Effects of freezing on plant mesophyll cells.

Freezing and thawing of leaves of herbaceous plants leads to damage when the freezing temperature falls below a certain tolerance limit, which depends on the plant species and state of acclimation. Such damage is expressed as an irreversible inhibition of photosynthesis observed after thawing. In frost-damaged leaves the capacity of photosynthetic reactions of the thylakoid membranes is impaired. Particularly, the water-oxidation system, photosystems II and I are inhibited. However, it appears that CO2 assimilation is more readily affected by freezing stress than the activity of the thylakoids. The inhibition of CO2 fixation seen in initial stages of damage seems to be independent of thylakoid inactivation. This can be shown by chlorophyll fluorescence analysis made simultaneously with measurement of CO2 assimilation. Fluorescence emission by leaves is strongly influenced by carbon assimilation activity, namely via the redox state of the photosystem II electron acceptor QA (QA-dependent quenching) and via energization of the thylakoid membranes depending on the transthylakoid proton gradient (energy-dependent quenching). Resolution of these components of fluorescence changes provides insight into alterations of the CO2 fixing capacity of the chloroplasts and properties of the thylakoids. The effects of freezing and thawing were studied in detail with isolated mesophyll protoplasts prepared from both non-hardened and cold-acclimated plants of Valerianella locusta L. Freezing damage was characterized by various parameters such as plasma membrane integrity, photosynthetic CO2 assimilation, chlorophyll fluorescence emission and activities of thylakoids isolated from the protoplasts. All tests indicated a substantially increased frost tolerance of protoplasts obtained from cold-acclimated as compared to non-hardened leaves. CO2 assimilation and related fluorescence changes were the most freezing-sensitive parameters in both types of protoplasts. Inactivation of CO2 assimilation was correlated neither to the disintegration of the plasma membrane nor to inactivation of the thylakoids. Experimental data indicate that freeze-thaw treatment affected the light-regulated enzymes of the carbon reduction cycle, such as fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase. Inhibition of light-activation of these enzymes may be based on altered properties of the chloroplast envelope.

Inactivation of the photosynthetic carbon reduction cycle in isolated mesophyll protoplasts subjected to freezing stress.

Isolated mesophyll protoplasts from Valerianella locusta L. were subjected to freeze-thaw cycles. Subsequently, steady-state pool sizes of (14)C-labeled intermediates of the photosynthetic carbon reduction cycle were determined by high performance liquid chromatography. Protoplasts in which CO2 fixation was inhibited by preceding freezing stress, showed a strong increase in the proportion of fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate and triose phosphates. These results indicate an inhibition of the activities of stromal fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase. Furthermore, freezing stress caused a slight increase in the proportion of labeled ribulose-1,5-bisphosphate, which may be based on an inhibition or ribulose bisphosphate carboxylase activity. It was shown earlier (Rumich-Bayer and Krause 1986) that freezing-thawing readily affects photosynthetic CO2 assimilation independently of thylakoid inactivation. The present results are interpreted in terms of an inhibition of the light-activation system of the photosynthetic carbon reduction cycle, caused by freezing stress.

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO2-dependent O2 evolution (representing net photosynthetic CO2 fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.