process, the onset of senescence requires the active transcription and translation of new gene products
and is thus under direct genetic control. It is definitely not a simple case of down-regulation of
genes that define a juvenile state, though such repression forms part of the process, but rather
progression into this final life phase requires the expression of new genes [3 – 5]. Some of these
senescence-associated genes have begun to be elu- cidated, and the known products of the more
abundantly expressed genes are those expected: nucleases [6,7], proteases [3,4], and nitrogen reas-
similation products [8], as well as some surprises [9]. The primary adaptive reason for the induced
degradation is to mobilize leaf nitrogen for trans- port to the developing seeds. With the induction of
the senescence program, the leaf no longer pro- vides new fixed carbon to the rest of the plant and
thus senescence involves a redistribution of re- sources from somatic tissue to the developing
progeny, a perfect example of parental altruism.
Several models have been put forth to explain the highly ordered and controlled nature of leaf
senescence. In many leguminous plants, the aging program appears to be controlled by the develop-
ing seeds [10]. Removal of floral structures extends the longevity of the leaves, suggesting the involve-
ment of a diffusible substance that initiates leaf senescence. The nature of this diffusible regulator
is still unknown. Plant-specific hormones also ap- pear to institute senescent phenotypes in many
systems. Since most studies using plant hormones involve their exogenous addition to various or-
gans, attached or detached, in many instances their endogenous role remains unclear. A common
molecular theme in plant senescence is evident in more downstream events, however. In most spe-
cies examined, ROS and their damage products increase during leaf aging. Thompson has sug-
gested that O
2 −
levels rise in senescing plant tis-
sues due to an increase in lipoxygenase activity, placing ROS production and damage at the same
location, cellular membranes [2]. Others have sug- gested, both in plants [11] and in animals [12] that
age-related ROS increases result from leaks in the normal electron transfer reactions of metabolic
pathways. Whatever their source, ROS are inti- mately associated with the degradative aspects of
senescence physiology in all plants that have been studied.
In this report, we test the hypothesis that age- dependent increases in ROS are the result of a
programmed downregulation of antioxidant en- zymes in Arabidopsis. Given the increasingly im-
portant role of H
2
O
2
as a signaling molecule, both in plant and animal systems, we decided to focus
on those enzymes that can modulate H
2
O
2
levels. We show that the transition from vegetative
growth to reproductive growth bolting is related to the longevity of rosette leaves as measured by
chlorophyll level and that this transition is associ- ated with ascorbate peroxidase APX decline and
lipoxygenase LOX activation.
2. Materials and methods
2
.
1
. Plant material and growth conditions Arabidopsis thaliana Columbia seeds Lehle
Seeds, Inc
were sown
in 2:1:1
potting soil:perlite:vermiculite following a 5-day imbibed
incubation in 0.1 agarose at 4°C. Nearly syn- chronous germination was encouraged by a 3-day
incubation of the pots at 4°C before transfer to growth chambers. The plants were irrigated
weekly with a nutrient solution [13] and kept in a growth chamber or environmental room at 23°C
under a 12 h photoperiod with a light fluence of 250 mmolem
2
per s. Plants were evenly spaced every 4 cm to encourage similar growth rates and
sizes.
2
.
2
. Extract preparations and enzyme acti6ity assays
Extracts were prepared by freezing leaf tissue in liquid N
2
and pulverizing to a fine powder before homogenization in with a Tissue Tearor in a
buffer solution [14]. The homogenate was cen- trifuged at 14 000 rpm for 20 min at 4°C and the
supernatant collected and stored at − 80°C until further analysis. Protein concentration was deter-
mined spectrophotometrically by the Bradford dye-binding assay using bovine serum albumin as
standard [15]. Protein extracts equal amounts per lane were subjected to Laemmli discontinuous
polyacrylamide gel electrophoresis under nondena- turing, nonreducing conditions. Gels for superox-
ide dismutase SOD and ascorbate peroxidase APX were run for 4 h at 4°C in a potassium
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
