The survivability and quality of micropropagated plants can be improved through mycorrhization. We consider that mycorrhization is important in supporting plants under stress conditions. The mechanism is not fully understood, but it seems that the enzymes involved in alleviating stress are important factors. We therefore studied superoxide dismutase (SOD; EC 1.15.1.1) isozymes. Insight is provided into the generation of superoxide radicals (SORs) and the detoxification role of SOD isozymes. Examples of how the expression of this enzyme changes in symbiotic processes are also given

Antonio Vilariňo

José M. Palma

Justo Arines

Agricultural and Food Science

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Research Note
Involvement of the superoxide dismutase enzyme in the
mycorrhization process
Justo Arines, Antonio Vilarino and José M. Palma
Arines, J., Vilarino, A'. & Palma, J. M. 2 1994. Involvement of the superoxide
dismutase enzyme in the mycorrhization process. Agricultural Science in Fin-
land 3: 303-307. ('lnstituto de Investigaciones Agrobiolögicas de Galicia (CSIC),
Apdo 122, 15080-Santiago de Compostela, Spain and, 2Estaci6n Experimental del
Zaidm (CSIC), Apdo 419, 18080-Granada, Spain.)
The survivability and quality of micropropagated plants can be improved through
mycorrhization. We consider that mycorrhization is important in supporting plants
under stress conditions. The mechanism is not fully understood, but it seems that
the enzymes involved in alleviating stress are important factors. We therefore
studied superoxide dismutase (SOD; EC 1.15.1.1) isozymes. Insight is provided
into the generation of superoxide radicals (SORs) and the detoxification role of
SOD isozymes. Examples of how the expression of this enzyme changes in symbi-
otic processes are also given.
Key words: activated oxygen species, arbuscular mycorrhizal fungi, isozymes, oxi-
dative stress, pathogenesis, superoxide radical
Introduction
Arbuscular mycorrhizal fungi (AMF) are known
to be able to colonize the roots of most vascular
plants and, under natural conditions, to provide a
partnership with a complex system of extraradi-
cal hyphae. This extraradical system contributes
to the uptake of water and nutrients and creates a
modified rhizosphere favourable for plant pro-
tection in stress situations (Sylvia and Williams
1992).
This is appreciated by mycorrhizologists, but
not by all plant growers, who still do not realize
that when cuttings are introduced into the soil
the rooted plants become mycorrhizal, and their
establishment and growth in the field are thus
improved.
The technique of ‘ in-vitro’ micropropagation
is of special interest in plant propagation. Exper-
iments made using this technique have shown
that inoculation of recently rooted plants at the
weaning stage improves the survival of the plants
because they are much better able to withstand
stress in a changing habitat. Growth differences
between mycorrhizal and non-mycorrhizal Pru-
nus cemsifera are very large (Fortuna et al.
1992), confirming the importance of mycorrhiza
inoculation of micropropagated plants. Mycorrhiza
appears to play a key role in improving the ‘ex-
vitro' development of micropropagated Avocado
plants (Azcon-Aguilar et al. 1992) and the sur-
vival of micropropagated Anthyllis cytisoides
(Salamanca et al. 1992). Moreover, the accli-
matization phase has been reduced to eight weeks
following mycorrhization with Anthyllis (Sala-
manca et al. 1992). As there is some degree of
specificity in the mycorrhization of micropropa-
gated pineapple plants (Guillemin et al. 1992),
303
Agricultural Science in Finland 3 (1994)
Research Note
the growth effects are not always comparable
because conditions for establishing the symbiot-
ic change within plant and fungus species vary.
This is probably a consequence of intrinsic fac-
tors and should be further investigated. The in-
volvement of various enzymes was recently re-
viewed by Gianinazzi (1991).
Enzymes participating in detoxification of su-
peroxide radicals (SORs) are important during
stress situations (Tsang et al. 1991). We, there-
fore, consider it necessary to study the extent of
superoxide dismutase (SOD) involvement in the
mycorrhization process.
Overview of stress-causing oxygen
derivatives
Although oxygen is an essential element for life,
its presence in living organisms implies that some
metabolic by-products can also be formed in the
cellular niche. In the presence of an adequate
electron donor and slight energy activation, the
reactive form ofthe SOR (O, ) is produced. There-
after, and under acidic conditions, the presence
of electron donors leads to generation of the per-
oxide anion, the hydroxyl radical and, finally,
