RESUMO
Abscisic acid-pretreated carrot (Daucus carota) somatic embryos survive dehydration upon slow drying, but fast drying leads to poor survival of the embryos. To determine whether the acquisition of desiccation tolerance is associated with changes in the physical stability of the cytoplasm, in situ Fourier transform infrared microspectroscopy was used. Although protein denaturation temperatures were similar in the embryos after slow or fast drying, the extent of the denaturation was greater after fast drying. Slowly dried embryos are in a glassy state at room temperature, and no clearly defined glassy matrix was observed in the rapidly dried embryos. At room temperature the average strength of hydrogen bonding was much weaker in the rapidly dried than in the slowly dried embryos. We interpreted the molecular packing to be "less tight" in the rapidly dried embryos. Whereas sucrose (Suc) is the major soluble carbohydrate after fast drying, upon slow drying the trisaccharide umbelliferose accumulates at the expense of Suc. The possibly protective role of umbelliferose was tested on protein and phospholipid model systems, using Suc as a reference. Both umbelliferose and Suc form a stable glass with drying: They depress the transition temperature of dry liposomal membranes equally well, they both prevent leakage from dry liposomes after rehydration, and they protect a polypeptide that is desiccation sensitive. The similar protection properties in model systems and the apparent interchangeability of both sugars in viable, dry somatic embryos suggest no special role of umbelliferose in the improved physical stability of the slowly dried embryos. Also, during slow drying LEA (late-embryogenesis abundant) transcripts are expressed. We suggest that LEA proteins embedded in the glassy matrix confer stability to these slowly dried embryos.
RESUMO
Isolated immature maize (Zea mays L.) embryos have been shown to acquire tolerance to rapid drying between 22 and 25 d after pollination (DAP) and to slow drying from 18 DAP onward. To investigate adaptations in protein profile in association with the acquisition of desiccation tolerance in isolated, immature maize embryos, we applied in situ Fourier transform infrared microspectroscopy. In fresh, viable, 20- and 25-DAP embryo axes, the shapes of the different amide-I bands were identical, and this was maintained after flash drying. On rapid drying, the 20-DAP axes had a reduced relative proportion of alpha-helical protein structure and lost viability. Rapidly dried 25-DAP embryos germinated (74%) and had a protein profile similar to the fresh control axes. On slow drying, the alpha-helical contribution in both the 20- and 25-DAP embryo axes increased compared with that in the fresh control axes, and survival of desiccation was high. The protein profile in dry, mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in the 20-DAP embryo axes and much less so in the 25-DAP axes. After slow drying, low plasma membrane permeability ensued in both the 20- and 25-DAP axes. We conclude that slow drying of excised, immature embryos leads to an increased proportion of alpha-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress.
