6 Combined Abiotic Stress in Legumes 129
6.3 Effect of Water Stress–Heat Stress Combination
on Different Plant Processes
L. corniculatus and Trifolium pratense are legumes used in agriculture as a forage
source. These species are both perennial herbaceous plants used in temperate grass-
land and can be nodulated by rhizobia. Nevertheless, lotus is better suited to soils
with water restriction and has a superior tolerance to water stress (Peterson et al.
1992 ). In the field, mainly during summer, these plants are commonly exposed to
environmental stresses such as water stress and high temperatures, which in fact are
considered to be the most important environmental factors limiting plant growth
and development (Berry and Bjorkman 1980 ; Yordanov et al. 1986 ; Sinsawat et al.
2004 ).
6.3.1 Proline Accumulation
The accumulation of proline is known to be a good indicator of water stress in
L. corniculatus (Díaz et al. 2005b). However, the responses to combination of
stresses are not a mere additive effect of the single stresses. For example, some
plants that tend to accumulate proline in water stress conditions replace it with
sucrose as the major osmoprotectant when subjected to a combination of water
stress and heat stress (Rizhsky et al. 2004 ). In L. corniculatus water stress and
heat individually produce proline accumulation, but concomitant imposition of
both stresses produced a higher accumulation of proline. In contrast, Trifolium
Pratense-accumulated proline in water stress conditions but not under heat stress
and the imposition of the combined stress produced only a slight increase in pro-
line concentration compared to unstressed plants (Signorelli et al. 2013b). There-
by, for L. corniculatus, proline accumulation is a parameter that can be used as
a stress marker to assess water stress and heat stress conditions, as well as the
combination of both. However, proline accumulation in legumes cannot always
be considered a good indicator of stress condition when two or more stresses are
present. It is also known that proline accumulation under heat stress decreases the
thermotolerance of the plant, probably because of an enhancement in the produc-
tion of ROS via the Pro/P5C cycle (Lv et al. 2011 ). In T. pratense, it was suggested
that blocking proline accumulation might be a strategy to avoid self-toxicity during
heat stress (Signorelli et al. 2013b). This hypothesis correlated with the lipid per-
oxidation estimated by thiobarbituric reactive substances (TBARS), as T. pratense
did not show an increase in lipid peroxidation under heat conditions. Moreover, T.
pratense has a lower lipid peroxidation content than L. corniculatus when water
stress and heat stress are combined—a treatment in which L. corniculatus accumu-
lates the highest levels of proline.
On the other hand, it has been demonstrated that proline can act as an osmo-
lyte under severe dehydration (Verslues and Sharp 1999 ). The non-accumulation
of proline and the greater leaf area of T. pratense are important disadvantages of