Báo cáo khoa học: L-Lactate metabolism in potato tuber mitochondria docx

L-Lactate metabolism in potato tuber mitochondria
Gianluca Paventi, Roberto Pizzuto, Gabriella Chieppa and Salvatore Passarella
Dipartimento di Scienze per la Salute, Universita
`
del Molise, Campobasso, Italy
According to the Davies–Roberts hypothesis, plants
primarily respond to oxygen limitation by a burst of
l-lactate production ([1] and refs there in). The acidifica-
tion of the cytoplasm during the first phase of anaerobi-
osis arising from lactic fermentation results in inhibition
of lactate dehydrogenase (LDH) and activation of
pyruvate decarboxylase [2]. As a result, a switch from
lactic to ethanolic fermentation occurs. In those organ-
isms that cannot switch to ethanolic fermentation, when
oxygen falls below 1%, glycolysis is stimulated and
l-lactate accumulates [3], leading to decreased cytoplasmic
pH and cell death [4,5]. Thus, according to the Davies–
Roberts concept, cytoplasmic acidification potentially
induces damage and death of intolerant plants.
Because of the damage that can arise from l-lactate
accumulation, a cellular safety valve to minimize that
damage is to be expected. It has been consistently repor-
ted that metabolism of l-lactate in potato after a period
of anoxia is accompanied by a two-fold increase in
LDH activity and by the induction of two LDH iso-
zymes [6]. These observations related to l-lactate meta-
bolism occurring in the cytoplasm involved pyruvate
formation via LDH, and further pyruvate metabolism,
both in mitochondria and in the cytoplasm. There is rea-
son to suspect, however, that mitochondria themselves
may be involved in l-lactate metabolism. This is based

on our previous work, which has shown that l-lactate is
transported into the organelles isolated from both rat
Keywords
L-lactate; L-lactate dehydrogenase;
mitochondrial transport; plant mitochondria;
shuttle
Correspondence
S. Passarella, Dipartimento di Scienze per la
Salute, Universita
`
del Molise, Via De
Sanctis, 86100 Campobasso, Italy
Fax: +39 0 874 404778
Tel: +39 0 874 404868
E-mail:
(Received 2 August 2006, revised 20
December 2006, accepted 10 January 2007)
doi:10.1111/j.1742-4658.2007.05687.x
We investigated the metabolism of l-lactate in mitochondria isolated from
potato tubers grown and saved after harvest in the absence of any chemical
agents. Immunologic analysis by western blot using goat polyclonal anti-
lactate dehydrogenase showed the existence of a mitochondrial lactate
dehydrogenase, the activity of which could be measured photometrically
only in mitochondria solubilized with Triton X-100. The addition of l-lac-
tate to potato tuber mitochondria caused: (a) a minor reduction of intra-
mitochondrial pyridine nucleotides, whose measured rate of change
increased in the presence of the inhibitor of the alternative oxidase salicyl
hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but
inhibited by salicyl hydroxamic acid; and (c) activation of the alternative
oxidase as polarographically monitored in a manner prevented by oxamate,

an l-lactate dehydrogenase inhibitor. Potato tuber mitochondria were
shown to swell in isosmotic solutions of ammonium l-lactate in a stereo-
specific manner, thus showing that l-lactate enters mitochondria by a pro-
ton-compensated process. Externally added l-lactate caused the appearance
of pyruvate outside mitochondria, thus contributing to the oxidation of
extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal
dependence on l-lactate concentration and was inhibited by phenylsucci-
nate. Hence, potato tuber mitochondria possess a non-energy-competent
l-lactate ⁄ pyruvate shuttle. We maintain, therefore, that mitochondrial
metabolism of l-lactate plays a previously unsuspected role in the response
of potato to hypoxic stress.
Abbreviations
AOX, alternative oxidase; COX IV, subunit IV of cytochrome oxidase; FCCP, carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; LDH,
L-lactate dehydrogenase; PTM, potato tuber mitochondria; SHAM, salicyl hydroxamic acid.
FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1459
heart [7] and liver [8] and metabolized there. Moreover,
a major role for the mitochondrial LDHs in the transfer
of reducing equivalents from the cytosol to the respirat-
ory chain (lactate shuttle) was also proposed [7].
In order to ascertain whether and how energy meta-
bolism, and in particular l-lactate metabolism, can
change as a result of spontaneous hypoxia in plants,
we used potato, which is an important crop whose
tubers show a high sensitivity to O
2
deprivation [3].
We show here for the first time the existence of LDH
in isolated potato tuber mitochondria (PTM). This
enzyme is localized in the inner mitochondrial
compartments and uses NADP