water (Elstner 1987). All these oxygen-activat-
ed forms can be detoxified by differentcompounds
usually present in the cell, the mechanisms cho-
sen being dependent on the particular situation
and cellular location.
Biologically, the most dangerous by-product is
the hydroxyl radical because of its high reactivi-
ty with chemical bonds, which causes oxidation
and cleavage of the unsaturated bonds in mem-
brane lipids termed lipid peroxidation. Hydroxyl
radicals are formed in living beings by a metal-
catalized Haber-Weiss reaction, where H 2 02 and
0
2
are precursors. Furthermore, the production
of H 202 is also partially dependent on either an
enzyme-dependent or an enzyme-independent dis-
mutation of the SOR. The oxidative stress gener-
ated by these radicals within the cell can be coun-
teracted by both enzymatic and non-enzymatic
mechanisms.
Superoxide radicals and their
dismutation
Superoxide radicals are biologically generated
during both mitochondrial respiration and photo-
synthesis. The steady-state level of SORs in in-
tact mitochondria, where SOD is present, is esti-
mated to be about 10"11 to KT 12 M. In washed
mitochondria from which SODs are removed,
SORs are estimated to account for about 4% of
total oxygen. These radicals are usually generat-
ed in mitochondria, chloroplasts, the microsomal
fraction, nuclei (Hassan and Scandalios 1990)
and peroxisomes (Del Rio et al. 1992).
Almost all aerobic living organisms are pro-
tected against the damaging action of SORs by
superoxide dismutases, metalloenzymes which
catalyze the conversion of O, to H,O, and 0,,
H 2 02 being then metabolized by catalase and/or
peroxidases. Three types of SOD have been char-
acterized on the basis of the metal accompanying
the protein: CuZn-SOD, Mn-SOD and Fe-SOD.
CuZn-SOD is the most abundant in eukaryotic
organisms and is mainly located in the cytosol
and in chloroplasts, whereas Mn-SOD is usually
located in mitochondria and in peroxisomes. Fe-
SOD was initially found in prokaryotes, but has
recently been detected in chloroplasts and in plant
peroxisomes. There is little difference between
organisms with regard to CuZn-SODs, and their
amino acid sequences show a high degree of sim-
ilarity (homodimer with 32 kDa MW). Mn-SODs
are mainly tetrameric with 75-95 kDa MW in
most organisms, and are phyllogenetically relat-
ed to Fe-SODs.
Role of superoxide dismutases in
symbiotic systems
In addition to their normal activity in plants, SODs
are also associated with stress situations. The reg-
ulation of SODs in plants exposed to environ-
mental stress has been studied by Tsang et al.
(1991) by determining the presence of mRNA
coding for different SODs in Nicotiana under dif-
ferent stress conditions (light, temperature, Para-
304
Agricultural Science in Finland 3 (1994)
Research Note
quat). Their conclusion was that oxidative stress
was a component of environmental stress. They
demonstrated that with exposure to Paraquat, the
chloroplastic Fe-SOD mRNA increased and that
it was not a general reflection of other photosyn-
thetic components. This finding suggests that the
Fe-SOD gene expression is controlled by the ox-
idative stress itself, and is not part of a global
response. Further, in mitochondrial Mn-SOD and
cytosolic CuZn-SOD, the abundance of mRNA
is also increased. Therefore, it is likely that, al-
though SORs are generated within a specific com-
partment, the damage can affect other compart-
ments of the cell. Of particular interest was the
finding that under illuminated conditions spray-
ing leaves with 5 x 10~5 M Paraquat increased the
quantity of Fe-SODchl, Mn-SODmit and CuZn-
SODcyt mRNAs about 40, 30 and 15-fold, re-
spectively. Light itself caused an increase in SODs
because SORs are generated primarily by the leak-
age of electrons from photosystem I and from
ferredoxin.
An interesting study has recently suggested that,
in pathogenic situations, the expression of induc-
ible SODs is related to resistance or susceptibili-
ty to rust in coffee plants (Daza et al. 1993).
Coffee leaves resistant to infection by the rust
Hemileia vastatrix show a different SOD pattern,
with two extra CuZn-SODs. This hypothesis is
supported by the fact that two different coffee
plant cultivars, which are resistant to infection
by Hemileia vastatrix, share the same SOD pat-
tern. A different strategy is adopted by some Pha-
seolus vulgaris cultivars, which are resistant to
Uromyces phaseoli (Buonario et al. 1987). An
increase in SOD activity has been detected in
susceptible and resistant plants, with selective
stimulation of SOD activity taking place: the man-
ganese-enzyme in the susceptible cultivar and the
cuprozinc-enzyme in the hypersensitive cultivars.
Thus an increase in the Mn enzyme seems to be
