phosphate buffer pH 7.0 with a constant current of 23 mA per gel. SOD activity was determined by
the negative staining procedure using nitroblue tetrazolium. For SOD isozyme identification, the
gels were incubated prior to activity staining in 5 mM H
2
O
2
or 2 mM KCN. Of the three isozymes that may be present, the Mn form is insensitive to
either treatment, while the CuZn isozyme is sensi- tive to CN
−
. The Fe isozyme is sensitive to H
2
O
2
[16]. APX activity was determined as described [14]. This assay is based on the ability of ascorbate to
reduce NBT, in the presence of tetramethylethylene- diamine to formazan. Ascorbate peroxidase pre-
vents the formation of formazan in the presence of H
2
O
2
, and thus the ascorbate peroxidase activity is seen as an achromatic band on a purple-blue
background. Activities
were quantified
using Molecular Analyst software BioRad, Inc. on dig-
ital images recovered from a CCD camera driven GelDoc 1000 system. The linear range of activity
was determined by loading increasing amounts of cell extract on the gels and comparing the pixel
density of the bands with the amounts loaded.
2
.
3
. Lipid peroxide and chlorophyll measurements Lipids were extracted from growing plant rosette
leaves at various stages following Bligh and Dyer’s method [17]. Lipid peroxides were quantified by the
FOX method [18] using the PeroXoquant™ Quan- titative Peroxide Assay from Pierce Chemical Co.
Samples 50 ml were incubated with 500 ml of working reagent which consists of 1 volume of 25
mM of Fe
2 +
and 2.5 M H
2
SO
4
, and 100 volumes of 4 mM BHT and 125 mM xylenol orange in
methanol. After a 20 min incubation at room temperature, the absorbances at 560 nm were read
on an HP 8452A diode array spectrophotometer. Freshly prepared hydrogen peroxide solutions were
used as standard.
A portion of total lipid extracts was used for chlorophyll determination. The lipid extracts were
diluted with N,N-dimethylformamide DMF. Ab- sorbances at 648 and 664 nm were recorded using
DMF as blank. The total chlorophyll content was calculated as described [19].
2
.
4
. Lipid peroxide characterization Standards were prepared by autoxidation of
linoleic and linolenic acids and subsequent reduc- tion to hydroxyl forms. In 10 ml of 0.2 mM
tBuOOH in 0.5 ethanol, linoleic or linolenic acid was dissolved at 4 mM final concentration. The
autoxidation was initiated by adding FeSO
4
to 0.2 mM. The reaction proceeded for 10 min at room
temperature. Reduction was fulfilled by introducing a molar excess of NaBH
4
in 5 N NaOH. The reaction mixture was acidified to pH 4 with 5 N HCl
and extracted by 1 ml hexane: diethyl ether 70:30, vv. After centrifugation, the upper layer was
collected and used for HPLC injection.
Hydroperoxy fatty acids were isolated and re- duced to their corresponding hydroxyl forms ac-
cording to Degousee et al. [20]. Briefly, fatty acids from Arabidopsis were prepared by homogeniza-
tion of 0.5 g of rosette leaves in 1 ml 5wv NaBH
4
in 0.2 N NaOH. After acidifying the ho- mogenate to pH 4 with 70 HClO
4
, 2.2 ml of CHCl
3
: CH
3
OH 50:50, vv were used to separate aqueous and organic phase. The lower phase was
collected after centrifugation for 5 min at 11 000 rpm 4°C. The upper phase was further extracted by
1.1 ml chloroform. All lower phases were combined following a 5 min 11 000 rpm 4°C centrifugation.
Solvent was removed and the residue dissolved in 0.5 ml of absolute ethanol and 0.5 ml of 3.5 N
NaOH. Following a 15 min reflux and cooling, 0.5 ml 3.5 N HClO
4
was used to acidify sample to pH 4. Phases were then separated with 0.3 ml hexane:
diethyl ether 70:30, vv. The upper layer was collected after a 10 min 10 000 rpm centrifugation
at 4°C. After filtering through a 0.45mm NY PP filter, the sample was used for HPLC analysis.
Isolated leaf fatty acids were characterized by analytical HPLC using a Rainin Dynamax instru-
ment equipped with a 4.6 × 250 mm Microsorb-MV column packed with 5 mm silica gel. Mobile phase
solvent consisted of hexane: diethyl ether: acetic acid
70:30:0.5, vvv.
Hydroxyl fatty
acid stereoisomers were separated by isocratic elution at
a flow rate of 0.5 mlmin. Absorbance at 234 nm was monitored with a photodiode array PDA-2
detector.
3. Results
3
.