+
as a cofactor, the
product, NADPH, being oxidized essentially by the
alternative oxidase (AOX), which is activated by pyru-
vate. The latter can also exit from the mitochondria in
a novel l-lactate ⁄ pyruvate shuttle operating in a non-
energy-competent manner.
Results
The existence of LDH in mitochondria isolated
from potato tubers
In order to verify the occurrence of LDH in PTM, use
was made of goat polyclonal antibodies raised against
LDH, which have already been shown to cross-react
with LDHs from different species [9–11]. Solubilized
mitochondrial proteins were analyzed by SDS ⁄ PAGE,
blotted onto poly(vinylidene difluoride) membrane, and
then probed with the antibody to LDH. In agreement
with Hondred & Hanson [12], LDH protein was visual-
ized as a single band with a molecular mass of about
39 kDa. A typical experiment is reported in Fig. 1,
which shows clearly the presence of LDH in the
mitochondrial fraction. Confirmation of this site of ori-
gin was provided by use of a specific antibody against
subunit IV of the cytochrome c oxidase (COX IV). A
band corresponding to a protein of molecular mass
35 kDa was observed; this is likely to arise from an
aggregate of COX IV (13 kDa [13]) and other unidenti-
fied protein ⁄ s, as already shown in pea mitochondria
[14]. The occurrence of respirasomes in potato mito-
chondria has been recently reported [15], making poss-

ible the occurrence of aggregates not separated in the
SDS ⁄ PAGE procedure. Whatever its origins, the lack
of this band in the cytosolic fraction showed that the
35 kDa band is specific for PTM and not a technical
artefact. In the same experiment, it was shown that the
PTM fraction did not contain b-tubulin, a protein
restricted to the cytoplasm, thus ruling out the possibil-
ity that the LDH detected arose from cytosolic contam-
ination. Contamination by other particulate ⁄ membrane
fractions was also ruled out, as we used purified mito-
chondria free of subcellular contamination (see Experi-
mental procedures).
The cytosolic fraction was free of mitochondrial
COX IV, showing that minimal rupture of PTM had
occurred during isolation. The intactness of the mit-
ochondrial outer membrane was measured as in Douce
et al. [16], and found to be 95%. In addition, we found
negligible fumarase activity, a plant mitochondrial
marker [17], in suspensions of mitochondria, thus fur-
ther confirming the intactness of the inner membrane.
To establish where LDH is localized within the
mitochondria and whether it is active, LDH was
assayed photometrically by measuring the absorbance
decrease of NADH [18] in the presence of pyruvate in
isolated PTM. When PTM (0.1 mg protein) were incu-
bated in the presence of NADH (0.2 mm), oxidation
occurred, catalyzed by external NADPH dehydro-
genases (Fig. 2A). The constant rate of decrease
in absorbance (about 130 nmolÆmin
)1

Æmg protein)
remained unchanged when pyruvate (10 mm) was
added; that is, the LDH was not accessible to sub-
strates. Consistently, no NADH formation was found
in the presence of 10 mml-lactate (not shown).
In order to rule out the possibility that l-lactate is
oxidized on the external face of the inner membrane,
with electrons transferred to the inner surface, intact
PTM were assayed for LDH activity by using phena-
zine methosulfate and dichloroindophenol (Fig. 2B), as
in Atlante et al. [19]. A negligible decrease in dichloro-
indophenol absorbance at 600 nm was found when
l-lactate (10 mm) was added to the PTM, either in the
absence or in the presence of 1 mm NAD
+
, confirming
the absence of LDH activity in the outer membrane, in
Fig. 1. Immunodetection of mitochondrial LDH. Solubilized protein
(30 and 40 lg) from both mitochondrial and cytosolic fractions was
analyzed by western blot as described in Experimental procedures.
Membrane blots were incubated with polyclonal anti-LDH, anti-
COX IV and anti-b-tubulin. COX IV and b-tubulin were used as mit-
ochondrial and cytosolic markers, respectively.
L-Lactate metabolism in PTM G. Paventi et al.
1460 FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS
the intermembrane space or on the outer side of the
mitochondrial inner membrane, or in any contamin-
ation of the mitochondrial suspension. Addition of
LDH externally produced a rapid decrease in absorp-
tion by dichloroindophenol. To validate the experi-

mental protocol that we had used, we confirmed that
addition of 0.3 mm glycerol 3-phosphate to PTM in
the presence of phenazine methosulfate and dichloroin-
dophenol resulted in a decrease of dichloroindophenol
absorbance with a rate of about 22 nmolÆmin
)1
Æmg
)1
protein, arising from the activity of glycerol 3-phos-
phate dehydrogenase (EC 1.1.1.8), which is located on
the outer side of the mitochondrial inner membrane
(Fig. 2B,a). On the other hand, no oxidation of succi-
nate by succinate dehydrogenase (which is located on
the matrix side of the inner mitochondrial membrane)
occurred with intact PTM. Oxidation did occur after
the addition of 0.1% Triton X-100, which solubilized
the mitochondrial membranes and allowed the interac-
tion between dichloroindophenol and the succinate
dehydrogenase complex (Fig. 2B,b).
To confirm that LDH is located in the internal mit-
ochondrial compartments, i.e. in the inner face of the
mitochondrial membrane or in the matrix, PTM were
solubilized with Triton X-100 (0.2%). Added NADH
(0.2 mm) was oxidized at a rate of about 105 nmolÆ
min
)1
Æmg
)1
protein, but when pyruvate was added, this
rate increased to about 170 nmolÆmin

