Cryopreservation of Chloroplasts and Thylakoids for Studies of Protein Import and Integration

Abstract

Furthermore, the ability to preserve active chloroplasts for extended periods will obviate the problem of seasonal variation in import competence of chloroplasts and permit investigators to comparatively analyze plastids isolated from different plant tissue at different times.Recent studies of plastid protein biogenesis have also focused on stromal factors necessary for assembly of thylakoid proteins (7,8,14). Purification schemes for the stromal factors require the reconstitution with active thylakoids for activity assays. Such assays are frequently conducted 1 d or several days after the original isolation of stroma and require the isolation of fresh chloroplasts for thylakoid preparation. Accordingly, it would be very helpful if thylakoids from the original chloroplast preparation could be frozen for later use.Methods for preservation of photosynthetically competent thylakoids have been available for several years (12,19,20), but until now, methods for preserving intact chloroplasts have not been described. The present communication describes protocols for the preservation of intact chloroplasts and thylakoids that are active for subsequent studies ofprotein import and integration, respectively.Recent advances in our understanding of the mechanisms of chloroplast protein biogenesis derive primarily from the availability of in vitro biochemical assays for chloroplast protein import and in organello protein synthesis. Active chloroplasts are prepared for these assays by homogenization of fresh tissue followed by a combination of differential and density gradient centrifugation (25). Chloroplasts are then incubated under conditions that either promote synthesis of proteins within the organelle (3, 17) or allow the import of cytoplasmically synthesized plastid proteins into the organelle (16). Isolation of chloroplasts from amenable plant species, such as spinach and peas, is not difficult but requires about two intensive hours from start to finish. This preparation time has become a limiting factor as investigations of transport mechanisms have become more sophisticated and the duration ofexperiments has increased. A ready source ofpreserved but active chloroplasts would greatly facilitate such studies.

Cryopreservation of Chlorophyll Synthesis and Apoprotein Stabilization in Barley Etioplasts

“…We further tested Suc concentrations between 20 and 50% (w/v) in washing buffer for a cryopreserving influence on etioplast intactness and found that these concentrations had no effect. Optimum cryopreservation of barley etioplasts and pea chloroplasts required the same concentration of EG, whereas a lower concentration of DMSO was sufficient to cryoprotect barley etioplasts (Yuan et al, 1991). Although cryopreservation of pea chloroplasts and barley etioplasts was poor in the presence of glycerol, twice the yield of etioplasts could be obtained in 20% glycerol.…”

“…DMSO and EG were shown to be most effective for the cryopreservation of photosynthetic activity (Farkas and Malkin, 1979), for protein import and integration in pea chloroplasts (Yuan et al, 1991), and for protein ímport and in organello protein synthesis in tobacco mitochondria (Schieber et al, 1994). Here we show that the synthesis of Chl and the Chl-dependent stabilization of Chl-apoproteins can be successfully studied in cryopreserved, intact barley etioplasts.…”

“…These data could indicate in general that the envelope membranes of plant mitochondria are less sensitive to interna1 ice crystal formation and osmotic imbalances during freezing than the envelope membranes of etioplasts and chloroplasts. This is also indicated by the finding that, upon freezing in the absence of cryoprotectants, neither barley etioplasts nor pea chloroplasts could be re-isolated intact, whereas about 50% of the tobacco mitochondria retained their intactness (Yuan et al, 1991;Schieber et al, 1994). Because of the poor cryopreservation of barley etioplasts in glycerol or SUC, only the plastids preserved in DMSO* or EG* were utilized in subsequent experiments.…”

“…Although there are still no absolute rules for cryopreservation, agents such as EG or DMSO, in combination with fast cooling rates, have been reported to be superior to glycerol and slow cooling rates (Farkas and Malkin, 1979;Yuan et al, 1991;Schieber et al, 1994).…”

Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70.

“…3H-labeled pLHCP was prepared either by in vitro transcription (13) and translation (12,18) or by overexpression in E. coli in the presence of [3H]leucine (38). Chloroplasts were isolated from 9-to 10-day-old pea (Laxton's Progress 9) seedlings as described (12,20) and were resuspended in import buffer (50 mM Hepes/KOH, pH 8/0.33 M sorbitol). Lysates, thylakoids, and SE were prepared from isolated chloroplasts (13,20).…”