Stress-induced expression of cyclophilins in proembryonic masses of Digitalis lanata does not protect against freezing thawing stress

Using proembryonic masses (PEMs) of Digitalis lanata Erh., it was demonstra ted that cold, hormonal or osmotic stress, which increased freezing toleran ce during cryopreservation, induced an increasing level of two peptidyl-pro lyl-cis/transisomerases (PPIases). The difference in pI (9.2 +/- 0.2 and 9. 5 +/- 0.2, +/- SD; n = 3) allowed the separation of the two enzymes by free -flow isoelectrophoresis. Both were inhibited by cyclosporin A and thus bel ong to the cyclophilin family of PPIases. The enzymes differed slightly in their substrate specificity and their relative molecular masses of 18038 +/ - 4 Da (D. lanataCyp18.0) and 18132 +/- 3 Da (D. lanataCyp18.1). Both cyclo philins were blocked N-terminally. Partial internal amino acid sequences fr om the two cyclophilins, with a length of 34 amino acids, displayed 82% seq uence identity to each other. Pretreatment of PEMs with abscisic acid, sorb itol or a combination of both substances led to a 270 +/- 30% elevation of the total cytosolic cyclophilin concentration determined with a cyclophylin affinity sensor. During the first 4 d of pretreatment, the total PPIase ac tivity was enhanced up to 230 +/- SD% compared with the control culture. Th e lag phase between maximal PPIase concentration after 4 d of pretreatment and maximal effect of freezing tolerance after 10 d of pretreatment indicat ed that increasing levels of cytosolic PPIases may be necessary to overcome the stress induced by hormones and osmotica during pretreatment but not to protect against freezing/thawing stress.