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
Trang 1According 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: passarel@unimol.it
(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 anti-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.
Trang 2heart [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 metameta-bolism, 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 O2 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)1Æmg 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 mm l-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 LDH,
anti-COX IV and anti-b-tubulin anti-COX IV and b-tubulin were used as
mit-ochondrial and cytosolic markers, respectively.
Trang 3the 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 dichloroindichloroin-dophenol
absorbance with a rate of about 22 nmolÆmin)1Æmg)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 succisucci-nate 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)1Æmg)1protein, but when pyruvate was added, this rate increased to about 170 nmolÆmin)1Æmg)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)1Æmg)1protein (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
(A600) was continuously monitored At the
times indicated by the arrows, L -lactate
( L -LAC, 10 m M ) 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)1Æmg)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 (A340) 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)1Æmg)1
protein.
Trang 4(Fig 3) Saturation kinetics were found with a Kmvalue
of 0.63 ± 0.14 mm; the Vmax value was 85 ± 7 nmolÆ
min)1Æmg)1sample 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 (Km¼ 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)1Æmg)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 addiconsump-tion 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 mm l-lactate resulted in oxygen uptake at a rate of 20 nmol O2Æmin)1Æmg)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 O2uptake 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)1Æmg)1protein.
Trang 5suspended 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 usswell-ing 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 mitomito-chondrial 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 m M ) were
added The numbers alongside the traces
refer to the rate of reduction of NAD(P) + in
nmolÆmin)1Æmg)1protein 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 m M ), D -lactate ( D -LAC, 10 m M ) 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 m M ), ADP
(0.2 m M ), cyanide (CN – , 1 m M ), L -lactate
( L -LAC, 10 m M ), pyruvate (PYR, 5 m M ), and
SHAM (1 m M ) Numbers along the curves
are rates of oxygen uptake expressed as
nmol O 2 Æmin)1Æmg)1mitochondrial protein.
Trang 6and 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) vi⁄ vo(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 K0.5of 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 mitomito-chondrial 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 C to
2 mL of sucrose (SUCR, 0.36 M ), ammonium L -lactate (NH 4 - L -LAC,
0.18 M ), ammonium D -Lactate (NH4- D -LAC, 0.18 M ), and ammonium
phosphate (NH4-Pi, 0.13 M ), and mitochondrial swelling was
monit-ored as described in Experimental procedures.
Trang 7As 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 (A340) was continuously
monit-ored L -Lactate (10 m M ) 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 m M ,
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)1Æmg)1protein Inset (b) shows the plot
of 1 ⁄ i against 1 ⁄ [phenylsuccinate], where
i ¼ 1 ) v i ⁄ v o , viand vobeing 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).
Trang 8mitochondrial 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.
Trang 9ADP, 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 4C, 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 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 mm 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 4C 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 25C 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)1Æmg)1protein (eNADH¼ 6.2 mm)1Æcm)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 25C 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
Trang 10expressed as nmol dichloroindophenol reducedÆmin)1Æmg)1
protein (edichloroindophenol¼ 21 mm)1Æcm)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 addiOxida-tion 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 oxidizedÆmin)1Æmg)1
mitochondrial protein (eNADH¼ 6.2 mm)1Æcm)1)
Oxygen uptake studies
Oxygen uptake measurements were carried out at 25C
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 MgCl2, 10 mm
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 kex334 nm
and kem456 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)1Æmg)1protein
Changes in mitochondrial membrane potential (DY) were
followed by monitoring safranin O fluorescence changes
(kex520 nm and kem570 nm) at 25C, 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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