A high affinity pyruvate decarboxylase is present in cottonwood leaf veins and petioles: A second source of leaf acetaldehyde emission?

Article abstract of DOI:10.1016/j.phytochem.2009.07.015

Considerable evidence indicates that acetaldehyde is released from the leaves of a variety of plants. The conventional explanation for this is that ethanol formed in the roots is transported to the leaves where it is converted to acetaldehyde by the alcohol dehydrogenase (ADH) found in the leaves. It is possible that acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase (PDC), an enzyme with an uncertain metabolic role, which has been detected, but not characterized, in cottonwood leaves. We have found that leaf PDC is present in leaf veins and petioles, as well as in non-vein tissues. Veins and petioles contained measurable pyruvate concentrations in the range of 2 mM. The leaf vein form of the enzyme was purified approximately 143-fold, and, at the optimum pH of 5.6, the K_m value for pyruvate was 42 μM. This K_m is lower than the typical millimolar range seen for PDCs from other sources. The purified leaf PDC also decarboxylates 2-ketobutyric acid (K_m = 2.2 mM). We conclude that there are several possible sources of acetaldehyde production in cottonwood leaves: the well-characterized root-derived ethanol oxidation by ADH in leaves, and the decarboxylation of pyruvate by PDC in leaf veins, petioles, and other leaf tissues. Significantly, the leaf vein form of PDC with its high affinity for pyruvate, could function to shunt pyruvate carbon to the pyruvate dehydrogenase by-pass and thus protect the metabolically active vascular bundle cells from the effects of oxygen deprivation.

Full text of DOI:10.1016/j.phytochem.2009.07.015

Phytochemistry 70 (2009) 1217–1221
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
A high affinity pyruvate decarboxylase is present in cottonwood leaf veins
and petioles: A second source of leaf acetaldehyde emission?
a
a,*
b
a
T. Nguyen , A.-M. Drotar , R.K. Monson , R. Fall
a
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, United States
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Considerable evidence indicates that acetaldehyde is released from the leaves of a variety of plants. The
conventional explanation for this is that ethanol formed in the roots is transported to the leaves where it
is converted to acetaldehyde by the alcohol dehydrogenase (ADH) found in the leaves. It is possible that
acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase (PDC), an enzyme with
an uncertain metabolic role, which has been detected, but not characterized, in cottonwood leaves. We
have found that leaf PDC is present in leaf veins and petioles, as well as in non-vein tissues. Veins and
petioles contained measurable pyruvate concentrations in the range of 2 mM. The leaf vein form of the
Received 10 December 2008
Received in revised form 24 June 2009
Available online 19 August 2009
Keywords:
Populus deltoides
Eastern cottonwood
Pyruvate decarboxylase
Enzyme purification
Acetaldehyde
Pyruvic acid
Veins
Petioles
Vascular bundles
enzyme was purified approximately 143-fold, and, at the optimum pH of 5.6, the K
was 42 M. This K is lower than the typical millimolar range seen for PDCs from other sources. The puri-
fied leaf PDC also decarboxylates 2-ketobutyric acid (K = 2.2 mM). We conclude that there are several
m
value for pyruvate
l
m
m
possible sources of acetaldehyde production in cottonwood leaves: the well-characterized root-derived
ethanol oxidation by ADH in leaves, and the decarboxylation of pyruvate by PDC in leaf veins, petioles,
and other leaf tissues. Significantly, the leaf vein form of PDC with its high affinity for pyruvate, could
function to shunt pyruvate carbon to the pyruvate dehydrogenase by-pass and thus protect the metabol-
ically active vascular bundle cells from the effects of oxygen deprivation.
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
Acetaldehyde is abundant and reactive in the atmosphere, and
tion stream transports ethanol from root to leaves to be oxidized
by ADH and reform acetaldehyde, an intermediate in acetate for-
mation (Harry and Kimmerer, 1991; Kreuzwieser et al., 1999a).
Some ethanol and acetaldehyde are lost to the air by evaporation
during their transport (Kreuzwieser et al., 1999b; Tadege et al.,
1999).
