Plant Molecular Biology 34: 209–221, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Characterization of nuclease activities and DNA fragmentation induced
upon hypersensitive response cell death and mechanical stress
and Eric Lam
Center for Agricultural Molecular Biology, Foran Hall, Dudley Road, Rutgers, The State University of New Jersey,
Cook College, P.O. Box 231, New Brunswick, NJ 08903-0231, USA (
author for correspondence);
address: Department of Plant Sciences, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel
Received 3 October 1996; accepted in revised form 12 February 1997
Key words: apoplast, extracellular, hypersensitive response, nuclease, programmed cell death, tobacco mosaic
virus, DNA fragmentation
Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading
pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate
the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic
virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments
of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of
nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities
did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD,
mechanical injury and inﬁltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an
increase in nuclease activities. Analysis of nuclease activitiesintotalleaf extracts, nuclear extracts, and intercellular
ﬂuid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of
theseatleastthree appearto besecreted intothe intercellularﬂuid. Althoughthelatterwere alsoinduced inresponse
to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during
late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by
the ﬁnding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the ﬁnding of
induced nuclease activities in the intercellular ﬂuid raises the possibility that they may serve a protective function
by degrading the DNA of invading pathogens.
The antimicrobial defense of multicellular organisms
in infected cells. The activation of this pathway, also
termed programmed cell death (PCD), is aimed at pre-
venting further proliferation of pathogenic microbes.
Thus cells that are initially infected undergo cell death
which restricts the invading pathogen to the site of
infection, and prevents systemic infection [27, 38]. In
some cases the cellular mechanisms which are activ-
ated during PCD are thought to result not only in the
killing of host cells butalso in the death of the invading
pathogen as well [1, 33].
ApoptosisisatypeofPCD which is associatedwith
the defenseofsome animalsagainstpathogens[15, 18,
35, 38, 45]. Death of cells during apoptosis is accom-
panied by several distinct biochemical and morpholo-
gical characteristics. These include shrinkage of cells,
condensation of chromatin, fragmentation of nuclei,
degradationofnuclear DNA and formation of apoptot-
ic bodieswhich are vesicles that containcellular debris
[17, 23]. Thus, infected cells which undergo apoptosis
bodies, and engulfed by macrophages.
One of the key aspects of apoptosis is the degrada-
tion of nuclear DNA . This process appears to result
from the combined effortof several nucleases [32, 33].
GR: 201001910, Pips nr. 135606 BIO2KAP
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