)1
Æmg
)1
protein
(Fig. 2C), showing that LDH is present in the inner
mitochondrial compartments.
The kinetic characteristics of the LDH reaction were
studied by determining the dependence of the rate of
oxidation of NADH on increasing concentrations of
externally added pyruvate in solubilized mitochondria
Fig. 2. Mitochondrial LDH activity assay in
PTM. (A) PTM (0.1 mg) were incubated in
2 mL of the standard medium (see Experi-
mental procedures) containing 200 l
M
NADH, and the absorbance (A
340
) was con-
tinuously monitored. Pyruvate (PYR, 10 m
M)
was added at the time indicated by the
arrow. The numbers alongside the traces
refer to the rate of oxidation of NADH in
nmolÆmin
)1
Æmg
)1
protein. (B) PTM (0.2 mg)
were incubated in 2 mL of standard medium
in the presence of phenazine methosulfate

(PMS) (30 l
M) plus dichloroindophenol
(50 l
M), either in the presence or in the
absence of NAD
+
, and the absorbance
(A
600
) was continuously monitored. At the
times indicated by the arrows,
L-lactate
(
L-LAC, 10 mM) and LDH (0.1 eu) were
added. The insets show control experi-
ments: at the times indicated by the arrows,
glycerol 3-phosphate (G3P, 0.3 m
M) (a) and
succinate (SUCC, 5 m
M) and Triton X-100
(0.2%) (b) were added to mitochondria trea-
ted with phenazine methosulfate and dichlo-
roindophenol. Numbers along the curves are
rates of
L-lactate, succinate or glycerol
3-phosphate oxidation expressed as nmol
dichloroindophenol reducedÆmin
)1
Æmg
)1

pro-
tein. (C) PTM solubilized with Triton X-100
(0.2%) were incubated in 2 mL of the stand-
ard medium, containing 200 l
M NADH, and
the absorbance (A
340
) was continuously
monitored. Pyruvate (1.5 m
M) was added at
the time indicated by the arrow. The num-
bers alongside the traces refer to the rate of
oxidation of NADH in nmolÆmin
)1
Æmg
)1
protein.
G. Paventi et al.
L-Lactate metabolism in PTM
FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1461
(Fig. 3). Saturation kinetics were found with a K
m
value
of 0.63 ± 0.14 mm; the V
max
value was 85 ± 7 nmolÆ
min
)1
Æmg
)1

sample protein.
Unfortunately, spontaneous oxidation of the NADH
formed during the oxidation of l-lactate prevented
assay with l-lactate and NAD
+
as the substrate pair.
L-Lactate metabolism in mitochondria
Uptake and metabolism of l-lactate was further inves-
tigated in a set of experiments carried out with isolated
coupled PTM. The assumption here is that the mitoch-
ondrial LDH is devoted to oxidation of l-lactate
rather than reduction of pyruvate, as the latter would
be immediately oxidized by the pyruvate dehydroge-
nase complex (K
m
¼ 0.06 mm [20]). l-Lactate metabo-
lism was monitored by determining the ability of
externally added l-lactate to reduce intramitochondrial
dehydrogenase cofactors. In this case, we resorted to
fluorimetric techniques that have previously been used
to monitor changes in the redox state of pyridine nu-
cleotides [21]. Reduction of mitochondrial NAD(P)
+
was found to occur at a rate of 0.19 nmolÆmin
)1
Æmg
)1
protein when l-lactate was added to PTM previously
incubated with or without the uncoupler carbonyl
cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP)

