Plant Science 159 2000 159 – 167
Redox state and peroxidase system in sunflower plants exposed to ozone
Annamaria Ranieri , Francesco Petacco, Antonella Castagna, Gian Franco Soldatini
Dipartimento di Chimica e Biotecnologie Agrarie, Uni6ersita` di Pisa, 6ia del Borghetto
80
,
56124
Pisa, Italy Received 22 May 2000; received in revised form 24 July 2000; accepted 24 July 2000
Abstract
Sunflower plants subjected to a short-term fumigation with O
3
150 ppb for 4 h repeated for 4 days exhibited an increase in total ascorbate content, accompanied by a marked oxidation of ascorbate, leading to a decrease in its redox state, either at
intracellular or extracellular level. O
3
exposure induced a rise in free extracellular peroxidase POD activity, assayed by syringaldazine as electron donor, as well as in the ionically and covalently cell wall bound PODs. On the contrary, the activity
of both extracellular and intracellular guaiacol – POD did not show significant changes as a consequence of the pollutant exposure. The stimulation of syringaldazine – POD activities may be related to the effect of ozone on the growth of the cells, inducing an
early senescence through the activation or acceleration of lignification processes. Beside, the reduced plasticity of the cell wall may oppose an unspecific mechanical resistance against the abiotic stress induced by the ozone exposure. © 2000 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords
:
Ascorbate; Ozone; Oxidative stress; Peroxidases; Redox state; Sunflower www.elsevier.comlocateplantsci
1. Introduction
Ozone O
3
is perceived to be one of the most ubiquitous and damaging air pollutant to which
vegetation is exposed [1]. Current ground-level concentrations of the pollutant are known to have
adverse effects on the vitality of natural and agro- nomic ecosystems in many parts of the industri-
alised world.
Ozone can affect biochemical processes even in plants showing no visible sign of injury [2]. The
influence of ozone on vegetation is dependent on concentration and the duration of exposure to the
pollutant, the genetic backgrounds and the growth phases of the plants [3,4]. The cuticle is practically
impermeable to O
3
, so the flux of the pollutant to the leaf interior is predominantly controlled by
stomatal aperture [5]. Once inside the leaf, ozone is in contact with the aqueous phase of the cell
wall the apoplast where the reaction of O
3
with constituents of the apoplast generates various re-
active oxygen species ROS like free radicals OH, O
2 −
and peroxides H
2
O
2
, R
2
O
2
[6 – 8], which can damage the components of plas-
mamembranes, such as proteins and lipids. Among ROS, the radical OH is the most reac-
tive one, able to attack all the biomolecular spe- cies. On the other side, O
2 −
and H
2
O
2
are less reactive but equally dangerous for their possibility
of diffusion in all cellular compartments. Thus, according to Luwe et al. [9], the aqueous
phase of the apoplast seems to represent the site where O
3
and its highly toxic reactive intermedi- ates must be detoxified if they are to be prevented
from reacting with the plasmamembranes and cy- toplasmic components. Detoxification systems in
this microenvironment, either enzymatic or non-
Corresponding author. Tel.: + 39-50-57155758; fax: + 39-50- 598614.
E-mail address
:
aranieriagr.unipi.it A. Ranieri. 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 5 2 - 6
enzymatic, able to prevent or to mitigate O
3
injury have been reported [10]. Among antioxidant
metabolites, ascorbic acid, in addition to playing a key role in detoxification of H
2
O
2
as a specific substrate for ascorbate peroxidase APX, is capa-
ble of direct scavenging of O
3
molecules and O
3
- derived ROS [7,8,11 – 13]. Although the pool of
ascorbate present in the apoplast is only a small proportion of the total leaf content, it is suggested
that its increase following ozone exposure may afford protection against the dangerous effect of
this pollutant [12,14].
In the process of detoxification of H
2
O
2
is par- ticularly important the large family of peroxidases
POD, which includes either the specific ascorbate peroxidase enzyme APX and the so called unspe-
cific peroxidases POD. Previous data, obtained in our laboratory on sunflower plants subjected to
O
3
exposure, have shown a differential stimulation of APX isoforms, indicating an enhancement in
the activity at both the apoplastic and symplastic level, while stromal and thylakoid-bound chloro-
plastic APX activity remained unchanged [15]. The unspecific POD, besides their antioxidant role, is
involved in many metabolic functions. In the cell wall, PODs are present in soluble, ionically- and
covalently-bound forms and, in addition to a detoxificant role as scavengers of H
2
O
2
, they have been reported to be involved in a number of
physiological processes which regulate cell growth by catalysing the formation of cross-links between
extensin and feruloyated polysaccarides and the polymerisation of lignin precursors [16 – 20]. The
cell wall stiffening has been attributed mainly to peroxidases whose activity can be detected by
using, in the enzymatic assay mixture, syringal- dazine as a specific substrate [16].
In the present work, the apoplastic ascorbate pool content of sunflower plants exposed to O
3
and the cell redox state, measured as reduced versus total ascorbate ratio, was determined. Be-
side, the behaviour of unspecific PODs following O
3
exposure was tested, measuring their activity by using different electron donors such as guaiacol
and syringaldazine, both in the extracellular IWF and in the intracellular RCM leaf fluids, as well
as in the soluble S, ionically IB and covalently CB cell wall-bound fractions. In addition, the
POD isoforms of each fraction, along with the apoplastic and symplastic ones, were separated by
isoelectrofocusing analysis.
2. Materials and methods
