Proline content in Trifolium shoot and root
The proline was extracted in 100 mM phosphate buffer (pH 7.8)
from 0.5 g of fresh leaves and root, previously immersed in liquid
N2 and stored at −80 ◦C according to Bates et al. (1973). The
homogenate was centrifuged at 18.000 × g for 10 min at 4 ◦C and
the supernatant was used for quantification of proline content. Proline
was estimated by spectrophotometric analysis at 520 nm using
the ninhydrin reaction (Bates et al., 1973).