and then treated with cyanide (CN
–
) (not shown). The
observed rate of reduction was, however, likely to be
underestimated, as the newly formed NAD(P)H would
be rapidly oxidized by the mitochondrial AOX, which
is usually activated by pyruvate, the product of l-lac-
tate metabolism. Hence, we checked whether inhibition
of AOX would cause an increase in the measured rate
of pyridine nucleotide reduction. To achieve this, use
was made of salicyl hydroxamic acid (SHAM, 1 mm),
an AOX inhibitor [22]. Addition of SHAM resulted in
a 150% increase in the measured rate of NAD(P)H
formation (Fig. 4A,a). Consistently, addition of l-lac-
tate to PTM previously incubated with SHAM caused
an increase in the rate of NAD(P)H formation
(Fig. 4A,b). In both cases, the addition of oxamate
(10 mm), an inhibitor of LDHs [23], completely
blocked the increase in fluorescence.
The failure of NADH, newly synthesized during
l-lactate oxidation, to be oxidized in the cytochrome
pathways was confirmed in another experiment (inset
to Fig. 4), in which we checked whether addition of
l-lactate to PTM could produce an increase in the
membrane potential as measured by using safranine O
as a fluorimetric probe. In contrast to succinate
(5 mm) and d-lactate (10 mm), l-lactate (10 mm) failed
to generate a change in electrical membrane potential,
DY. As expected, externally added FCCP (1 lm)
caused membrane potential collapse.

In the same experiment, we investigated, as in
Pastore et al. [24], whether l-lactate itself could activate
AOX, and obtained the results shown in Fig. 4B. In
this case, succinate was added to the mitochondria, fol-
lowed by ADP. Oxygen consumption via the electron
transfer chain was then blocked with CN
–
, and finally
l-lactate was added either in the absence (a) or presence
(b) of oxamate. In the former case, oxygen consump-
tion was restored, but in the latter, l-lactate addition
failed to restore oxygen uptake, thus showing that
l-lactate itself was not responsible for AOX activation.
It is likely that in the absence of oxamate, activation of
AOX was due to the newly formed pyruvate. In a par-
allel experiment, the ability of l-lactate to cause oxygen
uptake by PTM was investigated. We found that addi-
tion of 10 mml-lactate resulted in oxygen uptake at a
rate of 20 nmol O
2
Æmin
)1
Æmg
)1
protein. As expected,
this uptake was not stimulated by 0.2 mm ADP, and
was completely prevented following addition of SHAM
(not shown). Control experiments showed that SHAM
did not affect O
2

uptake due to either NADH or succi-
nate in the absence of CN
–
(not shown).
L-Lactate transport in PTM
The experiments reported above raise the question of
how l-lactate produced in the cytosol can cross the
mitochondrial membrane. To gain insight into this,
swelling experiments were carried out as in de Bari
et al. [25]; the results are shown in Fig. 5. PTM
Fig. 3. Assay of LDH activity in PTM solubilized with Triton X-100.
Pyruvate was added at the indicated concentrations to PTM treated
with Triton X-100 (0.2%). The rates (v
o
) of NADH oxidation, calcula-
ted as difference of rate in traces (b) and (a) of Fig. 2C, are
expressed as nmol pyruvate reducedÆmin
)1
Æmg
)1
protein.
L-Lactate metabolism in PTM G. Paventi et al.
1462 FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS
suspended in 0.18 m ammonium l-lactate showed
spontaneous swelling, but with a rate and to an extent
significantly lower than those found with ammonium
d-lactate, as judged by statistical analysis of five swell-
ing experiments using Student’s t-test (P<0.02). This
shows that both d-lactate and l-lactate can enter
PTM, but that the uptake is stereospecific. The results

indicate that l-lactate enters mitochondria in a proton-
compensated manner. The metabolite transport para-
digm proposed in Passarella et al. [21] suggests that
net carbon uptake by mitochondria is accompanied by
efflux of newly synthesized compound ⁄ s. We wished to
determine whether this applies in the case of l-lactate.
In particular, in the light of the occurrence of an l-lac-
tate ⁄ pyruvate shuttle in mammalian mitochondria, the
possible efflux of pyruvate as a result of l-lactate addi-
tion to PTM was investigated (Fig. 6A). The concen-
tration of pyruvate outside PTM was negligible, as
shown by the minimal change in absorbance at
334 nm found when commercial LDH was added
along with the NADH to complete the pyruvate-
detecting system (for details, see Experimental proce-
dures). On the other hand, in the presence of l-lactate
(10 mm), the absorbance at 334 nm decreased rapidly,
which is indicative of the appearance of pyruvate in
the extramitochondrial phase. This can be explained
on the basis that the l-lactate imported into the mito-
chondria forms pyruvate via the mitochondrial LDH,
Fig. 4. Effect of L-lactate addition to PTM.
Change in the redox state of pyridine nucle-
otides (A), failure to cause membrane poten-
tial generation (inset), and activation of AOX
(B). (A) PTM (0.2 mg protein) were incuba-
ted in 2 mL of the standard medium (see
Experimental procedures), and the fluores-
cence (k
ex