More recently, a second model supports the importance of PDC
activity in leaves, in addition to its role in metabolism of root-de-
rived ethanol. Our lab found that acetaldehyde is released from
cottonwood and sycamore leaves following light–dark transitions
(Karl et al., 2002). Graus et al. (2004) confirmed this observation
in poplar leaves. This release suggests a biochemical model relating
leaf acetaldehyde emission to the imbalance of cytosolic and mito-
chondrial pyruvate concentrations during light–dark transitions.
According to this model, following a sunfleck, photosynthesis and
mitochondrial pyruvate dehydrogenase (PDH), a light activated en-
zyme, are shut down in the shaded leaf. This could then lead to
cytosolic pyruvate accumulation, which can be detrimental to cel-
lular biological functions due to a concomitant drop in cytosolic
pH. PDC, in this situation, converts pyruvate to acetaldehyde and
it has been suggested that there are large unknown biogenic
sources of this aldehyde (Singh et al., 2001). Acetaldehyde emis-
sion is known to occur in leaves in response to flooding (reviewed
in Kreuzwieser et al. (1999b) and Tadege et al. (1999)) and during
light–dark transitions (Karl et al., 2002; Graus et al., 2004). In addi-
tion, the leaves of many higher plants under imposed anaerobic
conditions produce acetaldehyde (Kimmerer and Macdonald,
1
987). Two metabolic models have been proposed to explain leaf
acetaldehyde emission. In the most studied model, acetaldehyde
emission during flooding is primarily due to anoxic production of
ethanol in roots. During flooding, roots utilize glycolysis as the
main source of ATP, resulting in the formation of pyruvate. The
pyruvate is then decarboxylated by the root’s pyruvate decarboxyl-
ase (PDC) to form acetaldehyde, which is converted to ethanol by
+
alcohol dehydrogenase (ADH) to regenerate NAD . The transpira-
*
Corresponding author. Address: Department of Chemistry, Biochemistry, Uni-
2
CO and re-establishes the normal cellular pH. Therefore, elevation
of leaf cytosolic pyruvate concentration could lead to acetaldehyde
emission in a pathway independent of ethanol oxidation. Such a
versity of Colorado, Campus Box 215, Boulder, CO 80309, United States. Tel.: +1 303
4
0
92 7914; fax: +1 303 492 5894.
E-mail address: amtdrotar@gmail.com (A.-M. Drotar).
031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.07.015

1
218
T. Nguyen et al. / Phytochemistry 70 (2009) 1217–1221
pathway could be the PDH by-pass, which has been reviewed in
work by Gass et al. (2005) in their work on PDC activity in aerobic
pollen tissue.
established. SDS–PAGE analysis of different preparations of the
purified enzyme showed two major bands in the range of 67 kDa
(not shown). The PDC obtained could be stored in extract buffer
for 2 months at 4 °C with no loss of activity.
However, Graus et al. (2004) do not completely agree with the
sunfleck model. While they also showed increased acetaldehyde
emissions during light–dark transitions, when PDH was inhibited
or anaerobic metabolism was imposed (presumably increasing
cytosolic pyruvate concentrations), they did not observe increased
emissions during light–dark transitions when aldehyde dehydro-
genase was inhibited. Aldehyde dehydrogenase is a plant mito-
chondrial enzyme that oxidizes acetaldehyde for energy
metabolism as part of the PDH by-pass (Nakazono et al., 2000; Gass
et al., 2005).
2.2. The pH optimum of PDC
The effect of pH on kinetic behavior of leaf PDC was also inves-
tigated. Prior to these experiments, PDC was saturated with cofac-
tors by incubating in a buffer containing 2 mM TPP and 2 mM
MgCl2 to ensure co-factor saturation. It was then assayed in MES
buffers of varying pH, and the velocity of the reaction was plotted.
The enzyme had a pH optimum of 5.6 (Fig. 1).
PDC has been studied from many plant sources including: wheat
germ, maize kernels, rice, tobacco and pea, primarily from roots or
seeds (reviewed in Mucke et al. (1995). It has also been studied in
petunia and pollen tube tissues (Tadege and Kuhlemeier, 1997; Gass
et al., 2005), where the enzyme functions in an aerobic environment.
