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The Generation of Active Oxygen Species Differs in Tobacco and
Grapevine Mesophyll Protoplasts1
Anastasia K. Papadakis and
Kalliopi A. Roubelakis-Angelakis*
Department of Biology, University of Crete, P.O. Box 2280, 71 409 Heraklio, Greece
Our previous results have shown that
oxidative stress may reduce the regeneration potential of protoplasts,
but only protoplasts that are able to supply extracellularly
H2O2 can actually divide (C.I. Siminis, A.K.
Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87:
263-270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375-1383; A. de Marco, K.A.
Roubelakis-Angelakis [1996a] Plant Physiol 110: 137-145; A. de
Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149:
109-114). In the present study we have attempted to break down
the oxidative burst response into the individual active oxygen species
(AOS) superoxide (O2· ) and
H2O2, and into individual AOS-generating
systems during the isolation of regenerating tobacco (Nicotiana
tabacum L.) and non-regenerating grape (Vitis
vinifera L.) mesophyll protoplasts. Wounding leaf tissue or
applying purified cellulase did not elicit AOS production. However, the
application of non-purified cellulase during maceration induced a burst
of O2· and H2O2
accumulation in tobacco leaf, while in grape significantly lower levels
of both AOS accumulated. AOS were also generated when protoplasts
isolated with purified cellulase were treated with non-purified
cellulase. The response was rapid: after 5 min, AOS began to accumulate
in the culture medium, with significant quantitative differences
between the two species. In tobacco protoplasts and plasma membrane
vesicles, two different AOS synthase activities were revealed, one that
showed specificity to NADPH and sensitivity to diphenyleneiodonium
(DPI) and was responsible for O2·
production, and a second NAD(P)H activity that was sensitive to KCN and
NaN3, contributing to the production of both AOS. The first
activity probably corresponds to a mammalian-like NADPH oxidase and the
second to a NAD(P)H oxidase-peroxidase. In grape, only one
AOS-generating activity was detected, which corresponded to a NAD(P)H
oxidase-peroxidase responsible for the generation of both AOS.
1
This work was supported in part by the Interreg
II project (European Union and Greek Government).
*
Corresponding author; e-mail poproube{at}biology.uch.gr; fax
30-81-39459.
Plant Physiol. (1999) 121: 197-206
Copyright Clearance Center: 0032-0889/99/121//10
© 1999 American Society of Plant Physiologists
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