Mechanical freeze‐thaw damage and frost hardening in leaves and isolated thylakoids from spinach. I. Mechanical freeze‐thaw damage in an artificial stroma medium

Abstract

Freeze‐thaw damage to thylakoids in spinach leaves has been simulated in vitro, using a complex, defined artificial stroma medium. The resulting mechanical damage was quantified by measuring the loss of the marker protein plastocyanin from the thylakoid lumen, which is released as a result of membrane rupture. Loss of plastocyanin was already apparent at 0°C and became more severe at subzero temperatures. The time course of plastocyanin loss during freezing was biphasic: after an initial rapid loss, plastocyanin release was linearly dependent on incubation time. In short‐term experiments a linear dependence on freezing temperature was observed. Solute diffusion into the thylakoids, leading to influx of water and eventually membrane rupture, has been observed in vitro as well as after freezing of leaves.

Freezing of isolated thylakoid membranes in complex media. V. Inactivation and protection of electron transport reactions

“…This result is in agreement with data obtained during freezing of intact leaves (Heber et al 1973, Thebud and Santarius 1981, Klosson and Krause 1981, Krause et al 1982)and of isolated panied by release of plastocyanin which can be prevented by the addition of common lowmolecular-weight cryoprotectants prior to freezing , 1988.…”

Galactose-Specific Lectins Protect Isolated Thylakoids against Freeze-Thaw Damage

“…The first, rapid phase was not influenced. A slow, linearly time-dependent release of PC is also evident at O°C in the absence of ice formation (Hincha and Schmitt, 1988). This release was also reduced in the presence of 200 pg mL-' of agglutinin (Fig.…”

“…The prevention of the loss of PC from isolated thylakoids can be used as an indicator for the preseiice of cryoprotective proteins during freezing (Hincha and Schmitt, 1992b). If the membrane vesicles are suspended in a simplified artificial stroma medium (Hincha and Schmitt, 1988), the action of proteins can be assayed under conditions that are similar to those in vivo (Hincha and Schmitt, 1992a). Figure 1 shows that under these conditions the Gal-specific agglutinin from Abrus precatorius seeds was clearly a cryoprotective protein.…”

“…In the artificial stroma medium used in these experiments, freeze-thaw damage shows a characteristic biphasic time dependence (Hincha and Schmitt, 1988). The A. precatorius agglutinin only influenced the second, slow phase of PC release (Fig.…”

“…Freeze-thaw damage to thylakoids both in vitro and in vivo is accompanied by the release of the electron transport protein PC from the intramembrane spaces due to transient rupture of the membranes (Hincha et al, 1987;Hincha and Schmitt, 1988). This leads to an inactivation of photosyrithetic electron transport and light-driven ATP synthesis after a freeze-thaw cycle (for a review, see Hincha and Schmitt, 1992b).…”