Plant Science 158 2000 115 – 127
The developmental transition to flowering represses ascorbate peroxidase activity and induces enzymatic lipid peroxidation in
leaf tissue in Arabidopsis thaliana
Zhenzhen Ye, Roxana Rodriguez, Augustine Tran, Hoang Hoang, Darryn de los Santos, Shawn Brown, Robert Luis Vellanoweth
Department of Chemistry and Biochemistry, California State Uni6ersity Los Angeles, Los Angeles, CA
90032
-
8202
, USA Received 7 February 2000; received in revised form 31 May 2000; accepted 3 June 2000
Abstract
Leaf senescence in many plant species is associated with increased oxidative damage to cellular macromolecules by reactive oxygen species ROS. Since ROS levels and their damage products in many plants are known to increase during senescence, it
is possible that these changes are due to a decline in the levels of certain antioxidant enzymes. Using specific assays, we find that the developmental transition to bolting and flowering is associated with up to a 5-fold decline in ascorbate peroxidase activity and
an increase in chloroplastid superoxide dismutase. As expected, these changes are associated with a measured increase in lipid peroxidation products. By HPLC separation of the products, we identified the different positional isomers and find that
stereospecific lipid peroxidation occurs after the bolting transition. The product distribution suggests that enzyme-mediated lipid peroxidation, via a lipoxygenase, is responsible for the observed increase. Surprisingly, though consistent with the known
induction of antioxidant defenses by hydrogen peroxide, the activity of APX rebounds with further development reproduction and seed setting and this increase up to 5-fold is associated with declines in lipid peroxidation and with the onset of visible
senescence symptoms. Thus, in Arabidopsis, ROS increases are associated with the developmental transition to flowering, perhaps due to programmed declines in APX activity, and apparently lead to the oxidative activation of lipoxygenase and subsequent lipid
peroxidation. The reactivation of APX at later stages appears to help reduce the lipid peroxidation rate, although the senescence program continues unabated. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Ascorbate peroxidase; Lipid peroxidation; Reactive oxygen species; Senescence; Arabidopsis www.elsevier.comlocateplantsci
1. Introduction
During the final developmental phase in the life of an annual plant, reproduction and seed devel-
opment are accompanied by senescence of the leaves. This genetically programmed aging event is
an active process characterized by the degradation of leaf cell components. Metabolism in the leaves
is drastically altered. During vegetative growth, the primary function of the leaf is to provide fixed
carbon to the rest of the plant through the process of photosynthesis. However, as senescence pro-
ceeds, leaf photosynthetic capacity is diminished through a destruction of chlorophyll, a proteolytic
attack on the abundant CO
2
-fixing enzyme ribu- lose bisphosphate-1, 5-carboxylase, and a decline
in de novo synthesis of the photosynthetic appara- tus [1]. Protein and nucleic acid levels decline
through enhanced proteolysis and nuclease attack and membrane integrity is lost. A critical role for
reactive oxygen species ROS such as superoxide anion O
2
−
, hydrogen peroxide H
2
O
2
, and hy- droxyl radical HO throughout all stages of
senescence has been implicated in many plants, with the primary target of damage being the mem-
brane lipids [2]. Although apparently a destructive
Corresponding author. Tel: + 1-323-3432148; fax: + 1-323- 3436490.
E-mail address
:
vllnwthcalstatela.edu R.L. Vellanoweth. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 1 6 - 2
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