Notably, Kimmerer (1987) found PDC activity in cottonwood leaves,
determined that this enzyme is constitutive, but concluded that its
role in aerobic leaves is uncertain. There are multiple PDC genes in
some plants – for example, Arabidopsis has four PDC genes (Kurste-
iner et al., 2003), and in Arabidopsis PDC1 is the most abundant form
in seeds and their siliques, but the forms in leaves and stems are not
known. In petunia, analysis of the PDC gene expression showed that
Pdc2 transcripts dominated Pdc1 transcripts in leaves, similar to the
pattern inroots (Gass et al., 2005). A search of the annotatedP. tricho-
carpa genome (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.ho-
me.html) suggests that numerous PDC genes may be present in
poplar species.
The goal of this work was to increase our knowledge of leaf PDC,
one of the key enzymes involved in the biochemical scenarios pro-
posed for acetaldehyde emissions from vegetation. We focused on
characterizing this enzyme from leaves of a woody plant, cotton-
wood, that are known to contain constitutive levels of PDC as men-
tioned above (Kimmerer, 1987). The detection of PDC activity in
leave tissue and petioles was analyzed, and the enzyme was par-
tially purified from leaf veins. The kinetic behavior of the highly
purified enzyme was notable and these results are discussed in
relation to the possible role this enzyme plays in vascular bundle
metabolism.
2.3. The kinetic parameters of PDC
At the optimal pH, the Km value for pyruvic acid was measured.
The resultant initial velocity data was analyzed using Prism 3.0
(Fig. 2A). A Km value for pyruvic acid of 42 lM was obtained. The
pyruvate analog, 2-ketobutyric acid, was also tested. PDC activity
at varying concentrations of 2-ketobutyric acid gave a Km of
2.2 mM (Fig. 2B).
2.4. PDC activity: leaf vein vs. non-vein vs. petiole tissues
It is not known where PDC is located in leaf tissue. To determine
this, vein tissue was dissected from non-vein tissues. Petioles were
also removed from the leaves. Each tissue was extracted and as-
sayed for PDC activity. In these experiments, as shown in Fig. 3,
PDC activity is 5–6Â higher in vein compared to non-vein tissue.
In some experiments, extracts of the petioles were also made
and the activity determined. Fig. 3 shows the specific activity in
petiole extracts is 2.5Â that in vein extracts.
2.5. Pyruvate concentration in veins and petioles
The pyruvate concentration in the vascular bundle is not
known. To see if pyruvate could be detected, extracts from leaf vein
and petioles were boiled and centrifuged to remove protein. The
supernatant was assayed using lactate dehydrogenase and NADH.
The protein concentration of the original unboiled extract was
determined, and the amount of pyruvate detected per mg protein
was calculated. The amount of pyruvate detected was similar for
petiole and vein extracts, as shown in Table 2. Assuming 50% water
content in these tissues, concentrations of pyruvate were calcu-
lated to be 1.7–3.1 mM.
2
. Results
2.1. Purification of pyruvate decarboxylase
PDC was partially purified from crude extracts obtained from
the leaf veins of flooded cottonwood saplings. Flooded saplings
were used because in some experiments there was moderate
induction of leaf PDC activity. A 143-fold increase was seen in spe-
cific activity using the steps summarized in Table 1. It is notable
that preparations done during the summer showed higher, but var-
iable, purification factors, compared to preparations obtained dur-
ing other seasons. The reason for this variability was not fully
3. Discussion
The work presented here is to our knowledge one of the first
characterizations of leaf tissue PDC, an enzyme that converts pyru-
vate to acetaldehyde and CO . PDC has been studied extensively in
2
plant roots, especially during anoxia induced by flooding (Tadege
et al., 1998). Our interest in leaf PDC has been driven by our work
Table 1
a
Partial purification of leaf vein PDC from cottonwood.
