-6.0

-11.4

-4.2

-12.6

0

2mMSHAM

2mMSHAM

20µMAntimycin A

10mMSHAM

1mM KCN

1mMKCN

10mMKCN

20nmolO 2

1 min

Fig. 5 Effects of respiratory inhibitors on oxygen consump-

tion in 24 h old mycelium of P. blakesleeanus incubated in

20 lM antimycin A for 3 h. The numbers at the right of the

traces are O2 uptake rates in lmolO2 min-1 gDW-1. Arrowsindicate the moment of inhibitors addition, dashed linesindicate expected course of oxygen uptake in the absence of

inhibitors

Antonie van Leeuwenhoek (2009) 95:207–217 213

123

respiration. Preliminary results obtained from inves-

tigation of the oxygen regulation of CRR capacity

support this hypothesis. Namely, decrease of oxygen

concentration induced a substantial increase in CRR

capacity. Similar dependence on oxygen level was

shown in another zygomycetous fungus M. rouxii,

where mycelium grown under anaerobic conditions

expressed CRR, while that grown under aerobic

conditions did not (Salcedo-Hernandez et al. 1994).

Unexpected results concerning CRR activity in

P. blakesleeanus were obtained for activated spores.

Namely, in this phase of development the addition of

cyanide significantly increased respiration rate. Since

the respiration was inhibited by subsequent addition

of 2 mM SHAM (Fig. 3), the cyanide-mediated

stimulation indicated an increase in alternative respi-

ration. When KCN was added after SHAM (Fig. 3), it

demonstrated usual inhibitory effect. These results

could indicate that CRR in spores is more abundant

than the cytochrome pathway, but mainly inactive.

Therefore, activation of CRR with KCN, by one of

the mechanisms mentioned above, results in a net-

stimulation of respiration. The CRR capacity of fungal

spores has been investigated only in one more species,

M. rouxii, where similar results have been reported

(Salcedo-Hernandez et al. 1994). The fact that spores

of these two species possess respiratory pathways

capable of mutual compensation, suggests their greater

environmental adaptability compared to mycelium.

Since these two fungal species are closely related, no

general conclusion can be drawn, especially having in

mind the diversity of this phenomenon in plant seeds.

In seeds of Cicer arietinum during the initial 12 h of

germination, the respiration is predominantly cyanide

sensitive; showing a shift to CRR after that (Burguillo

and Nicolas 1974). Contrary, germinating seeds of

soybean show a transition from predominantly alter-

native respiration to a CSR between the 4th and the 8th

h of germination (Yentur and Leopold 1976). It is

likely that CSR meets high energy demands during

mobilization of food storage in seeds, while increased

CRR activity corresponds to low energy demand and

eventual elimination of ROS produced during oxida-

tive phosphorylation, as previously reported (Tommasi

et al. 2001).

It is well known that in fungi the inhibition of

cytochrome pathway by antimycin A or cyanide can

increase CRR capacity (Sherald and Sisler 1970,

1972; Sakajo et al. 1993). In P. blakesleeanus, SHAM

sensitive O2 uptake significantly increases in the first

80 min of incubation with antimycin A (Fig. 4). The

response of P. blakesleeanus to inhibition of core

pathway is faster than that of A. niger, since in

A. niger O2 uptake starts to increase after 3 h of

incubation (Kirimura et al. 1996). Taking into account

that the time frame for AOX nuclear induction to

insertion and activation requires between 3 and 5 h

(Vanlerberghe and McIntosh 1992; Mizutani et al.

1998), it can be assumed that mechanism faster than

nuclear induction is also involved. In order to test this

hypothesis, enhancement and repression of CRR were

examined by addition of metabolic inhibitors. Cyclo-

heximide and CCCP completely repressed CRR

(Fig. 4), as it was the case in A. niger (Kirimura

et al. 1996). As cycloheximide blocks protein synthe-

sis in the cytoplasm of eukaryotic cells, and CCCP,

the uncoupler, inhibits energy dependent import of

polypeptides into mitochondria (Joseph-Horne et al.

2001; Gasser et al. 1982; Nelson and Schatz 1979),

obtained results indicate that the enhancement of CRR

is a consequence of de novo synthesis of AOX in

cytosol and its transport to mitochondria. The expres-

sion of AOX in plants and fungi is controlled

primarily at the level of transcriptional activation

(Vanlerberghe and McIntosh 1997). However, acti-

nomycin D, the inhibitor of eukaryotic gene

transcription, shows only a slight effect on CRR in

P. blakesleeanus mycelium under given experimental

conditions. These results suggest that the synthesis of

AOX protein is not regulated at the level of gene

transcription. However, further measurements of

protein and mRNA levels of AOX as well as capacity

of the enzyme in isolated mitochondria are necessary

to confirm this assumption.

In 24 h old mycelium of P. blakesleeanus grown in

the absence of any drugs, CRR and CSR are consti-

tutive. The results obtained after incubation with

antimycin A indicated that P. blakesleeanus expresses

a type of respiration sensitive to cyanide and high

concentration of SHAM, but insensitive to antimycin

A and low concentration of SHAM, sufficient for

inhibition of CRR (Fig. 5). In C. parapsilosis, respi-

ration type insensitive to antimycin A and sensitive to

high concentration of SHAM was found (Milani et al.,

2001; Guerin and Camougrand 1994) and it was

proposed that it corresponds to activity of respiratory

chain parallel with classical one, with partitioning

point of electron flux at the level of ubiquinone (Milani

214 Antonie van Leeuwenhoek (2009) 95:207–217

123

et al. 2001). Also, In M. rouxii (Cano-Canchola et al.

1988) fungus closely related to P. blakesleeanus,

additional cytochrome (cyto), which is not the part of

classical respiratory chain, was detected. Having in

mind results presented for C. parapsilosis and

M. rouxii it may be assumed that P. blakesleeanus

expresses a third type of respiration, but further

investigations concerning molecular basis of the

observed respiration type should be performed.

Acknowledgments The authors are grateful to Prof. Paul

Galland for providing Phycomyces strains and critical reading

of this manuscript. This work was supported by Serbian

Ministry of Science grant No. 143016B.

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