132 S. Signorelli et al.
The differences detected between both species are mainly explained by changes
in the Cu/Zn- SOD isoforms. In T. pratense, H 2 O 2 accumulation showed the same
pattern; however, in L. corniculatus, the highest accumulation of ROS was observed
under water deficit. These results clearly demonstrate that combination of stress
situations cannot be always considered the additive responses of individual stresses.
L. corniculatus showed an increase in TBARS content as a consequence of wa-
ter deficit, heat stress and a combination of these. But T. pratense did not produce
any increase in TBARS content under heat stress. As proline antioxidant protection
function under stress conditions is now in discussion (Signorelli et al. 2013a), the
absence of proline accumulation in T. pratense may be an advantage under heat
stress by avoiding the Pro/P5C cycle which, as previosuly mentioned, could result
in higher ROS production via the Pro/P5CS cycle (Lv et al. 2011 ). However, proline
accumulation might be critical under combined stress because the osmolyte func-
tion seems to be important when water stress is established.
6.3.3 Photosynthesis
Water stress and heat combination affects the rate of photosynthesis due to an
increase in photoinhibition, a process that can be enhanced when more types of
abiotic stress coexist (Takahashi and Murata 2008 ). Under stress conditions, the
possibility of overexcitation of PSII increases. This can cause a decline in the
photosynthetic rate as the process of photoinhibition increases due to the neces-
sity to dissipate, through nonradiative processes, the excess of absorbed energy
(Takahashi and Murata 2008 ; Baker 2008 ). Because the capacity of photopro-
tection is limited, certain conditions can lead to damage and loss of active PSII
reaction centres. Under severely high temperatures, in combination with water
stress, the photosynthetic apparatus is the primary site of damage. On the con-
trary, photosystem I is more resistant to heat than PSII (Sayed et al. 1989 ; Hu
et al. 2004 ; Havaux 1993 ). Once photoinhibition is established, the PSII reaction
centre is simultaneously repaired via removal, synthesis and replacement of de-
graded D1 protein (Ohad et al. 1984 ; Kyle and Ohad 1986 ), a protein of reaction
centre of PSII (Fig. 6.1). The observed photoinhibitory damage is the net result of
a balance between photodamage and the repair process (Samuelsson et al. 1985 ;
Lidholm et al. 1987 ; Shyam and Sane 1989 ). Several studies have reported a good
correlation between changes in chlorophyll fluorescence parameters in response
to environmental stresses, such as heat, chilling, freezing and salinity (Bonnecar-
rére et al. 2011 ; Smillie and Hetherington 1983 ; Yamada et al. 1996 ; Hakam et al.
2000 ). Others authors have linked the decrease in the maximum quantum yield
of PSII ( FV/FM) to the physical dissociation of the PSII reaction centres that lead
to photoinhibition, and this assay was used to identify tolerant wheat cultivars
(Abdullah et al. 2011 ).
In L. corniculatus, no changes of the maximum quantum efficiency, evaluated
as FV/FM, were observed in any treatment until the 5th day, when the combined