6 Combined Abiotic Stress in Legumes 131
and this is likely because high temperature does not induce accumulation of this
ROS (Sainz et al. 2010 ).
In T. pratense, however, no changes were observed in the activities of any SOD
isoforms. The results of the quantitative enzyme activity assay demonstrated that
total SOD activity is 2.6-fold greater in L. corniculatus than in T. pratense, and it
is affected by the stress treatments. Heat did not modify the SOD activity in L. cor-
niculatus, but the combination with water stress led to same level activity observed
under water stress (Fig. 6.2). T. pratense showed a slight increase in the SOD activ-
ity by heat stress and combined stress (Fig. 6.2). In this case, for both legumes the
response of SOD activity in the combined stress was the addition of responses in the
individuals’ stresses. It could be concluded that if one of the stresses that produce
the induction of SOD activity is present, the induction of SOD activity will be war-
ranted in the combined stress. In L. japonicus, heat stress led to a decrease on Cu/
Zn-SOD contents, which also was observed under a combination of heat and water
deficit (Sainz et al. 2010 ).
In L. corniculatus, CAT activity only increases during the combination of wa-
ter stress and heat. However, in T. pratense, CAT enzyme activity increased with
reference to control in response to water deficit, heat stress and combined stress,
although no differences were observed among these stresses. In T. pratense, it was
observed that any stress was able to induce CAT activity and the combination of
both stresses did not lead to an additive effect on the enzyme activity. For L. cor-
niculatus, it seems that any individual stress is not sufficient to induce CAT activ-
ity; however, the combination of stresses led to the induction of CAT, suggesting
that more than one signal is required to induce this enzyme. In L. japonicus, the
combination of heat and water deficit led to an increase in CAT activity, that was
much higher than the activity observed when the stressors were imposed individu-
ally (Sainz et al. 2010 ).
Interestingly, the APX activity in L. corniculatus was inhibited by water stress
condition, while in T. pratense, this activity was inhibited only in the combined
stress treatment. This enzyme is inactivated by nitration (Begara-Morales et al.
2014 ), which is reported to occur under several abiotic stresses (Corpas et al. 2013 ).
For example, for L. japonicus, a closely related species, it was observed that water
deficit induces a nitro-oxidative stress that was also reducing APX activity (Si-
gnorelli et al. 2013c). We speculate that the different stressful situations are also
inducing nitro-oxidative stress in these plants, and this could explain the decay in
enzyme activity.
Both L. corniculatus and T. pratense leaves showed O 2 ●− accumulation only in
the water deficit–heat stress combination, as was previously observed in the model
legume L. japonicus (Sainz et al. 2010 ). The higher SOD activity in water stress
conditions with respect to controls, would allow this species to deal with the O 2 ●−
induced mainly by water stress. However, the increase of Mn-SOD and Fe-SOD
isoform activity by water stress was lost under high-temperature conditions, result-
ing in an increase of O 2 ●− in the combined treatment. In L. japonicus, similar results
were obtained with Cu/Zn-SOD, showing that deleterious effects of heat stress on
SOD activity might be a general response for this legume genus (Sainz et al. 2010 ).