334 nm, k
em
456 nm) was con-
tinuously monitored. At the times indicated
by the arrows,
L-lactate (10 mm), SHAM
(1 m
M), and oxamate (OXAM, 10 mM) were
added. The numbers alongside the traces
refer to the rate of reduction of NAD(P)
+
in
nmolÆmin
)1
Æmg
)1
protein. Inset: PTM
(0.2 mg of protein) were incubated in 2 mL
of the standard medium in the presence of
2.5 l
M safranin, and fluorescence
(k
ex
520 nm, k
em
570 nm), measured as
arbitrary units (a.u.), was continuously
monitored. Where indicated by S,
L-lactate
(

L-LAC, 10 mM), D-lactate (D-LAC, 10 mM)or
succinate (5 m
M) were added separately;
where indicated, FCCP (1 l
M) was added.
(B) PTM (0.2 mg protein) were suspended
at 25 °C in 1 mL of respiratory medium, and
the amount of residual oxygen was meas-
ured as a function of time. Where indicated,
the following additions were made: succi-
nate (SUCC, 5 m
M), oxamate (10 mM), ADP
(0.2 m
M), cyanide (CN
–
,1mM), L-lactate
(
L-LAC, 10 mM), pyruvate (PYR, 5 mM), and
SHAM (1 m
M). Numbers along the curves
are rates of oxygen uptake expressed as
nmol O
2
Æmin
)1
Æmg
)1
mitochondrial protein.
G. Paventi et al.
L-Lactate metabolism in PTM

FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1463
and that the pyruvate exits in exchange for further
l-lactate. As expected, externally added oxamate
(5 mm) was found to prevent pyruvate efflux, further
confirming that PTM produce pyruvate from l-lactate
via LDH. It was found that oxamate under the same
conditions did not impair pyruvate detection via the
pyruvate-detecting system. In the same experiment, the
addition of phenylsuccinate, a nonpenetrant compound
that inhibits a variety of carriers ([21] and refs therein),
resulted in strong inhibition of the rate of NADH
oxidation. In contrast, the pyruvate carrier inhibitor
a-cyanocinnamate did not affect pyruvate efflux, thus
ruling out the involvement of such a transporter in the
observed process. To find out whether the rate of
NADH oxidation mirrors the transport across the
mitochondrial membrane, we investigated the depend-
ence of the inhibition of the rate of NADH oxidation
on increasing phenylsuccinate concentration (Fig. 6A,a).
Significantly, the y intercept of the line fitting the
experimental points measured in the presence of the
inhibitor coincided with the experimental values meas-
ured in the absence of inhibitor. In accordance with
the control strength analysis [21], this shows that phe-
nylsuccinate controls the rate of the measured process;
that is, the rate of decrease of the absorbance of NADH
reflects the rate of pyruvate efflux. The data in the inset
were also plotted as 1 ⁄ i against 1 ⁄ [inhibitor], where the
fractional inhibition i is 1 ) v
i

⁄ v
o
(inset b). The y inter-
cept was 1, showing that phenylsuccinate could com-
pletely prevent l-lactate ⁄ pyruvate exchange, and that no
pyruvate efflux from mitochondria can occur either by
diffusion or via a carrier insensitive to phenylsuccinate.
Figure 6B shows the results of measurements of the
rate of pyruvate efflux as a function of increasing
l-lactate concentration. The dependence was sig-
moidal, with a K
0.5
of about 27 mm.
Discussion
In this article, we show for the first time the occur-
rence of mitochondrial l-lactate metabolism in plants
arising from the presence of a mitochondrial LDH. In
particular, we show that l-lactate can be transported
into mitochondria from potato tubers, and metabo-
lized therein. The sequence of events involved in
mitochondrial metabolism of l-lactate (Scheme 1) is
envisaged as: uptake into mitochondria of l-lactate,
synthesized in the cytosol by anaerobic glycolysis;
oxidation of the l-lactate to pyruvate by the mito-
chondrial LDH located in an inner mitochondrial
compartment; activation of AOX by the newly syn-
thesized pyruvate and oxidation of the intramito-
chondrial NAD(P)H via AOX; and efflux of pyruvate
via a putative l-lactate ⁄ pyruvate antiporter and the oxi-
dation of cytosolic NADH in a non-energy-competent