Step
Activity (nkatal/ml)
Protein (mg/ml)
Specific activity (nkatal/mg)
Purification factor
Yield (%)
1
2
3
4
5
. Crude extractb
10.4
8.28
8.33
13.3
4.16
0.615
0.382
0.189
0.157
0.0174
1.67
2.17
4.34
8.52
239
1
1.3
2.6
5.1
143
100
. Ammonium sulfate precipitation supernatant (0–30%)
. Ammonium sulfate precipitation supernatant (30–60%)
. Ammonium sulfate precipitation supernatant (60–90%)
. Phenyl Sepharose
57.9
26.6
20.4
9.43
a
Flooded cottonwood saplings were used, as explained in Section 2.1. One gram of vein tissue ground in 10 ml of extract buffer was used.
Crude extract included the Sephadex G-25 and protamine sulfate pretreatment as described in the text.
b

T. Nguyen et al. / Phytochemistry 70 (2009) 1217–1221
1219
1
.2
1
1.4
1.2
1
0
0
0
0
.8
.6
.4
.2
0
0
0
0
0
.8
.6
.4
.2
4
.5
5.5
6.5
7.5
pH
0
non-vein
vein
petiole
Fig. 1. Kinetic plot of PDC initial velocity vs. pH. The assay conditions are described
in the Section 5. Purified PDC was used for these assays, and although statistical
analysis was not carried out, each point represents the mean of triplicate
determinations.
Leaf Tissue
Fig. 3. PDC activity of vein, non-vein, and petiole tissue extracts from cottonwood
leaves. PDC specific activity was measured in crude extracts of the three different
leaf tissues. The graph includes the average and 95% confidence limits of
determinations from 28-different extracts of vein, 20-different extracts of petioles
and eight-different extracts of non-vein tissue. Each extract was assayed in
duplicate.
1
1
1
4
2
0
8
6
4
2
0
Table 2
Pyruvic acid determination from crude extracts of vein and petiole tissue.
A
Extract type
l
mol pyruvate/mg protein
Standard error of the mean
a
Vein
0.38
0.44
0.04
0.03
Petioleb
a
Six different vein extracts were assayed in duplicate.
Two different petiole extracts were assayed in duplicate.
b
0
1
2
3
[
Pyruvic acid] [mM]
2
004), but also because their leaves have large leaf veins that are
1
0
8
6
4
2
0
easy to dissect from the surrounding mesophyll cells. We discov-
ered that the specific activity of PDC in poplar vascular tissues,
such petioles and veins, is significantly higher than in the bulk tis-
sue of the leaf (Fig. 3); notably the non-vein tissue has low PDC
specific activity, but contains the bulk of assayable soluble PDC.
This suggests that PDC is enriched in the extractable protein frac-
tion of vascular tissues. Consistent with this idea, Kimmerer and
Stringer (1988) have also found PDC activity in the vascular cam-
bium of cottonwood stems.
The ability to easily isolate leaf veins greatly facilitated the puri-
fication of the enzyme. Although we did not achieve homogeneity,
we did purify leaf vein PDC approximately 143-fold, and deter-
mined that the enzyme had an apparent molecular mass of
B
0
5
10
15
20
[
2-Ketobutyric acid] (mM)
Fig. 2. Kinetic analysis of purified leaf vein PDC. Activity was measured at various
concentrations of pyruvic or 2-ketobutyric acid. (A) Velocity vs. pyruvic acid
concentration. (B) Velocity versus 2-ketobutyric acid concentration. Plot A sum-
marizes the mean values of 10 experiments (each in duplicate), and K
values were computed from a Prism 3.0 program; here the R value of the curve fit
6
7 kDa, similar to other plant PDCs (Mucke et al., 1995). This level
of purification allowed further characterization of the enzyme. Like
root PDCs, the leaf enzyme has a low pH optimum (pH 5.6). Unlike
m
and Vmax
Ó
2
root PDCs that have been studied, the K
(42 M); this compares to the higher K
(0.9 mM) (Lee and Langstonunkefer, 1985), wheat germ (3 mM)
Kuo et al., 1986) and yeast (1 mM) (Boiteux and Hess, 1970).
The low K for pyruvate in leaf PDC suggests that levels of pyru-
m
for pyruvate is quite low
was >0.95. For plot B results from only a single experiment are shown, again mean
Ó
l
m
s for PDC from maize seeds
values were used and K
m
and Vmax values were computed from a Prism 3.0
program; R2 value of the curve fit was also >0.95.
