6 Combined Abiotic Stress in Legumes 133
treatment showed a significant decrease in the FV/FM parameter. In contrast, in T.
pratense, this fluorescence parameter slightly decreased from the 1st day in the
heat and combined treatment, but no changes were observed under water stress
conditions.
L. corniculatus showed a slight decrease in the amount of D1 protein after water
stress treatment. However, there was no decrease in the protein content when the
control and heat conditions were compared. The D1-complex profile of T. pratense
was also analysed, and the western blot showed a very different result when com-
pared with the L. corniculatus profile. The total D1 protein content in T. pratense
did not change in any treatment, but a difference was found in the ratio between the
free protein and the complex form. In the treatments where heat was involved, an
increase in free D1 protein together with a decrease in the D1-complex form was
evident, but it should be considered that this result might be a consequence of the
high hydrophobicity of these complexes, which makes their isolation difficult. Re-
garding D2, in L. corniculatus, the results were similar to those observed with D1;
namely, when water stress was present in the treatments, a reduction in the amount
of D2 protein was observed. Surprisingly, in the combined treatment, the D2 protein
was not detected. In contrast, in T. pratense, no significant changes were observed
in D2 protein levels.
The chlorophyll fluorescence parameter that was evaluated showed that L. cor-
niculatus had a significant decrease in the maximum quantum efficiency at the end
of the combined treatment. The low FV to FM ratio indicated photoinhibition, a
process that can act as determinant of plant performance during a stress condition
(Abdullah et al. 2011 ). In T. pratense, only a small decrease in the FV/FM values was
observed from the 1st day and in the treatments involving heat stress.
Analysis of PSII proteins in the two legumes with contrasting water stress
responses shows an effect that is stress- and species-specific. The D1 and D2
subunit content is decreased in L. corniculatus in both treatments involving water
stress, showing certain adaptability in response to water stress. Interestingly, the
decrease in the D2 levels was pronounced in the combined treatment, and this is
well correlated with the decrease in the maximum quantum efficiency, suggesting
the presence of a disassembling process. The D2 subunit is of particular interest
because it represents the initial point for the assembly of the PSII as a whole (de
Vitry et al. 1989 ; Komenda et al. 2004 ; Minai et al. 2006 ). The expression of the
gene that encodes the D2 subunit of the PSII reaction centre is regulated post-
transcriptionally by an RNA-binding protein (Schwarz et al. 2007 ). Modifications
induced by the stress in this post-transcriptional regulation could be a possible
explanation for the absence of D2 in L. corniculatus subjected to the combined
stress treatment.
In T. pratense, the total content of D1 and D2 did not change, but we observed
an increase in the free form of D1 in treatments involving heat. One possible ex-
planation is that the turnover of D1 is taking place in the heat treatments, and this
is evident based on the increase of free D1 together with a reduction of the D1–D2
complex, as well as a decrease in the FV/FM values.