l-lactate ⁄ pyruvate shuttle.
The results that we have reported are entirely con-
sistent with this scheme. Existence of a mitochondrial
LDH has been shown both by western blotting and
by enzymatic assay (Figs 1 and 2). Note that the
occurrence of LDH in plant mitochondria cannot be
predicted by informatics analysis in Arabidopsis thali-
ana, in which the occurrence of LDH in chloroplasts
is suggested. As LDH activity can be assayed only
after addition of Triton X-100 to PTM, we conclude
that this enzyme is localized on the inner side of the
mitochondrial inner membrane or in the matrix
space. The experiment with dichloroindophenol in
Fig. 2 rules out the possibility that l-lactate is oxid-
ized on the external face of the inner membrane,
with electrons transferred to the inner surface. Mito-
chondria can take up l-lactate with net carbon
uptake in a proton-compensated manner, as shown
by swelling experiments. Whether l-lactate uptake
occurs in a carrier-mediated manner remains to be
established. The mitochondrial LDH is an NAD(P)-
dependent enzyme, as shown by reduction of the
intramitochondrial pyridine nucleotide. In this regard,
the LDH of PTM is similar to the enzymes found in
mitochondria from rat heart [7] and rat liver [8],
rather than to that from Euglena mitochondria [26],
with the major difference that in PTM, NAD(P)H is
not reoxidized in the cytochrome pathway, but by
the AOX.
Uptake of l-lactate by PTM was investigated using

spectroscopic techniques under conditions in which the
mitochondria were metabolically active; consequently,
mitochondrial reactions and traffic of newly synthes-
ized substrates across the mitochondrial membrane
could be monitored.
Fig. 5. Mitochondrial swelling in ammonium D-lactate and L-lactate
solutions. PTM (0.2 mg protein) were rapidly added at 25 °Cto
2 mL of sucrose (SUCR, 0.36
M), ammonium L-lactate (NH
4
-L-LAC,
0.18
M), ammonium D-Lactate (NH
4
-D-LAC, 0.18 M), and ammonium
phosphate (NH
4
-Pi, 0.13 M), and mitochondrial swelling was monit-
ored as described in Experimental procedures.
L-Lactate metabolism in PTM G. Paventi et al.
1464 FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS
As in Valenti et al. [7], the l-lactate ⁄ pyruvate shuttle
was reconstructed in vitro. At present, we would sug-
gest that the l-lactate ⁄ pyruvate shuttle makes use of
both cytosolic and mitochondrial LDHs and of a puta-
tive l-lactate ⁄ pyruvate carrier. Application of control
strength criteria showed that oxidation of the NADH
outside PTM was limited by the rate of pyruvate
efflux, at least at a lower l-lactate concentration
(10 mm). However, the dissection of the steps involved

in pyruvate efflux requires further work.
Whatever the detailed mechanism, our findings
that plant mitochondria can metabolize l-lactate
requires a detailed revision of all the metabolic path-
ways dealing with l-lactate metabolism in plants.
There are also obvious important implications for
understanding how plants respond to hypoxic stress.
In this regard, the reconstructed l-lactate ⁄ pyruvate
shuttle appears to have the unique characteristic of
providing a non-energy-competent mechanism for the
oxidation of cytosolic NADH, perhaps active under
hypoxic conditions. Under conditions that limit oxy-
gen availability, complete substrate oxidation is
restricted by the lack of an electron acceptor. Conse-
quently, oxygen deficiency causes a decrease of
Fig. 6. Appearance of pyruvate in the extra-
mitochondrial phase induced by the addition
of
L-lactate (L-LAC) to PTM. (A) PTM
(0.1 mg protein) were suspended at 25 °C
in 2 mL of standard medium in the pres-
ence of the pyruvate-detecting system
(0.2 m
M NADH plus LDH 2 eu), and the
absorbance (A
340
) was continuously monit-
ored.
L-Lactate (10 mM) was added both in
the absence and in the presence of phenyl-

succinate (PheSUCC, 10 m
M), or oxamate
(5 m
M), or a-cyanocinnamate (a-CCN
–
,
0.1 m
M). Inset (a) is a Dixon plot of the inhi-
bition by phenylsuccinate of the rate of
pyruvate efflux due to externally added
L-lac-
tate; the
L-lactate concentration was 10 mM,
and the rate of pyruvate appearance, meas-
ured as described above, was determined
as a function of increasing phenylsuccinate
concentrations and expressed as nmolÆ
min
)1
Æmg
)1
protein. Inset (b) shows the plot
of 1 ⁄ i against 1 ⁄ [phenylsuccinate], where
i ¼ 1 ) v
i
⁄ v
o
, v
i
and v

o
being the rate of
L-lactate uptake in the presence and in the
absence of phenylsuccinate, respectively.
(B) Dependence of the rate of pyruvate
efflux on increasing concentrations of
L-lac-
tate. The experiments and measurements
were carried out as in (A).
G. Paventi et al.
L-Lactate metabolism in PTM
FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1465
mitochondrial respiration, which is partly compensa-
ted by increased glycolytic flux. As a result, ATP
levels decrease and NADH levels increase. It is
tempting to propose that in addition to other proces-
ses, involving nitrate, nitric oxide and hemoglobin,
which contribute to plant adaptation to hypoxia, a
similar role is played by mitochondrial metabolism
of l-lactate [27].
Scheme 1. L-Lactate metabolism in PTM. For an explanation see the text. ALA, alanine; AOX, alternative oxidase; GLU, glutamate; GPT, glu-
tamate pyruvate transaminase; aKG, a-ketoglutarate; cLDH, cytosolic lactate dehydrogenase; mLDH, mitochondrial lactate dehydrogenase;
L-LAC, L-lactate; MAL, malate; ME, malic enzyme; mim, mitochondrial inner membrane; NAD(P)H DH int, internal NAD(P)H dehydrogenase;
PDH, pyruvate dehydrogenase; PYR, pyruvate; SHAM, salicyl hydroxamic acid; UQ, ubiquinone; +, activation; –, inhibition.
L-Lactate metabolism in PTM G. Paventi et al.
1466 FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS
Experimental procedures
Materials
ADP, antimycin A, BSA, CN
–