(
m
to understand acetaldehyde emissions from leaves and forest veg-
etation (Karl et al., 2002, 2003). The sources of this reactive alde-
hyde are of interest to atmospheric chemists. For example, Singh
et al. (2001) estimated that global sources of atmospheric acetalde-
vate in vascular bundle tissues could be relatively low. In other
studies by Tcherkez et al. (2005) the pyruvate concentration has
been estimated to be less than 2 mM in leaf cells of the French bean
(Phaseolus vulgaris). In this work, we measured similar levels of
pyruvate in both vein and petiole extracts, but the level of pyruvate
in the cellular compartment containing PDC is unknown. In addi-
tion, the cellular complexity of vascular bundles needs to be con-
sidered in future work on the metabolic role of the enzyme in
this tissue (Hibberd and Quick, 2002).
À1
hyde are 80–160 Tg yr , and also proposed that large biogenic
sources are missing from the global acetaldehyde inventory. Our
interest in characterizing leaf PDC is part of a larger effort to deter-
mine if enzymatic pathways utilizing PDC contribute to these
missing biogenic acetaldehyde sources.
We focused on cottonwood leaves as they are a significant
source of acetaldehyde emissions (Karl et al., 2002; Graus et al.,
Kimmerer and MacDonald (1987) suggested that the presence
of PDC in highly aerobic leaves is a paradox because this enzyme

1
220
T. Nguyen et al. / Phytochemistry 70 (2009) 1217–1221
is thought be part of a fermentative pathway (Kreuzwieser et al.,
999a). Recently however, it has been shown that oxygen
concentrations can fall to levels as low as 5–6% in phloem tissue
Geigenberger, 2003). Phloem tissue has a high metabolic rate,
but its cells are compact and it has few intracellular air spaces. It
is therefore possible, that the presence of PDC represents an adap-
tation to low oxygen concentrations in vascular bundle tissue. In
low oxygen environments an induction in PDC activity would be
a response of the plant to adopt energetically more efficient path-
ways that also conserve the available oxygen (van Dongen et al.,
pestle at 4 °C in an extract buffer at a ratio of 10 ml buffer per gram
of fresh plant tissue based on the method of Kimmerer (1987). Ex-
tract buffer contained 100 mM Hepes (pH 7.55), 2 mM MgCl ,
2
1 mM NAD, 2 mM thiamine pyrophosphate (TPP) and 2 mM dithi-
othreitol (DTT). One gram of poly(vinylpolypyrrolidone) (PVPP) per
gram of tissue was added. The enzyme extract was centrifuged at
10,000g for 20–30 min at 4 °C. The supernatant was isolated and
passed through a G-25 Sephadex column (5 ml) to remove low
molecular weight components. The protein fraction was collected
and kept at 4 °C.
1
(
2
003), such as the PDH by-pass discussed above. It is also possible
In some experiments a similar extraction method, except for
the Sephadex G-25 step, was used to assay PDC activity in
petioles.
that such a hypoxic response could explain in part the light-to-
dark releases we observed in these leaves (Karl et al., 2002), as dis-
cussed in the Introduction.
Aerobic PDC activity has also been observed in metabolically
active pollen tissue. Investigators working on developing pollen
tissue from petunia, have shown the presence of an aerobic
PDC (Tadege and Kuhlemeier, 1997; Gass et al., 2005). They sug-
gested that pollen has elevated glycolysis resulting in high pyru-
vate levels and flux through PDC. This leads to acetyl-CoA
formation independent of the mitochondrial pyruvate dehydro-
genase (PDH) reaction. This PDH by-pass pathway can be used
for energy and biosynthetic purposes, in these metabolically ac-
tive aerobic tissues.