more closely related to the biotrophic phase of
parasitism in the host cell, but an increase in the
CuZn enzyme to the necrotic process associated
with hypersensitivity (Buonario et al. 1987).
The first reference to SODs in symbiotic sys-
tems was by Becana et al. (1989) in their study
of free-living bacteria, bacteroids and nodules of
different legumes with Rhizobium or Bradyrhizo-
bium. Different patterns were found in each case.
With Rhizobium, the transformation into bacter-
oids induced the expression of a new Mn-SOD.
In our laboratory we recently studied the Tri-
folium pmtense-Glomus mosseae system (Palma
et al. 1993). G. mosseae spores contained only
one CuZn-SOD (G.m. CuZn-SOD); the non-my-
corrhizal root had one Mn-SOD (Mn-SOD I) and
two CuZn-SODs (CuZn-SOD 1 and CuZn-SOD
II). However, the mycorrhizal root had six SOD
isozymes; besides the plant SODs indicated above,
another Mn-SOD (Mn-SOD II) and two new
CuZn-SODs (G.m. CuZn-SOD and mycCuZn-
SOD) were detected. We propose that mycCuZn-
SOD and Mn-SOD II were induced by the sym-
biosis, although we could not determine the ori-
gin of these SODs. Furthermore, the activity of
CuZn-SOD I, which appeared in both kinds of
root, was strongly increased after mycorrhization.
Five isozymes were also detected in nodules of
red clover: the three plant SODs plus two new
Mn-SODs (nodMn-SOD and Mn-SOD II). nod-
Mn-SOD was exclusively expressed in nodules,
whereas nodule Mn-SOD II behaved like the Mn-
SOD II found in mycorrhizal roots, and we pos-
tulate that it may be a uniform response of the
plant to colonization by a foreign organism. We
think that in red clover, both symbioses induced
the expression of new SOD isozymes, suggesting
that activated oxygen species must be implicated
in the symbiosis. This was not the case for the
Pisum sativum-Glomus mosseae symbiosis. No
new isozymes were detected in mycorrhizal roots,
but total activity was higher, the extra activity
being accounted for by a CuZn-SOD (Arines et
al. 1994). Thus the symbiosis implies higher ac-
tivity in SODs, as do other stress situations, per-
haps because of the higher cellular activity asso-
ciated with this process.
Results obtained by Buonario et al. (1987)
and Daza et al. (1993) show that various strate-
gies may be adopted in the interaction between
pathogens and plants. In mycorrhizal symbiosis,
we have found that new isozymes are expressed
in T. pratense-G. mosseae but not in P. sativum-
305
Agricultural Science in Finland 3 (1994)
Agricultural Science in Finland 3 (1994)
G. mosseae. The difference may be related to the
different efficiency of the symbiosis in the two
plants: a higher efficiency in the colonization per-
centage was obtained with red clover, suggesting
that the expression of new isozymes may allow
the plant to cope with the excess SORs generated
during the entrance of a foreign organism. How-
ever, in peas, in which a lower percentage colo-
nization was determined, the plants’ own SOD
activity may be sufficient without inducing new
isozymes. We consider that the activity of SODs
is affected directly or indirectly by mycorrhiza-
tion, although, as happens in plant-pathogen re-
lationships, different strategies may be followed.
Further research is needed to understand the im-
portance of SOD in mycorrhization.
References
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1994. Protein patterns and superoxide dismutase activ-
ity in non-mycorrhizal and arbuscular mycorrhizal Pis-
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Vina, G. 1992. Further studies on the influence of
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Manuscript received January 1994
306
Research Note
SELOSTUS
Superoksidi-dismutaasientsyymin merkitys mykorritsan muodostuksessa
Justo Arines 1, Antonio Vilardmo 1 ja José M. Palma2
1 Instituto de Investigaciones Agrobiolögicas de Galicia (CSIC), Espanja ja 2 Estaciön Experimental
del Zaidin (CSIC), Espanja
Mykorritsan avulla voidaan lisätä mikrolisättyjen kasvien
eloonjäämistä ja parantaa kasvien laatua. Tiedetään, että
mykorritsa auttaa merkittävästi kasveja vaikeissa olosuh-
teissa, Tämän ilmiön mekanismia ei täysin tunneta, mutta
kasvien stressiä lievittävillä entsyymeillä näyttää olevan
siinä tärkeä merkitys. Tästä syystä tutkimme superoksidi-
dismutaasientsyymejä (SOD; EC 1.15.1.1). Superoksidi-
radikaalien (SOR) syntymistä ja SOD-isotsyymien merki-
tystä detoksifikaatiossa selvitettiin myös yksityiskohtai-
sesti. Myös esimerkkejä näiden entsyymien ilmenemisen
muuttumisesta symbioottisissa tapahtumissa on annettu.
307
Agricultural Science in Finland 3 (1994)Research Note


Involvement of the superoxide dismutase enzyme in the mycorrhization process

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