, FCCP, bovine heart LDH
(EC 1.1.1.27), dichloroindophenol, dithiothreitol, EDTA,
EGTA, mannitol, NADH, NAD
+
, phenazine methosul-
fate, phenylmethanesulfonyl fluoride, Tris, Triton X-100,
Tween-20, ascorbic acid, glycerol 3-phosphate, d-lactic
acid, l-lactic acid, pyruvic acid, SHAM and succinic acid
were obtained from Sigma-Aldrich Chemie (Steinheim,
Germany); phenylsuccinate and skimmed milk powder
were obtained from Fluka (Mallinckrodt, Buchs, Switzer-
land). Sucrose was obtained from Baker (Deventer, the
Netherlands).
All chemicals were of the purest grade available, and were
used as Tris salts at pH 7.0–7.4, adjusted with Tris or HCl.
SHAM, antimycin A and FCCP were dissolved in ethanol.
Both primary (goat polyclonal anti-LDH, goat polyclonal
anti-b-tubulin and rabbit polyclonal anti-COX IV) and
secondary (anti-goat and anti-rabbit horseradish per-
oxidase-conjugated) sera were obtained from Abcam plc
(Cambridge, UK).
Potato tubers were initially obtained either from local
farmers (who use no chemical additives during plant
growth and harvest) or from local markets. It was found,
however, that concurrent experiments carried out with
isolated mitochondria from potato tubers obtained from
the markets gave conflicting, often quantitatively differ-
ent, results. This could be attributed to either different
ages of the tubers or to the use of chemical agents in
their growth or harvest, or to both of these factors. For

this reason, we have preferred, in the work described
here, to use only tubers obtained from farmers who do
not use chemical agents in their production process. It
was also necessary to carry out experiments over a short
time interval (2–3 weeks), as the tubers showed significant
changes in l-lactate metabolism with the time postharvest
[28].
Isolation of PTM and preparation of the cytosolic
fraction
PTM were isolated as in Pastore et al. [29], free of sub-
cellular contamination as determined in Neuburger &
Douce [30], and checked for their intactness as in Douce
et al. [16]. Mitochondrial protein content was determined
by the method of Lowry as in Harris [31], using BSA as
a standard.
The cytosolic fraction was obtained by centrifugation
(105 000 g for 60 min at 4 °C, Kontron Ultracentrifuge
Centrikon T2170, fixed-angle rotor TFT 65.13) of the
supernatant obtained during isolation of PTM. Glucose-6-
phosphate dehydrogenase (EC 1.1.1.49) was assayed as in
Loh & Waller [32].
Immunoblot analysis
Immunoblot analysis was performed on total mitochondrial
or total cytosolic protein by using antibodies raised against
LDH, COX IV and b-tubulin. Polyclonal antibodies recog-
nizing COX IV and b-tubulin were used as markers of
mitochondria and cytosol, respectively.
Both purified PTM and cytosolic protein were solubi-
lized in 1% Triton X-100, 500 mm NaCl, 50 mm
Tris ⁄ HCl (pH 7.5), 1 mm EGTA, 1 mm EDTA, 0.5 mm

dithiothreitol and 0.1 m m phenylmethanesulfonyl fluoride
for 30 min on ice. Protein content was determined using
the Bradford reagent (Bio-Rad Laboratories, Hercules,
CA, USA), with BSA as a standard. Solubilized proteins
(30 and 40 lg) were subjected to electrophoresis on 12%
SDS ⁄ polyacrylamide gel [33]. Following electrophoresis,
protein blots were transferred to a poly (vinylidene
difluoride) membrane. The membrane was blocked with
5% nonfat milk in Tris buffer solution, and incubated
overnight with the corresponding primary antibodies in
the blocking solution at 4 °C. After being washed three
times with Tris buffer solution plus Tween-20 (0.3%), the
membrane was incubated at room temperature for 1 h
with horseradish peroxidase-conjugated secondary anti-
body. The detected protein signals were visualized with
enhanced chemiluminescence western blotting reagents
(Amersham, ECL, Little Chalfont, UK). Relative absor-
bances and areas of bands were quantified using a GS-
700 Imaging Densitometer implemented with molecular
analyst software (Bio-Rad Laboratories).
LDH activity and other photometric assays
The LDH assay was performed photometrically at 340 nm
in the pyruvate-to-lactate direction as in Hoffman et al.
[18], by means of a Jasco (Tokyo, Japan) V-560 spectro-
photometer. Briefly, Triton X-100-solubilized PTM were
incubated at 25 °C in 2 mL of the standard medium con-
sisting of 0.125 m mannitol, 65 mm NaCl, 2.5 mm sodium
phosphate, 0.33 mm Na-EGTA, and 10 mm Tris ⁄ HCl
(pH 7.20), in the presence of 0.2 mm NADH. LDH activity
was assayed by measuring the difference between the rate