5
.3. PDC purification
PDC purification is a multi-step process involving gel filtra-
tion, salting out, and hydrophobic interaction. All purification
steps, except for Phenyl Sepharose, were done at 4 °C. The first
step in PDC purification was to perform gel filtration using a
G-25 Sephadex column to remove low molecular weight sub-
stances; the bed volume was 5 ml per 0.5 ml of extract. The de-
salted pool was treated with protamine sulfate (2% w/v stock
solution) at a ratio of 1 ml protamine sulfate for every 20 ml
of protein extract. After stirring for 10 min and centrifugation
at 10,000g for 20 min, the pellet was discarded. The supernatant
was adjusted to 30% saturation with solid ammonium sulfate
(NH ) SO ), stirred for 60 min, and centrifuged (10,000g,
4
. Concluding remarks
For cottonwood leaves, the localization of a high affinity PDC in
4
2
4
the leaf veins and petioles is of interest, and it is possible that the
PDH by-pass could be present in these tissues. Future work should
establish if cottonwood vein cells contain the other enzymes of this
by-pass, and clarify the role of these cells in leaf acetaldehyde
emission. In addition, as discussed in the Introduction there is
growing evidence that there are multiple PDC genes in plants. Per-
haps the PDC purified from leaf veins, with its high affinity for
pyruvate, represents a unique form of the enzyme, and could be
a significant additional source of leaf acetaldehyde emission. It will
be of interest to see which PDC gene encodes this enzyme, if its
expression is tissue specific, to completely purify the enzyme,
and to characterize its metabolic role.
20 min). The supernatant was adjusted to 60% saturation, stirred
for another 60 min, and centrifuged (10,000g, 20 min). The
supernatant of this step was then adjusted to 90% salt satura-
tion, stirred for 60 min, and centrifuged; PDC remained in the
supernatant. Aliquots of the 90% (NH ) SO supernantant were
applied to a Phenyl Sepharose 4LB column in a ratio of 1 ml
supernatant to 20 ml bed volume (in different experiments); in
each case, the column was equilibrated with extract buffer.
Bound PDC was eluted by 50% (v/v) ethylene glycol. At each
stage, the fractions were tested for PDC activity. The purified en-
zyme was stored at 4 °C.
4
2
4
5.4. PDC enzyme assay
5
. Experimental
PDC activity was determined by a coupled assay (Kimmerer,
987). In this assay, pyruvate is first decarboxylated by PDC to
5.1. Plant materials and growth conditions
1
form acetaldehyde and carbon dioxide. Acetaldehyde is then re-
duced by alcohol dehydrogenase (ADH) using NADH to form etha-
nol. The assay buffer consisted of 100 mM MES (pH 5.6), 5 mM
Cottonwood leaves were cut from trees of Populus deltoides Barr.
S7c8 East Texas day-neutral clone) growing in glasshouses at Uni-
(
versity of Colorado, Boulder as previously described in Rosenstiel
et al. (2003). Trees were watered twice per day in the early morn-
ing (8 am) and early afternoon (2 pm). They were also fertilized 3Â
per week; 2Â per week they were fertilized with Scott’s General
Purpose fertilizer, and one day per week they were fertilized with
Scott’s Acid Special fertilizer to enhance growth. The trees were
also sprayed with pesticides on bimonthly intervals.
MgCl
7.85 mM NADH stock solution was added to 500
fer, followed by 310 l of H O, 100 l of protein extract, 30
equine liver ADH stock solution (2 units) purchased from Sigma–
Aldrich and 10 l of 200 mM pyruvate stock solution. NADH oxida-
tion activity was measured by a spectrophotometer at 340 nm at
5 °C for 3 min before the introduction of pyruvate. Decarboxyl-
2
, 1 mM DTT, and 1 mM TPP. To perform the assay, 10
l of assay buf-
l of
ll of
1
l
l
2
l
l
l
2
To flood plants to be used in the purification of PDC, greenhouse
grown trees (in 5 gal pots) were carefully moved into 50 l contain-
ers filled with water (taking care to insure that no air bubbles were
introduced). Plants were flooded for 3 days prior to leaf removal
and enzyme extraction.
ation of 2-ketobutyric acid was assayed, using the same method
by replacing pyruvate as the substrate. Kinetic determinations for
K
m
values were analyzed using Prism 3.0 software (GraphPad.com).