1
. Antioxidant enzyme acti6ities during the Arabidopsis life span
Numerous studies in several species have demon- strated a role for reactive oxygen species in
the progress of leaf senescence [2]. Their buildup is purported to lead to membrane leakage and
organellar disintegration during late stages of senescence. It is possible that a programmed
downregulation of antioxidant capacity might contribute to the observed ROS increase. We
focused our studies first on enzyme activity, as opposed to mRNA levels, since it is at this level
that changes would affect ROS metabolism. We thus prepared enzyme extracts from Arabidopsis
leaves at each important stage in the plant’s life history and measured the activities of two
antioxidant
enzymes. The
stages included
pre-bolting, i.e. before the transition from the vegetative juvenile phase to the reproductive
stage, short bolt B 1 cm, medium bolt \ 4 cm, long bolt \ 10 cm, flowering, seeding, and
senescing with rosette leaves at least 25 yellow. Equal amounts of protein isolated from the
various stages were loaded onto a native PAGE gel and stained for SOD activity. An inverted
image of one such gel is shown in Fig. 1. Slight variations in Mn-SOD activity as the life span
progressed were evident in this example, while Fe-SOD consistently displayed an increase at the
mid-bolt stage followed by a return to pre-bolt levels prior to senescence. The situation was even
more dramatic for ascorbate peroxidase APX. Fig. 2a shows the results of a similar experiment
for APX activity. The major band for APX activity declined precipitously as the plants entered
the reproductive phase of life. Thus, from pre-bolt 39 days after planting to short bolt 43 dap
APX levels declined more than fivefold. The decline was only transient, however, as the APX
level rebounded to pre-bolt levels at the long bolt stage 50 dap. APX levels were then found to
vary only slightly as the plants continued their life span through senescence. Data from six separate
experiments were compiled and are shown in Fig. 2b. The APX activity at each stage is reported as
the six-experiment average of the of the highest activity measured within any series. The standard
deviation of the data is shown as error bars in the figure. The transition to bolting at the meristem
was associated with an average two to threefold decrease in leaf APX activity. A very careful
selection of plants at identical developmental stages showed that this decline can reach fivefold
Fig. 2. In all cases, the APX level subsequently increased to the highest levels measured as the
bolts reached lengths of 10 cm. This dip and subsequent rebound of APX activity occurred
Fig. 1. Leaf superoxide dismutase activity as a function of life history stage. Inverted image of a native PAGE gel stained
for SOD activity. Extracts were prepared from plants of the indicated age in days and the following stages: PB, pre-bolt;
SB, short bolt; MB, medium bolt; LB, long bolt; FS, flower- ing and seeding; and SN, senescing. The positions of migra-
tion of Mn- and Fe-SOD are indicated.
Fig. 2. Leaf ascorbate peroxidase activity as a function of life history stage. a Inverted image of a native PAGE gel stained
for APX activity. Extracts were prepared from plants of the indicated age and stage. The arrow denotes an invariantly
stained activity. b The chart depicts a compilation of APX activity from six independent determinations. The data are
expressed as the average percent of maximum activity for a given generation. The error bars indicate the standard devia-
tion in the data.
Fig. 3. Leaf lipid peroxide level as a function of life history stage. Lipid peroxides columns were measured using the FOX assay and are expressed as mg of ROOH per mg of leaf tissue. The curve indicates the level of chlorophyll at the indicated stages. SN1,
leaf was approximately 20 yellow; SN2, 40 yellow; SN3, 60 yellow; SN4 80 yellow; and SN5, 100 yellow.
within a span of 10 days, or roughly one-eighth the total life span. As the plants entered senes-
cence, only a slight decrease in APX was mea- sured. Thus, in Arabidopsis it appears that APX
activity does decline in an apparently programmed manner, but its timing precedes considerably the
onset of visible symptoms of senescence.
3
.
2
. Leaf lipid peroxide le6els as a function of life span
The primary purpose of leaf APX is to scavenge hydrogen peroxide before it can react with cellular
biomolecules and cause damage. Our observations that leaf APX declines as Arabidopsis plants enter
the reproductive phase suggests that H
2
O
2
levels might increase during the 10-day period of low
activity and lead to biomolecular damage, a hall- mark of senescence in virtually all species [21]. To
test this possibility, we measured the levels of whole-cell lipid peroxides at the same stages used
above. Total lipid was extracted and lipid perox- ides were determined using the ferrous oxidation-
xylenol orange FOX method [18]. This assay method was used due to its higher specificity to-
wards lipid peroxides than the TBARS test. For each stage, only the 5th and 6th leaves to emerge
from the rosette were used for extract prepara- tions. Twenty samples were collected for each
stage in order to study the deviation in the data. The data in Fig. 3 show that lipid peroxides began
to increase at the bolting transition, as expected from the observed decline in APX, and reached a
maximum twofold higher level by the flowering stage. As the plants entered senescence though,
indicated by the decline in leaf chlorophyll at 64 dap, lipid peroxide levels also declined. By the
final stage of senescence 83 dap, leaves completely yellow but not dry, lipid peroxides were even
lower than at the young, pre-bolt stage. Since the data are reported as mg lipid peroxide per mg of
leaf tissue, the level of lipid peroxides on a per cell basis would be even lower since leaf mass had
declined due to senescence processes. The APX decline was therefore associated with an increase
in lipid peroxidation during the 10-day period of low APX. Lipid peroxides remained elevated,
however, for a further 10 – 15 days, suggesting that some mechanism other than simple chemical at-
tack of H
2
O
2
on membrane lipid double bonds was responsible for the elevated lipid peroxide
levels.