of decrease in absorbance at 340 nm due to the oxidation
of NADH before and after pyruvate addition. The activity
was expressed as nmol NADH oxidizedÆmin
)1
Æmg
)1
protein
(e
NADH
¼ 6.2 mm
)1
Æcm
)1
).
Glycerol-3-phosphate dehydrogenase and succinate dehy-
drogenase activities were checked photometrically at
600 nm as in Atlante et al. [19]. Briefly, PTM were incuba-
ted at 25 °C in 2 mL of the standard medium in the pres-
ence of 30 lm phenazine methosulfate and 50 lm
dichloroindophenol. Enzymatic activities were assayed by
measuring the decrease in absorbance at 600 nm due to the
reduction of dichloroindophenol that occurred when sub-
strates were added to the sample. The activities were
G. Paventi et al. L-Lactate metabolism in PTM
FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1467
expressed as nmol dichloroindophenol reducedÆmin
)1
Æmg
)1
protein (e

dichloroindophenol
¼ 21 mm
)1
Æcm
)1
).
Mitochondrial swelling was monitored photometrically at
546 nm. PTM (0.2 mg protein) were rapidly added to iso-
tonic solutions of ammonium salts, the pH values of which
were adjusted to 7.2, and the decrease in the absorbance
was continuously recorded.
Appearance of pyruvate outside the mitochondria was
monitored as in Valenti et al. [7] in 2 mL of standard
medium, using the pyruvate-detecting system consisting of
200 lm NADH plus 1 enzymatic unit (eu) of LDH. Oxida-
tion of NADH consequent on addition of l-lactate externally
was followed photometrically at 340 nm. l-Lactate itself had
no effect on the enzymatic reactions or on the absorbance
measured at 340 nm. Controls were carried out to ensure that
none of the compounds used affected the enzymes used to
reveal metabolite appearance outside mitochondria. The
rates of pyruvate efflux were obtained by the difference in
the oxidation rate of NADH before and after addition of
l-lactate, and are expressed as NADH oxidizedÆ min
)1
Æmg
)1
mitochondrial protein (e
NADH
¼ 6.2 mm

)1
Æcm
)1
).
Oxygen uptake studies
Oxygen uptake measurements were carried out at 25 °C
using a Rank Brothers Oxygraph (Cambridge, UK)
equipped with a Clark electrode in 1 mL of the respiratory
medium consisting of 0.3 m mannitol, 5 mm MgCl
2
,10mm
NaCl, 0.1% (w ⁄ v) defatted BSA, and 10 mm sodium phos-
phate buffer (pH 7.20).
Fluorimetric assays
Changes in the redox state of mitochondrial nicotinamide
nucleotide were monitored fluorimetrically at k
ex
334 nm
and k
em
456 nm, as in Valenti et al. [7]. PTM (0.2 mg of
protein) were incubated in 2 mL of standard medium,
either in the presence or in the absence of 1 lm FCCP, and
then treated with 1 mm CN
–
. NAD(P)
+
reduction due to
l-lactate addition was observed as fluorescence increase,
and the rate of reaction was calculated as the tangent to

the initial part of the progress curve and expressed as nmol
NAD(P)
+
reducedÆmin
)1
Æmg
)1
protein.
Changes in mitochondrial membrane potential (DY) were
followed by monitoring safranin O fluorescence changes
(k
ex
520 nm and k
em
570 nm) at 25 °C, as in Moore & Bon-
ner [34], by means of a Perkin-Elmer (Beaconsfield, UK)
LS50B spectrofluorimeter in 2 mL of standard medium
containing 2.5 lm safranin O and PTM (0.2 mg of protein).
Acknowledgements
The authors thank Professor Shawn Doonan for his
critical reading. This work was partially financed by
Fondi di Ricerca di Ateneo del Molize to SP and by
PRIN 2004 ‘Cross talk between organelles in response
to oxidative stress and programmed cell death in
plants.
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