The relative recovery of PDC activity from different leaf tissue
extracts is difficult to assess; for example, there may be different
levels of polyphenols that may inactivate the enzyme. This was
partly addressed by mixing extracts to determine if a soluble inhib-
itor was present. No inhibition was detected when extracts from
vein and non-vein tissue were mixed. Inactivation during extrac-
tion is more difficult to determine. Thus we report the amount of
extractable enzyme recovered only.
5.2. Enzyme extraction
The veins or mesophyll cells of the leaves were removed with a
razor blade. The total tissue mass was quantified and then frozen in
liq. N
. Subsequently, they were then ground using a mortar and
2

T. Nguyen et al. / Phytochemistry 70 (2009) 1217–1221
1221
5.5. Pyruvate determination
Geigenberger, P., 2003. Response of plant metabolism to too little oxygen. Curr.
Opin. Plant Biol. 6, 247–256.
Graus, M., Schinitzler, J., Hansel, A., Cojocariu, C., Rennenberg, H., Wisthaler, A.,
Kreuzwieser, J., 2004. Transient release of oxygenated volatile organic
compounds during light–dark transitions in grey poplar leaves. Plant Physiol.
The concentration of pyruvate in some of the leaf vein and pet-
iole extracts was determined using rabbit muscle lactate dehydro-
genase (LDH; Sigma–Aldrich) was diluted 1/100 in 0.2 M HEPES pH
135, 1967–1975.
Harry, D.E., Kimmerer, T.W., 1991. Molecular genetics and physiology of
alcohol dehydrogenase in woody plants. Forest Ecol. Manage. 43, 251–
272.
Hibberd, J.M., Quick, W.P., 2002. Characteristics of C-4 photosynthesis in stems and
petioles of C-3 flowering plants. Nature 415, 451–454.
Karl, T., Curtis, A.J., Rosenstiel, T.N., Monson, R.K., Fall, R., 2002. Transient releases of
acetaldehyde from tree leaves – products of a pyruvate overflow mechanism?
Plant Cell Environ. 25, 1121–1131.
Karl, T., Guenther, A., Spirig, C., Hansel, A., Fall, R., 2003. Seasonal variation of
biogenic VOC emissions above a mixed hardwood forest in northern Michigan.
Geophys. Res. Lett. 30. doi:10.1029/2003GL018432.
7
K
.3. The assay was performed in 0.2 M HEPES pH 7.3. To determine
and Vmax of the purchased enzyme under the prevailing condi-
tions, 1 ml buffer plus varying concentrations of pyruvate were
added to a cuvette, then 10 l of 17.85 mM NADH was added plus
l (0.5 units) of the diluted LDH to initiate the reaction. The de-
m
l
4
l
crease in absorbance at 340 nm was determined. K
m
for lactate
was found to be 1.5 mM.
The assay was used to determine pyruvate concentrations in
vein and petiole extracts. The extracts were made, leaving out
Kimmerer, T.W., 1987. Alcohol dehydrogenase and pyruvate decarboxylase activity
in leaves and roots of eastern cottonwood (Populus deltoides Bartr) and soybean
+
NAD from the extraction buffer and were not run over a Sephadex
(Glycine max L.). Plant Physiol. 84, 1210–1213.
G-25 column. After centrifugation, they were placed in boiling
water for 3 min. and then centrifuged at 10,000g for 20 min. Levels
Kimmerer, T.W., Macdonald, R.C., 1987. Acetaldehyde and ethanol biosynthesis in
leaves of plants. Plant Physiol. 84, 1204–1209.
Kimmerer, T.W., Stringer, M.A., 1988. Alcohol dehydrogenase and ethanol in the
stems of trees – evidence for anaerobic metabolism in the vascular cambium.
Plant Physiol. 87, 693–697.
of 100 or 200
of pyruvate. The background was determined before adding LDH.
Since the pyruvate concentration was far below its apparent K
the concentration of pyruvate was determined using K (v/
max) = [S]. The protein concentrations of the extracts before boil-
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5
.6. Protein concentration
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(
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Acknowledgements
5
This work was supported by a grant from NSF (IOB-0543895).
We would like to thank Todd Rosenstiel for advice and encourage-
ment of this work, Pete Casey for growth of cottonwood saplings,
and Kolby Jardine for helpful discussions.
2
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