3
.
3
. Isomeric composition of leaf lipid peroxides The reaction of H
2
O
2
with plant membrane lipids would be expected to generate a multitude
of products which vary both in the position of the
added peroxy group on the fatty acid chain and in the number of double bonds and chain length
reflecting the natural diversity of cellular lipids. The major lipid fraction in Arabidopsis leaves is
composed of linoleic and linolenic acids, however [22]. Thus, a determination of the peroxy position
in cellular lipids is manageable by HPLC isomeric composition analysis. A set of standards was first
prepared by autoxidation and enzymatic genera- tion of lipid hydroperoxides to identify the peaks
on the chromatogram. Fig. 4a and b show results from t-butly hydroperoxide-catalyzed autoxida-
tion of linoleic and linolenic acid, respectively, while a mixture of the two is shown in Fig. 4. The
conjugated diene of the reduced products was followed by A
234
. Compared with published work [20,23] under similar HPLC conditions, the peaks
were identified and are listed in the Fig. 4 legend. The amounts of the positional isomers were not
equal. Thus, 13-HODE and 9-HODE represented about 85 of the four identified isomers from
linoleic acid while 16-HOTE and 9-HOTE ac- counted for about 75 of the four isomers from
linolenic acid. Other peaks that eluted prior to 12 min were not identified but were also present in
the pre-autoxidation material. Standards were also prepared from enzymatic peroxidation of linoleic
acid using soybean lipoxygenase LOX and sub- jected to HPLC separation. Unlike autoxidation,
13-HODE was the predominant product with more than 90 of the total as shown in Fig. 5.
Next, lipid peroxides from different-aged Ara- bidopsis leaves were prepared and analyzed by
HPLC following reduction to their corresponding hydroxyl forms. The chromatographic profile Fig.
6 between 15 and 25 min showed considerably less complexity in the leaf-derived sample than the
autoxidation standards Fig. 4a and b and was rather similar to the enzymatically generated stan-
dard Fig. 5. In the natural sample, the second peak, 13-HOTE, showed the greatest increase in
amount following the transition from a pre-bolted stage to the reproductive phase. The level of 13-
HOTE slowly began to decrease as the plants entered senescence, paralleling the kinetics of lipid
peroxide changes found by the FOX assay Fig. 3. In addition to the major increase in 13-HOTE,
smaller increases were observed for 13-HODE, 13-HODE trans, 16-HOTE, and 9-HODE. These
isomers followed the same kinetics as 13-HOTE and thus contributed to the overall pattern of lipid
peroxidation changes during the life span.
3
.
4
. Leaf longe6ity and flowering We have made casual observations in our lab
that the timing of the developmental transition to the reproductive state was associated with the
timing of visible senescence symptoms in leaves. We have observed this phenomenon in the Colum-
bia ecotype, whereas others have reported that late-flowering mutants exhibit wild-type leaf senes-
cence timing in the Landsberg ecotype, suggesting a lack of association between flowering and
Fig. 4. HPLC separation of reduced hydroperoxy fatty acid standards generated by autoxidation of a linoleic and b
linolenic acid. A mixture of the two is shown in panel c. The isomers indicated by letters are: a, 13-HODE, 13-hydroxy-
9,11Z,Eoctadecadienoic acid; b, 13-HOTE, 13-hydroxy- 9,11,15Z,E,Zoctadecatrienoic
acid; c,
12-HOTE, 12-hydroxy-9,13,15Z,E,Zoctadecatrienoic acid; d, 13-HODE
trans, 13-hydroxy-9,11E,Eoctadecadienoic acid; e, 9-HODE, 9-hydroxy-10,12Z,Eoctadecadienoic acid; f, 16-HOTE, 16-
hydroxy-9,12,14Z,Z,Eoctadecatrienoic acid; g, 9-HOTE, 9- hydroxy-10,12,15E,Z,Zoctadecatrienoic
acid; and
h, 9-HODE tran, 9-hydroxy-10,12E,Eoctadecadienoic acid.
Fig. 5. HPLC separation of products generated by incubation of linoleic acid with soybean lipoxygenase and reduced to their hydroxy derivatives. The products indicated by letter are: a, 13-HODE; and e, 9-HODE.
4. Discussion
